Pharmacology of neurosteroid biosynthesis. Role of the mitochondrial DBI receptor (MDR) complex.
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Animals↗
Biomedical subjects
D R Grayson
Publications and source records attributed to D R Grayson.
Changes in gamma-aminobutyrate type A receptor subunit mRNAs, translation product expression, and receptor function during neuronal maturation in vitro.
The amounts of mRNAs encoding alpha 1, alpha 6, beta 2, beta 3, gamma 2, and delta subunits of gamma-aminobutyrate type A (GABAA) receptors and the gold immunolabeling density of their translation products were monitored during the growth of neonatal rat granule cells in primary culture. We investigated possible correlations (i) between temporal changes in mRNA content and expression density of their respective translation products and (ii) between the quantitative changes of receptor subunit expression, the GABA EC50 for Cl- channel activation, and diazepam efficacy in modulating GABA action on the Cl- channels. At 3 days in vitro, the amount of GABAA receptor subunit mRNAs and the expression of their respective translation products were very low. During the next 2 weeks both parameters for every subunit studied increased asynchronously; moreover, at 14 days in vitro the sum of gamma 2 and delta subunit expression was smaller than the expression of the alpha 1 or alpha 6 or beta 2/beta 3 subunits. This suggests that during in vitro maturation each subunit may be regulated independently and invites speculation as to possible changes in specific GABAA receptor subtype abundance during development in vitro. The maximal current intensity elicited by GABA failed to increase from 5 to 14 days in vitro, though the amount of mRNA encoding various subunits and the expression density of their respective translation products increased. Thus, qualitative changes in the GABAA receptor subtypes expressed and/or abnormalities in the subunit assembly very likely account for the uniformity of the maximal current intensity elicited by GABA during in vitro development. Also, during maturation of neuronal cultures from 5 to 20 days in vitro the extent of the positive modulation of GABA action by diazepam decreased dramatically. This finding might be related to an increase in the abundance of GABAA receptors including the alpha 6 subunit and/or to the expression, during granule cell maturation in vitro, of GABAA receptors devoid of gamma 2 subunits.
Animals↗
Acute and long-term inhibition of agonist-stimulated phosphoinositide hydrolysis by pulse treatment of cerebellar granule cells with TPA.
Acute pretreatment (30 min) of primary cultures of cerebellar granule cells with TPA (10 nM) resulted in a decrease in carbachol-and glutamate-stimulated phosphoinositide hydrolysis, but not in basal levels of PI hydrolysis. To investigate the mechanism of TPA action, phospholipase C was assayed in membranes prepared from cerebellar granule cells acutely treated with TPA. TPA had no effect on basal, GTP gamma S-, NaF-, and calcium-stimulated phospholipase C when compared with membranes prepared from vehicle-treated cells. The effects of pulsing with TPA (30-min pulse, 10 nM) on agonist-stimulated PI hydrolysis were studied 1, 3, and 5 or 6 d after TPA treatment. TPA treatment results in a statistically significant decrease in glutamate-stimulated PI hydrolysis, and a slight reduction of carbachol-stimulated PI hydrolysis when compared to temporally matched controls. Measurements in membranes prepared from TPA-treated vs control cells 1, 3, and 5 d after treatment showed that calcium- and NaF-stimulated phospholipase C activity was significantly decreased at all days tested, whereas GTP gamma S-stimulated phospholipase C activity was significantly decreased only at d 3. These data demonstrate differences in the acute vs long-term effects of TPA treatment on agonist-stimulated PH hydrolysis, and suggest that the acute effects may be mediated at the level of the receptor, whereas long-term effects of TPA on PI hydrolysis may be mediated by deficits in effector function.
Animals↗
Temporal and depolarization-induced changes in the absolute amounts of mRNAs encoding metabotropic glutamate receptors in cerebellar granule neurons in vitro.
Cerebellar granule neurons in primary culture express metabotropic glutamate receptors (mGluRs) coupled to the stimulation of phosphoinositide hydrolysis and to the inhibition of cyclic AMP (cAMP) formation. To evaluate which mGluR mRNAs are expressed in granule neurons under different depolarizing conditions, we measured the absolute amounts of selected receptor mRNAs in neurons cultured for 3-13 days in the presence of either 10 or 25 mM KCl. mGluR-specific primer pairs and internal standards, corresponding to unique regions of mGluR1a, mGluR2, mGluR3, mGluR4, and mGluR5, were constructed and used in a competitive PCR-derived assay to quantify the corresponding mRNA levels. For phosphoinositide-coupled receptors, the absolute content of mGluR1a mRNA was three to 10 times higher than the content of mGluR5 mRNA. The expression of mGluR5 mRNA increased up to 9 days in vitro and was much higher in 10 mM than in 25 mM KCl. For the cAMP-coupled receptors, there was a large amount of mGluR4 mRNA and a much smaller content of the mGluR3 and mGluR2 mRNAs. Maintaining the granule neurons in vitro in 10 mM KCl increased the absolute amount of mRNAs encoding mGluR2 and mGluR4 at 9 and 13 days in vitro. In contrast, the content of the mGluR3 mRNA was consistently higher in neurons cultured in 25 mM KCl. These data are consistent with the possibility that in primary cultures of cerebellar neurons, phosphoinositide responses may be predominantly mediated by mGluR1a, rather than mGluR5, and that cAMP inhibition involves preferentially mGluR4 and mGluR3.
Animals↗
Quantitative changes in alpha 1 and alpha 5 gamma-aminobutyric acid type A receptor subunit mRNAs and proteins after a single treatment of cerebellar granule neurons with N-methyl-D-aspartate.
It was previously demonstrated that daily administration of N-methyl-D-aspartate (NMDA) to primary cultures of cerebellar granule neurons for 5 days in vitro mediates an increase in the relative content of mRNAs encoding selected subunits of the gamma-aminobutyric acid (GABA)A receptor. This analysis was extended using a competitive polymerase chain reaction assay with internal standards to quantitate changes that occur in the absolute amounts of selected GABAA receptor subunit mRNAs in cerebellar granule neurons in vitro after the single administration of nontoxic doses of either NMDA or glutamate. For these studies, we focused on the alpha 1, alpha 5, and alpha 6 receptor subunit mRNAs and examined their absolute contents in cultures maintained in low KCl (12.5 mM), maintained in low KCl and treated with NMDA (10 microM) for 24 hr, or maintained in high KCl (25 mM). The absolute amounts of each mRNA varied in these paradigms; whereas the alpha 1 and alpha 5 receptor subunit mRNAs increased in response to NMDA-selective glutamate receptor stimulation, the alpha 6 receptor subunit mRNA did not. The time course of the alpha 1 and alpha 5 mRNA increases, dose dependence, and effects of glutamate in the presence or absence of MK-801 were also analyzed. Treatment of cultures maintained in 12.5 mM KCl with 5 microM glutamate resulted in comparable changes in the alpha 1 and alpha 5 receptor subunit mRNA contents, and a somewhat smaller increase in the alpha 6 mRNA content was observed. Using corresponding GABAA receptor subunit-specific antibodies, it was shown that the observed mRNA changes are accompanied by increased expression of alpha 1- and alpha 5-like receptor subunit immunoreactivity. Collectively, these data demonstrate that signal transduction mechanisms triggered by NMDA-selective glutamate receptor stimulation differentially modulate the levels of selected GABAA receptor subunit mRNAs and the corresponding proteins they encode.
Amino Acid Sequence↗
Developmental expression of the alpha 6 GABAA receptor subunit mRNA occurs only after cerebellar granule cell migration.
Using a competitive polymerase chain reaction (PCR) and appropriate internal standards, we have analyzed absolute amounts of the alpha 6 GABAA receptor subunit mRNA in the postnatally developing cerebellum and neocortex. The PCR data have shown that absolute amounts of the alpha 6 receptor subunit mRNA in the cerebellum increase dramatically (nearly 100-fold) during the second postnatal week, reaching maximal levels by postnatal day 21 (1 fmol/microgram total RNA). The absolute amount of the alpha 6 GABAA receptor subunit mRNA in the cortex at postnatal day 1 was 2 amol/microgram total RNA and increased to 7 amol/micrograms total RNA by postnatal day 14. No further increase in alpha 6 mRNA expression in the adult cortex was observed. Microscopic analysis of emulsion coated and counterstained sections indicated that alpha 6 GABAA receptor subunit mRNA labeling was only detected in the internal granule cell layer and not in either the external granule cell layer or in migrating granule cells. The alpha 1 GABAA receptor subunit mRNA increased in the cerebellar cortex with a similar temporal profile, although its distribution extended to additional cell types (Purkinje cells, stellate/basket cells and possibly cerebellar astrocytes). The temporal expression of these two GABAA receptor subunit mRNAs is coincident with the formation of synaptic contacts in the granule cell dendrites suggesting that afferent pathways innervating these neurons following cell migration may play a critical role in increasing the expression of mRNAs encoding the alpha 1 and alpha 6 GABAA receptor subunits.
Aging↗
Expression of c-fos mRNA following seizures evoked from an epileptogenic site in the deep prepiriform cortex: regional distribution in brain as shown by in situ hybridization.
Using in situ hybridization histochemistry for the detection of c-fos mRNA, we examined the pathways activated by seizures evoked by a focal application of bicuculline into a highly discrete epileptogenic site in the deep prepiriform cortex, the area tempestas (AT). Thirty minutes after the initiation of limbic motor seizures evoked by bicuculline in AT, a marked increase in c-fos mRNA was detected in the hippocampal formation, amygdala, olfactory bulb, piriform cortex, and entorhinal cortex. The increase of c-fos mRNA was strictly dependent upon the infusion of the drug in AT. Infusions of bicuculline in the same dose outside the AT did not increase c-fos mRNA levels. The extent to which the mapping pattern of c-fos mRNA expression was specific to limbic seizures was evaluated by examining another focally evoked seizure model involving the application of bicuculline bilaterally into the inferior colliculus. The absence of any detectable c-fos induction in the limbic system after explosive running-bouncing clonic seizures evoked by bicuculline injected into the inferior colliculus indicates that the pattern of activation that we found in the AT-evoked seizure model is not common to all forms of convulsive activity. Furthermore these observations suggest that the pattern of activation we have observed is seizure dependent and not stress induced. Our results indicate that c-fos mRNA expression is useful in the functional mapping of pathways involved in seizure propagation and that the anatomic pattern of activation is selectively related to the type of seizure evoked.
Animals↗
Temporal and spatial patterns of expression of c-fos, zif/268, c-jun and jun-B mRNAs in rat brain following seizures evoked focally from the deep prepiriform cortex.
Using in situ hybridization, we previously investigated (17) the regional pattern of c-fos mRNA increases in the brain following convulsive seizures elicited from a highly circumscribed epileptogenic site located in the deep prepiriform cortex. In this paper, we focus on the hippocampus and examine mRNAs encoding other immediate early genes (IEGs), namely c-jun, jun-B, and zif/268, for changes following the focally evoked seizures. Although the anatomic distribution of increases in each IEG mRNA was qualitatively comparable, the temporal analysis indicated that increases in zif/268 mRNA appeared prior to the other genes studied. Each of the mRNAs reached a maximum increase by 30 min and declined to basal levels within 3 h following seizure initiation. The data indicate that these four IEGs respond in a coordinated fashion to propagated seizure activity with increases in mRNA and, furthermore, that increased expression of all four genes appears to occur in the same cell types in the hippocampus.
Animals↗
Regional distribution in the rat central nervous system of a mRNA encoding a portion of the cardiac sodium/calcium exchanger isolated from cerebellar granule neurons.
The cardiac Na+/Ca2+ exchanger is a bidirectional electrogenic ion transporter that exchanges three Na+ ions for each Ca2+ ion and plays a critical role in returning sarcolemma Ca2+ concentrations to their resting levels. Because of the importance that the Na+/Ca2+ exchanger may play in maintaining neuronal Ca2+ homeostasis in the central nervous system, we subcloned a 456 bp portion of the Na+/Ca2+ exchanger cDNA from RNA isolated from primary cultures of rat cerebellar granule neurons using the polymerase chain reaction (PCR). This cDNA fragment was sequenced and shown to share 91.4% sequence identity with the human and 88% sequence identity with the canine cardiac Na+/Ca2+ exchangers. The PCR amplification product was used to analyze the distribution of this portion of the Na+/Ca2+ exchanger mRNA in various regions of the CNS by both Northern blotting and in situ hybridization histochemistry. The Northern analysis showed that the rank order of abundance of this mRNA was: hippocampus > cortex > cerebellum > hypothalamus > midbrain > striatum. The in situ hybridization data indicated that the corresponding mRNA containing this portion of the exchanger was present in numerous brain regions including multiple cortical layers, the hippocampus, septal nuclei, various thalamic nuclei, cerebellum, hypothalamus, olfactory bulb, brainstem, in various regions of the thoracic spinal cord and to a lesser extent in the striatum. The differential distribution of the mRNA as revealed by the in situ hybridization pattern suggests that either additional molecular variants exist or that different Na+/Ca2+ exchange mechanisms may be operative in those cell types that contain low amounts of this fragment of the exchanger mRNA.
Animals↗
Structure of the rat gene encoding the mitochondrial benzodiazepine receptor.
The gene encoding the rat mitochondrial benzodiazepine receptor (MBR) was cloned and characterized. Hybridization of a previously cloned cDNA for MBR to genomic Southern blots indicated that the gene was probably present at one copy per haploid genome. Rapid amplification of cDNA ends with rat adrenal RNA was used to obtain 47 nt of additional sequence upstream from our previously cloned MBR cDNA proving to be a crucial step in cloning the first exon of this gene. The MBR gene is comprised of four exons spanning approx. 10 kb. The first intron, contained within a 8-kb stretch of this gene, is located within the 5'-untranslated sequence, whereas the remaining two introns are much shorter (641 and 854 bp) and interrupt the coding sequence. The third intron contains sequences homologous to rodent B1 repetitive elements and a novel sequence closely resembling part of a repetitive element belonging to the Alu family in humans. The transcription start point was mapped by S1 nuclease protection assays suggesting that the first exon is just 56 bp in length. The sequence upstream from this region contains three GC boxes but lacks other known consensus recognition sites for sequence-specific transcription factors.
Amino Acid Sequence↗
Pharmacological characterization of regulation of phosphoinositide metabolism by recombinant 5-HT2 receptors of the rat.
Transfection of 5-HT2 receptor cDNA in 293 cells induced the expression of a protein binding domain, exhibiting the classical 5-HT2 receptor transduction mechanism. Both [3H]DOB and [3H]spiperone high affinity binding sites were present in membranes of sense but not of antisense, 5-HT2 receptor cDNA transfected cells. Addition of 1 microM 5-HT induced a time-dependent increase of phosphoinositide (PI) metabolism in sense but not in antisense, 5-HT2 receptor cDNA transfected cells. Graded concentrations of 5-HT and of different serotonergic agonists showed different potencies (DOI greater than 5-HT greater than quipazine greater than DOM greater than alpha-methyl-5-HT greater than 8-OH-DPAT greater than 2-methyl-5-HT greater than CGS-12066B) in stimulating turnover of PI in cells transfected with cDNA encoding for 5-HT2 receptors of the rat. The ability of different antagonists to inhibit 5-HT-stimulated turnover of PI bore a direct relationship with their potency to inhibit 5-HT2 receptor binding in cells transfected with 5-HT2 receptor cDNA (spiperone greater than ketanserin greater than ritanserin greater than mianserin greater than haloperidol). Preincubation of transfected 293 cells with pertussis toxin failed to modify either 5-HT- or DOI-induced activation of metabolism of PI. Pretreatment of transfected 293 cells with DOI (100 nM) for 2 hr or more, significantly reduced activation of turnover of PI elicited by graded doses of 5-HT. When the transfected 293 cells were exposed to DOI (100 nM) for 12 hr and the challenge was performed after a 2-hr wash-out period, the desensitization of the response to 5-HT was virtually abolished.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
Distinct developmental patterns of expression of rat alpha 1, alpha 5, gamma 2S, and gamma 2L gamma-aminobutyric acidA receptor subunit mRNAs in vivo and in vitro.
We have quantitated the alpha 1, alpha 5, gamma 2S, and gamma 2L gamma-aminobutyric acidA (GABAA) receptor subunit mRNAs in the maturing cerebellum in vivo and in cerebellar granule neurons differentiating in vitro. Absolute amounts of mRNA were measured by reverse transcription and competitive polymerase chain reaction (PCR) analysis with appropriate internal standards. The alpha 1 and gamma 2L mRNA content increased continuously during postnatal cerebellar maturation and their changes with time matched very closely those of the cerebellar granule cells differentiating in vitro. The gamma 2S subunit mRNA showed a relatively constant pattern of expression both in vivo and in vitro, with comparable absolute concentrations in both developmental paradigms. The alpha 5 mRNA was initially high in vivo and decreased (eight-fold) to adult levels as postnatal cerebellar development progressed. In vitro the amount of alpha 5 GABAA receptor subunit mRNA was higher than in vivo at 3 days, increased by more than twofold by 8 days, and declined to approximately the initial values at 23 and 28 days in vitro. Collectively, the results indicate that the alpha 1, alpha 5, gamma 2S, and gamma 2L GABAA receptor subunit mRNAs are regulated differentially in a temporal manner during in vivo and in vitro maturation. Moreover, a comparison of the ontogenetic profiles of the gamma 2S and gamma 2L mRNAs indicates that alternative splicing of the gamma 2 primary RNA transcript is regulated developmentally during postnatal maturation of the rat cerebellum.
Animals↗
Site-directed mutagenesis of hepatocyte nuclear factor (HNF) binding sites in the mouse transthyretin (TTR) promoter reveal synergistic interactions with its enhancer region.
The transthyretin (TTR) gene is regulated by two DNA regions which elicit hepatocyte-specific expression: a proximal promoter and distal enhancer. The TTR promoter and enhancer are composed of at least eight DNA binding sites for three different hepatocyte nuclear factors (HNF), CCAAT/enhancer binding protein (C/EBP), and AP-1/cJun. Site directed mutations within each of the HNF binding sites in the TTR promoter were introduced to evaluate their contribution to transcriptional activity in hepatoma cells. The data indicate that the strong affinity HNF-3-S binding site (-106 to -94) is absolutely required for TTR promoter activity since several mutations in this site eliminate TTR expression in the context of its enhancer. Conversion of a second weak affinity HNF3-W site (-140 to -131) in the TTR promoter to a high affinity site resulted in higher levels of expression. TTR mutations that disrupted several weak affinity sites (HNF1, HNF3-W, and HNF4) only slightly diminished expression levels in the presence of the TTR enhancer. In contrast, when we deleted the TTR enhancer from these HNF mutant constructs, TTR expression decreased to undetectable levels. This result suggests cooperation between the factors binding to the TTR promoter and enhancer regions. These results also demonstrate that the HNF3-S site alone is not sufficient to activate TTR transcription, but rather requires the participation of three cell-specific factors to elicit minimal promoter activity. The complexity of this promoter design and the requirement for a minimal number of cell-specific factors to achieve transcription allows us to propose a model which may explain the maintenance of tissue-specific expression of TTR.
Animals↗
Nonselective inhibition by antisense oligonucleotides of cytosine arabinoside action.
Antisense oligonucleotides can be used in cell cultures to inhibit biosynthesis of neurotransmitter receptors. Hence, they operate as highly specific pharmacological antagonists. In obtaining a pure neuronal primary culture the suppression of non-neuronal cell proliferation is required; usually 1-beta-D-arabinofuranosylcytosine (AraC) is used. We report that in primary cultures of rat cerebellar cells, oligonucleotides, targeted to: (1) glutamate receptor, (2) the seven transmembrane spanning region of receptors coupled to GTP binding proteins, and (3) beta-adrenergic receptor kinase, nonspecifically inhibit the cell incorporation of 3H-AraC and curtail its antiproliferative action. This nonspecific action might occur at the level of the mechanism of action of AraC and should be taken into account when antisense probes as pharmacological antagonists are used.
Animals↗
Regulation of gamma-aminobutyric acidA receptor subunit expression by activation of N-methyl-D-aspartate-selective glutamate receptors.
Exposure of primary cultures of rat cerebellar granule cells to specific antagonists of the N-methyl-D-aspartate (NMDA)-selective glutamate receptor reduces the steady state levels of mRNAs encoding various gamma-aminobutyric acidA (GABAA) receptor subunits. These neurons are glutamatergic and require a depolarizing concentration of K+ (25 mM) for optimal development and survival. When the neuronal differentiation rate is retarded by lowering of the extracellular [K+] (to 12.5 mM), a persistent stimulation of the same glutamate receptors with nonneurotoxic doses of NMDA increases the expression of these GABAA receptor subunits. This suggests that the lowered K+ concentration reduces neuronal depolarization and the consequent release of glutamate from the cells. These results show that the neuronal content of selected GABAA receptor subunit mRNAs is optimized by certain levels of glutamate in the culture medium, suggesting a neurotrophic action of this neurotransmitter at certain developmental stages of granule cells in culture.
6-Cyano-7-nitroquinoxaline-2,3-dione↗
Purification, cloning, and expression of a peripheral-type benzodiazepine receptor.
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Amino Acid Sequence↗
Transcriptional program coordination by N-methyl-D-aspartate-sensitive glutamate receptor stimulation in primary cultures of cerebellar neurons.
In primary cultures of rat cerebellar neurons, a brief stimulation of glutamate receptors results in coordinated activation of a programmed early gene response involving increases in the amount of c-fos, c-jun, jun-B, and zif/268 mRNAs. Each of these genes was induced to a different extent and showed a temporal pattern characterized by either a monophasic "early" response, occurring within 30 min of glutamate addition, or a biphasic response (c-jun), lasting for up to 6 to 8 hr after the initial stimulus. The early phase of the glutamate-induced gene expression was prevented by 3-(2-carboxypiperazin-4-yl)propyl-1-phosphonic acid, a highly selective isosteric antagonist of the N-methyl-D-aspartate (NMDA)-sensitive glutamate receptor (NMDA receptor). The second phase of the c-jun response was not blocked when the NMDA receptors were completely inhibited after the initial pulse of agonist or when the quisqualate-kainate receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione was added, suggesting that a brief NMDA receptor stimulation triggers a cascade of events critical for the manifestation of the delayed c-jun expression. Furthermore, gel retardation assays demonstrated that NMDA receptor activation results in a prolonged increase in nuclear DNA-binding activity specific for the AP-1 transcriptional regulatory element. Protein immunoblot analysis showed that the composition of this nucleoprotein complex changes as a function of time, reflecting a cascade that involves an increased translation of Fos and several Fos-related proteins. The coordinated induction of several different transcription factors and the variations in transcriptional complex formation initiated by NMDA receptor stimulation may be a key mechanism in the orchestration of specific target gene expression that underlies various aspects of neuronal function, including plasticity responses.
6-Cyano-7-nitroquinoxaline-2,3-dione↗
Molecular cloning and expression of cDNA encoding a peripheral-type benzodiazepine receptor.
This report describes the cloning of a full length cDNA encoding PKBS, a protein of approximately 17 kDa associated with peripheral-type benzodiazepine binding sites. Cyanogen bromide digestion of purified PKBS yielded several peptide fragments which were subjected to gas-phase sequencing. Based on these partial amino acid sequences, oligonucleotide probes were used to screen a rat adrenal cDNA library. Several hybridizing clones were isolated which were found to contain overlapping sequences. The longest cDNA spanned 781 base pairs and specified an open reading frame of 169 amino acids which matched all of the peptide sequences. Northern analysis with this PKBS cDNA probe in different rat tissues revealed one RNA species of approximately 850 nucleotides exhibiting relative abundances qualitatively comparable with the densities of peripheral-type benzodiazepine binding sites in each tissue. The PKBS cDNA was cloned into an eukaryotic expression vector placing it under transcriptional control of the beta-globin promoter and SV40 enhancer. Transfection of the transformed human kidney 293 cell line with this recombinant vector resulted in stoichiometric increases of about 900 fmol/mg of protein in binding capacities for Ro5-4864 (4'-chlorodiazepam) and PK 11195, two peripheral-type benzodiazepine ligands. These increases were accounted for by the expression of binding sites with approximate dissociation constants of 5 nM for PK 11195 and 8 nM for Ro5-4864, thereby distinguishing the expressed binding sites as being characteristic of the receptor from rat origin rather than of the host human-derived cell line. The rank order of potency of different ligands to compete against [3H]Ro5-4864 binding in the transfected cells was PK 11195 greater than Ro5-4864 greater than diazepam greater than protoporphyrin IX greater than clonazepam, consistent with the specificity characteristic of rat peripheral-type benzodiazepine binding sites. These studies suggest that PKBS comprises binding domains for benzodiazepines and isoquinoline carboxamides and hence is apparently responsible for the manifestation of peripheral-type benzodiazepine recognition sites.
Adrenal Glands↗