Potassium Ion Facilitation of Phosphoinositide Turnover Activation by Muscarinic Receptor Agonists in Rat Brain

In rat hippocampal slices kept in Krebs-Henseleit medium, an increase of K+ ions to 12 mM potentiates the stimulation of phosphoinositide turnover elicited by carbachol and (+/-)-cis-methyldioxolane. Oxotremorine is inactive if tested in Krebs-Henseleit medium but it stimulates by 220% the phosphoinositide turnover when K+ is increased to 12 mM. The K+ facilitation of the carbachol stimulation of phosphoinositide turnover was blocked by pirenzepine, a muscarinic antagonist. This drug was equally potent in inhibiting the carbachol stimulation of phosphoinositide turnover both in normal and 12 mM K+ Krebs medium. This facilitatory effect of K+ appears to be preferential for muscarinic receptors, since it failed to increase the activation of phosphoinositide breakdown induced by norepinephrine and histamine. The K+ potentiation of the muscarinic stimulation of phosphoinositide turnover is not mediated by a release of one of the endogenous neurotransmitters stored in these slices because such a facilitation occurs in Ca2+-deprived Krebs-Henseleit medium and failed to occur following a depolarizing dose of veratrine. Our experiments excluded that K+ facilitates carbachol stimulation of phosphoinositide turnover because it modifies the binding characteristics of muscarinic receptors; however, they cannot exclude that K+ acts at the receptor transducer coupling.     

Tryptamine Induces Phosphoinositide Turnover and Modulates Adrenergic and Muscarinic Cholinergic Receptor Function in Cultured Cerebellar Granule Cells

Abstract: Tryptamine dose-dependently increased phosphoinositide (PI) hydrolysis by approximately fourfold in primary cultures of rat cerebellar granule cells (EC₅₀ = 56 µ M ). The PI response stimulated by tryptamine was dependent on the presence of extracellular Ca²⁺ and Na⁺. Tryptamine-induced PI breakdown could be partially inhibited by pretreatment with 4β-phorbol 12-myristate 13-acetate but not pertussis toxin. The presence of tryptamine markedly attenuated PI responses induced by norepinephrine (NE) and carbachol, with no apparent effect on the responses to 5-hydroxytryptamine and glutamate. The inhibition of NE- and carbachol-induced PI turnover by tryptamine was dose dependent with IC₅₀ values of ∼0.4 and ∼2.5 m M , respectively. Pretreatment of cells with tryptamine (0.5 m M ) also attenuated NE- and carbachol-induced PI turnover, but failed to affect 5-hydroxytryptamine- and glutamate-induced responses. Furthermore, ketanserin, atropine, and prazosin did not have any effect on inositol phosphate formation induced by tryptamine. These observations indicate that tryptamine markedly increased Ca²⁺- and Na⁺-dependent PI turnover in cerebellar neurons and selectively inhibited NE- and carbachol-induced PI hydrolysis.     

Sequestration of Muscarinic Cholinergic Receptors in Permeabilized Neuroblastoma Cells

Abstract: The feasibility of using a permeabilized preparation of human SH-SY-5Y neuroblastoma cells for studies of muscarinic acetylcholine receptor (mAChR) sequestration has been evaluated. Exposure of cells permeabilized with digitonin, streptolysin-O, or the α-toxin from Staphylococcus aureus to oxotremorine-M (Oxo-M) for 30 min resulted in a 25–30% reduction in the number of cell surface mAChRs, as monitored by the loss of N[³H]methyl- scopolamine ([³H]NMS) binding sites. The corresponding value for intact cells was 40%. For cells permeabilized with 20 μM digitonin, the Oxo-M-mediated reduction in [³H]NMS binding was time (t_1/2～ 5 min) and concentration (EC₅₀～ 10 μM) dependent and was agonist specific (Oxo M > bethanechol = arecoline = pilocarpine). In contrast, no reduction in total mAChR number, as monitored by the binding of [³H]quinuclidinyl benzilate, occurred following Oxo-M treatment. The loss of [³H]NMS sites observed in the presence of Oxo-M was unaffected by omission of either ATP or Ca²⁺, both of which are required for stimulated phosphoinositide hydrolysis, but could be inhibited by the inclusion of guanosine 5′-O-(2-thiodiphosphate). mAChRs sequestered in response to Oxo-M addition were unmasked when the cells were permeabilized in the presence of higher concentrations of digitonin (80 μM). The results indicate (a) that permeabilized SH-SY-5Y cells support an agonist-induced sequestration of mAChRs, the magnitude of which is ～ 65–70% of that observed for intact cells, (b) that when internalized, mAChRs are located in a cellular compartment to which [³H]NMS has only a limited access despite the removal of the plasma membrane barrier, and (c) that the production of phosphoinositide-derived second messengers is not a prerequisite for mAChR sequestration.     

Activation of Adenosine A2 Receptors Stimulates Phosphoinositide Metabolism in Rat Peripheral Nerve

Abstract: The effects of adenosine analogues on phosphoinositide metabolism in rat sciatic nerve were examined. Sciatic nerve segments were prelabeled with [³H]-cytidine and incubated in the presence of LiCl and varying concentrations of adenosine analogues. The formation of [³H]cytidine monophosphate phosphatidic acid ([³H]-CMP-PA) was determined as an index of phosphoinositide breakdown. Liponucleotide accumulation was elevated significantly in the presence of 5'- N -ethylcarboxamidoadenosine (NECA), a nonselective analogue, and two different A₂-selective analogues, N ⁶-[2-(3,5-dimethoxyphenyl)-2-(2-methylphenyl)ethyl]adenosine and 2- p -(2-carboxyethyl)phenethylamino-NECA (CGS 21680), but not by N ⁶-cyclopentyladenosine, an A₁-selective analogue. The stimulatory action of CGS 21680 was blocked by the A₂-selective adenosine receptor antagonists 3,7-dimethyl-1-propargylxanthine (DMPX) and 1,3-dipropyl-7-methylxanthine. Inositol phosphate formation was also stimulated to a comparable degree by CGS 21680 and this response was antagonized by DMPX. Carbamylcholine, which was previously shown to stimulate phosphoinositide breakdown, also enhanced the accumulation of CMP-PA. When adenosine analogues and carbamylcholine were simultaneously present, their effects were additive. Taken together, these data suggest that an adenosine receptor, possibly of the A₂ subtype, is coupled to enhanced phosphoinositide hydrolysis in peripheral nerve. However, adenosine-receptor activation does not appear to modulate phosphoinositide hydrolysis stimulated via muscarinic receptors.     

Effect of Electrical Stimulation on Phosphoinositide Metabolism in Rat Sciatic Nerve In Vivo

The metabolism of phosphoinositides in rat sciatic nerves in vivo during electrical stimulation was studied. Nerves were prelabeled by injection of [2-3H]-myo-inositol alone for periods of 2 and 20 h or together with [32P]orthophosphate for 2 h and then electrically stimulated (100 Hz) for 5 or 20 min. Contralateral unstimulated nerve served as the control. When tritiated myo-inositol was used alone for prelabeling the nerves, approximately 6% and 14% of the label was incorporated into lipids after 2 h and 20 h, respectively. Both 5 and 20 min of electrical stimulation caused an insignificant change in the percentage of radioactivity recovered in lipids from the nerves prelabeled with either myo-inositol or with a mixture of myo-inositol and phosphate. The proportion of label associated with phosphoinositides of nerves prelabeled with myo-inositol for both 2 h and 20 h showed an increase in phosphatidyl-inositol-4-phosphate at the expense of phosphatidylinositol in stimulated nerves. Similar results were obtained with nerves prelabeled for 2 h with a mixture of [32P]orthophosphate and [2-3H]myo-inositol. No significant changes in the radioactivity associated with water-soluble inositol phosphates were found in stimulated versus control nerves.     

Relationship of ATP Turnover, Polyphosphoinositide Metabolism, and Protein Phosphorylation in Sciatic Nerve and Derived Peripheral Myelin Subfractions from Normal and Streptozotocin Diabetic Rats

Sciatic nerve from streptozotocin-induced diabetic rats has previously been shown to incorporate more 32P into phosphatidylinositol-4,5-bisphosphate (PIP2) and the principal myelin proteins than normal nerve. In the present study, labeling of ATP and PIP2 was compared. Using nerve segments, [gamma-32P]ATP specific activity reached a plateau after incubation for 4 h with [32P]orthophosphate, whereas the specific activity of [32P]PIP2 rose much more slowly and was still increasing after 8 h. The rate of disappearance of radioactivity from prelabeled ATP was biphasic, with 75% being lost within 30 min and the remainder declining much more slowly for several hours thereafter. In contrast, no decrease in prelabeled PIP2 radioactivity could be detected for up to 4 h. The kinetics of ATP metabolism were not appreciably different for normal and diabetic nerve. However, after incubation with [32P]orthophosphate for 2 h, the specific activity of PIP2 was 50-120% higher in diabetic nerve. This phenomenon, therefore, cannot be ascribed to altered specific activity of the ATP precursor pool. Greater labeling of PIP2 in 32P-labeled diabetic nerve was present in purified myelin isolated using a simple discontinuous sucrose density gradient, but not in a "nonmyelin" fraction. When nerve homogenate was fractionated on a more complex gradient, three myelin-enriched subfractions were obtained which were heterogeneous as judged by morphological appearance, protein profile, and lipid metabolic activity. The proportion of total lipid radioactivity accounted for by PIP2 was elevated in all the subfractions relative to the homogenate. As compared to myelin subfractions from normal nerve, an increased percentage of 32P in PIP2 was obtained only in the major myelin subfraction from diabetic nerve. The phosphorylation of P0 relative to the other myelin proteins was also enhanced in this subfraction in nerve from diabetic animals.     

Cholinergic Dilatation and Constriction of Feline Cerebral Blood Vessels Are Mediated by Stimulation of Phosphoinositide Metabolism via Two Different Muscarinic Receptor Subtypes

Abstract: The muscarinic receptors involved in phosphoinositide (PI) hydrolysis have been pharmacologically characterized in cat cerebral blood vessels. Carbachol elicited a concentration-dependent increase in inositol phosphate accumulation [inositol monophosphate, bisphosphate, trisphosphate (IP₃) and tetrakisphosphate] in both major cerebral arteries and small pial vessels, which reached 140–280% of baseline at 10^?3M carbachol (referred to as maximal effect). However, the inositol phosphate accumulation response was found to be biphasic with a submaximal effect (30–50% of the maximal stimulation) obtained at low carbachol concentrations (<10^?5M). Endothelial denudation induced a virtual disappearance of the submaximal PI response without affecting that elicited by high concentrations of carbachol. The pharmacology of the two carbachol-induced PI responses was investigated by comparing the potency of selected muscarinic antagonists to block the IP₃ accumulation induced by 10^?7M (endothelium-dependent submaximal effect) and 10^?4M (endothelium-independent near-maximal effect) carbachol. In both major arteries and pial vessels, the activation of IP₃ production by 10^?4M carbachol was similarly inhibited by muscarinic antagonists with the following averaged rank order of potency (in -log IC₅₀): 4-diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP; 8.65) > pirenzepine (8.28) > 6-chloro-5,10-dihydro-5-[(1-methyl-4-piperidinyl)acetyl]-11H-dibenzo[b,e][1,4]diazepine-11-one (UH-AH 371; 7.87) > 11-[[2-[(diethylamino)methyl]-1-piperidinyl]acetyl]-5,-11-dihydro-6H-pyridol[2,3-b][1,4]benzodiazepine-6-one (AF-DX 116; 6.62), a pharmacological profile compatible with an M₁ receptor subtype. In contrast, the submaximal stimulation of the PI metabolism elicited by 10^?7M carbachol in major arteries was blocked by the same antagonists with the following order of potency (in -log IC₅₀): 4-DAMP (8.38) > pirenzepine (7.25) > UH-AH 371 (6.25) > AF-DX 116 (5.72), which was reminiscent of an M₃ pharmacological profile. These findings indicate that stimulation of cerebrovascular muscarinic receptors is accompanied by PI hydrolysis via two distinct receptors, most probably the M₁ and M₃ subtypes that have been associated with constriction and dilatation, respectively, of cat cerebral arteries. Furthermore, these results provide strong evidence for an endothelial localization of the M₃ dilatatory receptors within the vessel wall.     

Effects of Muscarinic Agonists and Depolarizing Agents on Inositol Monophosphate Accumulation in the Rabbit Vagus Nerve

The effects of muscarinic agonists and depolarizing agents on inositol phospholipid hydrolysis in the rabbit vagus nerve were assessed by the measurement of [3H]inositol monophosphate production in nerves that had been preincubated with [3H]inositol. After 1 h of drug action, carbachol, oxotremorine, and arecoline increased the inositol monophosphate accumulation, though the maximal increase induced by these agonists differed. Addition of the muscarinic antagonists atropine or pirenzepine shifted the carbachol dose-response curves to the right, without decreasing the carbachol maximal stimulatory effects. The KB for pirenzepine was 35 nM, which is characteristic of muscarinic high-affinity binding sites coupled to phosphoinositide turnover and often associated with the M1 receptor subtype. On the other hand, agents known to depolarize or to increase the intracellular Ca2+ concentration, e.g., elevated extracellular K+, ouabain, Ca2+, and the Ca2+ ionophore A23187, also increased inositol monophosphate accumulation. These effects were not mediated by the release of acetylcholine, as suggested by the fact that they could not be potentiated by the addition of physostigmine nor inhibited by the addition of atropine. The Ca(2+)-channel antagonist Cd2+, also known to inhibit the Na+/Ca2+ exchanger, was able to block the effects of K+ and ouabain, but did not alter those of carbachol. These results suggest that depolarizing agents increase inositol monophosphate accumulation in part through elevation of the intracellular Ca2+ concentration and that muscarinic receptors coupled to phosphoinositide turnover are present along the trunk of the rabbit vagus nerve.     

Changes in Nicotinic and Muscarinic Cholinergic Receptors in Alzheimer-Type Dementia

Nicotinic and muscarinic cholinergic receptors were studied in autopsied brains from four histologically normal controls and five histopathologically verified cases of Alzheimer-type dementia (ATD), using ligand binding techniques. Nicotinic and muscarinic cholinergic receptors were assessed by (-)-[3H]nicotine and [3H]quinuclidinyl benzilate [( 3H]QNB), respectively. Compared with the controls, (-)-[3H]nicotine binding sites in the ATD brain regions examined were significantly reduced in the putamen and the nucleus basalis of Meynert (NbM). [3H]QNB binding was significantly reduced in the hippocampus and NbM. These findings suggest that there are significant changes of nicotinic and muscarinic cholinergic receptors in selected regions of ATD brains.     

Identification of G Protein Subtypes in Peripheral Nerve and Cultured Schwann Cells

In this study, we investigated the expression of various G proteins in whole sciatic nerves, in myelin and nonmyelin fractions from these nerves, and in membranes of immortalized Schwann cells. In myelin, nonmyelin, and Schwann cell membranes we detected two 39-40-kDa pertussis toxin substrates that were resolved on separation on urea-gradient gels. Two cholera toxin substrates with apparent molecular masses of 42 and 47 kDa were present in nerve and brain myelin and in Schwann cell membranes. In these membranes, a third 45-kDa cholera toxin substrate, which displayed the highest labeling, was also present. Immunoblotting with specific antisera allowed the identification of G(o) alpha, Gi1 alpha, Gi2 alpha, Gi3 alpha, Gq/G11 alpha, and the two isoforms of Gs alpha in nerve homogenates, nerve, and brain myelin fractions. In Schwann cell membranes we identified G(o) alpha, Gi2 alpha, Gi3 alpha, and proteins from the Gq family, but no immunoreactivity toward anti-Gi1 alpha antiserum was detected. In these membranes, anti-Gs alpha antibody recognized the three cholera toxin substrates mentioned above, with the 45-kDa band displaying the highest immunoreactivity. Relative to sciatic nerve myelin, the Schwann cell membranes revealed a significantly higher expression of Gi3 alpha and the absence of Gi1 alpha. The different distribution of G proteins among the different nerve compartments might reflect the very specialized function of Schwann cells and myelin within the nerve.     

Enhanced Coupling of Neonatal Muscarinic Receptors in Rat Brain to Phosphoinositide Turnover

The relationship between the density of the muscarinic receptor in developing rat cerebral cortex and its coupling to phosphoinositide turnover is examined. Tissue slices from rats of various ages were incubated with myo-[2-3H]inositol, and the effect of carbamoylcholine on the release of total inositol phosphates was determined. Binding of [3H]quinuclidinyl benzilate was determined in the same tissue. Although muscarinic receptor density in day-18 embryonic cortex was only 5% of that in the adult, the maximal response of stimulated phosphoinositide turnover to carbamoylcholine (1-10 mM) was at the adult level (i.e., three-fold increase). Comparison of the dependence of the turnover on carbamoylcholine concentration revealed that in neonates, the dose-response curve was shifted to the left, giving a half-maximal effect at concentrations approximately tenfold lower than that in the adult. In addition, the partial muscarinic agonists oxotremorine-2 and bethanechol were both more efficacious in young rats than in adults. The differences could not be accounted for either by alterations in agonist affinity for the receptor or by the presence of "spare" muscarinic receptors. These results indicate that muscarinic receptors in fetal and newborn rat cerebral cortex are more efficiently coupled to stimulation of phosphoinositide turnover than in the adult.     

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.     

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.     

Maitotoxin Induces Phosphoinositide Turnover and Modulates Glutamatergic and Muscarinic Cholinergic Receptor Function in Cultured Cerebellar Neurons

Maitotoxin (MTX) stimulated inositol phosphate (IP) formation in primary cultures of rat cerebellar granule cells. MTX-induced IP production was dependent on extracellular Ca2+ but independent of extracellular Na+. The stimulation of IP formation elicited by MTX was unaffected by pretreatment of cells with phorbol dibutyrate, pertussis toxin, and a variety of Ca2+ entry blockers, such as nimodipine, nisoldipine, Co2+, and Mn2+. The presence of MTX markedly attenuated IP production induced by carbachol and glutamate, with no apparent effect on the responses to norepinephrine (NE), histamine, 5-hydroxytryptamine (5-HT), and endothelin-1. The inhibition of the carbachol- and glutamate-induced responses by MTX was dose dependent with IC50 values of 1.2 and 0.5 ng/ml, respectively. Pretreatment of cells with a lower concentration of MTX (0.3 ng/ml) also attenuated carbachol- and glutamate-induced IP formation, in a time-dependent manner, with a decrease observed after 30 min prestimulation, but failed to affect NE-, histamine-, 5-HT-, endothelin-1, and sarafotoxin S6b-induced responses. Thus, MTX elicited a marked Ca2(+)-dependent phosphoinositide (PI) turnover in cerebellar granule cells and selectively inhibited carbachol- and glutamate-induced PI hydrolysis. Possible mechanisms underlying these selective modulations are discussed.     

Interactions of Muscarinic Receptors with the Heterotrimeric G Proteins Gq and G12: Transduction of Proliferative Signals

Abstract: The proliferative and transforming properties of m2 and m5 muscarinic acetylcholine receptors and a series of wild-type, chimeric, and mutant G proteins were measured alone or in combination in NIH 3T3 cells to determine which G proteins mediate these signals and to what extent these signals can be influenced by changing the stoichiometry of receptors and G proteins. Responses were measured using the focus-forming assay and a novel assay called R-SAT (R eceptor S election and A mplification T echnology) in which proliferative responses are monitored using a reporter gene. Individually, GTPase-deficient mutants (^?) of Gαq and Gα12, wild-type Gαq, and m5 were active in R-SAT. Gα12^? and m5 also induced focus formation. m2 was inactive in both assays. The ability of m5 to induce foci was significantly reduced by coexpression of Gαq^?. Synergistic effects of receptor/G protein combinations were not observed in focus-forming assays but were readily detected by R-SAT. Coexpression of Gαq with m5 induced constitutive activity in R-SAT and increased the potency of agonists at m5 by 90-fold. Gαq also evoked agonist-dependent responses from m2 but not constitutive activity. Agonist potency was increased 10-fold at m2 and decreased 15-fold at m5 when these receptors were coexpressed with Gαqi5, a chimeric G protein containing the five C-terminal residues of Gαi2, compared with coexpression with Gαq. Both Gαq and Gαqi5 had biphasic effects on the proliferative responses to m5 and m2, respectively, inhibiting responses at high agonist concentrations. Coexpression of Gα12 or Gα12i5 had no effect on the concentration-response relationships of m5, but both elicited weak responses from m2. We conclude that although Gα12 is a more potent oncogene, Gαq transduces m5-driven cellular responses. The demonstrations that proliferative responses can be elicited from a nonmitogenic receptor by altering the type and concentration of available G proteins and that constitutive responses can be induced by G proteins imply that both the magnitude and type of receptor-initiated signal can be regulated at the level of G proteins in vivo.     

Structural Microheterogeneity of the Muscarinic Cholinergic Receptor Is Not Related to Functional Diversity Identified by Differences in Affinity for Pirenzepine

The muscarinic receptor for acetylcholine shows a diversity in ligand binding properties and effector mechanisms which have suggested the existence of two subtypes (M1 and M2), to which the selective antagonist pirenzepine binds with markedly different affinities. The receptor from rat brain, covalently labelled with the alkylating antagonist tritiated propylbenzilylcholine mustard, displays a structural microheterogeneity on electrophoresis, covering the region of apparent molecular weight 66,000-76,000, with dominant components at 68,000 and 73,000. Selective inhibition by pirenzepine of labelling of the M1 receptor with tritiated mustard has been analysed on fluorographs of sodium dodecyl sulphate-polyacrylamide gels and shown to cause a uniform reduction in radioactive labelling of the broad receptor peak, rather than selectively inhibiting either the high- or low-molecular-weight regions of the band. It is further shown that although this receptor microheterogeneity is found for each of four brain regions studied, it is not found for the heart receptor, which gives a discrete labelled band of apparent molecular weight 72,000. It is therefore suggested that the structural microheterogeneity is the result of tissue-specific, posttranslational modification of the molecule, such as glycosylation, and is not directly related to the functional diversity of the receptor.     

Muscarinic Cholinergic Stimulation of Exogenous Phosphatidylinositol Hydrolysis Is Regulated by Guanine Nucleotides in Rabbit Brain Cortical Membranes

Rabbit brain cortical membranes, which have been extracted with 2 M KCl, hydrolyze exogenously added [3H]phosphatidylinositol [( 3H]PI) in a guanine nucleotide- and carbachol-dependent manner. Both oxotremorine-M and carbachol are full agonists with EC50 values of 8 and 73 microM, respectively. Pirenzepine and atropine inhibit carbachol-stimulated [3H]PI hydrolysis. The hydrolysis-resistant guanine nucleotide analog guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) is the most potent in supporting carbachol-stimulated hydrolysis of PI. There is no effect of carbachol in the absence of guanine nucleotides or in the presence of 100 microM adenosine 5'-O-(3-thiotriphosphate), adenosine-5'-(beta, gamma-imido)triphosphate, or sodium pyrophosphate. Guanylyl-5'-(beta,gamma-imido)triphosphate [Gpp(NH)p] in the presence of carbachol also stimulates PI hydrolysis although much less than that seen with GTP gamma S. GDP and Gpp(NH)p are potent antagonists of the GTP gamma S-dependent carbachol response. Optimal stimulation by carbachol and GTP gamma S was observed at 0.3-1 microM free Ca2+ and 6 mM MgCl2. Limited trypsinization resulted in loss of receptor-regulated PI breakdown and a slight decrease in basal activity. These results demonstrate that phospholipase C hydrolysis of exogenous PI by rabbit cortical membranes may be stimulated by carbachol in a guanine nucleotide-dependent manner.     

α2-Adrenergic and Muscarinic Cholinergic Receptors Have Oposing Actions on Cyclic AMP Levels in SK-N-SH Human Neuroblastoma Cells

Forskolin and vasoactive intestinal polypeptide (VIP) were shown to increase cyclic AMP accumulation in a human neuroblastoma cell line, SK-N-SH cells. The alpha 2-adrenergic agonist UK 14304 decreased forskolin-stimulated cyclic AMP levels by 40 +/- 2%, with an EC50 of 83 +/- 20 nM. This response was blocked by pretreatment with pertussis toxin (PT) (EC50 = 1 ng/ml) or by the alpha 2-antagonists yohimbine, idazoxan, and phentolamine. Antagonist IC50 values were 0.3 +/- 0.1, 2.2 +/- 0.3, and 1.4 +/- 0.1 microM, respectively. This finding suggests the presence of normal inhibitory coupling of SK-N-SH cell alpha 2-adrenergic receptors to adenylate cyclase via the inhibitory GTP-binding protein species, Gi. Muscarinic receptors in many target cell types are coupled to inhibition of adenylate cyclase. However, in SK-N-SH cells, muscarinic agonists synergistically increased (67-95%) the level of cyclic AMP accumulation elicited by forskolin or VIP. EC50 values for carbamylcholine (CCh) and oxotremorine facilitation of the forskolin response were 1.2 +/- 0.2 and 0.3 +/- 0.1 microM, respectively. Pharmacological studies using the muscarinic receptor subtype-preferring antagonists 4-diphenylacetoxy-N-methylpiperidine, pirenzepine, and AF-DX 116 indicated mediation of this response by the M3 subtype. IC50 values were 14 +/- 1, 16,857 +/- 757, and 148,043 +/- 16,209 nM, respectively. CCh-elicited responses were unaffected by PT pretreatment. Muscarinic agonist binding affinity was indirectly measured by the ability of CCh to compete for [3H]quinuclidinyl benzilate binding sites on SK-N-SH cell membranes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Stimulation of Phospholipase D Activity in Human Neuroblastoma (LA-N-2) Cells by Activation of Muscarinic Acetylcholine Receptors or by Phorbol Esters: Relationship to Phosphoinositide Turnover

We have investigated the coupling of muscarinic acetylcholine receptors (mAChR) to phospholipid hydrolysis in a human neuroblastoma cell line, LA-N-2, by measuring the formation of 3H-inositol phosphates (3H-IP) and of [3H]phosphatidylethanol ([3H]PEt) in cells prelabeled with [3H]inositol and [3H]oleic acid. The muscarinic agonist carbachol (CCh) stimulated the phospholipase C (PLC)-mediated formation of 3H-IP in a time- and dose-dependent manner (EC50 = 40-55 microM). In addition, in the presence of ethanol (170-300 mM), CCh elevated levels of [3H]PEt [which is regarded as a specific indicator of phospholipase D (PLD) activity] by three- to sixfold. The effect of CCh on PEt formation also was dose dependent (EC50 = 50 microM). Both effects of CCh were antagonized by atropine, indicating that they were mediated by mAChR. Incubation of LA-N-2 cells with the phorbol ester phorbol 12-myristate 13-acetate (PMA, 0.1 microM; 10 min) increased [3H]PEt levels by up to 10-fold. This effect was inhibited by the protein kinase C (PKC) inhibitor staurosporine (1 microM) or by pretreatment for 24 h with 0.1 microM PMA, by 74% and 65%, respectively. In contrast, the effect of CCh on PEt accumulation was attenuated by only 28% in the presence of staurosporine (1 microM). In summary, these results suggest that, in LA-N-2 neuroblastoma cells, mAChR are coupled both to phosphoinositide-specific PLC and to PLD. PKC is capable of stimulating PLD activity in these cells; however, it is not required for stimulation of the enzyme by mAChR activation.     

Effects of Systemically Administered Lithium on Phosphoinositide Metabolism in Rat Brain, Kidney, and Testis

A single subcutaneous dose of 10 mEq/kg LiCl gives rise to an increase in the cerebral cortex level of myo-inositol-1-P (I1P) that closely follows cortical lithium levels and, at maximum, is 40-fold above the control value. Kidney and testis show smaller increases in I1P level following LiCl administration. The I1P level is still sixfold greater than that of untreated rat cortex 72 h later. In cortex, parallel increases also occur in myo-inositol-4-P (I4P) and myo-inositol 1,2-cyclic-P (cI1,2P), whereas myo-inositol-5-P (I5P) remains unchanged. The cortical increases in I1P and I4P levels are partially reversed by administering 150 mg/kg of atropine 22 h after the LiCl, treatment that does not affect cI1,2P. When doses of LiCl from 2 to 17 mEq/kg are given, the cerebral cortex levels of I1P and myo-inositol, measured 24 h later, are found to reach a plateau at about 9 mEq/kg of LiCl, whereas cortical lithium levels continued to increase with greater LiCl doses. Levels of all three of the brain phosphoinositides are unchanged by the 10 mEq/kg LiCl dose, as is the uptake of 32Pi into these lipids. Chronic dietary administration of LiCl for 22 days showed that the effects of lithium on I1P and myo-inositol levels persist for that period. Over the course of the chronic administration of the lithium, levels of I1P, myo-inositol, and of lithium in cortex remained significantly correlated. We believe that these increases in inositol phosphates result from endogenous phosphoinositide metabolism in cerebral cortex and that lithium is capable of modulating that metabolism by reducing cellular myo-inositol levels. The size of the effect is a function of both lithium dose and the degree of stimulation of receptor-linked phosphoinositide metabolism. This property of lithium may explain part of its ability to moderate the symptoms of mania. Our chronic study suggests that prolonged administration of LiCl does not result in compensatory changes in myo-inositol-1-P synthase or myo-inositol-1-phosphatase.