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Biomedical subjects

P Skolnick

Publications and source records attributed to P Skolnick.

At least 199 records · Page 11Linked to original sources
PubMed1988

Tissue specific regulation of "peripheral-type" benzodiazepine receptor density after chemical sympathectomy.

The characteristics of [3H]Ro 5-4864 binding to "peripheral" benzodiazepine receptors (PBR) in the central nervous system and peripheral tissues were examined after chemical sympathectomy with 6-hydroxydopamine (6-OHDA). One week after the intracisternal administration of 6-OHDA, the number of [3H]Ro 5-4864 binding sites (Bmax) in the hypothalamus and striatum increased 41 and 50%, respectively, concurrent with significant reductions in catecholamine content. An increase (34%) in the Bmax of [3H]Ro 5-4864 to cardiac ventricle was observed one week after parenteral 6-OHDA administration. In contrast, the Bmax of [3H]Ro 5-4864 to pineal gland decreased 48% after 6-OHDA induced reduction in norepinephrine content. The Bmax values for [3H]Ro 5-4864 binding to other tissues (including lung, kidney, spleen, cerebral cortex, cerebellum, hippocampus and olfactory bulbs) were unaffected by 6-OHDA administration. The density of pineal, but not cardiac PBR was also reduced after reserpine treatment, an effect reversed by isoproterenol administration. These findings demonstrate that alterations in sympathetic input may regulate the density of PBR in both the central nervous system and periphery in a tissue specific fashion.

Animals↗
PubMed1988

Asymmetrical activation of GABA-gated chloride channels in cerebral cortex.

A pronounced left/right asymmetry of GABA-gated chloride channels was observed in rat cerebral cortex. This asymmetry was manifest as a higher apparent affinity and density of binding sites for the "cage" convulsant [35S]t-butylbicyclophosphorothionate (TBPS) in the right compared to left cortex. Asymmetries in [35S]TBPS binding were not observed in other brain areas, and were restricted to the occipital and entorhinal/pyriform areas of cerebral cortex. Evaluation of other components of the benzodiazepine/GABA receptor chloride ionophore complex in cerebral cortex suggests that this asymmetry is not present in either benzodiazepine receptors or that population of GABA receptors linked to benzodiazepine receptors. Brief restraint-stress significantly increased both the apparent affinity and number of [35S]TBPS binding sites in cortex, but the left/right asymmetry was no longer apparent. These findings indicate a time-dependent, asymmetric response of cortical GABA-gated chloride channels to stress rather than an anatomical hemispheric difference, and suggest that GABA-gated chloride channels may be involved in a differential processing of stressful stimuli by the cerebral hemispheres.

Animals↗
PubMed1988

Characterization of stress-induced alterations in [3H] Ro5-4864 binding to peripheral benzodiazepine receptors in rat heart and kidney.

Inescapable tailshock has been shown to elicit a tissue specific decrease in the density of peripheral benzodiazepine receptors (PBR). We have now explored possible mechanisms that may be responsible for this phenomenon. An 80 minute session of inescapable tailshock produced a reduction in the binding of [3H] Ro5-4864 to renal membranes at 0, 1 and 2 hr after stress, with values returning to control (naive) levels within 24 hr. In cardiac membranes, statistically significant reductions were observed only at 2 and 4 hr after stress. The role of the pituitary-adrenal axis and the sympathetic nervous system in this phenomenon was assessed by subjecting adrenalectomized, hypophysectomized, 6-OHDA-treated or control (sham-operated or saline-treated) rats to inescapable shock. Neither adrenalectomy, hypophysectomy, nor 6-OHDA pretreatment altered the stress-induced reduction in renal PBR. However, the stress-induced decrease in renal PBR was blocked by pretreatment with clonazepam (1 mg/kg), a potent anxiolytic with low affinity for PBR.

Adrenalectomy↗
PubMed1988

Synthesis of novel 3-substituted beta-carbolines as benzodiazepine receptor ligands: probing the benzodiazepine receptor pharmacophore.

The 3-substituted beta-carbolines 2-4 and 5-7 were prepared from 3-amino-beta-carboline (8) in one step via diazotization, followed by reaction with the appropriate nucleophile in order to determine their binding affinity for benzodiazepine receptors (BzR). All three of the 3-alkoxy-beta-carbolines 2 (IC50 = 124 nM), 3 (IC50 = 24 nM), and 4 (IC50 = 11 nM) have high affinities for BzR. The beta-carbolines substituted with electron-withdrawing groups including 5 (Cl; IC50 = 45 nM), 6 (NO2; IC50 = 125 nM), and 7 (N = C = S; IC50 = 8 nM) also had high affinities for BzR. The affinities of 5-8 clearly indicate that a carbonyl moiety at position 3 of a beta-carboline is not required for high-affinity binding to BzR. These findings have led to the development of a model for the binding of ligands to an inverse agonist domain at BzR. This model is supported by the recent synthesis of 3-ethoxy-beta-carboline (3), a potent, long-lived partial inverse agonist, and 7, an irreversible BzR ligand.

Animals↗
PubMed1988

Differences in the biophysical properties of the benzodiazepine/gamma-aminobutyric acid receptor chloride channel complex in the long-sleep and short-sleep mouse lines.

Significant differences in the thermal stability of benzodiazepine receptors were found in cerebral cortical membranes prepared from the long-sleep (LS) and short-sleep (SS) selected mouse lines. Thus, benzodiazepine receptors from LS mice were heat inactivated (55 degrees C) at a significantly faster rate than those from SS mice. Although gamma-aminobutyric acid (GABA) reduced the rate of heat inactivation in both lines, the more rapid rate of inactivation in the LS line was maintained. Furthermore, the potency of GABA to enhance [3H]flunitrazepam binding decreased threefold in membranes from LS mice as the incubation temperature was increased from 0 degrees to 37 degrees C, but was unaltered in membranes from SS mice. These differences in the biophysical properties of the benzodiazepine/GABA receptor chloride channel complex ("supramolecular complex"), together with a higher KD for t-[35S]butylbicyclophosphorothionate in membranes from LS compared to SS mice, suggest that the supramolecular complex may modulate the differential sensitivity to some depressants and convulsants in these lines.

Animals↗
PubMed1988

Modulation of the benzodiazepine/gamma-aminobutyric acid receptor chloride channel complex by inhalation anesthetics.

Inhalation anesthetics, such as diethyl ether, halothane, and enflurane, increase 36Cl- uptake into rat cerebral cortical synaptoneurosomes in a concentration-dependent, picrotoxin-sensitive fashion. At concentrations consistent with those that stimulate 36Cl- uptake, inhalation anesthetics also inhibit the binding of t-[35S]butylbicyclophosphorothionate ([35S]TBPS) to well-washed cortical membranes. Scatchard analysis of [35S]TBPS binding indicates that these agents reduce the apparent affinity of this radioligand and have little effect on the Bmax. The ability of inhalation anesthetics to directly stimulate 36Cl- uptake and inhibit [35S]TBPS binding is a property shared by nonvolatile anesthetics. Nonetheless, there are differences between nonvolatile agents (such as barbiturates and alcohols) and inhalation anesthetics, because the former compounds augment muscimol (a GABAmimetic) stimulated 36Cl- uptake, whereas the latter group (such as ether and enflurane) inhibit this effect. These findings demonstrate that therapeutically relevant concentrations of inhalation anesthetics perturb the benzodiazepine/gamma-aminobutyric acid receptor chloride channel complex, and suggest this oligomeric protein may be a common mediator of some aspects of anesthetic action.

Anesthesia, Inhalation↗
PubMed1988

Regulation of gamma-aminobutyric acid/barbiturate receptor-gated chloride ion flux in brain vesicles by phospholipase A2: possible role of oxygen radicals.

Preincubation of brain membranes with phospholipase A2 (PLA2) has been shown previously to affect the binding characteristics of various recognition sites associated with the gamma-aminobutyric acid (GABA) receptor complex. In the present study, we have investigated the effects of PLA2 (from Naja naja siamensis venom) on the functional activity of the GABA receptor/chloride ion channel. PLA2 (0.001-0.02 U/mg protein) preincubation decreased pentobarbital-induced 36Cl- efflux and muscimol-induced 36Cl- uptake in rat cerebral cortical synaptoneurosomes. The effect of PLA2 was prevented by EGTA and two nonselective PLA2 inhibitors, mepacrine and bromophenacyl bromide. The removal of free fatty acids by addition of bovine serum albumin both prevented and reversed the effect of PLA2. Products of the catalytic activity of PLA2, such as the unsaturated free fatty acids, arachidonic and oleic acids, mimicked the effect of PLA2. However, the saturated fatty acid, palmitic acid, and lysophosphatidyl choline had no effect on pentobarbital-induced 36Cl- efflux. Because unsaturated free fatty acids are highly susceptible to peroxidation by oxygen radicals, the role of oxygen radicals was investigated. Xanthine plus xanthine oxidase, a superoxide radical generating system, mimicked the effect of PLA2, whereas the superoxide radical scavenger, superoxide dismutase, diminished the effects of PLA2 and arachidonic acid on pentobarbital-induced 36Cl- efflux. Similarly, the effect of PLA2 was also inhibited by methanol (1 mM), a scavenger of the hydroxyl radical, and by catalase. These data indicate that exogenously added PLA2 induces alterations in membrane phospholipids, possibly promoting the generation of oxygen radicals and fatty acid peroxides which can ultimately modulate GABA/barbiturate receptor function in brain.

Acetophenones↗
PubMed1988

Enhancement of t-[35S]butylbicyclophosphorothionate and [3H]strychnine binding by monovalent anions reveals similarities between gamma-aminobutyric acid- and glycine-gated chloride channels.

The characteristics of [3H]strychnine and t-[35S]-butylbicyclophosphorothionate ([35S]TBPS) binding to sites associated with glycine- and gamma-aminobutyric acid (GABA)-gated chloride channels were compared in the presence of a series of anions with known permeabilities through these channels. Good correlations were found between (a) the potencies (EC50) of these anions to stimulate radioligand binding and their permeabilities relative to chloride; (b) the affinities (KD) of these radioligands in the presence of fixed concentrations of these anions and their relative permeabilities; (c) the potencies (EC50) of these anions to stimulate [35S]TBPS and [3H]strychnine binding; and (d) the affinities (KD) of [3H]strychnine and [35S]TBPS measured at a fixed concentration of these anions. These studies support electrophysiological and biochemical observations demonstrating similarities between glycine- and GABA-gated chloride channels, and suggest that anions enhance [3H]strychnine and [35S]TBPS binding through specific anion binding sites located at the channels.

Animals↗
PubMed1988

Anion regulation of agonist and inverse agonist binding to benzodiazepine receptors.

Binding of the benzodiazepine inverse agonist [3H]methyl-6,7-dimethoxy-4-ethyl-beta-carboline-3-carboxylate [( 3H]DMCM) and the agonist [3H]flunitrazepam [( 3H]FNZ) was compared in rat cortical membranes. Halide ions enhanced [3H]DMCM binding three- to fourfold, increasing both the apparent affinity and the number of binding sites for this radioligand. The effect was present at both 0 and 37 degrees C. In contrast, the magnitude of halide stimulation of [3H]FNZ binding was much smaller, resulting solely from an increase in the apparent affinity for this radioligand, and was not observed at 37 degrees C. The potencies but not the efficacies of a series of anions to stimulate both [3H]DMCM and [3H]FNZ binding to benzodiazepine receptors were highly correlated with their relative permeabilities through gamma-aminobutyric acid (GABA)-gated chloride channels. Two stress paradigms (10 min of immobilization or ambient-temperature swim stress), previously shown to increase significantly the magnitude of halide-stimulated [3H]FNZ binding, did not significantly affect [3H]DMCM binding. Phospholipase A2 treatment of cortical membrane preparations was equipotent in preventing the stimulatory effect of chloride on both [3H]DMCM and [3H]FNZ binding. These data strongly suggest that anions modify the binding of [3H]DMCM and [3H]FNZ by acting at a common anion binding site that is an integral component of the GABA/benzodiazepine receptor chloride channel complex.

Animals↗
PubMed1988

Anion regulation of [3H]strychnine binding to glycine-gated chloride channels is explained by the presence of two anion binding sites.

The effects of six monovalent anions (chloride, bromide, iodide, nitrate, perchlorate, and thiocyanate) on [3H]strychnine binding to glycine-gated chloride channels were examined. These anions have previously been shown to permeate glycine-gated chloride channels and stimulate [3H]strychnine binding. Whereas low concentrations (10-200 mM) of all these anions enhanced [3H]strychnine binding, higher concentrations (0.2-3 M) of thiocyanate, perchlorate, and iodide produced a robust inhibition of radioligand binding, and a more modest inhibition was observed with the same concentrations of nitrate and bromide. The presence of one binding site for anions at glycine-gated chloride channels can account for either the activation or the inhibition phase, but not both. However, these biphasic effects can be explained by the presence of two binding sites for anions at these channels. Two models with two anion binding sites were considered, the first assuming both allosteric activation and inhibition of the binding of the ligand, and the other explained by allosteric activation combined with competitive inhibition. Mathematical expressions for both models were formulated, and the equations obtained yielded satisfactory fitting to the results obtained with all anions tested in both concentration-response and saturation experiments. These equations also permitted the calculation of several parameters describing the interaction of the anions with these channels. The main difference in the behavior of these anions relates to the extent to which they produce activation of [3H]strychnine binding and to their cooperative interaction at the two putative anion binding sites. Thus, a strong negative cooperativity was observed for the simultaneous binding of two molecules of chloride, bromide, or nitrate, but not for the simultaneous binding of thiocyanate, perchlorate, or iodide. This latter property may be related to the conductance of these anions through glycine-gated chloride channels.

Allosteric Regulation↗
PubMed1988

Picrate and niflumate block anion modulation of radioligand binding to the gamma-aminobutyric acid/benzodiazepine receptor complex.

The organic anions picrate (2,4,6-trinitrophenol) and niflumate (2-[[3-(trifluoromethyl)phenyl]-amino]-3-pyridinecarboxylate) were examined for their effects on radioligand binding to the gamma-aminobutyric acid (GABA)/benzodiazepine receptor complex. Neither organic anion produced the enhancement of [35S] t-butylbicyclophosphorothionate (TBPS) binding characteristic of anions (such as Cl- and Br-) known to permeate GABA-gated chloride channels. However, both picrate and niflumate potently (IC50 values between 66 and 531 and 30 and 155 microM, respectively) inhibited the effect of 100-200 mM concentrations of anions (I-, Br-, Cl-, SCN-, and F-) to increase the binding of [35S]TBPS to GABA-gated chloride channels. This inhibition resulted from a decrease in both the maximum number of binding sites and the apparent affinity (increased Kd) of [35S]TBPS. Niflumate was consistently more potent than picrate, but both organic anions exhibited the same sequence of relative potencies against smaller anions (I- greater than Br- greater than Cl- greater than SCN- greater than F-). This sequence was similar to that described for the relative permeabilities of these anions through GABA-gated chloride channels. Niflumate and picrate were potent inhibitors of Cl-, but not GABA-modulated radioligand binding to benzodiazepine receptors. These findings suggest that picrate and niflumate bind with high affinity at or near an anion binding site that may regulate the movement of anions through GABA-gated chloride channels and radioligand binding at this "supramolecular complex."

Animals↗
PubMed1988

Differential responsiveness of cerebellar Purkinje neurons to GABA and benzodiazepine receptor ligands in an animal model of hepatic encephalopathy.

The role of the GABA-benzodiazepine receptor complex in the pathogenesis of hepatic encephalopathy was investigated by recording the electrophysiological responses of single cerebellar Purkinje neurons from rabbits with hepatic encephalopathy due to galactosamine-induced fulminant hepatic failure. Both the GABAmimetic muscimol and the benzodiazepine receptor agonist flunitrazepam were 3-4 times more potent in depressing the spontaneous activity of Purkinje neurons from rabbits with hepatic encephalopathy than from control animals. Furthermore, qualitatively different responses of Purkinje neurons to benzodiazepine receptor antagonists (Ro 15-1788 and Ro 14-7437) were found in controls and rabbits with hepatic encephalopathy. These compounds markedly excited Purkinje neurons from rabbits with hepatic encephalopathy, but had either no effect (Ro 14-7437) or partially suppressed (Ro 15-1788) the spontaneous activity of neurons from control animals. In addition, incubation of Purkinje neurons from rabbits with hepatic encephalopathy with subthreshold concentrations of Ro 14-7437 reduced their sensitivity to muscimol, whereas treatment of control neurons with Ro 14-7437 had no effect on their sensitivity to muscimol. Finally, Purkinje neurons from hepatic encephalopathy and control rabbits displayed no difference in sensitivity to the depressant actions of the alpha-adrenoceptor agonist phenylephrine. These findings demonstrate a differential responsiveness of Purkinje neurons from an animal model of hepatic encephalopathy to ligands that interact with the GABA-benzodiazepine receptor complex. Furthermore, the observations made in this experimental model are consistent with the involvement of the GABA-benzodiazepine receptor complex in mediating hepatic encephalopathy, and provide a potential explanation for the reported efficacy of benzodiazepine receptor antagonists in ameliorating this syndrome.

Animals↗
PubMed1987

Acute stress enhances the activity of the GABA receptor-gated chloride ion channel in brain.

The effect of acute swim stress on the functional activity of the gamma-aminobutyric acid (GABA) receptor/chloride ion channel was studied using an assay to measure 36chloride (36Cl-) uptake into rat brain synaptoneurosomes. Muscimol-stimulated 36Cl- uptake in cerebral cortex, hippocampus, but not cerebellum, was enhanced (increased potency and efficacy) in rats subjected to 10 min of swimming, compared to non-stressed, control rats. The effect of swim stress on the activity of the GABA receptor/Cl ion channel was prevented by adrenalectomy.

Adrenalectomy↗
PubMed1987

Ontogeny of susceptibility to the convulsant, Ro 5-4864, and its relationship to audiogenic seizure susceptibility in inbred mice.

The postnatal development of susceptibility to the convulsant effects of Ro5-4864 (4'-chlorodiazepam) was characterized in two inbred mouse strains (DBA/2J and BALB/c ByJ) which as adults differ markedly in their response to this convulsant. Onset of susceptibility to a dose of Ro5-4864 which caused a high frequency of clonic seizures in adults was observed at 10 days of age in DBA/2 mice, but not until 35 days in BALB/c By mice. At 14 days of age an abrupt increase in susceptibility to Ro5-4864-induced tonic seizures was found in DBA/2 but not BALB/c By mice. Both the peak of tonic seizure susceptibility (21 days) and the time course of its subsequent age-dependent decline closely paralleled the change in audiogenic seizure susceptibility in the DBA/2 strain. PK11195 (40 mg/kg) blocked Ro5-4864 (25 mg/kg)-induced, age-dependent tonic seizures but had no effect on clonic seizure induction in the same mice. These observations establish that both the susceptibility to Ro5-4864 in adult mice and the postnatal time course for development of susceptibility to this convulsant are inherently different in these two strains of mice. The lack of coincidence between the developmental onset of susceptibility to Ro5-4864-induced seizures and the onset of supersensitivity to Ro5-4864-induced tonic seizures during the period of peak audiogenic seizure susceptibility in DBA/2 mice implies that more than one neurochemical mechanism is involved in the ability of Ro5-4864 to induce seizures in this strain. However, the blockade of Ro5-4864-induced tonic seizures by PK11195 suggests that peripheral type benzodiazepine receptors may mediate this effect.

Acoustic Stimulation↗
PubMed1987

Heterogeneity of beta-adrenoreceptors in guinea pig alveolar type II cells.

[3H]Dihydroalprenolol ([3H]DHA) binding to guinea pig alveolar type II cell membrane revealed the presence of both high (KD = 0.38 nM) and low (KD = 4.2 nM) affinity beta-adrenoreceptors. The low affinity site had a higher binding capacity (Bmax = 245.6 fmol/mg protein) than the high affinity site (Bmax = 71.7 fmol/mg protein). Displacement of [3H]DHA by practolol, a selective beta 1 agent, confirmed the existence of two species of adrenoreceptors, corresponding to 21% high affinity (beta 1) and 79% low affinity (beta 2), respectively.

Animals↗
PubMed1987

'Peripheral-type' benzodiazepine receptors in the kidney: regulation of radioligand binding by anions and DIDS.

The high density of 'peripheral-type' benzodiazepine receptors (PBR) in the thick ascending loop of Henle (TAL) of the kidney prompted an evaluation of the effects of anions and anion channel blockers on these sites. [3H]Ro 5-4864 binding to rat kidney membranes was inhibited by halides in a concentration dependent fashion. Significant differences were observed in the efficacies (i.e. maximal effect achieved) of these halides to inhibit [3H]Ro 5-4864 binding. The effects of halides to inhibit this binding was manifest as a reduction in the apparent affinity of this radioligand with no effect on the maximum number of binding sites. The concentration necessary to achieve half maximal inhibition was around the physiologic range for Cl- in all extracellular body fluids. A strong correlation (r = 0.95; P less than 0.001) was obtained between the permeability of anions relative to Cl- and their efficacies to inhibit [3H]Ro 5-4864 binding. DIDS (4,4'-diisothiocyanostilbene-2,2'-disulfonic acid), an ion transport blocker, inhibited [3H]Ro 5-4864 binding to a maximum of 80%. The presence of Na2SO4 enhanced the potency of DIDS up to five-fold giving an IC50 of 43 +/- 6 microM. In contrast, the binding of [3H]PK 11195 was unaffected by DIDS and only slightly inhibited by I-. These data demonstrate that characteristics of radioligand binding to peripheral-type benzodiazepine receptors in the kidney are regulated by anions and anion transport blockers and suggest that the PBR may play an important role in anion transport. Additionally, these data suggest that the sites at which [3H]Ro 5-4864 and [3H]PK 11195 bind on the PBR may be overlapping, but not identical.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1987

Regulation of dihydropyridine calcium antagonist binding sites in the rat hippocampus following neurochemical lesions.

The effects of catecholaminergic, cholinergic, serotonergic, and glutaminergic terminal destruction and neurotransmitter depletion on [3H]nitrendipine binding to rat brain membranes were determined using the neurotoxins 6-hydroxydopamine, 5,7-dihydroxytryptamine, and kainic acid and the neurotransmitter-depleting agent reserpine. Following intracisternal injection of 6-hydroxydopamine there were time-dependent increases (14-23%) in the density but not change in the affinity of hippocampal [3H]nitrendipine binding sites. 6-Hydroxydopamine significantly increased [3H]nitrendipine binding in the hippocampus 4 and 10 days following injection. However, no significant change in binding was observed at 16 and 26 days. [3H]Nitrendipine binding in the cerebral cortex, striatum, cerebellum, and brain stem was unaffected by 6-hydroxydopamine. Neither 5,7-dihydroxytryptamine nor kainic acid affected [3H]nitrendipine binding in the hippocampus and cerebral cortex. Acute and chronic reserpinization also did not affect [3H]nitrendipine binding in the hippocampus and cerebral cortex. These results indicate that dihydropyridine calcium antagonist bindings sites in rat brain are subject to brain region-specific regulation following neurochemical lesions and may be present in their largest densities on postsynaptic membranes.

Adrenergic Fibers↗