Carbachol-stimulated Phosphoinositide Turnover Research Articles

Arecoline desensitizes carbachol-stimulated phosphatidylinositol breakdown in rat brain cortices.

To understand the effects of arecoline administration on the muscarinic cholinergic signaling pathway, rats were injected with arecoline, 10 mg/kg i.p., and the carbachol-stimulated phosphoinositide breakdown in rat brain cortical slices was examined. In vivo administration of arecoline resulted in inhibition of carbachol-stimulated phosphoinositide turnover in rat brain cortical slices. Arecoline was a partial agonist with peak effects of 30% of the maximum as obtained with carbachol. Coaddition of arecoline inhibited the carbachol-stimulated phosphoinositide breakdown. Pretreatment of rat brain cortical slices with arecoline in vitro resulted in a dose-dependent inhibition of carbachol-stimulated [3H]inositol monophosphate accumulation. The inhibition occurred rapidly, with half-maximal inhibition occurring at 15 min and maximal inhibition achieved within 60 min. The inhibition of phosphoinositide breakdown was recovered 1 h after arecoline was removed. When synaptoneurosomes were used for the ligand binding studies, arecoline pretreatment was found to have decreased the maximal ligand binding (Bmax) without inducing any marked change in binding affinity (K(D)). The influence could be recovered by incubating the synaptoneurosomes in the absence of arecoline for 2 h. Taken together, these data suggest that the underlying mechanism by which phosphoinositide turnover is inhibited is arecoline-induced receptor sequestration.

Modulation of phosphoinositide turnover by chronic nicergoline in rat brain

Basal and agonist-stimulated phosphoinositide (PI) turnover and inositol 1,4,5 -trisphospate (InsP 3) content in rat brain were investigated after chronic nicergoline (SERMION®) treatment. Oral administration of nicergoline (5 mg/kg b.i.d. for 7 weeks) enhanced the basal turnover of PI in the cerebral cortex compared to controls. This effect was paralleled by a significant rise of cortical InsP 3 levels. No significant changes of noradrenaline- or carbachol-induced accumulation of [ 3H]-inositol-1-phophate ([ 3H]-InsP 1) were found in cortices from nicergoline-treated rats. On the contrary, in the striatum nicergoline significantly potentiated the responsiveness of noradrenaline- and carbachol-stimulated PI turnover, leaving unchanged the basal production of [ 3H]-InsP 1 and InsP 3 levels. The results suggest that the interaction of nicergoline with PI transducing pathway might have relevance to the mechanisms of action of nicergoline.

Developmental changes in carbachol-stimulated phosphoinositide turnover in synaptoneurosomes of the robust nucleus of the archistriatum in the zebra finch.

The effects of acetylcholine (ACh) on synaptoneurosomes of the robust nucleus of the archistriatum (RA), one of the song control nuclei, were investigated during the sensitive period of song learning in the zebra finch. Carbachol (an ACh agonist) stimulated phosphoinositide (PI) turnover at three different ages. Corresponding to a marked increase in ACh levels in the RA, carbachol-stimulated PI turnover peaked during the most sensitive learning period in the life of the zebra finch. These findings suggest that ACh contributes to the regulation of synaptic plasticity in RA neurones involved with song learning in the zebra finch.

Hippocampal phosphoinositide turnover is altered by hippocampal sympathetic ingrowth and cholinergic denervation

Cholinergic denervation of the hippocampus, by medial septal (MS) lesions, results in an unusual neuronal rearrangement in which peripheral sympathetic nerves, which originate from the superior cervical ganglia, grow into the hippocampal formation. To assess the functional significance of hippocampal sympathetic ingrowth (HSI), hydrolysis of phosphoinositides was examined in three groups: control, MS lesions + sham ganglionectomy (HSI group); and MS lesions + ganglionectomy (MSGx; no ingrowth). Four months after surgery, both norepinephrine (NE) and carbachol were found to produce a dose-dependent increase in the hydrolysis of hippocampal phosphoinositides in all groups. However, the presence of HSI, when compared to control and MSGx groups, significantly enhanced the turnover of phosphoinositides when stimulated by carbachol, but not NE. In further studies, the time course of this effect was studied. One week after surgery, carbachol-stimulated phosphoinositide turnover was equivalent among all groups; by 2 weeks, phosphoinositide turnover was enhanced in the HSI and MSGx group; by 4 weeks. PI turnover was markedly diminished in the MSGx group when compared to both the HSI and control groups, which were equivalent to each other. To ensure that the ganglionectomy alone did not alter phosphoinositide turnover, a ganglionectomy-alone group was studied at the 4-week time point. In this group, phosphoinositide turnover was equivalent to controls, suggesting no influence of the superior cervical ganglia on this response. In all groups, atropine inhibited carbachol-stimulated phosphoinositide turnover. These results suggest that both cholinergic denervation (i.e., MSGx group) and HSI produce marked functional alterations in hippocampal metabolic activity.

Effect of the putative 5-HT 1A receptor antagonist NAN-190 on rat brain serotonergic transmission

Based on the fact that 1-(2-methoxyphenyl)-4(4-(2-phtalimido)butyl)piperazine (NAN-190), a high-affinity ligand for 5-HT 1A and α 1-adrenoceptors, antagonizes the behavioural effects of the 5-HT 1A receptor agonist 8-OH-DPAT (8-hydroxy-2-(di-n-propylamino) tetralin), it has been suggested that this drug behaves as a 5-HT 1A receptor antagonist. In the present study we examined the effects of this putative 5-HT 1A receptor antagonist on rat brain serotonergic neurotransmissicn. In hippocampal slices of immature rats, NAN-190 but not prazosin potently antagonized ( IC 50 = 29 nM) the inhibitory effect of 8-OH-DPAT (1 μM) on carbachol-stimulated phosphoinositide turnover but (up to 1 μM) failed to alter the carbachol response. Similarly, NAN-190 (0.1 μM) almost totally prevented the inhibition by 8-OH-DPAT (1 μM) of forskolin-stimulated adenylate cyclase activity in adult rat hippocampal slices but, per se, was without effect on the forskolin response. These results indicate that NAN-190 is a potent antagonist at postsynaptic 5-HT 1A receptors in vitro. However, NAN-190 also potently antagonized ( IC 50 = 0.16 nM) the stimulation by norepinephrine of phosphoinositide turnover in rat cortical slices. In this respect NAN-190 was a 250-fold more potent antagonist than prazosin ( IC 50 = 49 nM). Thus, in addition to its 5-HT 1A receptor antagonist properties, NAN-190 has potent α 1-adrenoceptor blocking properties. Systemic administration of NAN-190 to adult rats caused a diminution of serotonin synthesis in the frontal cortex (−39% at l mg/kg i.p.), of extracellular 5-hydroxyindoleacetic acid levels (as measured by voltammetry) in the dorsal raphe (−50% at 0.3 mg/kg i.p.) and of extracellular serotonin levels (as measured by microdialysis) in the hippocampus (−45% at 0.3 mg/kg s.c.) as did 8-OH-DPAT. The α 1-adrenoceptor antagonist prazosin (0.3–1 mg/kg) reproduced the effects of NAN-190 on the above-mentioned parameters. It is concluded that while NAN-190 blocks postsynaptic 5-HT 1A receptor-mediated responses in the hippocampus, it reduces serotonin turnover in the hippocampus and frontal cortex, probably as a consequence of its α 1-adrenoceptor-blocking properties.

Inhibition of carbachol-stimulated phosphoinositide turnover by U-50,488H in rat hippocampus — involvement of GTP-binding protein

The effect of U-50,488H, a selective kappa-opioid agonist, on carbachol-stimulated phosphoinositide (PI) turnover response in rat hippocampal slices was examined. U-50,488H which stimulates PI turnover response in this preparation (Periyasamy and Hoss, 1990, Life Sci. 47, 219), inhibited carbachol-stimulated PI turnover in a concentration-dependent manner with an IC 50 value of 33±9.0 μM. The inhibitory effect of U-50,488H was not blocked by the kappa-selective antagonists, e.g., nor-binaltorphimine (10 μM), and MR2266 (10 μM), or tetrodotoxin (1 μM) suggesting that the effect of U-50,488H was mediated neither through the kappa-receptors nor through the release of an endogenous neurotransmitter(s). A Lineweaver-Burke plot of the stimulation of PI turnover by carbachol in the presence and absence of U-50,488H showed that the K m was not changed (11.4±3.4 and 11.5±2.6 μM) whereas the V max was reduced from 3849±460 to 1534±31 cpm indicating that the inhibition was non-competitive. U-50,488H also inhibited guanosine 5′-[β,γ-imido]triphosphate (Gpp[NH]p)-stimulated PI turnover in rat hippocampal membranes in a concentration-dependent manner with an IC 50 value of 33±12 μM. However, U-50,488H had no effect on exogenously added phospholipase-C-stimulated PI turnover. In receptor binding studies. U-50,488H displaced the non-selective muscarinic antagonist [ 3H]quinuclidinyl benzilate ([ 3H]QNB) binding in a concentration-dependent manner with an IC 50 value of 4.5±1.5 μM. A Scatchard plot of [ 3H]QNB binding in the presence and absence of U-50,488H showed that the K d was changed from 883±250 to 364±147 pM whereas the B max remained the same (1.66±0.16 and 1.65±0.18 pmol/mg protein) suggesting that the inhibition was competitive. These data indicate that the inhibitory effect of U-50,488H on carbachol-stimulated PI turnover was mediated through the interaction with the G-protein but not through kappa-receptors or release of neurotransmitter(s).

Biochemical and behavioral characterization of a novel cholinergic agonist, SR 95639

Selective M1 cholinergic agonists may be useful in treating dementias due to cholinergic hypofunction. SR 95639 has recently been described as such a compound. We found the compound to have affinity for M1 sites ( K i = 2.1 μM) which was approximately 3-fold higher than its affinity for M2 sites. Functional partial agonism was suggested by an inconsistent increase in phosphoinositide (PI) turnover in rat hippocampal slices, combined with blockade of carbachol-stimulated PI turnover. In vivo M2-mediated effects were absent. Scopolamine-induced hyperactivity was attenuated by SR 95639 and scopolamine-impaired inhibitory avoidance and radial maze performance were improved. The compound appears to be a weakly selective M1 partial agonist with potential advantages over existing compounds.

Pertussis toxin-sensitive G-protein mediates galanin's inhibition of scopolamine-evoked acetylcholine release in vivo and carbachol-stimulated phosphoinositide turnover in rat ventral hippocampus

The effects of intracerebroventricular (i.c.v.) injections of pertussis toxin were investigated on the inhibitory action of galanin on acetylcholine release and phosphoinositide breakdown stimulated by muscarinic agents in rat ventral hippocampus. Pertussis toxin (0.6 μg, i.c.v., 96 h) counteracted the in vitro inhibitory effect of galanin (3.1 nmol) on phosphoinositide breakdown stimulated by carbachol without altering the stimulatory action of the cholinergic agonist on signal transduction, in miniprisms from rat ventral hippocampus. Pertussis toxin also abolished the in vivo effect of galanin on scopolamine-stimulated acetylcholine release in vivo but did not affect basal acetylcholine release. The results indicate that pertussis toxinsensitive G-protein(s) mediates the galanin receptor regulation of pre- and postsynaptic cholinergic functions in the ventral hippocampus.

Potential Mechanisms Involved in the Negative Coupling Between Serotonin 5‐HT1A Receptors and Carbachol‐Stimulated Phosphoinositide Turnover in the Rat Hippocampus

Serotonin 5-HT1A receptors have been reported to be negatively coupled to muscarinic receptor-stimulated phosphoinositide turnover in the rat hippocampus. In the present study, we have investigated further the pharmacological specificity of this negative control and attempted to elucidate the mechanism whereby 5-HT1A receptor activation inhibits the carbachol-stimulated phosphoinositide response in immature or adult rat hippocampal slices. Various 5-HT1A receptor agonists were found to inhibit carbachol (10 microM)-stimulated formation of total inositol phosphates in immature rat hippocampal slices with the following rank order of potency (IC50 values in nM): 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT) (11) greater than ipsapirone (20) greater than gepirone (120) greater than RU 24969 (140) greater than buspirone (560) greater than 1-(m-trifluoromethylphenyl)piperazine (1,500) greater than methysergide (5,644); selective 5-HT1B, 5-HT2, and 5-HT3 receptor agonists were inactive. The potency of the 5-HT1A receptor agonists investigated as inhibitors of the carbachol response was well correlated (r = 0.92) with their potency as inhibitors of the forskolin-stimulated adenylate cyclase in guinea pig hippocampal membranes. 8-OH-DPAT (10 microM) fully inhibited the carbachol-stimulated formation of inositol di-, tris-, and tetrakisphosphate but only partially antagonized (-40%) inositol monophosphate production. The effect of 8-OH-DPAT on carbachol-stimulated phosphoinositide turnover was not prevented by addition of tetrodotoxin (1 microM), by prior destruction of serotonergic afferents, by experimental manipulations causing an increase in cyclic AMP levels (addition of 10 microM forskolin), or by changes in membrane potential (increase in K+ concentration or addition of tetraethylammonium). Prior intrahippocampal injection of pertussis toxin also failed to alter the ability of 8-OH-DPAT to inhibit the carbachol response. Carbachol-stimulated phosphoinositide turnover in immature rat hippocampal slices was inhibited by the protein kinase C activators phorbol 12-myristate 13-acetate (10 microM) and arachidonic acid (100 microM). Moreover, the inhibitory effect of 8-OH-DPAT on the carbachol response was blocked by 10 microM quinacrine (a phospholipase A2 inhibitor) but not by BW 755C (100 microM), a cyclooxygenase and lipoxygenase inhibitor. These results collectively suggest that 5-HT1A receptor activation inhibits carbachol-stimulated phosphoinositide turnover by stimulating a phospholipase A2 coupled to 5-HT1A receptors, leading to arachidonic acid release. Arachidonic acid could in turn activate a gamma-protein kinase C with as a consequence an inhibition of carbachol-stimulated phosphoinositide turnover. This inhibition may be the consequence of a phospholipase C phosphorylation and/or a direct effect on the muscarinic receptor.(ABSTRACT TRUNCATED AT 400 WORDS)

Interaction of σ‐Compounds with Receptor‐Stimulated Phosphoinositide Metabolism in the Rat Brain

Sigma-receptors are nonopioid, nondopaminergic receptors that bind with high affinity several antipsychotic drugs and appear to be involved in regulation of posture and movement. The second messenger system coupled to these receptors is still unknown. Recently, an inhibitory effect of various sigma-compounds on carbachol-stimulated phosphoinositide metabolism has been reported. We have investigated the effect of six sigma-compounds on carbachol- and norepinephrine-stimulated 3H-inositol phosphate accumulation in rat cerebral cortex slices. All compounds tested had a dose-dependent inhibitory effect on both systems, although their order of potency differed between neurotransmitters. Pentazocine and 1,3-di-o-tolylguanidine were the most potent inhibitors of carbachol-stimulated phosphoinositide turnover (IC50 = 31.5 and 45.7 microM, respectively), while haloperidol showed the greatest potency on the norepinephrine-coupled system (IC50 = 3.5 microM). In the presence of IC50 concentrations of these inhibitors, the dose-response curves for the agonists were shifted to the right and the EC50 values were significantly increased. Sigma-compounds also inhibited the binding of [3H]quinuclidinyl benzilate to muscarinic receptors and of [3H]prazosin to alpha 1-adrenoceptors in cortical membranes. In the presence of IC50 concentration (11 microM) of 1,3-di-o-tolylguanidine, no differences were found in the maximal number of muscarinic binding sites, whereas the dissociation constant increased approximately fivefold, indicating a decrease of the radioligand's affinity for the receptor. These results indicate that sigma-compounds, at micromolar concentrations, inhibit muscarinic and alpha 1-adrenergic receptor-coupled phosphoinositide metabolism, probably through an interaction with the neurotransmitter recognition sites.

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.

Effect of in vivo exposure to hypoxia on muscarinic cholinergic receptor-coupled phosphoinositide turnover in the rat brain

We examined the effects of a transient exposure of rats to severe (5% O 2, 95% N 2 for 30 min) or mild (10% O 2, 90% N 2 for 30 min) hypoxia and a chronic exposure to mild hypoxia (10% O 2, 90% N 2 up to 48 h) on carbachol-stimulated phosphoinositide (PI) turnover and [ 3H]quinuclidinyl benzilate (QNB) binding in 4 regions of the brain (cerebral cortex, striatum, hippocampus and cerebellum). When the rats were exposed to hypoxia transiently, significant changes were caused only by severe hypoxia. In all 4 regions examined, basal incorporation of [ 3H]inositol into inositol phosphates (IPs) in the absence of carbachol was significantly increased following transient severe hypoxia and remained high for 48 h. Carbachol stimulation of PI turnover (% of basal) was significantly enhanced only in the hippocampus. The increase in carbachol stimulation in the hippocampus was exposure to room air following the severe hypoxia and lasted for at least 48 h. In Ca 2+-free incubation medium, the increase in basal incorporation of [ 3H]inositol into IPs elicited by severe hypoxia was not observed but carbachol still had an enhanced stimulatory effect. Binding studies showed that maximum binding capacity ( B max values) of [ 3H]QNB binding was significantly increased only in the hippocampus and only after a 6-h exposure to room air following the severe hypoxic exposure and remained increased for 48 h. K d values showed no significant change. While a transient exposure of rats to mild hypoxia caused no significant change either in carbachol-stimulated PI turnover or in [ 3H]QNB binding parameters, a chronic exposure to mild hypoxia for more than 6 h caused a significant increase in [ 3H]QNB binding capacity localized in the hippocampus, which was accompanied by an enhanced stimulatory effect of carbachol on PI turnover. These changes observed with mild hypoxia, however, were transient and tended to return to control levels following a 48-h exposure. These results suggest an up-regulation of muscarinic cholinergic receptor-coupled PI turnover in the rat brain caused either by transient severe hypoxia or by chronic mild hypoxia.

Inhibition of phosphoinositide turnover by selective muscarinic antagonists in the rat striatum: Correlation with receptor occupancy

In the rat corpus striatum, receptor occupancy and the inhibition of phosphoinositide turnover by muscarinic antagonists have been examined under very similar conditions with respect to tissue preparation and buffer composition. The results suggest a good correlation between receptor occupancy and inhibition by muscarinic antagonists, of the carbachol-stimulated turnover of inositol phospholipids, measured by the accumulation of [ 3H]inositol phosphates in the presence of 5 mM LiCl. In the presence of 10 mM carbachol (CCh), the accumulation of labeled inositol phosphates was increased 8-fold above basal levels ( ec 50 = 95 μ M ). Inclusion of antagonists resulted in a dose-dependent inhibition of the 0.1 mM CCh-stimulated inositol phosphate accumulation, with a rank order of potency of atropine > trihexyphenidyl > pirenzepine ⪢ gallamine. Radioligand binding studies with [ 3H]- l-quinucliclinyl benzilate ([ 3H]QNB) in a cell aggregate preparation revealed a single class of saturable, high affinity [ 3H]QNB binding sites exhibiting a K d of 74 pM and a B max, of 2.85 pmol/mg protein. The antagonists examined were able to inhibit the binding of [ 3H]QNB with the same rank order of potency as for the inhibition of carbachol-stimulated phosphoinositide turnover (atropine > trihexyphenidyl > pirenzepine ⪢ gallamine). Although the inhibition of phosphoinositide turnover and [ 3H]QNB binding by the nonselective antagonist atropine was best described by interaction at a single site, inhibition of phosphoinositide turnover and [ 3H]QNB binding by both pirenzepine, which is selective for M1 receptors, and gallamine, which is selective for M2 receptors, is complex. Pirenzepine was much more potent than gallamine for both binding to receptors and inhibiting phosphoinositide turnover. Nonlinear curve-fitting analysis indicated that slope factors for inhibition of phosphoinositide turnover (analogous to Hill coefficient for binding) by only subtype selective antagonists were significantly less than unity. The above-mentioned antagonist interactions together with the apparently multicomponent stimulation of phosphoinositide turnover by carbachol suggest that phosphoinositide turnover may be coupled to more than one muscarinic receptor subtype in the corpus striatum.

Differential effects of phenylmethanesulfonyl fluoride (PMSF) on carbachol and potassium stimulated phosphoinositide turnover and contraction in longitudinal smooth muscle of guinea pig ileum

Phenylmethanesulfonyl fluoride (PMSF) (2 mM), a putative inhibitor of phosphatidylinositol-specific phospholipase C, almost completely inhibited carbachol-stimulated inositol incorporation into phosphatidylinositol (PI) of longitudinal smooth muscle of guinea pig ileum, while it had no effect on potassium-stimulated inositol incorporation. This suggests that the two stimuli may affect phosphoinositide turnover by different mechanisms, distinguishable by PMSF. In contrast to its specific inhibition of carbachol-stimulated phosphoinositide turnover, PMSF produced a transient inhibition of contraction by both carbachol and potassium. The non-selective effect of PMSF on contraction suggests that it is not the result of its inhibitory effect on phosphoinositide breakdown. PMSF (2 mM) inhibited carbachol-stimulated inositol phosphate accumulation in the presence of Li + by only 15%–19%, indicating that PMSF inhibition of phosphoinositide turnover was not due to its inhibition of phosphoinositide phosphodiesterase, but to one or more steps following phosphoinositide breakdown.