Long-term atropine treatment lowers the efficacy of carbachol to stimulate phosphatidylinositol breakdown in the cerebral cortex and hippocampus of rats.

The effect of long-term treatment with atropine, a muscarinic antagonist, known to cause up-regulation of receptor numbers, was examined on the muscarinic-receptor-mediated stimulation of phosphoinositide breakdown in the rat cerebral cortex and hippocampus. Although the numbers of both M1 muscarinic receptors, as measured by [3H]pirenzepine binding, and M1 and M2 receptors increased in both brain regions, the maximal breakdown of myo-[3H]inositol-labelled phosphoinositides was unaltered in the presence of carbachol at a saturating concentration (10(-2) M). In fact the efficacy of carbachol was decreased in slices from atropine-treated cerebral cortex [EC50 (concentration producing half-maximal effect) = 93 microM] as compared with the saline-treated control (EC50 = 23 microM)(P less than 0.005). Similarly the EC50 value (23 microM) in hippocampal slices from saline-treated rats increased in atropine-treated rats to 126 microM (P less than 0.005). This lowered efficacy of muscarinic stimulation could not be explained in terms of residual atropine in the tissue from treated rats. The noradrenaline- or serotonin (5-hydroxytryptamine)-stimulated breakdown or the K+ potentiation of the muscarinic-receptor-stimulated breakdown of [3H]phosphoinositides was not affected by the atropine treatment. Chromatography of the released [3H]inositol phosphates shows that atropine treatment did not cause any qualitative change in the pattern of [3H]inositol phosphates released by carbachol stimulation.     

The effects of fatty acids on phosphoinositide synthesis and myo-inositol accumulation in exocrine pancreas

The effects of arachidonic acid (20:4) on phosphoinositide turnover were examined in rat pancreatic acinar cells prelabeled with myo-[3H]inositol. Arachidonic acid (50 microM) increased the accumulation of myo-[3H]inositol, but not that of [3H]inositol monophosphate, [3H]inositol bisphosphate, or [3H]inositol trisphosphate. By contrast, 10 microM carbamoylcholine increased the accumulation of all four compounds. A combination of arachidonic acid plus carbamoylcholine caused a selective and marked accumulation of myo-[3H]inositol, which was abolished by 10 mM LiCl. Arachidonic acid (10-100 microM) produced a concentration-dependent inhibition of myo-[3H]inositol incorporation into phosphoinositides and markedly depressed carbamoylcholine-induced increases in myo-[3H]inositol incorporation into inositol phospholipids. Several other unsaturated and saturated fatty acids failed to elicit a synergistic response with carbamoylcholine in stimulating myo-[3H]inositol accumulation and did not retard the incorporation of myo-[3H]inositol into phosphoinositides. The fact that eicosapentaenoic acid (20:5), but not arachidic acid (20:0), mimicked the depressant effect of arachidonate on phosphoinositide labeling suggests that the degree of unsaturation of the fatty acid, rather than chain length, is important for inhibition of phosphoinositide synthesis. The arachidonate-induced decrease in myo-[3H]inositol incorporation was accompanied by a reduction in the steady state level of [32P]phosphatidylinositol 4,5-bisphosphate. The mass of arachidonic acid liberated in response to carbamoylcholine was measured by gas chromatography-mass spectrometry, and the time course of stimulated arachidonate accumulation paralleled that of inositol phosphate accumulation and amylase release. These observations suggest that in exocrine pancreas, endogenous arachidonic acid serves as a negative feedback regulator of phosphoinositide turnover.     

In Vivo Determination of 5-Hydroxytryptamine Receptor-Stimulated Phosphoinositide Turnover in Rat Brain

Phosphoinositide turnover stimulated by 5-hydroxytryptamine (5-HT) receptors in the intact rat brain was studied using an in vivo method. Phosphoinositides in the rat brain were prelabeled with [3H]inositol injected into the lateral cerebral ventricles. The rats were killed by microwave irradiation after 48 h and the contents in the frontal cortex of 3H-inositol phosphates, [3H]inositol-1-monophosphate [( 3H]IP1), [3H]inositol-1,4-bisphosphate [( 3H]IP2), and a mixture of [3H]inositol-1,4,5-trisphosphate and [3H]inositol-1,3,4-trisphosphate [( 3H]IP3) were assayed by HPLC. Lithium treatment (10 mEq/kg, i.p., 2 h before) increased the content of [3H]IP1 and [3H]IP2. 5-Methoxy-N,N-dimethyltryptamine (5-MeODMT) and quipazine, 5-HT agonists, significantly increased the amount of 3H-inositol phosphates under lithium pretreatment. The response to 5-MeODMT was inhibited by ritanserin, a 5-HT2 antagonist, but not by (-)-propranolol, a 5-HT1 antagonist. These results suggest that phosphoinositide turnover in the rat frontal cortex in vivo is stimulated by 5-HT2 receptor activation. It is considered that this method will be useful for measurement of 5-HT2 receptor-stimulated phosphoinositide turnover in vivo to examine the in vivo effects of various psychotropic drugs such as antidepressants.     

Time-, concentration-, and age-dependent inhibition of muscarinic receptor-stimulated phosphoinositide metabolism by ethanol in the developing rat brain

We have previously reported that administration of ethanol (EtOH; 4 g/Kg/day) to rats from postnatal day 4 to day 10 causes microencephaly and decreases muscarinic receptor-stimulated inositol metabolism on days 7 and 10 (1). An identical exposure to EtOH of adult rats, which resulted in similar blood EtOH concentrations, did not have any effect on the same system. Initial in vitro studies have shown the presence of a differential sensitivity to EtOH of the phosphoinositide system coupled to muscarinic receptors during development (2). In the present study we have expanded these findings by investigating the concentration-, time-, and age-dependent effects of EtOH on accumulation of [³H]inositol phosphates ([³H]InsPs) in brain slices. EtOH caused a dose-dependent inhibition of carbachol-stimulated phosphoinositide metabolism in cerebral cortex slices from 7 day-old rats. When the time of incubation with EtOH was increased to 90 minutes, concentrations as low as 50 mM, which are reached following in vivo administration of EtOH, significantly inhibited the muscarinic response. The effect of EtOH was rather specific for the muscarinic receptors, since, even with longer incubation times, the accumulation of [³H]InsPs induced by norepinephrine or serotonin was inhibited only at concentrations of 150–500 mM. The effect of EtOH was more pronounced in cerebral cortex, hippocampus and cerebellum, and less in the brainstem. The potency of EtOH in inhibiting carbachol-stimulated phosphoinositide metabolism was also dependent on the age of the animals. Its effect was maximal in the 7 day-old rat and less pronounced in younger and older animals. These results confirm that the phosphoinositide system coupled to muscarinic receptors might represent a relevant target for the developmental neurotoxicity of EtOH.Presented in part at the 29th Annual Meeting of the Society of Toxicology (Toxicologist 1990; 10: 273).     

Comparison of muscarinic and alpha-adrenergic receptors in rat atria based on phosphoinositide turnover

The effects of muscarinic and alpha-adrenergic receptor stimulation on phosphoinositide turnover in rat atria have been compared. Despite the similar densities of muscarinic receptors in rat left and right atria, 0.1 mM carbachol increased [32P]phosphate incorporation into phosphatidylinositol (PI) by 35% (p less than 0.05) in left atria but had no effect in right atria. By contrast to the small muscarinic receptor effect, stimulation of alpha 1-adrenergic receptors by 0.1 mM methoxamine produced a more than two fold increase in [32P]phosphate incorporation into PI in both left and right atria, despite the reported smaller density of alpha-adrenergic receptors in rat atria compared to muscarinic receptors. Enhanced phosphate labelling by methoxamine did not occur in phospholipids other than PI, and was blocked by the alpha-adrenergic antagonist, phentolamine (20 microM). The results indicate that the majority of the muscarinic receptors in rat atria are not coupled to phosphoinositide turnover. If indeed the observed enhancement in [32P]-phosphate labelling by carbachol reflects phosphoinositide turnover, and assuming equal coupling efficiencies of muscarinic and adrenergic receptors, it is calculated that not more than 2% of the muscarinic receptors in rat left atria are coupled to this response.     

Subacute reserpine treatment reveals preferential coupling between the M3 muscarinic receptor subtype and phosphatidylinositol turnover.

Muscarinic receptors in the rat cerebral cortex, cardiac atria and vas deferens were identified, quantitated, and characterized relative to phosphatidylinositol (PI) turnover as the functional response to stimulation of specific receptor subtypes. Receptor densities as determined by 3H-QNB binding were ranked: cerebral cortex greater than vas deferens greater than heart. Using displacement of 3H-QNB binding by the selective M1 and M2 muscarinic receptor antagonists pirenzepine and 11[[2-[(diethylamino)methyl]-1-piperidinyl]acetyl]-5,11-dihydro- 6H-pyrido [2,3-b] [1,4] benzodiazepine-6-one (AF-DX 116) respectively, heterogeneous populations were found in the cerebral cortex and vas deferens. The M1 receptor subtype predominated in the former and the M2 predominated in the latter. An homogeneous M2 receptor population was present in the heart. Methacholine-stimulated accumulation of 3H inositol-1-phosphate was greater in the vas deferens than in the cerebral cortex, whereas PI turnover was not enhanced in cardiac atria. Reserpine treatment of rats (0.5 mg kg-1 day-1 for 7 days) increased muscarinic receptor density in the vas deferens coincident with a shift in the low affinity pKi for AF-DX 116 to a value comparable to high affinity binding, and abolished the enhanced PI hydrolysis. In the cerebral cortex, reserpine treatment shifted only the early portion of the methacholine dose-response curve to the right. These results are judged to be supportive of preferential coupling between the M3 muscarinic receptor subtype and PI turnover.     

Increased Intracellular Calcium Stimulates 3H-Inositol Polyphosphate Accumulation in Rat Cerebral Cortical Slices

Agents that increase the intracellular Ca2+ concentration have been examined for their ability to stimulate 3H-inositol polyphosphate accumulation in rat cerebral cortex slices. Elevated extracellular K+ levels, the alkaloid sodium channel activator veratrine, the calcium ionophore ionomycin, and the marine toxin maitotoxin were all able to stimulate phosphoinositide metabolism. Certain features appear common to the agents studied. Thus, although [3H]inositol monophosphate, [3H]inositol bisphosphate ([3H]InsP2), and [3H]inositol trisphosphate were all stimulated, a proportionally greater effect was observed on [3H]InsP2 in comparison to stimulation by the muscarinic receptor agonist carbachol. However, only an elevated K+ level stimulated [3H]inositol tetrakisphosphate ([3H]InsP4) accumulation alone or produced marked synergy with carbachol on the formation of this polyphosphate. The results suggest that agents that elevate the cytoplasmic Ca2+ concentration in cerebral cells can increase the hydrolysis of membrane polyphosphoinositides. The pattern of the response differs from that produced by muscarinic receptor agonists and indicate that Ca2(+)-dependent hydrolysis may involve different pools of lipids, phosphoinositidase C enzymes, or both. However, clear differences in the ability of these agents to stimulate InsP4, alone or in the presence of muscarinic agonist, suggest that factors other than a simple elevated intracellular Ca2+ concentration are implicated.     

Potassium ions potentiate the muscarinic receptor-stimulated phosphoinositide metabolism in cerebral cortex slices: A comparison of neonatal and adult rats

Activation of cholinergic muscarinic receptors results in an increased turnover of membrane inositol phospholipids. In rat cerebral cortex slices, carbachol- and acetylcholine-induced inositol phosphates ([³H]InsPs) accumulation is maximal in 7 day-old rats and lowest in adults, while the density of muscarinic binding sites increases gradually with age, suggesting the presence of a more effective receptor-effector coupling during neonatal life. In the process of investigating the nature of such differential stimulation, we have studied the effects of potassium ions on muscarinic receptor-stimulated phosphoinositide metabolism during development. Increasing the concentration of K⁺ from 6 to 12 mM potentiated the stimulating effect of carbachol by 80–100% in adult animals, as previously shown, but only 10–20% in 7 day-old animals, without altering its EC₅₀ values. The differential potentiation by K⁺ at these two ages was specific for muscarinic receptors, since norepinephrine-stimulated accumulation was potentiated only 18% and 12% in adult and 7 day-old rats, respectively. Two other monovalent cations, rubidium and cesium, had the same effect as K⁺ on carbachol-stimulated [³H]-InsPs accumulation. The effect of K⁺ was not antagonized by the K⁺ channel blocker 4-aminopyridine, but was antagonized by tetraethylammonium (TEA). TEA, however, also interacted with muscarinic binding sites. Omission of calcium from the incubation medium did not influence the potentiating effect of 12 mM K⁺. However, when EDTA (1 mM) was added, the stimulating effect of carbachol alone or carbachol + K⁺ was almost completely prevented. The potentiating effect of K⁺ during development was inversely proportional to the stimulation of phosphoinositide metabolism induced by carbachol. These results suggest that the mechanism responsible for the potentiating effect of K⁺ in adult rats might be already operating in neonatal animals.     

Age-Related Alterations In Pre-Synaptic and Receptor-Mediated Cholinergic Functions In Rat Brain

Fractional [³H]acetylcholine (ACh) release and regulation of release process by muscarinic receptors were studied in corpus striatum of young and aged rat brains. [³H] Quinuclidinyl benzilate (QNB) binding and carbachol stimulated phosphoinositide turnover, on the other hand, were compared in striatal, hippocampal and cortical tissues. High potassium (10 mM)-induced fractional [³H]ACh release from striatal slices was reduced by aging. Although inhibition of acetylcholinesterase with eserine (20 M) significantly decreased stimulation-induced fractional [³H]ACh release in two groups of rats, this inhibition slightly lessened with aging. Incubation of striatal slices with muscarinic antagonists reversed eserine-induced inhibition in fractional [³H]ACh release with a similar order of potency (atropine = 4-DAMP > AF-DX 116 > pirenzepine) in young and aged rat striatum, but age-induced difference in stimulated ACh release was not abolish by muscarinic antagonists. These results suggested that fractional [³H]ACh release from striatum of both age groups is modulated mainly by M₃ muscarinic receptor subtype. Although both muscarinic receptor density and labeling of inositol lipids with [myo-³H]inositol decreased with aging, carbachol-stimulated [³H]myo inositol-1-fosfat (IP₁) accumulation was found similar in striatal, cortical and hippocampal slices.     

Effect of prenatal treatment with methylazoxymethanol on carbachol-, norepinephrine- and glutamate-stimulated phosphoinositide metabolism in the neonatal,young, and adult offspring

Carbachol-, norepinephrine- and glutamate-stimulated phosphoinositide metabolism was investigated in the neonatal, young and adult cerebral cortex slices of rats prenatally treated with methylazoxymethanol (MAM) on gestational day 15 (GD15) or GD19. In rat offspring treated on GD15 there was a significant reduction in the accumulation of [³H]inositol phosphates induced by carbachol and a significant increase in the accumulation of [³H]inositol phosphates induced by norepinephrine on day 7, whereas no changes were observed at the other ages. No significant changes, on the other hand, were observed for glutamate-stimulated phosphoinositide metabolism in GD15 treated rats and for carbachol-, norepinephrine- and glutamate-stimulated phosphoinositide metabolism in animals treated on GD19 at any of the different ages evaluated. These results indicate that treatment with MAM on GD15, which results in a marked microencephaly, causes a marked alteration of muscarinic and ₁-adrenergic receptor-stimulated phosphoinositide metabolism during brain development and that these alterations undergo adaptive changes in the adult brain.     

Stereoselective binding and activity of oxotremorine analogs at muscarinic receptors in rat brain.

The activities of the enantiomers of BM-5 were examined to measure muscarinic cholinergic selectivity in the central nervous system. Autoradiographic studies assessed the ability of each enantiomer to inhibit the binding of [3H]-(R)-quinuclidinyl benzilate ([3H]-(R)-QNB) to muscarinic receptors in the rat brain. (+)-(R)-BM-5 inhibited [3H]-(R)-QNB binding to rat brain sections at concentrations below 1.0 microM, while 100-fold higher concentrations of (-)-(S)-BM-5 were required for comparable levels of inhibition. Analysis of the autoradiograms indicated that both stereoisomers had a similar distribution of high affinity binding sites. Each enantiomer displayed higher affinity for muscarinic receptors in the superior colliculi and lower affinity for receptors in the cerebral cortex and hippocampus. (+)-(R)-BM-5 and oxotremorine inhibited adenylyl cyclase activity in the cerebral cortex with efficacies comparable to that for acetylcholine. (+)-(R)-BM-5 was 26-fold more potent than (-)-(S)-BM-5 in inhibiting adenylyl cyclase. Oxotremorine-M and carbamylcholine stimulated phosphoinositide turnover in the cerebral cortex. Oxotremorine had lower activity and (+)-(R)-BM-5 was essentially inactive at comparable concentrations. The difference in activity of the two enantiomers indicates a remarkable stereochemical selectivity for muscarinic receptors. The stereoselectivity index is comparable for both the autoradiographic assays (48) and measures of adenylyl cyclase activity (26) in the cerebral cortex.     

Electroconvulsive Shock Increases α1b-but Not α1a-Adrenoceptor Binding Sites in Rat Cerebral Cortex

Repeated administration of electroconvulsive shock (ECS) increases [3H]prazosin binding to alpha 1-adrenoceptors in rat cerebral cortex. In contrast, [3H]WB4101 binding in cortex has been reported to be unchanged after ECS. [3H]Prazosin labels two alpha 1-adrenoceptor subtypes, termed alpha 1a and alpha 1b, whereas [3H]WB4101 labels the alpha 1a subtype preferentially. The purpose of this study was to determine whether ECS increases one or both alpha 1-adrenoceptor subtypes in rat cerebral cortex. We found that treatment of rats with ECS once daily for 10-12 days increased [3H]prazosin binding in cortex by about 25% but did not significantly alter [3H]WB4101 binding to alpha 1-adrenoceptors. Measurement of alpha 1a and alpha 1b receptors by competition analysis of the selective alpha 1a antagonist 5-methylurapidil against [3H]prazosin and measurement of [3H]prazosin binding in homogenates preincubated with chlorethylclonidine, which alkylates alpha 1b binding sites, also indicated that the ECS-induced increase in alpha 1-adrenoceptors is confined to the alpha 1b subtype. In contrast to its effect on [3H]prazosin binding, ECS did not increase phosphoinositide hydrolysis as measured by [3H]inositol 1-phosphate accumulation in slices of rat cerebral cortex stimulated by either norepinephrine or phenylephrine. The failure of ECS to increase [3H]inositol 1-phosphate accumulation stimulated by phenylephrine, which is a partial agonist for this response, suggests that spare receptors do not account for the apparent absence of effect of ECS on alpha 1-adrenoceptor-mediated phosphoinositide hydrolysis.     

Selective interaction of tricyclic antidepressants with a subclass of rat brain cholinergic muscarinic receptors

Experiments were undertaken to determine whether the anticholinergic actions of tricyclic antidepressants are mediated by a selective interaction with a subclass of muscarinic receptors. To this end, the potencies of these antidepressants to inhibit [3H]-QNB binding to rat brain cerebral cortical membranes was compared to their potencies as antagonists of carbachol-stimulated inositol phosphate accumulation in cerebral cortical slices and carbachol-induced inhibition of GTP-stimulated adenylate cyclase in striatal membranes. Whereas amitriptyline was more potent than pirenzepine, a selective muscarinic M1 receptor antagonist, in competing for [3H]-QNB binding sites and as an antagonist of carbachol-induced inhibition of adenylate cyclase, pirenzepine was substantially more active (ten-fold) than amitriptyline in blocking carbachol-stimulated phosphatidyl inositol turnover. Atropine was more potent than all other agents in these assays, failing to display any significant degree of selectivity. The results suggest that the tricyclic antidepressants, in particular amitriptyline, appear to be selective antagonists for muscarinic receptors associated with adenylate cyclase in striatal membranes. Given the current classification of cholinergic receptors, these findings indicate that the tricyclic antidepressants may be useful for defining the properties of M2 receptors in brain.     

Cholinergic hyperinnervation in the cerebral cortex of microencephalic rats does not result in muscarinic receptor down-regulation or in alteration of receptor-stimulated phosphoinositide metabolism

Administration of methylazoxymethanol (MAM; 25 mg/kg) to pregnant rats at gestational day 15 (GD 15) induces a marked reduction of telencephalic areas of the offspring brain. Previous neurochemical studies demonstrated a marked cholinergic hyperinnervation in the cerebral cortex of microencephalic rats. In this study we have evaluated whether this cholinergic hyperinnervation could result in altered functionality of muscarinic receptors. Acetylcholinesterase activity (AChE) was increased by 69% in the cerebral cortex of MAM treated rats confirming a relative hyperinnervation, whereas in the hippocampus and striatum no significant changes were observed. Despite the marked hyperinnervation, in the cerebral cortex of microencephalic rats neither muscarinic receptor-stimulated phosphoinositide metabolism nor muscarinic, receptor density were altered. No differences in receptor density were also observed in the hippocampus and striatum. Chronic diisopropylfluorophosphate (DFP) administration induced a marked decrease of AChE activity and down-regulation of muscarinic receptors whereas atropine administration resulted in receptor up-regulation in cerebral cortex, striatum and hippocampus of both control and MAM rats. The results confirm a relative cholinergic hyperinnervation in the cerebral cortex of microencephalic rats and demonstrate that the regulation of muscarinic receptor-stimulated phosphoinositide metabolism and muscarinic receptor plasticity is not modified in a condition of increased cholinergic presynaptic terminals.     

Heterogeneity of the M1 muscarinic receptor subtype between peripheral lung and cerebral cortex demonstrated by the selective antagonist AF-DX 116

Recent studies have demonstrated that the majority of muscarinic receptors in rabbit peripheral lung homogenates bind pirenzepine with high affinity (putative M1 subtype). In experiments of AF-DX 116 inhibiting [3H](-)quinuclidinyl benzilate or [3H]pirenzepine, we found similar inhibitory constants for AF-DX 116 binding in rat heart and rabbit peripheral lung that were 4-fold smaller (i.e. of higher affinity) than the inhibitory constant for rat cerebral cortex. This result demonstrates heterogeneity of the M1 muscarinic receptor subtype between peripheral lung and cerebral cortex.     

Muscarinic-agonist and guanine nucleotide activation of polyphosphoinositide phosphodiesterase in isolated islet-cell membranes.

Stimulated hydrolysis of the inositol phospholipids phosphatidylinositol 4-phosphate (PtdIns4P) and phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] was investigated by studying the phosphoinositides produced in a suspended preparation of plasma membranes by transference of 32P from [gamma-32P]ATP. At basal Ca2+ concentration (calculated free Ca2+, 150 nM) phospholipid hydrolysis was stimulated either by the muscarinic agonists carbamoylcholine and bethanecol or by the addition of the non-hydrolysable analogue of GTP, guanosine 5'-[beta gamma-imido]triphosphate [p(NH)ppG]. GTP was without effect on basal hyrolysis. Both GTP and p(NH)ppG enhanced the rapid (within 10 s) hydrolysis of PtdIns4P and PtdIns(4,5)P2 induced by carbamoylcholine in a dose-dependent manner. A rightward shift in the competition curve of carbamoylcholine for bound L-[3H]quinuclidinyl benzilate was seen on addition of GTP or p(NH)ppG (100 microM) under phosphorylating conditions. Pretreatment of intact islet cells with Bordetella pertussis toxin, islet-activating protein (IAP) or treatment of membranes with IAP under conditions which elicited ADP-ribosylation of a protein of Mr 41,000 was without effect on muscarinic binding, phosphoinositide phosphorylation or subsequent hydrolysis by carbamoylcholine. The findings indicate the involvement of a GTP-binding protein in the coupling of the muscarinic receptor to phosphoinositide hydrolysis in the islet cell and suggest that this is distinct from the GTP-binding regulatory component of adenylate cyclase which is covalently modified by IAP.     

Pertussis toxin does not inhibit muscarinic-receptor-mediated phosphoinositide hydrolysis or calcium mobilization.

Pertussis toxin was used to examine the role of the inhibitory guanine nucleotide regulatory protein, Ni, in muscarinic-receptor-mediated stimulation of phosphoinositide turnover and calcium mobilization. In cultured chick heart cells, pertussis-toxin treatment inhibited muscarinic-receptor-mediated attenuation of isoprenaline-stimulated cyclic AMP accumulation. This finding is consistent with the proposal that pertussis toxin blocks the capacity of Ni to couple muscarinic receptors to adenylate cyclase. In contrast, treatment of chick heart cells or 1321N1 human astrocytoma cells with pertussis toxin did not block muscarinic-receptor-mediated stimulation of phosphoinositide hydrolysis, as measured by [3H]inositol phosphate accumulation in the presence of Li+. Pertussis-toxin treatment also had little effect on basal and muscarinic-receptor-stimulated phosphatidylinositol synthesis, as measured by the incorporation of [3H]inositol into phosphatidylinositol. Activation of muscarinic receptors also enhances the rate of unidirectional 45Ca2+ efflux in 1321N1 cells; this response, like phosphoinositide hydrolysis, was not prevented by pertussis-toxin treatment. Our data suggest that muscarinic receptors are not coupled to phosphoinositide hydrolysis or calcium mobilization through Ni.     

Lithium Modulation of Phosphoinositide Signaling System in Rat Cortex: Selective Effect on Phorbol Ester Binding

Abstract— Recent work indicates that the therapeutic action of lithium may be mediated through perturbation of postreceptor second messenger systems. To elucidate further the postreceptor cellular sites of action(s) of lithium, the effect of chronic lithium treatment on various components of the receptor-activated phosphoinositide pathway was investigated. We found that chronic administration of lithium (0.2% LiCI, 21 days) to adult male rats did not significantly affect phosphoinositide hydrolysis in cerebral cortical slices induced by carbachol (1 m M ) or NaF (10 m M ). Nor did the same treatment alter the carbachol (1 m M ) potentiation of guanosine 5'-(γ-thio)triphosphate (30 μ M ) stimulation of phosphoinositide hydrolysis (an index of receptor/G protein coupling) in cortical membranes. Immunoblotting studies revealed no changes in the levels of Gα_q/11 immunoreactivity in the cortex after chronic lithium treatment. The levels of protein kinase C, as revealed by specific binding of [³H]phorbol dibutyrate ([³H]PDBu), were significantly reduced in the cytosolic fraction and increased in the particulate fraction of rat cortex after chronic lithium, whereas the K _D of [³H]PDBu binding remained relatively constant. A small and insignificant decrease in the density of [³H]inositol 1,4,5-trisphosphate binding was also found in the cortex. The above data suggest that chronic lithium treatment affects neither the muscarinic cholinergic-linked phosphoinositide turnover nor the putative G protein α subunit (Gα_q/11) responsible for phospholipase C activation. However, a possible translocation and activation of protein kinase C activity may be significant in the therapeutic effect of this mood-stabilizing agent.     

Activation of Muscarinic and of α1-Adrenergic Receptors on Astrocytes Results in the Accumulation of Inositol Phosphates

Astrocyte-enriched cultures prepared from the newborn rat cortex incorporated [3H]myo-inositol into intracellular free inositol and inositol lipid pools. Noradrenaline and carbachol stimulated the turnover of these pools resulting in an increased accumulation of intracellular [3H]inositol phosphates. The effects of noradrenaline and carbachol were dose-dependent and blocked by specific alpha 1-adrenergic and muscarinic cholinergic receptor antagonists, respectively. The increase in [3H]inositol phosphate accumulation caused by these receptor antagonists was virtually unchanged when cultures were incubated in Ca2+-free medium, but was abolished when EGTA was also present in the Ca2+-free medium. Cultures of meningeal fibroblasts, the major cell type contaminating the astrocyte cultures, also accumulated [3H]myo-inositol, but no increased accumulation of [3H]inositol phosphates was found in response to either noradrenaline or carbachol.     

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.