Peripheral-type benzodiazepine receptors in human blood cells of patients affected by migraine without aura

The kinetic parameters at equilibrium of peripheral benzodiazepine receptors in platelets, lymphocytes and granulocytes of 15 patients affected by migraine without aura were tested using [3H]PK 11195, a specific radioligand for this receptor and compared with the same number of healthy controls: a statistically significant increase (platelets 212%, lymphocytes 203%, granulocytes 171%, as absolute percentage) in the maximal number of binding sites (B(max)) in all three patient samples, compared with healthy controls was detected; on the contrary, the values of the dissociation constant (K(d)) at equilibrium do not show any statistically significant variations between the two groups. These data further confirm the presence of peripheral biochemical alterations in migraine without aura. As peripheral benzodiazepine receptors appear to be involved in the regulation of the mitochondrial respiratory chain, the observed increase in B(max) might be related to the mitochondrial anomalies found in migraine disorders.     

Diethylpyrocarbonate modification of benzodiazepine receptors from calf cerebral cortex

Diethylpyrocarbonate (DEP), an amino acid modifying reagent, causes complete inactivation of particulate and deoxycholate-solubilized benzodiazepine-receptors from calf cerebral cortex. No heterogeneity was observed in DEP-sensitivity of the receptors. Protection from DEP-induced inactivation was provided by the centrally active benzodiazepines, diazepam and nitrazepam, but not by the peripherally active Ro5-4864, suggesting that DEP modifies a residue which is essential for the central actions of benzodiazepines. GABA did not protect against inactivation or influence the protection afforded by diazepam, indicating that the DEP-modifiable residue is independent of GABA binding sites, or that GABA binding sites are also sensitive to DEP. DEP-induced inactivation of benzodiazepine-receptors proceeds much faster at pH 10.1 than at pH 8.1 or 6.0, indicating the modification of a high pK_a side group, possibly the phenol of a tyrosyl residue. This postulation is in accord with our previous findings with the modifying reagents tetranitromethane and N-acetylimidazole.     

Properties of H-diazepam binding to benzodiazepine receptors in rat cerebral cortex

The recently discovered specific binding sites for benzodiazepines in the central nervous system have been characterized further. Specific ³H-diazepam binding to synaptosomal preparations from rat cerebral cortex is saturable with the dissociation constant for diazepam K_D = 3.6 ± 0.1 nM. The maximal amount of specifically bound ³H-diazepam is 0.88 ± 0.05 pmole per mg protein. In the binding assay equilibrium between the specific binding sites and ³H-diazepam is reached within 15 min. Specific ³H-diazepam binding shows a linear dependency on protein up to 3.0 mg protein per assay and a sharp pH-optimum between pH 7.0 to pH 7.4 ³H-diazepam binding is temperature dependent with the highest amount bound at 4° C. Specific ³H-diazepam binding is altered by treating the synaptosomal preparations with proteolytic enzymes: trypsin and -chymotrypsin lead to the complete disappearance of specific ³H-diazepam binding; carboxypeptidase A or B decrease specific ³H-diazepam binding by 36 %, while leucineaminopeptidase was without effect.     

Heterogeneity in the physicochemical properties of deoxycholate-solubilized benzodiazepine receptors from calf cerebral cortex

The hydrodynamic behaviour of benzodiazepine receptors solubilized by deoxycholate from calf cerebral cortex reveals two molecular forms. The Stokes radii are 46.5 A and 67.2 A, and the sedimentation coefficients are 10.9 S and 14.6 S. The calculated apparent molecular weights and frictional ratios suggest either two nearly globular proteins of ca. 200K and 400K daltons each, or two ca. 300K daltons proteins which differ significantly in their degree of asymmetry. The benzodiazepine binding site is located on ca. 51K daltons component(s) in both forms.     

Temperature effect on subclasses of muscarinic receptors in rat colon, heart and cerebral cortex

The high-affinity muscarinic antagonist /3H/-Quinuclidinyl benzilate (/3H/-QNB) has been used to label muscarinic receptors in a crude membrane fraction of rat cerebral cortex, colon and heart. The inhibition of /3H/-QNB binding by Atropine, Oxotremorine and Pirenzepine was investigated at three temperatures: 37 degrees C, 22 degrees C and 10 degrees C. The IC50 values and the proportion of high (Rt1) and low (Rt2) affinity binding sites were determined for the three compounds. When the temperature were lowered from 37 degrees C to 10 degrees C, in the agonist and antagonist dissociation constants decreased in all tissues. Changes in temperature did not modify Rt1 or Rt2 values for Oxotremorine and Pirenzepine. The results show marked temperature-dependent modifications of IC50 values for muscarinic receptors of high- and low-affinity sites in rat cerebral cortex, colon or heart.     

Peripheral-type benzodiazepine receptors are involved in the regulation of cholesterol side chain cleavage in adrenocortical mitochondria

In an attempt to elucidate the physiological relevance of the peripheral type of benzodiazepine receptor in adrenocortical mitochondria, we examined the effect of three different benzodiazepines (diazepam, Ro5-4864, and chlordiazepoxide) on the conversion of cholesterol to pregnenolone, the rate-limiting step in steroidogenesis, by using cholesterol-loaded mitochondria from bovine adrenal zona fasciculata. These benzodiazepines, except chlordiazepoxide, caused a dose-dependent stimulation of the cholesterol side chain cleavage in the mitochondria. The stimulatory effect of Ro5-4864 was approximately 10 times more potent than that of diazepam. No inhibitory effect of YM-684 (Ro15-1788), a potent antagonist to central-type benzodiazepine receptors, was observed in the stimulation induced by diazepam and Ro5-4864. Both external calcium ion and voltage-dependent calcium channel blocker, (+)-PN200-110, were without effect on the diazepam-induced steroidogenesis. By contrast, pretreatment of mitochondria with digitonin abolished the stimulatory effect of diazepam on the mitochondrial steroidogenesis. The present results indicate that the peripheral-type benzodiazepine receptor of adrenocortical mitochondria plays an essential role in regulating cholesterol side chain cleavage without any change of calcium channels.     

Peripheral-type benzodiazepine receptors mediate translocation of cholesterol from outer to inner mitochondrial membranes in adrenocortical cells

In previous studies we demonstrated that peripheral-type benzodiazepine receptors (PBR) were coupled to steroidogenesis in several adrenocortical and Leydig cell systems (Mukhin, A.G., Papadopoulos, V., Costa, E., and Krueger, K.E. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 9813-9816; Papadopoulos, V., Mukhin, A.G., Costa, E., and Krueger, K.E. (1990) J. Biol. Chem. 265, 3772-3779). The current study elucidates the specific step in the steroid biosynthetic pathway by which PBR mediate the stimulation in steroid hormone production. The adrenocorticotropin (ACTH)-responsive Y-1 mouse adrenocortical cell line was used to compare the mechanisms by which ACTH and PK 11195 (a PBR ligand) stimulate steroidogenesis. The effects of these agents were studied at three stages along the steroid biosynthetic pathway: 1) secretion of 20 alpha OH-progesterone by Y-1 cell cultures; 2) pregnenolone production by isolated mitochondrial fractions; 3) quantities of cholesterol resident in outer and inner mitochondrial membrane fractions. Steroid synthesis stimulated by ACTH was blocked by cycloheximide, an effect documented by other laboratories characterized by an accumulation of mitochondrial cholesterol specifically in the outer membrane. In contrast, PK 11195-stimulated steroidogenesis was not inhibited by cycloheximide, and the magnitude of the stimulation was markedly enhanced when the cells were pretreated with cycloheximide and ACTH. When isolated mitochondria were used, stimulation of pregnenolone production by PK 11195 was largely independent of exogenously supplied cholesterol, indicating that PBR act on cholesterol already situated within the mitochondrial membranes. This phenomenon was found to be the result of a translocation of cholesterol from outer to inner mitochondrial membranes induced by the PBR ligand. These studies therefore suggest that mitochondrial intermembrane cholesterol transport in steroidogenic cells is mediated by a mechanism coupled to PBR.     

Aging and benzodiazepine binding in the rat cerebral cortex

The specific binding of ³H-diazepam in the cerebral cortex was investigated in membrane preparations from 6 and 30 month old Fischer-344 rats. No age-related differences in the association, equilibrium, or dissociation binding characteristics were observed. The increased sensitivity of the elderly to the central sedative effects of the benzodiazepines does not, therefore, appear to involve changes in binding to the receptor site located in the cortex.     

Photoaffinity labeling of the benzodiazepine receptor in bovine cerebral cortex

Clonazepam, nitrazepam and flunitrazepam were found to engage in an irreversible interaction with benzodiazepine binding sites in bovine cerebral cortex homogenates upon irradiation with ultraviolet light. Photoaffinity labeling with [³H]flunitrazepam could be substantially (approx. 85%) inhibited by a number of different benzodiazepines, including clonazepam, lorazepam, Ro5-3027, and non-radioactive flunitrazepam. Spiroperidol, atropine, naltrexone, propranolol and GABA had no effect on irreversible [³H]flunitrazepam binding, indicating that this binding is to the benzodiazepine receptor as defined in previous studies.     

Thromboxane receptors in human kidney tissues.

Thromboxane (TX) A2 effects in the kidneys include contraction of glomerular mesangial cells and intrarenal vascular tissue. A kidney cDNA encoding a TX receptor expressed in rat renal glomeruli and rat renal arterial smooth muscle cells has been reported. However, TXA2 receptors in human kidneys have not been documented. The purpose of this study was to identify and characterize TXA2 receptors in glomeruli and intrarenal arteries isolated from human kidneys. Normal kidneys, not used for transplant because of technical reasons, were kept at -70 degrees C and used for research purposes. The glomeruli and intrarenal arteries were isolated from renal cortical tissue by a mechanical sieving technique. The equilibrium dissociation constant and receptor number were determined by nonlinear analysis of binding inhibition data. The data were generated in radioreceptor assays using [125I]-BOP, a stable analog of TXA2. The dissociation constants (mean +/- SEM) for binding of I-BOP to human glomeruli and intrarenal arterial membranes were 6.6 +/- 1.1 nM (n = 7) and 20 +/- 6 nM (n = 7), respectively (p < 0.05). The receptor number was 311 +/- 91 fmol/mg protein (n = 7) in glomeruli and 74 +/- 16 fmol/mg protein (n = 7) in intrarenal arterial membranes (p < 0.04). The order of specificity of TXA2 analogs for [125I]-BOP binding sites was similar in glomeruli and in arterial membranes and was I-BOP > or = U46619 > or = pinane TXA2 > or = carbocyclic TXA2 > or = PGH2. These findings provide direct evidence for the presence of specific, high-affinity [125I]-BOP binding sites in human renal glomeruli and extraglomerular vascular tissue. These data also indicate that the human binding sites have higher affinity for the TXA2 agonist I-BOP than for PGH2.     

Polarized expression of human P2Y receptors in epithelial cells from kidney, lung, and colon

Eight human G protein-coupled P2Y receptors (P2Y₁, P2Y₂, P2Y₄, P2Y₆, P2Y₁₁, P2Y₁₂, P2Y₁₃, and P2Y₁₄) that respond to extracellular nucleotides have been molecularly identified and characterized. P2Y receptors are widely expressed in epithelial cells and play an important role in regulating epithelial cell function. Functional studies assessing the capacity of various nucleotides to promote increases in short-circuit current (I_sc) or Ca²⁺ mobilization have suggested that some subtypes of P2Y receptors are polarized with respect to their functional activity, although these results often have been contradictory. To investigate the polarized expression of the family of P2Y receptors, we determined the localization of the entire P2Y family after expression in Madin-Darby canine kidney (MDCK) type II cells. Confocal microscopy of polarized monolayers revealed that P2Y₁, P2Y₁₁, P2Y₁₂, and P2Y₁₄ receptors reside at the basolateral membrane, P2Y₂, P2Y₄, and P2Y₆ receptors are expressed at the apical membrane, and the P2Y₁₃ receptor is unsorted. Biotinylation studies and I_sc measurements in response to the appropriate agonists were consistent with the polarized expression observed in confocal microscopy. Expression of the G_q-coupled P2Y receptors (P2Y₁, P2Y₂, P2Y₄, P2Y₆, and P2Y₁₁) in lung and colonic epithelial cells (16HBE14o– and Caco-2 cells, respectively) revealed a targeting profile nearly identical to that observed in MDCK cells, suggesting that polarized targeting of these P2Y receptor subtypes is not a function of the type of epithelial cell in which they are expressed. These experiments highlight the highly polarized expression of P2Y receptors in epithelial cells. Madin-Darby canine kidney; 16HBE14o–; Caco-2; confocal microscopy; polarized targeting     

Pipecolic acid receptors in rat cerebral cortex

Identification of [¹⁴C]pipecolic acid (PA) receptors was attempted in the solubilized membrane fraction from rat cerebral cortex. Specific binding proteins for both PA and muscimol, a potent -aminobutyric acid (GABA) agonist, were detected in the same preparation. Separation of labeled PA and GABA binding proteins by glycerol gradient centrifugation has shown labeled protein bands of similar sedimentation rates, suggesting that PA and GABA may be binding to identical proteins. It seems likely that the PA binding receptor either may possess the same sedimentation characteristics as that of the GABA receptor, or both GABA and PA which is an endogenous and weak GABA agonist may bind to the same receptor complex, if not to the same binding site.     

Laminar distribution of the multiple opioid receptors in the human cerebral cortex

Quantitative autoradiographic assessment of cerebral cortical laminar distribution of μ, δ and κ opioid receptors was carried out in coronal sections of five post-mortem human brains obtained at autopsy. The cortical areas studied were: cingulate, frontal, insular, parietal, parahippocampal, temporal, occipitotemporal, occipital and striate area. In general, the laminar patterns of distribution for the three types of receptors are distinctive. Peak levels of δ opioid binding are in laminae I, II, and IIa. μ-Receptors are located in lamina III followed by I and II in cingulate, frontal, insular and parietal cortices and lamina IV in temporal and occiptotemporal cortices. κ-Receptors are found concentrated in laminae V and VI. The patterns of opioid binding in cortical laminae showed remarkable consistency in all five brains examined. In contrast to other cortical areas, the parahippocampal gyrus, at the level of the amygdaloid formation, demonstrated peak κ receptor density in laminae I, II and III. μ-Opioid binding was undetectable in the lateral occipital cortex and in the striate area. Special issue dedicated to Dr. Eric J. Simon.     

Laminar distribution of benzodiazepine receptors in visual cortex of adult rat

The characteristics and distribution of benzodiazepine receptors in individual layers of the visual cortex of adult rats were examined with the 3H-flunitrazepam binding technique employed on intact tissue slices. The different visual cortical layers were separated by cutting serial cryocut sections horizontally to the cortical surface and collecting the slices from each individual cortical layer under anatomical control. Highest benzodiazepine receptor densities were found in layers IV and VI. A moderate receptor density was detected in layer V (80% of highest density). The lowest receptor binding was observed in cortical layers I and II/III, still representing 66% of the highest receptor density. Binding affinities varied slightly between layers with dissociation constants somewhat higher for layers IV to VI in comparison to layers I and II/III. The distinct laminar pattern of benzodiazepine receptors in rat visual cortex suggests a differential neuromodulatory significance of these receptors in each individual cortical layer.     

Peripheral benzodiazepine receptors in isolated human pancreatic islets

Peripheral benzodiazepine receptors have been shown in some endocrine tissues, namely the testis, the adrenal gland, and the pituitary gland. In this work we evaluated whether peripheral benzodiazepine receptors can be found in the purified human pancreatic islets and whether they may have a role in insulin release. Binding of the isoquinoline compound [³H]1-(2-chlorophenyl-N-methyl-1-methyl-propyl)-3-isoquinolinecarboxamide ([³H]PK-11195), a specific ligand of peripheral benzodiazepine receptors, to cellular membranes was saturable, and Scatchard's analysis of the saturation curve demonstrated the presence of a single population of binding sites, with an affinity constant value of 9.20 ± 0.80 nM and a maximum number of binding sites value of 8913 ± 750 fmol/mg of proteins. PK-11195 and 7-chloro-1,3-dihydro-1-methyl-5-(p-chlorophenyl)-2H-1,4-benzodiazepin-2-on (Ro 5-4864) significantly potentiated insulin secretion from freshly isolated human islets at 3.3 mM glucose. These results show the presence of peripheral benzodiazepine receptors in purified human pancreatic islets and suggest their role in the mechanisms of insulin release. J. Cell. Biochem. 64:273–277. © 1997 Wiley-Liss, Inc.     

Melatonin reverses pinealectomy-induced decrease of benzodiazepine binding in rat cerebral cortex

Pinealectomy of rats resulted in significant depression of benzodiazepine receptors (assessed by [³H]flunitrazepam binding) in cerebral cortex 3–14 days after surgery without affecting their affinity significantly. A single s.c. injection of melatonin (800 μg/kg body wt) restored the depressed brain benzodiazepine receptor sites. Single melatonin injections (up to 1600 μg/kg) to intact rats did not affect brain benzodiazepine binding when injected at either morning or evening hours. Daily melatonin treatment to intact rats for 5 days augmented benzodiazepine receptor density in brain (morning injections) or its dissociation constant (evening injections). Melatonin added in vitro to rat cerebral cortex membranes only slightly depressed [³H]flunitrazepam binding at 100 μM concentrations. These results point out a link between pineal activity and benzodiazepine receptor function in rats. They also indicate that pharmacological doses of melatonin affect benzodiazepine binding sites in rat cerebral cortex.     

Expression of P-glycoprotein in human cerebral cortex microvessels.

P-Glycoprotein (P-gp) is an ATP-dependent efflux transporter that extrudes non-polar molecules, including cytotoxic substances and drugs, from the cells. It was initially found in cancer cells and then was shown to be a normal component of complex transport systems working at the blood-brain barrier (BBB). Previous studies have demonstrated that, in the brain, P-gp is localized on the luminal plasmalemma of BBB endothelial cells and that it may interact with the caveolar compartment of these cells. The aim of this study was to identify the site of cellular expression of P-gp in human brain in situ and to morphologically determine whether an association may exist between P-gp and caveolin-1, a structural and functional protein of the caveolar frame. The study was carried out on human cerebral cortex by immunoconfocal microscopy with antibodies to both P-gp and caveolin-1. The results show that P-gp marks the microvessels of the cortex and that the transporter is localized in the luminal endothelial compartment, where it co-localizes with caveolin-1. The demonstration of this co-localization of P-gp with caveolin-1 contributes a morphological backing to biochemical studies on P-gp/caveolin-1 relationships and leads us to suggest that interactions between these molecules may occur at the BBB endothelia.     

Telocytes in the human kidney cortex

Renal interstitial cells play an important role in the physiology and pathology of the kidneys. As a novel type of interstitial cell, telocytes (TCs) have been described in various tissues and organs, including the heart, lung, skeletal muscle, urinary tract, etc. ( www.telocytes.com ). However, it is not known if TCs are present in the kidney interstitium. We demonstrated the presence of TCs in human kidney cortex interstitium using primary cell culture, transmission electron microscopy (TEM) and in situ immunohistochemistry (IHC). Renal TCs were positive for CD34, CD117 and vimentin. They were localized in the kidney cortex interstitial compartment, partially covering the tubules and vascular walls. Morphologically, renal TCs resemble TCs described in other organs, with very long telopodes (Tps) composed of thin segments (podomers) and dilated segments (podoms). However, their possible roles (beyond intercellular signalling) as well as their specific phenotype in the kidney remain to be established.     

A gamma-aminobutyric acid/benzodiazepine receptor complex of bovine cerebral cortex

The gamma-aminobutyric acid/benzodiazepine receptor from bovine cerebral cortex was solubilized with sodium deoxycholate and purified by affinity chromatography on benzodiazepine-agarose and ion exchange chromatography. The benzodiazepine binding protein was enriched 1800-fold. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and dithiothreitol showed the presence of two major bands of Mr = 57,000 and 53,000. [3H]Flunitrazepam, after UV irradiation, was incorporated irreversibly into both bands of the isolated protein. A high affinity binding site for gamma-aminobutyric acid was co-purified with the benzodiazepine binding site and the two sites were shown to reside on the same physical structure. The dissociation constants were 10 +/- 4 nM for [3H] flunitrazepam and 12 +/- 3 nM for the gamma-aminobutyric acid agonist [3H]muscimol. The maximum specific activity for [3H] muscimol binding was 4.3 nmol/mg of protein. The ratio of [3H]muscimol to [3H]flunitrazepam binding sites was between 3 and 4. Gel filtration and sucrose density gradient sedimentation studies gave a Stokes radius of 7.3 +/- 0.5 nm and a sedimentation coefficient of 11.1 +/- 0.3 S, respectively. The purified complex had a pharmacological profile that corresponds to the receptor specificity found in membranes and crude soluble extracts.