Involvement of protein kinase C and protein kinase A in the muscarinic receptor signalling pathways mediating phospholipase C activation, arachidonic acid release and calcium mobilisation.

The involvement of protein kinase C (PKC) and protein kinase A (PKA) in cholinergic signalling in CHO cells expressing the M3 subtype of the muscarinic acetylcholine receptor was examined. Muscarinic signalling was assessed by measuring carbachol-induced activation of phospholipase C (PLC), arachidonic acid release, and calcium mobilisation. Carbachol activation of PLC was not altered by inhibition of PKC with chelerythrine chloride, bisindolylmaleimide or chronic treatment with phorbol myristate acetate (PMA). Activation of PKC by acute treatment with PMA was similarly without effect. In contrast, inhibition of PKC blocked carbachol stimulation of arachidonic acid release. Likewise, PKC inhibition resulted in a decreased ability of carbachol to mobilise calcium, whereas PKC activation potentiated calcium mobilisation. Inhibition of PKA with H89 or Rp-cAMP did not alter the ability of carbachol to activate PLC. Similarly, PKA activation with Sp-cAMP or forskolin had no effect on PLC stimulation by carbachol. Carbachol-mediated release of arachidonic acid was decreased by H89 but only slightly increased by forskolin. Forskolin also increased calcium mobilisation by carbachol. These results suggest a function for PKC and PKA in M3 stimulation of arachidonic acid release and calcium mobilisation but not in PLC activation.

M3 muscarinic acetylcholine receptors regulate cytoplasmic myosin by a process involving RhoA and requiring conventional protein kinase C isoforms.

Although muscarinic acetylcholine receptors (mAChR) regulate the activity of smooth muscle myosin, the effects of mAChR activation on cytoplasmic myosin have not been characterized. We found that activation of transfected human M3 mAChR induces the phosphorylation of myosin light chains (MLC) and the formation of myosin-containing stress fibers in Chinese hamster ovary (CHO-m3) cells. Direct activation of protein kinase C (PKC) with phorbol 12-myristate 13-acetate (PMA) also induces myosin light chain phosphorylation and myosin reorganization in CHO-m3 cells. Conventional (alpha), novel (delta), and atypical (iota) PKC isoforms are activated by mAChR stimulation or PMA treatment in CHO-m3 cells, as indicated by PKC translocation or degradation. mAChR-mediated myosin reorganization is abolished by inhibiting conventional PKC isoforms with Go6976 (IC50 = 0.4 microM), calphostin C (IC50 = 2.4 microM), or chelerythrine (IC50 = 8.0 microM). Stable expression of dominant negative RhoAAsn-19 diminishes, but does not abolish, mAChR-mediated myosin reorganization in the CHO-m3 cells. Similarly, mAChR-mediated myosin reorganization is diminished, but not abolished, in CHO-m3 cells which are multi-nucleate due to inactivation of Rho with C3 exoenzyme. Expression of dominant negative RhoAAsn-19 or inactivation of RhoA with C3 exoenzyme does not affect PMA-induced myosin reorganization. These findings indicate that the PKC-mediated pathway of myosin reorganization (induced either by M3 mAChR activation or PMA treatment) can continue to operate even when RhoA activity is diminished in CHO-m3 cells. Conventional PKC isoforms and RhoA may participate in separate but parallel pathways induced by M3 mAChR activation to regulate cytoplasmic myosin. Changes in cytoplasmic myosin elicited by M3 mAChR activation may contribute to the unique ability of these receptors to regulate cell morphology, adhesion, and proliferation.

Ethanol disrupts carbamylcholine-stimulated release of arachidonic acid from Chinese hamster ovary cells expressing different subtypes of human muscarinic receptor.

Ethanol disrupts signal transduction mediated by a variety of G-protein coupled receptors. We examined the effects of ethanol on arachidonic acid release mediated by muscarinic acetylcholine receptors. Chinese hamster ovary (CHO) cells transfected with the different subtypes of human muscarinic receptors (M1 to M5) were incubated with [3H]arachidonic acid ([3H]AA) for 18 hr, washed, and exposed to the cholinergic agonist carbamylcholine for 15 min. Carbamylcholine induced [3H]AA release from CHO cells expressing M1, M3, or M5, but not M2 or M4, muscarinic receptors. Dose response curves revealed that carbamylcholine stimulated [3H]AA release by up to 12-fold with an ECo of approximately 0.4 microM; maximal responses were obtained with 10 microM carbamylcholine. Exposure of M1-, M3-, or M5-expressing cells to ethanol for 5 min before stimulating with carbamylcholine reduced [3H]AA release by 40 to 65%; 50% of the maximal inhibition was obtained with an ethanol concentration of 30 to 50 mM. Ethanol did not affect basal [3H]AA release measured in the absence of carbamylcholine. Dose response curves suggest that ethanol acts as a noncompetitive inhibitor of muscarinic receptor-induced [3H]AA release insofar as maximal [3H]AA release was depressed in the presence of ethanol with no apparent change in the EC50 for stimulation by carbamylcholine. Exposure of CHO cells to 38 mM ethanol for 48 hr increased [3H]AA release induced by carbamylcholine without affecting basal [3H]AA release or altering the EC50 for carbamylcholine. These results indicate that ethanol acutely inhibits muscarinic receptor signaling through the arachidonic acid pathway in a noncompetitive manner, but chronically enhances muscarinic signaling through the same pathway.

Influence of acute and chronic ethanol treatment on muscarinic responses and receptor expression in Chinese hamster ovary cells.

The influence of ethanol on the muscarinic receptor-mediated release of inositol phosphate from Chinese hamster ovary (CHO) cells stably transfected with one of the five subtypes of muscarinic acetylcholine receptor was determined. In CHO cells expressing M3 muscarinic receptors (CHO-M3), carbamylcholine increased muscarinic receptor-induced release of inositol phosphate by 150-350% following a 15-min incubation with an EC50 of approximately 30 microM. Maximal responses were obtained with 1 mM carbamylcholine, while responses to 10 mM carbamylcholine were somewhat less than maximal. Preincubation with atropine for 10 min inhibited the response with an IC50 of approximately 30 nM. CHO cells transfected with M1, M3, and M5 receptors displayed a similar pattern of activity; CHO cells transfected with M2 and M4, as well as untransfected cells, were unresponsive to carbamylcholine. Ethanol acutely inhibited the response of CHO-M3 cells to carbamylcholine by 15% at 18 mM and by 47% at 180 mM (the highest concentration examined). CHO-M3 cells were incubated with 50 mM ethanol for 48 hr. This treatment did not affect the number of cells or their protein content (113 pg/cell). The expression of M3 muscarinic receptors (determined using [3H]N-methylscopolamine) increased from 1.34 +/- 0.23 to 1.75 +/- 0.16 pmol/mg protein (P < 0.05). In contrast, carbamylcholine-stimulated release of inositol phosphate was depressed by 40-70% in four experiments. Concentration-response analyses indicated a non-competitive inhibitory mechanism. This dissociation of muscarinic receptor expression and muscarinic signaling suggests a compensatory increase in receptor expression in response to chronic inhibition of muscarinic signaling by ethanol.

Inhibition of M3 muscarinic acetylcholine receptor-mediated Ca2+ influx and intracellular Ca2+ mobilization in neuroblastoma cells by the Ca2+/calmodulin-dependent protein kinase inhibitor 1-[N,O-bis(5-isoquinolinesulfonyl)-N-methyl-L-trosyl]-4-phenylpiperazin e (KN-62).

The role of Ca2+/calmodulin-dependent protein kinase (CaM kinase; EC 2.7.1.123) in the generation of Ca2+ signals by muscarinic acetylcholine receptors (mAChR) was studied. Changes in intracellular Ca2+ concentrations ([Ca2+]i) induced by mAChR activation were monitored in SK-N-SH human neuroblastoma cells using the dye Fura-2. SK-N-SH cells express M3 mAChR, as well as CaM kinase types II and IV, which are specifically inhibited by the CaM kinase antagonist KN-62 (1-[N,O-bis(5-isoquinolinesulfonyl)-N-methyl-L-tyrosyl]-4-phenylpiperazi ne). Carbamylcholine (100 microM) elicited an initial transient peak in [Ca2+]i due to mobilization of Ca2+ from internal stores, followed by a sustained elevation in [Ca2+]i that depended on the influx of extracellular Ca2+ and which was inhibited by EGTA and Ni2+. These mAChR-induced Ca2+ signals were diminished to an equal extent by preincubating the cells with 0.01 to 100 microM KN-62. KN-62 inhibited mAChR-induced Ca2+ influx and mobilization from internal stores by about 25-30%, producing a half-maximal effect at approximately 1 microM. In contrast, KN-62 (25 microM) almost completely abolished carbamylcholine-stimulated entry of divalent cations through Mn2+-permeant channels, as revealed by Mn2+ quenching of Fura-2 fluorescence. KN-62 also almost completely abolished Ca2+ influx induced by depolarization of the cells with 25 mM K+ (IC50 = 3 microM). These results suggest that CaM kinases regulate both the mobilization of intracellular Ca2+ and the stimulation of Ca2+ influx that are induced by mAChR activation, and indicate that the mAChR-induced influx of Ca2+ occurs through Ca2+ channels other than, or in addition to, the voltage-gated calcium channels or Mn2+-permeant channels which are inhibited by KN-62.

Characterization of the muscarinic receptor subtypes in the bovine corneal epithelial cells.

Muscarinic receptor subtypes in the bovine corneal epithelial cells (BCE) were characterized on the basis of their: 1) ligand binding properties, 2) linkage to Ca2+ and cAMP cell signaling pathways, and 3) gene transcripts. Receptor subtypes, m1 and m2, are indicated by competition experiments using subtype-selective muscarinic receptor ligands. [3H]N-methylscopolamine ([3H]-MS) binding was displaced with IC50s of: 1) 1 microM for the m1 antagonist, pirenzipine; 2) 51 microM for the competitive m2 antagonist, AFDX-116; 3) 100 microM for the competitive m3 antagonist, 4-DAMP. In fural2 loaded BCE, carbachol (0.001 - 100 microM) increased intracellular Ca2+ concentration ([Ca2+]i), and these responses were significantly suppressed if they were preincubated with either atropine (1 microM) or 1 microM pirenzipine. In the absence of extracellular Ca2+, these carbachol-induced increases in [Ca2+]i were depressed. A considerable fall occurred with the presence of extracellular Ca2+ and 1 microM verapamil, an L-type Ca2+ channel blocker. These responses suggest that carbachol increases Ca2+ influx through an L-type Ca2+ channel in the plasma membrane, in addition to mobilizing Ca2+ from an intracellular store. BCE also possessed muscarinic receptors which were negatively linked to cAMP production insofar as: 1) preincubation with 10 microM carbachol significantly suppressed the increases in cAMP accumulation induced by isoproterenol (1 - 25 microM); 2) this blunting effect of carbachol on cAMP production was eliminated when the BCE were preincubated with either 1 microM AFDX-116, or 100 ng/ml pertussis toxin. The results of probing for muscarinic receptor gene expression are partially consistent with the ligand binding and functional assays. Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis revealed the presence of m2 but not m1, m3 or m4 gene transcripts. In summary, we obtained pharmacological and functional evidence for m1 and m2 receptors in BCE. However, only the m2 gene transcript could be detected.

Spermidine attenuation of volatile anesthetic inhibition of glutamate-stimulated [3H](5D,10S)-(+)-methyl-10,11-dihydro-5H- dibenzo[a,d]cyclohepten-5,10-imine ([3H]MK-801) binding to N-methyl-D-aspartate (NMDA) receptors in rat brain.

The influence of spermidine, a polyamine agonist, on volatile anesthetic inhibition of N-methyl-D-aspartate (NMDA) receptor activation, as indicated by glutamate stimulation of [3H]MK-801 ([3H](5D,10S)-(+)-methyl-10,11-dihydro-5H- dibenzo[a,d]cyclohepten-5,10-imine) binding, was studied in rat brain. Spermidine reserved the inhibition caused by four volatile anesthetics (enflurane, halothane, methoxyflurane and chloroform) at the same concentrations (EC50 approximately 3 microM) at which it potentiated glutamate opening of the NMDA ion channel. The anesthetics had no effect on the direct stimulation of channel opening by spermidine, which occurred at concentrations of spermidine greater than 30 microM in the absence of receptor agonist. In these actions, spermidine closely resembled the allosteric co-agonist glycine. The present results suggest that anesthetic action on NMDA receptors involves a set of sites on the channel complex that is distinct from the recognition sites for glutamate, glycine, and channel blockers, and are consistent with the idea that blockade of NMDA receptors contributes to the development of the anesthetic state.

Volatile anesthetics and glutamate activation of N-methyl-D-aspartate receptors.

Several studies have indicated important functional interactions between volatile anesthetics and the N-methyl-D-aspartate (NMDA) class of glutamate receptors. In the present study, we examined the effects of diethyl ether, chloroform, methoxyflurane, halothane, enflurane, and isoflurane on (1) glutamate activation of the NMDA receptor complex, including glycine reversal of anesthetic action, as revealed by [3H]-(5R, 10S)-(+)methyl-10,11-dihydro-5H-dibenzo [a,d]cyclohepten-5,10-imine, dizocilpine (MK-801) binding to the cation channel, and (2) [3H]cis-4-( phosphonomethyl)piperidine-2-carboxylic acid (CGS 19755) binding to the glutamate recognition site of the NMDA receptor In agreement with previous studies, glutamate increased the binding of 1 nM [3H]MK-801, measured after a 1-hr incubation at 37 degrees, by up to several hundred fold. This stimulation was blocked by glutamate antagonists and potentiated by glycine with an EC50 of approximately 0.03 muM. Glycine also had a direct stimulatory effect on [3H]MK-801 binding at much higher concentrations ( > or = 10 muM). All of the anesthetics examined depressed glutamate stimulation of [3H]MK-801 binding in a concentration-dependent manner with the following order of potency: halothane > or = enflurane > methoxyflurane > chloroform > diethyl ether. This inhibition of [3H]MK-801 binding was observed at concentrations that are routinely attained in the cerebrospinal fluid during surgical anesthesia. Moreover, the inhibition was reversed rapidly following removal of the anesthetics from the assay medium. Inclusion of glycine in the incubation medium markedly attenuated anesthetic-induced inhibition of glutamate-sensitive [3H]MK-801 binding with an EC50 of between 0.1 and 1 muM. Thus, this reversal by glycine correlated with its potentiating as opposed to its direct stimulatory, effect on NMDA receptors. Anesthetic inhibition of [3H]MK-801 binding could not be overcome by raising the glutamate concentration (i.e. the interaction did not appear to be competitive with respect to glutamate) unless glycine was present. Binding of [3H]CGS 19755 to the glutamate recognition site was also inhibited by each of the anesthetics examined. However, with the exception of chloroform, all of the anesthetics were more potent inhibitors of glutamate-stimulated [3H]MK-801 binding than they were of [3H]CGS 19755 binding. [3H]CGS 19755 binding saturation curves in the presence of halothane and enflurane indicated a decrease in the density of [3H]-CGS 19755 binding sites with no change in binding affinity (i.e. the inhibition did not appear to be competitive). These findings support the idea that anesthetic drugs disrupt NMDA receptor transmission through multiple allosteric effects on the receptor-channel activation mechanisms and the glutamate binding site.

Volatile anesthetics inhibit NMDA-stimulated 45Ca uptake by rat brain microvesicles.

We have previously shown that volatile anesthetics inhibit glutamate-stimulated [3H]MK-801 binding to the ionophore of NMDA receptor complexes in rat brain. In the present study, we examined the influence of enflurane and halothane on NMDA-stimulated 45Ca uptake by a microvesicle fraction isolated from rat brain. NMDA stimulated 45Ca uptake (30 sec) by rat brain microvesicles by up to 70% with an EC50 of 1.4 +/- 0.5 microM. The NMDA-stimulated 45Ca uptake was inhibited by MK-801 and D-AP-5 with IC50's of approximately 10 microM. Enflurane and halothane inhibited 45Ca uptake stimulated by 100 microM NMDA by as much as 60-80% with IC50's of 0.2-0.3 mM, concentrations achieved during routine clinical use. Basal 45Ca uptake measured in the absence of agonist was not affected by the anesthetics. Glycine did not affect the level of NMDA-stimulated 45Ca uptake, but markedly reduced the inhibition of uptake caused by enflurane and halothane. Preincubation of microvesicles with NMDA resulted in a desensitization of NMDA-stimulated 45Ca uptake, with a t1/2 of approximately 20 sec. Enflurane and halothane diminished both the extent and rate of development of this desensitization, as did glycine. These findings support the idea that volatile anesthetic interference with neurotransmission at NMDA receptor complexes contributes to the development of the anesthetic state.

Role of prostanoids in the regulation of central cholinergic receptor sensitivity.

Tachyphylaxis develops to the hypertensive response to central (i.c.v.) injection of carbachol in conscious rats. This pressor response exhibits tachyphylaxis if the injection is repeated within 8 hr of the first injection. Blockade of brain prostaglandin synthesis with indomethacin does not inhibit the pressor response to carbachol in naive rats, but eliminates the pressor response to carbachol when the muscarinic agonist is repeated within a few hours of the first injection. If the time interval is extended to permit return of the full response (i.e., 24 hr later), indomethacin no longer inhibits the pressor response. The related cyclooygenase inhibitor meclofenamate produced effects which were identical to those of indomethacin, but at approximately 10-fold higher doses. When shorter acting drugs (duration of action < 30 min), physostigmine or arecoline, were used according to the same paradigm, indomethacin was less effective at inhibiting the pressor response to the second injection, even when the two agonist injections were spaced only 30 min apart. The ability of indomethacin to enhance central muscarinic receptor tachyphylaxis was also observed in carbachol-induced hypothermia. The density of diencephalic muscarinic receptors was estimated by using N-[3H]methylscopolamine as a probe. Carbachol-induced a down-regulation of muscarinic receptors, and indomethacin increased the extent of this down regulation. These findings suggest that prostaglandins play a role in the development of tachyphylaxis to brain muscarinic receptor stimulation: activation of prostaglandin synthesis may decelerate the development of desensitization to muscarinic agonists.

Retinoic acid-induced differentiation of a human neuroblastoma cell line alters muscarinic receptor expression.

Muscarinic receptor density increased by approximately 36% after differentiation induced by retinoic acid (Bmax, control = 126 +/- 13 fmol/mg protein; Bmax, retinoic acid-treated = 170 +/- 17 fmol/mg protein; P < 0.05), corresponding to a 170% increase in receptor content per cell. The affinity of [3H]NMS for the receptors was somewhat lower in the retinoic acid-treated cells (Kd, control = 0.14 +/- 0.04 nM; Kd, retinoic acid-treated = 0.25 +/- 0.04 nM; P < 0.05). Reverse transcriptase/polymerase chain reaction analysis using subtype-specific primers revealed that undifferentiated Sk-N-SH cells transcribed mRNA for all 5 receptor subtypes; this pattern was not affected by retinoic acid treatment. [3H]NMS displacement curves with subtype selective receptor ligands (pirenzepine, m1; AFDX-116, m2; 4-DAMP, m3) indicated the predominant expression of m3 and m1 receptor subtypes, and differentiation did not affect the pharmacological profile of the expressed receptor populations. The present results indicate that differentiation induces selective changes in the expression and activity of muscarinic receptors in a neuronal cell line.

Test-tube equilibration of volatile anaesthetic concentrations.

We have measured concentrations of volatile anaesthetics after injection into test-tube assay solutions. In non-agitated tubes, aqueous anaesthetic concentrations decreased significantly throughout 40 min of incubation. Agitation (20-s vortex) of the tubes decreased the aqueous anaesthetic concentration by approximately 80% from concentrations in non-agitated tubes and resulted in more stable liquid anaesthetic concentrations over time. A decrease of less than 15% occurred between 10 and 50 min of incubation.

A novel agonist binding site on nicotinic acetylcholine receptors.

This report provides evidence that physostigmine (Phy) and benzoquinonium (BZQ) are able to activate nicotinic acetylcholine receptors (nAChRs) through binding site(s) distinct from those of the natural transmitter, ACh. Such findings are in agreement with a second pathway of activation of nAChRs. Receptor activation may be modulated through the novel site, and, consequently, physiological processes involving nicotinic synapses could be controlled. Using patch clamp techniques, single channel currents activated by ACh and anatoxin were recorded from frog interosseal muscle fibers under cell-attached condition and outside-out patches excised from cultured rat hippocampal neurons. Whole cell nicotinic currents were also studied in the cultured neurons. In most of the neurons, nicotinic responses were blocked by the nicotinic antagonists methyllycaconitine (MLA) and alpha-bungarotoxin (alpha-BGT). Evaluation of the effects of Phy and BZQ on the muscle and on the alpha-BGT- and MLA-sensitive neuronal nAChRs demonstrated that both compounds were open channel blockers at these receptors. Furthermore, at low micromolar concentrations, Phy and BZQ activated the nAChRs of all preparations tested, such an effect being unexpectedly resistant to alpha-BGT or MLA. Thus, the nAChRs could be activated via two distinct binding sites: one for ACh and the other for Phy and BZQ. These findings and previous biochemical results led us to suggest that a putative endogenous ligand could bind to the new site and thereby regulate the activation of nAChRs in nicotinic synapses.

Effect of colchicine and taxol on stimulation of G protein GTPase activity in anterior pituitary lobe of rats by gonadotrophin- and thyrotrophin-releasing hormones.

Colchicine and taxol, which are known to influence tubulin function, were used to delineate the possible role of tubulin in signal transduction in the anterior pituitary lobe. Anterior pituitary lobes, obtained from adult male rats, were processed by discontinuous sucrose gradient centrifugation to obtain plasma membranes. The low Km GTPase activity (EC 3.6.1.) was assayed in 5 micrograms membrane protein using [gamma-32P]GTP at 37 degrees C in an ATP-regenerating buffer containing 1 mumol unlabelled GTP l-1. Ten nmol l-1 each of colchicine, lumicolchicine and taxol maximally stimulated the GTPase activity by about 40% (P < 0.05). A time-course study revealed that 100 nmol colchicine l-1 stimulated the enzyme activity by 55, 74, 89 and 53% at 5, 10, 20 and 30 min, respectively (P < 0.05); lumicolchicine (100 nmol l-1) stimulated the GTPase activity by 44, 36, 11 and 55% at 5, 10, 20 and 30 min, respectively (P < 0.05). Taxol (100 nmol l-1) stimulated the enzyme activity by 39 and 25% at 20 and 30 min, respectively (P < 0.05). Gonadotrophin-releasing hormone (GnRH) and thyrotrophin-releasing hormone (TRH) stimulated the low Km GTPase activity in a concentration-dependent manner, by up to 40-60% (P < 0.05). In the presence of 100 nmol colchicine l-1, the ability of GnRH or TRH to stimulate the GTPase activity was inhibited.(ABSTRACT TRUNCATED AT 250 WORDS)

Substance P receptor desensitization in the dorsal horn: possible involvement of receptor-G protein complexes.

The repeated administration of a high dose of substance P (SP) onto the spinal cord has been shown to attenuate behavioral responses to an intense heat (tail-flick) or noxious mechanical stimulus (paw pressure). Studies performed to investigate the action of spinal SP have suggested that changes in behavioral responses involve endogenous opiate or neurokinin systems. This study was performed to investigate whether the binding characteristics of SP receptors in the dorsal horn are altered following successive administration of SP. Two populations of [3H]-SP binding sites were distinguished on the basis of their binding affinity. Gpp(NH)p, a stable analogue of GTP, decreased the size and affinity of the high affinity binding component selectively labelled with [125I]-Bolton Hunter-SP. Repeated intrathecal administration of SP (15 micrograms) which reduced behaviors also reduced the number and affinity of high affinity binding sites. Thus, attenuated behaviors in response to repeated administration of SP are paralleled by an alteration of SP binding in the dorsal horn. The altered agonist affinity seen under desensitizing conditions raises the possibility that SP receptor desensitization involves an uncoupling of receptor-G protein complexes.

Stimulation of the release of [32P]guanosine 5'-diphosphate from G proteins in the rat anterior pituitary lobe by gonadotrophin-releasing and thyrotrophin-releasing hormones.

Plasma membranes (1-2 mg protein) purified from the anterior pituitary lobes of adult male rats were incubated with 0.6 mumol [alpha-32P]guanosine 5'-triphosphate (GTP) l-1 in an ATP-regenerating buffer at 37 degrees C for 60 min; during this incubation the [32P]GTP was hydrolysed and the nucleotide that was predominantly bound to the membranes was [32P]GDP. The release of [32P]GDP from the membranes was monitored at 37 degrees C; the amount released was proportional to the protein concentration and increased as a function of time. 5'-Guanylylimidodiphosphate (Gpp(NH)p) increased [32P]GDP release by up to 30% at 0.1 mumol l-1. Although 10 nmol Gpp(NH)p l-1 had no effect on GDP release, it appeared to stabilize the hormonal effect by blocking further GDP-GTP exchange. Gonadotrophin-releasing hormone (GnRH) agonist and thyrotrophin-releasing hormone (TRH), at 0.1 mumol l-1 caused a maximum increase in the release of [32P]GDP of 31-38%. The GnRH agonist (0.1 mumol l-1) stimulated GDP release by 21%, 24%, 17% and 14% at 30 s, 1, 2 and 5 min, respectively. TRH (0.1 mumol l-1) stimulated GDP release by 38%, 30%, 17% and 16% at 30 s, 1, 2 and 5 min, respectively. A GnRH antagonist also stimulated [32P]GDP release, albeit less effectively than GnRH agonist; the antagonist did not inhibit agonist stimulation of GDP release. These results indicate that ligand binding to the GnRH and TRH receptors results in interaction of the receptor with a guanine-nucleotide-dependent transducer protein (G protein) and activation of GTP-GDP exchange.(ABSTRACT TRUNCATED AT 250 WORDS)

Pyrazole, an alcohol dehydrogenase inhibitor, has dual effects on N-methyl-D-aspartate receptors of hippocampal pyramidal cells: agonist and noncompetitive antagonist.

Electrophysiological and biochemical studies demonstrated that pyrazole, an inhibitor of alcohol dehydrogenase and a proposed therapeutic agent for treatment of alcoholic intoxication, activated and blocked the N-methyl-D-aspartate (NMDA) receptor and did not interact significantly with the end-plate nicotinic acetylcholine receptor (AChR). Pyrazole, at concentrations as low as 0.5 microM, applied to outside-out patches excised from the membrane of cultured rat hippocampal neurons, elicited single-channel currents of 48 pS which were blocked by DL-2-amino-5-phosphorovaleric acid, a competitive antagonist of NMDA. In addition, binding studies showed that pyrazole displaced 1-(cis-2-carboxypiperidine-4-yl)methyl-1-phosphoric acid from the agonist recognition site of the NMDA receptor in a concentration-dependent manner and enhanced the binding of (+)-5-methyl-10,11-dihydro-5H- dibenzo[a,d]cyclohepten-5,10-imine to this complex. These data indicate that pyrazole is an agonist at NMDA receptors. However, at higher concentrations, open and burst times as well as the frequency of single-channel currents activated by pyrazole were reduced significantly, a finding which suggests that this compound is also an open channel blocker. In agreement with these results, it was shown biochemically that pyrazole was able to stimulate influx of Ca++ into rat brain microsomes via NMDA receptors and on the other hand to block the influx of Ca++ induced by NMDA. Pyrazole was unable to affect the neuromuscular transmission of frog sartorius muscle-sciatic nerve preparations. Additionally, pyrazole did not interact either with the agonist recognition site or with noncompetitive sites of the AChR. However, this drug had a very weak agonist-like action on the AChR of the Torpedo electric organ, most likely via binding sites different from those described previously for acetylcholine. Therefore, the therapeutic efficacy of pyrazole may be related at least in part to its effects on the NMDA receptor. Furthermore, this compound, because of the small size and rigidity of its molecular structure, becomes a promising drug for the study of the NMDA receptor. Indeed its use may allow a better understanding of the physiological and pathological processes involving this receptor.

Progesterone, testosterone and estradiol-17 beta inhibit gonadotropin-releasing hormone stimulation of G protein GTPase activity in plasma membranes from rat anterior pituitary lobe.

In order to understand the biochemical mechanisms underlying the rapid, non-genomic effects of gonadal steroids on gonadotropin secretion, we examined the effects of progesterone, testosterone and estradiol-17 beta on the low Km GTPase activity associated with transducer G proteins coupled to gonadotropin-releasing hormone (GnRH) receptors. Homogenates of anterior pituitary lobes from adult male rats were processed by discontinuous sucrose gradient centrifugation to isolate plasma membranes. The low Km GTPase activity (EC 3.6.1.-) was assayed in 5 micrograms membrane protein using [gamma-32P]GTP at 37 degrees C in an ATP-regenerating buffer containing 1 mumol/l unlabeled GTP. One hundred nmol/l each of progesterone, testosterone and estradiol-17 beta maximally stimulated low Km GTPase activity by 61%, 59% and 45%, respectively (p less than 0.05). Time course studies revealed that 100 nmol/l progesterone stimulated the enzyme activity by 93% and 62% at 5 and 30 min, respectively; 100 nmol/l testosterone stimulated GTPase activity by 100% and 72% at 5 and 30 min, respectively; 100 nmol/l estradiol-17 beta stimulated GTPase activity by 80% and 70% at 5 and 30 min, respectively. GnRH stimulated the low Km GTPase activity by about 60% in a concentration-dependent manner. In the presence of the gonadal steroids, the ability of GnRH to stimulate the GTPase activity was inhibited. For example, stimulation ranged from 36% to 60% with 0.1-100 nmol/l GnRH alone, but only from 7% to 20% in the presence of GnRH and 100 nmol/l progesterone (p less than 0.05). Similarly, in the presence of 100 nmol/l estradiol-17 beta, GnRH stimulation of the enzymatic activity ranged from 12% to 19%.(ABSTRACT TRUNCATED AT 250 WORDS)

Muscarinic binding sites on bovine pulmonary arterial endothelial cells in culture.

We have investigated the presence and nature of muscarinic binding sites on membranes from cultured bovine pulmonary arterial endothelial cells (BPAE). BPAE were harvested and subcultured nonenzymatically; experiments were performed 3-5 days postconfluence and between 10 and 25 passage numbers. Utilizing radioligand binding techniques with the muscarinic receptor antagonists [3H]3-quinuclidinyl benzilate ([3H]QNB) and [3H]N-methylscopolamine ([3H]MS) as probes, we identified a small population of atropine-sensitive muscarinic sites (1,800-2,000 sites/cell or 7-8 fmol/mg protein). Muscarinic binding sites on BPAE membranes resembled classical muscarinic receptors in that (a) the binding of 2 nM [3H]QNB was inhibited by muscarinic agonists and antagonists, (b) [3H]QNB binding was 30 times more sensitive to R(-)- than to S(+)-QNB, (c) binding of the muscarinic receptor agonist carbamylcholine involved high and low affinity components, (d) the stable GTP analog, Gpp(NH)p (100 microM) shifted agonist binding curves to the right by a factor of three, and (e) the high affinity binding of the agonist [3H]oxotremorine-M to muscarinic receptors was depressed by Gpp(NH)p. On the other hand, gallamine, which allosterically regulates muscarinic receptor binding in other tissues, did not affect the rates of dissociation of [3H]QNB, [3H]MS or [3H]oxotremorine-M from BPAE binding sites. We concluded that BPAE in culture exhibit muscarinic binding sites which possess many but not all of the properties associated with classical muscarinic receptors.