Density Of Specific Binding Sites Research Articles

The role of glutamate and GABA receptors in the anticonvulsive effects of levetiracetam and a 4-phenylpirrolidone derivative (GIZh-290) in rats

The method of radioligand binding ex vivo has been used to evaluate the involvement of the ionotropic and metabotropic glutamate and GABA receptors in the mechanism of anticonvulsant action of levetiracetam and the original compound GIZh-290 (2-oxo-4-phenylpyrrolidin-1-yl) acetic acid) in the rat brain. We found that at the peak of lithium-pilocarpine seizures, the Bmax value (the density of specific binding sites) decreases for [3H]-SR 95531, [3H](–)baclofen, [3Н](+)MK-801, and [3H]LY 354740 by 30–50% from the control level. The pre-treatment with levetiracetam (600 mg/kg) 40 minutes before pilocarpine kept the number of GABA- and NMDA-type receptors at the control level without affecting the metabotropic receptors. The administration of GIZh-290 (5.0 mg/kg), in contrast, maintained the density of GABAA receptors at the control level, while the density of other types of receptors remained decreased at the peak of seizures. Thus, the anticonvulsant effect of levetiracetam and GIZh-290 has different mechanisms.

Electropolymerized molecularly imprinted polypyrrole film for sensing of clofibric acid.

Piezoelectric quartz crystals and analogous gold substrates were electrochemically coated with molecularly imprinted polypyrrole films for pulsed amperometric detection (PAD) of clofibric acid, a metabolite of clofibrate. Cyclic voltammetry data obtained during polymerization and deposited weight estimations revealed a decrease of the polymerization rate with increasing clofibric acid concentration. XPS measurements indicated that clofibric acid could be removed after imprinting with an aqueous ethanol solution, which was further optimized by using PAD. Zeta potential and contact angle measurements revealed differences between molecularly imprinted (MIP) and non-imprinted polymer (NIP) layers. Binding experiments with clofibric acid and other substances showed a pronounced selectivity of the MIP for clofibric acid vs. carbamazepine, but the response of MIP and NIP to 2,4-dichlorophenoxyacetic acid was higher than that for clofibric acid. A smooth surface, revealed by AFM measurements, with roughness of 6–8 nm for imprinted and non-imprinted layers, might be a reason for an excessively low density of specific binding sites for clofibric acid. Furthermore, the decreased polymerization rate in the presence of clofibric acid might not result in well-defined polymer structures, which could be the reason for the lower sensitivity.

Effect of meta-chlorobenzhydryl urea (m-ClBHU) on benzodiazepine receptor system in rat brain during experimental alcoholism.

Chronic alcohol intake induces neuroadaptive changes in benzodiazepine receptors modulating GABAA receptors that promote alcohol addiction. Analysis of benzodiazepine receptors in the brain of Wistar rats differing by alcohol preference has demonstrated that affinity of [(3)H]flunitrazepam and [(3)H]Ro5-4864 binding with membrane fraction was reduced, while the density of specific binding sites in the brain cortex of heavy drinking and low drinking rats was increased in comparison with rats nonpreferring alcohol. Administration of anticonvulsant meta-chlorobenzhydryl urea increased affinity of benzodiazepine receptors in the brain cortex of heavy drinking rats, which improved GABA neurotransmission in the brain of these animals and reduced alcohol consumption.

Autoradiography of 2‐[18F]F‐A‐85380 on nicotinic acetylcholine receptors in the porcine brain in vitro

Noninvasive molecular imaging of subtypes of neuronal nicotinic acetylcholine receptors (nAChRs) will provide information on the role of these receptors in neurodegenerative diseases. The binding of the positron emission tomography ligand 2-[18F]F-A-85380 to nAChRs was investigated in the porcine brain by quantitative autoradiography in vitro. The high-affinity binding of 2-[18F]F-A-85380 to each of the investigated 12 brain areas was saturable and apparently monophasic (e.g., apparent KD value of 1.72 nM in the thalamus). The highest density of specific binding sites was observed in the thalamus (1,158 fmol/mg protein), and the lowest density was measured in the cerebellar gray matter (11 fmol/mg protein). An attempt to assess nAChR subtype specificity of 2-[18F]F-A-85380 was made by competitive autoradiography. Binding of 2-[18F]F-A-85380 coincubated with 2-F-A-85380, epibatidine, cytisine, or methyllycaconitine, respectively, indicated a specificity of 2-[18F]F-A-85380 to beta2-containing nAChRs in the porcine brain. The autoradiographic data confirmed the suitability of swine as a model for the evaluation of radioligands designed for imaging of nAChR subtypes in the living brain.

Characterization of angiotensin II–receptor subtypes in podocytes

Glomerular podocytes play a key role in maintaining the integrity of the glomerular filtration barrier. This function may be regulated by angiotensin II (Ang II) through activation of cell-surface receptors. Although studies suggest that podocytes express receptors for Ang II, the Ang II binding site has not been characterized with radioligand binding techniques. We therefore used iodine 125–labeled Ang II to monitor Ang II–receptor density during differentiation of a mouse podocyte cell line. Scatchard analyses of equilibrium binding data revealed a single class of high-affinity binding sites (dissociation constant ∼ 3 nmol/L) in both differentiated and nondifferentiated cells. During differentiation, the density of Ang II–receptor sites increased roughly 15-fold in differentiated podocytes (maximal density of specific binding sites 881 fmol/mg protein) compared with that in nondifferentiated cells (52 fmol/mg protein; P < .005). Glomerular podocytes expressed messenger RNA for AT1A, AT1B, and AT2 receptor subtypes, and competitive binding studies found that differentiated podocytes expressed mostly AT1 receptors (∼75%) with lesser amounts of AT2 (∼25%). Up-regulation of Ang II–receptor number was associated with increased Ang II–receptor responsiveness, as evidenced by enhanced Ang II–stimulated inositol phosphate (IP) generation and incorporation of tritiated thymidine. Both [ 3H]thymidine incorporation and IP generation were mediated by AT1-receptor activation. These data suggest that glomerular podocytes express a high-affinity binding site for Ang II with pharmacologic characteristics of both AT1 and AT2 receptors. This receptor site is up-regulated during podocyte differentiation, and receptor activation induces both IP generation and DNA synthesis by AT1-dependent mechanisms. We speculate that activation of podocyte Ang II receptors contributes to glomerular damage in disease states.

Melanin concentrating hormone increase hippocampal synaptic transmission in the rat

A retrograde facilitation has been demonstrated in the one trial step-down inhibitory avoidance of melanin-concentrating hormone (MCH), when it was infused into rat hippocampal formation. Considering the high density of specific binding sites for the MCH peptide on the hippocampus and the participation of this structure on learning and memory processes we have studied the effects of MCH on the hippocampal synaptic transmission. For this purpose, slices of rat hippocampus were perfused with different concentration of MCH. The main result of the present study was a long-lasting potentiation on the hippocampal evoked response on dentate gyrus induced by MCH (4–11 μM) at 30, 60 and 120 min with a maximum effect at 120 min. Previous perfusion of DL - 2- amino - 5 phosphonovaleric acid (APV, 20 μM) was unable to impair the increased hippocampal evoked response induced by MCH 4 μM. On the other hand, the channel blocker Dizocilpine (MK-801, 10 μM) completely impaired the increased hippocampal synaptic plasticity induced by MCH perfusion. We postulate the increased hippocampal synaptic efficacy induced by MCH as one of the mechanisms underlying the retrograde facilitation on the inhibitory avoidance paradigm, observed after MCH hippocampal microinjection. We cannot rule out other MCH neurochemical mechanism and other areas of the brain involved in the MCH effects.

Neuropeptide FF, pain and analgesia.

Neuropeptide FF (Phe-Leu-Phe-Gln-Pro-Gln-Arg-Phe-NH2) and the octadecapeptide neuropeptide AF (Ala-Gly-Glu-Gly-Leu-Ser-Ser-Pro-Phe-Trp-Ser-Leu-Ala-Ala-Pro-Gln-Arg-Phe -NH2) were isolated from bovine brain, and were initially characterized as anti-opioid peptides. They can oppose the acute effects of opioids and an increase in their brain concentrations may be responsible for the development of tolerance and dependence to opioids. Numerous experiments suggest a possible neuromodulatory role for neuropeptide FF. A precursor protein has been identified, in particular in human brain. Neuropeptide FF immunoreactive neurons are present only in the medial hypothalamus, and the nucleus of the solitary tract, and in the spinal cord in the superficial layers of the dorsal horn and areas around the central canal. Depolarization induces a Ca2+-dependent release of neuropeptide FF immunoreactivity from the spinal cord. Neuropeptide FF acts through stimulation of its own receptors and high densities of specific binding sites are found in regions related either to sensory input and visceral functions or to the processing of nociceptive messages. In both isolated dorsal root ganglion neurons and CA1 pyramidal neurons of the hippocampus, neuropeptide FF has little effect of its own but reverses the effects of mu-opioid receptor agonists. In agreement with the hypothesized anti-opioid role of neuropeptide FF, supraspinal injection lowers the nociceptive threshold and reverses morphine-induced analgesia in rats. Furthermore, immunoneutralization of neuropeptide FF increases endogenous and exogenous opioid-induced analgesia. Similarly, microinfusion of neuropeptide FF or neuropeptide FF analogs into the nucleus raphe dorsalis, the parafascicular nucleus, or the ventral tegmental area has no effect on the nociceptive threshold but inhibits the analgesia induced by co-injected morphine. Furthermore, infusion of neuropeptide FF into the parafascicular nucleus or the nucleus raphe dorsalis reverses the analgesic effect of morphine infused into the nucleus raphe dorsalis or the parafascicular nucleus, respectively, demonstrating remote interactions between neuropeptide FF and opioid systems. By contrast, intrathecal administration of neuropeptide FF analogs induces a long lasting, opioid-dependent analgesia and potentiates the analgesic effect of morphine. Analgesic effects of neuropeptide FF after supraspinal injection could also be observed, for example during nighttime. In young mice, (1DMe)Y8Famide (D.Tyr-Leu-(NMe)Phe-Gln-Pro-Gln-Arg-Phe-NH2), a neuropeptide FF analog, increases delta-opioid receptor-mediated analgesia. These findings indicate that neuropeptide FF constitutes a neuromodulatory neuronal system interacting with opioid systems, and should be taken into account as a participant of the homeostatic process controlling the transmission of nociceptive information.

Hair cycle-dependent expression of corticotropin-releasing factor (CRF) and CRF receptors in murine skin.

We demonstrate the presence and hair cycle-dependent expression of corticotropin-releasing factor (CRF) and CRF receptors (CRF-R) in C57BL/6 mouse skin. To correlate this with a physiological, developmentally controlled tissue remodeling process, we have analyzed CRF and CRF-R expression during defined stages of the murine hair cycle with its rhythmic changes between growth (anagen), regression (catagen), and resting (telogen). Using reversed-phase HPLC combined with two independent anti-CRF radioimmunoassays, we have identified CRF in murine skin. Maximal CRF levels were found in anagen III-IV skin, and minimal values were detected in catagen and telogen skin. By immunofluorescence, maximal CRF immunoreactivity (CRF-IR) was seen in the basal epidermis, nerve bundles of skin, the outer root sheath and matrix region of anagen IV-VI follicles, and in defined sections of their perifollicular neural network, whereas catagen and telogen skin displayed minimal CRF-IR. Using quantitative autoradiography and 125I-CRF as a tracer, high-affinity binding sites for CRF were detected in murine skin. The highest density of specific binding sites was detected in the panniculus carnosus, the epidermis, and the hair follicle. CRF-R type 1 (CRF-R1) IR was detected by immunohistology mainly in the outer root sheath, hair matrix, and dermal papilla of anagen VI follicles, as well as in the inner and outer root sheaths of early catagen follicles. CRF-R1 expression was also hair cycle dependent. Therefore, in normal murine skin, the CRF-CRF-R signaling system may operate as an additional neuroendocrine pathway regulating skin functions, possibly in the context of cutaneous stress responses.

Characterization of CB1 receptors on rat neuronal cell cultures: binding and functional studies using the selective receptor antagonist SR 141716A.

This study was undertaken to characterize further the central cannabinoid receptors in rat primary neuronal cell cultures from selected brain structures. By using [3H]SR 141716A, the specific CB1 receptor antagonist, we demonstrate in cortical neurons the presence of a high density of specific binding sites (Bmax = 139 +/- 9 fmol/mg of protein) displaying a high affinity (KD = 0.76 +/- 0.09 nM). The two cannabinoid receptor agonists, CP 55940 and WIN 55212-2, inhibited in a concentration-dependent manner cyclic AMP production induced by either 1 microM forskolin or isoproterenol with EC50 values in the nanomolar range (4.6 and 65 nM with forskolin and 1.0 and 5.1 nM with isoproterenol for CP 55940 and WIN 55212-2, respectively). Moreover, in striatal neurons and cerebellar granule cells, CP 55940 was also able to reduce the cyclic AMP accumulation induced by 1 microM forskolin with a potency similar to that observed in cortical neurons (EC50 values of 3.5 and 1.9 nM in striatum and cerebellum, respectively). SR 141716A antagonized the CP 55940- and WIN 55212-2-induced inhibition of cyclic AMP accumulation, suggesting CB1 receptor-specific mediation of these effects on all primary cultures tested. Furthermore, CP 55940 was unable to induce mitogen-activated protein kinase activation in either cortical or striatal neurons. In conclusion, our results show nanomolar efficiencies for CP 55940 and WIN 55212-2 on adenylyl cyclase activity and no effect on any other signal transduction pathway investigated in primary neuronal cultures.

609 The physiological roles of serine protease, neuropsin, in the hippocampus

Presence and localization of the prostacyclin receptor in the rat brain and spinal cord were examined by in vitro autoradiography using [3H]iloprost, a highly specific and stable agonist for this receptor. Density of specific binding sites for iloprost was generally high in four regions of the lower brain stem, that is, the medial and commissural subnuclei of the nucleus tractus solitarius, the area postrema, superficial layers of the spinal trigeminal nucleus caudalis and dorsal horn. Moderate density was found in the thalamus, cerebral cortex, hippocampus, striatum and dorsal cochlear nucleus. The distribution pattern was distinct from those of other prostanoid binding sites previously studied except that prostaglandin E2 binding sites were also abundant in the nucleus tractus solitarius, spinal trigeminal nucleus caudalis and dorsal horn. Even in these regions, binding sites for iloprost had several features clearly different from those for prostaglandin E2. First, within the medial and commissural subnuclei of the nucleus tractus solitarius, iloprost binding sites were distributed preferentially in the dorsal part, while those for prostaglandin E2 were located more ventrolaterally. Second, on postnatal day 0, iloprost binding sites have already been expressed in large amounts in the nucleus tractus solitarius, spinal trigeminal nucleus caudalis and dorsal horn of rats, while prostaglandin E2 binding sites are negligible at this stage. Thirdly, the binding of 10 nM [3H]iloprost in these three regions was almost completely displaced by 10 μM unlabelled iloprost but only slightly by 10 μM unlabelled prostaglandin E2. Unilateral nodose ganglionectomy or dorsal rhizotomy decreased the density of iloprost binding sites in the nucleus tractus solitarius or dorsal horn, respectively, with a greater decrease in the operated side. Ligation of the vagus either central or peripheral to the nodose ganglion resulted in an accumulation of iloprost binding sites proximal to the ligation.These results suggest that specific binding sites for iloprost, presumably prostacyclin receptor, are present in the nervous system and, in particular, that the iloprost binding sites in the nucleus tractus solitarius, dorsal horn and possibly in the superficial layers of the spinal trigeminal nucleus caudalis are produced in their sensory ganglia and transported to central terminals of the primary sensory afferents as well as to their peripheral terminals.

Photoaffinity labeling of protoporphyrinogen oxidase, the molecular target of diphenylether-type herbicides.

Diphenylether-type herbicides are extremely potent inhibitors of protoporphyrinogen oxidase, a membrane-bound enzyme involved in the heme and chlorophyll biosynthesis pathways. Tritiated acifluorfen and a diazoketone derivative of tritiated acifluorfen were specifically bound to a single class of high-affinity binding sites on yeast mitochondrial membranes with apparent dissociation constants of 7 nM and 12.5 nM, respectively. The maximum density of specific binding sites, determined by Scatchard analysis, was 3 pmol.mg-1 protein. Protoporphyrinogen oxidase specific activity was estimated to be 2500 nmol protoporphyrinogen oxidized h-1.mol-1 enzyme. The diazoketone derivative of tritiated acifluorfen was used to specifically photolabel yeast protoporphyrinogen oxidase. The specifically labeled polypeptide in wild-type mitochondrial membranes had an apparent molecular mass of 55 kDa, identical to the molecular mass of the purified enzyme. This photolabeled polypeptide was not detected in a protoporphyrinogen-oxidase-deficient yeast strain, but the membranes contained an equivalent amount of inactive immunoreactive protoporphyrinogen oxidase protein.

Photoaffinity Labeling of Protoporphyrinogen Oxidase, the Molecular Target of Diphenylether‐Type Herbicides

Diphenylether‐type herbicides are extremely potent inhibitors of protoporphyrinogen oxidase, a membrane‐bound enzyme involved in the heme and chlorophyll biosynthesis pathways. Tritiated acifluorfen and a diazoketone derivative of tritiated acifluorfen were specifically bound to a single class of high‐affinity binding sites on yeast mitochondrial membranes with apparent dissociation constants of 7 nM and 12.5 nM, respectively. The maximum density of specific binding sites, determined by Scatchard analysis, was 3 pmol · mg−1 protein. Protoporphyrinogen oxidase specific activity was estimated to be 2500 nmol protoporphyrinogen oxidized · h−1· mol−1 enzyme. The diazoketone derivative of tritiated acifluorfen was used to specifically photolabel yeast protoporphyrinogen oxidase. The specifically labeled polypeptide in wild‐type mitochondrial membranes had an apparent molecular mass of 55 kDa, identical to the molecular mass of the purified enzyme. This photolabeled polypeptide was not detected in a protoporphyrinogen‐oxidase‐deficient yeast strain, but the membranes contained an equivalent amount of inactive immunoreactive protoporphyrinogen oxidase protein.

Prostacyclin receptor in the brain and central terminals of the primary sensory neurons: An autoradiographic study using a stable prostacyclin analogue [3H]iloprost

Presence and localization of the prostacyclin receptor in the rat brain and spinal cord were examined by in vitro autoradiography using [3H]iloprost, a highly specific and stable agonist for this receptor. Density of specific binding sites for iloprost was generally high in four regions of the lower brain stem, that is, the medial and commissural subnuclei of the nucleus tractus solitarius, the area postrema, superficial layers of the spinal trigeminal nucleus caudalis and dorsal horn. Moderate density was found in the thalamus, cerebral cortex, hippocampus, striatum and dorsal cochlear nucleus. The distribution pattern was distinct from those of other prostanoid binding sites previously studied except that prostaglandin E2 binding sites were also abundant in the nucleus tractus solitarius, spinal trigeminal nucleus caudalis and dorsal horn. Even in these regions, binding sites for iloprost had several features clearly different from those for prostaglandin E2. First, within the medial and commissural subnuclei of the nucleus tractus solitarius, iloprost binding sites were distributed preferentially in the dorsal part, while those for prostaglandin E2 were located more ventrolaterally. Second, on postnatal day 0, iloprost binding sites have already been expressed in large amounts in the nucleus tractus solitarius, spinal trigeminal nucleus caudalis and dorsal horn of rats, while prostaglandin E2 binding sites are negligible at this stage. Thirdly, the binding of 10 nM [3H]iloprost in these three regions was almost completely displaced by 10 microM unlabelled iloprost but only slightly by 10 microM unlabelled prostaglandin E2. Unilateral nodose ganglionectomy or dorsal rhizotomy decreased the density of iloprost binding sites in the nucleus tractus solitarius or dorsal horn, respectively, with a greater decrease in the operated side. Ligation of the vagus either central or peripheral to the nodose ganglion resulted in an accumulation of iloprost binding sites proximal to the ligation. These results suggest that specific binding sites for iloprost, presumably prostacyclin receptor, are present in the nervous system and, in particular, that the iloprost binding sites in the nucleus tractus solitarius, dorsal horn and possibly in the superficial layers of the spinal trigeminal nucleus caudalis are produced in their sensory ganglia and transported to central terminals of the primary sensory afferents as well as to their peripheral terminals.

Identification of specific binding sites for glucagon-like peptide-1 on the posterior lobe of the rat pituitary.

Glucagon-like peptide-1 (GLP-1) immunoreactivity has been found in autonomic and neuroendocrine brain regions, whereas only limited data are available regarding the characterization and localization of brain GLP-1 receptors. In the present study, using quantitative in vitro autoradiography, a high density of specific binding sites for GLP-1 was characterized on sections of the posterior pituitary lobe of the rat. Low specific binding of radiolabeled GLP-1 was found in the anterior lobe and no specific binding in the intermediate lobe. To examine the specificity of GLP-1 binding sites, sections of the posterior lobe were incubated with radiolabeled GLP-1 in the presence of various peptides. Radiolabeled [Tyr39]exendin-4, a specific GLP-1 agonist, bound to these receptor sites with the same affinity as GLP-1, while glucagon and vasoactive intestinal peptide (VIP) were unable to displace 125I-GLP-1. Both unlabeled exendin-4 and GLP-1 inhibited this binding with equally high affinity. Using 125I-[Tyr39]exendin-4 as radiolabel, the concentration of biding sites was found to be 7.8 +/- 0.4 fmol/mg tissue. Further analysis of the binding data from experiments with tissue slices revealed the presence of high and low affinity binding sites. In experiments with unlabeled [Tyr39]exendin-4, the KdS were 6.2 +/- 1.4 x 10(-12) and 9.3 +/- 1.5 x 10(-10) M, respectively, and in experiments with unlabeled GLP-1, 3.4 +/- 1.8 x 10(-12) and 5.9 +/- 1.5 x 10(-10) M, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Multiple cholinergic markers are unexpectedly not altered in the rat dentate gyrus following entorhinal cortex lesions

Since major cholinergic deficits are observed in Alzheimer's disease, the development of models to study possible cholinergic plasticity has generated great interest. In this regard, it has been shown that lesions of the entorhinal cortex, which sends glutamatergic projections to the hippocampus, promote the sprouting and plasticity of presumptive cholinergic septohippocampal fibers in the dentate gyrus, as revealed by AChE histochemistry. This sprouting was reported to be evident at 8 d and up to 30 d postlesion (DPL) and is now widely used as a model of cholinergic neuronal plasticity. In the present study, unilateral lesions of the entorhinal cortex were made in adult rats, and the status of various putative pre- and postsynaptic cholinergic markers was assessed after 2, 4, 8, 14, and 30 DPL. As expected, AChE was increased in the outer molecular layer of the ipsilateral dentate gyrus from 8 to 30 DPL. In contrast, the activity of ChAT, the enzyme responsible for the synthesis of ACh, and the densities of specific binding sites for 3H-AH5 183/vesamicol (blocker of the ACh vesicular transport sites), 3H-hemicholinium-3 (blocker of the high-affinity choline uptake sites), muscarinic-M2 (3H-AF-DX 384 and 3H-ACh), muscarinic-M1 (3H-pirenzepine), and nicotinic (3H-N-methylcarbamylcholine) cholinergic receptors were not increased on the ipsilateral molecular layer of the dentate gyrus, as compared to their contralateral controls. We conclude that the increase in AChE staining in the molecular layer of the dentate gyrus following entorhinal cortex lesions may be due to changes in noncholinergic neurons.

Multiple [ 3H]DTG binding sites in guinea pig cerebellum: evidence for the presence of non-specific binding

The characteristics of the low affinity component of 1,3-di(2- 3H]tolyl)guanidine binding to the guinea pig cerebellum were investigated. Saturation binding assays where σ 1 receptors were masked with dextrallorphan indicated that 1,3-di(2-[5- 3H]tolyl)guanidine bound to cerebellar membranes in a fashion best described by a 1 site + non-specific binding model with a low density of specific binding sites ( B max ≈ 200 fmol/mg protein). Boiling the cerebellar membranes before addition to the saturation assay had no effect on the density of 1,3-di(2-[5- 3H]tolyl)guanidine binding. In contrast, both the K d and B max for 1,3-di(2-[5- 3H]tolyl)guanidine binding to liver membranes was significantly reduced by boiling, as was the density of [ 3H](+)-pentazocine binding to cerebellum and liver. Thus, a substantial component of 1,3-di(2-[5- 3H]tolyl)guanidine binding in the guinea pig cerebellum is to non-specific, proteinaceous binding sites with some of the pharmacological characteristics of the σ 2 binding site.

Evaluation of .ALPHA.1-Adrenoceptor Subtypes in Human Hypertrophied Prostate Using (3H)YM617, an .ALPHA.1-Selective Antagonist.

The purpose of this study was to demonstrate the distribution of α1-adrenoceptors in the human hypertrophied prostate with [3H]YM617 (a newly synthesized α1-blocker), and to determine the receptor subtype of [3H]YM617 binding sites by the use of chlorethylclonidine dihydrochloride (CEC). The slices of prostate, removed en bloc in 7 patients with benign prostatic hypertrophy and sectioned vertically to the urethra, were preincubated with or without CEC (10μM). The slices were then incubated with [3H]YM617 (1nM) in the presence or absence of phentolamine (100μM). Specific binding of [3H]YM617 was seen relatively homogeneous, and distributed in the periglandular interstitium (mainly corresponding to smooth muscles) of the prostate. The CEC-pretreated specimens showed a decrease in density of specific binding sites (30.9% mean decrease compared with the CEC non-pretreated specimens on quantitative autoradiogram) (p<0.05), but localization was almost the same as in the CEC non-pretreated specimens. Membrane assay gave similar results. These experiments indicate that ca 70% of α1-adrenoceptor binding sites in the hypertrophied prostate are insensitive to CEC, while ca 30% of that are sensitive to CEC.

Quantitative autoradiography reveals higher densities of specific calcitonin-gene-related peptide receptors in small intramyocardial compared with large epicardial coronary arteries

1. Binding sites for calcitonin-gene-related peptide were localized and characterized in porcine coronary arteries using quantitative autoradiography, and the density of binding sites was compared between large epicardial and small intramyocardial coronary arteries. 2. A single class of binding sites for calcitonin-gene-related peptide with a dissociation constant of 2.1 +/- 0.2 nmol/l was detected in both the large and small coronary arteries. The density of specific binding sites was higher (maximum binding site density 231 +/- 14 fmol/mg of protein) in the small coronary arteries than in the large epicardial coronary arteries (maximum binding site density 108 +/- 5 fmol/mg of protein). beta-Human calcitonin-gene-related peptide showed higher affinity than alpha-human calcitonin-gene-related peptide for the binding sites. Most of the specific binding sites for both peptides in the large coronary artery were localized in the intima and media. 3. In coronary artery from patients with coronary heart disease, there were more binding sites for calcitonin-gene-related peptide in the smooth muscle layer of atheromatous segments (7.2 +/- 0.7 amol/mm2) than in that of normal segments (3.0 +/- 0.3 amol/mm2, P < 0.002). 4. The present findings lend further support to the theory of regional variation in the vasodilator response to calcitonin-gene-related peptide in porcine coronary arteries, which seems to be due to different densities of a single type of receptor for calcitonin-gene-related peptide.

Species differences in characteristics and distribution of [ 3H]α,β-methylene ATP binding sites in urinary bladder and urethra of rat, guinea-pig and rabbit

The characteristics and distribution of [ 3H]α, β-methylene ATP ([ 3H]α, β-MeATP, a radioligand for P 2x-purinoceptors) binding sites in the urinary bladder of rat, guinea-pig and rabbit were examined. Autoradiographic localization of [ 3H]α, β-MeATP binding sites was also carried out on the urethra of the three species. Receptor binding assay showed that the rat bladder possesses the highest density of specific binding sites, followed by rabbitand guinea-pig bladder. Semi-quantitative analysis of autoradiograms showed that the grain densities in the bladders of rat, guinea pig, and rabbit were parallel to those obtained from receptor binding assay. The grain densities were greatly reduced in the presence of β,γ-methylene ATP (β,γ-MeATP). No significant specific binding was detected in the smooth muscle of rat and guinea-pig urethra, while a very low level of specific binding was observed in the rabbit urethra. Differences of grain densities different regions (dome, body, and trigone) of the same bladder were also observed, but they were not as remarkable as those between species. The results of this study demonstrate species differences of P 2x-purinoceptor densities in the urinary bladder, which may reflect differing degrees of purinergic neurotransmitter control of the bladder detrusor muscle.

Specific binding sites for bifemelane in the hippocampus of the guinea pig, relevant to its pharmacological actions

Bifemelane has an anti-amnesic effect, produces the translocation of protein kinase C in the hippocampal CA3 region but not in CA1 and enhances long-term potentiation in the mossy fibre-CA3 system but not in the Schaffer collateral-CA1 system. The present study examined the specific binding of [ 3H]bifemelane in membrane preparations of guinea pig hippocampus and regional differences in such a binding. The binding of [ 3H]bifemelane was reversible and greater when incubated at 4°C than at 25 or 37°C. The binding of [ 3H]bifemelane appeared to be composed of at least 2 different affinity components. Imipramine significantly suppressed the binding of [ 3H]bifemelane at 1 μM and, in the presence of 1 μM imipramine, the low-affinity component of the binding of [ 3H]bifemelane was eliminated. The density of specific binding sites for 1 nM [ 3H]bifemelane was significantly higher in the hippocampal CA3 region than in the CA1. The specific binding of 1 nM [ 3H]bifemelane was not inhibited by other nootropic drugs, such as idebenone, calcium hopantenate, vinpocetine, indeloxazine and piracetam. The present results suggest that there are specific binding sites for bifemelane in hippocampus, which are different from those for other nootropic drugs tested and that the regional differences in the pharmacological susceptibilities to bifemelane are at least, in part, attributed to those in the density of binding sites for bifemelane.