(+)-PN200-l 10 and Ouabain Binding Sites in Purified Bovine Adrenomedullary Plasma Membranes and Chromaffin Cells

Bovine adrenal medulla plasma membranes were purified by a differential centrifugation procedure using sucrose and Urografin discontinuous density gradients; the membranes were enriched 10-12-fold in acetylcholinesterase activity and [3H]ouabain binding sites. Specific (+)-[3H]PN200-110 binding to these membranes amounted to 90% of total binding and was saturable and of high affinity (KD = 41 pM; Bmax = 119 fmol/mg of protein) with a Hill coefficient close to 1, a result suggesting the presence of a single, homogeneous population of dihydropyridine receptors. The association and dissociation rate constants were, respectively, 7.5 X 108 M-1 min-1 and 0.023 min-1. Unlabeled (+)-PN200-110 displaced (+)-[3H]PN200-110 binding with a potency 100-fold higher than (-)-PN200-110 (IC50,0.5 and 45nM, respectively). Although the two enantiomers of BAY K 8644 completely displaced (+)-[3H]PN200-110 binding, they exhibited no stereoselectivity (IC50, 69 and 83 nM,respectively). Whereas ( +/- )-nitrendipine very potently displaced (+)-[3H]PN200-110 binding (IC50 = 1.3 nM) verapamil and cinnarizine displaced the binding by only 30 and 40% at 1 microM, and diltiazem increased it by 20% at 10 microM. [3H]Ouabain bound to plasma membranes with a KD of 34 nM and a Bmax of 9.75 pmol/mg of protein, a figure 80-fold higher than the Bmax for (+)-PN200-110. [3H]Ouabain also bound to intact chromaffin cells with a Bmax of 244 fmol/10(6) cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Adrenergic receptors in the liver parenchyma in children with chronic hepatitis]

In the present study adrenergic receptors have been investigated in liver parenchyma, obtained at the resection of extrahepatic portal hypertension children without parenchymal affection (control group, n-7) and the resection of children in parenchymal affection (group of chronic hepatitis children, n-6). It has been shown, that the binding of beta-adrenergic radioligand 3H-dihydroalprenolol (3H-DHA) in liver parenchyma membranes of both control and chronic hepatitis groups was saturable and showed high affinity. The Scatchard analysis of the binding data indicated that the binding site was characterized by Kd and Bmax of 1.2 +/- 0.5 nM, 261.2 +/- 50 fmol/mg, respectively, for the control group; and 0.9 +/- 0.15 nM, 68.5 +/- 18.8 fmol/mg, respectively, for the group of chronic hepatitis patients; (mean+SEM). The binding of alpha 1-adrenergic antagonist 3H-prazosin (3H-PRZ) in liver parenchyma was also saturable and showed high affinity. The binding site is characterized by Kd = 0.6 +/- 0.12 nM, Bmax = 92.8 +/- 8.0 fmol/mg, for the control group; and Kd = 0.8 +/- 0.15 nM, Bmax = 195.0 +/- 22.0 fmol/mg, for the group of chronic hepatitis. It has been found that the number of binding sites of 3H-DHA significantly decreased and the number of binding sites of 3H-PRZ did not change in chronic hepatitis liver parenchyma in comparison with the control group. The results obtained suggest the important role of beta-adrenergic receptors in the pathogenesis of chronic hepatitis and in liver regeneration in children.     

Benzodiazepine receptors on human blood platelets

Binding studies conducted on membrane preparation from human platelets using (3H) Ro5-4864 and (3H) diazepam showed specific and saturable binding. Scatchard analysis revealed a single class of binding sites with KD = 10.8 +/- 0.9 nM and Bmax = 775 +/- 105 fmol/mg protein for (3H) Ro5-4864 and KD = 10.5 +/- 1.1 nM and Bmax = 133 +/- 19 fmol/mg for (3H) diazepam. We were unable to detect any GABA binding site on crude membrane preparation, nor did GABA enhance the binding of (3H) Ro5-4864 or (3H) diazepam. This suggests that benzodiazepine receptors are uncoupled to GABA system on human platelets. Ro15-1788, a specific antagonist for "central type" benzodiazepine (BDZ) binding sites was inactive in displacing (3H) Ro5-4864 from membrane receptors, while PK 11195 (a specific ligand for the "peripheral type" receptor) was the most potent of the drugs tested in inhibiting (3H) Ro5-4864 binding. These results indicate that human blood platelets bear "peripheral-type" BDZ receptor. Moreover, we could not detect any (3H) propyl beta carboline specific binding on platelet membranes. Results on benzodiazepine receptors on human circulating lymphocytes are also reported and similarity in pharmacological properties with platelet benzodiazepine receptors is suggested.     

Effects of chronic nicotine treatment on nicotinic receptors in the rabbit urinary bladder

Nicotine induced a phasic contraction in the rabbit urinary bladder. The response was abolished by hexamethonium and partially reduced by atropine and capsaicin. Simultaneous atropine and capsaicin treatment did not abolish the contraction. These findings suggest that the response to nicotine is due to acetylcholine, tachykinins, and unknown mediator release. In contrast, nicotine-induced contraction diminished following the chronic nicotine treatment without a change of its pharmacological properties. These results suggest the possibility that chronic nicotine treatment causes a decrease in nicotinic receptor numbers. Therefore, the binding properties of (-)-[3H]nicotine on rabbit urinary detrusor muscle membrane fractions were studied to evaluate the effects of chronic nicotine treatment on nicotinic receptors. Specific (-)-[3H]nicotine binding reached saturation and Scatchard plots were curvilinear, suggesting the existence of two different affinity sites for (-)-[3H]nicotine. Dissociation constants (KD) and maximum binding sites (Bmax) were KD1 = 4.91 +/- 1.88 nM, Bmax1 = 2.42 +/- 0.22 fmol/mg protein and KD2 = 263 +/- 56 nM, Bmax2 = 25.0 +/- 4.3 fmol/mg protein. In urinary bladder membrane fractions from chronic nicotine-treated rabbits, KD and Bmax values were KD1 = 3.96 +/- 0.38 nM, Bmax1 = 1.07 +/- 0.25 fmol/mg protein and KD2 = 249 +/- 12 nM, Bmax2 = 10.8 +/- 1.5 fmol/mg protein. Dissociation constants for both sites following chronic nicotine treatment did not change but maximum binding site numbers for both sites significantly decreased (p less than 0.05). These results suggest that the decrease in contractile response evoked by nicotine after chronic nicotine treatment in rabbit urinary bladder is due to a decrease in numbers of nicotinic receptors.     

Characterization of Opioid Receptors in Cultured Neurons

The appearance of mu-, delta-, and kappa-opioid receptors was examined in primary cultures of embryonic rat brain. Membranes prepared from striatal, hippocampal, and hypothalamic neurons grown in dissociated cell culture each exhibited high-affinity opioid binding sites as determined by equilibrium binding of the universal opioid ligand (-)-[3H]bremazocine. The highest density of binding sites (per mg of protein) was found in membranes prepared from cultured striatal neurons (Bmax = 210 +/- 40 fmol/mg protein); this density is approximately two-thirds that of adult striatal membranes. By contrast, membranes of cultured cerebellar neurons and cultured astrocytes were devoid of opioid binding sites. The opioid receptor types expressed in cultured striatal neurons were characterized by equilibrium binding of highly selective radioligands. Scatchard analysis of binding of the mu-specific ligand [3H]D-Ala2,N-Me-Phe4,Gly-ol5-enkephalin to embryonic striatal cell membranes revealed an apparent single class of sites with an affinity (KD) of 0.4 +/- 0.1 nM and a density (Bmax) of 160 +/- 20 fmol/mg of protein. Specific binding of (-)-[3H]bremazocine under conditions in which mu- and delta-receptor binding was suppressed (kappa-receptor labeling conditions) occurred to an apparent single class of sites (KD = 2 +/- 1 nM; Bmax = 40 +/- 15 fmol/mg of protein). There was no detectable binding of the selective delta-ligand [3H]D-Pen2,D-Pen5-enkephalin. Thus, cultured striatal neurons expressed mu- and kappa-receptor sites at densities comparable to those found in vivo for embryonic rat brain, but not delta-receptors.     

Physicochemical Properties of Serotonin 5-HT3 Binding Sites Solubilized from Membranes of NG 108-15 Neuroblastoma-Glioma Cells

Specific binding sites with pharmacological properties typical of serotonin 5-HT3 receptors were identified in membranes of the murine hybridoma cell line NG 108-15, using [3H]zacopride as a ligand. Optimal solubilization of these sites (yield, 50%) could be achieved using the detergent 3-[3-(cholamidopropyl)dimethylammonio]-1-propane sulfonate (CHAPS) at 24 mM plus 0.5 M NaCl in 25 mM Tris-HCl, pH 7.4. Specific [3H]zacopride binding to soluble sites in the 100,000-g CHAPS extract was saturable and showed characteristics (Bmax = 425 +/- 81 fmol/mg of protein; KD = 0.19 +/- 0.02 nM) closely related to those of membrane-bound sites (Bmax = 932 +/- 183 fmol/mg of protein; KD = 0.60 +/- 0.03 nM). Determination of association (k+1 = 0.17 nM min-1) and dissociation (k-1 = 0.02 min-1) rate constants for the soluble sites gave a KD value of 0.12 nM, a result consistent with that calculated from saturation studies. As assessed from the displacement potencies (IC50) of 10 different drugs, the pharmacological profile of [3H]zacopride specific binding sites was essentially the same (r = 0.99) in the CHAPS-soluble extract and in cell membranes, although some increase in the affinity for 5-HT3 antagonists (zacopride, ICS 205-930, and MDL 72222) and decrease in the affinity for 5-HT3 agonists (2-methyl-5-hydroxytryptamine and phenylbiguanide) were noted for the soluble sites. Sucrose density gradient sedimentation of the CHAPS-soluble extract gave a Svedberg coefficient of 12S for the material with [3H]zacopride specific binding capacity. Chromatographic analyses using Sephacryl S-400 and wheat germ agglutinin-agarose columns indicated marked enrichment (by 2.5- and 10-fold, respectively) in [3H]zacopride specific binding activity in the corresponding eluates compared with the starting soluble extract, a finding suggesting that both steps are of potential interest for the partial purification of solubilized 5-HT3 receptors. Two soluble materials with apparent molecular masses of approximately 600 and approximately 36 kDa were found to bind [3H]zacopride specifically in the Sephacryl S-400 eluate. Interestingly, molecular mass determination by radiation inactivation of [3H]zacopride binding sites in frozen NG 108-15 cells gave a value of approximately 35 kDa.     

Binding properties of beta-adrenergic receptors in early human fetal lung

The characteristics of beta-adrenergic receptors in human fetal lung were examined using the beta-adrenoceptor antagonist 3H-dihydroalprenolol /DHA/. Steady-state binding was reached by 15 min at 25 degrees C, and the association and dissociation rate constants were 0.0422 nM-1 min-1 and 0.0874 min-1, respectively. From saturation experiments, Bmax of 82.0 +/- 38 fmol/mg protein and KD = 1.85 +/- 0.92 nM were calculated. Inhibition of 3H-DHA binding by beta-1 /metoprolol/ and beta-2 /zinterol, IPS-339, fenoterol/ selective drugs resulted in biphasic displacement curves with slope factors less than 1.0. Analysis of these curves revealed a beta-1: beta-2 ratio of 40:60 in human fetal lung. The presence of 3H-DHA binding sites in early human lung may have a developmental importance which is not yet understood.     

Interaction of sarcolysine with beta-adrenoreceptors in tumor cells

The sites of specific binding of 3H-L-dihydroalprenolol (3H-DHA) were identified on the surface of ascites sarcoma 37 cells, using competitive displacement and binding of the beta-adrenergic antagonists, 3H-DHA and L-propranolol. These binding sites possessed the properties of beta-adrenergic receptors coupled with adenylate cyclase. Analysis of 3H-DHA binding by the Scatchard method revealed the presence of beta-adrenergic receptors of two types, i. e., with a high (Kd = 0.9-1.0 nM) and low (Kd = 15-20 nM) affinity for 3H-DHA. The number of high affinity receptors was (5.0-7.5) X 10(3); that of low affinity receptors was (20-30) X 10(3) on a per cell basis. Sarcolysine at concentrations of 1-10 microM displaced receptor-bound 3H-DHA, competed with the ligand for the common binding sites and caused, similar to isoproterenol, a short-term elevation of the intracellular cAMP content. Sarcolysine within the same concentration range (2.5-25 microM) caused non-competitive inhibition of the cAMP phosphodiesterase (PDE2) activity of plasma membranes isolated from ascites sarcoma 37 cells. The data obtained point to the functional coupling between beta-adrenergic receptors, adenylate cyclase and membraneous PDE2 of tumour cells as well as to its possible role in the antitumour effect of sarcolysine.     

Correlation of changes in cardiac calcium channels with hemodynamics in Syrian hamster cardiomyopathy and heart failure

We compared hemodynamics with [3H]nitrendipine (calcium channel) binding to cardiac membranes from Bio 14.6 cardiomyopathic Syrian hamsters at 4 and 10 months with their F1B controls. A 50% increase in the number (Bmax) of nitrendipine binding sites (calcium channels) was seen only in the 4 month old myopathic vs controls (Bmax = 468 +/- 11 vs 309 +/- 10 fmol/mg prot with no change in affinity (KD) (KD = .65 +/- .12 vs .75 +/- .14 nM), while no differences in Bmax or KD were seen at 10 months (Bmax = 375 +/- 9 vs 362 +/- 7 fmol/mg prot/KD = .82 +/- .18 vs .89 +/- .17 nM) myopathic vs control respectively. Hemodynamic studies revealed no significant differences in cardiac output, cardiac index, stroke volume, heart rate, mean arterial pressure, peripheral resistance, body weight, heart weight at 4 months, but a significant decrease in peripheral resistance (1120 +/- 360 vs 2080 +/- 240) increase in body weight (118 +/- 2 vs 94 +/- 2 grams) and heart weight (97 +/- 5 vs 78 +/- 2 gms/100 gms body weight) in 10 month myopathic vs control animals. We conclude that the onset of cardiomyopathy at 4 months is associated with a selective increase in calcium channel binding sites and heart failure at 10 months is associated with a relative decrease in these sites.     

Solubilized Rat Brain Adenosine Receptors Have Two High-Affinity Binding Sites for l, 3-Dipropyl-8-Cyclopentylxanthine

The specific binding of L-N6-[3H]phenylisopropyladenosine (L-[3H]PIA) to solubilized receptors from rat brain membranes was studied. The interaction of these receptors with relatively low concentrations of L-[3H]PIA (0.5-12.0 nM) in the presence of Mg2+ showed the existence of two binding sites for this agonist, with respective dissociation constant (KD) values of 0.24 and 3.56 nM and respective receptor number (Bmax) values of 0.28 +/- 0.03 and 0.66 +/- 0.05 pmol/mg of protein. In the presence of GTP, the binding of L-[3H]PIA also showed two sites with KD values of 24.7 and 811.5 nM and Bmax values of 0.27 +/- 0.09 and 0.93 +/- 0.28 pmol/mg of protein for the first and the second binding site, respectively. Inhibition of specific L-[3H]PIA binding by 1,3-dipropyl-8-cyclopentylxanthine (DPCPX) (0.1-300 nM) performed with the same preparations revealed two DPCPX binding sites with Ki values of 0.29 and 13.5 nM, respectively. [3H]DPCPX saturation binding experiments also showed two binding sites with respective KD values of 0.81 and 10.7 nM and respective Bmax values of 0.19 +/- 0.02 and 0.74 +/- 0.06 pmol/mg of protein. The results suggest that solubilized membranes from rat brain possess two adenosine receptor subtypes: one of high affinity with characteristics of the A1 subtype and another with lower affinity with characteristics of the A3 subtype of adenosine receptor.     

Preponderance of alpha 2- over beta 1-adrenergic receptor sites in human fat cells is not predictive of the lipolytic effect of physiological catecholamines

Adrenergic control of human fat cell lipolysis is mediated by two kinds of receptor sites that are simultaneously stimulated by physiological amines. To establish a correlation between the binding characteristics of the receptor and biological functions, the ability of physiological amines to stimulate or inhibit isolated fat cell lipolysis in vitro was compared to the beta- and alpha 2-adrenoceptor properties of the same fat cell batch. The beta-selective antagonist (-)[3H]dihydroalprenolol ([3H]DHA) and the alpha 2-selective antagonists [3H]yohimbine ([3H]YOH) and [3H]rauwolscine ([3H]RAU) were used to identify and characterize the two receptor sites. Binding of each ligand was rapid, saturable, and specific. The results demonstrate 1) the weaker lipolytic effect of epinephrine compared with norepinephrine. This can be explained by the equipotency of the amines at the beta 1-sites and the higher affinity of epinephrine for alpha 2-adrenergic receptors. 2) The preponderance of alpha 2-adrenergic receptor sites labeled by [3H]YOH (Bmax, 586 +/- 95 fmol/mg protein; KD, 2.7 +/- 0.2 nM) or [3H]RAU (Bmax, 580 +/- 100 fmol/mg protein; KD, 3.7 +/- 0.1 nM). These two ligands can be successfully used to label alpha 2-adrenergic receptor sites. 3) The beta 1-adrenergic receptor population labeled by [3H]DHA(Bmax, 234 +/- 37 fmol/mg protein; KD, 1.8 +/- 0.4 nM), although a third as numerous as the alpha 2-adrenergic population, is responsible for the lipolytic effect of physiological amines and is weakly counteracted by simultaneous alpha 2-adrenergic receptor stimulation under our experimental conditions. It is concluded that, in human fat cells, the characterization of beta 1- and alpha 2-adrenergic receptors by saturation studies or kinetic analysis to determine affinity (KD) and maximal number of binding sites (Bmax) is not sufficient for an accurate characterization of the functional adrenergic receptors involved in the observed biological effect.     

[3H]5-Hydroxytryptamine Binding to Brain Astroglial Cells: Differences Between Intact and Homogenized Preparations and Mature and Immature Cultures

[3H]5-hydroxytryptamine ([3H]serotonin) binds with high affinity (KD 2-12 nM) to a finite number of sites on brain astroglial cells. The number of binding sites in the C6 glioma line is decreased significantly (Bmax = 315 versus 30 fmol/mg) by homogenization. In intact primary cultures, derived from newborn rat brain, the number of binding sites is far greater in cultures of immature astrocytes than in cultures treated with dibutyryl cyclic AMP (Bmax = 1,520 versus 580 fmol/mg). A role for these receptors in development is suggested.     

3H]mepyramine binding to histamine H1 receptors in bovine retina

The promethazine-sensitive [3H]mepyramine binding was used to determine the presence of histamine H1 receptors in membranes from bovine retina. Specific mepyramine binding to retinal membranes was reversible, saturable and of high affinity. The apparent dissociation constant (KD = 2.2 +/- 0.4 nM) and the density of binding sites (Bmax = 60.9 +/- 5.1 fmol/mg protein), obtained in equilibrium studies, were similar to those found in bovine brain cortex. Binding was stereospecific and the inhibitory potencies of H1 and H2 antagonists indicated that [3H] mepyramine binding sites in the retina have characteristics of H1 receptors.     

Effect of chronic capsaicin treatment on tachykinin NK1 binding sites in the rat.

Binding sites for [125I]-Bolton-Hunter substance P (BHSP) were investigated in homogenates of rat submandibular gland, colon smooth muscle, and urinary bladder. In vehicle-treated animals, the equilibrium dissociation constant (KD) was similar for both submandibular gland (0.46 +/- 0.03 nM) and colon (0.57 +/- 0.04 nM), although the maximum density of binding sites (Bmax) was about six-fold higher in submandibular gland compared with colon. These binding parameters remained unchanged in capsaicin-pretreated animals (140 mg/kg IP). In contrast, capsaicin pretreatment reduced (p less than 0.05) the Bmax in urinary bladder by twenty-five percent (0.56 fmol/mg wet weight) when compared to vehicle-treated controls (0.73 fmol/mg wet weight), although the KD was unchanged (vehicle, 0.29 +/- 0.08 nM; capsaicin, 0.24 +/- 0.04 nM). These data demonstrate that the NK1 receptors in submandibular gland and colon smooth muscle are not associated with or dependent upon intact primary afferent sensory neurons. However, a minority of NK1 receptors in the urinary bladder were lost after capsaicin, indicating that these receptors are located on sensory terminals, or may be dependent on growth factors or other chemicals released from these nerves.     

Specific Binding of Insulin to Membranes from Dendrodendritic Synaptosomes of Rat Olfactory Bulb

The external plexiform layer of the olfactory bulb is among the brain regions where insulin receptors are most abundant. In vitro binding of porcine 125I-insulin to membranes of dendrodendritic synaptosomes isolated from adult rat olfactory bulbs was studied to test the hypothesis that dendrodendritic synapses are major insulin-receptive sites in the external plexiform layer of olfactory bulbs. Of the specific insulin binding sites present in a total particulate fraction from the olfactory bulbs, approximately half were recovered in the dendrodendritic synaptosome fraction. The only other subcellular fraction to which substantial insulin binding was observed was the conventional (axodendritic/axosomatic) synaptosome fraction. Analysis of equilibrium binding of insulin to dendrodendritic synaptosomal membranes, at total insulin concentrations of 0.5-1,000 nM, revealed binding site heterogeneity consistent with a two-site model for insulin binding to a high-affinity (KD = 6 nM), low-capacity (Bmax = 110 fmol/mg of protein) site and a low-affinity (KD = 190 nM), high-capacity (Bmax = 570 fmol/mg of protein) site. The results indicate that the intense labeling of the external plexiform layer of the olfactory bulb in autoradiographic studies of insulin binding can be attributed to insulin receptors on dendrodendritic synaptic membranes in this region.     

Binding characteristics and affinity labeling of protein constituents of the human IM-9 lymphoblast receptor for substance P

The neuropeptide substance P (SP) stimulates human T-lymphocyte function in vitro. Human blood T-lymphocytes and cultured human IM-9 B-lymphoblasts express 7,000-10,000 and 25,000-30,000 substance P receptors per cell, respectively. The specific binding of 125I-SP is retained in IM-9 lymphoblast membranes solubilized in 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid (CHAPS) at a detergent-to-protein ratio of 1.0. In addition, specific and reversible SP binding to soluble IM-9 cell membrane proteins is demonstrated by gel filtration. The saturation of binding of 125I-SP to both intact and solubilized IM-9 cell membranes attained a steady state after 40-50 min at 4 degrees C. Scatchard analysis of the concentration dependence of 125I-SP binding to IM-9 cell membranes revealed a KD of 0.87 +/- 0.8 nM (mean +/- S.D., n = 4), which is similar to that observed in intact cells, and a density of receptors of 21 +/- 3 fmol/mg of membrane protein (mean +/- S.D.). Binding of 125I-SP to solubilized membranes demonstrated a KD of 0.75 +/- 0.33 nM (mean +/- S.D., n = 3) and a density of receptors of 3.7 +/- 1.5 fmol/mg of membrane protein (mean +/- S.D., n = 3). Affinity cross-linking of 125I-SP by disuccinimidyl suberate to intact IM-9 cells and membranes revealed specifically labeled proteins of Mr 58,000 and 33,000 in cells, and 58,000, 33,000, and 16,000 in membranes by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under both reducing and nonreducing conditions. Competitive effects of substituent peptides of SP on cross-linking and 125I-SP binding to membranes demonstrated that the SP receptor recognized the carboxyl-terminal domain of the peptide. Membranes from cells preincubated in vitro for 12 h at 37 degrees C with 10(-8) M SP demonstrated a decrease in SP receptor density to 13 +/- 2 fmol/mg (mean +/- S.D., n = 2), and a parallel diminution in the specific labeling of membrane proteins of Mr 58,000 and 33,000. These observations suggest that solubilization in CHAPS preserves the binding characteristics of the IM-9 lymphoblast receptor for SP, and that affinity cross-linking techniques identify by sodium dodecyl sulfate-polyacrylamide gel electrophoresis membrane proteins that are specifically labeled by SP.     

Presynaptic Cholinergic Mechanisms in the Rat Cerebellum: Evidence for Nicotinic, but Not Muscarinic Autoreceptors

The present study shows that N-[3H]methylcarbamylcholine ([3H]MCC) binds to a single population of high-affinity/low-density (KD = 5.0 nM; Bmax = 8.2 fmol/mg of protein) nicotinic binding sites in the rat cerebellum. Also, there exists a single class of high-affinity binding sites (KD = 4.8 nM; Bmax = 24.2 fmol/mg of protein) in the cerebellum for the M1 specific muscarinic ligand [3H]pirenzepine. In contrast, the M2 ligand, [3H]AF-DX 116, appears to bind to two classes of binding sites, i.e., a high-affinity (KD = 3 nM)/low-capacity (Bmax = 11.7 fmol/mg of protein) class, and a second class of lower affinity (KD = 28.4 nM) and higher capacity (Bmax = 36.3 fmol/mg of protein) sites. The putative M3 selective ligand [3H]4-diphenylacetoxy-N-methylpiperidine also binds to two distinct classes of binding sites in cerebellar homogenates, one of high affinity (KD = 0.5 nM)/low capacity (Bmax = 19.5 fmol/mg of protein) and one of low affinity (KD = 57.5 nM)/high capacity (Bmax = 140.6 fmol/mg of protein). In experiments which tested the effects of cholinergic drugs on acetylcholine release from cerebellar brain slices, the nicotinic agonist MCC enhanced spontaneous acetylcholine release in a concentration-dependent manner, and the maximal increase in acetylcholine release (59.0-68.0%) occurred at 10(-7) M. The effect of MCC to increase acetylcholine release was Ca2+-dependent and tetrodotoxin-insensitive, suggesting an action on cholinergic terminals. Also, the MCC-induced increase in acetylcholine release was effectively antagonized by dihydro-beta-erythroidine, d-tubocurarine, and kappa-bungarotoxin, but was insensitive to either atropine or alpha-bungarotoxin.(ABSTRACT TRUNCATED AT 250 WORDS)     

Flunitrazepam Binding to Intact and Homogenized Astrocytes and Neurons in Primary Cultures

[3H]Flunitrazepam binds to intact and homogenized mouse astrocytes and neurons in primary cultures. In intact cells, the binding is to a single, high-affinity, saturable population of benzodiazepine binding sites with a KD of 7 nM and Bmax of 6,033 fmol/mg protein in astrocytic cells and a KD of 5 nM and Bmax of 924 fmol/mg protein in neurons. After homogenization, the Bmax values decrease drastically in both cell types, but most in astrocytes. The temperature and time dependency are different for the two cell types, with a faster association and dissociation in astrocytes than in neurons and a greater temperature sensitivity in the astrocytes. Moreover, flunitrazepam binding sites on neuronal and astrocytic cells have different pharmacological profiles. In intact astrocytic cells, Ro 5-4864 (Ki = 4 nM) is the most potent displacing compound, followed by diazepam (Ki = 6 nM) and clonazepam (Ki = 600 nM). In intact neurons, the relative order of potency of these three compounds is different: diazepam (Ki = 7 nM) is the most potent, followed by clonazepam (Ki = 26 nM) and Ro 5-4864, which has little effect. After homogenization the potency of diazepam decreases. We conclude that both neuronal and astrocytic cells possess high-affinity [3H]flunitrazepam binding sites. The pharmacological profile and kinetic characteristics differ between the two cell types and are further altered by homogenization.     

High-affinity prostaglandin E receptors attenuate adenylyl cyclase activity in isolated bovine myometrial membrane

Purified bovine myometrial plasma membranes were used to characterize prostaglandin (PG) E2 binding. Two binding sites were found: a high-affinity site with a dissociation constant (KD) of 0.27 +/- 0.08 nM and maximum binding (Bmax) of 102.46 +/- 8.6 fmol/mg membrane protein, and a lower affinity site with a KD = 6.13 +/- 0.50 nM and Bmax = 467.93 +/- 51.63 fmol/mg membrane protein. Membrane characterization demonstrated that [3H]PGE2 binding was localized in the plasma membrane. In binding competition experiments, unlabelled PGE1 displaced [3H]PGE2 from its receptor at the same concentrations as did PGE2. Neither PGF2 alpha nor PGD2 effectively competed for [3H]PGE2 binding. Adenylyl cyclase activity was inhibited at concentrations of PGE2 that occupy the high-affinity receptor. These data demonstrate that two receptor sites, or states of binding within a single receptor, are present for PGE2 in purified myometrial membranes. PGE2 inhibition of adenylyl cyclase activity support the view that cAMP has a physiological role in the regulation of myometrial contractility by PGE2.     

Evidence that the potential antipsychotic agent rimcazole (BW 234U) is a specific, competitive antagonist of sigma sites in brain

Rimcazole (BW 234U) is a potential antipsychotic agent which in open-clinical trials appears to be effective in acute schizophrenic patients. In the present study, rimcazole was found to block the specific binding of [3H]-(+)-SKF 10,047 to sigma sites in rat and guinea pig brain (IC50 = 5.0 X 10(-7) M). The compound was 100 times weaker as a blocker of phencyclidine sites (IC50 = 4.3 X 10(-5) M). At 1 X 10(-5) M, rimcazole had only weak effects on mu, delta, kappa and epsilon opioid receptors. Scatchard analysis of the binding data from guinea pig brain revealed an apparent KD for [3H]-(+)-SKF 10,047 of 85 +/- 5 nM and a Bmax of 824 +/- 27 fmole/mg protein. In the presence of 5 X 10(-7) M BW 234U, the apparent KD was 165 +/- 35 nM, but the Bmax (892 +/- 146 fmoles/mg protein) was not affected. This suggests that rimcazole is a competitive inhibitor of sigma sites. The agent was also capable of blocking sigma sites in vivo (ID50 = 6 mg/kg i.p., mice) as judged by an in vivo sigma receptor binding assay. Thus, if the antipsychotic activity of rimcazole is confirmed in double-blind, placebo-controlled trials, it would be the first compound whose mechanism of antipsychotic activity may best be explained by a direct blockade of sigma sites and not by a direct blockade of dopamine (D2) receptors in brain.