Functional and morphological changes associated with the ageing of primary cultures of embryonic adrenal gland cells derived from the Pekin duck. The morphological and functional changes associated with ageing were studied in adrenal steroidogenic cells derived from duck embryos. Cells grown for not more than three days had structural characteristics similar to their counterparts in vivo; they contained numerous lipid droplets and mitochondria, an abundant smooth endoplasmic reticulum, an even network of microtubules, and microfilaments that formed extensive and elaborate systems of parallel stress fibers. After the 3rd day of growth in culture, many of the cells started to decrease in size and become elongated; the older cells showed less well-defined actin filaments and contained elongated mitochondria, fewer lipid droplets, less smooth endoplasmic reticulum, and swollen cisternae of rough endoplasmic reticulum. The proliferative capacity of the cells was the same when they were cultured in either the presence or the absence of 1-24 ACTH. After the first day of growth in culture, the steroidogenic capacity of the cells declined and the addition of 1-24 ACTH to the growth medium did not prevent changes in their structure and function. The decline in steroidogenic capacity occurred both in terms of the amount of hormone released into the culture medium and in the ability of the cells to respond when incubated in buffer containing 1-24 ACTH. Since the basal unstimulated rates of corticosteroid production also declined as the cells aged, it is probable that the steroidogenic deficiency occurs at a site distal to the corticotropin receptor; this is also consistent with the ultrastructural observations that suggest a relationship between the morphological changes and the decline in steroidogenic capacity as the cells age.


[Isolation of HAV from sea clam].

[Isolation of HAV from sea clam]. Three strains of HAV were isolated directly from clams (Arca Subcrensta Lisckke) collected from sea with cell culture and identified by electron microscopy, immunofluorescent test, ELISA, HAV-cDNA hybridization and neutralization test. The strains were stably passed in 2BS cells with the titer of 4.0-5.0 TCID50/ml. Results indicated that the clams were contaminated by HAV and may cause an outbreak of hepatitis A.


Treatment of migraine attacks with subcutaneous sumatriptan: first placebo-controlled study. The Subcutaneous Sumatriptan International Study Group. The results of the very first large-scale placebo-controlled dose-response trial with the novel selective 5-hydroxytryptamine1-like (5HT1-like) receptor agonist sumatriptan are presented. We studied the efficacy and tolerability of subcutaneous injections of 1 mg, 2 mg and 3 mg of sumatriptan in alleviating migraine attacks in a double-blind, placebo-controlled, parallel-group, multicentre clinical trial. Six-hundred and ninety patients were randomized and 685 received study medication. At 30 min, reduction of headache severity to mild or none (primary efficacy endpoint) was achieved in 22% (95% CI: 15-28%) of placebo-treated patients and in 39% (CI: 31-46%) of patients treated with 1 mg sumatriptan, 44% (CI: 36-51%) treated with 2 mg sumatriptan and 55% (CI: 48-63%) treated with 3 mg sumatriptan. Differences from placebo were 17% (CI: 8-27%) for 1 mg sumatriptan, 22% (CI: 13-32%) for 2 mg sumatriptan and 34% (CI: 24-44%) for 3 mg sumatriptan (p < 0.001 for all three comparisons). Other migraine symptoms were also more effectively treated by sumatriptan than by placebo. Subsequently, an open-label 3 mg dose subcutaneous sumatriptan was given to partial or non-responders. Thirty minutes after this open dose the response rate to sumatriptan had improved to between 70 and 80%. Adverse events after sumatriptan were minor and short-lived. We conclude that subcutaneous sumatriptan is well tolerated in doses up to 3 + 3 mg and may rapidly abort migraine attacks.


The role of topoisomerase IV in partitioning bacterial replicons and the structure of catenated intermediates in DNA replication. Mutants in bacterial topoisomerase (topo) IV are deficient in chromosomal partitioning. To investigate the basis of this phenotype, we examined plasmid DNA topology in conditionally lethal topo IV mutants. We found that dimeric catenated plasmids accumulated in vivo after topo IV inhibition. The catenanes were supercoiled, contained from 2 to > 32 nodes, and were the products of DNA synthesis. Electron microscopy and recombination tests proved that the catenanes have the unique structure predicted for replication intermediates. These data provide strong evidence for a model in which unlinking of the double helix can occur in two stages during DNA replication and for the critical role of topo IV in the second stage. The interlocks in the catenanes appear to be sequestered from DNA gyrase, perhaps by compartmentalization in an enzyme complex dedicated to partitioning.


Activation of the MAP kinase pathway by the protein kinase raf. Both MAP kinases and the protein kinase p74raf-1 are activated by many growth factors in a c-ras-dependent manner and by oncogenic p21ras. We were therefore interested in determining the relationship between MAP kinases and raf. The MAP kinase ERK2 is activated by expression of oncogenically activated raf, independently of cellular ras. Overexpressed p74raf-1 potentiates activation of ERK2 by EGF and TPA. MAP kinase kinase inactivated by phosphatase 2A treatment is phosphorylated and reactivated by incubation with p74raf-1 immunoprecipitated from phorbol ester-treated cells. We conclude that raf protein kinase is upstream of MAP kinases and is either a MAP kinase kinase kinase or a MAP kinase kinase kinase kinase.


Inactivation of MyoD in mice leads to up-regulation of the myogenic HLH gene Myf-5 and results in apparently normal muscle development. The myogenic basic HLH transcription factor family of genes, composed of MyoD, myogenin, Myf-5, and Myf-6, are thought to regulate skeletal muscle differentiation. To understand the role of MyoD in myogenesis, we have introduced a null mutation of MyoD into the germline of mice. Surprisingly, mice lacking MyoD are viable and fertile. Histological examination of skeletal muscle failed to reveal any morphological abnormalities in these mice. Furthermore, Northern analysis revealed normal levels of skeletal muscle-specific mRNAs. Significantly, Myf-5 mRNA levels are elevated in postnatal mutant mice. Normally, Myf-5 expression becomes markedly reduced at day 12 of gestation when MyoD mRNA first appears. This suggests that Myf-5 expression is repressed by MyoD. Our results indicate that MyoD is dispensable for skeletal muscle development in mice, revealing some degree of functional redundancy in the control of the skeletal myogenic developmental program.


Detection and biochemical characterization of the mouse mammary tumor virus 7 superantigen (Mls-1a). Mouse mammary tumor viruses encode superantigens that bind to class II major histocompatibility complex proteins and engage T cells that bear particular V beta s. Among these superantigens is the long known, but previously uncharacterized, Mls-1a product, encoded by Mtv-7. Using a monoclonal antibody, we detect the Mtv-7 superantigen on the surface of activated B cells, but not on T cells or resting B cells. The superantigen is synthesized as a 45 kd transmembrane glycoprotein precursor, but is proteolytically processed to yield an 18.5 kd surface protein that we suggest is the functional form of the superantigen.


Yeast pheromone receptor endocytosis and hyperphosphorylation are independent of G protein-mediated signal transduction. When alpha factor binds to the yeast alpha factor receptor a signal is transmitted via a tripartite G protein that prepares the cell for conjugation. As a result of alpha factor binding the receptor also undergoes a regulated internalization and hyperphosphorylation. Using cells that lack activity of this tripartite G protein, we show that G protein-mediated pheromone signal transduction is not necessary for regulation of receptor internalization or hyperphosphorylation. Therefore, the processes of signal transduction and down regulation can be uncoupled. We propose that binding of alpha factor to its receptor results in a receptor conformation change that permits receptor hyperphosphorylation and interaction with the endocytic machinery.


TNF activates NF-kappa B by phosphatidylcholine-specific phospholipase C-induced "acidic" sphingomyelin breakdown. In this paper, we describe a phospholipid transmission pathway mediating tumor necrosis factor (TNF) activation of the nuclear transcription factor kappa B (NF-kappa B). Central to this TNF signaling route is the second messenger-like molecule ceramide, which is generated by sphingomyelin (SM) breakdown catalyzed by a sphingomyelinase (SMase). SMase activation is secondary to the generation of 1,2-diacylglycerol (DAG) produced by a TNF-responsive PC-specific phospholipase C (PC-PLC). The functional coupling of these two C type phospholipases is revealed by D609, a selective inhibitor of PC-PLC. SMase itself, or SMase-inducing regimens such as exogenous PLC or synthetic DAGs, induces NF-kappa B activation at pH 5.0, suggesting the operation of an acidic SMase. A model is proposed in which a TNF-responsive PC-PLC via DAG couples to an acidic SMase, resulting in the generation of ceramide, which eventually triggers rapid induction of nuclear NF-kappa B activity.


The high mobility group protein HMG I(Y) is required for NF-kappa B-dependent virus induction of the human IFN-beta gene. In this paper, we show that both NF-kappa B and the high mobility group protein I(Y) (HMG I(Y)) are required for virus induction of the human interferon-beta (IFN-beta) gene. NF-kappa B binds to the terminal regions of a 10 bp regulatory sequence through contacts in the major groove. while HMG I(Y) recognizes the central region of the same sequence through contacts in the minor groove. Mutations that interfere with binding of either protein decrease the level of virus induction, and activation of the gene can be blocked by either NF-kappa B or HMG I(Y) antisense RNA. HMG I(Y) stimulates the binding of NF-kappa B to the IFN-beta promoter, and it may also function as a promoter-specific accessory factor for NF-kappa B transcriptional activity.


The capture of a DNA double helix by an ATP-dependent protein clamp: a key step in DNA transport by type II DNA topoisomerases. The binding of linear and circular forms of DNA to yeast DNA topoisomerase II or its complex with AMPPNP, the nonhydrolyzable beta,gamma-imido analog of ATP, was carried out to probe the ATP analog-induced conformational change of the enzyme. Binding of the ATP analog is shown to convert the enzyme to a circular clamp with an annulet, through which only a linear DNA can pass; subsequent circularization of the bound linear DNA forms a salt-stable catenane between the protein circular clamp and the DNA ring. Analysis of catenane formation between a small DNA ring originally bound to the topoisomerase and a large DNA ring subsequently added, under conditions such that the two do not exchange, supports a model in which a second DNA double-helix can enter the open jaws of a DNA-bound protein clamp, and the closure of the jaws upon ATP-binding traps the second duplex and transports it through an enzyme-operated gate in the first DNA duplex.


CD28 and staphylococcal enterotoxins synergize to induce MHC-independent T-cell proliferation. The bacterial exotoxins staphylococcal enterotoxin A and B (SEA and SEB) mediate disease through their effects on T lymphocytes. In this manuscript we have demonstrated that both SEA and SEB can directly activate purified T cells in the absence of accessory cells as determined by a transition from G0 to G1 and induction of IL-2 receptor expression. However, neither SEA nor SEB alone was sufficient to result in T-cell proliferation. The induction of T-cell proliferation by SEB or SEA required the addition of a second costimulatory signal. This could be provided by either accessory cells or monoclonal antibody stimulation of CD28. As previously reported, T-cell proliferation induced by enterotoxin in the presence of accessory cells was partially inhibited by a blocking antibody against class II MHC. In contrast, in purified T cells when costimulation was provided through CD28, proliferation was not inhibited by class II antibody, and HLA-DR expression was not detectable. In addition, costimulation through CD28 was partially resistant to the effects of cyclosporin A. These results demonstrate that CD28 costimulation is sufficient to induce proliferation of enterotoxin-activated T cells, and that this effect is independent of class II MHC expression.


Blocking the interleukin-1 receptor inhibits leukotriene B4 and prostaglandin E2 generation in human monocyte cultures. Interleukin-1 is a potent stimulator of arachidonic acid (AA) metabolism and this activity could be attributed to the activation of the prostaglandin-forming enzyme cyclooxygenase or of the arachidonic-releasing enzyme phospholipase A2 or both. Prostaglandin E2 (PGE2), a cyclooxygenase product, and LTB4 (5-(S),12-(R)-dihydroxy-6,14-cis-8,10-trans-eicosatetraenoic acid), a lipoxygenase product, are potent mediators of inflammation. Recently a new cytokine produced by macrophages and named interleukin-1 receptor antagonist (IL-1ra) (MW 22,000 Da) which specifically binds and blocks IL-1 receptors, has proven to be a potent inflammatory inhibitor. In our studies we found that monocyte suspensions, pretreated with hrIL-1ra at increasing concentrations (0.25-250 ng/ml) for 10 min and then treated with LPS in an overnight incubation inhibits, in a dose-dependent manner, the generation of LTB4 as measured by the highly sensitive radioimmunoassay method. In monocytes pretreated with hrIL-1ra (250 ng/ml) for 10 min and treated with arachidonic acid (10(-5)-10(-9) M) and LPS overnight, the release of LTB4 was partially inhibited when compared to hrIL-1ra-untreated cells. Moreover, hrIL-1ra (250 ng/ml) caused a partial inhibition of monocyte LTB4 production when the cells were activated with AA (10(-7) M) and then treated with IL-1 beta (5 ng/ml) overnight or 24 hr incubation. In addition, human monocytes pretreated for 10 min with increasing doses of hrIL-1ra (0.25-250 ng/ml) and then treated with hrIL-1 alpha (5 ng/ml) or beta (5 ng/ml) for 18 hr, also resulted in the inhibition of PGE2 generation as measured by RIA when compared with hrIL-1ra-untreated cells. When the cells were treated with hrIL-1ra (250 ng/ml) and activated for 18 and 48 hr with increasing doses of hrIL-1 beta a strong inhibitory effect was found on PGE2 production. HrIL-1ra used at 15 ng/ml gave a partial inhibition of LTB4 generation, after LPS (1-100 ng/ml) treatment, while NDGA totally blocked the production of LTB4. Moreover, PGE2 released by macrophages activated with LPS (100 ng/ml) or hrIL-1 beta (5 ng/ml) at 18 hr incubation time was strongly inhibited when hrIL-1ra (250 ng/ml) was used. These data suggest that the inhibition of LTB4 and PGE2 by this new macrophage-derived monokine IL-1ra occurs through the block of the IL-1 receptor, rather than phospholipase A2, and thus IL-1ra may offer a potential therapeutic approach to inflammatory states.


Enhanced antiviral activity and altered subcellular distribution of magnesium/poly r(A-U) combinations. When Mg2+ or ethidium bromide (EB) were combined with poly r(A-U) at a ligand/ribonucleotide ratio of 1/4, the antiviral activity of the Mg2+ and EB increased 136-fold and 154-fold. Eriochrome Blue SE was employed to visualize the subcellular distribution of Mg2+ following co-incubation of Human Foreskin Fibroblasts (HSF) with Mg2+ alone or with the Mg2+/poly r(A-U) combination. Phase contrast micrographs of these Mg(2+)-treated HSF cells as well as phase contrast and fluorescence micrographs of EB-treated or EB/poly r(A-U)-treated HSF cells illustrated that the Mg2+ (or EB)/poly r(A-U) combinations display altered subcellular distribution with the Mg2+ and EB being localized in the nucleoli and chromatin of the HSF cells. These results suggest that modulation of nuclear processes may be responsible for the enhanced antiviral activity.


Effect of hydroxyl radical on Na(+)-K(+)-ATPase activity of the brain microsomal membranes. Sphingomyelin liposomes and brain microsomes were oxidized by exposure to hydrogen peroxide and ferrous ion. Lipid peroxidation were measured by the formation of thiobarbituric acid- reactive substances (TBAR). Hydroxyl radical was detected using the spin- trapping technique. Incubation of sphingomyelin liposomes with H2O2-Fe2+ resulted in an increase in the formation of TBAR. Na(+)-K(+)-ATPase activity was markedly inhibited and the SH group content decreased during incubation of microsomes in the presence of H2O2-Fe2+. Sodium ferulate effectively inhibited TBAR formation, protected Na(+)-K(+)-ATPase activity and prevented the oxidative modification of SH groups. Spin-trapping experiments showed that sodium ferulate effectively scavenged the hydroxyl radicals.


Control of organelle transport in melanophores: regulation of Ca2+ and cAMP levels. Melanophores of the cichlid Tilapia mossambica can be induced to aggregate pigment by addition of epinephrine to the medium, suggesting adrenergic control of this transport. The melanophore response to adrenergic stimulation was examined using agonists and antagonists that are highly specific for each alpha-adrenoceptor subclass. The signal transduction mechanism of each subclass is unique: stimulation of alpha 1 receptors results in a rise in intracellular free Ca2+, while alpha 2 stimulation results in decreased cAMP levels [Exton, 1985: Am. J. Physiol. 248:E633-E647]. Each alpha 1 or alpha 2 specific agonist tested showed a dose dependent ability to induce aggregation and each was able to effect complete aggregation of pigment, suggesting that aggregation can be mediated either by elevating Ca2+ or by lowering cAMP. However, in the presence of either an alpha 1 or an alpha 2 receptor antagonist, none of the agonists were able to induce significant aggregation, suggesting that changes in levels of both messengers are required for pigment aggregation in the melanophores. Moreover, experiments in which intracellular levels of Ca2+ or cAMP were perturbed, using BAPTA and forskolin, respectively, indicated that elevating Ca2+ in the presence of high cAMP is not sufficient to induce aggregation and, conversely, that lowering cAMP levels in the presence of reduced Ca2+ is not sufficient to induce pigment aggregation. These data indicate that the concentrations of both cAMP and Ca2+ are important in regulating pigment aggregation in teleost melanophores, and suggest that maximal aggregation of pigment requires altering the levels of both messengers.


Neuropharmacology of sleep induction by benzodiazepines. Although benzodiazepines (BZs) have been the most widely used sedative/hypnotics for many years, the mechanism by which they induce sleep and the neuroanatomic site(s) at which they act have remained poorly understood. Recent characterization of the central BZ-GABAA receptor complex using molecular biological techniques and sleep studies employing new ligands have begun to elucidate these issues. Although alterations in GABAergic activity are involved in the anticonvulsant and myorelaxant properties of BZs, the significance of GABA function in their hypnotic effects is less clear. The pharmacologic significance of receptor subtypes also remains uncertain. A growing body of evidence indicates that the hypnotic effects of BZs involve alterations in potential-dependent calcium ion flux. In terms of neuroanatomy, BZ effects on sleep may result from actions in the anterior hypothalamus as well as brainstem structures including the dorsal raphe nuclei.


A circadian clock of Drosophila: effects of deuterium oxide and mutations at the period locus. Mutations at the period (per) locus (1:1.3; 3B1-2) in Drosophila melanogaster lengthen (perL), shorten (pers), or abolish (per0) overt circadian rhythmicity. Deuterium oxide lengthens the free-running circadian period. We tested the effects of deuterium on three mutants of the per gene (pers, perL, and per0) and wild-type Drosophila melanogaster (per+) to assess interactions. With increasing concentrations of deuterium, the free-running circadian period of locomotor activity rhythms increased. The dose-response was linear in all genotypes tested. With increasing dosages of deuterium, circadian rhythms became weaker as evidenced by the signal-to-noise ratio (SNR). Genotype and deuterium changed circadian period length independently and additively, showing no interaction. SNRs for all genotypes converged on a low level as deuterium concentration increased. Deuterium increased life span, except at high concentrations (40 and 50%).


Identification of UAS elements and binding proteins necessary for derepression of Saccharomyces cerevisiae fructose-1,6-bisphosphatase. Fructose-1,6-bisphosphatase is a key enzyme in gluconeogenesis and the FBP1 gene is not transcribed during growth with glucose. Genetic analysis indicated a positive regulation of FBP1 expression after exhaustion of glucose. By linker-deletion analysis, two upstream activation sites (UAS1 and UAS2) were localized and the respective UAS-binding factors (DAP I and DAP II for derepression activating protein) were identified by gel retardation. UAS1 and UAS2 span about 30 bp each, and are separated by approximately 30 bp. Both UAS sites act synergistically. Although UAS1 showed some similarities to the DNA-binding consensus for the general yeast activator Rap1, competition experiments and DEAE-chromatography proved that DAP I and Rap1 correspond to different proteins. Gel retardation by DAP I depended on carbon sources and did not occur in cells growing logarithmically with glucose, whereas a strong retardation signal was obtained with ethanol-grown cells. The present results suggest that DAP I and DAP II are the final regulatory elements for glucose derepression.


Effects of cadmium, copper and metallothionein synthesis inhibiting and stimulating compounds on zinc uptake and accumulation in rat hepatoma HTC cells. Experiments were carried out to investigate the uptake and accumulation of Zn in rat hepatoma HTC cells, as affected by interfering metals (Cd, Cu), metallothionein synthesis inhibiting compounds (Actinomycin D, cycloheximide) and metallothionein synthesis stimulating compounds (dexamethasone, dibu-cAMP). Cell viability was tested under all experimental conditions by the measurement of LDH leakage, K+ uptake and total cell protein. Determinations of Zn were performed by AAS (total Zn) or by gamma-ray spectrometry (65Zn). Metallothionein analysis was carried out by Cd-saturation tests. The results indicate that cellular responses in rat hepatoma HTC cells with respect to the uptake and accumulation of 65Zn are fully comparable with literature data existing for 65Zn accumulation in rat hepatocytes, under all experimental conditions applied. Cu2+ and dibutyryl-cAMP did not significantly affect rates of 65Zn accumulation. Cd2+, Actinomycin D and cycloheximide reduced 65Zn uptake, but dexamethasone additions resulted in increased 65Zn accumulation in the cells. Effects on 65Zn were shown both in cytosolic and in the membranes/organelles cell fractions. HPLC chromatography in control cells suggested that newly accumulated cytosolic 65Zn was predominantly MT-associated. Dexamethasone-induced 65Zn accumulation could not be related to elevated cellular MT levels, nor were the total cytosolic Zn levels significantly affected. Non-specific attenuations in MT levels (Actinomycin D, cycloheximide and dibu-cAMP) yielded linear relations between cytosolic 65Zn and MT levels, without any change in cytosolic Zn (AAS). Combined addition of Cd and dexamethasone yielded elevated MT levels, but severely reduced total cytosolic Zn and 65Zn concentrations. The results further indicate the non-Zn-specific nature of dexamethasone-action and suggest the relatively easy Zn-complexing and Zn-release of MT. The simultaneous determinations of total cytosolic zinc and cytosolic 65Zn levels showed that the application and sole measurement of radiotracers may yield only one-sided views of what is actually present or occurring in the cells.


Perturbation effect of eight calcium channel blockers on liposomal membranes prepared from rat brain total lipids. EPR spectroscopy of phosphatidylcholine or stearic acid labeled at the doxyl group at the 16-carbon position was used to compare the perturbation effect of eight calcium channel blockers (CB) on overall dynamics/disorder of the hydrophobic part of liposome membranes prepared from rat brain total lipids at the drug/lipid molar ratio of 1/2. Nifedipine (NIF), nimodipine, niludipine and nitrendipine had a minor effect on the dynamics/disorder of the liposome membranes, whereas the disordering effect of verapamil (VER), mepamil, gallopamil and diltiazem was more pronounced. Concentration dependence of the overall disordering effect of VER on liposomal membranes was found at the VER/lipid ratio greater than 0.02 and for the tranquilizer thioridazine greater than 0.005. VER exerted a disordering effect at the hydrophobic part of synaptosomal membranes at concentrations greater than or equal to 0.32 mmol/l, whereas NIF did not exhibit a disordering effect even at concentrations of 10-20 mmol/l.


Rational design and synthesis of phospholipids for the two-dimensional crystallization of DNA gyrase, a key element in chromosome organization. Properties required of lipids for two-dimensional crystallization of proteins on lipid layers at the air/water interface are discussed in terms of molecular structure. These properties are related to essential features of the overall system such as (i) the fluidity and stability of the lipid film, (ii) the affinity of the protein to be crystallized for the lipids and (iii) the accessibility of the protein to the ligand in the lipid layer as well as (iv) technical constraints of the crystallization technique. The resulting ideas were tested through the rational design and synthesis of original phospholipid structures linked to novobiocin subsequently used in the production of two-dimensional crystals of DNA gyrase (B subunit), a prokaryotic type II DNA topoisomerase.


Studies on glutathione transferases belonging to class pi in cell lines with different capacities for conjugating (+)-7 beta, 8 alpha-dihydroxy-9 alpha, 10 alpha-oxy-7,8,9,10-tetrahydrobenzo[a]pyrene. The glutathione transferases (GST) belonging to class pi are primarily responsible for the intracellular detoxification of the highly mutagenic and carcinogenic compound (+)-7 beta, 8 alpha-dihydroxy-9 alpha, 10 alpha-oxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE). The aim of the present investigation was to study the nature and function of the GST pi gene in relation to the mutagenicity of BPDE in different cell lines. The studies were performed on three cell lines commonly used in toxicological studies, i.e. rat hepatoma cells (H4IIE), human mammary carcinoma cells (MCF-7) and Chinese hamster lung fibroblasts (V79). Western blotting with antisera against GST pi revealed a high level of reaction with cytosol from V79 and H4IIE cells. Furthermore, cytosol from the V79 cells demonstrated low levels of GSTs belonging to the alpha and mu classes, suggesting that a considerable portion of the total capacity of these cells to conjugate chlorodinitrobenzene (CDNB) was provided by GST pi. The level of mRNA for GST pi, as measured by Northern blots, was high in V79 and H4IIE and undetectable in the MCF-7 cell line. Analysis of the DNA fragment patterns using a series of restriction enzymes, revealed that all three cell lines have the pi class gene, although with different band patterns. The findings with H4IIE and MCF-7 cells with respect to their expression of the GST pi gene and their ability to conjugate BPDE were in agreement with the mutagenic effects of BPDE, produced by metabolic activation of (-)-7 beta, 8 alpha-dihydroxybenzo[a]-pyrene in the cells. In contrast, V79 cells although expressing high levels of GST pi, showed no ability to conjugate BPDE or to inhibit the mutagenicity of this compound. Based on these results, we suggest that V79 Chinese hamster lung cells contain a GST pi with a different substrate specificity from those of the human and rat GST pi enzymes.


The effect of sodium selenite on cell proliferation and transformation of primary rat tracheal epithelial cells. The effects of sodium selenite (Na2SeO3) on cell proliferation and the development of preneoplastic transformed variants were studied in primary cultures of rat tracheal epithelial cells. Results revealed a biphasic effect of Na2SeO3 on cell proliferation: at concentrations between 6 x 10(-8) and 6 x 10(-6) M, it stimulated and at concentrations of approximately 2 x 10(-5) and above it inhibited cell proliferation (presumably due to toxicity). Nontoxic concentrations of Na2SeO3 (6 x 10(-8) -6 x 10(-7) M) significantly reduced the spontaneous transformation frequency. Transformation induced by the tobacco-specific nitrosamine 4-(methyl-nitrosamino)-1-(3-pyridyl)-1-butanone (NNK) was effectively inhibited by nontoxic as well as toxic concentrations of Na2SeO3. Treatment of cultures with Na2SeO3 after cessation of NNK exposure, i.e. during the selection period, also significantly reduced the transformation frequency. These experiments show that the inhibition of transformation by Na2SeO3 is not the result of an antiproliferative effect. They further indicate that the inhibitory effect occurs even when the chemical treatment occurs during the 'postinitiation' phase. Thus the inhibition of transformation by Na2SeO3 cannot solely be explained by its effects on drug metabolism.


Reduced proliferative activity of polyploid cells in primary hepatocellular carcinoma. The proliferative activity of tumor cells differing in DNA content (ploidy) and nuclearity was investigated in primary hepatocellular carcinomas of the rat by flow cytometric analysis of collagenase-isolated cells immunostained after labelling with bromodeoxyuridine (BrdU) in vivo. The diploid cell fraction in these euploid tumours was higher than in normal liver, and the rate of binucleation as well as the proliferative activity of the binuclear cells was very low. The highest proliferative activity (BrdU labelling index) was found among the diploid tumour cells. The activity in mononuclear tetraploid and octoploid cells was reduced in inverse proportion to their increasing DNA content, possibly suggesting a loss of proliferative potential associated with polyploidization. There was a significant correlation between the proliferative activity of hepatocellular carcinoma cells and nonparenchymal liver cells in the different tumours, indicating that different cell types within a tumour may respond to common growth stimuli. Treatment of tumour-bearing rats with a promoting carcinogen (2-acetylaminofluorene) resulted in significant stimulation of tumour cell proliferation (all ploidy classes), whereas the proliferation of non-parenchymal (stromal) cells in the tumour was slightly inhibited.


Vitamin E protection against nitrosamine-induced esophageal tumor incidence in mice immunocompromised by retroviral infection. Retrovirally induced immunosuppression may elevate the incidence of chemically induced cancers. A proposed hypothesis to explain this relationship is the increased free radical activity observed during retroviral infection and carcinogen activation. We previously found that vitamin E retarded growth of esophageal tumors accompanied by reductions of free radical products. This study investigated the contribution that retroviral immunosuppression has on esophageal cancer induced by the carcinogen N-nitrosomethylbenzylamine (NMBzA), and the response that increased levels of dietary vitamin E has on this induced carcinogenesis. Female C57BL/6 mice received NMBzA or vehicle (corn oil) i.p. weekly for 3 weeks. Then some of the mice were infected with LP-BM5 murine retrovirus and fed diets containing 30 IU vitamin E or 172 IU vitamin E/kg of diet. As an assessment of free radical activity, exhaled ethane was measured prior to killing the animals at 26 weeks. Esophagi from the various mice groups were assessed for size and frequency of tumors. Livers homogenates were analyzed for vitamins A and E, lipid fluorescence, conjugated dienes and malondialdehyde. Hepatic levels of vitamin A and E were decreased (P < 0.05) and indices of lipid peroxidation were greater (P < 0.05) in NMBzA-treated mice relative to controls. Lipid peroxidation and serum transaminases (ALT and AST) were greatest in mice given NMBzA and infected with the retroviruses. Incidence of esophageal tumors were also greatest in the NMBzA-treated, immunocompromised animals. Mice fed vitamin E-supplemented diets showed increased (P < 0.05) hepatic concentrations of vitamin E and vitamin A, decreased activities of serum transaminases, decreased indices of lipid peroxidation, and decreased size and frequency of esophageal tumors in both the immunocompromised and non-immunocompromised mice. These results suggest that vitamin E plays an antioxidant function that retards the incidence of esophageal cancers in immunocompromised and non-immunocompromised animals.


An alternative theory of tissue specificity by tumor promotion of okadaic acid in glandular stomach of SD rats. To challenge the theory of tissue specificity of tumor promoters, the biochemical and tumor promoting effects of okadaic acid (OA), a potent tumor promoter on mouse skin, were studied in the mucosa of rat glandular stomach. OA strongly inhibited protein phosphatases 1 and 2A, and increased 4-fold the phosphorylation of elongation factor 2 in vitro in the mucosa. Intubation of 10 micrograms (12.4 nmol) OA induced ornithine decarboxylase in the mucosa. Tumor promotion of OA was studied in the glandular stomach initiated with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) in a two-stage carcinogenesis experiment. OA in drinking water, 10 micrograms (12.4 nmol) per rat per day from weeks 9-55 of the experiment, and 20 micrograms (24.8 nmol) from weeks 56-72, significantly enhanced development of the neoplastic changes in the glandular stomach (P < 0.05). The neoplastic changes included adenomatous hyperplasias and adenocarcinomas, both of which correspond to papillomas and carcinomas in a two-stage mouse skin carcinogenesis experiment. The percentages of neoplastic change-bearing rats of the groups treated with MNNG plus OA, MNNG alone or OA alone were 75.0, 46.4 and 0% respectively. OA enhanced tumorigenesis in the MNNG-initiated glandular stomach of rats through the same mechanisms of action as in mouse skin. The OA pathway mediated through inhibition of protein phosphatases 1 and 2A is applicable to various organs as a general mechanism of tumor promotion.


The onset of oncogene hypomethylation in the livers of rats fed methyl-deficient, amino acid-defined diets. This study examines proto-oncogene hypomethylation in rat livers during the early stages of hepatocarcinogenesis by dietary methyl deprivation in the presence and absence of initiation by diethylnitrosamine (DEN). Male weanling F344 rats were fed a complete diet, or a diet deficient in methionine and choline (MDD). Half the animals in each dietary group were given a single initiating dose of DEN (20 mg/kg). Animals from each of the treatment groups were killed at 1, 3, 8, 16 and 32 weeks, and hepatic DNA was isolated. This DNA was digested with the restriction enzymes MspI and HpaII to determine the extent of methylation of the CCGG sequences in c-Ha-ras, c-Ki-ras and c-fos proto-oncogenes. The results indicate that the administration of the MDD produced hypomethylation of these proto-oncogenes at all times investigated, independent of DEN initiation. The methylation changes in the c-Ha-ras gene increased in intensity throughout the experiment until at 32 weeks they were similar to the patterns seen in both neoplastic and preneoplastic livers of rats fed the deficient diet for 18 months. These results demonstrate that early, selective hypomethylation of some, but not all, CCGG sites occurs in rats undergoing hepatocarcinogenesis by dietary methyl deprivation.


Tumor-promoting and hepatocarcinogenic effects of 1,4-bis[2-(3,5-dichloropyridyloxy)]benzene (TCPOBOP) in DBA/2NCr and C57BL/6NCr mice and an apparent promoting effect on nasal cavity tumors but not on hepatocellular tumors in F344/NCr rats initiated with N-nitrosodiethylamine. The tumor-promoting and carcinogenic effects of 1,4-bis[2-(3,5-dichloropyridyloxy)]benzene (TCPOBOP) in the liver and in other organs were quantified and compared to those of phenobarbital (PB) in two inbred strains of mice (C57BL/6NCr, DBA/2NCr) and in F344/NCr rats initiated at 5 weeks of age with N-nitrosodiethylamine (NDEA; 90 mg/kg in mice, 75 mg/kg in rats). Two weeks later animals were placed on a regimen of TCPOBOP once every 2 weeks (administered i.p. or i.g.) or on a diet containing 500 p.p.m. PB as a positive control for the duration of the experiment. Mice were administered TCPOBOP (3.0 mg/kg/dose) for 30 weeks followed by control diet, while rats were given the TCPOBOP regimen (3.0 or 30 mg/kg/dose) for the full 78 weeks of the experiment. TCPOBOP was a complete carcinogen and an extremely potent promoter in both strains of mice, particularly the DBA strain in which NDEA followed by TCPOBOP (i.p.) resulted in death of all the animals within 30 weeks from multiple hepatocellular tumors. TCPOBOP alone induced 100% tumor incidence in DBA mice within 60 weeks. In addition, in both strains of mice, a high proportion of those animals with liver tumors had metastases to the lungs. In contrast, TCPOBOP was ineffective as a liver tumor promoter in F344 rats at even 10 times the dose administered to mice. Interestingly however, TCPOBOP, when given subsequent to NDEA, caused a significant increase in nasal cavity tumors in F344 rats. PB was an effective liver tumor promoter in male DBA mice and male F344 rats, but was relatively ineffective as a promoter in C57 mice. When tumor-promoting activity and induction of cytochrome P450 IIB1 were compared, good agreement between these two parameters was observed. PB was an effective inducer of P450 IIB1 in the rats and in both strains of mice and a potent liver tumor promoter in both DBA mice and F344 rats, whereas TCPOBOP was a potent inducer and tumor promoter in both strains of mice but was negligibly effective as either an inducer or a promoter in F344 rats at even 10-fold higher dosage.


Expression of topoisomerase II, catalase, metallothionein and thymidylate-synthase in human squamous cell lung carcinomas and their correlation with doxorubicin resistance and with patients' smoking habits. Forty-eight human squamous cell lung carcinomas of previously untreated patients were analyzed for resistance to doxorubicin and for the presence of topoisomerase II (Topo II), metallothionein (MT), thymidylate synthase (TS) and catalase (Cat). Significant correlations exist between resistance to doxorubicin measured by the in vitro short-term test and overexpression of MT and TS measured by immunohistochemistry. No significant correlation was found between resistance and expression of Topo II or Cat. No significant interrelationship between smoking habits of patients and expression of Topo II, MT, TS or Cat was found.


Sequential combined tumorigenic effect of HPV-16 and chemical carcinogens. We immortalized oral keratinocytes by transfection with recombinant human papillomavirus type 16 (HPV-16) DNA and established two cell lines, human oral keratinocytes-16A (HOK-16A) and -16B (HOK-16B). These cell lines were morphologically different from the normal counterpart, contained HPV-16 DNA as integrated form and expressed numerous viral genes. However, these cells proliferated only in culture medium containing low calcium (0.15 mM) and are not tumorigenic in nude mice. To test the hypothesis that tumors can be developed by sequential combined effect of human papillomavirus and chemical carcinogens in the oral cavity, these immortalized cell lines were chemically transformed by exposure to either benzo[a]pyrene or methanesulfonic acid ethyl ester. Such transformants proliferated in medium containing physiological calcium levels (1.5 mM) and demonstrated enhanced growth potential in nude mice, whereas primary human oral keratinocytes treated with these chemical carcinogens failed to show any evidence of transformation. Chemically transformed cells contained integrated, intact HPV-16 sequences and transcribed significantly higher amount of HPV-16 E6/E7 messages and transforming growth factor-alpha (TGF-alpha) compared with the immortalized oral keratinocytes. Like the HPV-immortalized cell lines, the chemically transformed oral keratinocytes contained lower levels of newly synthesized, wild-type p53 proteins compared to normal cells, and expressed wild-type c-Ha-ras. These results indicate that this in vitro system is useful for investigating the mechanisms of multistep oral carcinogenesis.


Fiber-induced hydroxyl radical formation: correlation with mesothelioma induction in rats and humans. As part of a program to study the effects of inhaled fibers, we characterized the capacity of various fibers to initiate hydroxyl radical (.OH) formation from hydrogen peroxide in a non-cellular system. We studied five natural fibers (erionite, crocidolite, amosite, anthophyllite and chrysotile) and two man-made fibers (JM code 100 glass fibers and glass wool). The fibers were incubated for 5 min at 37 degrees C with hydrogen peroxide and salicylic acid in pH 7.0 aqueous solutions. The salicylate reacted with any .OH formed in these mixtures to produce stable addition products. The amount of .OH addition products formed during the incubations was determined by the salicylate assay which uses HPLC with electrochemical detection. Erionite, JM code 100 and glass wool were the most effective initiators of .OH formation, followed, in order, by crocidolite, amosite and chrysotile. When the capacity of the natural fibers to initiate .OH formation was plotted versus either the values for tumor rates of rats that received pleural inoculations of fibers or the literature values for the human mesothelioma mortality rates, positive correlations (r2 > or = 0.896) were found. Similar correlations with man-made fibers were not found. No positive correlation could be made between .OH formation capacity versus the tumor rates of rats that received peritoneal injections of either type of fibers.


In vivo formation of chromium(V) in chick embryo liver and red blood cells. In order to understand the possible role of reactive intermediates in the formation of tissue-specific DNA damage by chromium(VI), electron paramagnetic resonance spectroscopy was used to study the in vivo formation of chromium(V) in the liver and red blood cells of 14 day chick embryos following treatment with chromium(VI). In vivo administration of sodium dichromate onto the inner shell membrane of 14 day chick embryos resulted in the formation of a persistent chromium(V) species in liver cells (g = 1.987). The intensity of the chromium(V) signal in liver cells plateaued at 70 min and persisted for 240 min after treatment with chromium(VI). The dependence of chromium(V) formation on the dose of sodium dichromate administered to the embryo was clearly different in liver versus red blood cells. Chromium(V) was detected in red blood cells only at high doses of sodium dichromate (0.50-0.60 mmol/kg), whereas chromium(V) was undetectable in red blood cells at lower doses of sodium dichromate (0.10-0.30 mmol/kg) which produced clear evidence for chromium(V) in liver. Uptake studies showed that total chromium levels in red blood cells were 10-fold greater than in liver cells, and that up to 10% of the total chromium existed as chromium(V) in liver and red blood cells in vivo. Depletion of glutathione by pretreatment of embryos with L-buthionine-S,R-sulfoximine (BSO) for 24 h prior to treatment with a high dose of sodium dichromate (0.60 mmol/kg) caused both a decrease in the levels of chromium(V) species produced and a decrease of chromium uptake into red blood cells 50 min after treatment. At this high dose of chromium(VI), BSO pre-treatment had no effect on the level of the chromium(V) or on chromium uptake into liver cells after a 70 min incubation period. Thus, the concentration of chromium(V) inside the cell correlated with the levels of chromium taken up into the cell. Chromium(V) may be the form of chromium which is responsible for induction of DNA damage following in vivo administration of sodium dichromate.


Effect of dietary fiber on cytochrome P450IA1 induction in rat colonic mucosa. The effect of dietary fiber on the induction of cytochrome P450IA1 in rat colonic mucosa after a single intragastric injection of 3-methylcholanthrene (3MC, 20 mg/kg) was investigated by examining the drug-metabolizing enzyme activity, immunoblotting for cytochrome P450IA1 and immunohistochemistry. 7-Ethoxycoumarin-O-deethylase activities were approximately 20-fold higher in microsomes from both proximal and distal portions of the colonic mucosa of control diet-fed 3MC-treated rats compared with those of control diet-fed untreated rats. Strong immunofluorescence for cytochrome P450IA1 was localized in the cytoplasm of the colonic mucosa surface epithelium from the control diet-fed 3MC-treated rats. 7-Ethoxycoumarin-O-deethylase activity and cytochrome P450IA1 content determined by immunoblotting were significantly lower in wheat bran-fed 3MC-treated rats than in control diet-fed 3MC-treated rats. Immunohistochemical analysis showed much weaker immunofluorescence for cytochrome P450IA1 in the surface epithelium of the colonic mucosa of the wheat bran-fed 3MC-treated rats. These observations suggested that dietary fiber can affect the induction of cytochrome P450IA1 in colonic mucosa by dietary inducers or carcinogens.


Inhibitory effects of crude soybean trypsin inhibitor on pancreatic ductal carcinogenesis in hamsters after initiation with N-nitrosobis(2-oxopropyl)amine. The effects of soybean trypsin inhibitor (SBTI) administration during the promotion phase of pancreatic carcinogenesis were investigated. Female Syrian golden hamsters were given three weekly s.c. injections of N-nitrosobis(2-oxopropyl)amine (BOP) each at a dose of 10 mg/kg and then administered 5% SBTI diet for the following 37 weeks. Additional groups of animals received the BOP injection alone or the 5% SBTI diet alone as controls. At week 40 of the experiment, all surviving animals were killed and development of pancreatic lesions was assessed histopathologically. The results showed that the incidence of dysplastic lesions in hamsters of the BOP/SBTI group was significantly decreased as compared to that of the BOP group (P < 0.01). A similar but not significant tendency was also found for pancreatic adenocarcinomas. In addition, the number of dysplastic lesions in the pancreas head portion in the BOP/SBTI group were significantly decreased as compared to the BOP group value (P < 0.05). Furthermore, atrophic changes of the pancreatic exocrine tissue were more severe in the BOP group than in the BOP/SBTI group (P < 0.01), indicating that SBTI treatment gave effective protection against the replacement process of acinar cell induced by BOP. Thus, the present experiment demonstrated that SBTI can inhibit hamster pancreatic ductal carcinogenesis when given in the promotion phase, in clear contrast to the enhancing effects reported for preneoplastic acinar lesion development in rats.


Caffeine-derived N-nitroso compounds--I: Nitrosatable precursors from caffeine and their potential relevance in the etiology of oesophageal and gastric cancers in Kashmir, India. Salted tea prepared in Kashmir by adding sodium bicarbonate shows high methylating activity (equivalent to 3 p.p.m. N-methylnitrosourea) upon in vitro nitrosation. Pure caffeine treated under conditions of the tea preparation formed caffeidine and caffeidine acid. We report here the formation of two new compounds, mononitrosocaffeidine, an asymmetric nitrosamine, and dinitrosocaffeidine, a N-nitrosamide, on in vitro nitrosation of caffeidine. Mononitrosocaffeidine is also found after nitrosation of the typical Kashmir tea. The nitrosation of caffeidine acid produced N,N'-dimethyl-parabanic acid, mononitrosocaffeidine and N,N'-dimethyl-N-nitrosourea. In view of the well-known structure-activity relationships of these N-nitroso compounds, their possible endogenous formation due to high consumption of salted tea may be a critical risk factor for the high occurrence of oesophageal and gastric cancers in Kashmir.


Mechanism of citrinin-induced dysfunction of mitochondria. II. Effect on respiration, enzyme activities, and membrane potential of liver mitochondria. The mycotoxin citrinin, depressed the phosphorylation efficiency of liver mitochondria as deduced from a decrease of respiratory coefficient and of the ADP/O ratio. Citrinin (1.0 mM) inhibited some enzymes linked to the respiratory chain, namely NADH oxidase and NADH cytochrome c reductase involved with complex I. The activities of enzymes related with other enzymatic complexes of the respiratory chain were either unaffected or enhanced. ATPase activity was inhibited by the mycotoxin. Malate, glutamate, and 2-oxoglutarate dehydrogenases were also inhibited. The transmembrane potential (delta psi), developed by energized mitochondria and depolarization on the addition of ADP, was decreased. The results suggest that citrinin promotes a partial dissipation of the transmembrane potential, different from that resulting from a classical uncoupler such as 2,4-dinitrophenol.


Lysophosphatidylcholine inhibits bradykinin-induced phosphoinositide hydrolysis and calcium transients in cultured bovine aortic endothelial cells. Vascular endothelium, which produces endothelium-derived relaxing and constricting factors, plays an important role in regulating the vascular tone. We recently demonstrated that oxidized low density lipoprotein inhibited endothelium-dependent relaxation and that lysophosphatidylcholine accumulated during the oxidative modification of low density lipoprotein was the essential substance for the inhibition of endothelium-dependent relaxation. To clarify the mechanisms of the inhibitory effect of lysophosphatidylcholine, we used a bioassay system to investigate the effect of lysophosphatidylcholine on the production and/or release of endothelium-derived relaxing factor and its effect on the cytosolic Ca2+ level ([Ca2+]i) and phosphoinositide hydrolysis in cultured bovine aortic endothelial cells. [Ca2+]i was monitored by the fura 2 method, and the accumulation of inositol phosphates in cells labeled with myo-[2-3H]inositol was measured. Bioassay experiments showed that lysophosphatidylcholine inhibited the production and/or release of endothelium-derived relaxing factor from cultured endothelial cells. Lysophosphatidylcholine (5-20 micrograms/ml) induced a biphasic increase in [Ca2+]i, which consisted of a rapid increase followed by a sustained increase, and the initial component was a result of mobilization from intracellular Ca2+ stores without detectable synthesis of inositol 1,4,5-trisphosphates. Furthermore, lysophosphatidylcholine (5-20 micrograms/ml) dose-dependently inhibited both phosphoinositide hydrolysis and the increases in [Ca2+]i evoked by bradykinin. These results indicate that the impairment of endothelium-dependent relaxation induced by lysophosphatidylcholine is due to the inhibition of phosphoinositide hydrolysis and the subsequent increases in [Ca2+]i in endothelial cells. Lysophosphatidylcholine that accumulates in oxidized low density lipoprotein and atherosclerotic arteries may play an important role in the modification of endothelial function.


Reperfusion-induced arrhythmias. A role for washout of extracellular protons? Rapid washout of extracellular H+ accumulated during preceding ischemia (i.e., the abrupt restoration of extracellular pH) has been implicated as an arrhythmogenic factor during reperfusion. Therefore, we hypothesized that by limiting the rate at which extracellular pH was restored during early reperfusion it should be possible to protect against reperfusion-induced arrhythmias. To test this, we used isolated rat hearts (n = 12 per group) and a novel dual coronary perfusion cannula that permitted the induction of regional ischemia (10 minutes) and the selective reperfusion (8 minutes) of the ischemic zone with modified solutions. We examined the antiarrhythmic efficacy of 1) acidic (pH 6.6) reperfusion with stepwise restoration of extracellular pH to 7.4 (stepped pH) and 2) transient (2-minute) acidic (pH 7.1, 6.8, 6.6, or 6.4) reperfusion with subsequent abrupt restoration of extracellular pH to 7.4. Hearts in two contemporary control groups were reperfused with solution at pH 7.4 throughout. In all groups, 100% of hearts exhibited ventricular tachycardia (VT) on reperfusion. VT degenerated into ventricular fibrillation (VF) in 100% of hearts in the control group but in only 42% of hearts in the stepped-pH group (p < 0.05). In the groups subjected to transient acidic reperfusion, there was a pH-dependent prolongation of VT cycle length (measured at 15 seconds of reperfusion), which was 47.1 +/- 3.9, 51.1 +/- 5.5, 56.0 +/- 1.9, 60.4 +/- 2.8 (p < 0.05), and 68.8 +/- 5.0 (p < 0.05) msec in the pH 7.4 (control), 7.1, 6.8, 6.6, and 6.4 groups, respectively. In these groups, VT degenerated into VF in 92%, 92%, 83%, 42% (p < 0.05), and 33% (p < 0.05) of hearts, respectively. In conclusion, limiting the rate at which extracellular pH is restored during early reperfusion does not affect the rapid induction of VT but inhibits the degeneration of VT into VF and promotes spontaneous reversion to normal sinus rhythm. This is consistent with a major arrhythmogenic role, during uncontrolled reperfusion, for the rapid washout of extracellular H+.


Alpha 1-adrenoceptor stimulation enhances the delayed rectifier K+ current of guinea pig ventricular cells through the activation of protein kinase C. The effect of alpha 1-adrenoceptor stimulation on the delayed rectifier K+ current (IK) was examined in isolated guinea pig ventricular cells by use of the patch-clamp method. IK was evoked by a 3-second depolarizing pulse from a holding potential of -30 mV in a Na(+)- and K(+)-free solution containing 3 microM nifedipine. Phenylephrine (30 microM) in the presence of propranolol (1 microM) produced an increase in IK. In five cells, phenylephrine increased the tail current of IK by 23 +/- 5%. This effect of phenylephrine was blocked by prazosin (0.3 microM), a selective alpha 1-blocker. Phenylephrine produced only a small effect on the voltage and time dependence of IK. Pretreatment with 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H-7, 10 microM) abolished the phenylephrine-induced increase in IK. In addition, pretreatment with a maximally effective concentration of 12-O-tetradecanoylphorbol 13-acetate (100 nM) abolished the phenylephrine-induced increase in IK. In conclusion, alpha 1-adrenoceptor stimulation increases IK in guinea pig cardiomyocytes. This alpha 1-adrenoceptor-mediated response may be related to an activation of protein kinase C. The increase in IK may explain a shortening of action potential duration observed after alpha 1-adrenoceptor stimulation in guinea pig cells.


Regulation of Na,K-ATPase gene expression by thyroid hormone in rat cardiocytes. Synthesis and activity of the enzymatic equivalent of the sodium pump, Na,K-ATPase, are regulated by thyroid hormone in responsive tissues. The purpose of this study was to determine whether triiodothyronine (T3) regulates the level of the messenger RNA (mRNA) coding for Na,K-ATPase alpha- and beta-subunits in the heart. The expression of Na,K-ATPase mRNAs in in vitro myocardial cells was directly assayed by Northern and slot blot hybridization using Na,K-ATPase alpha- and beta-isoform-specific cDNA probes. Exposure of cultured neonatal rat cardiocytes to 10(-8) M T3 resulted in 1) threefold to fourfold increase in alpha 1- and beta 1-mRNA accumulation, with a maximum elevation at 48 hours, 2) sevenfold increase in alpha 2-mRNA accumulation with a peak elevation at 72 hours, and 3) transient threefold increase in alpha 3-mRNA within the first 24 hours followed by a deinduction thereafter. The increase in alpha 1-mRNA accumulation by T3 occurred over the physiological T3 concentration range with an EC50 of 5 x 10(-10) M. This was associated with a twofold increase in alpha 1-subunit protein accumulation and an increase in Na,K-ATPase transport activity. The half-life of alpha 1-mRNA analyzed by actinomycin D chase was less than 3 hours and was not affected by T3. Transfection experiments with the luciferase reporter gene revealed that thyroid hormone response sequences are located within the 5'-flanking regions of each alpha-isoform gene.(ABSTRACT TRUNCATED AT 250 WORDS)


Transcriptional regulation of left ventricular beta-adrenergic receptors during chronic hypoxia. beta-Adrenergic receptor downregulation is the end result of cellular adaptation to prolonged agonist exposure. The factors mediating receptor downregulation include receptor phosphorylation, receptor movement from the plasma membrane to intracellular sites, and alterations in nascent receptor synthesis. We have previously demonstrated a downregulation of the left ventricular beta-receptor during chronic hypoxia in vivo. To determine the mechanism of this downregulation, we produced chronic hypoxia in seven newborn lambs by creating right ventricular outflow obstruction and an atrial septal defect. Oxygen saturation was reduced to 65-74% for 2 weeks. Six lambs served as normoxic controls. Sarcolemmal membrane and cytosolic fractions were prepared from left ventricular free wall samples. beta-Receptor density in each fraction was determined with the radioligand [125I]iodocyanopindolol. Steady-state levels of beta-receptor mRNA were determined by Northern blot analysis using a beta 1-adrenergic receptor cDNA probe. During chronic hypoxia, left ventricular membrane beta-adrenergic receptor density decreased by 55% (153 +/- 28 fmol/mg for hypoxic lambs versus 342 +/- 79 fmol/mg for control lambs, p < 0.05). There was no corresponding increase in beta-receptor density in the cytosolic fraction (23 +/- 3 fmol/mg for hypoxic lambs versus 33 +/- 9 fmol/mg for control lambs, p = NS), nor was there a significant change in the ratio of beta 1-receptor/beta 2-receptor subtypes as assessed by radioligand binding (beta 1 subtype, 84.1 +/- 10.1% for hypoxic lambs versus 93.2 +/- 8.8% for control lambs; p = NS).(ABSTRACT TRUNCATED AT 250 WORDS)


A prospective study of nutritional factors and hypertension among US men. An effect of diet in determining blood pressure is suggested by epidemiological studies, but the role of specific nutrients is still unsettled. The relation of various nutritional factors with hypertension was examined prospectively among 30,681 predominantly white US male health professionals, 40-75 years old, without diagnosed hypertension. During 4 years of follow-up, 1,248 men reported a diagnosis of hypertension. Age, relative weight, and alcohol consumption were the strongest predictors for the development of hypertension. Dietary fiber, potassium, and magnesium were each significantly associated with lower risk of hypertension when considered individually and after adjustment for age, relative weight, alcohol consumption, and energy intake. When these nutrients were considered simultaneously, only dietary fiber had an independent inverse association with hypertension. For men with a fiber intake of < 12 g/day, the relative risk of hypertension was 1.57 (95% confidence interval, 1.20-2.05) compared with an intake of > 24 g/day. Calcium was significantly associated with lower risk of hypertension only in lean men. Dietary fiber, potassium, and magnesium were also inversely related to baseline systolic and diastolic blood pressure and to change in blood pressure during the follow-up among men who did not develop hypertension. Calcium was inversely associated with baseline blood pressure but not with change in blood pressure. No significant associations with hypertension were observed for sodium, total fat, or saturated, transunsaturated, and polyunsaturated fatty acids. Fruit fiber but not vegetable or cereal fiber was inversely associated with incidence of hypertension. These results support hypotheses that an increased intake of fiber and magnesium may contribute to the prevention of hypertension.


Differential effects of captopril and enalapril on tissue renin-angiotensin systems in experimental heart failure. Angiotensin converting enzyme (ACE) inhibitor therapy elicits beneficial responses from patients with heart failure. We hypothesized that a major site of action of these drugs is tissue ACE and that ACE inhibitors might differ in their ability to inhibit tissue ACE. To test this hypothesis, we assessed the effects of captopril and enalapril on blood pressure and renal function and on serum and tissue ACE activities in sham-operated rats and rats with heart failure induced by coronary artery ligation. During short-term (1-week) treatment, captopril (200 mg.kg-1.day-1) and enalapril (25 mg.kg-1.day-1) elicited equipotent effects on blood pressure and inhibition of serum ACE activity (85%). The effects of long-term treatment (47 days) were then studied beginning 45 +/- 5 days after coronary ligation in four treatment groups: sham-operated, vehicle (n = 14); heart failure, vehicle (n = 10); heart failure, captopril (n = 8); and heart failure, enalapril rats (n = 7). During long-term treatment, captopril and enalapril caused comparable falls of 12-18 mm Hg in blood pressure (p < 0.01 compared with vehicle treatment). There was no change in urine volume or sodium or potassium excretion in vehicle- or captopril-treated heart failure rats; in contrast, enalapril-treated heart failure rats demonstrated 83% and 10% increases in urine volume and daily sodium excretion, respectively, compared with vehicle-treated rats (both p < or = 0.01). No significant changes in blood urea nitrogen or creatinine were observed with either treatment. Enalapril but not captopril elicited a significant decrease in serum and lung ACE activities. Captopril but not enalapril inhibited aortic ACE activity. Both agents caused a comparable inhibition of renal ACE activity. The magnitude of inhibition of renal ACE activity but not serum and vascular (aortic) ACE activities correlated with the long-term blood pressure response. Enalapril but not captopril normalized renal angiotensinogen expression; the magnitude of this effect correlated with the increase in daily urinary sodium excretion (r = -0.43; p < or = 0.005). These data suggest that chronic treatment with these two agents elicits differential effects on tissue ACE activities and renal angiotensinogen regulation. The differential renal effects of these agents may be important in the treatment of heart failure.


Effects of beta-adrenergic blockade on papillary muscle function and the beta-adrenergic receptor system in noninfarcted myocardium in compensated ischemic left ventricular dysfunction. beta-Adrenergic receptor blockade has been reported to improve hemodynamics and beta-adrenergic receptor-adenylate cyclase function in idiopathic dilated cardiomyopathy. The purpose of this study was to determine the effects of beta-adrenergic receptor blockade on the beta-adrenergic receptor system and myocardial function in a model of compensated ischemic heart failure. We examined the effects of propranolol treatment on the beta-adrenergic receptor-adenylate cyclase system and isolated papillary muscle isometric function in noninfarcted left ventricular myocardium in rats after coronary artery ligation. In untreated rats with large myocardial infarction (MI), developed tension (DT) (3.0 +/- 0.7 versus 5.1 +/- 1.1 g/mm2), peak rate of tension rise (+dT/dt) (40.3 +/- 9.5 versus 71.2 +/- 12.0 g/mm2/sec), and peak rate of tension fall (-dT/dt) (24.4 +/- 5.0 versus 38.2 +/- 6.0 g/mm2/sec) were decreased (p < 0.05). In addition, DT, +dT/dt, and -dT/dt of untreated MI rats demonstrated an impaired response to isoproterenol stimulation compared with controls. beta-Adrenergic receptor density (Bmax) measured by [125I]iodocyanopindolol (ICYP) binding was decreased 23% after infarction (9.3 +/- 0.6 versus 12.0 +/- 1.8 fmol/mg protein [prot]) (p < 0.05); however, the dissociation constant (Kd) for ICYP was not changed (24.1 +/- 5.7 versus 33.2 +/- 12.1 pM). Adenylate cyclase activity in the presence of 10(-2) M MgCl2 was reduced (p < 0.05) in MI rats (30.3 +/- 10.8 versus 45.9 +/- 12.5 pmol cAMP/min/mg prot). Maximal isoproterenol (52.5 +/- 7.3 versus 79.9 +/- 10.0 pmol cAMP/min/mg prot), guanyl-5'-imidodiphosphate (GppNHp) (95 +/- 8 versus 141 +/- 25 pmol cAMP/min/mg prot) and forskolin (503 +/- 76 versus 753 +/- 157 pmol cAMP/min/mg prot) stimulation of adenylate cyclase was also decreased (p < 0.05). In addition, manganese-stimulated adenylate cyclase activity was depressed (p < 0.05) in MI rats compared with controls (23.5 +/- 2.8 versus 52.1 +/- 9.0 pmol cAMP/min/mg prot). Chronic propranolol treatment in MI rats improved DT (4.1 +/- 0.9 versus 3.0 +/- 0.7 g/mm2) and +dT/dt (54.4 +/- 11.3 versus 40.5 +/- 9.5 g/mm2/sec) (p < 0.05); however, isoproterenol-stimulated isometric function remained impaired. Propranolol treatment normalized Bmax (11.9 +/- 1.7 versus 9.3 +/- 0.6 fmol/mg prot) (p < 0.05), whereas adenylate cyclase activity remained depressed. After large MI in rats, there is impaired papillary muscle function with decreased beta-adrenergic receptors and adenylate cyclase activity in the noninfarcted myocardium. Propranolol treatment improved basal isometric muscle function and beta-adrenergic receptor density in rats after myocardial infarction but did not improve adenylate cyclase activity or isoproterenol-stimulated muscle function. These data suggest that there is a primary defect in adenylate cyclase function that persists despite upregulation of receptors with propranolol treatment.


Persistence of viral genome into late stages of murine myocarditis detected by polymerase chain reaction. Enteroviruses have been considered as the most common etiologic agents in clinical myocarditis and dilated cardiomyopathy; however, their pathogenetic role remains unknown. Hence, the relation of viral replication and development of cardiomyopathy has been determined in a murine model of myocarditis by evaluating the persistence of viral genome during acute and chronic stages of myocarditis by means of Northern blot hybridization and polymerase chain reaction (PCR). DBA/2 mice (n = 146) were injected peritoneally with 10 plaque-forming units of encephalomyocarditis (EMC) virus, and the control mice (n = 33) were injected with normal saline. Animals were randomly killed at 4, 7, 10, 14, 21, 28, 35, and 42 days after infection. Histology revealed acute myocardial necrosis with massive inflammatory cell infiltrate peaking on day 14 followed by increasing fibrosis and declining chronic inflammation features compatible with dilated cardiomyopathy between days 21 and 42. Northern blot analysis of control and infected hearts showed detectable viral RNA in the infected hearts initially at day 4, peaking by day 7, diminishing between day 7 and day 14, and absent at day 21 and day 28. However, potential viral remnants present in low quantities and undetectable by Northern blot were further detected by PCR followed by confirmation with an internal oligonucleotide probe after day 14 up to day 42. Viral RNA signals on Northern blot showed a strong correlation with massive myocyte necrosis on day 14, but the viral RNA fragment was consistently detectable into late stages of cardiomyopathy on days 21, 28, 35, and 42 by PCR. This indicated that the mature virions are fully developed early in infection and are capable of persisting in the myocardium after virus-mediated myocytolysis stage. Therefore, PCR is an extremely sensitive method for detecting residual viral genome and viral persistence in the myocardium and may offer insights into the pathogenesis of chronic myocarditis leading to dilated cardiomyopathy.


The epidemiology of diet and blood pressure. Epidemiological studies of diet and blood pressure support the concept that some dietary nutrients are strongly related to blood pressure and are amenable to modifications, with a likely impact on the incidence of hypertension and of related vascular events. For other potential nutrients, the evidence is more difficult to obtain. Further studies are required to establish 1) the practical value of assessment of individual nutrients versus dietary patterns in risk prediction, 2) the effects of changes of these individual nutrients versus dietary patterns on blood pressure, and 3) whether long-term changes in these individual nutrients or dietary patterns reduce the incidence of hypertension and can be implemented at a population level.


Calcium channels expressed in vascular smooth muscle. Regulation of intracellular calcium levels is known to activate signal transduction pathways, leading to well-defined patterns of gene expression. Among the calcium-responsive genes are those involved in the growth and proliferative responses of vascular smooth muscle cells. Cytoplasmic calcium also plays a role in activating a host of cellular functions including smooth muscle contraction and growth factor release. Calcium channels participate in the regulation of cytoplasmic calcium concentration in vascular smooth muscle. Two major classes of calcium channels are expressed in vascular smooth muscle cells: voltage-dependent calcium channels on the plasmalemma and intracellular calcium release channels on the endoplasmic reticulum. The voltage-dependent calcium channel is activated by depolarization of the plasmalemma. This calcium channel belongs to the super gene family that includes the voltage-dependent potassium and sodium channels. These three cation channels share a common transmembrane topography. The major intracellular calcium release channel in vascular smooth muscle is the inositol 1,4,5-trisphosphate receptor (IP3R) on the endoplasmic reticulum. The IP3R is activated by IP3, a second messenger generated at the plasmalemma, which mediates numerous cellular responses including smooth muscle contraction. Also present in smooth muscle cells is the ryanodine receptor (RYR)/calcium release channel of the sarcoplasmic reticulum. The RYR is the major intracellular calcium release channel of striated muscles and is expressed in relatively low levels in vascular smooth muscle. The IP3R and RYR are members of a gene family encoding intracellular calcium release channels with characteristic fourfold symmetric structures.


Experimental models that mimic the differentiation and dedifferentiation of vascular cells. Endothelial and smooth muscle cells normally exist in a quiescent differentiated state. After injury to the vessel, these cells dedifferentiate, migrate, and proliferate as needed for repair. In culture on plastic, both endothelial and smooth muscle cells exhibit the dedifferentiated phenotype. We have found that laminin and reconstituted basement membrane proteins (Matrigel) induce a very rapid cessation of endothelial cell proliferation followed by alignment and subsequent reorganization into tubelike structures. We have also found that smooth muscle cells in culture exhibit a differentiated phenotype when exposed to Matrigel. The molecular mechanisms involved in smooth muscle differentiation resemble those of skeletal muscle, in which proliferation and differentiation appear to be mutually exclusive states controlled by both positive and negative transcriptional regulators. The dedifferentiated smooth muscle cells produce proteases and exhibit a migratory and invasive phenotype capable of destroying normal tissue architecture. These studies suggest that the modulation of endothelial and smooth muscle cells between a differentiated and dedifferentiated phenotype is regulated by extracellular matrix components as well as by cytokines. Model systems such as those described here should allow the identification of molecular events controlling the differentiation of vascular cells and facilitate the development of therapeutic agents that maintain healthy vessels.


Seizure activity-induced changes in polyamine metabolism and neuronal pathology during the postnatal period in rat brain. Systemic injection of kainic acid (KA) does not cause neuronal pathology in limbic structures in rat brain prior to postnatal day (PND) 21. The present study tested if the development of the pathogenic response is associated with the maturation of a link between seizure activity and polyamine metabolism. Pathology was assessed with histological techniques and with the binding of [3H]Ro5-4864, a ligand for the peripheral type benzodiazepine binding sites (PTBBS), a marker of glial cell proliferation. In agreement with previous results, peripherally administered kainate at doses sufficient to induce intense behavioral seizures produced a loss of Nissl staining in hippocampus after PND 21 but not at earlier ages. The pattern of neuronal damage observed after PND 21 resembled that found in adult animals: extensive losses of Nissl staining in area CA3 of hippocampus and in piriform cortex, more modest effects in CA1 and sparing of the granule cells of the dentate gyrus. Similarly, no increase in [3H]Ro5-4864 binding as a result of KA administration was observed in hippocampus and piriform cortex until PND 21. Ornithine decarboxylase (ODC) activity and putrescine levels were high in the neonatal brain and decreased to reach adult values by PND 21. KA-induced seizure activity did not significantly alter both variables until PND 21. After PND 21, ODC activity and putrescine levels markedly increased 16 h after KA-induced seizure activity in hippocampus and piriform cortex. The magnitude of the effects increased between PND 21 and PND 30, at which point the changes in both parameters were comparable to those found in adults. Polyamines stimulate the activity of the calcium-dependent proteases calpain in brain fractions and may increase calpain-mediated proteolysis in situ. In accord with this, kainate-induced breakdown of spectrin, a preferred substrate of calpain, measured 16 h after KA injection followed a developmental curve parallel to that for kainate-induced increases in putrescine levels. These results indicate that the onset of vulnerability to seizure activity triggered by kainic acid is correlated with the development of an ODC/polyamine response to the seizures and further support a critical role for the ODC/polyamine pathway in neuronal pathology following a variety of insults.


Temporal and compartmental restriction of neuron-specific enolase expression in the rat mesostriatal system. The striatum and the mesencephalic dopamine neurons which innervate it, are each organized into developmentally and biochemically distinct compartments. Striatal patches, characterized in the neonate by high concentrations of opiate receptors and substance P, are innervated prenatally by fibers originating in one group of midbrain dopamine neurons, the ventral tier. By the third postnatal day, a dense dopamine projection from neurons in the dorsal tier of the mesostriatal group innervates non-patch areas of the striatum, i.e. the matrix, and is followed by the appearance there of neurotensin, somatostatin and calcium binding protein. We have recently observed that the period of establishment of connections between dorsal tier dopamine neurons and their target cells in the striatal matrix is accompanied by a surge in expression of the gene coding for tyrosine hydroxylase (TH). In order to determine the overall metabolic state of mesencephalic and striatal neurons during the period of up-regulation of TH gene expression, we have applied immunocytochemistry for neuron specific enolase (NSE), and cytochrome oxidase histochemistry, known markers for neuronal activity, as well as TH immunohistochemistry to the mesencephalon and striatum of postnatally developing rats. At birth, both NSE and cytochrome oxidase were expressed almost exclusively in the patches, appearing in the matrix only after the 2nd postnatal day. Patches of NSE remained visible thru the 14th day. In the mesencephalon, cytochrome oxidase and immunoreactive NSE cells in adjacent sections, were present only in the pars reticulata (i.e. ventral tier). By day 8, both techniques identified nigral cells in the dorsal as well as ventral tiers.(ABSTRACT TRUNCATED AT 250 WORDS)


Physiological properties of isolated motor units in normal and bilaterally innervated Xenopus gastrocnemius muscles. Physiological properties of isolated gastrocnemius motor units were measured in normal juvenile postmetamorphic Xenopus frogs and in a group of juvenile animals with a single bilaterally innervated hindlimb. The aim of this study was to evaluate changes to the organisation of the motor unit when the muscle is hyperinnervated. Animals with a single bilaterally innervated hindlimb have previously been shown to support up to twice the normal number of motoneurons projecting into a single hindlimb. Under these circumstances there was a lowering of average neuromuscular efficacy (as judged by motor unit twitch/tetanus ratio) in comparison with normal age-matched siblings. Motor units with neurons in the lateral motor column contralateral to the remaining hindlimb were indistinguishable from those originating ipsilaterally. There is a wide range of safety margins for neuromuscular transmission at the various terminals of individual frog motor units, and comparison of motor unit contraction times with twitch/tetanus ratios showed that under pressure of hyperinnervation, motoneurons tend to retain their safest terminals on muscle fibres with fast contraction times.


Transient cyclic AMP accumulation mediated by dopamine D1 receptors in the chick embryo optic lobe. [3H]SCH 23390 bound with high affinity (Kd = 0.6 nM) and in a saturable manner (Bmax = 130 fmol/mg protein) to membrane preparations of the chick optic lobe. Pharmacological experiments, using several dopaminergic ligands, revealed that [3H]SCH 23390 bound stereospecifically to dopaminergic receptors of the D1 type in this tissue. Other experiments revealed that dopamine was able to induce cyclic AMP accumulation in the optic lobe (ED50 = 3 microM), an effect that was blocked by fluphenazine, a potent D1 antagonist (IC50 = 1.8 microM). The developmental profile of tissue dopamine-dependent cyclic AMP accumulation, however, was quite different from the differentiation pattern of [3H]SCH 23390 specific binding sites. While [3H]SCH 23390 binding sites increased 4-fold after the 12th embryonic day (E12), dopamine-dependent cyclic AMP accumulation was maximal in earlier stages, decreasing progressively after E10. In tissues from embryos at E16 or older, no difference was observed between basal and dopamine-stimulated levels of cyclic AMP. These data suggest that D1 receptors are coupled to adenylate cyclase in a limited period of the development of the optic lobe and that D1 receptors not coupled to the enzyme can be a common feature in the CNS.


Rapid progression of HIV-1 infection to AIDS. Homosexually acquired human immunodeficiency virus type 1 infection is usually slowly progressive, and reports of its rapid progression to acquired immunodeficiency syndrome are rare. We present a case of acute human immunodeficiency virus type 1 and cytomegalovirus coinfection that progressed to acquired immunodeficiency syndrome and death in 7 months. The factors that determine the clinical outcome of human immunodeficiency virus type 1 infection are poorly defined; however, coinfection with other agents, such as cytomegalovirus, may influence its natural history.


Effects of angiotensinase inhibitors on plasma protein binding and IC50 determinations of renin inhibitors. To establish whether the use of proteinase inhibitors in the routine determination of in vitro plasma renin activity overestimates the potency of renin inhibitors in vivo, we examined the effects of phenylmethylsulfonyl fluoride and 8-hydroxyquinoline sulfate on the binding to plasma proteins and the respective IC50 values (50% inhibiting concentrations) of three renin inhibitors. All three renin inhibitors, A-64662, A-65317, and A-74273, bound (> 60%) to plasma proteins at both pH 6.0 and 7.4, with slightly greater binding at pH 7.4. Phenylmethylsulfonyl fluoride (1.45 mmol/L) had no significant effect on the protein binding at either pH 6.0 or 7.4; 8-hydroxyquinoline sulfate (3.4 mmol/L) caused a modest dissociation (10-30%) of the renin inhibitors from plasma proteins at both pH values; and the effects of both proteinase inhibitors together were similar to those of 8-hydroxyquinoline alone. At pH 7.4, phenylmethylsulfonyl fluoride increased the potencies of the three renin inhibitors slightly (< or = 43%), whereas IC50 values determined in the presence of 8-hydroxyquinoline decreased by 1.5- to 3.7-fold. The greatest increase in potency occurred with the most hydrophilic compound, and with both angiotensinase inhibitors the effect was no greater than that of 8-hydroxyquinoline alone. The results show that any dissociation of the hypotensive activity measured in vivo from the plasma renin activity measured in vitro is not simply an artifact in the plasma renin activity assay stemming from the use of these angiotensinase inhibitors, especially if only phenylmethylsulfonyl fluoride is used.


Calcium-channel antagonists: mechanisms of action, vascular selectivities, and clinical relevance. The calcium-channel antagonists represent three separate structural categories of drugs. They share a common action--the blockade of calcium-ion flow through one specific type of calcium channel. The chemical heterogeneity of these agents is reflected in their pharmacologic and therapeutic diversity. The calcium-channel antagonists enjoy significant use in cardiovascular medicine for the treatment of hypertension, angina, and some cardiac arrhythmias. However, the 1,4-dihydropyridines, the most potent antihypertensive calcium-channel blockers, lack antiarrhythmic properties. The selectivity of action of calcium-channel antagonists rests upon a number of factors, including pathways of calcium mobilization, types of channel activated, state-dependent interactions, and the pathological state of the tissue. An understanding of these factors is important to the rational application of these drugs and to the development of newer agents with different specificities.


Concentration of serum laminin and type IV collagen in liver diseases assayed by a sandwich enzyme-immunoassay using monoclonal antibodies. Serum laminin (P1 fragment) and type IV collagen levels were determined in patients with hepatic disorders. The method was based on a sandwich enzyme-immunoassay using two monoclonal antibodies that recognize different epitopes of either laminin or type IV collagen molecule. Laminin and type IV collagen levels in the serum of patients with chronic hepatic disorders were higher as compared with those in healthy control subjects, with the increment of serum type IV collagen being far greater than that of laminin. Since type IV collagen and laminin are major basement membrane components, it is suggested that the higher levels of these peptides may reflect a so-called capillarization of the perisinusoidal wall encountered in hepatic fibrogenesis. The assay system used in this experiment is simple and sensitive and can be applied to clinical evaluation of hepatic fibrosis.


Comparison of urinary excretion of four lysosomal hydrolases in healthy elderly and young adults. The activities of four lysosomal enzymes and creatinine levels were measured in the plasma and urine of 17 healthy elderly and 7 young adults. Fractional enzyme excretion (FE ENZ) values for beta-hexosaminidase (N-acetylglucosaminidase), alpha-galactosidase, beta-galactosidase and beta-glucuronidase were calculated and compared between the two groups of subjects. FE ENZ was calculated as the ratio of enzyme clearance to creatinine clearance. The FE ENZ values for alpha-galactosidase, beta-galactosidase and beta-glucuronidase between the elderly and young populations were not statistically different; however, relative to the young control group, the FE ENZ value for beta-hexosaminidase was elevated approximately 2-fold in the elderly population (P = 0.06). The mean urinary alpha-galactosidase activity for the elderly population, when expressed on the basis of creatinine, was 50% lower than that of the control group (P = 0.03), whereas the mean urinary beta-hexosaminidase activity for the elderly was significantly higher compared to the control group (P = 0.008). When data for all subjects was analyzed, no correlation was observed between the urinary excretion of beta-hexosaminidase or alpha-galactosidase and glomerular filtration rate. These data indicate that with advancing age there are changes in the tubular secretion or reabsorption of selective lysosomal enzymes, particularly beta-hexosaminidase and alpha-galactosidase. These biochemical changes may provide a means of assessing subtle progressive deterioration of renal function.


Interaction of oxidized low density lipoproteins with both apo B,E and scavenger receptors. A model for its production in vitro. Oxidation of low density lipoprotein (LDL) has been demonstrated in vivo and directly implicated in the process of foam cell formation. Consequently, a considerable research effort has been devoted to the assessment of the metabolic behaviour of oxidized LDL. We have developed a simple and reproducible model to obtain oxidized LDL consisting of the dialysis of LDL (4 g/l) contained in a cellulose bag against 5 litres of 0.15 M NaCl, 5 microM CuSO4, 0.6 mM FeCl3, pH 7.4, 37 degrees C with constant oxygen bubbling. While the resulting particles have a number of physicochemical properties suggesting lipid oxidation, neither apo B fragmentation nor modification in the size and shape were observed. This oxidized LDL showed internalization into cells through both the apo B,E and the scavenger receptors and the rate of removal from the plasma in injected rats was faster than that observed for normal LDL. We suggest that these particles may represent an equivalent to the circulating oxidized LDL postulated in humans.


The incidence of late-onset congenital adrenal hyperplasia due to 3 beta-hydroxysteroid dehydrogenase deficiency among hirsute women. The present study was designed to determine the incidence of 3 beta-hydroxysteroid dehydrogenase deficiency (3 beta-HSD) in adult women with hyperandrogenism. In 78 hirsute patients and 30 normal women in the same age range, an ACTH stimulation test was performed on day 5 of the cycle by administration of a single bolus of 0.25 mg ACTH-(1-24) at 0800 h. The following steroids were measured before, 30 and 60 minutes after ACTH injection: delta 5-pregnenolone (delta 5-P), 17-hydroxy-delta 5-pregnenolone (17-OH delta 5-P), dehydroepiandrosterone (DHEA), delta 5-androstenediol, progesterone (P), 17-hydroxyprogesterone (17-OHP), androstenedione (A), testosterone (T) and cortisol. Maximum ACTH-stimulated values of delta 5-steroids were in excess of the 90% confidence limits of the control group in 19 hirsute women. Ten patients had an isolated increase in delta 5-P, 17-OH delta 5-P, DHEA or delta 5-androstenediol. Nine patients had an increase in two delta 5 steroids and none had increased values of three or four delta 5 steroids. The ratios of 17-OH delta 5-P to 17-OHP, DHEA to A, delta 5-P to P and delta 5-androstenediol to T were increased in 5, 1, 1 and 1 patients respectively. No patient had elevated values of more than one ratio. Using stringent diagnostic criteria, partial 3 beta-HSD deficiency was excluded in all 78 patients and therefore appears to be a rare disorder.


Investigation of adrenal function in women with oligomenorrhoea and hirsutism (clinical PCOS) from the north-east of England using an adrenal stimulation test. To determine the prevalence of adrenal enzyme dysfunction in women presenting with oligomenorrhoea and hirsutism, two clinical features of polycystic ovary syndrome (PCOS). A prospective study of women attending outpatient clinics with these complaints. Androstenedione, dehydroepiandrosterone (DHEA), 17-hydroxyprogesterone (17-OHP), 11-deoxycortisol and cortisol were measured before and after overnight dexamethasone suppression and at 60 minutes after adrenal stimulation by ACTH injection. Fifty women with clinical features of PCOS and 37 control women with regular cycles and normal hair distribution from the catchment area of the Royal Victoria Infirmary which includes Newcastle upon Tyne, Co. Durham, Cleveland, Cumbria and Northumberland. Number of women with steroid responses to ACTH beyond the normal range, as defined by the responses of the control group and in previous studies. Nineteen women (38%) were found to have some abnormality. One woman (2%) was identified with 21-hydroxylase (21-OHase) deficiency and a second (2%) had an increase in 17-OHP compatible with the heterozygote state for 21-OHase deficiency. Four women (8%) had isolated elevations in the DHEA response consistent with minimal 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) deficiency. Thirteen women (26%) showed increases in both androstenedione and DHEA, or androstenedione alone, compatible with enhanced 17-20 lyase activity. Twelve per cent of the group showed evidence consistent with an adrenal enzyme deficiency; 26% had results in keeping with increased adrenal androgen production without an enzyme deficiency. These findings may be of relevance both in the pathogenesis of the features of PCOS and in determining appropriate treatment for individual patients.


Adrenocortical dysfunction in paracoccidioidomycosis: comparison between plasma beta-lipotrophin/adrenocorticotrophin levels and adrenocortical tests. Paracoccidioidomycosis is an important cause of Addison's disease in South America. We have carried out an extensive and prospective study on paracoccidioidomycosis comparing glucocorticoid, mineralocorticoid and androgen function with adrenal regulators, ACTH/beta-LPH and plasma renin activity (PRA). Forty-seven male patients with active paracoccidioidomycosis were studied consecutively together with 20 healthy controls. On day 1, plasma aldosterone and PRA levels were measured in blood samples obtained from patients in the supine and erect position. On day 2 at 0900 h, baseline plasma samples were taken for ACTH, beta-lipotrophin (beta-LPH), cortisol, corticosterone, aldosterone, androstenedione (delta 4-A) and dehydroepiandrosterone sulphate (DHEA-S). ACTH 1-24 (250 micrograms) was given i.v. and blood samples for these steroid assays were taken at 1 and 2 hours. Five patients (10%) had Addison's disease with high basal plasma ACTH and beta-LPH, and low cortisol levels after the ACTH test. In the remaining 42 patients, baseline ACTH and beta-LPH levels and plasma cortisol after ACTH were within the normal range. A high percentage of patients presented with reduced corticosterone (21% of patients) and aldosterone (23%) secretion and increased PRA (31%). Plasma delta 4-A (19%) and DHEA-S (50%) levels were also reduced. The frequency of Addison's disease among our patients with paracoccidioidomycosis was 10%. In addition, a subset of patients presented with adrenal dysfunction detected by mineralocorticoid or androgen tests. In parallel to pathological lesions a functional adaptation may occur during adrenal involvement in paracoccidioidomycosis.


Mediation of gonadotrophin-stimulated growth and differentiation of human granulosa cells by adenosine-3',5'-monophosphate: one molecule, two messages. To determine how the second messenger adenosine-3',5'-monophosphate (cyclic AMP) is able to mediate divergent actions of FSH and LH on granulosa cell growth and differentiation in human ovaries. Human granulosa cells were cultured for 96 hours in serum-free medium 199 containing increasing doses of either FSH, LH or dibutyryl cyclic AMP. Extra and intra-cellular cyclic AMP levels were determined by radioimmunoassay. Tritiated thymidine uptake and cell number were measured as indices of cell growth, and spent medium was assayed for steroids (oestradiol and progesterone) to reflect differentiation. 'Mature' granulosa cells were aspirated from preovulatory follicles in the ovaries of clomiphene-stimulated patients undergoing laparoscopic sterilization; 'luteinized' granulosa cells were aspirated from periovulatory follicles in the ovaries of gonadotrophin-stimulated in-vitro fertilization patients. LH consistently inhibited, whereas FSH maintained or stimulated, basal granulosa cell numbers. Steroidogenesis was dose-dependently increased by both gonadotrophins, with LH having the significantly greater effect over the entire dose-response range (1-100 micrograms/l). LH also induced significantly more cyclic AMP production than FSH, both intra and extra-cellularly, providing a basis for differential post-receptor signalling via a common second messenger. Addition of dibutyryl cyclic AMP, at low concentrations (10-250 mumol/l) to the cultured cells in the absence of gonadotrophins mimicked FSH effects with stimulation/maintenance of cell numbers and moderate steroidogenesis. High concentrations of dibutyryl cyclic AMP (500-1000 mumol/l) caused a significant inhibition of cell numbers together with maximal steroidogenesis, simulating LH action. These results suggest that granulosa cell maturation in the follicular phase of the menstrual cycle (controlled by FSH) is associated with a low cyclic AMP tone that favours cell growth and expression of aromatase activity in the developing preovulatory follicle. During the early luteal phase (dominated by LH), the intracellular cycle AMP tone increases to allow maximal progesterone production and inhibition of cell growth in the corpus luteum. Thereby one second messenger can mediate divergent gonadotrophic effects on granulosa cell growth and differentiation in the human ovary.


The early mineralocorticoid effector mechanism, the sodium-proton exchanger, is sensitized in lymphocytes from patients with Cushing's syndrome. In-vitro binding of aldosterone to mineralocorticoid receptors on human mononuclear leucocytes and its effects on the intracellular sodium and potassium concentrations, the sodium-proton exchanger and cell volume of human mononuclear leucocytes have been described for normals. In the present paper this easily accessible human cell model was studied in Cushing's syndrome to detect abnormalities of the mineralocorticoid effector mechanism. The rate of cell swelling in isotonic sodium propionate reflecting the activity of the sodium-proton exchanger and the stimulatory activity of 1.4 nM aldosterone were determined in a Coulter Channelyzer. Nine female patients with pituitary-dependent (7) and adrenal hypercortisolism (2) were included in the study. Compared with controls from matched normals, the cell volume of human mononuclear leucocytes in a physiological buffer was significantly increased in the patients. The increment of cell size in isotonic sodium propionate was elevated in the presence of 1.4 nM aldosterone only. These findings are equivalent to an excess stimulation of the sodium-proton exchanger by aldosterone in these patients. Plasma cortisol was inversely correlated with the cell swelling in propionate. These data indicate that the early mineralocorticoid effector mechanism in human mononuclear leucocytes from patients with Cushing's syndrome has an increased sensitivity to aldosterone compared with that from normals. This could reflect an adaptation of the cellular electrolyte metabolism to the decreased mineralocorticoid activity balancing the increased glucocorticoid activity. If representative of other cell systems, e.g. renal tubular cells, these findings would identify accompanying electrolyte disorders in these patients not as a side-effect of glucocorticoids, but as a result of an increased sensitivity to endogenous mineralocorticoids.


Plasma 19-hydroxy-androstenedione (19-OH-A) in essential hypertension. 19-hydroxy-androstenedione (19-OH-A), a C19 steroid, is an amplifier of the sodium retaining action of aldosterone under the control of ACTH and renin-angiotensin system. These findings suggest that 19-OH-A may be involved in the regulation of hydroelectrolyte balance and blood pressure. Aim of the present study was to examine the behaviour of 19-OH-A in normal volunteers (N) and in patients with Essential Hypertension (EH) in basal conditions and after dynamic tests such as postural changes, physical exercise and ACTH administration. The significant increase in 19-OH-A after ACTH confirms its adrenal origin. During bicycle exercise the significant increase in plasma catecholamines, renin-activity, aldosterone, blood pressure and heart rate at maximum effort was not associated with a parallel increase in 19-OH-A. No significant differences were found in plasma 19-OH-A levels between N and EH patients both in basal conditions and after dynamic tests. Therefore, our findings seem to exclude an important role of 19-OH-A in the pathogenesis of EH.


Efficacy and tolerability of lisinopril compared with extended release felodipine in patients with essential hypertension. Danish Cooperative Study Group. The antihypertensive effects of lisinopril 10-20 mg once daily and felodipine (extended release formulation) 5-10 mg once daily were compared in a double-blind, parallel group study of eight weeks duration involving 219 patients with mild to moderate hypertension. On lisinopril treatment sitting blood pressure fell from 166.3/102.9 +/- 17.5/5.8 mmHg to 146.7/89.7 +/- 19.5/8.7 mmHg and on felodipine blood pressure fell from 166.7/103.3 +/- 18.3/5.4 mmHg to 153.6/92.3 +/- 15.9/7.9 mmHg. The decreases in sitting systolic and diastolic blood pressures were significantly greater on lisinopril than on felodipine treatment (p = 0.019 and p = 0.033). A subgroup analysis in elderly patients (age > or = 65 years) showed that lisinopril and felodipine were equally effective in reducing blood pressure. In young subjects (age < 65 years) felodipine treatment lowered systolic blood pressure less than did lisinopril treatment (p = 0.001). Lisinopril was better tolerated than felodipine. On lisinopril treatment, reports of headache and dizziness were reduced while that of cough increased. On felodipine treatment, dizziness was reduced but reports of flushing and oedema were increased. The results show a better antihypertensive effect and better tolerability for lisinopril compared with extended release felodipine.