Tissue disposition of glyceryl trinitrate, 1,2-glyceryl dinitrate, and 1,3-glyceryl dinitrate in tolerant and nontolerant rats.

The tissue distribution of glyceryl trinitrate (GTN) and its two dinitrate metabolites 1,2-glyceryl dinitrate (1,2-GDN) and 1,3-glyceryl trinitrate (1,3-GDN), was studied in GTN-tolerant and nontolerant male Sprague-Dawley rats. The concentrations of GTN, 1,2-GDN, and 1,3-GDN were measured in plasma, heart, brain, liver, aortic tissue, and adipose tissue at various time points after a subcutaneous dose of GTN (50 mg/kg). At the first time point (5 hr), concentrations of GTN, 1,2-GDN, and 1,3-GDN in plasma were equal for tolerant and nontolerant rats, but the elimination rate was altered for the tolerant rats as compared with nontolerant rats. In adipose tissue, the concentration of GTN was significantly higher as compared with concentrations of the dinitrate metabolites. In contrast, the other tissues studied showed significantly higher concentrations of the GDNs when compared with GTN. The 1,3-GDN/1,2-GDN ratio decreased with time for both tolerant and nontolerant rats. This study indicates that long-term GTN administration results not only in tolerance development, but also in altered pharmacokinetics of GTN, 1,2-GDN, and 1,3-GDN. The results also show that the 1,3-GDN/1,2-GDN ratio is dependent on the GTN concentration.


Pharmacokinetic analysis of enterohepatic circulation of 4-[2-(4-isopropylbenzamido)ethoxy]benzoic acid. Effect of intramolecular rearrangement of its acyl glucuronide.

The enterohepatic circulation of 4-[2-(4-isopropylbenzamido)ethoxy]benzoic acid (PBAB) was studied in rats after an iv administration of 30 mg/kg. After the bolus injection, the PBAB concentrations in the plasma decreased rapidly, then increased to a peak concentration at 4 hr. Over a 6-hr period, 52% of the dose (Fe) was excreted in the bile as 1 beta-O-acyl glucuronide of PBAB (1 beta-PG). Elimination of PBAB from the plasma of bile duct-cannulated rats was more rapid than for the sham-operated rats. These results suggest that PBAB undergoes enterohepatic circulation. The equation for an enterohepatic circulation model was fitted to the plasma PBAB concentrations for intact rats using the program MULTI (FILT) to estimate the single circulating fraction (Fc) (bile----intestine----systemic circulation). The Fc value was 0.072, which means that 7.2% of the dose was reabsorbed to systemic circulation during the first cycle. The fraction (Fa) reabsorbed from small intestine to systemic circulation during first cycle can be estimated by the formula Fa = Fo/Fa. The Fa value obtained was 14% of the dose, which was smaller than the Fa' (26% of the dose) standing for systemic availability of PBAB after oral dosing. When 1 beta-PG was incubated with bile for 2 hr, 79% was transformed to beta-glucuronidase-resistant isomers by intramolecular acyl migration. We consider that the acyl migration of 1 beta-PG in the small intestine or bile causes the difference between Fa and Fa' values, and decreases the enterohepatic circulation of PBAB.


Correlation between N-acetyltransferase activities in uroepithelia and in vivo acetylator phenotype.

The relationship between in vivo acetylator phenotype of individuals and N-acetyltransferase (NAT) activity in the cytosol of their cultured uroepithelia was examined in four urology patients. In vivo acetylator phenotypes were assigned by determining the ratio of N-acetyl vs. total [N-acetyl+free] sulfamethazine in urine and blood following a single oral dose (1 gm) of sulfamethazine. From the same patients, a surgical specimen of the ureter was obtained, uroepithelial cells were cultured in vitro, and the cytosols prepared. NAT activities were determined by measuring the amount of 4-acetylaminobiphenyl formed from incubation of uroepithelial cytosol with the substrate, 4-aminobiphenyl, and the cofactor [14C]acetyl coenzyme A. The two individuals phenotyped as "slow acetylators" by the in vivo method had NAT activities of 8.3 and 16.2 pmol 4-acetylaminobiphenyl/mg protein/min. In contrast, the two individuals phenotyped as "rapid acetylators" showed activities of 50.9 and 109.5 pmol 4-acetylaminobiphenyl/mg protein/min. The rapid acetylators exhibit about 6-fold greater uroepithelial NAT activities than slow acetylators, thus showing a direct correlation between the NAT activity in the uroepithelium, the target tissue of the human bladder carcinogen 4-aminobiphenyl, and the in vivo acetylator phenotype. These results imply that susceptibility of individuals to arylamine-induced bladder cancer might be associated with NAT activities in their target cells and that in vivo acetylator phenotyping could serve as a useful and relevant biochemical screening marker to assess the risk of developing bladder cancer.


3'-azido-3'-deoxythymidine drug interactions. Screening for inhibitors in human liver microsomes.

Zidovudine is a widely used antiretroviral drug active against human immunodeficiency virus. The drug interactions of this compound, which are primarily eliminated as a glucuronide, have not yet been extensively studied. Because zidovudine is frequently combined with other drugs, complete knowledge of interactions is essential to optimize AIDS therapy. We therefore screened the effect of 55 molecules, representative of 20 different therapeutic classes, on 3'-azido-3'-deoxythymidine (AZT) glucuronidation by human liver microsomes. We demonstrate that many drugs caused more than 15% inhibition of AZT glucuronidation in vitro, whereas major antibiotics (ceftazidine, isoniazid, aminoglycosides, macrolides, and sulfamides), antivirals (2',3'-dideoxycytidine, 2',3'-dideoxyinosine, and acyclovir), flucytosine, metronidazole, acetaminophen, and ranitidine had no effect. For compounds that appeared to inhibit AZT glucuronidation, extrapolation to the clinical situation must take into account both the in vitro apparent Ki values and the usual expected plasma level for the coadministered drug. By considering these parameters, this work indicates that clinically relevant inhibition of AZT glucuronidation may be observed with the following drugs: cefoperazone, penicillin G, amoxicilin, piperacillin, chloramphenicol, vancomycin, miconazole, rifampicin, phenobarbital, carbamazepine, phenytoin, valproic acid, quinidine, phenylbutazone, ketoprofen, probenecid, and propofol. Complementary clinical and pharmacokinetic studies should be performed to validate these assumptions.


Identification of urinary metabolites of 8-methyl-8-azabicyclo-[3,2,1] octan-3-yl 3,5-dichlorobenzoate (MDL 72,222) in the dog and monkey.

The metabolism of 8-methyl-8-azabicyclo- 3,2,1]octan-3-yl 3,5-dichlorobenzoate (MDL 72,222) was studied in the dog and monkey. Four urinary metabolites were detected by HPLC, HPLC/MS, and GC/MS, and were identified by comparison to authentic standards. The major metabolite in the dog, approximately 41% of the administered dose excreted between 0 and 120 hr, was the MDL 72,222-N-oxide. On the other hand, the major metabolite in the monkey was the glycine conjugate of 3,5-dichlorobenzoic acid (greater than 56% of the dose). Seven percent of the dose in the monkey urine was free 3,5-dichlorobenzoic acid. N-Desmethyl MDL 72,222 was present at 2.5 and 1% in the dog and monkey, respectively. Very little (less than 1%) of the parent compound was found in urine. The major pathways of metabolism of MDL 72,222 are N-oxidation, N-demethylation, ester hydrolysis, and amino acid conjugation.


Pharmacokinetics and tissue distribution of human urinary tumor necrosis factor binding protein in mice.

Iodinated natural human urinary tumor necrosis factor binding protein I (125I-uTBP) was iv injected into BALB/c mice, and its pharmacokinetics and tissue distribution were assessed during a short-term (0-1 hr) and for a long-term (0-24 hr) period. The blood 125I-uTBP concentration displayed a biphasic pattern that was adequately described by a biexponential function with estimated half-lives of 0.1 and 3.8 hr. The apparent volume of distribution (Vc) of the central compartment was 3 ml, which approximated the mouse blood volume. The clearance (CL) derived either from a model-dependent or a model-independent method of analysis was 2.5 and 2.9 ml/hr, respectively. One hr after the iv administration of 125I-uTBP, the radioactivity accumulated in the major organs and tissues. The highest concentrations in terms of pg per organ were seen in the skin and in the liver. When expressed as pg 125I-uTBP per mg organ, the distribution was the highest in the gallbladder, bladder, kidneys, and lungs. At 24 hr, the distribution of 125I-uTBP represented about 10% of the amount measured at 1 hr. The rank order of accumulation of the radiolabeled uTBP in the major organs, expressed as pg per organ at 24 hr was skin greater than liver greater than kidneys greater than lungs greater than gut greater than spleen greater than gallbladder.


Pharmacokinetics and metabolism of the 5-hydroxytryptamine antagonist tropisetron after single oral doses in humans.

Tropiestron is a potent and selective antagonist of 5-hydroxytryptamine receptors. Tropisetron was developed for the indication of cancer chemotherapy-induced emesis. The pharmacokinetic and metabolic dispositions of tropisetron were studied in 12 healthy male volunteers receiving a single oral dose of 62 or 312 mumol (20 or 100 mg) of [14C]tropisetron. Serial plasma samples and complete urine and feces were collected for 120 hr postdose. Whereas the absorption of oral doses of 62-312 mumol tropisetron was rapid and complete, bioavailability was estimated to be only 66% for the 312 mumol dose and 52% for the 62 mumol dose, apparently because of saturable first-pass metabolism. Maximal concentrations of tropisetron averaged 87 and 608 nM after doses of 62 and 312 mumol, respectively, and the parent drug accounted for 21 and 36% of the radioactivity in AUC0-24 hr pools. Approximately 90% of the drug was metabolized before excretion, and approximately 70% of the dose was recovered in the urine. Following both the 62 and 312 mumol doses, the terminal half-life of tropisetron averaged 6-7 hr and that of total radioactivity was 10-11 hr. Tropisetron and its metabolites in plasma and urine were separated by gradient elution reversed-phase HPLC. Structures of eight metabolites were assigned on the bases of NMR and MS data. Tropisetron was metabolized by oxidative hydroxylation of the indole ring at positions 5, 6, and 7. The hydroxylated derivatives are further conjugated with glucuronic acid and sulfate. N-Oxygenation and oxidative N-demethylation at the tropinyl nitrogen also occur in trace amounts.(ABSTRACT TRUNCATED AT 250 WORDS)


Renal handling of alendronate in rats. An uncharacterized renal transport system.

Alendronate (4-amino-1-hydroxybutylidene-1,1-bisphosphonate), an antiosteolytic agent, is currently under investigation in the treatment of a variety of bone diseases. Earlier studies from this laboratory have demonstrated that systemically administered alendronate is rapidly either taken up by bone tissues or excreted by the kidney, and that renal excretion is the only route of elimination. The purpose of this study is to characterize the renal handling of alendronate in rats by standard clearance procedures with inulin as a marker of glomerular filtration rate. Alendronate is highly bound to rat serum protein. The excretion of alendronate by the kidney is concentration-and dose-dependent, and saturable, indicating that it is secreted by an active transport mechanism. The secretory mechanism exhibits limitation of transport, with an apparent Tm of approximately 25 micrograms/min/kg. However, high doses of cimetidine, quinine, probenecid, and p-aminohippuric acid had no effect on the renal excretion of alendronate, suggesting that alendronate is not secreted by anionic or cationic transport systems. In contrast, alendronate clearance is inhibited by etidronate, another bisphosphonate, in a dose-dependent manner, implying that these two bisphosphonates compete for an as yet uncharacterized renal transport system. As expected, the renal excretion of alendronate is drastically reduced in rats with acute renal failure. As a consequence of renal impairment, alendronate accumulates in plasma, and the concentration of the drug in bone tissues increases significantly.


Tetrahydrobiopterin-dependent functional recovery in 6-hydroxydopamine-treated rats by intracerebral grafting of fibroblasts transfected with tyrosine hydroxylase cDNA.

Fibroblasts (NRK-49F) were transfected with human type 2 tyrosine hydroxylase (TH; EC 1.14.16.29) cDNA, to clarify the mechanism involved in amelioration of parkinsonism by intracerebral grafting of catecholaminergic neurons and to investigate its possible use as a donor material. These genetically manipulated fibroblasts did not develop into a mass of tissue, and survived well in the host striatum. Expression of the TH minigene in the cells was successful even when they were transplanted into the host brain. Intracerebral microdialysis revealed that a measurable amount of L-3,4-dihydroxyphenylalanine (L-DOPA) was not spontaneously released from the implanted cells into the host striatum. However, release of a large amount of L-DOPA from the cells was observed when (6R)-L-erythro-5,6,7,8-tetrahydrobiopterin (BH4) was perfused through a dialysis probe. Finally, we investigated whether these BH4-dependent L-DOPA-secreting fibroblasts are able to ameliorate the abnormal behavior of 6-hydroxydopamine-treated rats. Apomorphine-induced rotating behavior was not reversed by the grafting alone, whereas a marked reduction in drug-induced circling was observed temporarily after BH4 was perfused around the implanted cells. These findings indicate that TH cDNA-transfected non-neuronal cells might be able to be used as donor material for intracerebral grafting and ameliorate the abnormal behavior of rats with experimental Parkinson's disease.


The fimbriae-like structures on V. cholerae isolated in Kenya.

For Vibrio cholerae 01 to overcome the normal intestinal clearing mechanisms and facilitate colonization in the host gut, fine filamentous structures covering the surface of all the cell must exist. These fimbriae-like structures were investigated in this study. Selection of the K23 V. cholerae 01 biotype el-tor isolated in Kisumu, Kenya, was based on its proteinase activity and ability to agglutinate 0.2M ammonium sulphate. This strain was cultured in modified tryptone broth pH 8.4, at 37 degrees C overnight. Electron microscopy of the strain revealed the presence of fimbriae-like structures which varied in number. Some cells showed more than 200, whereas in other cells only several filamentous structures were detected. It is suggested that these structures could be a possible candidate for a cholera vaccine.


Reversal of flutamide-induced cryptorchidism by prenatal time-specific androgens.

The recent discovery of time-specific antiandrogens (flutamide) to induce undescent of the testes has provided us the ability to study androgen therapy for cryptorchidism in the first specific antiandrogen animal model for testicular descent. Postpartal administration of pharmacological doses of testosterone, dihydrotestosterone, or human chorionic gonadotropin failed to reverse antiandrogen-induced cryptorchidism. Reduction in the frequency of testicular undescent occurred only when these agents were administered before parturition. Indeed, statistically significant reductions in the incidence of undescended testes occurred only when androgens were administered simultaneously with the antiandrogens on gestational days 16-17. However, the successful ability of prenatal dihydrotestosterone or testosterone to prevent cryptorchidism was associated with significant complications of prenatal androgen therapy. Specifically, 100% (46 of 46) of the animals that had inhibition of flutamide-induced cryptorchidism manifested findings of hypogonadotropic hypogonadism. The findings of descended diminutive testes and epididymis in these latter animals did, however, substantiate that testicular weight does not play a significant role in descent of this organ. In summary, these studies delineate a narrow window of time in which androgens act to effect testicular descent that is independent of testicular size.


Dopamine and somatostatin inhibition of prolactin secretion from MMQ pituitary cells: role of adenosine triphosphate-sensitive potassium channels.

The sulfonylurea glibenclamide, which is known to block ATP-sensitive potassium channels, increases, in a dose-dependent manner, the release of PRL from MMQ pituitary cells. Glibenclamide does not reduce the dopaminergic inhibition of forskolin-stimulated PRL secretion; conversely it almost completely abolishes the inhibitory effect of somatostatin (SRIF) on this parameter. The sulfonylurea dose dependently increases basal [Ca++]i, without affecting the increase in [Ca++]i induced by high concentrations of extracellular potassium. Glibenclamide does not modify dopamine-induced [Ca++]i reduction, whereas it abolishes the inhibitory effect of SRIF on basal [Ca++]i. In the presence of diazoxide, an opener of ATP-sensitive potassium channels, which lowers basal [Ca++]i, dopamine still reduces [Ca++]i whereas SRIF does not induce a further decrease. Glibenclamide induces the depolarization of the cell membrane and prevents the SRIF-evoked hyperpolarization. The hyperpolarization of the cell membrane induced by dopamine is not modified by glibenclamide. Diazoxide induces a cell membrane hyperpolarization that is enhanced by dopamine but not by SRIF. Finally, glibenclamide does not affect basal and stimulated adenylate cyclase activity. In conclusion, our findings show that, in MMQ cells, glibenclamide stimulates PRL release, suggesting an involvement of ATP-sensitive potassium channels in the regulation of PRL secretion. The reversal by glibenclamide of the effects of SRIF on calcium homeostasis, membrane potential, and PRL release suggests that this type of potassium channel participates to the somatostatinergic inhibition of PRL secretion. Conversely, we found that glibenclamide does not modify the dopaminergic inhibition of PRL secretion and second messenger systems, suggesting that ATP-sensitive potassium channels may not be involved in the inhibitory effect of dopamine on PRL release.


Endoscopic laser lithotripsy with an automatic stone recognition system for basket impaction in the common bile duct.

In a patient with a common bile duct stone 28 mm in diameter, the traction wires of two basket catheters fractured during endoscopic mechanical lithotripsy. Disintegration of the concrement and removal of the impacted baskets failed even after extracorporeal application of 8,000 shockwaves. Pulsed dye laser lithotripsy was carried out via a 250 microns fiber which was advanced to the stone through a 6 French ERCP guiding catheter. Lithotripsy could be safely performed under fluoroscopic control since the laser used provides an automatic cut-out system upon tissue contact. 3,600 of 11,800 applied pulses were emitted with the total power setting and complete disintegration of the calculus was achieved. The baskets and the fragments could be removed endoscopically in the same session. Laser lithotripsy with a stone recognition system would seem to improve the applicability and safety of intracorporeal lithotripsy even when performed without direct visual guidance.


Molecular biological approaches to genetic disorders in prenatal diagnosis.

DNA analysis of Duchenne muscular dystrophy (DMD) and hemophilia A was performed with the aim of establishing the most efficient and reliable method for carrier and prenatal diagnosis in the Japanese population.


In vitro and in vivo characterization of the NMDA receptor-linked strychnine-insensitive glycine site.

Modulation of the NMDA receptor by the strychnine-insensitive glycine site was studied both in vitro and in vivo. In vitro the glycinergic stimulation of [3H]MK801 binding was measured in three different rat forebrain membrane preparations. An increased association rate of [3H]MK801 in the presence of glycine was observed. The binding of the radioligand was also enhanced by D-serine, whereas L-serine was less potent. The concentration-effect curves were shifted to the right by the glycine antagonist 7-chlorokynurenic acid (7CKA). In vivo modulation of the N-methyl-D-aspartate (NMDA) receptor was studied using NMDA induced convulsions in 7 day old rats. The NMDA effect was blocked by (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten,5,10-imine maleate (MK801) and D-(-)-2-amino-5-phosphono-pentanoic acid (AP5). The effect of a submaximal dose of NMDA was dose-dependently potentiated by 1-10 mg/kg D-serine, whereas higher doses of L-serine were needed to obtain a similar effect. 7CKA did not affect NMDA-induced convulsions but reduced the D-serine potentiation of NMDA responses. This study illustrates the ability of the strychnine-insensitive glycine site to modulate the NMDA receptor function both in vitro and in vivo.


The contribution of AMPA and NMDA receptors to graded bursting activity in the hippocampal CA1 region in an acute in vitro model of epilepsy.

The AMPA/KA receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) and the NMDA receptor antagonist 2-amino-5-phosphonovalerate (D-APV) were used to investigate the contribution of excitatory amino acid (EAA) receptors to graded bursting activity recorded in the CA1 region of the rat hippocampal slice following bath application of the convulsant drug bicuculline methiodide (BIC, 2-3 microM). CNQX (5-9 microM) significantly antagonised the burst in a reversible, concentration-dependent manner (n = 5). The effect involved a reduction in the amplitude but not the number of population spikes of the burst and also a depression of the underlying burst excitatory post-synaptic potential (EPSP). D-APV (5-25 microM), in contrast, reduced the amplitude and number of spikes in the burst but had no effect on the burst EPSP (n = 5). Following a single concentration of CNQX (5 microM), applied in the presence of bicuculline, it was observed that the components of epileptiform response which remained could be completely abolished with D-APV (10 microM; n = 10). It was also shown that, following elimination of synaptic transmission with CNQX (5 microM), application of bicuculline (2-3 microM) induced a small burst that could be reversibly antagonised with D-APV (10 microM). These results show that evoked epileptiform activity witnessed in the presence of bicuculline involves the activation of both AMPA and NMDA receptors, the AMPA receptor activation making the major contribution. The burst mediated by NMDA receptors is not dependent on prior activation of AMPA receptors.


Sharp anterior boundary of homeotic gene expression conferred by the fushi tarazu protein.

Parasegmental boundaries in the Drosophila embryo are delimited by the products of the fushi tarazu (ftz) and even-skipped (eve) genes. We show here that these act through particular key control regions of the homeotic gene Ultrabithorax (Ubx) to generate ftz- or eve-like stripe patterns of beta-galactosidase expression. Footprint analysis and tests in transformed embryos of constructs bearing mutated footprint regions suggest that ftz protein acts directly as a transcriptional activator of Ubx. Its activity outside the Ubx expression domain is suppressed by hunchback (hb), a repressor of Ubx. Some DNA binding sites for ftz protein are adjacent to, others overlap binding sites for hb protein, and we provide evidence that ftz protein competes with hb protein for DNA binding and/or for transcriptional activation. This competition mechanism results in a sharp anterior expression boundary. Direct activation of homeotic gene control regions by ftz (or eve) protein may be a regulatory step which is generally used to align expression of homeotic genes with parasegmental boundaries.


Two regulatory proteins that bind to the basic transcription element (BTE), a GC box sequence in the promoter region of the rat P-4501A1 gene.

The cDNAs for two DNA binding proteins of BTE, a GC box sequence in the promoter region of the P-450IA1(CYP1A1) gene, have been isolated from a rat liver cDNA library by using the BTE sequence as a binding probe. While one is for the rat equivalent to human Sp1, the other encodes a primary structure of 244 amino acids, a novel DNA binding protein designated BTEB. Both proteins contain a zinc finger domain of Cys-Cys/His-His motif that is repeated three times with sequence similarity of 72% to each other, otherwise they share little or no similarity. The function of BTEB was analysed by transfection of plasmids expressing BTEB and/or Sp1 with appropriate reporter plasmids into a monkey cell line CV-1 and compared with Sp1. BTEB and Sp1 activated the expression of genes with repeated GC box sequences in promoters such as the simian virus 40 early promoter and the human immunodeficiency virus-1 long terminal repeat promoter. In contrast, BTEB repressed the activity of a promoter containing BTE, a single GC box of the CYP1A1 gene that is stimulated by Sp1. When the BTE sequence was repeated five times, however, BTEB turned out to be an activator of the promoter. RNA blot analysis showed that mRNAs for BTEB and Sp1 were expressed in all tissues tested, but their concentrations varied independently in tissues. The former mRNA was rich in the brain, kidney, lung and testis, while the latter was relatively abundant in the thymus and spleen.(ABSTRACT TRUNCATED AT 250 WORDS)


An autoregulatory element of the murine Hox-4.2 gene.

Hox-4.2 promoter activity was assayed by transient expression assays in P19 embryonal carcinoma (EC) cells. Cotransfection of a luciferase reporter gene construct driven by Hox-4.2 upstream sequences with an expression vector for the Hox-4.2 gene product resulted in a 20-fold increase in luciferase activity. This activity was specific in that the Hox-1.6 gene product had no effect in the same assay. Mutational analysis defined a cis-acting element with enhancer function which conferred most of this increase. Activation was largely dependent on two TAAT/ATTA motifs within this 217 bp fragment and HOX-4.2 bound specifically to both of these motifs. The 217 bp element maps within a highly conserved region of the human Hox-4.2 gene (HOX4B) which has been shown to display spatial enhancer activity in mice and flies. These findings suggest a conserved autoregulatory mechanism for the control of Hox-4.2 expression.


A single amino acid substitution is sufficient to modify the mitogenic properties of the epidermal growth factor receptor to resemble that of gp185erbB-2.

The epidermal growth factor (EGF) receptor (EGFR) and the erbB-2 gene product, gp185erbB-2, exhibit distinct abilities to stimulate mitogenesis in different target cells. By using chimeric molecules between these two receptors, we have previously shown that their intracellular juxtamembrane regions are responsible for this specificity. Here we describe a genetically engineered EGFR mutant containing a threonine for arginine substitution at position 662 in the EGFR juxtamembrane domain, corresponding to threonine 694 in gp185erbB-2. This mutant, designated EGFRThr662, displayed affinity for EGF binding and catalytic properties that were indistinguishable from those of the wild type EGFR. However, EGFRThr662 behaved much as gp185erbB-2 in a number of bioassays which readily distinguish between the mitogenic effects of EGFR and gp185erbB-2. Moreover, significant differences were detected in the pattern of intracellular proteins phosphorylated on tyrosine in vivo by EGFR and EGFRThr662 in response to EGF. Thus, small differences in the primary sequence of two closely related receptors have dramatic effects on their ability to couple with mitogenic pathways.


Differential DNA sequence recognition is a determinant of specificity in homeotic gene action.

The homeotic genes of Drosophila encode transcriptional regulatory proteins that specify distinct segment identities. Previous studies have implicated the homeodomain as a major determinant of biological specificity within these proteins, but have not established the physical basis of this specificity. We show here that the homeodomains encoded by the Ultrabithorax and Deformed homeotic genes bind optimally to distinct DNA sequences and have mapped the determinants responsible for differential recognition. We further show that relative transactivation by these two proteins in a simple in vivo system can differ by nearly two orders of magnitude. Such differences in DNA sequence recognition and target activation provide a biochemical basis for at least part of the biological specificity of homeotic gene action.


A POU-A related region dictates DNA binding specificity of LFB1/HNF1 by orienting the two XL-homeodomains in the dimer.

LFB1/HNF1 regulates the hepatocyte-specific transcription of several genes, binding as a dimer to cis-acting elements that match the inverted palindrome GTTAATNATTAAC. The DNA binding domain of LFB1/HNF1 is characterized by a unique tripartite structure that includes an unusually long homeodomain (domain C), a region related to the POU-specific A-box (domain B) and a short N-terminal dimerization domain (domain A). We report that a recombinant peptide corresponding to the isolated homeodomain of LFB1/HNF1 binds as a monomer to a half-palindrome binding site, but shows diminished sequence specificity. Domain B, in addition to the homeodomain, is required and sufficient for proper recognition of LFB1/HNF1-responsive sites. A protein consisting of only these latter two domains is a monomer in solution, but forms dimers upon DNA binding. The protein-protein contacts established within the bound dimer restrain the orientation of the two homeodomains with respect to one another, thus contributing in a critical fashion to the recognition of the dyad symmetry-related LFB1/HNF1 sites. The DNA-independent dimerization domain (domain A) is required to increase the affinity of DNA binding, but does not influence the dimer geometry.


Induction of the three peroxisomal beta-oxidation enzymes is synergistically regulated by dexamethasone and fatty acids, and counteracted by insulin in Morris 7800C1 hepatoma cells in culture.

This work describes the molecular mechanism of hormonal modulation of fatty-acid peroxisomal beta oxidation in liver. Morris 7800C1 hepatoma cells and isolated hepatocytes were cultured in the presence of myristic acid (1 mM) and tetradecylthioacetic acid, a 3-thia fatty acid (50 microM), separately or in combination with dexamethasone (0.25 microM) or insulin (0.4 microM). Myristic acid stimulated acyl-CoA oxidase and a synergistic action was observed with dexamethasone. Parallel changes were recognized in enzyme protein and mRNA levels as quantified from immunoblots and Northern analyses. Myristic acid and tetradecylthioacetic acid had similar effects on this enzyme, while insulin inhibited the basal activity and blocked all inductions by the fatty acids and dexamethasone. Parallel mRNA and immunoblot analyses of the subsequent enzymes in the peroxisomal beta-oxidation pathway, enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase/delta 3,delta 2-enoyl-CoA isomerase and 3-oxoacyl-CoA thiolase, showed an even stronger induction by tetradecylthioacetic acid and dexamethasone, while the counteraction by insulin was maintained in both 7800C1 hepatoma cells and hepatocytes. In hepatoma cells, the thiolase always showed the most pronounced induction (about 40-fold) after 14 days, with parallel changes in protein and mRNA levels. The results suggest that the changes in peroxisomal beta-oxidation enzymes in 7800C1 hepatoma cells are due to a major effect on steady-state mRNA levels giving rise to corresponding alterations in enzyme protein. These results may be explained by regulation at the level of transcription of corresponding genes, but mRNA stability changes and/or translational effects may also be of importance.


Regulation of recombinant human tyrosine hydroxylase isozymes by catecholamine binding and phosphorylation. Structure/activity studies and mechanistic implications.

Three isozymes of human tyrosine hydroxylase (hTH1, hTH2 and hTH4) were expressed in Escherichia coli and purified to homogeneity. Natural catecholamines and related synthetic compounds were found to be potent inhibitors, competitive to the tetrahydrobiopterin cofactor, of all the isozymes. Combining visible spectroscopy and equilibrium-binding studies, it was found that catecholamines bind to hTH1 and hTH2 with a stoichiometry of about 1.0 mol/mol enzyme subunit, interacting with the catalytic iron at the active site. All the isozymes tested were excellent substrates for cAMP-dependent protein kinase (Km = 5 microM, Vmax = 9.5 mumol.min-1.mg kinase-1). The incorporation of about 1.0 mol phosphate/subunit at Ser40 decreased the affinity of dopamine binding by a factor of 10. Conversely, the addition of stoichiometric amounts of Fe(II) and dopamine to the apoenzymes reduced both the affinity and stoichiometry of phosphorylation by cAMP-dependent protein kinase by 2-3-fold. These data provide evidence for a mutual interaction between the presumed regulatory and catalytic domains of hTH, and show that activation of the enzyme by phosphorylation and inactivation by binding of catecholamines are related events, which probably represent important mechanisms for the regulation of the enzyme activity in vivo.


Evidence for an impaired long-chain fatty acid oxidation and ketogenesis in Fao hepatoma cells.

Fatty acid metabolism has been studied in Fao rat hepatoma cells. In basal conditions of culture, [1-14C]oleate is mainly esterified (85% of oleate uptake) in Fao cells, phospholipids being the most important esterified products (60% of oleate esterified). Addition of N6,O2'-dibutyryl-adenosine 3',5'-monophosphate (0.1 mM) in Fao cells does not change the metabolic fate of oleate whereas it induces gluconeogenesis and phosphoenolpyruvate carboxykinase mRNA accumulation. It is shown that the limitation of oleate oxidation is located at the level of the entry into mitochondria since octanoate is actively oxidized in Fao cells. Neither the activities of carnitine palmitoyltransferase (CPT) I and II nor the CPT II protein amount are affected by cAMP addition. The limitation of oleate oxidation in Fao cells results from (a) a high rate of lipogenesis and a high malonyl-CoA concentration, (b) a CPT I very sensitive to malonyl-CoA inhibition. The presence of an active oleate oxidation in mitochondria isolated from Fao cells confirms that CPT I is the limiting step of oleate oxidation. Moreover, Fao cells are unable to perform ketogenesis. This particular feature results from a specific deficiency in mitochondrial hydroxymethylglutaryl-CoA synthase protein, activity and gene expression. The metabolic characteristics observed in Fao cells could be a common feature in hepatoma cell lines with regard to the low capacity for long-chain fatty acid oxidation and ketone body production observed in the rat H4IIE and the human HepG2 cells.


Lithium, an inhibitor of cAMP-induced inositol 1,4,5-trisphosphate accumulation in Dictyostelium discoideum, inhibits activation of guanine-nucleotide-binding regulatory proteins, reduces activation of adenylylcyclase, but potentiates activation of guanylyl cyclase by cAMP.

Li+ drastically alters pattern formation in Dictyostelium by inhibiting cAMP-induced prespore-gene expression and promoting cAMP-induced prestalk-gene expression. We reported previously that Li+ inhibits inositol monophosphatases in this organism and strongly reduces basal and cAMP-stimulated inositol 1,4,5-trisphosphate levels. We show here that Li+ also reduces cAMP-induced accumulation of cAMP, but promotes cAMP-induced accumulation of cGMP. This effect is not due to inhibition of cGMP hydrolysis or inhibition of adaptation and may therefore reflect stimulation of guanylyl-cyclase activation. Li+ does not affect the binding of cAMP to surface receptors but interferes with the interaction between receptors and guanine-nucleotide-binding regulatory (G) proteins. These effects are complex; in the absence of Mg2+, Li+ increases guanosine 5'-[gamma-thio]triphosphate(GTP[S])-binding activity to similar levels as 1 mM Mg2+. However, while Mg2+ potentiates cAMP-induced stimulation of GTP[S]-binding activity, Li+ effectively inhibits stimulation. Li+ also inhibits cAMP-stimulated, but not basal high-affinity GTP-ase activity, indicating an inhibitory effect on cAMP-induced activation of G-proteins. Our data suggest that in addition to inositolphosphate metabolism, the activation of G-proteins may be a second biochemical target for Li+ effects on pattern formation and signal transduction in Dictyostelium.


Purification, cDNA cloning and Northern-blot analysis of mitochondrial chaperonin 60 from pumpkin cotyledons.

Two different cDNA clones, pMCPN60-1 and pMCPN60-2, encoding the mitochondrial homologues of chaperonin 60 (Cpn60) were isolated from a cDNA library of germinating pumpkin cotyledons by use of mixtures of synthetic oligonucleotides based on the N-terminal amino acid sequence of the protein. Determination of the complete nucleotide sequences of the two cDNA revealed that pMCPN60-1 and pMCPN60-2 each contain one open reading frame that encodes a protein of 575 amino acids with molecular masses of 61052 Da and 61127 Da, respectively. The deduced amino acid sequences of the two polypeptides include a 32-residue N-terminal putative mitochondrial presequence attached to the mature polypeptides, and they are 95.3% identical. From a comparison of deduced amino acid sequences with other Cpn60, it appears that the mature polypeptides of pumpkin mitochondrial Cpn60 are 44-59% identical to the other Cpn60, namely, GroEL of Escherichia coli, the 60-kDa heat-shock protein (Hsp60) of mitochondria in the yeast Saccharomyces cerevisiae, P1 protein of mammalian mitochondria and the Ribulose-1,5-bisphosphate carboxylase/oxygenase subunit-binding proteins alpha and beta of plastids in higher plants. Genomic Southern-blot analysis identified at least two copies of the gene for mitochondrial Cpn60 in the pumpkin genome. The levels of mRNA for mitochondrial Cpn60 in cotyledons, hooks and hypocotyls of pumpkin seedlings increased in response to heat stress, as deduced from Northern-blot analysis, indicating that pumpkin mitochondrial Cpn60 is a heat-induced stress protein.


Haemodynamic effects of different doses of dopexamine hydrochloride in low cardiac output states following cardiac surgery.

We studied the haemodynamic effects of dopexamine hydrochloride, a beta 2-adrenergic agonist with dopaminergic (DA1) activity, in 20 patients with low cardiac output following surgery for coronary artery bypass grafting and/or valve replacement or repair. Following titration of four doses (1, 2, 4 and 6 micrograms.kg-1.min-1), the dose producing the optimal response was infused for up to 48 h (five patients). During the infusion, significant increases in cardiac index and stroke volume were accompanied by significant decreases in systemic vascular resistance. Heart rate increased significantly up to 6 h and thereafter returned to control levels. Mean blood pressure was reduced but did not fall below 60 mmHg. However, in five patients treated for 48 h mean blood pressure had returned to control levels. Unwanted effects (tachycardia and hypotension) were seen chiefly at higher doses, leading us to conclude that infusion rates of 4 micrograms.kg-1.min-1 or less will be useful in the treatment of low cardiac output following cardiac surgery.


The role of beta receptor blockade in preventing sudden death.

The high mortality rate from coronary heart disease in hypertensives can only be substantially reduced if sudden coronary death rates can be decreased. The aim of this review is to discuss how treatment may be tailored to reduce the risk of sudden death in high-risk patients. Clinical trials have not yet produced long-term primary prevention data on the effects of angiotensin converting enzyme (ACE) inhibitors, calcium antagonists or alpha-blockers on cardiovascular complications and sudden death in hypertensive patients. Further, the conclusion from large-scale secondary preventive studies presently available on ACE inhibitors and calcium antagonists is that their impact on sudden death has been disappointing. By contrast, some beta-blockers have reduced sudden death and other coronary events both in primary and secondary preventive studies. The benefits have been attributed to beta 1-blockade, and seem to be independent of blood pressure control. It cannot be assumed that all beta-blockers are equally effective in preventing ventricular fibrillation, sudden death and other coronary events. To date, the best documented data cover the lipophilic beta-blockers and it is speculated that by increasing levels of cardiac vagal tone and electrical stability in the heart, beta 1-blockade in the brain might contribute to the reduction in sudden death risk seen with these beta-blockers.


Pharmacokinetics and first clinical experiences with an antihypertensive dopamine (DA2) agonist.

The pharmacokinetic properties and first clinical experiences with the antihypertensive dopamine (DA2) agonist, carmoxirole, are summarized. In man carmoxirole was rapidly absorbed. On oral administration the maximum plasma concentration was reached after 2-3 h. The drug was metabolized, mainly to an ester-type glucuronide, and was excreted (unchanged carmoxirole plus glucuronide) largely by the kidneys. The plasma half-life of the parent compound was 5.5 h. For the dose range tested (0.5 to 1.5 mg) the pharmacokinetics were linear. The drug was rapidly distributed in animals but only very small amounts penetrated the blood-brain barrier. Carmoxirole did not affect supine blood pressure in healthy subjects, but under the conditions of the Schellong's test some orthostatic reactions occurred with high doses. In patients the blood pressure was reduced for at least 8 h after single oral doses. On repeated administration for several weeks a relevant antihypertensive effect was still measurable 12 and 24 h after dose. The most frequently reported adverse events have been headache, dizziness, tiredness, nausea, and gastric disorders. These symptoms are considered to be mainly due to blood pressure reduction, as is frequently observed at the beginning of antihypertensive therapy. In patients the incidence of orthostatic reactions is appreciably lower than in healthy subjects, and in both change of position was sufficient to relieve the symptoms.


Pharmacological basis for antihypertensive therapy with a novel dopamine agonist.

In the past, nearly all major mechanisms involved in the regulation of blood pressure have become targets of antihypertensive drugs. They include the brain stem with its neuronal circuits of central cardiovascular regulation, the sympathetic neuro-effector system, the kidney, the renin angiotensin aldosterone system and the vascular smooth muscle cell. There are various ways of influencing the function of the sympathetic nervous system, but the clinical potential of one mechanism of action has not yet been explored in detail. Drugs that inhibit noradrenaline release through stimulation of inhibitory receptors located at adrenergic nerve terminals in the cardiovascular system (inhibitory presynaptic receptors) are not available for the treatment of hypertension. Among the multiple presynaptic receptors, dopamine receptors which belong to the dopamine2 subtype, are of particular interest. Carmoxirole is a novel indole derivative with a potent agonist action selective for dopamine2-receptors of the periphery. Experimental evidence shows that carmoxirole lowers blood pressure in various models of hypertension mainly or exclusively through inhibition of noradrenaline release from sympathetic nerve endings. This effect of carmoxirole is mediated by presynaptic dopamine receptors with the characteristic that release inhibition is restricted to low rates of sympathetic nerve discharge.


Molecular analysis of patients with Wiedemann-Beckwith syndrome. I. Gene dosage on the short arm of chromosome 11.

Wiedemann-Beckwith syndrome (WBS) is characterised by a specific group of congenital malformations associated with an increased concurrent risk for development of a defined group of childhood neoplasms. The mode of inheritance is complex, but recently compiled family data suggest that it is an autosomal dominant trait of varying expression. It has previously been suggested that major rearrangements on the short arm of chromosome 11 may be involved in the aetiology of the disease, particularly in the region of the insulin like growth factor II (IGF-II) gene (11p15.5). This gene is thought to be parentally imprinted in the mouse and it has been suggested that in the human, duplication of the non-imprinted locus in WBS patient might lead to diploid expression of the gene and consequent general hyperplasia. This model predicts that there should be both frequent and parental origin specific duplication of the IGF-II gene in the patients. It was the aim of this study to examine the IGF-II locus and its surrounding chromosomal environment for such lesions in a large number of WBS patients. Using restriction fragment length polymorphism analysis for four linked markers on 11p and genomic clones internal to the IGF-II locus we could find no evidence of alteration or amplification of this area in any of the 11 patients investigated. In one patient who developed a Wilms tumour we could find no evidence for loss of any material on the short arm of chromosome 11 as reported previously.(ABSTRACT TRUNCATED AT 250 WORDS)


Molecular analysis of patients with Wiedemann-Beckwith syndrome. II. Paternally derived disomies of chromosome 11.

In Wiedemann-Beckwith syndrome (WBS) a putative disease gene resides at the tip of the short arm of chromosome 11 in the region of the insulin growth like factor II (IGF-II) gene. Whilst changes in gene dosage in this area do not appear to be common in the syndrome, in familial cases the lesion appears to be dominant only when inherited through the female line. We undertook to examine the parental origin of the copies of chromosome 11 in a large group of WBS patients using a series of restriction fragment length polymorphisms (RFLPs) on 11p, and report here that in one sporadic case of WBS out of 14 both copies of chromosome 11 are derived from the father and are present in a normal dosage. This suggests that at least one mode of expression of the lesion is modified by genomic imprinting.


Regulation of interleukin-4 production in human mononuclear cells.

Interleukin (IL)-4 is a cytokine with a broad range of effects on immune cells, however, little is known regarding the regulation of its production in freshly isolated human peripheral blood mononuclear cells (PBMC). Here we report the production of IL-4 in such cells following stimulation with monoclonal antibodies (mAb) directed against different cell surface antigens. We show that triggering via CD2 is more efficient for IL-4 production than triggering via the CD3 complex. The addition of a CD28 mAb enhances IL-4 production approximately threefold. Cell depletion experiments show that among CD2 plus CD28-stimulated PBMC the production of IL-4 is restricted to the CD8-CD45RA-T cell subpopulation. mAb interfering with the binding of IL-2 to its receptor can inhibit the production of IL-4 in CD2 plus CD28-stimulated PBMC. As IL-2 induces cell proliferation and production of interferon-gamma, but not production of IL-4, it follows that IL-2 is necessary but not sufficient for IL-4 production.


The substituted amphetamines 3,4-methylenedioxymethamphetamine, methamphetamine, p-chloroamphetamine and fenfluramine induce 5-hydroxytryptamine release via a common mechanism blocked by fluoxetine and cocaine.

The abilities of the substituted amphetamines 3,4-methylenedioxymethamphetamine (MDMA), methamphetamine, p-chloroamphetamine (PCA) and fenfluramine to induce synaptosomal [3H]serotonin (5-HT) release were compared using a novel microassay system. The rank order of release potencies was found to be (+/-)PCA congruent to (+)-fenfluramine greater than (+)-MDMA much greater than (+)-methamphetamine. Combination of two drugs at their EC50 did not cause more release than either drug alone at an equivalent concentration. In addition, the 5-HT uptake blockers fluoxetine and cocaine inhibited the release induced by MDMA, methamphetamine, PCA and fenfluramine to the same percentage. However, threshold concentrations of the substituted amphetamines known to inhibit uptake did not attenuate the release caused by higher concentrations of these compounds. These results suggests that MDMA, methamphetamine, PCA and fenfluramine cause 5-HT release via a common mechanism. Furthermore, these results indicate that the 5-HT uptake blockade induced by these substituted amphetamines in vitro is different from that induced by either fluoxetine or cocaine.


Biochemical and pharmacological studies on pramipexole, a potent and selective dopamine D2 receptor agonist.

Pramipexole (SND 919; 2-amino-4,5,6,7-tetrahydro-6-propyl-amino-benzthiazole- dihydrochloride) was tested for its agonistic activity at pre- and postsynaptic dopamine (DA) receptors. L-Dihydroxyphenylalanine (L-dopa) accumulation in the rat striatum and limbic system and the alpha-methyltyrosine-induced reduction of DA were inhibited. Both effects were fully antagonized by haloperidol but not by the selective DA D1 receptor antagonist SCH 23390. Pramipexole decreased the levels of DA metabolites dose dependently, whereas striatal DA levels remained unchanged. In mice, pramipexole (0.001-1 mg/kg s.c.) reduced exploratory locomotor activity. In rats with unilateral striatal lesions, only weak ipsilateral rotation was produced by pramipexole at the highest dose. However, in rats with unilateral lesions of the medial forebrain bundle, pramipexole potently induced contralateral circling (ED50 0.026 mg/kg s.c.). In the N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) monkey model, pramipexole also had potent stimulatory effects. Finally, in haloperidol-sensitized monkeys, the substance did not elicit dyskinesia/dystonia when given alone, but rather inhibited those symptoms which had been induced by haloperidol (ED50 0.116 mg/kg i.m.). It is concluded that pramipexole has therapeutic potential for schizophrenic patients, as a result of its autoreceptor agonistic effects and its weak effects at normosensitive postsynaptic DA receptors. Furthermore, its potent stimulatory effects in DA-depleted animals suggest a possible use in the treatment of Parkinson's disease.


Effects of i.c.v. lithium chloride administration on monoamine concentration in rat mediobasal hypothalamus.

We investigated the acute effects of a single i.c.v. injection of lithium chloride (LiCl) the neuroamine content of the rat mediobasal hypothalamus (MBH). The effects of lithium on amine synthesis and degradation enzymes were also studied in vitro. Noradrenaline (NA), dopamine (DA), serotonin (5-HT) and 5-hydroxyindoleacetic acid (5-HIAA) concentrations were reduced 10 min after i.c.v. injection of 24 nmol of LiCl and returned to control values 30 min after the injection. Two nmol of LiCl reduced the concentration of DA (10 and 30 min after injection) and 5-HIAA (30 min after injection). LiCl (0.5-10 mM) inhibited tyrosine hydroxylase activity (catecholamine synthesis) in vitro in a concentration dependent manner. The i.c.v. administration of a high dose of LiCl reduced the content of neuroamines in the MBH. This might result from and inhibition of synthesis. A possible link between the observed changes and some reported side effects of lithium therapy is discussed.


The behavioural effects of MK-801 in rats: involvement of dopaminergic, serotonergic and noradrenergic systems.

The non-competitive NMDA receptor antagonist, MK-801 (dizocilpine), induces in rats a characteristic behavioural syndrome with ataxia, stereotypies and hyperlocomotion. At least part of this behavioural syndrome is thought to be related to interactions between glutamatergic and dopaminergic neurotransmission. Based on recent biochemical evidence that serotonin (5-HT) might also be involved in the effects of MK-801 several 5-HT receptor ligands were tested for effects on MK-801-induced behaviours. The 5-HT1A receptor ligands, ipsapirone and NAN-190, which are known to display antagonist-like properties in functional models of postsynaptic 5-HT1A receptor activity attenuated or blocked the hyperlocomotion and head weaving observed after administration of MK-801, whereas the 5-HT2 receptor antagonist, ritanserin, was ineffective in this respect. The dopamine receptor antagonist, haloperidol, and the alpha 1-adrenoceptor antagonist, prazosin, also attenuated behaviours induced by MK-801. In contrast to its effects on stereotypies induced by MK-801, ipsapirone potentiated rather than attenuated the stereotyped behaviour induced by the dopamine receptor agonist, apomorphine, indicating that antagonism of MK-801-induced stereotypies by ipsapirone may not be related to the dopaminergic system. The data indicate that, in addition to catecholaminergic systems, serotonergic neurotransmission is significantly involved in the mechanisms by which MK-801 alters behaviour in rats.


Resistance of beta 2-adrenoceptor-mediated responses of lung strips to desensitization by long-term agonist exposure--comparison with atrial beta 1-adrenoceptor-mediated responses.

In vitro desensitization of beta 2-adrenoceptor-mediated relaxation of guinea-pig isolated parenchymal strips was examined. Concentration-response curves for isoprenaline were obtained and after long-term incubation with isoprenaline, followed by washout, a second curve was obtained. Correction for time-dependent loss of sensitivity was made from time-matched controls. After incubation with 10(-5) M isoprenaline for 0.5, 1, 2, 4 and 8 h, loss of responsiveness of carbachol-contracted lung strips was observed after 4h as a reduced post-incubation maximum response. When the concentration was reduced to 10(-6) M, a 4 h incubation with 1 h washout no longer induced a shift of the post-incubation curve in carbachol-contracted lung strips. In contrast, lung strips with intrinsic tone displayed reduced responsiveness to isoprenaline after 4 h incubation with 10(-6) M isoprenaline. Incubation of the tissue for 4 h with lanthanum (1.4 x 10(-6) M), a relaxant not operating through beta 2-adrenoceptors or their effector coupling, had the same effect upon isoprenaline concentration-response curves as incubation with isoprenaline. This was irrespective of whether intrinsic tone (10(-6) M isoprenaline) or carbachol-contracted (10(-5) M isoprenaline) lung strips were used. It was concluded that the loss of beta 2-adrenoceptor responsiveness after incubation with 10(-6) M isoprenaline was due to the prolonged maximal relaxation of the tissue for 4 h rather than desensitization of the beta 2-adrenoceptor. Indeed, after correction for maximal relaxation and for time, no significant change in beta 2-adrenoceptor sensitivity of the lung occurred after incubation with 10(-6) M isoprenaline for 4 h. This contrasts with significant rightwards shifts of the concentration-response curves for the beta 1-adrenoceptor-mediated increases in rate and tension of guinea-pig right and left atria after identical incubation conditions. Thus whereas beta 1-adrenoceptor-mediated responses displayed desensitization after long-term in vitro agonist exposure, the beta 2-adrenoceptor-mediated responses were resistant.


Effects of beta-adrenoceptor blockers on mitochondrial ATPase activity in guinea pig heart preparations.

The effects of three beta-adrenoceptor blockers atenolol, indenolol and nadolol on myocardial mitochondrial ATPase (ATP: phosphohydrolase EC 3.6.1.3) activity were evaluated and compared with that of propranolol in guinea pig heart preparations. Propranolol and indenolol inhibited ATPase activity with IC50 values of 4.4 +/- 0.5 and 5.3 +/- 0.4 mM, respectively. In contrast, however, nadolol and atenolol markedly enhanced mitochondrial ATPase activity. Atenolol increased the enzyme activity by approximately 5, 240 and 950%, while nadolol enhanced it by 13, 280 and 2800% at 100 microM, 1.0 mM and 10.0 mM, respectively. The results indicate that these drugs exhibit two modes of interaction with the mitochondrial ATPase: inhibition by propranolol and indenolol and stimulation by atenolol and nadolol. The inhibitory actions are probably related to the membrane-stabilizing effects and therefore antiarrhythmic actions of the two drugs, while the stimulatory effects of atenolol and nadolol are probably a result of interactions with some component of oxidative phosphorylation or the respiratory chain.


Beta-adrenergic regulation of amine precursor amino acid transport across the blood-brain barrier.

Beta-Adrenoceptor agonists were administered i.p. into rats and amino acid levels in brain and plasma were then determined to assess the effects on transport across the blood-brain barrier. Isoproterenol (10 mumol/kg) caused significant increases in aromatic amino acid (tyrosine, phenylalanine and tryptophan) levels in cerebral cortex and decreases in almost all amino acid concentrations in plasma. This effect of isoproterenol on brain tyrosine level was dose-dependent with an ED50 of 0.25 mumol/kg. Salbutamol (beta 2-adrenoceptor agonist, 10 mumol/kg) showed similar effects, but dobutamine (beta 1-adrenoceptor agonist, 50 mumol/kg) failed to increase brain amino acid levels. When 1-threo-3,4-dihydroxyphenylalanine (L-DOPA, 100 mumol/kg) was i.p. loaded, beta-adrenoceptor agonists promoted the transport of L-DOPA into brain without increasing the clearance rate of plasma L-DOPA. Moreover, significant increases in dopamine and its metabolites were observed in rat brain. These findings suggest that the transport of aromatic amino acids across the blood-brain barrier may be regulated through beta 2-adrenoceptors and that co-administration of beta 2-adrenoceptor agonists with L-DOPA may enhance the therapeutic efficacy of L-DOPA.


Evidence that an alpha 2A-adrenoceptor subtype mediates antinociception in mice.

In the hot-plate test in mice, the antinociceptive action of the alpha 2-adrenoceptor agonist, UK 14,304, was abolished by the alpha 2-adrenoceptor antagonist, idazoxan, the potent alpha 2A-adrenoceptor antagonist, RX 821002 and the preferential alpha 2A-adrenoceptor antagonist, BRL 44408. In contrast, the preferential alpha 2B- (and alpha 2C)-adrenoceptor ligands ('antagonists'), ARC-239, BRL 41992 and prazosin were inactive. The preferential alpha 2A-adrenoceptor partial agonist, guanfacine, partially inhibited UK 14,304-induced antinociception. Further, guanfacine BRL 44408 reversibly elicited submaximal antinociception. It is concluded that alpha 2A-adrenoceptors mediate antinociception in mice.


In vitro study of a novel atypical beta-adrenoceptor agonist, SM-11044.

SM-11044 (L-threo-3-(3,4-dihydroxyphenyl)-N-[3-(4-fluorophenyl) propyl] serine pyrrolidine amide hydrobromide) stimulated the relaxation of guinea pig ileum (EC50: 3.0 x 10(-8) M), trachea (EC50: 1.3 x 10(-7) M), lung parenchyma (EC50: 2.1 x 10(-6) M) and the increase in right atrial rate (EC50: 6.9 x 10(-6) M). It also induced lipolysis of rat white adipocytes (EC50: 1.2 x 10(-6) M). Both the relaxant response of ileum and the lipolytic response of adipocytes to SM-11044 were resistant to inhibition by propranolol (10(-6) M), but were antagonized by cyanopindolol (10(-6) M). In contrast, the responses to SM-11044 in trachea, lung parenchyma and right atrium were almost completely abolished by propranolol (10(-6) M). Furthermore, the selectivity of SM-11044 relative to isoproterenol was ileum greater than adipocytes greater than trachea (beta 2) greater than lung (beta 2) greater than atrium (beta 1). These results suggest that SM-11044 is a selective agonist of atypical beta-adrenoceptors that are resistant to antagonism by propranolol but sensitive to cyanopindolol. The receptor binding and adenylate cyclase stimulating activity of SM-11044 were also examined on transfected Chinese hamster ovary cells expressing human beta 1-, beta 2- or beta 3-adrenoceptors. SM-11044 inhibited the binding of [125I]iodocyanopindolol to the three types of receptors in a concentration-dependent manner. The selectivity in terms of Ki values was beta 3 (Ki: 1.3 x 10(-6) M) greater than beta 2 (Ki: 4.1 x 10(-6) M) greater than beta 1 (Ki: 18.1 x 10(-6) M)-adrenoceptors.(ABSTRACT TRUNCATED AT 250 WORDS)


NMDA receptor antagonists block cardiovascular responses to intrathecal administration of D-baclofen in the rat.

In previous studies we found that D and L-baclofen have different effects on sympathetic output when administered intrathecally, yet the actions of both enantiomers are blocked by intrathecal administration of phaclofen. The present experiments were done to determine the mechanism by which D-baclofen expresses its effects. In urethane-anaesthetized Sprague-Dawley rats, when D-baclofen was given intrathecally at the T9 spinal level following pretreatment with 2 nmol of the NMDA receptor antagonist, DL-2-amino-5-phosphonovaleric acid (APV), it increased systolic and diastolic arterial pressures (n = 7), as in the previous studies. However, after intrathecal administration of 10 nmol of APV, administration of D-baclofen had no effect on these parameters (n = 7). Intravenous administration of ketamine (7.5 mg/kg), another NMDA receptor antagonist, also blocked the effect of D-baclofen (n = 6) but it had no effect on the pressor responses produced by intrathecal administration of carbachol (27.4 nmol; n = 6). In additional experiments, L-baclofen (70 nmol) had no effect on the increases in heart rate and arterial pressure produced by N-methyl-D-aspartic acid (NMDA) (2 nmol; n = 8). These results indicate that D-baclofen increases arterial pressure via an NMDA receptor-mediated mechanism, perhaps by provoking the release of an endogenous ligand which activates these receptors.


Effects of alpha-adrenoceptor antagonists and clonidine on the haemodynamic response to acute hypovolaemia in conscious rabbits.

In conscious rabbits an inferior vena caval cuff was progressively inflated so cardiac output fell at a constant approximately 8% of its baseline value. There was a biphasic haemodynamic response, consisting of an initial compensatory phase during which there was progressive systemic vasoconstriction and tachycardia, followed by a decompensatory phase in which systemic vasoconstriction failed abruptly, blood pressure plummeted and heart rate declined. We tested the effects on the haemodynamic response of prior 4th ventricular, and in some cases intravenous, infusions of saline, yohimbine, clonidine, yohimbine plus clonidine, and bunazosin. From the results we conclude that a yohimbine-sensitive mechanism in the brainstem, possibly alpha 2-adrenoceptor-mediated, may be an essential element of the cardiac receptor-mediated decompensatory phase of acute central hypovolaemia, but does not contribute to the arterial baroreflex-mediated compensatory phase.


Stimulation of bicarbonate secretion by atypical beta-receptor agonists in rat cecum in vitro.

This study examined the effects of beta-adrenoceptor agonists on bicarbonate secretion by the rat cecum in vitro. Isoprenaline, the beta 2-selective agonist salbutamol and the 'atypical' beta-agonist SR58611A stimulated bicarbonate secretion in a concentration related manner. Another atypical agonist, BRL 37344, also stimulated. Responses to isoprenaline were antagonised by alprenolol and propranolol (both 20 microM) but not the selective antagonists practolol (10 microM) or ICI 1185511 (1 microM). Responses to SR 58611A were only antagonised by alprenolol. Replacement of Cl- by NO3- on the mucosal surface reduced basal secretion and abolished the response to isoprenaline. Exposure to a single concentration of atypical agonist resulted in desensitisation to a second application and to isoprenaline. There was no evidence of desensitisation with isoprenaline or salbutamol. The results show that beta-adrenoceptor agonists stimulated bicarbonate secretion in contrast to the previously described inhibitory effect of cholinergic drugs in this tissue. Stimulation was mediated by beta-adrenoreceptors, which had properties consistent with the atypical receptors described in gut smooth muscle and in adipose tissue. Both adrenergic and cholinergic drugs may act on the same mechanism of secretion which may involve an exchange of HCO3- for mucosal Cl-.


Dose-dependent conditioned place preference produced by etonitazene and morphine.

The conditioned place preference (CPP) paradigm was used to study the reinforcing properties of etonitazene in comparison with those of morphine. Increasing doses of etonitazene (2.5-15 micrograms/kg i.p.) and morphine (1-80 mg/kg i.p.) induced a dose-dependent CPP. High doses of etonitazene (25-40 micrograms/kg) did not elicit CPP. In addition, these reinforcing properties were related to behavioral modifications such as analgesia, assessed with the tail-flick method, and increased catalepsy, evaluated by a scoring system. It is concluded that neither the strong behavioral effects induced by etonitazene nor tolerance to such effects account for the results. These findings are discussed with regard to the possibility that etonitazene could interfere with associative learning motivated by reward.


Presynaptic inhibition by clonidine of neurotransmitter amino acid release in various brain regions.

The release of endogenous aspartic acid (Asp), glutamic acid (Glu) and gamma-aminobutyric acid (GABA) was investigated in synaptosomes prepared from various regions of the rat brain. The basal release of Asp, Glu and GABA from various regions was 12-35, 24-107 and 15-43 pmol/min per mg protein, respectively. Exposure to a depolarizing concentration of KCl (30 mM) resulted in 1.7 to 3.6-fold increases in Asp, Glu and GABA release. When clonidine (10(-4) M) was added to the perfusion medium, the K(+)-evoked overflow of both Asp and Glu was inhibited by 50-90% in the anterior cortex, thalamus and hypothalamus. Clonidine inhibited the K(+)-evoked Glu overflow by 30-40% in the posterior cortex and hippocampus. No significant effects were observed in the other brain regions (olfactory bulb, striatum, midbrain, cerebellum, pons, medulla oblongata). The inhibitory effects of clonidine were counteracted by an alpha 2-adrenoceptor antagonist, rauwolscine. The data suggest that the basal and K(+)-evoked release of Asp, Glu and GABA from nerve terminals is different in rat brain regions and that the presynaptic alpha 2-adrenoceptors which regulate the release of excitatory amino acids are mainly distributed in the anterior cerebral cortex, thalamus and hypothalamus of the rat brain.


Behavioral effects of modulators of ATP-sensitive K+ channels in the rat dorsal pallidum.

The effects of the potent ATP-sensitive K+ channel blocker glipizide were measured on the locomotor activity of rats after bilateral intracerebral administration into the dorsal pallidum. Glipizide (10 pmol) was found to reduce spontaneous locomotor activity measured during the night cycle of the rats, whereas the ATP-sensitive K+ channel activator (-)-cromakalim (5 fmol) enhanced spontaneous locomotor activity. Glipizide (0.5, 2.5 and 10 pmol) was also found to depress noticeably d-amphetamine-induced locomotor activity (1 mg/kg s.c.). These results are in agreement with the idea that ATP-dependent potassium channels within the dorsal pallidum are involved in controlling motor activity in the rat.


Effects of inhibitory and excitatory drugs on the metabolic rhythm of the hamster suprachiasmatic nucleus in vitro.

In order to elucidate the role of excitatory and inhibitory transmitters within the suprachiasmatic nucleus (SCN) in the circadian change of 2-deoxyglucose (2-DG) uptake in this nucleus, the effects of 8-hydroxy-2-(di-n-propylamino) tetralin hydrobromide (8-OH-DPAT), muscimol, flurazepam, pentobarbital and glutamate on uptake of 2-DG by hamster SCN were examined in hypothalamic slice preparations. 2-DG uptake in the SCN was high during the subjective day and low during the subjective night. The high uptake of 2-DG in the SCN during the daytime was inhibited by the superfusion of 8-OH-DPAT, muscimol, flurazepam and pentobarbital in a dose-dependent manner, but the low uptake of 2-DG during the night was unaffected. The low uptake during the night was significantly increased by treatment with glutamate, whereas 2-DG uptake during the day was unaffected. In contrast to the above results, 20 mM KCl and 1 microM tetrodotoxin increased and decreased 2-DG uptake during both the day and night, respectively. The present results strongly suggest that agonists of 5-HT1A receptors and GABAA-benzodiazepine-barbiturate complex receptors regulate the function of the SCN through their inhibitory action on 2-DG uptake during the day, and that glutamate also regulates SCN function through it stimulatory action on 2-DG uptake during the night.


Involvement of serotonin receptors in methionine sulfoximine-induced hypothermia in the rat.

L-Methionine-D,L-sulfoximine (MSO), intraperitoneally (i.p.) or intracerebroventricularly (i.c.v.) (third ventricle) injected at a convulsant dose, induced a centrally mediated body hypothermia in the restrained rat maintained at an ambient temperature of 23 degrees C. Pretreatment with (+/-)-pindolol (1.5-3 mg/kg s.c.) significantly attenuated MSO-induced hypothermia, but at a dose of 6 mg/kg s.c. hypothermia developed without any modification of its characteristics. Pretreatment with (-)-propranolol (16-25 mg/kg i.p.) potentiated MSO-induced hypothermia, but pretreatment of MSO-treated rats with ketanserin (0.7-4 mg/kg i.p.) did not significantly modify hypothermia. Selective antagonists for beta-adrenoceptors were used and their effects on MSO-induced hypothermia were compared with those of pindolol and propranolol. Pretreatment with betaxolol (1.5-4 mg/kg s.c.) did not modify the hypothermia following administration of MSO, but potentiation of hypothermia was recorded in rats pretreated with ICI 118,551 (2.26 mg/kg i.p.) then i.p. injected with MSO. These findings favour a control exerted by 5-HT1 receptors in the central development of MSO-induced hypothermia in the restrained rat.


Alpha 2-adrenoceptor and catecholamine-insensitive binding sites for [3H]rilmenidine in membranes from rat cerebral cortex.

The kinetic and pharmacological characteristics of the binding of the oxazoline antihypertensive drug, [3H]rilmenidine, to membranes of rat cerebral cortex have been determined. Computerised resolution of curvi-linear, equilibrium binding isotherms was consistent with the existence of two distinct binding sites for [3H]rilmenidine: Kd 17.3 +/- 7.41 nM, Bmax 0.197 +/- 0.06 pmol/mg protein and Kd 254 +/- 48 nM, Bmax 1.59 +/- 0.08 pmol/mg protein. Moreover, the resolution of two association and dissociation rates also suggested the existence of two binding site populations. Drug inhibition studies revealed that specific binding of [3H]rilmenidine (2 nM) was only inhibited by a maximum of 50% by the catecholamines, adrenaline and noradrenaline, but was completely inhibited by some oxazolines, by guanabenz (a guanidino drug) and by several imidazoline compounds including naphazoline, oxymetazoline and clonidine. Binding isotherms for these drugs were also best fit by a two-site model. The relative Ki values at the high affinity site for [3H]rilmenidine and the number of these high affinity sites are consistent with this site being an alpha 2-adrenoceptor. The high affinity of oxymetazoline and low affinity of prazosin for high affinity [3H]rilmenidine binding sites together with the rank order of potency of oxymetazoline greater than phentolamine greater than SKF 104078 greater than ARC-239 greater than prazosin suggest that [3H]rilmenidine binds to the alpha 2A sub-type of adrenoceptor. Computer-resolved Ki values for drugs at the larger number of lower affinity binding sites were very similar to Ki values determined in the presence of 10 microM adrenaline (used to block alpha 2-adrenoceptor binding).(ABSTRACT TRUNCATED AT 250 WORDS)


Thiokynurenates prevent excitotoxic neuronal death in vitro and in vivo by acting as glycine antagonists and as inhibitors of lipid peroxidation.

Several derivatives of kynurenic and thiokynurenic acids were synthesized and tested for their ability to protect primary cultures of cerebellar granule cells against excitotoxic damage, and to affect the binding of [3H]glycine ([3H]Gly), [3H]alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid ([3H]AMPA), [3H]3-(2-carboxypiperazine-4-yl-)propyl-1-phosphonic acid ([3H]CPP), [3H]kainic acid and [3H]N-[1-(2-thienyl)cyclohexyl]-3,4-piperidine ([3H]TCP) to rat cortical membranes. Kynurenic and thiokynurenic acid derivatives with one or two halogens in position 5 or 7 were selective glycine antagonists, failing to affect N-methyl-D-aspartate (NMDA), kainate or AMPA sites at micromolar concentrations. 7-Cl-kynurenic, 7-Cl-thiokynurenic, 5,7-diCl-kynurenic and 5,7-diCl-thiokynurenic acids had similar IC50s for displacing [3H]Gly from its strychnine-insensitive site and for reducing the stimulated (0.5 microM NMDA and 1 microM glycine) [3H]TCP binding to cortical membranes. However, 7-Cl-thiokynurenic acid was particularly potent to prevent excitotoxic neuronal death in cultured cerebellar granule cells. This action may be ascribed to inhibition of lipid peroxidation, a property which was demonstrated for the 5- or 7-Cl derivatives of thiokynurenic acid. Furthermore, 7-Cl-thiokynurenic acid reduced excitotoxic damage caused by the injection of quinolinic acid in the rat striatum. Thus, 7-Cl-thiokynurenic acid appears to be a new compound with interesting antiexcitotoxic properties both in vitro and in vivo.


The interaction of R(+)- and S(-)-zacopride with PCPA to modify rodent aversive behaviour.

The interaction of R(+)- and S(-)zacopride (0.00001-10 mg/kg i.p.) with parachlorophenylalanine (PCPA, 3 day treatment 100 mg/kg i.p.) to modify behaviour in an aversive situation was investigated in the mouse black and white test box and rat social interaction test. R(+)-Zacopride (but not S(-)zacopride) and PCPA had an anxiolytic profile of action to reduce aversive responding in both species. Their established anxiolytic profiles were abolished by a subsequent treatment with S(-)zacopride. In contrast, S(-)-zacopride was less or ineffective if administered simultaneously with R(+)-zacopride. A co-treatment of PCPA with R(+)-zacopride also inhibited the anxiolytic profiles observed to the individual treatments. It is concluded that there is a complex interaction between the isomers of zacopride to modify responding to an aversive situation that is dependent on the temporal sequence of drug administration, and which may be modified by changes in basal 5-hydroxytryptamine (5-HT) tone and agonist, partial agonist and antagonist effects at the 5-HT3 receptor.


Profiles of interaction of R(+)/S(-)-zacopride and anxiolytic agents in a mouse model.

The mouse black and white test box was used to measure changes in behaviour in an aversive situation where the administration of R(+)-zacopride (but not S(-)-zacopride) alone decreased aversive responding to the white area. A similar anxiolytic profile of action was observed using parachlorophenylalanine (PCPA), whose effects were antagonised by a co-treatment with R(+)-zacopride and reversed by S(-)-zacopride to an exacerbation of the aversive response. An anxiolytic profile of action was also observed using ondansetron, granisetron, chlordiazepoxide, diazepam, ritanserin, 8-OH-DPAT (8-hydroxy-2-(di-n-propylamino)tetralin), E4424 (2-[4-[4-(4-chloro-l-pyrazoyl)butyl]-l-piperazinyl]-pyrimidine), umepsirone, DuP753 (2-n-butyl-4-chloro-5-hydroxy-methyl-1-[2(1H-tetrazol-5-yl) biphenyl-4-yl)methyl)]-imidazole), SQ29,852 ((S)-1-[6-amino-2[hydroxy)(4-phenyl-butyl)phosphinyl]-oxy)-1- nexy]-2-proline), devazepide and guanfacine, and this was retained following co-treatment with PCPA. The anxiolytic profile of action of PCPA was also retained following co-treatment with renzapride which when administered alone failed to modify behaviour. However, the ability of chlordiazepoxide, diazepam, ondansetron and E4424 (but not devazepide, DuP753 or SQ29,852) to reduce aversive responding was inhibited by co-treatment with R(+) and/or S(-)-zacopride. It is concluded that the reduction in aversive responding caused by pharmacological manipulation at the benzodiazepine, 5-HT receptor subtypes 5-HT1A, 5-HT1C/5-HT2 and 5-HT3 (but not at the cholecystokin CCKA or angiotensin receptors or inhibition of angiotensin converting enzyme) can be inhibited by R(+) and S(-)-zacopride. The data is discussed in terms of zacopride having an agonist or partial agonist effect at the 5-HT3 receptor.


NMDA receptor-mediated depolarizing action of proline on CA1 pyramidal cells.

This study investigated the actions of proline on CA1 hippocampal pyramidal cells with use of slice preparations. Bath-applied L-proline first induced these cells to fire multiple orthodromic population spikes in response to a single stimulus and then blocked their response to both orthodromic and antidromic stimulation. These effects could be explained by postsynaptic depolarization followed by depolarization block. Grease-gap studies confirmed that L-proline depolarizes CA1 pyramidal cells. D-Proline was inactive in these tests. Excitatory amino acid antagonists reduced depolarizing responses to proline and N-methyl-D-aspartate (NMDA) in parallel. Mn2+ failed to attenuate proline-evoked depolarizations at concentrations that substantially inhibited synaptic transmission, but at a higher concentration it reduced responses to both proline and NMDA. These results suggest that proline depolarized CA1 pyramidal cells mainly by activating postsynaptic NMDA receptors. The neuroexcitatory and neurotoxic actions of proline in the hippocampus may contribute to the seizures and mental retardation associated with hyperprolinemia.


Effects of cortical ablation on apomorphine- and scopolamine-induced changes in dopamine turnover and ascorbic acid catabolism in the rat striatum.

Levels of dopamine (DA), 3,4-dihydroxyphenylacetic acid (DOPAC), ascorbic acid and dehydroascorbic acid (DHAA) were measured by HPLC in the striatum of rats whose fronto-parietal cortex had been unilaterally ablated after a single injection of apomorphine (1 mg/kg s.c.), scopolamine (0.6 mg/kg s.c.) or L-glutamate (500 mg/kg i.p.). Unilateral cortical ablation decreased striatal levels of glutamate in both striata ipsilateral (35%) and contralateral (17-25%) to the lesion. Apomorphine and scopolamine significantly increased (+94 and +122%, respectively) the DHAA/ascorbic acid ratio in the striata ipsilateral to the lesion in unoperated and sham-operated rats (+72 and +34%, respectively), but both drugs failed to increase it in ablated rats. L-Glutamate significantly increased the DHAA/ascorbic acid ratio in unoperated (+53%) and ablated rats (+37%). The increase in sham-operated rats (+34%) did not reach statistical significance. Apomorphine and scopolamine significantly decreased the DOPAC/DA ratio in the striata ipsilateral to the lesion of unoperated, sham-operated and ablated rats. The decrease in the DOPAC/DA ratio induced by apomorphine and scopolamine was greater in ablated rats than in sham-operated rats. L-Glutamate induced only minor changes in striatal DA and DOPAC levels. We conclude that the apomorphine- and scopolamine-induced increase in ascorbic acid oxidation in the striatum requires intact cortico-striatal glutamatergic pathways. Cortical ablation potentiates the apomorphine- and scopolamine-induced inhibition of striatal DA turnover.


[125I]thienylphencyclidine, a novel ligand for the NMDA receptor.

We have monitored the binding of [125I]thienylphencyclidine ([125I]TCP), a novel high affinity radioiodinated ligand that specifically recognizes the NMDA (N-methyl-D-aspartate) receptor in rat brain membranes. [125I]TCP binds with an affinity of about 30 nM, and recognizes a similar number of binding sites to previously employed ligands for this receptor. [125I]TCP binding is characterized by slow association and dissociation rates, and the latter can be modified by the addition of Mg2+ or Zn2+, as previously described for [3H]dizocilpine ([3H]MK801). Other phencyclidine-like ligands displaced [125I]TCP binding with the order of potency dizocilpine greater than thienylphencyclidine greater than ITCP greater than phencyclidine greater than ketamine. The binding of [125I]TCP was also increased by NMDA and glycine-site agonists and inhibited by antagonists of these sites. Surprisingly, however, the polyamines spermidine and spermine did not increase [125I]TCP, even though the polyamine antagonist arcaine was an effective inhibitor of binding. These results show that [125I]TCP is a useful ligand for the NMDA receptor complex that binds to the receptor in a manner that is qualitatively distinct from previously described ligands.


Demonstration of inhibition of cyclic AMP accumulation in brain by very low concentrations of lithium in the presence of alpha-adrenoceptor blockade.

In the present paper, we have studied the effect of lithium on cAMP levels induced by isoprenaline and norepinephrine in the presence of alpha- or beta-adrenoceptor antagonists. Our results show that low lithium concentrations, starting at 0.3 x 10(-3) M, have a significant inhibitory effect on cAMP content induced by isoprenaline in brain tissue pretreated with the alpha-adrenoceptor blocker phenoxybenzamine. On the other hand, the inhibitory effect of lithium on cAMP levels induced by norepinephrine when beta-adrenoceptors are blocked with propranolol, is observed at concentrations starting at 2.5 x 10(-3) M. These results show that in the presence of alpha blockade, low lithium concentrations which are within the therapeutic plasma range for treatment of manic patients, are able to act on an adenylate cyclase-cAMP system coupled to beta-adrenoceptors.


Developmental changes in the effect of atrial natriuretic peptide on tissue cyclic GMP content and particulate guanylate cyclase activity of aorta, kidney and lung of rats.

To investigate possible developmental changes in the physiological effect of atrial natriuretic peptide (ANP) after birth, we studied the effect of ANP on the slice cGMP content and the particulate guanylate cyclase activity of aorta, kidney and lung in neonate, 2-week-old and adult rats of both sexes. Incubation with human ANP(99-126) (hANP) increased significantly the slice cGMP content of aorta, kidney and lung in three ages of rats. The hANP-stimulated fraction of cGMP contents of kidney decreased, that of lung increased with development, whereas that of aorta showed no significant change. Consistently, the hANP-responsive particulate guanylate cyclase activity decreased in kidney, increased in lung during development, without significant developmental change in aorta. These results indicate a differential change in the effect of hANP on the slice cGMP content among tissues during development. The developmental change in the effect of hANP on slice cGMP content is probably caused by the ontogenetic change in activation of ANP receptor-linked guanylate cyclase.


A toxin (Aga-GI) from the venom of the spider Agelenopsis aperta inhibits the mammalian presynaptic Ca2+ channel coupled to glutamate exocytosis.

Venom of the funnel web spider Agelenopsis aperta was fractionated and screened for activity against the mammalian presynaptic, voltage-dependent Ca2+ channel coupled to glutamate exocytosis. A purified toxin (Aga-GI) from this venom inhibits glutamate exocytosis evoked by elevated potassium or by 4-aminopyridine but is without effect on ionomycin-evoked release. At the same time a partial inhibition of the depolarisation-evoked elevation of cytoplasmic free Ca2+ is seen. The toxin does not inhibit 4-aminopyridine- or potassium-evoked depolarisation, or block Ca(2+)-dependent, potassium-evoked [3H]noradrenaline release. The results indicate that the venom contains a toxin capable of inhibiting the presynaptic voltage-dependent Ca2+ channel coupled to glutamate exocytosis in the mammalian central nervous system. This channel is resistant to block by either omega-conotoxin GVIA or nifedipine. Thus Aga-GI is a novel tool with which to probe this elusive neuronal calcium channel.


The neuroprotective properties of ifenprodil, a novel NMDA receptor antagonist, in neuronal cell culture toxicity studies.

We investigated the effect of ifenprodil on excitotoxic cell death induced by acute exposure to glutamate receptor agonists in primary cultures of foetal mouse cerebral cortex. L-Glutamate and N-methyl-D-aspartate (NMDA) but not kainate and quisqualate-induced toxicity was attenuated in the presence of ifenprodil. In addition, ifenprodil and MK-801 blocked NaCN-induced toxicity. It is concluded that the cerebro-protective properties of ifenprodil in these models are mediated by NMDA receptor antagonism.


Stimulation of cyclic AMP production in human alveolar macrophages induced by inflammatory mediators and beta-sympathicomimetics.

We have investigated the effects of inflammatory mediators and beta-adrenoceptor agonists on the adenylyl cyclase responsiveness in alveolar macrophages from control subjects, patients suffering from chronic obstructive pulmonary disease (COPD) and asthmatics. Basal cyclic AMP (cAMP) levels in alveolar macrophages from COPD patients were significantly elevated (plus 42%) as compared to controls. In addition, the adenylyl cyclase responsiveness to prostaglandin E2, histamine and the beta-adrenoceptor agonist salbutamol was significantly impaired in alveolar macrophages from COPD patients and asthmatics. The lipid mediator platelet activating factor showed no effect on cAMP production in all three alveolar macrophage populations. Furthermore, the cAMP-enhancing effects of isoprenaline, salbutamol and histamine appeared to be mediated via beta 2-adrenoceptors and histamine H2-receptor subtypes respectively. Taken together, these data suggest an intrinsic desensitization phenomenon in alveolar macrophages from COPD patients and asthmatics.


Cyanide selectively augments kainate- but not NMDA-induced release of glutamate and taurine.

The effect of cyanide on the kainate-, quisqualate- and N-methyl-D-aspartate (NMDA)-induced release of several amino acids from cerebellar granule neurons was studied. Cyanide, 100 microM, augmented the kainate- and quisqualate-induced release of glutamate and taurine in neurons but had no effect on the NMDA-induced release of these excitatory amino acids. In addition to the interaction with the above excitatory amino acids, cyanide had effects on several amino acids independent of excitatory amino acid stimulation; cyanide treatment resulted in a significant elevation over saline controls of arginine and taurine, but not alanine, aspartate+asparagine or glycine. With the exception of taurine, this pattern was not apparent in cells treated with any of the above excitatory amino acid.


Surface distribution and internalization of erbB-2 proteins.

We report the localization over the cell surface and the early steps of antibody-induced internalization of the product of the erbB-2 proto-oncogene, structurally related to the epidermal growth factor receptor (EGFR). We show that erbB-2/p 185 is mostly excluded from endocytic pits on the cell surface. Incubation at 37 degrees C with an anti-erbB-2/p185 monoclonal antibody induces the rapid entry of the protein into the cell. Similar internalization is shown by a chimeric molecule EGFR/erbB-2 in response to EGF. Both the timing and the pathway of internalization followed by the erbB-2/p185 appear totally similar to those described for the EGFR. At variance with the normal erbB-2/p185, two mutant activated erbB-2 proteins are frequently localized within endocytic pits of the cell surface, indicating that mutations in the transmembrane regions may determine constitutive internalization of the protein.


Regulation of transglutaminase 1 gene expression by 12-O-tetradecanoylphorbol-13-acetate, dexamethasone, and retinoic acid in cultured human keratinocytes.

Transglutaminase 1 (TG1) is an enzyme that is expressed at the late stage of terminal differentiation of keratinocytes and catalyzes the epsilon-(gamma-glutamyl)lysine cross-linking reaction to form a highly insoluble cell envelope. To elucidate the mechanism of TG1 gene expression in keratinocytes, we examined the effects of 12-O-tetradecanoylphorbol-13-acetate (TPA), dexamethasone, 1,25-dihydroxyvitamin D3, and retinoic acid on the levels of TG1 mRNA in cultured normal human epidermal keratinocytes (NHEK). Treatment of NHEK with TPA, up to 10 nM, markedly increased the levels of TG1 mRNA in a dose-dependent manner. The effect by treatment with 1 nM TPA reached a peak after 16 h of incubation (20-fold above the basal level). In contrast, phorbol had no effect on TG1 gene expression. The induction of TG1 mRNA expression by TPA was inhibited by 1-(5-isoquinolinylsulfonyl)-2-methyl-piperazine (H-7) and staurosporine. Dexamethasone at a concentration of 1 microM also increased the TG1 mRNA levels, but the maximum induction was observed (3-fold above the basal level) after 72 h of incubation. The effect of dexamethasone was not suppressed by H-7. Moreover, 1 microM of retinoic acid completely inhibited the induction of TG1 mRNA by both TPA and dexamethasone. 1,25-Dihydroxyvitamin D3 showed no effect on the TG1 mRNA levels. From these results, we suggest that the expression of TG1 gene may be upregulated by protein kinase C and glucocorticoid receptor systems and down-regulated by the retinoic acid receptor system.


Intracellular distribution of a nuclear localization signal binding protein.

The transport of proteins into the nucleus requires the recognition of a nuclear localization signal sequence. Several proteins that interact with these sequences have been identified, including one of about 66 kDa. We have prepared antibodies that recognize the 66-kDa nuclear localization signal binding protein (NLSBP) and inhibit nuclear localization in vitro. By immunofluorescence, it is seen that the NLSBP is predominantly cytoplasmic and is distributed peripherally around the nucleus and the microtubule organizing center. There is also a weak punctate staining of the surface of the nucleus. Methanol-fixed cells can also be stained directly with fluorescently labeled karyophilic proteins. These stains reveal the same cytoplasmic structures as anti-NLSBP. The expression of the NLSBP is growth dependent. When cells grown to confluence are examined, the cytoplasmic staining is greatly reduced, leaving the punctate nuclear staining as the predominant feature. In serum-starved cells, very little staining of either the cytoplasm or the nucleus can be seen. Upon simulation by the addition of serum, the original cytoplasmic and nuclear envelope staining is restored. Cells grown in the presence of colchicine or taxol have an altered NLSBP distribution but apparently normal cytoplasmic nuclear transport.


Phylogenetic and epidemiological analysis of Neisseria meningitidis using DNA probes.

The genetic relationships between various serotypes and serogroups of meningococcal strains were investigated by restriction fragment-length polymorphism (RFLP) analysis using a number of random DNA probes and a probe containing a truncated copy of the meningococcal insertion sequence IS1106. The data were used to estimate genetic distance between all pairs of strains and to construct phylogenetic trees for meningococcal strains. B15:P1.16R strains isolated from cases of systemic meningococcal disease in two health districts with a high incidence of disease were clonal in contrast to similar strains from cases occurring in other parts of the UK. Strains from these areas, which contain a similar genomic deletion, were found to be derived from two distinct lineages within the B15:P1.16R phylogenetic group. RFLP data demonstrated that present serological typing systems for the meningococcus do not necessarily reflect true genetic relationships.


Molecular epidemiology of Shigella infections in Israel.

The DNAs of Shigella sonnei or Shigella dysenteriae type 1 strains isolated in outbreaks of shigellosis or in sporadic cases were analysed by restriction fragment length polymorphism (RFLP). Southern blots of the DNAs of 36 S. sonnei isolates digested by 8 restriction enzymes were hybridized with an Escherichia coli rRNA probe. The S. sonnei strains were unexpectedly diverse in their RFLP. Antibiotypes of the same isolates showed clusters of strains corresponding to the various outbreaks. On the other hand, RFLP analysis suggested concomitant multiple sources of infection rather than a common source and thereby introduced a new insight in the epidemiology of shigellosis. RFLP was also used to trace S. dysenteriae type 1 transmission in a recent cluster of clinical cases. Although antibiotic resistance patterns indicated the presence of more than one strain, RFLP analysis showed that the six isolates were identical clones and suggested the loss of an R episome after one person-to-person passage.


Antifungal properties of taxol and various analogues.

The antimitotic agent taxol was tested for toxicity towards fungi from different taxonomic groups and found to be particularly active against oomycete fungi. In germinating zoospore cysts of the oomycete Phytophthora capsici the mechanism of action of taxol was shown to involve inhibition of mitosis, presumably resulting from an effect on microtubules. Various taxol analogues with deleted A-ring C-13 side chain substituents were tested for toxicity towards P. capsici and Aphanomyces cochlioides to provide insight into structural features required for activity. The importance of the side chain was shown by the much lower activity as compared to taxol of analogues lacking all or part of the side chain. The effect of stereochemistry at the C-2' position on fungitoxicity towards oomycetes was similar to that reported previously on mammalian microtubule assembly.


Control of the cardiovascular system of Aplysia by identified neurons.

The neural network that controls the cardiovascular system of Aplysia adapts cardiovascular function to a variety of different physiological and behavioral situations. It (1) coordinates the cardiovascular system with the renal and respiratory systems; (2) modifies both systemic and regional blood flow during food-elicited arousal and feeding; and (3) changes the tension of longitudinal vascular muscle to adapt the arterial tree to changes in body shape. Indirect evidence suggests that the cardiovascular control circuit may also play a role in maintaining homeostasis during egg laying. Several putative neurotransmitters, including acetylcholine, serotonin, R15 alpha 1 and R15 alpha 2 peptides, have been localized to identified neurons in this circuit.


Aberrations in cerebral vascular functions due to Plasmodium yoelii nigeriensis infection in mice.

Plasmodium yoelii nigeriensis infection in mice caused an increase in uptake of 125I-labeled bovine serum albumin, 51Cr-labeled erythrocytes and Evans blue dye from peripheral circulation into the brain. Isolated cerebral microvessels which were characterized in terms of their morphology under scanning electron microscope and enhancement of the specific activities of biochemical markers, viz. alkaline phosphatase, gamma-glutamyl transpeptidase, and monoamine oxidase, showed significant decrease in these activities due to P. yoelii nigeriensis infection. On the other hand, relatively minor (statistically insignificant) changes occurred in the first two enzyme specific activities in the cerebral cortex and monoamine oxidase registered an increase in this tissue due to infection. Histological examination of the cerebral tissue of infected animals by light and electron microscopy showed broken blood vessel walls and leakage of erythrocytes into extravascular space, some of which contained intraerythrocytic malarial parasite in a state of cell division.


The secretion of aspartyl proteinase, a virulence enzyme, by isolates of Candida albicans from the oral cavity of HIV-infected subjects.

Prevalence, serotype and in vitro secretion of aspartyl proteinase, a virulence enzyme, were studied in Candida isolates from the oral cavity of 337 HIV-infected subjects. Controls were 95 age-sex-matched HIV- (seronegative) subjects, belonging to either HIV-risk categories (47) or to the normal, general population (48). Fungi were isolated from 155 HIV+ subjects. C. albicans was the most prevalent species (85.8% of all isolates). 94.6% of C. albicans isolates were serotype A and all were agglutinated by a monoclonal antibody (AF1) directed against a major mannoprotein immunogen of the candidal cell wall, confirming previous results with C. albicans isolates from non-immunodeficient subjects. With regard to the stage of HIV infection, there were no statistically significant differences in the incidence of oral Candida carriage between asymptomatic (stage II) HIV+ and HIV- subjects, and between stage II and lymphadenopathic (stage III) individuals. Also, the low (3.8%) incidence of oral candidiasis in the subjects of the latter stage was insignificant with respect to stage II subjects. However, the incidence of C. albicans in stage IV (AIDS) subjects (46.8%) was significantly higher than in all other subjects, and in almost all cases, fungal isolation was accompanied by oral thrush and lower CD4+ lymphocyte counts (less than 400 x 10(6)/L). All isolates of C. albicans were proteolytic in vitro, as assessed by scoring the proteinase activity on BSA agar and monitoring the secreted proteinase antigen by a highly sensitive (1 ng) and specific immunoenzymatic assay.(ABSTRACT TRUNCATED AT 250 WORDS)