Asymmetric distribution and down-regulation of the muscarinic acetylcholine receptor in rat cerebral cortex

The distribution and down-regulation of the muscarinic acetylcholine receptor (mAChR) were studied in dissociated cells from right (RCC) and left (LCC) cerebral cortex. For this purpose [³H]quinuclidinyl benzilate (QNB) and [³H]pirenzepine (Pz), two muscarinic antagonists, were used. The mAChR binding sites detected with [³H]QNB were asymmetrically distributed between the two hemispheres, the majority being found in the RCC. Asymmetry was also evident in the distribution of the mAChR subtypes (M₁ and M₂) detected with [³H]Pz. Under basal conditions the RCC had roughly 50% more M₁ subtype than the LCC. The pharmacological and kinetic parameters were similar for both antagonists in RCC and LCC, indicating that the observed lateralization was due to a different density of the receptor rather than to different kinetics of binding of the two radioligands. After sustained stimulation with the agonist carbamoylcholine, the receptor sites detected with [³H]Pz, i.e. the M₁ subtype of mAChR, decreased at a higher rate in the RCC (44%) than in the LCC (25% of controls), demonstrating that the down-regulation process is more active in the right than in the left cortex, and thus implying that there is better coupling between the stimulated mAChR and its effector system in the former.     

Phospholipid metabolism under muscarinic cholinergic stimulation exhibits brain asymmetry

In order to characterize some of the lateralized biochemical events promoted in brain upon massive neurotransmitter release, the labeling of lipids under specific stimulation of the muscarinic acetylcholine receptor (mAChR) has been studied in synaptosomes obtained from right and left cerebral cortex (RCC and LCC respectively). Synaptosomes were incubated with [³²P]phosphate in the absence and in the presence of the cholinergic agonist carbamoylcholine and the muscarinic antagonist atropine. Binding of the agonist to the mAChR promoted an enhanced labeling of polyphosphoinositides, such effect being considerably more pronounced in the LCC than in the RCC. The differences observed could be due to a higher mAChR-elicited activity of phospholipase C in the RCC than in the LCC. The results show that mAChR stimulation activates the turnover of inosítol lipids to a different extent in the two hemispheres, indicating either an uneven distribution of the receptor in brain and/or dissimilarities in the degree of coupling of the mAChR with its corresponding transmembrane signaling system in each hemicortex.     

AFDX-116 discriminates between muscarinic M2 receptors of the heart and the iris smooth muscle

Summary Studies with the atypical muscarinic antagonist pirenzepine provide convincing evidence for the classification of muscarinic acetylcholine receptors (mAChRs) into two subtypes, M₁ and M₂. The present study examines the heterogeneity of the M₂ subtype employing the newly developed competitive muscarinic antagonist, AFDX-116. Comparison of the binding affinities of pirenzepine, atropine, and AFDX-116 to mAChRs in microsomes from the rabbit cerebral cortex, heart, and iris smooth muscle shows that iris mAChRs, which are pharmacologically of the M₂ subtype, can be distinguished from M₂ cardiac receptors based on their affinity for AFDX-116. These results are consistent with the hypothesis that the M₂ receptor subtype consists of a heterogeneous population of receptors.Abbreviations mAChRs Muscarinic Acetylcholine Receptors - CCh Carbachol - NMS N-Methylscopolamine - AFDX-116 11-[[2-[(diethylamino)methyl]-1-piperidinyl]acetyl]-5,11-dihydro-6Hpyrido[2,3-b][1,4]benzodiazepine-6-one     

A Point Mutation in the Coding Sequence of the β-Hexosaminidase α Gene Results in Defective Processing of the Enzyme Protein in an Unusual GM2-Gangliosidosis Variant

The M1-selective (high affinity for pirenzepine) muscarinic acetylcholine receptor (mAChR) antagonist pirenzepine displaced both N-[3H]methylscopolamine [( 3H]NMS) and [3H]quinuclidinylbenzilate from intact human SK-N-SH neuroblastoma cells with a low affinity (Ki = 869-1,066 nM), a result indicating the predominance of the M2 or M3 (low affinity for pirenzepine) receptor subtype in these cells. Whereas a selective M2 agent, AF-DX 116 [11-2[[2-[(diethylamino)methyl]-1-piperidinyl]- acetyl]-5,11-dihydro-6H-pyrido[2,3-b][1,4]benzodiazepin-6-one) bound to the mAChRs with a very low affinity (Ki = 6.0 microM), 4-diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP), an agent that binds with high affinity to the M3 subtype, potently inhibited [3H]NMS binding (Ki = 7.2 nM). 4-DAMP was also 1,000-fold more effective than AF-DX 116 at blocking stimulated phosphoinositide (PPI) hydrolysis in these cells. Covalent labeling studies (with [3H]propylbenzilycholine mustard) suggest that the size of the SK-N-SH mAChR (Mr = 81,000-98,000) distinguishes it from the predominant mAChR species in rat cerebral cortex (Mr = 66,000), an M1-enriched tissue. These results provide the first demonstration of a neural M3 mAChR subtype that couples to PPI turnover.     

Expression and Agonist-Induced Down-Regulation of mRNAs of m2- and m3-Muscarinic Acetylcholine Receptors in Cultured Cerebellar Granule Cells

The regulation and expression of muscarinic acetylcholine receptor (mAChR) mRNA was studied in cultured cerebellar granule cells using Northern blot hybridization, mRNA species for m2- and m3-mAChRs but not m1- and m4-mAChRs were detected in these cells. The expression of mRNAs of both m2- and m3-mAChRs reached a maximum on the tenth day in culture but their expression patterns differed. Treatment of cerebellar granule cells after 8 days in culture with 100 microM carbachol led to differential down-regulation of the mRNA species of both mAChR subtypes present. Muscarinic receptor antagonists, atropine (1 microM) and pirenzepine (10 microM), prevented carbachol-induced m3-mAChR mRNA down-regulation observed at 8 h. However, exposure to either atropine or pirenzepine alone for 8 h led to a significant up-regulation of m3-mAChR mRNA. Thus, the mRNA species for both m2- and m3-mAChR subtypes are differentially expressed in culture and down-regulated by agonist stimulation. The loss of these mRNA species may play a role in the down-regulation of mAChR binding sites that occurs after desensitization.     

Upregulation of mRNA Encoding the M5 Muscarinic Acetylcholine Receptor in Human T- and B-Lymphocytes During Immunological Responses

Lymphocytes possess an independent, non-neuronal cholinergic system. Moreover, both T- and B-lymphocytes express multiple muscarinic acetylcholine receptors (mAChR). To obtain a better understanding of the regulatory mechanisms governing mAChR gene expression in the lymphocytic cholinergic system, we examined the effects of lymphocyte activation on expression of mAChR mRNA. Stimulation of T- and B-lymphocytes, respectively, with T-cell activator phytohemagglutinin and B-cell activator Staphylococcus aureus Cowan I upregulated M₅ mAChR mRNA expression in the CEM human leukemic T-cell line and in the Daudi B-cell line, which served as models of lymphocytes. In striking contrast, M₃ and M₄ mAChR mRNA expression was not affected in either cell line. Nonetheless, stimulating lymphocytes with phorbol 12-myristate 13-acetate, a protein kinase C activator, plus ionomycin, a calcium ionophore, upregulated expression of both M₃ and M₅ mAChR mRNA. This represents the first demonstration that immunological stimulation leads to M₅ mAChR gene expression in lymphocytes.     

Activation of protein kinase C induces rapid internalization and subsequent degradation of muscarinic acetylcholine receptors in neuroblastoma cells

The tumor-promoting phorbol ester 4 beta-phorbol 12 beta-myristate 13 alpha-acetate (PMA), which activates protein kinase C, acted synergistically with A23187 to decrease muscarinic acetylcholine receptor (mAChR) number in neuroblastoma cells (clone N1E-115) as determined by a filter binding assay using [3H]quinuclidinyl benzilate in membrane homogenates. After a 6-h incubation, 10(-7) M PMA and 3 X 10(-7) M A23187 reduced mAChR number 30-40%, compared to the 40-50% reduction observed after treatment with 10(-3) M carbachol, a muscarinic agonist. Incubation with 3 X 10(-7) M A23187 and 10(-7) M 4 alpha-phorbol 12,13-didecanoate, an inactive phorbol ester, did not alter mAChR number. The addition of PMA and A23187 to cultures incubated with 10(-3) M carbachol caused only a modest 6% further reduction in mAChR number as compared to incubation with carbachol alone. The kinetics of the decrease in mAChR number produced by PMA/A23187 were similar to those seen after carbachol treatment. Recovery of mAChR number after treatment with either carbachol or PMA/A23187 was blocked by treatment with the protein synthesis inhibitor cycloheximide. Intact cell binding studies employing [3H]N-methylscopolamine showed that treatment with either PMA/A23187 or carbachol caused a rapid (within 15 min) loss of receptors from the cell surface prior to the decrease in total mAChR number. PMA (10(-7) M), but not 4 alpha-phorbol 12,13-didecanoate, promoted the translocation of protein kinase C activity from the cytosol to the membrane. Incubation with carbachol increased membrane-associated protein kinase C activity within 5 min with an EC50 of 3 X 10(-6) M. This increase persisted for at least 60 min in the continued presence of carbachol and was blocked by simultaneous incubation with atropine. These results suggest that activation of protein kinase C may be involved in the regulation of mAChR number in response to agonist.     

Separation of Muscarinic Acetylcholine Receptor Entities from Rat Cerebral Cortex by Ion Exchange Chromatography

Abstract

Digitonin solubilized muscarinic acetylcholine receptor (mAChR) of rat cerebral cortex membranes were chromatographed on the FPLC anion exchanger Mono Q. [³H]QNB (quinuclidinyl benzilate) and [³H]PZ (pirenzepine) binding activity was retarded from a NaCl free elution buffer and thereby separated from a part of the accompanying proteins. Elution of the column with a continuously increasing NaCl concentration desorbed the radioligand binding activities forming several peaks, two of which were nearly completely separated. Our data show that the mAChR in rat cerebral cortex consists of several entities with different electrical charges.     

Phorbol diesters and dioctanoylglycerol stimulate accumulation of both diacylglycerols and alkylacylglycerols in human neutrophils

Human neutrophils treated with phorbol 12-myristate 13-acetate (PMA) or dioctanoylglycerol exhibited a large (10-fold), sustained accumulation of the mass of diradylglycerol, beginning 1 min after stimulation and continuing for 30 to 60 min. Phorbol dibutyrate was less potent than PMA in stimulating diradylglycerol accumulation, whereas the 4-alpha analogs of PMA and phorbol dibutyrate were inactive. Submaximal concentrations of PMA (0.5 to 2.5 nM) plus the calcium ionophore, ionomycin (15 to 60 nM), led to synergistic accumulation of diradylglycerols. Chlorpromazine and sphingosine, inhibitors of protein kinase C, blocked PMA-stimulated accumulation of diradylglycerol with IC50 concentrations of 32 and 9 microM, respectively, paralleling their inhibition of PMA-stimulated O2- production. These compounds also inhibited the ionomycin-stimulated accumulation of diradylglycerols. A third protein kinase C inhibitor, H-7, was less effective, inhibiting PMA-stimulated accumulation of diradylglycerol by 25% at 100 microM. Differential sensitivity to alkaline hydrolysis suggests that diradylglycerols that accumulate in response to PMA or ionomycin stimulation are composed of a mixture of two distinct diglyceride species, diacylglycerols and alkylacylglycerols. Whereas diacylglycerol may activate cellular protein kinase C, the importance of the production of alkylacylglycerols is uncertain.     

Control of glycogen phosphorylase interconversion by phorbol esters, diacylglycerols, Ca2+ and hormones in isolated rat hepatocytes.

In isolated rat hepatocytes: phosphorylase activation by the ionophore A23187 was enhanced in the presence of tumour-promoting phorbol esters and 1,2- (but not 1,3-) diacylglycerols (dioleoyl- and oleoylacetyl-glycerol), with a similar dose-dependency; the activation of phosphorylase by phenylephrine (1 microM) (but not by vasopressin or glucagon) was inhibited both by tumour-promoting phorbol esters and diacylglycerols, but with a different dose-dependency: complete inhibition was achieved with concentrations of phorbol esters two orders of magnitude lower than those of diacylglycerol; binding of the alpha 1-adrenergic antagonist [3H]prazosin and its displacement by unlabelled prazosin was not significantly affected in the presence of the phorbol esters. The possible involvement of protein kinase C in the control of phosphorylase interconversion is discussed.     

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.     

Regional Differences in the Coupling of Muscarinic Receptors to Inositol Phospholipid Hydrolysis in Guinea Pig Brain

The differential effects of muscarinic agents on inositol phospholipid hydrolysis and the role in this process of putative muscarinic receptor subtypes (M1 and M2) were investigated in three regions of guinea pig brain. Addition of the agonist oxotremorine-M to slices of neostriatum, cerebral cortex, or hippocampus incubated in the presence of myo-[2-3H]inositol and Li+ resulted in a large accumulation of labeled inositol phosphates (733, 376, and 330% of control, respectively). In each tissue, the principal product formed was myo-inositol 1-phosphate (59-86%), with smaller amounts of glycerophosphoinositol and inositol bisphosphate. Only trace amounts of inositol trisphosphate could be detected. Regional differences were observed in the capacity of certain partial agonists to evoke inositol lipid hydrolysis, the most notable being that of bethanechol, which was four times more effective in the neostriatum than in either the cerebral cortex or hippocampus. In addition, the full agonists, oxotremorine-M and carbamoylcholine, were more potent stimulators of inositol phosphate release in the neostriatum than in the cerebral cortex. The putative M1 selective agonist 4-m-chlorophenylcarbamoyloxy-2-butynyl trimethyl ammonium chloride had little stimulatory effect in any brain region, whereas the putative M1 selective antagonist pirenzepine blocked the enhanced release of inositol phosphates with high affinity in the cerebral cortex and hippocampus (Ki = 12.1 and 13.9 nM; "M1") but with a lower affinity in the neostriatum (Ki = 160 nM; "M2"). In contrast to its differential effects on stimulated inositol lipid hydrolysis, no regional differences were observed in the capacity of pirenzepine to displace [3H]quinuclidinyl benzilate, a muscarinic antagonist, bound to membrane fractions. Atropine, an antagonist that does not discriminate between receptor subtypes, inhibited the enhanced release of inositol phosphates with similar affinities in the three regions (Ki = 0.40-0.60 nM). The results indicate that by measurement of inositol lipid hydrolysis, regional differences in muscarinic receptor coupling characteristics become evident. These differences, which are not readily detected by radioligand binding techniques, might be accounted for by either the presence of functionally distinct receptor subtypes, or alternatively, by regional variations in the efficiency of muscarinic receptor coupling to inositol lipid hydrolysis.     

Antibodies against cerebral M1 cholinergic muscarinic receptor from schizophrenic patients: molecular interaction

We demonstrated the presence of circulating Abs from schizophrenic patients able to interact with cerebral frontal cortex-activating muscarinic acetylcholine receptors (mAChR). Sera and purified IgG from 21 paranoid schizophrenic and 25 age-matched normal subjects were studied by indirect immunofluorescence, flow cytometry, immunoblotting, dot blot, ELISA, and radioligand competition assays. Rat cerebral frontal cortex membranes and/or a synthetic peptide, with an amino acid sequence identical with that of human M(1) mAChR, were used as Ags. By indirect immunofluorescence and flow cytometry procedures, we proved that serum-purified IgG fraction from schizophrenic patients reacted to neural cell surfaces from rat cerebral frontal cortex. The same Abs were able to inhibit the binding of the specific M(1) mAChR radioligand [(3)H]pirenzepine. Immunoblotting experiments showed that IgG from schizophrenic patients revealed a band with a molecular mass coincident to that labeled by an anti-M(1) mAChR Ab. Using synthetic peptide for dot blot and ELISA, we demonstrated that these Abs reacted against the second extracellular loop of human cerebral M(1) mAChR. Also, the corresponding affinity-purified antipeptide Ab displayed an agonistic-like activity associated to specific receptor activation, increasing cyclic GMP production and inositol phosphate accumulation, and protein kinase C translocation. This paper gave support to the participation of an autoimmune process in schizophrenia.     

(-)-Indolactam V activates protein kinase C and induces changes in muscarinic receptor functions in SH-SY5Y human neuroblastoma cells

The effects of a synthetic protein kinase C (PKC) activator, (-)-indolactam V (ILV), were studied in SH-SY5Y human neuroblastoma cells. (-)-ILV induced a translocation of PKC from cytosol to plasma membrane and displaced 3H-phorbol dibutyrate binding in the micromolar range. In addition, (-)-ILV caused a decreased sensitivity of cells to muscarinic agonist-induced Ca2+ mobilization measured with quin-2 and induced a down-regulation of cell surface muscarinic receptors. All the changes induced by (-)-ILV were similar in magnitude to those seen with the phorbol ester tetradecanoyl phorbol acetate (TPA). The results suggest that (-)-ILV is a full activator of PKC and a promising alternative to phorbol esters in studies on mechanism of actions of PKC.     

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.     

Role of protein kinase C in transmembrane signaling

Many extracellular signals elicit Ca2+ mobilization and diacylglycerol formation in their target cells. Diacylglycerol is derived from the receptor-linked phosphoinositide turnover and serves as a second messenger for the activation of protein kinase C in the presence of Ca2+ and phosphatidylserine. Unique diacylglycerols such as 1-oleoyl-2-acetyl-glycerol, which activate intracellular protein kinase C when added to intact cells, have been synthesized. Tumor-promoting phorbol esters substitute for such diacylglycerols and directly activate protein kinase C in both intact cell and cell-free systems. Under appropriate conditions, the synthetic diacylglycerols and phorbol esters induce protein kinase C activation without Ca2+ mobilization, whereas Ca2+ ionophore A23187 induces Ca2+ mobilization without protein kinase C activation. Using these substances, we have obtained evidence that both protein C and Ca2+ are involved in and play a synergistic role in exocytosis, cell division, and other cellular functions. In this article, the role of protein kinase C in transmembrane signaling is discussed.     

MUSCARINIC ACETYLCHOLINE RECEPTOR SUBTYPE mRNAs IN THE HUMAN AND RAT VESTIBULAR PERIPHERY

The expression of the five muscarinic acetylcholine receptor (mAChR) subtypes (m1–m5) in the vestibular end-organs and in the primary afferent vestibular ganglia of the human and rat was studied using RT-PCR from the two tissue populations from both species. In the human, although all five mAChR subtypes were expressed in brain, only the m1, m2, and m5 mAChR subtypes were amplified from both the vestibular ganglia and the vestibular end-organs, while in the rat, all five mAChR subtypes were expressed. These data suggest that the efferent cholinergic axo-dendritic and axo-somatic synapses have a muscarinic component and that there are pharmacologic implications for patients with vestibular dysfunction.     

Activation of muscarinic cholinergic receptors enhances the volume-sensitive efflux of myo-inositol from SH-SY5Y neuroblastoma cells

A mechanism used by cells to regulate their volume under hypo-osmotic conditions is the release of organic osmolytes, one of which is myo-inositol. The possibility that activation of phospholipase-C-linked receptors can regulate this process has been examined for SH-SY5Y neuroblastoma cells. Incubation of cells with hypo-osmolar buffers (160-250 mOsm) led to a biphasic release of inositol which persisted for up to 4 h and could be inhibited by inclusion of anion channel blockers - results which indicate the involvement of a volume-sensitive organic anion channel. Inclusion of oxotremorine-M, a muscarinic cholinergic agonist, resulted in a marked increase (80-100%) in inositol efflux under hypo-osmotic, but not isotonic, conditions. This enhanced release, which was observed under all conditions of hypo-osmolarity tested, could be prevented by inclusion of atropine. Incubation of the cells with either the calcium ionophore, ionomycin, or the phorbol ester, phorbol 12-myristate 13-acetate, partially mimicked the stimulatory effect of muscarinic receptor activation when added singly, and fully when added together. The ability of oxotremorine-M to facilitate inositol release was inhibited by removal of extracellular calcium, depletion of intracellular calcium or down-regulation of protein kinase C. These results indicate that activation of muscarinic cholinergic receptors can regulate osmolyte release in this cell line.     

Muscarinic activation of mitogen-activated protein kinase in rat thyroid epithelial cells

Carbachol (Cch), a muscarinic acetylcholine receptors (mAChR) agonist, produces time- and dose-dependent increases in mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) phosphorylation in nondifferentiated Fischer rat thyroid (FRT) epithelial cells. Cells pretreatment with the selective phospholipase C inhibitor U73122 resulted in a decrease of Cch-stimulated ERK1/2 phosphorylation. These data indicated that the effect of mAChR on ERK activation could be mediated through agonist-induced Ca(2+) mobilization or PKC activation. Phosphorylation of ERK1/2 was mimicked by the protein kinase C (PKC) activator phorbol 12-myristate acetate (PMA), but was not altered either by PKC inhibitor GF109203X or by down-regulation of PKC. Phosphorylation of ERK1/2 was elevated by a direct [Ca(2+)](i) increase caused by thapsigargin or ionophore. Additionally, Cch-induced ERK1/2 phosphorylation was reduced after either inhibition of Ca(2+) influx or intracellular Ca(2+) release. Nevertheless, Cch-mediated ERK1/2 activation was genistein sensitive, indicating the involvement of protein tyrosine kinases on the downstream signalling of mAChR. Pretreatment of the cells with PP2 markedly decreased Cch-induced ERK1/2 phosphorylation, suggesting a role of Src family of tyrosine kinases in the signal transduction pathway involved in ERK1/2 activation by mAChR. To test the biological consequences of ERK activation, we examined the effect of mAChR on cell functions. Cch stimulation of FRT cells did not affect cell proliferation, but increased protein synthesis. This effect was significantly attenuated by PD98059, a selective inhibitor of mitogen-activated protein kinase kinase (MAPKK/MEK). This study demonstrated that muscarinic receptor-mediated increase in the ERK1/2 phosphorylation was dependent on [Ca(2+)](i) but independent of PKC and was mediated by the Src family of tyrosine kinases. Our results also supported the idea that the protein synthesis stimulated by mAChR in polarized FRT epithelial cells was regulated by the ERK1/2 phosphorylation pathway.     

Synergism between diacylglycerols and calcium ionophore in the induction of human B cell proliferation mimics the inositol lipid polyphosphate breakdown signals induced by crosslinking surface immunoglobulin

Resting human tonsillar B cells were stimulated to divide by heat killed Staphylococcus aureus Cowan strain 1 which was shown to induce hydrolysis of phosphatidylinositol 4, 5-bisphosphate known to give rise to diacylglycerol and an increase in cytosolic free calcium. Addition of the diacylglycerols, 1-oleoyl-2 acetyl glycerol or sn-1, 2-dioctanoylglycerol, together with the calcium ionophore ionomycin to B cell cultures induced marked cell proliferation whereas these agents were ineffective when used alone. Both diacylglycerols were shown to compete with [3H] phorbol 12,13 dibutyrate in binding to protein kinase C. These data support the hypothesis that synergism between cytosolic calcium and endogenous diacylglycerol, which activates protein kinase C, is involved in signal transduction in the proliferation of human B cells.