Hyperinsulinemia and its impact on obesity and insulin resistance. The impact of hyperinsulinemia on the establishment of insulin resistance was investigated. This was done by treating normal rats with insulin for 3-4 days via osmotic minipumps, and by comparing them with saline-treated controls. Hyperinsulinemia produced by prior insulin treatment (i.e. prior insulinization of the normal rats) resulted in a well tolerated hypoglycemia, increased food intake and body weight gain. Euglycemic-hyperinsulinemic clamps were carried out at the end of the insulinization to assess the acute effects of insulin in control and insulinized rats. It was found that prior insulinization of normal rats resulted in increases in total insulin-stimulated glucose utilization and hepatic lipogenesis, while hepatic glucose production (HGP) was normally suppressed by the hormone. Glucose utilization index by individual tissues was then measured (labelled 2-deoxy-D-glucose method). Prior insulinization of normal rats resulted in increased insulin-stimulated glucose utilization index of white adipose tissue, accompanied by increased insulin-stimulated de novo lipogenesis and glycogen synthesis. In contrast, prior insulinization of normal rats resulted in a decreased insulin-stimulated glucose utilization index of most muscles studied. The decreased insulin-stimulated muscle glucose utilization index brought about by prior insulinization persisted in adrenomedullectomized or propranolol-treated rats, ruling out a role of catecholamines in the effects observed. It is concluded that hyperinsulinemia is a pathological driving force in producing both incipient obesity by overstimulating white adipose tissue and liver metabolic activity, and concomitantly producing incipient muscle insulin resistance.


Obesity increases risk for diabetes.

Obesity increases risk for diabetes. The relationship of obesity to the development of diabetes has been reviewed. Older individuals are more obese and have more diabetes. Both the duration and magnitude of the obesity increase risk of diabetes. Family history of diabetes mellitus is also important in whether obesity leads to diabetes. Abdominal body fat increases the risk of diabetes. These factors help in understanding the reasons why obesity poses such a high risk for developing diabetes mellitus.


Regional fat distribution--implications for type II diabetes. Abdominal obesity is closely associated with risk factors for cardiocerebrovascular disease and NIDDM and the precipitation of these diseases. Together, they seem to constitute a metabolic syndrome where hyperinsulinaemia, insulin resistance, hyperlipidaemia, hypertension, visceral fat accumulation, cardiocerebrovascular disease and NIDDM are the individual constituents. The background to this syndrome might be a primary aberration expressing itself as an increased sensitivity of the hypothalamo-adrenal axis, and subsequent inhibition of sex steroid hormone secretions. This in turn will probably be followed by metabolic derangements, primarily peripheral insulin resistance, as well as by visceral fat accumulation by mechanisms which are partially visualized by recent work in the field. Visceral fat accumulation may then amplify the metabolic aberrations via hepatic effects of excessive concentrations of portal FFA, producing hyperproteinaemia, hyperglycaemia, hyperinsulinaemia and, perhaps, hypertension. The background to the central endocrine aberration remains more speculative, but factors leading to increased cortisol production, including specific stress reactions, tobacco smoking and alcohol may turn out to be important. The tentative conclusion provides a hypothesis for further work, and has recently obtained considerable support from further observations in humans in other than the endocrine and metabolic areas, as well as from studies in experimental animal models, where such factors can be studied under fully controlled conditions, which is not possible in humans for ethical reasons.


Risk of obesity in type II diabetes mellitus. The obvious syntropy of obesity and type II (non-insulin dependent) diabetes mellitus has always suggested a causal inter-relationship between the two diseases. However, the actual pathophysiological connection still remains to be elucidated. Recent findings have suggested that insulin resistance and hyperinsulinaemia might link glucose intolerance/type II diabetes mellitus, hypertension and hyperlipoproteinaemia in the context of a hypothetical 'syndrome X' characterized by an excessive risk constellation for the development of atherosclerosis. However, as to the practical consequences of the ('diabesity') syndrome of type II diabetes mellitus and structured programmes for effective therapy, very little new information has been gathered during the past 100 years.


An update on the pharmacology of serotoninergic appetite-suppressive drugs. D-Fenfluramine, fluoxetine and sertraline are considered to be serotoninergic appetite-suppressive drugs. These three agents have been compared in fasted mice, rats and guinea pigs for their activity as food intake inhibitors. D-Fenfluramine is the most effective drug in the three animal species followed by fluoxetine and then sertraline. All three compounds are metabolized to N-dealkylated metabolites which accumulate in the brain and are themselves effective in reducing food intake. At anorectic doses the brain levels of the drugs and their metabolites are compatible with the concentrations able to block serotonin (5-HT) uptake and to release brain 5-HT from brain synaptosomes. However only the anorectic activity of D-fenfluramine is antagonized by the previous administration of 5-HT antagonists. These results cast some doubts on the role of brain 5-HT in explaining the anorectic activity of fluoxetine and sertraline.


The human pharmacology of fluoxetine.

The human pharmacology of fluoxetine. Fluoxetine is a potent and specific inhibitor of 5-HT uptake even after prolonged dosing. Both fluoxetine and norfluoxetine, an active metabolite, have a long plasma half life. The drug is extensively metabolized and undergoes renal elimination. Impairment of renal function, increasing age or obesity do not appear to alter the disposition of fluoxetine, although hepatic impairment predictably decreases the clearance of the drug. Recent data suggests that fluoxetine is an inhibitor of cytochrome P450-II-D6 in man and has been shown to inhibit the metabolism of a number of drugs which are substrates for this isoenzyme. It does not, however, affect the elimination of ethanol and does not enhance the effect of ethanol on psychometric testing. Fluoxetine has no effect on normal blood pressure although mean heart rate is slightly reduced. It does not appear to have a clinically significant effect on pituitary function. Although there is psychometric and EEG evidence to suggest a vigilance enhancing property, fluoxetine does not exhibit stimulant activity of the amphetamine type and has been shown to be well tolerated at doses clinically useful for appetite suppression in extensive clinical trials and widespread clinical use since marketed in 1987 for depressive illness.


Fluoxetine increases insulin action in obese type II (non-insulin dependent) diabetic patients. Insulin resistance contributes to the metabolic defects in non-insulin dependent diabetes mellitus (NIDDM). Anorectic agents have been shown to improve insulin action in NIDDM, irrespective of weight reduction. In a double-blind placebo-controlled cross-over study, we examined hepatic and peripheral insulin action by the sequential hyperinsulinaemic-euglycaemic clamp technique with infusion of 3-[3H]-glucose in eight obese NIDDM patients and in eight obese non-diabetics, matched for age, sex and body mass index. Body weight was kept constant. After 14 days of fluoxetine, 60 mg daily, in NIDDM half-maximal peripheral glucose uptake was achieved at a lower insulin level than after placebo (ED50pgu: 180.5 +/- 25.8 vs. 225.3 +/- 39.9 mU/l, P < 0.05), but not in non-diabetics (140 +/- 15.3 vs. 135.3 +/- 22.2 mU/l, n.s.). Maximal peripheral glucose uptake (Vmaxpgu) did not change significantly. Multivariate analysis disclosed no differences in the effect of fluoxetine between NIDDM and non-diabetics. When non-diabetics and NIDDM were considered together, only the most insulin-resistant individuals demonstrated a decrease in ED50pgu (P < 0.001). Likewise, only the individuals with the most outspoken hepatic insulin resistance demonstrated a decrease in insulin level, at which hepatic glucose production (HGP) is completely suppressed (HGP0) (P < 0.01). In conclusion, fluoxetine improves peripheral and hepatic insulin action in obese insulin-resistant subjects irrespective of its weight lowering effect.


Fluoxetine therapy in obese diabetic and glucose intolerant patients. A double-blind placebo-controlled trial was conducted, involving 97 obese diabetic and glucose intolerant patients receiving either 60 mg fluoxetine daily (47 patients) or a placebo (50 patients); a similar calorie-restricted diet was prescribed to all patients. Weight loss was significantly higher in the fluoxetine-treated patients, whose diabetic status improved. Drop-out rate was not significantly different for both groups of patients.


A randomized double-blind clinical trial of fluoxetine in obese diabetics. Fluoxetine is an inhibitor of serotonin re-uptake which has been found to produce weight loss in humans and animals. To test the effects of this drug in obese diabetic subjects, 48 male and female, obese type II non-insulin dependent (NIDDM) diabetics who were being treated with insulin were randomized to receive either fluoxetine 60 mg or a placebo once daily in a randomized double-blind fashion for 24 weeks. A four week single-blind placebo lead-in period preceded and a six week single-blind placebo period followed the double-blind treatment period. Subjects performed daily home glucose monitoring and were given instruction in a 1200kcal American Diabetic Association (ADA) diet. Subjects treated with fluoxetine achieved a maximum 8 kg greater weight loss on average than the placebo-treated subjects. At the end of active treatment, fluoxetine-treated subjects had significantly lower glycohaemoglobin levels than the placebo-treated group (9.72 vs. 10.76%, P < 0.05). In addition, fluoxetine-treated subjects showed a greater decrease in total daily insulin dose than placebo-treated subjects (44.5 vs. 20.1% decrease at the end of active treatment, P < 0.05). These results suggest that fluoxetine may be of benefit in the treatment of obese patients with type II non-insulin dependent diabetes mellitus.


A hospital based case-control study of hepatocellular carcinoma. A hospital based case-control study was conducted to define the role of some of the factors predisposing to hepatocellular carcinoma (HCC). It included 62 HCC cases and 62 age, sex and occupation matching control. Selected past history like schistosomal infection, jaundice, blood transfusion, contraceptive pills use, alcohol consumption and family history of cancer were examined. The history of schistosomiasis and that of jaundice show statistical significant differences.


Decreased phosphodiesterase activity in cholera toxin-induced hypersecretion in suckling rats. During cholera toxin (CT)-induced hypersecretion in suckling rats, the rise in the intestinal cAMP concentration was found to be accompanied by a decrease in the cAMP-phosphodiesterase activity. The results suggest the involvement of phosphodiesterase (PDE) as one of the factors governing the rise of cAMP.


Poliovirus surveillance: isolation of polioviruses in Japan, 1980-1991. A report of the National Epidemiological Surveillance of Infectious Agents in Japan. This report presents an overall distribution of poliovirus isolations in Japan, where poliomyelitis has been under control over two decades as a result of legal administration of two doses of the trivalent live oral poliovirus vaccine of the Sabin strains (OPV) to children under 48 months of age. During the past 12 years from 1980 through 1991, a total of 1,126 poliovirus isolations from humans and 268 isolations from sewage/river water were reported by respectively 49 and nine of the participating laboratories. Type 2 was most frequently isolated from children after administration of one dose of OPV, followed by type 1 and type 3. On the contrary, after the second dose of OPV, the rate of isolation of type 3 exceeded those of type 2 and type 1. Seasonal and age distribution of poliovirus isolations from both humans and sewage/river water paralleled the OPV vaccination schedule in Japan. One percent of the isolations were, however, from infants younger than the vaccination-scheduled ages and 5% were from children older than those ages, including one each from 15 and 16 years olds. The data indicate that the poliovirus has silently been disseminated from vaccinated children to others and the community, thus suggesting repeated transmission of the viruses. The fact that some elder children had poliovirus colonization in their alimentary tracts indicates a potential risk of infection of such a population when exposed to a wild virus and of becoming a source of transmission to others.


Disseminated intravascular coagulation (DIC) in rabbit haemorrhagic disease. Seven rabbits experimentally infected with rabbit haemorrhagic disease virus were examined haematologically and histologically. Haematologically, activated partial thromboplastin time and prothrombin time were markedly prolonged in the terminal phase of the disease, just prior to death (all the animals died between 27 and 40 hr after inoculation with rabbit haemorrhagic disease virus). There was an increase in the titre of fibrin degradation products and a decrease in antithrombin III activity during the same interval. Acute necrotic hepatitis and disseminated intravascular coagulation (DIC) in many organs, including the lung, kidney, spleen and heart were the characteristic histopathological changes. Thus, the haematological and histological changes suggested that DIC was induced by rabbit haemorrhagic disease virus infection. Severe liver necrosis was considered to be a factor causing DIC by inducing a hypercoagulable condition in the systemic blood circulation.


[Pregnancy in patients with essential thrombocythemia. Three cases]. The authors report 3 pregnancies in 3 patients with essential thrombocythemia. In the 3 cases, the course of the pregnancy was successful, with normal delivery in 2 cases, and premature delivery of a live fetus because of an abruptio placentae in the third case. From these 3 cases and a review of the literature, the authors analyse the fetal and maternal risks of pregnancy in these patients and the potential usefulness of preventive treatments (platelet antiaggregating drugs, anticoagulants). Besides, in these 3 cases a steadily decrease in blood platelet counts was observed during pregnancy with a nadir just before delivery followed by a sharp increase in the days following delivery. These findings may give some useful information on the regulation of thrombopoiesis.


[Effect of heavy water on the growth, glucose assimilation and stability of Escherichia coli to freezing-thawing]. Growth and resistance to freezing--thawing of Escherichia coli B-1640 were investigated during cultivation in synthetic media prepared with H2O and D2O. It is found that during cultivation in D2O the maximum specific growth rate decreases and the duration of the exponential growth phase increases. During the growth in D2O the glucose consumption rate drops in the exponential growth phase, the lactate content in the culture liquid is lower by two orders than that in H2O; the resistance to freezing--thawing is lower than that in H2O. After leaving the exponential phase the culture in D2O restores specific growth rate, glucose consumption rate and resistance to freezing--thawing up to the values obtained during the growth in H2O. The translation ability of ribosomes isolated from cells grown in D2O and H2O is the same. We conclude that the culture adapts to D2O during the exponential growth phase. It is suggested that during the adaptation the second carbon source is used which compensates the consequences of the disturbances of glucose metabolism and transport caused by deuteration of the cell content in the adaptation to D2O.


Calcitonin gene-related peptide: multiple actions, multiple receptors. Calcitonin gene-related peptide (CGRP) shows diversity both in its effects and its receptors. It is likely to have roles as a neurotransmitter, neuromodulator, local hormone and trophic factor. Its effects include rapid changes in neuronal activity, relaxation of many types of smooth muscle, actions on metabolism and changes in gene expression. Receptor heterogeneity has been revealed from experiments comparing agonist potency ratios and antagonist affinities. The evidence from these approaches is reviewed in this article and a speculative receptor classification scheme is proposed. Some of the likely future directions for CGRP research are discussed.


High performance liquid chromatographic (HPLC) measures of hydrophobicity as determined by means of new HPLC columns. Two new commercially available reversed phase high performance liquid chromatographic (HPLC) columns were tested from the view point of their usefulness for determination of hydrophobicity by means of partition chromatography. One column comprised a specially prepared hydrocarbon-bonded silica stationary phase material. The other column was packed with a polybutadiene-coated alumina (PBCA) phase. As reference served a regular commercial octadecylsilica (ODS) column. A series of test solutes were pyrazine CH- and NH-acids--the compounds which are able to take part in specific as well as in nonspecific intermolecular interactions. Unique properties of the new hydrocarbonaceous column manifested themselves in regular linear relationships between logarithms of capacity factors and volume percent of methanol in aqueous eluent. The main advantage of the PBCA column is a possibility of performing chromatography at alkaline pH. Both new columns are superior to regular ODS columns in respect of providing reliable chromatographic measures of hydrophobicity. It was observed that hydrophobicity parameters determined in individual HPLC systems are not highly intercorrelated and hence can reflect different structural features of solutes.


Neoadjuvant chemotherapy with cisplatin, epirubicin and VP-16 for stage IIIA-IIIB non-small-cell lung cancer: a pilot study. Twenty patients with stage IIIA-IIIB non-small-cell lung cancer were treated with cisplatin, epirubicin and VP-16 (PEV) neoadjuvant chemotherapy (CDDP, 70 mg/m2, i.v., d 1; EDX, 60 mg/m2, i.v., d 1; VP-16, 100 mg/m2, i.v., d 1-2-3; every 3 weeks). A partial response was obtained in 11 cases (55%), stable disease in 3 cases (15%), and progressive disease in 6 cases (30%). After chemotherapy, 8 (40%) patients, all achieving a partial response, were elegible for surgery: 5 (25%) had a complete resection (4 IIIA and 1 IIIB) and 3 (15%) an incomplete resection. The treatment was well tolerated. These data show that PEV is an active regimen for neoadjuvant chemotherapy in NSCLC and recommend this therapeutic approach for stage IIIA patients.


Small cell carcinoma of the urinary bladder. Report of two cases and review of the literature. Undifferentiated small cell carcinoma of the bladder is a rare but aggressive subset of urinary tract neoplasms. Analogous to small-cell carcinoma of the lung, this tumor frequently exhibits neuroendocrine differentiation. We report the 92nd and 93rd case of small cell carcinoma of the bladder reported in the literature with characteristic cytologic, histologic, histochemical, and ultrastructural features. The patients were treated initially with chemotherapy, but after a brief clinical course died for progression of disease and for myocardial infarction, respectively. The pathologic and clinical features and therapeutic options of the cases described in the literature are reviewed.


Effect of monensin on fermentation of hay and wheat bran investigated by the Rumen Simulation Technique (Rusitec). 1. Basal parameters of fermentation. An experiment was performed with Rumen Simulation Technique (Rusitec) in which the fermentation of mixed ration of hay (12.8 g/d) and wheat bran (3.2 g/d) was compared with the fermentation of the same diet in the presence of 2, 5 and 10 mg of monensin/d. The duration of the experiment was 12 days. During the first six days the fermentation conditions in Rusitec were stabilized. Monensin significantly depressed the digestibility of dry matter and fibre digestion--neutral detergent fibre (NDF), acid detergent fibre (ADF) and cellulose but only after using 2 mg monensin/d. Addition of monensin depressed the production of VFAs--acetate, butyrate and isovalerate and acetate:propionate ratio. Production of propionate was increased and production of methane and CO2 was decreased in the presence of monensin. It can be explained by changes in the production of VFAs and redistribution of metabolic hydrogen. The recovery of nitrogen was satisfactory (about 100%) and its distribution in the effluent was increased by monensin. The recovery of protein (measured as alpha--NH2 groups) and distribution of protein in the effluent and residues was increased with the increasing dose of monensin.


Effect of monensin on fermentation of hay and wheat bran investigated by the Rumen Simulation Technique (Rusitec). 2. End-products of fermentation and protein synthesis. An experiment was performed with Rumen Simulation Technique (Rusitec) in which the fermentation of mixed ration of hay (12.8 g/d) and wheat bran (3.2 g/d) was compared with the fermentation of the same diet supplemented with 2.5 and 10 mg monensin. The duration of the experiment was 12 days. During the first six days the fermentation conditions in Rusitec were stabilised. The end products of fermentation and indices of protein synthesis were determined. The energy efficiency of volatile fatty acids (VFA), proportion of fermented hexose energy on VFA energy and on energy of bacterial matter were increased in the presence of monensin. The proportion of fermented hexose energy in methane energy was decreased. Utilization of glucose and production of adenosine triphosphate were not affected. The balance of metabolic H2 was reduced and this was manifested in decreasing production, utilization and recovery of metabolic H2. Microbial efficiency expressed per mol ATP (g/mol) was increased by monensin from 7.8 to 12.6. The indices of protein synthesis--protein conversion ratio, degradability of protein and microbial protein synthesis efficiency were increased and non protein utilization ratio decreased in the presence of monensin.


Influence of yeast (Saccharomyces cerevisiae as Yea-Sacc or Levaferm) on in Sacco dry matter degradability and ruminal parameters of variously fed small ruminants. Two series of experiments with rumen fistulated castrated male sheep and goats were carried out. In experiment I three sheep each consumed rations rich in concentrate (700 g concentrate, 200 g chopped wheat straw) or roughage (700 g artificially dried ryegrass, 200 g chopped wheat straw per animal per day) and supplemented with 0, 1, 2 or 4 g Yea-Sacc (Saccharomyces cerevisiae; USA) per sheep per day. In experiment II three sheep were fed with 1000 g artificially dried ryegrass and 200 g concentrate, three goats consumed 750 g ryegrass and 150 g concentrate. 0, 0.5, 1 or 2 g Levaferm (Saccharomyces cerevisiae; Germany) per animal per day were added. Rations of all animals were supplemented with minerals and vitamins. After 14 days of feeding wheat straw, ammonia treated wheat straw and artificially dried grass (exp. I) or wheat straw and artificially dried grass (exp. II) were incubated in nylon bags in the rumen for 6, 12, 24, 48 and 72 hours. At the end of the experiments rumen fluid was taken via cannulae and parameters of rumen fermentation were measured. Higher levels of added Yea-Sacc decreased in sacco dry matter degradability of all incubated feeds. Depression was much higher if Yea-Sacc was added to the concentrate ration (overall mean for 24, 48 and 72 h incubation time: 55.1, 47.1, 46.1 and 44.5 for 0, 1, 2 and 4 g Yea-Sacc) than to the roughage diet (58.7, 56.3, 55.0 and 54.1%). Levaferm did not significantly influence the rumen dry matter degradability of incubated feeds (overall mean for 24, 48, and 72 h incubation time: 64.0; 64.9; 64.9 and 64.2% for sheep; 63.0; 63.2; 63.2 and 61.6% for goats, if added with 0, 0.5, 1 and 2 g Levaferm per animal per day). Rumen pH, concentration of volatile fatty acids and molar concentration of fatty acids in rumen fluid were not significantly influenced by added yeasts. More research seems necessary to find out the mode of action of yeast and to quantify and to reproduce the effects of added yeast.


The influence of contamination with separate mycotoxins (aflatoxins, ochratoxin A, citrinin, patulin, penicillic acid or sterigmatocystin) on the in vitro dry matter and organic matter digestibilities of some roughages (berseem hay and wheat straw). In vitro study on berseem hay and wheat straw was undertaken to investigate the the effect of mycotoxin contamination on dry matter and organic matter digestibilities. The data revealed a negative effect of most studied mycotoxins on the materials digestibility. Among the investigated mycotoxins, penicillic acid with its two concentrations (5 and 10 nmol) was the most negative, affecting digestibilities of both feed materials. Wheat straw digestibility was more influenced than berseem hay by the ochratoxin A, citrinin and sterigmatocystin (besides the penicillic acid) particularly with their high level (10 nmol). Yet, some mycotoxins act as antibiotics which may affect only the harmful flora but encourage the rumen microflora resulting in slight improvement of digestibility. The rumen conditions were able to metabolize or deform the used levels of all mycotoxins studied. Thus, there were no detectable residues of these mycotoxins in the digestion media after the in vitro fermentation.


Expression of a mutant regulatory subunit of cAMP-dependent protein kinase in the Caco-2 human colonic carcinoma cell line. Caco-2 human colonic carcinoma cells were transfected with an expression vector encoding a mutant form of RI (regulatory subunit of the type 1 cAMP-dependent protein kinase), driven by the metallothionein 1 promoter. A stable transformant was isolated that expressed the mutant RI gene in a Zn(2+)-inducible manner. The consequences of the RI mutation on cAMP-dependent protein kinase activity, cell division, and regulation of chloride efflux were examined. When grown in the absence of ZnSO4, protein kinase activity in the transformant was stimulated 2.5-fold by cAMP and approached the levels of cAMP-dependent protein kinase activity seen in parental Caco-2 cells; when treated with ZnSO4, cAMP-dependent protein kinase activity in the transformant was inhibited by 60%. In the absence of ZnSO4 the transformant grew with the same doubling time and to the same saturation density as the untransformed parent. In the presence of ZnSO4 the transformant exhibited a cAMP-reversible inhibition of cell division, indicating that a functional cAMP-dependent protein kinase was required for the growth of these cells in culture. Induction of the mutant RI gene also abolished forskolin-stimulated chloride efflux from these cells, suggesting obligatory roles for cAMP and cAMP-dependent protein kinase in forskolin's actions on chloride channel activity. We anticipate that this transformant will be useful for further studies on the roles of cAMP and cAMP-dependent protein kinase in the regulation of intestinal epithelial cells, including regulation of cell proliferation and differentiation, and regulation of chloride channel activity by neurohormones and neurotransmitters.


Regulation of the gene encoding the p24 actin-binding protein in Dictyostelium discoideum. We have isolated cDNA clones on the basis of sequence similarity to the gene encoding the cyclic cAMP-binding protein CABP1 of Dictyostelium discoideum. The predicted amino acid sequence of the cloned cDNAs shows that the homology to CABP1 is restricted to a region rich in proline, glycine, glutamine, and tyrosine. Sequence comparison indicates that the cloned cDNAs encode the actin-binding protein p24. We have examined by RNA blot hybridization the expression of the gene encoding p24. For cells developed in suspension, the levels of p24 mRNA increase rapidly during early development, reaching a peak at 3-4 h. Addition of high concentrations of exogenous cAMP during the first 4 h of development produced little or no effect on the accumulation of p24 mRNA. Treatment with cAMP during subsequent stages of development reduced the levels of p24 mRNA. We attempted to determine if the synthesis of new proteins during early development is a requirement for the reduction in p24 mRNA levels by treating the cells with protein synthesis inhibitor. Unexpectedly, the addition of the inhibitor cycloheximide resulted in an increase in the level of p24 mRNA. The roles of cycloheximide and cAMP on the expression of the p24 gene are discussed.


Regulation of mammalian ribonucleotide reductase by the tumor promoters and protein phosphatase inhibitors okadaic acid and calyculin A. A rapid elevation of ribonucleotide reductase activity was observed with BALB c/3T3 fibroblasts treated with 10 nM okadaic acid, a nonphorbol ester tumor promoter and protein phosphatase inhibitor. Northern blot analysis of the two components of ribonucleotide reductase (R1 and R2) showed a marked elevation of R1 and R2 mRNA expression. Western blot analysis with R1 and R2 specific monoclonal antibodies indicated that the increase in ribonucleotide reductase activity was primarily due to the elevation of the R2 rather than the R1 protein during treatment with okadaic acid. The okadaic acid induced elevations in R1 and R2 message levels occurred without a detectable change in the proportion of cells in S phase and were blocked by treatment of cells with actinomycin D, indicating the importance of the reductase transcriptional process in responding to the action of okadaic acid. Furthermore, down-regulation of protein kinase C with 12-O-tetradecanoylphorbol-13-acetate pretreatment abrogated the okadaic acid mediated elevation of ribonucleotide reductase mRNAs, consistent with the involvement of this signal pathway in the regulation of ribonucleotide reductase and the effects of okadaic acid. Treatment of cells with 2.5 nM calyculin A, another non-phorbol ester tumor promoter and protein phosphatase inhibitor, resulted in a rapid elevation of both R1 and R2 mRNA levels within 10 min of treatment.(ABSTRACT TRUNCATED AT 250 WORDS)


Spinal and cortical potentials evoked by tibial nerve stimulation in humans: effects of sex, age and height. Somatosensory evoked potentials by posterior tibial nerve stimulation at the ankle were performed in 74 healthy volunteers (36 females and 38 males) aged 14-76 years. Cortical potentials were obtained in all subjects and spinal potentials (N22) in 71 subjects. All parameters were related to subject's age, height and sex. Sex influenced only P40-N50 amplitude, which was greater in females. All latencies of spinal and cortical components increased in a similar manner with subject's height (about 0.16-0.18 ms per cm), whereas the N22-P40 interpeak latency was independent from height, but related to T12-Cz distance. Absolute latencies of the spinal and of most cortical components, but not interpeak latencies, increased with subject's age (about 0.06-0.09 ms per year). The parameters to compute normative data (according to univariate or bivariate regression models) are furnished. Limits of right-left differences are reported.


Toxin resistance and reduced secretion in a mouse L-cell mutant defective in herpes virus propagation. The mouse L-cell mutant gro29 was selected originally for its inability to propagate herpes simplex virus; it shows severe defects in virus egress and the transport and processing of viral glycoproteins after infection. In this report, we show that uninfected gro29 cells display pleiotropic changes in protein secretion, oligosaccharide processing, and sensitivity to the toxins ricin and modeccin. Specifically, the rate of secretion of a nonglycosylated protein, human growth hormone, was reduced 70% in gro29 cells compared with the parental L cells. A direct measurement of the transport capacity of Golgi membranes in a cell-free assay suggests that gro29 cells contain less functional Golgi than parental cells. Despite this deficiency, N-linked oligosaccharides were processed efficiently in mutant cells, although there were differences in the structure of the mature forms. Lectin intoxication assays revealed that gro29 cells were cross-resistant to killing by the cytotoxic lectins ricin and modeccin, but not to wheat germ agglutinin, Ricinus communis agglutinin RCA120, or leucoagglutinin. Fluorescence labeling using fluorescein-conjugated lectins showed that uninfected gro29 cells expressed relatively few ricin-binding molecules, suggesting a possible mechanism for toxin resistance. These studies provide evidence that the processes of protein secretion, lectin intoxication, and herpes virus maturation and egress may share a common cellular component.


Hepatocellular carcinoma and preneoplastic lesions of the liver: evaluation of argyrophilic nucleolar organizer regions (AgNORs). The authors applied a silver colloid technique to identify Argyrophilic Organiser Region (AgNOR) to 8 groups of hepatic lesions: alcoholic hepatitis with dysplasia (3 cases); chronic active hepatitis with dysplasia (4 cases); cirrhosis with dysplasia (5 cases); focal nodular hyperplasia (4 cases) and hepatocellular carcinomas (3 cases of grade I, 3 cases of grade II and 5 cases of grade III of Edmondson). Four cases of non-specific reactive hepatitis were used as control. This work suggests the simplicity and utility of simultaneous application of clumps per cell, AgNORs per clump and total AgNORs counts in the evaluation of neoplastic and preneoplastic lesions of the liver. The results show, in hepatocellular carcinomas, a relationship between the number of clumps, the AgNORs per clump, the total number of AgNORs and the grading of Edmondson. The nodular lesions that can be considered in the differential diagnosis with carcinoma are sufficiently well discriminated using the two parameters AgNORs per clump and total number of AgNORs.


Disruption of the coordinate expression of muscle genes in a transfected BC3H1 myoblast cell line producing a low level of the adenovirus E1A transforming protein. Mouse BC3H1 myoblasts were stably transfected with the adenovirus 5 E1A gene. One clonal line, BC3E7, was found to differ in some important respects from those previously reported for E1A-transformed myoblasts. In contrast to BC3H1 cells which differentiate when confluent in medium containing 0.5% fetal calf serum (FCS), BC3E7 cells failed to elongate and align, to express acetylcholine receptor and creatine kinase, and to down-regulate expression of beta- and gamma-actins and tropomyosin isoform (TM) 1. However, increased synthesis of TMs 2, 3, and 4, and myosin light chain 1 associated with differentiation in BC3H1 still occurred in BC3E7 cells, and most surprisingly, alpha-actin was produced at a significant level in both proliferating and confluent BC3E7 cells. Interestingly, myogenin was expressed in confluent BC3E7 cells in 0.5% FCS, but not in 20%. The level of E1A expression in BC3E7 cells was found to be very low by analysis of mRNA, by immunoprecipitation of E1A protein, and by the ability of BC3E7 cells to complement the E1A-deficient adenovirus mutant dl312. These results suggest that different levels of E1A may be needed to repress different promoters and that E1A does not block myogenic differentiation by repressing myogenin expression, but represses each muscle gene independently.


cAMP-dependent protein kinase, but not the cGMP-dependent enzyme, rapidly phosphorylates delta-CREB, and a synthetic delta-CREB peptide. Phosphorylation of the cAMP response element binding protein (CREB) by the catalytic subunit of cAMP-dependent protein kinase (cAK) has been implicated in the cAMP-dependent stimulation of gene transcription. delta-CREB, a spliced variant of CREB, and CREBtide (KRREILSRRPSYR), a synthetic peptide based on the phosphorylation sequence in delta-CREB, were tested as substrates of cAK. Phosphorylation of delta-CREB (0.17 microM) was stoichiometric within 30 s when using a concentration of cAK which approximated the intracellular level (0.2 microM). The rate of phosphorylation of delta-CREB was comparable to the rates of the best physiological substrates of cAK tested. The rate of CREBtide phosphorylation was at least as great as that of delta-CREB, indicating that the peptide retained the determinants of delta-CREB which were responsible for substrate efficacy. The apparent Km of CREBtide phosphorylation by cAK was 3.9 microM, which is 10-fold lower than that of kemptide (Km = 39 microM), the synthetic peptide substrate most often employed for cAK measurement. The Vmax values were 12.4 mumol/(min.mg) for CREBtide and 9.8 mumol/(min.mg) for kemptide. The apparent Km of CREBtide phosphorylation by cGMP-dependent protein kinase (cGK) was 2.9 microM and the Vmax value was 3.2 mumol/(min.mg). Both delta-CREB and CREBtide were phosphorylated at a much slower rate by cGK as compared with cAK, implying that the high cAK/cGK specificity exhibited by delta-CREB was retained by the peptide. Taken together, the results indicated that delta-CREB and CREBtide are among the best substrates tested for cAK and suggested that phosphorylation of CREB by this enzyme could occur in intact cells.(ABSTRACT TRUNCATED AT 250 WORDS)


How to make a blastocyst.

How to make a blastocyst. Several of the new reproductive technologies have been cultivated from our current understanding of the genetic programming and cellular processes that are involved in the major morphogenetic events of mammalian preimplantation development. Research directed at characterizing the patterns of gene expression during early development has shown that the embryo is initially under maternal control and later superseded by new transcriptional activity provided by the activation of the embryonic genome. Several embryonic transcripts encoding: (i) growth factors, (ii) cell junctions, (iii) plasma membrane ion transporters, and (iv) cell adhesion molecules have been identified as contributing directly to the progression of the embryo through the preimplantation interval of development. In this brief review, we have outlined the patterns of expression and the integral roles that these gene families play in the morphogenetic events of compaction and cavitation. Research of this type has greatly facilitate our understanding of the control processes that underlie preimplantation development and represent but one area of this exciting and vigorous field of research.


Patterning of development in Dictyostelium discoideum: factors regulating growth, differentiation, spore dormancy, and germination. Cellular communication dictates all stages of growth and development in the cellular slime molds. Dictyostelium discoideum utilizes a number of signal molecules for cell-to-cell communication, including growth and density factors, cAMP, ammonia, differentiation-inducing factor, discadenine, and spore autoactivator. A source and sink model is presented in which the assimilation of ammonia plays a major role in determining cell fate and pattern formation. This model emphasizes a recycling of ammonia by prespore cells, the accumulation of free hydrophilic and neutral amino acids, and their incorporation into proteins associated with sporulation and (or) germination. If spore cAMP signalling is regulated by the relative concentrations of discadenine and autoactivator, and its disruption triggers the initiation of the spore germination cascade, then the accumulation of intracellular cAMP may be necessary for both sporulation and dormancy maintenance.


Activities of scavenging enzymes of oxygen radicals in early maturation stages of Paragonimus westermani. In early maturation stages of Paragonimus westermani (metacercariae, 4-, 8-, 12-week old worms), activities of antioxidant enzymes, such as superoxide dismutase, catalase, peroxidase and glutathione peroxidase, were examined. Specific activity of catalase was the highest in metacercariae and decreasing with age. That of superoxide dismutase was higher in metacercariae and 4-week worms. Specific activity of peroxidase was at its peak in 4-week worms while that of glutathione peroxidase was in 8-week worms. Specific activities of all these antioxidant enzymes were decreased to their lowest in 12-week old adults.


Dissociation of magnitude of relaxation from cyclic AMP levels in rabbit urinary bladder smooth muscles. The effects of forskolin and isoproterenol on contractile force and cyclic AMP levels were compared in smooth muscle strips from rabbit urinary bladder dome. Both of forskolin and isoproterenol produced the time- and the dose-dependent relaxation and the time- and the dose-dependent increases in cAMP levels. The relaxant response to forskolin caused more slowly than that to isoproterenol. The two agents caused almost the same relaxant responses at same concentration. However, cAMP levels induced by forskolin was much more than those induced by isoproterenol. These results that amounts of cAMP in urinary bladder don't correlate with the relaxation responses in urinary bladder smooth muscle strips suggest that some forms of functional compartmentalization of cAMP may exist in the urinary bladder.


[New findings on calcium antagonism].

[New findings on calcium antagonism]. The family of calcium antagonist substances is continuously increasing. Often it is difficult, if not impossible, to have clear, objective criteria to differentiate between the available molecules. We have considered some molecular aspects of calcium channels and of the effects of calcium antagonists which may be relevant for the clinical utilization of these drugs. In particular, differences between L and T type of calcium channels in the myocytes and between VOC and ROC calcium channels in the smooth muscle are described. Then we have considered the main differences in the mechanism of action and clinical use of the three prototypes of calcium antagonists: phenilalkilamines, dihydropyridines and benzothiazepines. Finally, we have synthetically depicted the characteristic of the second generation agents such as nisoldipine, amlodipine, felodipine, isradipine, lacidipine and gallopamil.


[Cellular calcium, vasoconstriction, hypertension]. The control of vasomotion is a central issue in blood pressure regulation and is a primary goal of antihypertensive therapy. Calcium is the final messenger in the contractile mechanism of vascular smooth and cardiac muscle. Vasoconstrictor agents enhance the entry of Ca++ into vascular myocytes; vasodilators usually depress it. The most recent findings, based on direct measures of intracellular Ca++, have also highlighted the importance of calcium-sensitization mechanisms: vasoconstrictors sensitize the contractile apparatus to Ca++; vasodilators have an opposite effect. Cell calcium control alterations have been reported in different forms of hypertension. An increase in vascular myoplasmic Ca++ and a higher rate of calcium influx through specific, dihydropiridine-sensitive calcium channels have been found in genetic or secondary animal models. In hypertensive patients, an elevation of cytoplasmic Ca++ was noted in the platelets. Since Ca++ is a ubiquitous intracellular messenger, these changes may have profound implications for the pathophysiology of hypertension.


Determination of T cell monoclonality in non-Hodgkin's lymphoma by frozen section immunohistology. The SWOG Central Repository Members. Monoclonal antibodies (mAb) reactive with seven distinct T cell receptor (TcR) alpha/beta variable region (V) families have become available. We investigated the potential utility of these mAb to establish T cell clonality (restrictive expression of one single V region family type) by frozen section immunohistology. We studied 40 non-Hodgkin's lymphomas (NHL) previously classified, immunophenotypically and genotypically by the South Western Oncology Group (SWOG) as 20 B and 20 T cell NHL. Frozen sections of each neoplasm were immunostained with the following mAb: beta-V5a, beta-V5b, beta-V6a, beta-V8a, beta-V12a, alpha/beta-Va and alpha-V2a. The large atypical lymphocytes of 18 of 20 T cell NHL showed no reactivity with the seven V region family mAb and only two showed exclusive immunoreactivity (one with anti-alpha V2a and the other with anti-beta V6a). All large atypical B cells in the 20 B cell NHL were non-reactive with the V region family mAb and each of the 40 neoplasms disclosed no or a trace reactivity in small host T cells. The results show that clonality can be determined in only a small percentage of T cell NHL (Sensitivity 10%, specificity 100%). Therefore, until new mAb become available, genotypic analysis remains the most sensitive and reliable method to establish T cell clonality.


Hormones and immune responsiveness in chronic lymphocytic leukemia. Chronic lymphocytic leukemia (CLL) is a B cell disorder with multiple abnormalities in T-cell function. The status of the immune system will depend to some extent upon the net effect of the changes in the equilibrium of various hormones. In order to investigate the association of the defects in the cellular immunity and hormonal dysregulation in CLL, studies were performed in 130 CLL patients with that disorder. Decreased lymphocyte proliferation in response to mitogen stimulation appears to be an early event in CLL pathogenesis and is not always influenced by the clinical stage of the disease or the specific treatment. The dysfunction of T-lymphocytes was accompanied by increased serum cortisol (C) concentrations. Elevated levels of follicle stimulating hormone (FSH), luteinizing hormone (LH), 17 beta-estradiol (E) and testosterone (T) ratio were found in male CLL patients, but not in female patients. In view of our findings, it is conceivable that there are a number of disturbances in the lymphocyte-microenvironmental regulation, which may be responsible for immuno-hormonal imbalance in some patients with CLL.


[The cluster headache: a clinical model of immunologic receptor pathology?]. It is well established that cluster headache shows impaired functions at their neuroimmunomodulatory system level. Defect in receptor expression for 5-HT, IL-1 and IL-2 have been found in these patients. Sumatriptan, a molecule with agonistic activity for 5-HT1D receptor, truncates cluster headache attacks in 74% of patients. Flow cytometric analysis of monocytes expressing 5-HT receptor in cluster headache patients showed different trends clearly correlated with the clinical response to sumatriptan. Our findings strongly support the concept that cluster headache patients who are non responders to sumatriptan could present a block in their 5-HT receptor possibly due to specific autoantibodies for this receptor site.


Adaptation of the herpetic virus on human diploid and monkey kidney cells. Viral suspensions of herpetic virus were inoculated on different cell cultures: human diploid cells (HDC) and monkey kidney cells--primary culture and cell line--for adapting the virus on these cells and obtaining several high constant titers. The human diploid cells and the monkey kidney cell line have proved to be the most suitable cells for the virus growth; thus an optimum development of the virus with a high constant titer was obtained starting with the 4th passage (10(5.7)-10(6)CPD/50/ml). The optimum conditions provided for the herpetic virus culture have allowed to obtain a herpetic virus stock used as control preparation for testing the efficacy of the "OFTALMOHERPIN" biological preparation for current use in the prophylaxis and therapy of ocular herpes.


Non radioactive in situ hybridization for detection of human papilloma virus DNA in squamous cell carcinoma of tongue. Previous studies indicate that HPV type 16 and 18 (HPV) are associated with squamous cell carcinoma of the head and neck. In this investigation we evaluate in our hospital 253 patients with oral squamous cell carcinoma of the tongue not related to tobacco or alcohol with a tumor index of T2 NO MO between 1981 and 1991. From 12 patients we were able to obtain tissue. For detection of human papilloma virus, DNA sequences 6, 11, 16, 18, 31 and 33 in paraffin-embedded human tissue biopsies a non-radioactive in situ hybridization procedure was utilized. Approximately 60% of the carcinoma of the tongue are positive for episomal viral DNA 6, 11, 16 and 18. These results confirms that HPV infection may play a possible role in the multifactorial etiology of carcinogenesis of squamous cell carcinoma of the tongue and probably acting synergistically with other carcinogenesis.


Evaluation of the biological activity of unmodified synthetic eel calcitonin rectal capsules. Comparison with intramuscular administration and placebo. Biological activity of eel calcitonin 100 IU administered by the rectal route was evaluated in healthy volunteers by measuring plasmatic variations of cyclic adenosine monophosphate (cAMP) after a single dose and during a 21-day treatment. This formulation appears to allow a bioavailability of the drug which is half of the same dose administered intramuscularly, and to cause a prompt and significant increase of plasma cAMP, with minimal variations of calcaemia. Repeated administration shows the persistence in time of this effect. The overall conclusion is that the amount of calcitonin absorbed through the rectal mucosa seems sufficient to induce a typical biological response related to the interaction of the hormone with its specific receptors in bone.


Functional conservation of nematode and vertebrate myogenic regulatory factors. The Caenorhabditis elegans protein, CeMyoD, is related to the vertebrate myogenic regulatory factors MyoD, myogenin, MRF-4 and Myf-5. Like its vertebrate counterparts, CeMyoD accumulates in the nucleus of striated muscle cells prior to the onset of terminal differentiation. CeMyoD also shares functional similarities with the vertebrate myogenic regulatory factors. Viral LTR driven expression of CeMyoD in mouse 10T1/2 cells can convert this cell line into myoblasts as well as efficiently trans-activate mouse muscle-specific promoters. Furthermore, mouse MyoD expression can activate a CeMyoD-beta-galactosidase reporter construct in a 10T1/2 co-transfection assay.


Retinoid receptors and binding proteins. Retinoids, in particular all-trans retinoic acid (T-RA), are essential for normal development and homeostasis of vertebrates. Although many effects of retinoids, particularly with regard to teratogenicity, have been described in the literature, the mechanisms by which these simple signalling molecules work has only recently begun to be elucidated. We now recognize at least two classes of retinoid-binding proteins and two families of retinoid receptors. The ultimate interpretation of the retinoid signal within a given cell is probably the result of a complex series of interactions between these proteins, yet little is understood concerning the role each member of this signalling pathway plays. It is therefore imperative to dissect the molecular mechanisms which transduce the effects of these ligands, both in vivo and in isolated systems. One approach we are employing is gene targeting of retinoic acid receptors (RARs) and cellular retinoid-binding proteins to generate mice in which one or more of these genes has been functionally inactivated.


In vitro effect of opioid agonist and antagonist on superoxide release by granulocytes. The effect of the opioid agonist morphine and of the (-) and (+) stereoisomers of the antagonist naloxone were studied on the O2-generation from human granulocytes. Morphine or naloxone had no effect on basal or phorbol myristate acetate (PMA) stimulated O2-generation, while equimolar (-) naloxone and morphine concentrations (1 x 10-13 - 1 x 10-7 M) inhibited the stimulated O2-generation. The effect of (-) naloxone was stereospecific, suggesting the involvement of opioid receptors. The unmasking of non opioid effects of morphine could be responsible for the inhibition of O2-generation. It is suggested that the opioid control of oxidative metabolism in human granulocytes could involve multiple receptors mediating opposite effect.


Neural plasticity and memory: towards an integrated view. The search for the neural determinants of learning and memory has recently focused on phenomena of neural plasticity. The present paper will try to review and relate the functional significance of such specific plastic changes within neural elements to the process of learning in several species and cell types. Pioneer work proposed in simple organisms and recent emerging evidence on the role of the NMDA receptor complex and calcium-related events will be analyzed in the light of experimental findings suggesting that common biochemical events may underlie different forms of experience-dependent neural adaptation.


A perspective on narcolepsy.

A perspective on narcolepsy. Neurochemical analyses were performed on brains obtained at post mortem from 3 patients with narcolepsy. The salient findings were an increase in noradrenergic and serotonergic neuronal activity, a decrease in dopaminergic neuronal activity, and, in 2 of the 3 brains, a decrease in the number of alpha-1-noradrenergic receptors in the frontal cortex and amygdala. A pathophysiological model for narcolepsy is developed on the basis of these findings. The neurobiology of narcolepsy is compared with that of depression. The role of MHC--class II gene products in the genesis of narcolepsy is discussed. Some treatment implications follow.


Serum B12 binding proteins versus seminal plasma binder. It was well confirmed that B12 binding protein in human serum and body fluids largely consists of two kinds of binding proteins, physiologically B12 transporting transcobalamin and haptocorrins which does not involve in B12 transport. In seminal plasma, very potent B12 binding protein, in quantity as much as 23 times of normal serum, was identified. Separation characteristics, gel filtration, CM cellulose column chromatography and column IEF all agreed as one of the haptocorrins. One peculiar feature of seminal plasma binder is marked heterogeneity of much lower pI regions suggesting the presence of increased amount of sialic residues on the molecule. The preliminary data from this laboratory support the view, however, physiological role such as the relationship to the spermatogenesis is not known. The most recent findings in clinical observation on B12 is the treatment of male infertility using methycobalamin. The detailed analysis of seminal binders may open up new arena of B12 bioeffect.


Effect of a centrally-acting muscle relaxant, eperisone hydrochloride, on muscle sympathetic nerve activity in humans. The sympatho-modulating effects of eperisone hydrochloride, a centrally acting muscle relaxant, on microneurographically recorded muscle sympathetic nerve activity (MSA) were analyzed in human volunteers. A single dose of 300 mg of eperisone was orally administered to 19 healthy subjects aged between 19 and 27, and effects on 1) spontaneous MSA in 30 degrees head-up tilted position, 2) resting MSA and responsiveness to standing, 3) exercise-induced enhancement of MSA were observed. Eperisone has a sympatho-suppressive action in resting skeletal muscles, but has no effect on MSA in actively contracting muscles, e.g. standing, hand-gripping. The sympatho-suppressive effect of eperisone may be related to the drug-induced increase of blood low in the resting skeletal muscles; also it may be one more mechanism through which the drug can exert its muscle relaxant action.


Vitamin A: differentiating action on gastric cancer cells and gastric mucosal cells in elderly people. Since RA is effective only in gastric mucosal cells but not in isolated parietal cells, it is strongly suggested that RA acts on multi-potential stem cells in the gastric mucosa, facilitating their differentiation into mature parietal cells. Moreover, although RA was very effective in newborn rats as well as in young people, responsiveness to RA is decreased in the elderly people especially with atrophic gastritis. This unresponsiveness in the elderly people might be involved in the development of gastric cancer.


Hydroxyl radical generation from heart mitochondria damaged by ischemia. The hydroxyl radicals are known to be a biologically active oxygen species. A remarkable enhancement of hydroxyl radical generation was observed in the mitochondria obtained from ischemic myocardium. The hydroxyl radicals are the primary reactive species leading to cellular damages, such as membrane damages, DNA damages, enzyme inactivation, protein denaturation and so forth. The origin of the hydroxyl radicals appeared in mitochondria was discussed.


Imaging of neurotransmitter receptors in the living human brain by positron emission tomography (PET). More than 30 receptors or subtypes will be investigated in various neurological and psychiatric disorders using PET in the near future. However, the data obtained by PET should be carefully evaluated, because the PET method is based on the ligand-receptor binding in vivo. Extensive studies on animals and autopsied human brain samples are important to assess the significance of the findings revealed by PET technique to substantiate its validity.


Renal osteodystrophy and vitamin D derivatives: cellular mechanisms of hyperparathyroidism in uremia. In the early phase of chronic renal failure, physiological concentrations of 1,25(OH)2D in circulation may become insufficient to regulate parathyroid cell function normally. As a result, the set-point of PTH secretion to plasma Ca ion concentration shifts to the right, PTH synthesis becomes enhanced at transcriptional level, and parathyroid cells begin to proliferate. This resistance of the parathyroid cells to the physiological concentration of 1,25(OH)2D may be in part due to the reduction of 1,25(OH)2D receptor density in parathyroid cells. However, the mechanism by which the number of 1,25(OH)2D receptors decreases in parathyroid cells in early renal failure remains to be elucidated.


Some properties of Ca(2+)-induced Ca2+ release mechanism in single visceral smooth muscle cell of the guinea-pig. 1. Late transient outward Ca(2+)-dependent K+ current (ILTO) correlated with Ca(2+)-induced Ca2+ release mechanism was studied in relation to the calcium inward current (ICa) in single isolated smooth muscle cells of the guinea-pig ileum using the whole-cell patch-clamp technique. 2. The voltage dependencies of peak ICa and ILTO were both bell shaped. However, the I-V curve of the outward current was shifted toward more positive potentials by about 60 mV in comparison to that for ICa. 3. Reduction in the external Ca2+ concentration resulted in a decrease of peak amplitude of both ICa and ILTO. However, caffeine-induced outward current was also decreased abruptly suggesting a rapid loss of stored Ca2+ upon lowering the external Ca2+ concentration. 4. Investigation of the relation of ILTO to partially inactivated ICa showed that inactivation of ICa by approximately 65, 80 or 84% of control (produced by prepulse to -20 mV for 2 s, shifting the holding potential to -20 mV for 30 s or by the ramp voltage command from -50 to +10 mV, respectively) was without detectable effect on the ILTO generation. 5. Bath application of the Ca2+ antagonist nifedipine (300 nM) inhibited ICa by 81% without affecting ILTO peak amplitude (92.0 +/- 5.6% of control in six cells). The mean concentration-response curve for ICa inhibition was sigmoidal with the apparent dissociation constant of 86.9 nM, whereas that for the ILTO had a characteristic sharp transition indicating a definite threshold of Ca2+ influx for ILTO generation. 6. Application of Ca(2+)-free external solution during 500 ms of the time when ICa peaked inhibited the current by about 76% whereas the ILTO during such an intervention remained virtually unchanged. 7. In double-pulse experiments, with conditioning and test pulses to +10 mV from -50 mV and an interpulse interval of 600 ms, most of the cells (about 80%) showed larger outward current at the test pulse suggesting continued Ca2+ release triggered by Ca2+ influx during a short (50-200 ms) depolarizing prepulse. The outward current could also be evoked at large positive potentials (presumably near the calcium equilibrium potential) where it did not occur normally by a prepulse to +10 mV for 50 ms. The charge transferred by Ca2+ current necessary to activate Ca2+ release in most of the cells was estimated to be from 6 to 20 pC. 8. The data are interpreted to suggest that the Ca(2+)-induced Ca2+ release mechanism operates in single ileal cells in a regenerative manner.(ABSTRACT TRUNCATED AT 400 WORDS)


Calcium-induced calcium release in crayfish skeletal muscle. 1. Cut crayfish skeletal muscle fibres were mounted in a triple Vaseline-gap voltage clamp with the Ca(2+)-sensing dye Rhod-2 allowed to diffuse in via the cut ends. Ca2+ currents across the surface/T-tubule membranes (ICa) were recorded simultaneously with changes in myoplasmic Ca2+ concentration (Ca2+ transients). 2. Excitation-contraction coupling in crayfish skeletal muscle fibres is abolished when calcium in the extracellular solution is replaced by Mg2+. 3. The amplitude of the Ca2+ transients elicited by voltage clamp pulses closely followed the amplitude of the peak calcium currents recorded simultaneously across the surface/T-tubule membranes. This included decreases in both parameters as the pulse potential approached ECa (reversal potential for Ca2+), as well as secondary Ca2+ transients accompanying large tail calcium currents occurring upon repolarization from very large depolarizations. 4. A large contribution of sarcoplasmic reticulum (SR) Ca2+ release to the Ca2+ transients was revealed by a large decrease in the transient caused by the calcium-induced calcium release (CICR) blockers procaine and tetracaine. 5. Short pulses which interrupted the calcium current while SR Ca2+ release was in progress at high rates caused the Ca2+ transient to stop rising nearly immediately after the end of the pulse in most fibres. In about 15% of the fibres the Ca2+ transients continued to rise, albeit at a slower rate, for 10-20 ms after the end of the pulse, as if released Ca2+ was able to elicit some further Ca2+ release from the SR for a while. 6. Even with fibres displaying little sign of continued release after termination of short pulses under control conditions, procaine accelerated the decay of Ca2+ transients elicited by short pulses, indicating that continued release was taking place even as the transient was declining. 7. These results suggest that CICR in crayfish fibres is more closely controlled by a small entry of Ca2+ via surface/T-tubule membrane Ca2+ current than by a larger amount of Ca2+ released from the SR. The limited positive feedback of released Ca2+ on further Ca2+ release allows CICR to remain graded (according to ICa) rather than all-or-none.


Modulation of beta-adrenergic responses of chloride and calcium currents by external cations in guinea-pig ventricular cells. 1. The catecholamine-induced Cl- current and the Ca2+ current were recorded in the single ventricular cells of guinea-pig hearts, using the whole-cell patch clamp technique combined with internal perfusion. Dependence of the beta-adrenergic responses on external monovalent cations was investigated. The Cl- current was recognized by measuring the reversal potential of the agonist-induced current. 2. The amplitude of the Cl- current, activated by 1 microM adrenaline or 0.01-0.1 microM isoprenaline, was decreased when the external Na+ concentration ([Na+]o) was reduced by replacement with Tris+. The conductance of the catecholamine-induced Cl- current was proportional to the logarithm of the [Na+]o over a range of 15-140 mM. When the conductance was plotted against the concentration of Tris+, a dose-dependent inhibition of the Cl- response by Tris+ was suggested with a half-maximum concentration of 95 mM. 3. The inhibitory effect of the Na+ substitute TEA+ on the Cl- current was not affected by either increasing the buffer for the internal Ca2+ (10 mM BAPTA) or for the pH (50 mM HEPES). 4. In the relationship between agonist concentration and the Cl- conductance, the half-maximum concentration (K1/2) of isoprenaline was 0.013 microM in the control Na+ solution, and was shifted to 0.07, 0.08, 0.1 and 0.3 microM in the Li+, Cs+, TEA+ and Tris+ external solutions, respectively. The maximum slope conductance was not significantly affected, except for a slight depression on the Tris+ solution. When the current was induced by adrenaline, qualitatively the same finding was obtained; K1/2 was 0.15 and 3.2 microM in the Na+ and Tris+ solutions, respectively. 5. As a substitute for the external Na+, sucrose seemed to be inert. The activation of the inward Cl- current was conserved in the 300 mM sucrose solution ([Cl-]o = 8 mM) with a K1/2 value of 0.015 microM isoprenaline. 6. The Cl- current, when activated by either an external application of forskolin (0.2-10 microM) or an internal perfusion of cyclic AMP (100-500 microM), was not affected by replacing external Na+ with other cations. Activation of the Cl- current by 0.2-5 microM histamine was also insensitive to a substitution of Na+. These findings indicate that the inhibition by the Na+ substitute is at a point before the activation of GTP-binding protein. 7. The effects of Na+ substitution were not affected by varying the Na+ concentration (0-115 mM) in the internal solution, excluding an involvement of a change in the [Na+]i.(ABSTRACT TRUNCATED AT 400 WORDS)


Expression and characterization of a canine hippocampal inwardly rectifying K+ current in Xenopus oocytes. 1. An inwardly rectifying potassium current expressed in Xenopus laevis oocytes injected with canine hippocampal poly(A)+ RNA was investigated with the two-microelectrode voltage clamp technique. 2. Xenopus oocytes injected with canine hippocampal poly(A)+ RNA expressed a current activated by hyperpolarization. This current contained an instantaneous and a time-dependent component. Both components were inwardly rectifying and could be blocked by extracellular Cs+ or Ba2+. 3. The expressed current was carried mainly by K+. Its reversal potential measured in different [K+]os could be fitted by the Nernst equation with a slope of -50.7 per tenfold change in [K+]o. Extracellular Cl- and Na+ made minimal contributions to the current. 4. The activation of the expressed current depended on both voltage and [K+]o. Activation started near EK and the activation curve shifted along the voltage axis in parallel with EK when [K+]o was altered. 5. The activation time constants of the expressed current also depended on both voltage and [K+]o. The voltage dependence of the time constants was bell-shaped and the peak value was at a potential 30-50 mV more negative than EK. The voltage dependence of the time constants shifted along the voltage axis when EK was changed. 6. The poly(A)+ RNA extracted from canine hippocampus was fractionated in a 10-31% linear sucrose gradient. The size of the mRNA required to express the inwardly rectifying current was estimated to be around 4 kb. 7. In conclusion, the expressed current is an inwardly rectifying potassium current. The canine hippocampal mRNA should be an excellent source for expression-cloning of the inward rectifier channel.


Calcium currents at motor nerve endings: absence of effects of adenosine receptor agonists in the frog. 1. The effects of adenosine (50 microM) and 2-chloroadenosine (1-25 microM) were studied on Ca2+ currents in frog motor nerve endings. 2. Ca2+ currents associated with the synchronous, neurally evoked release of acetylcholine (ACh) were measured using either perineural or patch recording methods. Tetraethylammonium and/or 3,4-diaminopyridine were employed to block K+ currents. 3. Ca2+ currents were depressed by omega-conotoxin (1.5-2.5 microM), Cd2+ (100 microM-2 mM), Co2+ (500 microM-5 mM) or by a reduction of the extracellular calcium concentration. Such currents were also observed when Sr2+ was substituted for Ca2+. Both ACh release and Ca2+ currents at motor nerve endings have been reported to be insensitive to 1,4-dihydropyridine antagonists in this species. 4. Adenosine receptor agonists did not affect Ca2+ currents at concentrations that produced maximal inhibition of ACh release. 5. The effects of adenosine receptor agonists were examined on asynchronous K(+)-dependent ACh release under conditions in which the Ca2+ concentration gradient is likely to be reversed (Ca(2+)-free Ringer solution containing 1 mM EGTA). ACh release was measured by monitoring the frequency of occurrence of miniature endplate potentials (MEPPs). In Ca(2+)-free solutions containing 1 mM EGTA, high K+ depolarization caused a decrease in MEPP frequency, presumably because it elicits the efflux of Ca2+ from the nerve ending via membrane Ca2+ channels in a reverse Ca2+ gradient. 6. The Ca2+ channel blocker Co2+, which blocks the exit of Ca2+ from the nerve ending, increased the frequency of MEPPs in a concentration-dependent manner in a reverse Ca2+ gradient. 7. Adenosine or 2-chloroadenosine inhibited ACh release in a reverse Ca2+ gradient. 8. The results suggest that blockade of Ca2+ entry is not responsible for the inhibitory effects of adenosine at frog motor nerve endings.


Membrane properties and synaptic responses of Golgi cells and stellate cells in the turtle cerebellum in vitro. 1. Intracellular recordings from anatomically identified Golgi cells and deep stellate cells were obtained in a slice preparation of the turtle cerebellar cortex. 2. Golgi cells and stellate cells had very similar firing patterns, which differed from those of Purkinje cells. In the interneurones, a short time constant and a high input resistance ensured a short response time. A pronounced spike after-hyperpolarization (spike AHP) participated in the rapid repolarization following a depolarizing input. The active and passive membrane properties of the interneurones ensured a very tight temporal coupling between input and output. 3. TTX abolished both the action potentials and a subthreshold depolarizing response. The Na+ excitability was increased by addition of Mn2+ or Co2+ to block calcium channels, or by addition of potassium channel blockers. 4. Ca2+ spikes and a Ca2+ plateau could be evoked following addition of potassium channel blockers. A partly 4-aminopyridine (4-AP)-sensitive transient hyperpolarization was found to control Ca2+ excitability in Golgi cells. It is suggested that this hyperpolarization is due to an A-like conductance. 5. A strong anomalous rectification was activated just below spike threshold, and dominated the subthreshold membrane potential at time scales longer than ca 100 ms. The anomalous rectification was partly blocked by Cs+. 6. Temporal integration over time scales up to ca 25 s was provided by activity-dependent adaptation in firing frequency and a long-lasting after-hyperpolarization (AHPL), which had both TTX-sensitive, Ca(2+)-independent, and Ca(2+)-dependent components. 7. Spontaneous IPSPs and EPSPs were abundant. The IPSPs were abolished by bicuculline. EPSPs were easily evoked by parallel fibre stimulation, had a shorter time course than in Purkinje cells, and were suppressed by the spike AHP. 8. Due to a short response time and a relatively short overall time frame for temporal integration, cerebellar interneurones operate on a faster time scale than the Purkinje cells, the output neurones of the cerebellar cortex. 9. It is suggested that information from shared sources, e.g. the parallel fibres, is distributed onto dynamically different cellular populations based on differences in the intrinsic membrane properties of the postsynaptic neurones.


Hyperthyroidism selectively modified a transient potassium current in rabbit ventricular and atrial myocytes. 1. Transient outward potassium currents (I(t)) were compared in single cardiac myocytes obtained from normal and hyperthyroid rabbits. Currents were recorded using the suction electrode whole-cell voltage clamp technique. 2. In ventricular myocytes from hyperthyroid animals (at 22 degrees C and a stimulation rate of 0.2 Hz), I(t) was 4- to 5-fold larger than in normal myocytes, in a potential range of -20 to +60 mV. As in normal myocytes, I(t) in hyperthyroid myocytes was calcium insensitive, and was more than 90% suppressed by 2 mM 4-aminopyridine. 3. The increase in I(t) was observed over a wide range of stimulation rates, even at rates sufficiently slow to enable complete reactivation of the I(t) channels. However, there was a major change in the rate dependence of I(t) in hyperthyroid myocytes, with significant I(t) current still present at rates (e.g. 1-2 Hz) at which it is normally completely suppressed. 4. The augmentation of I(t) in the hyperthyroid myocytes could not be accounted for by changes in the voltage dependence or the kinetics of channel activation or inactivation. There was no change in the reversal potential of I(t), implying no change in the selectivity of the channel. 5. Single-channel activity was recorded using the cell-attached mode of recording. In myocytes from hyperthyroid rabbit we observed the following: (a) active patches (often containing two channels) were obtained more frequently in comparison to control; (b) the unitary conductance of the channel was the same; (c) single-channel openings persisted at high stimulation rates. 6. In contrast to hyperthyroid ventricular cells, I(t) in atrial cells from the same hearts was not substantially changed. 7. The rate dependence of I(t) in atrial cells was also unaffected by hyperthyroidism, in contrast to the large changes observed in ventricular cells. Thus, in atrial cells from hyperthyroid hearts the current was totally suppressed at rates of 1-2 Hz, as in euthyroid conditions. 8. Single-channel recordings in the cell-attached mode showed a unitary conductance similar to that found in normal atrial cells. Channel activity was suppressed at 2 Hz, in contrast to hyperthyroid ventricular cells. 9. In conclusion, I(t) is drastically changed in hyperthyroid rabbit ventricle cells. The changes are in the magnitude of the macroscopic current and its rate dependence. Since the unitary conductance is unchanged (and the peak open probabilities are normally high at positive membrane potential(s) the number of active channels in the membrane must be increased. In atrial cells from the same hyperthyroid hearts no changes are apparent.(ABSTRACT TRUNCATED AT 400 WORDS)


Presynaptic histamine H1 and H3 receptors modulate sympathetic ganglionic synaptic transmission in the guinea-pig. 1. To study the effects of histamine on the efficacy of sympathetic ganglionic synaptic transmission, extracellular recordings of the postganglionic compound action potential (CAP) and intracellular recordings of excitatory postsynaptic potentials (EPSPs) elicited by preganglionic electrical stimulation were obtained from isolated guinea-pig superior cervical ganglia (SCG). 2. In different preparations, superfusion with histamine (0.1-100 microM) either potentiated or depressed the postganglionic CAP elicited by electrical stimulation of the cervical sympathetic trunk (0.2-3.0 Hz). The direction of response produced by histamine did not depend on stimulation frequency or histamine concentration; potentiation and depression both showed concentration dependence over the range of histamine concentrations tested. 3. Experiments employing a variety of histamine receptor agonists or antagonists revealed that histamine-induced potentiation of the postganglionic CAP could be attributed to histamine H1 receptor activation, and depression to H3 receptor activation. 4. Histamine similarly potentiated or depressed the intracellularly recorded EPSP. However, these opposite effects occurred at different synapses. In agreement with the studies on the postganglionic CAP, histamine H1 antagonists prevented histamine-induced potentiation of the EPSP and H3 receptor antagonists prevented histamine-induced depression. 5. Direct quantal analyses of histamine-induced synaptic potentiation and depression were implemented to determine the pre- and postsynaptic components of these effects. Quantal size was estimated by measuring the amplitude of spontaneous miniature EPSP amplitudes. Histamine-induced potentiation and depression of the evoked EPSP were found to be accompanied by increased or decreased quantal content respectively, and unchanged quantal size, providing evidence that presynaptic mechanisms were involved in mediating both effects. 6. Some guinea-pigs were actively sensitized to ovalbumin. Subsequent exposure of the isolated SCG from these animals to the sensitizing antigen produced changes in the EPSP amplitude that correlated significantly to the response produced by exogenously applied histamine at the same synapse. 7. The correspondence between the effects of specific antigen challenge and exogenous histamine on evoked EPSPs at a synapse provides evidence that endogenous histamine released during an immunological response to antigen challenge can activate histamine H1 and H3 receptors to modulate synaptic efficacy in sympathetic ganglia.


Ca2+ currents in single myocytes from human mesenteric arteries: evidence for a physiological role of L-type channels. 1. Voltage-gated Ca2+ currents (ICa) in isolated human mesenteric arterial cells were characterized in solutions containing normal (1.5 mM) Ca2+ and elevated concentrations of divalent cations using the conventional whole-cell patch clamp technique. 2. In normal Ca2+ solution, depolarization beyond -40 mV elicited a slowly decaying ICa which reached a maximum at +10 mV and appeared to reverse between +40 and +50 mV. The amplitude of this current in a group of cells correlated with cell membrane capacitance. 3. In two of thirty-three cells a small transient component of inward current was detected in the voltage range between -40 and -10 mV when cells were held at -80 mV. This current was abolished at a holding potential of -40 mV, while the current at 10 mV was not affected. These currents were referred to as T- and L-type Ca2+ respectively. 4. Elevation of the extracellular Ca2+ concentration to 20 mM shifted the voltage dependencies of Ca2+ current activation and inactivation by approximately +20 mV; a small T-current component was then observed in seven of nine cells held at -60 mV. 5. Replacement of 1.5 mM Ca2+ with 10 mM Ba2+ increased the amplitude of the current elicited at +10 mV by a factor of 3.7 and a small barium current (IBa) through T-type Ca2+ channels was also observed in most cells studied. Activation and steady-state inactivation curves for L-type current were found to be almost identical in both solutions. The steady-state inactivation for the T-type IBa was, however, more than 30 mV more negative (half-inactivation potential of -62.6 mV) of that for L-current in 1.5 mM Ca2+ and 10 mM Ba2+ solutions (-30.4 and -24.9 mV respectively). 6. A sustained inward Ca2+ channel current was recorded in the presence of normal Ca2+ and high divalent cation concentrations during 30 s depolarizations. The amplitude of this sustained current was found to be similar to the theoretical 'window current' predicted by the overlap of the activation and inactivation functions in these solutions. 7. Examination of the inactivation of the L-type current using a two-pulse protocol with a 240 ms prepulse revealed a U-shaped potential dependency for ICa, but not for IBa, suggesting the presence of a Ca(2+)-dependent component of the inactivation process. 8. These cells resemble other arterial smooth muscle cells previously studied in that they demonstrate both T- and L-components of ICa.(ABSTRACT TRUNCATED AT 400 WORDS)


Guanosine 3',5'-cyclic monophosphate regulates calcium channels in neurones of rabbit vesical pelvic ganglia. 1. The effects of dibutyryl guanosine 3',5'-cyclic monophosphate (db-cyclic GMP) were studied in vitro on calcium channels of neurones in rabbit vesical parasympathetic ganglia, using intracellular and single-electrode voltage-clamp recordings. 2. Db-cyclic GMP (100 microM) caused membrane depolarization associated with a decrease in membrane input resistance and an after-hyperpolarization associated with an increase in membrane input resistance. 3. Db-cyclic GMP (0.01-1 mM) caused a concentration-dependent, transient inward current followed by a long-lasting outward current. Membrane conductance was increased and decreased during the inward and outward currents, respectively. 4. The db-cyclic GMP-induced inward current was depressed in nominally calcium-free solutions, by cobalt (1 mM) and nicardipine (10 microM). The mean reversal potentials of the inward current were +42 and -20 mV in the presence and absence of calcium in the external solution, respectively. 5. The db-cyclic GMP-induced inward current was not altered by lowering the external sodium concentration, raising external potassium concentration or by intracellular injection of caesium. 6. A calcium-insensitive component of the db-cyclic GMP-induced current was increased by lowering the external chloride concentration and blocked by 4-acetamido-4'-isothiocyanostilbene-2,2'-disulphonic acid, a chloride channel blocker. 7. Voltage-dependent, high-threshold calcium currents were depressed during the db-cyclic GMP-induced inward current and facilitated during the outward current. 8. Cyclic GMP was less potent than db-cyclic GMP in causing both inward and outward currents or modulation of calcium currents. GTP, GDP, GMP, guanosine, 8-bromoadenosine 3',5'-cyclic monophosphate and forskolin did not alter the holding current or voltage-dependent calcium currents. 9. It is concluded that intracellular cyclic GMP causes not only activation of resting calcium and chloride channels but also a transient depression followed by long-lasting facilitation of voltage-dependent calcium currents in neurones of vesical parasympathetic ganglia.


Inactivation characteristics of a sustained, Ca(2+)-independent K+ current of rat hippocampal neurones in vitro. 1. Current or voltage clamp recordings from CA3 neurones of the adult rat hippocampal slice were performed to study the inactivation properties of a slow outward K+ current identified as the delayed rectifier (IK). 2. In current clamp experiments, burst firing evoked from resting membrane potential by intracellular current injection was reduced or blocked by conditioning hyperpolarizing pre-pulses of 20-40 mV amplitude. This effect was inhibited by tetraethylammonium (TEA; 20 mM) but was unaffected by Cs+ (3 mM), 4-aminopyridine (4-AP; 2 mM), carbachol (30-50 microM), mast cell degranulating peptide (MCDP; 300 nM), thyrotrophin releasing hormone (TRH; 1 microM) or by a Ca(2+)-free solution containing Mn2+ or Co2+ (2 mM). 3. Single-electrode voltage clamp experiments were carried out on neurones superfused with Ca(2+)-free solution, containing tetrodotoxin (TTX; 1 microM), Mn2+ or Co2+ (2 mM), 4-AP (2 mM), Cs+ (3 mM) and carbachol (30 microM). Step depolarizations from a holding potential of -55 mV activated an outward current which reached a plateau after 200 ms, followed by an outward tail current. Such an outward current had the characteristics of IK. 4. The outward currents were significantly potentiated by conditioning hyperpolarizing pre-pulses suggesting the IK was reduced by a voltage-dependent inactivation process. Removal of inactivation was a function of the amplitude of the conditioning hyperpolarizing pre-pulse. At a holding potential of -55 mV removal of inactivation was time dependent with a time constant of 211 ms. High K+ (12.5 or 21.5 mM) solutions did not affect the inactivation characteristics of IK. 5. Tetraethylammonium (20 mM) or low concentrations of Ba2+ (0.1 mM) readily depressed the outward current without significantly affecting the inactivation process. Dendrotoxin (200 nM) also depressed such a slow current but, in addition, increased the inactivation process of IK. 6. It is suggested that removal of inactivation of IK by hyperpolarization can modulate cell excitability by fully restoring the ability of IK to inhibit burst firing of CA3 hippocampal neurones.


Voltage-dependent ionic currents in dissociated paratracheal ganglion cells of the rat. 1. Conventional whole-cell voltage-clamp technique was used to study the electrophysiological and pharmacological properties of voltage-dependent Na+, K+ and Ca2+ channels in parasympathetic neurones enzymatically dissociated from the paratracheal ganglia of rat trachea. The voltage-dependent Na+, K+ and Ca2+ currents (INa, IK and ICa) were separated by the use of ion subtraction and pharmacological treatments. 2. INa was activated by a step depolarization more positive than -50 mV and fully activated at positive potentials more than +10 mV. The inactivation phase of INa consisted of fast and slow exponential components (tau if and tau is, respectively). The tau if and tau is were voltage dependent and decreased with a more positive step pulse. 3. The time course for recovery of INa from the complete inactivation exhibited two exponential processes. 4. The reversal potential of INa was equal to the Na+ equilibrium potential (ENa) and resembled a simple Na+ electrode depending only on external Na+ concentration. 5. Tetrodotoxin (TTX) reduced INa without affecting the current kinetics in a concentration-dependent manner, and the concentration of half-maximal inhibition (IC50) was 6 x 10(-9) M. There was no TTX-resistant component of INa in any of the cells tested. 6. Scorpion toxin increased the peak amplitude of INa and prolonged the inactivation phase in a time- and concentration-dependent manner. In the presence of toxin, both tau is and the fractional contribution of the slow current component to total INa increased concentration dependently. 7. High-threshold (L-type) ICa was activated by a step depolarization more positive than -30 mV and reached a peak at near 0 mV in the external solution with 2.5 mM Ca2+. The current was inactivated to only a small extent (< 10%) during 100 ms of depolarizing step pulse. There was no low-threshold (T-type) ICa in this preparation. 8. The maximum ICa in individual current-voltage (I-V) relationships was saturated by an increase in extracellular Ca2+ concentration ([Ca2+]o). The I-V relationships were also shifted along the voltage axis to the more positive potential with increasing [Ca2+]o. 9. The inactivation process of the L-type ICa was dependent on Ca2+ influxes (ICa-dependent inactivation). 10. Relative maximum peak currents of divalent cations passing through the L-type Ca2+ channels were in the order of IBa > ICa > ISr. 11. Organic and inorganic Ca2+ antagonists blocked the ICa in a concentration-dependent manner.(ABSTRACT TRUNCATED AT 400 WORDS)


Modulation of contraction by intracellular Na+ via Na(+)-Ca2+ exchange in single shark (Squalus acanthias) ventricular myocytes. 1. The effect of direct alteration of intracellular Na+ concentration on contractile properties of whole-cell clamped shark ventricular myocytes was studied using an array of 256 photodiodes to monitor the length of the isolated myocytes. 2. In myocytes dialysed with Na(+)-free solution, the voltage dependence of Ca2+ current (ICa) and contraction were similar and bell shaped. Contractions activated at all voltages were completely suppressed by nifedipine (5 microM), and failed to show significant tonic components, suggesting dependence of the contraction on Ca2+ influx through the L-type Ca2+ channel. 3. In myocytes dialysed with 60 mM Na+, a ICa-dependent and a ICa-independent component of contraction could be identified. The Ca2+ current-dependent component was prominent in voltages between -30 to +10 mV. The ICa-independent contractions were maintained for the duration of depolarization, increased with increasing depolarization between +10 to +100 mV, and were insensitive to nifedipine. 4. In such myocytes, repolarization produced slowly decaying inward tail currents closely related to the time course of relaxation and the degree of shortening prior to repolarization. 5. With 60 mM Na+ in the pipette solution, positive clamp potentials activated decaying outward currents which correlated to the size of contraction. These outward currents appeared to be generated by the Na(+)-Ca(2+)-exchanger since they depended on the presence of intracellular Na+, and were neither suppressed by nifedipine nor by K+ channel blockers. 6. The results suggest that in shark (Squalus acanthias) ventricular myocytes, which lack functionally relevant Ca2+ release pools, both Ca2+ channel and the Na(+)-Ca2+ exchanger deliver sufficient Ca2+ to activate contraction, though the effectiveness of the latter mechanism was highly dependent on the [Na+]i.


Modulation of calcium current gating in frog skeletal muscle by conditioning depolarization. 1. Ca2+ inward currents were measured by voltage clamping cut skeletal muscle fibres of the frog (Rana esculenta) in a double-Vaseline-gap system. 2. In order to study the basis of the previously described fast gating mode induced in the Ca2+ inward current by a conditioning depolarization we quantitatively analysed the response to differing features of the conditioning prepulse. 3. The faster activation seen during the second of two depolarizations was confined to the component of the inward current which could be blocked by 5 to 10 microM nifedipine. 4. By applying depolarizing conditioning pulses of gradually increasing length the time course of the transition to the fast gating mode could be determined. 5. Both the transition to the fast gating mode (point 4) caused by a depolarization and the slow inward current activated during the same depolarization showed similar voltage-dependent kinetics. 6. The kinetic change of the test current appeared to be equal when the same fractional activation was achieved at the end of the conditioning pulse independent of its duration or amplitude. 7. Flash photolysis of nifedipine in the interval between conditioning and test pulse showed that the predepolarization causes a rate-enhancing effect even though the slow channels were blocked by nifedipine during the conditioning pulse. 8. We conclude that the transition of the calcium channel from its slow to its fast gating mode is determined by the slow voltage-dependent reaction which limits the rate of channel opening under control conditions. This reaction is apparently not prevented by the binding of nifedipine and the block of current flow through the channel.


Muscarinic inhibition of M-current and a potassium leak conductance in neurones of the rat basolateral amygdala. 1. Voltage-clamp recordings using a single microelectrode were obtained from pyramidal neurones of the basolateral amygdala (BLA) in slices of the rat ventral forebrain. Slow inward current relaxations during hyperpolarizing voltage steps from a holding potential of -40 mV were identified as the muscarinic-sensitive M-current (IM), a time- and voltage-dependent potassium current previously identified in other neuronal cell types. 2. Activation of IM was voltage dependent with a threshold of approximately -70 mV. At membrane potentials positive to this, the steady-state current-voltage (I-V) relationship showed substantial outward rectification, reflecting the time- and voltage-dependent opening of M-channels. The underlying conductance (gM) also increased sharply with depolarization. 3. The reversal potential for IM was -84 mV in medium containing 3.5 mM K+. This was shifted positively by 27 mV when the external K+ concentration was raised to 15 mM. 4. The time courses of M-current activation and deactivation were fitted by a single exponential. The time constant for IM decay, measured at 24 degrees C, was strongly dependent on membrane potential, ranging from 330 ms at -40 mV to 12 ms at -100 mV. 5. Bath application of carbachol (0.5-40 microM) inhibited IM, as evidenced by the reduction or elimination of the slow inward M-current relaxations evoked during hyperpolarizing steps from a holding potential of -40 mV. The outward rectification of the steady-state I-V relationship at membrane potentials positive to -70 mV was also largely eliminated. The inhibition of IM by carbachol was dose dependent and antagonized by atropine. 6. Carbachol produced an inward current shift at a holding potential of -40 mV that was only partially attributable to inhibition of IM. An inward current shift was also produced by carbachol at membrane potentials negative to -70 mV, where IM is inactive. These effects were dose dependent and antagonized by atropine. They were attributed to the muscarinic inhibition of a voltage-insensitive potassium leak conductance (ILeak). 7. In most cells, carbachol reduced the slope of the instantaneous I-V relationship obtained from a holding potential of -70 mV so that it crossed the control I-V plot at the reversal potential for ILeak. This was found to be -108 mV in 3.5 mM K+ saline, shifting to -66 mV in 15 mM K+ saline.(ABSTRACT TRUNCATED AT 400 WORDS)


Survival of human monoclonal anti-Rho (D) antibodies in the rhesus monkey. In vivo half-life of a 125I-labeled human anti-D monoclonal antibody (mAb) and that of 131I-labeled Rho-GAM was assessed in a rhesus monkey injected simultaneously with both reagents. The half-life of the mAb was 7.9 days, compared to 17 days of Rho-GAM. Survival of the second dose of mAb, given 34 days after the first injection, was identical to that of the first dose, thus showing that the human mAb did not elicit an immune response. The in vitro produced human mAbs appear to be an alternative, unlimited source of anti-D antibodies for possible use in prevention of feto-maternal Rh immunization.


A simple and rapid in vitro test system for the screening of histamine H3 ligands. A simple and rapid functional test system for the screening of histamine H3 ligands is described. It is based on the inhibitory effect of histamine H3 agonists on electrically-evoked contractile response of isolated guinea pig intestine. Whole jejunum segments are continuously stimulated maximally (15 V) by electrical pulses with a frequency of 0.1 Hz and a duration of 0.5 msec. The resulting twitches are recorded isotonically (1.0 g) and can be completely abolished by atropine (0.1 mcM).


Protective actions of S-nitroso-N-acetylpenicillamine (SNAP) in a rat model of hemorrhagic shock. The anti-shock effects of an organic nitric oxide donor, S-nitroso-N-acetylpenicillamine (SNAP), were tested in a rat model of hemorrhagic shock. Administration of SNAP at a dose of 10 mcg/kg injection followed by 10 mcg/kg/h infusion neither significantly decreased mean arterial blood pressure (MABP) nor significantly altered bleedout volumes in hemorrhagic rats, indicating that SNAP did not modify the severity of the shock protocol. However, hemorrhaged rats treated with SNAP maintained post-reinfusion MABP at significantly higher values than hemorrhaged rats receiving 0.9% NaCl (final MABP 81 +/- 3.0 mmHg vs. 54 +/- 1.1 mmHg, respectively; p < 0.001). SNAP also significantly increased survival times following hemorrhagic shock (113 +/- 4 min in SNAP treated rats compared with 70 +/- 4.5 min in vehicle treated rats, p < 0.001). The overall survival rates were 87.5% when treated with SNAP and 0% with 0.9% NaCl (p < 0.01). In hemorrhagic shock rats receiving only vehicle, a significant accumulation of neutrophils in intestinal tissue occurred as indicated by a higher MPO activity in intestinal tissue (MPO activity, 1.26 +/- 0.31 vs. 0.14 +/- 0.05U/100 mg in sham hemorrhagic shock rats, p < 0.02). Administration of SNAP significantly attenuated the neutrophil accumulation in the intestinal tissue (MPO activity, 0.42 +/- 0.09U/100 mg, p < 0.05 compared with hemorrhagic rats receiving only the vehicle). Moreover, endothelial dysfunction of superior mesenteric artery rings occurred in hemorrhagic shock rats given only 0.9% NaCl.(ABSTRACT TRUNCATED AT 250 WORDS)


Interaction of Klebsiella capsule type 7 with human polymorphonuclear leucocytes. Klebsiella serotype K7 is found among the capsule types that are most prevalent in respiratory tract isolates. To evaluate the significance of the K7 antigen in bacteria-leucocyte interactions, K7-encapsulated Klebsiella pneumoniae strains and their non-capsulate mutants were investigated. The K7 isolates were compared to K2-capsulate strains and their respective K- derivatives. K7-capsulate bacteria were less hydrophilic, and more readily phagocytosed and killed by human polymorphonuclear leucocytes (PMNL) than K2 strains. Loss of the K7 antigen resulted in increased surface hydrophobicity but did not affect phagocytosis and killing, whereas loss of the K2 capsule caused greater susceptibility to the phagocytic and killing action of PMNL. Both the K7 and K2 antigen stimulated the extracellular release of lysozyme from neutrophils but not of myeloperoxidase, indicating degranulation of only secondary granules. All K- mutants induced the release of both lysozyme and myeloperoxidase. Our results suggest that, in contrast to the K2 antigen, the K7 capsular polysaccharide does not confer antiphagocytic properties on bacteria. However, the K7 antigen is able to impede the extracellular release of primary granule enzymes.


Suppression of LPS-inducible cytotoxicity in cytomegalovirus-infected THP-1 monocytic cell cultures does not correlate with a decrease in TNF-alpha antigen. We document suppression of tumor necrosis factor alpha (TNF-alpha)-associated cytotoxic activity in a human monocytic cell line (THP-1) infected with the mycoplasma free human cytomegalovirus (CMV) strain AD169. Addition of lipopolysaccharide (LPS) to cell cultures that had been infected with CMV for 24 h resulted in a significant reduction in released cytotoxic activity to mouse L929 cells at 3-22 h post-LPS compared with mock-infected cultures. However, using an ELISA to measure TNF-alpha antigen levels in these culture supernatants, we found infected cultures had significantly higher TNF-alpha antigen levels than in mock-infected cultures following LPS induction. CMV alone also induced TNF-alpha release and possibly TNF-alpha inhibitor(s) which may have blocked TNF-alpha associated cytotoxic activity in CMV-infected THP-1 cell culture supernatants.