Muscarinic Receptor-mediated Phosphoinositide Research Articles

Modulation of β 2- and β 3-adrenoceptor-mediated relaxation of rat oesophagus smooth muscle by protein kinase C

Although a prominent role for protein kinase C (PKC) in the cross-talk between the phosphoinositide pathway and β 2-adrenoceptor signalling has been indicated, modulation of β 3-adrenoceptor function by PKC has not been studied thus far. In the present study, we have compared the relative capacity of PKC in modulating β 2- and β 3-adrenoceptor-mediated relaxation of methacholine-contracted rat oesophagus smooth muscle. To this purpose the effects of the PKC-inhibitor GF 109203X (2-[1-(3-dimethylaminopropyl)-1H-indol-3-yl]-3-(1H-indol-3-yl)-maleimide) on relaxation induced by fenoterol, formoterol, (−)-noradrenaline, BRL 35135 (4-[2-[(2-hydroxy-2-(chlorophenyl)ethyl)amino]-propyl]-phenoxyacetic-acidmethylester) and IBMX (3-isobutyl-1-methyl-xanthine) were studied, in the absence and presence of the selective β 2-adrenoceptor antagonist ICI 118,551 (erythro-1(7-methylindan-4-yloxy)-3-(isopropylamin)-butan-2-ol). Our results show that inhibition of PKC resulted in differential augmentation of both β 2- and β 3-adrenoceptor-mediated relaxation. In contrast, relaxation induced by IBMX was not influenced at all by GF 109203X. The β 2-adrenoceptor bears phosphorylation sites for several kinases, including PKC. Since the β 3-adrenoceptor lacks these consensus sites, the results may also indicate that PKC-mediated Gα s phosphorylation is involved in the cross-talk between the muscarinic receptor-mediated phosphoinositide pathway and β 2- and, particularly, β 3-adrenoceptor signalling.

Visualization of muscarinic receptor-mediated phosphoinositide turnover in the hippocampus of young and aged, learning-impaired Long Evans rats.

Hippocampal receptor-mediated phosphoinositide (PI) turnover is severely blunted in aged rats that demonstrate cognitive deficits in the Morris water maze. To further examine the anatomical localization of this deficit, we examined the topography of muscarinic receptor-mediated PI turnover in young and aged-learning impaired rats by taking advantage of an autoradiographic method that visualizes PI turnover by measuring the diacylglycerol (DAG) branch of the PI turnover signal transduction system. Using this method, muscarinic cholinergic receptors were stimulated in hippocampal slices with agonist, and the receptor-mediated incorporation of [3H] cytidine into [3H]CDP-DAG was subsequently quantified in subregions of the hippocampus using film autoradiography. Our results show a significant decrease in basal incorporation of [ 3H]CDP-DAG in the subiculum and in the dentate gyrus in the aged rats. The muscarinic receptor-mediated [3H]CDP-DAG response was significantly blunted in the aged rats in subiculum, CA3, and CA1. In contrast, the receptor-mediated response was maintained in the dentate gyrus and hilus. These results indicate that the age-associated impairment in receptor-mediated PI turnover differs regionally, with a reduction in the subiculum and hippocampus proper that is pronounced relative to the hilus and dentate gyrus.

Contrasting effects of phorbol ester and agonist-mediated activation of protein kinase C on phosphoinositide and Ca2+ signalling in a human neuroblastoma

The effects of protein kinase C (PKC) activation on muscarinic receptor-mediated phosphoinositide and Ca2+ signalling were examined in the human neuroblastoma, SH-SY5Y. Carbachol evoked rapid transient elevations of Ins(1,4,5)P3 and intracellular [Ca2+] followed by lower sustained elevations. Phorbol 12,13-dibutyrate (PDBu) preferentially attenuated transient phases. Removal of the transplasmalemmal Ca2+ gradient coupled with depletion of intracellular Ca2+ stores with thapsigargin also reduced carbachol-mediated Ins(1,4,5)P3 accumulation. Under these conditions, PDBu virtually abolished Ins(1,4,5)P3 responses to carbachol thereby implicating both Ca(2+)- and PKC-sensitive components. PDBu also reduced agonist-mediated accumulation of inositol phosphates and depletion of lipids, thereby eliminating an effect of PKC on Ins(1,4,5)P3 metabolism or phosphoinositide synthesis. In electroporated cells, PDBu inhibited Ins(1,4,5)P3 accumulation mediated by carbachol or guanosine 5'-[gamma-thio]-triphosphate, the latter indicating that some PDBu-sensitive elements were downstream of the receptor. The PKC inhibitor, Ro-318220, protected against PDBu but did not enhance responses to maximal concentrations of carbachol, indicating no feedback inhibition by agonist-activated PKC. Muscarinic antagonist activity of Ro-318220 complicated such assessment at low agonist concentrations. Carbachol or PDBu induced cytosol to membrane translocation of PKC alpha. This was faster and possibly greater with PDBu, which may explain the lack of feedback by agonist-activated PKC. These results indicate that, in SH-SY5Y cells, PDBu activation of PKC preferentially inhibits rapid muscarinic receptor-mediated phosphoinositide and Ca2+ responses via suppression of PtdIns(4,5)P2 hydrolysis. This is at least partially through inhibition of Gq-protein/phosphoinositidase C coupling. However, at least at high agonist concentrations, a major agonist-mediated PKC feedback is not present in these cells.

Inhibition of muscarinic stimulated phosphoinositide hydrolysis in the rat parotid gland by cAMP

The ability of agents that increase or mimic cAMP to affect muscarinic receptor mediated phosphoinositide hydrolysis was investigated in the rat parotid gland. Forskolin (10 μM) and isoproterenol (10 μM) elevated cAMP in the parotid gland by 2-fold and 7-fold, respectively, and these agents also inhibited oxotremorine-M (3 H-M) mediated phosphoinositide hydrolysis by 14% and 26%, respectively. Forskolin (1,4.3, 18, and 75 μM) increased cAMP accumulation and inhibited PIP 2 hydrolysis in a concentration-dependent manner. Forskolin (75 μM) shifted the concentration-response curve for the full agonist oxotremorine-M rightward by 4.2-fold. Pre-treatment with the phosphodiesterase inhibitor isobutylmethylxanthine (1 mM) reduced the maximum effect of oxotremorine-M by 31%. The inhibitory effect of isoproterenol and forskolin on muscarinic receptor-mediated phosphoinositide hydrolysis was unaffected by the removal of extracellular Ca 2+. Moreover, isoproterenol and forskolin dampened sodium fluoride and oxotremorine-M mediated phosphoinositide hydrolysis to the same extent suggesting that the inhibitory effect of cAMP is downstream from the muscarinic receptor.

Effects of chronic nicotine and haloperidol administration on muscarinic receptor-mediated phosphoinositide turnover in rat brain slices.

Muscarinic receptor-mediated phosphoinositide (PI) turnover in rat brain slices was assessed after chronic administration of nicotine (12 mg/kg/day) or haloperidol decanoate (1.5 mg/kg/day), either alone or in combination, for 6 weeks. Nicotine alone did not significantly alter carbachol-induced inositol monophosphate (IP1) accumulation in the frontal cortex, but did result in a significant increase in the hippocampus, and in a decrease in the striatum. Haloperidol alone attenuated carbachol-stimulated IP1 accumulation in all three brain regions. Chronic treatment with combined nicotine and haloperidol resulted in no significant change in carbachol-sensitive IP1 accumulation in either the frontal cortex or hippocampus but did result in a decrease in the striatum. The results suggest significant cross-talk between cholinergic and dopaminergic systems in affecting PI metabolism.

Effect of the 5-HT 1A agonist 8-OH-DPAT on muscarinic receptor-mediated phosphoinositide breakdown in human neuroblastoma cells

Effect of the 5-HT 1A agonist 8-OH-DPAT on muscarinic receptor-mediated phosphoinositide breakdown in human neuroblastoma cells

Differential enhancement by potassium ions of M1-type and M2-type muscarinic receptor-mediated phosphoinositide breakdown in the rat brain

Raising the assay [K+] from 6 to 18 mM enhances the inositol phospholipid breakdown response to carbachol in rat brain miniprisms. In the frontal cortex, the degree of enhancement by K+ was independent of the carbachol concentration used, whereas in the striatum, a significantly higher degree of enhancement was seen at 1000 than at 50 microM carbachol. The carbachol-stimulated inositol phospholipid breakdown was antagonized by pirenzepine at both [K+] with potencies suggesting involvement of M1-type muscarinic receptors in the frontal cortex and both M1- and M2-type muscarinic receptors in the striatum. It is suggested that the response mediated by the M1-type receptors is enhanced to a greater degree by raised [K+] than that mediated by the M2-type receptors.

Relationship between the partial inhibition of muscarinic receptor-mediated phosphoinositide hydrolysis by phorbol esters and tetrodotoxin in rat cerebral cortex

Our results demonstrate that phorbol esters and tetrodotoxin (TTX) partially inhibit muscarinic receptor-mediated increase in phosphoinositide (PI) hydrolysis in rat cerebral cortex cell aggregates; this inhibition was observed using several muscarinic agonists. While these effects were not accompanied by major changes in the total muscarinic receptor population, phorbol esters, but not TTX, reduced the relative concentration of the high affinity binding sites of the M 1-selective ligands pirenzepine and telenzepine. In contrast, the binding of a muscarinic agonist to multiple receptor conformations was not influenced by either phorbol esters or TTX. Our data also show that the partial inhibition of the PI response by these agents is not due to a selective effect on the response mediated by a certain muscarinic receptor subtype or a receptor population which is more sensitive to agonist-induced desensitization. Evidence is provided that the effects of both phorbol esters and TTX might be mediated largely, although not entirely, by a common mechanism.

Differential desensitization of muscarinic receptor-stimulated phosphoinositide turnover in the rat brain

Desensitization of the muscarinic receptor-mediated phosphoinositide (PI) turnover response in the striatum and cortex of the rat brain was examined. The rate of accumulation of inositol phosphates during carbachol-stimulated PI turnover was constant for at least 60 min in the cortex, but decreased with time in the striatum. The effects of preincubation in the presence of the muscarinic agonist carbachol on muscarinic receptor-mediated PI turnover in slices and subsequent receptor binding in cell aggregates prepared from the slices were measured. Preincubation of striatal tissue produced a significant and sustained loss of responsiveness, which reached a minimum after 2 h; whereas, a transient and much weaker loss of responsiveness was observed in the cortex. Preincubation did not affect the number of muscarinic receptors as measured by the binding of [ (3)H ]l- quinuclidinyl benzilate or [ (3)H ]N- methylscopolamine in either cortex or striatum. The data suggest that the muscarinic receptor-stimulated PI turnover response can be more readily desensitized in striatum compared with cortex.

Mixed Competitive and Allosteric Antagonism by Gallamine of Muscarinic Receptor‐Mediated Second Messenger Responses in N1E‐115 Neuroblastoma Cells

The antagonistic effects of gallamine on muscarinic receptor-linked responses were investigated in N1E-115 neuroblastoma cells. M1 muscarinic receptor-mediated phosphoinositide hydrolysis induced by carbamylcholine was antagonized by gallamine, with a Ki value of 33 microM. By comparison, gallamine was four- to fivefold less potent in blocking noncardiac M2 muscarinic receptor-mediated inhibition of cyclic AMP formation, with a Ki value of 144 microM. The resulting Arunlakshana-Schild plots of the antagonism of both responses by gallamine were linear and exhibited slopes not differing from 1, a result indicative of a competitive mechanism. To elucidate further the nature of gallamine's inhibitory actions, experiments were performed where the effects of gallamine in combination with the known competitive muscarinic antagonist, N-methylscopolamine (NMS), were studied. In the presence of both antagonists, a supraadditive shift in the carbamylcholine dose-response curve was demonstrated for the two responses, a result suggestive of an allosteric mode of interaction between gallamine and NMS binding sites. Confirmation that gallamine allosterically modifies the muscarinic receptor was provided by radioligand binding studies. Gallamine competition curves with either [N-methyl-3H]scopolamine methyl chloride ([3H]NMS) or [N-methyl-3H]quinuclidinyl benzilate methyl chloride ([3H]NMeQNB) were unusually shallow. Furthermore, gallamine decelerated the rate of dissociation of receptor-bound [3H]NMS greater than [3H]NMeQNB in a dose-dependent manner. The present study demonstrates that whereas gallamine antagonizes carbamylcholine-mediated responses in N1E-115 cells in a competitive manner, an allosteric component of its action is revealed in the presence of muscarinic antagonists such as NMS.

Lack of alterations in muscarinic receptor subtypes and phosphoinositide hydrolysis upon acute DFP treatment

There was a 25 and 27% reduction in the density of mouse brain muscarinic acetylcholine receptors 18 and 24 h following a single injection of the organophosphate diisopropylfluorophosphate (DFP) when the muscarinic antagonist [ 3H]N-methylscopolamine ([ 3H]NMS) was used as the ligand. Down-regulation of specific [ 3H]NMS binding was rapidly reversible reaching control levels 36 h after DFP administration. Carbamylcholine and pirenzepine competition for the specific binding of either [ 3H]NMS or [ 3H]quinuclidinyl benzilate ([ 3H]QNB) in brain homogenates from untreated and DFP-treated mice demonstrated that the alteration in muscarinic receptor density following acute DFP treatment was not accompanied by a change in a particular muscarinic receptor binding conformation. Furthermore, the magnitude of muscarinic receptor-mediated phosphoinositide hydrolysis was unchanged following short-term DFP treatment suggesting that a physiological desensitization in this response does not accompany acute down-regulation of [ 3H]NMS binding sites.