Involvement of phosphatidylcholine-specific phospholipase C in thromboxane A2-induced activation of mitogen-activated protein kinase in astrocytoma cells

Thromboxane A2 (TXA2) receptor-mediated signal transduction was investigated in 1321N1 human astrocytoma cells. 9,11-Epithio-11,12-methano-TXA2 (STA2), a TXA2 receptor agonist, induced Ca2+ mobilization and phosphoinositide hydrolysis in a concentration-dependent manner. These responses were inhibited by treatment with U73122, an inhibitor of phosphatidylinositol-specific phospholipase C, or by culturing in 0.5% fetal calf serum containing 0.5 mM dibutyryladenosine 3',5'-cyclic monophosphate (dbcAMP) for 2 days. However, the dbcAMP treatment augmented the TXA2 receptor-mediated phosphorylation of mitogen-activated protein kinase (MAPK). These results were confirmed by a functional MAPK assay measuring the incorporation of 32P into the MAPK substrate peptide. The TXA2 receptor-mediated MAPK activation was inhibited by SQ29548, a TXA2 receptor antagonist, and GF109203X, an inhibitor of protein kinase C. Although U73122 did not inhibit or only slightly inhibited the activation of MAPK, D-609, an inhibitor of phosphatidylcholine-specific phospholipase C, potently attenuated the activation in a concentration-dependent manner. Furthermore, STA2 accelerated the release of [3H]choline metabolites from the cells prelabeled with [3H]choline chloride. This release was inhibited by treatment with D-609. These results suggest that phosphatidylcholine-specific phospholipase C and protein kinase C, but not phosphatidylinositol-specific phospholipase C, are involved in TXA2 receptor-mediated MAPK activation in 1321N1 human astrocytoma cells.     

Increased expression of Galpha(q/11) and of phospholipase-Cbeta1/4 in differentiated human NT2-N neurons: enhancement of phosphoinositide hydrolysis

The CNS is enriched in phosphoinositide-specific phospholipase C (PLC) and in the G proteins linked to its activation. Although the regional distributions of these signaling components within the brain have been determined, neither their cell type-specific localizations (i.e., neuronal versus glial) nor the functional significance of their high expression has been definitively established. In this study, we have examined the expression of phosphoinositide signaling proteins in human NT2-N cells, a well characterized model system for CNS neurons. Retinoic acid-mediated differentiation of NT2 precursor cells to the neuronal phenotype resulted in five- to 15-fold increases in the expression of PLC-beta1, PLC-beta4, and Galpha(q/11) (the prime G protein activator of these isozymes). In contrast, the expression of PLC-beta3 and PLC-gamma1 was markedly reduced following neuronal differentiation. Similar alterations in cell morphology and in the expression of PLC-beta1, PLC-beta3, and Galpha(q/11) expression were observed when NT2 cells were differentiated with berberine, a compound structurally unrelated to retinoic acid. NT2-N neurons exhibited a significantly higher rate of phosphoinositide hydrolysis than NT2 precursor cells in response to direct activation of either G proteins or PLC. These results indicate that neuronal differentiation of NT2 cells is associated with dramatic changes in the expression of proteins of the phosphoinositide signaling system and that, accordingly, differentiated NT2-N neurons possess an increased ability to hydrolyze inositol lipids.     

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.     

Phorbol ester inhibits phosphoinositide hydrolysis and calcium mobilization in cultured astrocytoma cells

In cultured human 1321N1 astrocytoma cells, muscarinic receptor stimulation leads to phosphoinositide hydrolysis, formation of inositol phosphates, and mobilization of intracellular Ca2+. Treatment of these cells with 1 microM 4 beta-phorbol 12 beta-myristate 13 alpha-acetate (PMA) completely blocks the carbachol-stimulated formation of [3H]inositol mono-, bis-, and trisphosphate ( [3H]InsP, [3H]InsP2, and [3H]InsP3). The concentrations of PMA that give half-maximal and 100% inhibition of carbachol-induced [3H]InsP formation are 3 nM and 0.5 microM, respectively. Inactive phorbol esters (4 alpha-phorbol 12,13-didecanoate and 4 beta-phorbol), at 1 microM, do not inhibit carbachol-stimulated [3H]InsP formation. The KD of the muscarinic receptor for [3H]N-methyl scopolamine is unchanged by PMA treatment, while the IC50 for carbachol is modestly increased. PMA treatment also abolishes carbachol-induced 45Ca2+ efflux from 1321N1 cells. The concomitant loss of InsP3 formation and Ca2+ mobilization is strong evidence in support of a causal relationship between these two responses. In addition, our finding that PMA blocks hormone-stimulated phosphoinositide turnover suggests that there may be feedback regulation of phosphoinositide metabolism through the Ca2+- and phospholipid-dependent protein kinase.     

Evidence that muscarinic cholinergic receptors selectively interact with either the cyclic AMP or the inositol phosphate second-messenger response systems.

The relative capacities of muscarinic cholinergic receptor (MR) and bradykinin (BK)-receptor activation to increase phosphoinositide hydrolysis and to increase cytosolic Ca2+ were compared in NG108-15 neuroblastoma x glioma and 1321N1 human astrocytoma cells. In 1321N1 cells, the muscarinic cholinergic agonist carbachol and BK each stimulated a concentration-dependent accumulation of inositol phosphates (K0.5 approximately 10 microM and approximately 10 nM respectively) and a rapid increase in cytosolic Ca2+ as determined by quin2 fluorescence. In NG108-15 cells, BK alone stimulated a pertussis-toxin-insensitive accumulation of inositol phosphates (K0.5 approximately 10 nM) under conditions in which pertussis toxin completely inhibited MR-mediated inhibition of adenylate cyclase. BK also stimulated a rapid increase in cytosolic Ca2+ in NG108-15 cells. In contrast, no MR-mediated increase in phosphoinositide hydrolysis or change in cytosolic Ca2+ concentration was observed in NG108-15 cells. These results support the idea that MR selectively interact with either the cyclic AMP or the inositol phosphate second-messenger systems.     

Coupling of the thrombin receptor to G12 may account for selective effects of thrombin on gene expression and DNA synthesis in 1321N1 astrocytoma cells.

In 1321N1 astrocytoma cells, thrombin, but not carbachol, induces AP-1-mediated gene expression and DNA synthesis. To understand the divergent effects of these G protein-coupled receptor agonists on cellular responses, we examined Gq-dependent signaling events induced by thrombin receptor and muscarinic acetylcholine receptor stimulation. Thrombin and carbachol induce comparable changes in phosphoinositide and phosphatidylcholine hydrolysis, mobilization of intracellular Ca2+, diglyceride generation, and redistribution of protein kinase C; thus, activation of these Gq-signaling pathways appears to be insufficient for gene expression and mitogenesis. Thrombin increases Ras and mitogen-activated protein kinase activation to a greater extent than carbachol in 1321N1 cells. The effects of thrombin are not mediated through Gi, since ribosylation of Gi/Go proteins by pertussis toxin does not prevent thrombin-induced gene expression or thrombin-stimulated DNA synthesis. We recently reported that the pertussis toxin-insensitive G12 protein is required for thrombin-induced DNA synthesis. We demonstrate here, using transfection of receptors and G proteins in COS-7 cells, that G alpha 12 selectively couples the thrombin receptor to AP-1-mediated gene expression. This does not appear to result from increased mitogen-activated protein kinase activity but may reflect activation of a tyrosine kinase pathway. We suggest that preferential coupling of the thrombin receptor to G12 accounts for the selective ability of thrombin to stimulate Ras, mitogen-activated protein kinase, gene expression, and mitogenesis in 1321N1 cells.     

Guanine nucleotide regulation of agonist binding to muscarinic cholinergic receptors. Relation to efficacy of agonists for stimulation of phosphoinositide breakdown and Ca2+ mobilization.

The efficacies of a series of six muscarinic cholinergic receptor agonists for stimulation of phosphoinositide breakdown and unidirectional efflux of 45Ca2+ in 1321N1 human astrocytoma cells were compared with the relative capacity of these agonists for formation of a GTP-sensitive high-affinity binding state in washed membranes. Carbachol and methacholine were 'full' agonists as regards phosphoinositide breakdown and Ca2+ mobilization, whereas bethanechol, arecoline and oxotremorine were 'partial' agonists for these two responses. Pilocarpine was the least efficacious of the six drugs tested. Except for pilocarpine, competition curves generated with the agonists and [3H]quinuclidinyl benzilate did not follow the Law of Mass Action for ligand interaction at a single site. Non-linear regression analyses of these data indicated that the data significantly better fit a two-, rather than a single-, site model with a high- and a low-affinity binding component. Competition curves generated in the presence of GTP were shifted to the right, and the extent of receptors in the high-affinity agonist-binding state was decreased. The relative efficacies of the six agonists for stimulation of phosphoinositide breakdown and Ca2+ mobilization were significantly correlated with the difference in affinities (KL/KH) between the two affinity states for each agonist. The relative efficacy of the agonists for stimulation of Ca2+ mobilization also was significantly correlated with the extent of receptors in the high-affinity state (%H) for each agonist. The results suggest that interaction with an as-yet unidentified guanine nucleotide regulatory protein is important in the mechanism whereby muscarinic receptors stimulate phosphoinositide breakdown in 1321N1 astrocytoma cells.     

Guanine nucleotide-dependent pertussis-toxin-insensitive stimulation of inositol phosphate formation by carbachol in a membrane preparation from human astrocytoma cells.

The efficacy of muscarinic-receptor agonists for stimulation of inositol phosphate formation and Ca2+ mobilization in intact 1321N1 human astrocytoma cells is correlated with their capacity for formation of a GTP-sensitive high-affinity binding complex in membranes from these cells [Evans, Hepler, Masters, Brown & Harden (1985) Biochem. J. 232, 751-757]. These observations prompted the proposal that a guanine nucleotide regulatory protein serves to couple muscarinic receptors to the phospholipase C involved in phosphoinositide hydrolysis in 1321N1 cells. Inositol phosphate (InsP) formation was measured in a cell-free preparation from 1321N1 cells to provide direct support for this idea. The formation of InsP3, InsP2 and InsP1 was increased in a concentration-dependent manner (K0.5 approximately 5 microM) by guanosine 5'-[gamma-thio]triphosphate (GTP[S]) in washed membranes prepared from myo-[3H]inositol-prelabelled 1321N1 cells. Both GTP[S] and guanosine 5'-[beta gamma-imido]triphosphate (p[NH]ppG) stimulated InsP formation by 2-3-fold over control; GTP, GDP and GMP were much less efficacious. Millimolar concentrations of NaF also stimulated the formation of inositol phosphates in membrane preparations from 1321N1 cells. In the presence of 10 microM-GTP[S], the muscarinic cholinergic-receptor agonist carbachol stimulated (K0.5 approximately 10 microM) the formation of InsP above that achieved with GTP[S] alone. The effect of carbachol was completely blocked by atropine. The order of potency of nucleotides for stimulation of InsP formation in the presence of 500 microM-carbachol was GTP[S] greater than p[NH]ppG greater than GTP = GDP. Pertussis toxin, at concentrations that fully ADP-ribosylate and functionally inactivate Gi (the inhibitory guanine nucleotide regulatory protein), had no effect on InsP formation in the presence of GTP[S] or GTP[S] plus carbachol. These data are consistent with the idea that a guanine nucleotide regulatory protein that is not Gi is involved in receptor-mediated stimulation of InsP formation in 1321N1 human astrocytoma cells.     

Expression of c-kit protooncogene is stimulated by cAMP in differentiated F9 mouse teratocarcinoma cells.

Protooncogene c-kit, a transmembrane tyrosine kinase receptor, was recently shown to map to the dominant white spotting locus (W) of the mouse. W mutations affect melanogenesis, gametogenesis, and hematopoiesis during development and in adult life. In order to determine the regulation of the c-kit gene in cell differentiation, we investigated its expression during the differentiation of F9 cells. Undifferentiated F9 cells and F9 cells treated with retinoic acid (RA) alone or dbcAMP alone showed little expression of c-kit mRNA if any. The subsequent addition of dbcAMP to F9 cells treated with RA markedly increased the expression of c-kit mRNA. Furthermore, the effect of dbcAMP on c-kit expression is reversible. In differentiated cells treated with RA, c-kit gene expression is induced by agents such as forskolin or theophylline, which are known to elevate cellular cAMP level. These results indicate that the expression of the c-kit gene is regulated by the level of intracellular cAMP in differentiated F9 cells induced by RA.     

Calcium mobilization by muscarinic receptors in human astrocytoma cells: measurements with quin 2

Activation of muscarinic cholinergic receptors on 1321N1 human astrocytoma cells leads to Ca2+ mobilization as measured by quin 2 fluorescence. Acetylcholine and methacholine were full and potent agonists, while carbachol and muscarine, were fully efficacious but 6- and 10-fold less potent than acetylcholine. The carbachol-induced Ca2+ response was also observed in absence of extracellular Ca2+ and was blocked by muscarinic receptor antagonists but not by organic Ca2+ channel blockers, tetrodotoxin (TTX), tetraethylammonium (TEA) or metal cations, suggesting that Ca2+ is mobilized from intracellular storage sites rather than through plasma membrane ion channels. Muscarinic receptor-mediated Ca2+ release was also detected in kidney epithelial cells but not in rat fibroblasts, glial cells or differentiated neuroblastoma x glioma hybrid cells.     

Involvement of aquaporin in thromboxane A2 receptor-mediated, G12/13/RhoA/NHE-sensitive cell swelling in 1321N1 human astrocytoma cells

The physiological role of the thromboxane A₂ (TXA₂) receptor expressed on glial cells remains unclear. We previously reported that 1321N1 human astrocytoma cells pretreated with dibutyryl cyclic AMP (dbcAMP) became swollen in response to U46619, a TXA₂ analogue. In the present study, we examined the detailed mechanisms of TXA₂ receptor-mediated cell swelling in 1321N1 cells. The cell swelling caused by U46619 was suppressed by expression of p115-RGS, an inhibitory peptide of Gα_12/13 pathway and C3 toxin, an inhibitory protein for RhoA. The swelling was also inhibited by treatment with Y27632, a Rho kinase inhibitor and 5-(ethyl-N-isopropyl)amiloride (EIPA), a Na⁺/H⁺-exchanger inhibitor. Furthermore, cell swelling was suppressed by the pretreatment with aquaporin inhibitors mercury chloride or phloretin in a concentration-dependent manner, suggesting that aquaporins are involved in U46619-induced 1321N1 cell swelling. In fact, U46619 caused [³H]H₂O influx into the cells, which was inhibited by p115-RGS, C3 toxin, EIPA, mercury chloride and phloretin. This is the first report that the TXA₂ receptor mediates water influx through aquaporins in astrocytoma cells via TXA₂ receptor-mediated activation of Gα_12/13, Rho A, Rho kinase and Na⁺/H⁺-exchanger.     

Muscarinic receptor activation of phosphatidylcholine hydrolysis. Relationship to phosphoinositide hydrolysis and diacylglycerol metabolism

We examined the relationship between phosphatidylcholine (PC) hydrolysis, phosphoinositide hydrolysis, and diacylglycerol (DAG) formation in response to muscarinic acetylcholine receptor (mAChR) stimulation in 1321N1 astrocytoma cells. Carbachol increases the release of [3H]choline and [3H]phosphorylcholine ([3H]Pchol) from cells containing [3H]choline-labeled PC. The production of Pchol is rapid and transient, while choline production continues for at least 30 min. mAChR-stimulated release of Pchol is reduced in cells that have been depleted of intracellular Ca2+ stores by ionomycin pretreatment, whereas choline release is unaffected by this pretreatment. Phorbol 12-myristate 13-acetate (PMA) increases the release of choline, but not Pchol, from 1321N1 cells, and down-regulation of protein kinase C blocks the ability of carbachol to stimulate choline production. Taken together, these results suggest that Ca2+ mobilization is involved in mAChR-mediated hydrolysis of PC by a phospholipase C, whereas protein kinase C activation is required for mAChR-stimulated hydrolysis of PC by a phospholipase D. Both carbachol and PMA rapidly increase the formation of [3H]phosphatidic acid ([3H]PA) in cells containing [3H]myristate-labeled PC. [3H]Diacylglycerol ([3H]DAG) levels increase more slowly, suggesting that the predominant pathway for PC hydrolysis is via phospholipase D. When cells are labeled with [3H]myristate and [14C]arachidonate such that there is a much greater 3H/14C ratio in PC compared with the phosphoinositides, the 3H/14C ratio in DAG and PA increases with PMA treatment but decreases in response to carbachol. By analyzing the increase in 3H versus 14C in DAG, we estimate that the DAG that is formed in response to PMA arises largely from PC. Muscarinic receptor activation also causes formation of DAG from PC, but approximately 20% of carbachol-stimulated DAG appears to arise from hydrolysis of the phosphoinositides.     

Cross-desensitization among CXCR1, CXCR2, and CCR5: role of protein kinase C-epsilon

The IL-8 (or CXCL8) chemokine receptors, CXCR1 and CXCR2, activate protein kinase C (PKC) to mediate leukocyte functions. To investigate the roles of different PKC isoforms in CXCL8 receptor activation and regulation, human mononuclear phagocytes were treated with CXCL8 or CXCL1 (melanoma growth-stimulating activity), which is specific for CXCR2. Plasma membrane association was used as a measure of PKC activation. Both receptors induced time-dependent association of PKCalpha, -beta1, and -beta2 to the membrane, but only CXCR1 activated PKCepsilon. CXCL8 also failed to activate PKCepsilon in RBL-2H3 cells stably expressing CXCR2. DeltaCXCR2, a cytoplasmic tail deletion mutant of CXCR2 that is resistant to internalization, activated PKCepsilon as well as CXCR1. Expression of the PKCepsilon inhibitor peptide epsilonV1 in RBL-2H3 cells blocked PKCepsilon translocation and inhibited receptor-mediated exocytosis, but not phosphoinositide hydrolysis or peak intracellular Ca(2+) mobilization. epsilonV1 also inhibited CXCR1-, CCR5-, and DeltaCXCR2-mediated cross-regulatory signals for GTPase activity, Ca(2+) mobilization, and internalization. Peritoneal macrophages from PKCepsilon-deficient mice (PKCepsilon(-/-)) also showed decreased CCR5-mediated cross-desensitization of G protein activation and Ca(2+) mobilization. Taken together, the results indicate that CXCR1 and CCR5 activate PKCepsilon to mediate cross-inhibitory signals. Inhibition or deletion of PKCepsilon decreases receptor-induced exocytosis and cross-regulatory signals, but not phosphoinositide hydrolysis or peak intracellular Ca(2+) mobilization, suggesting that cross-regulation is a Ca(2+)-independent process. Because DeltaCXCR2, but not CXCR2, activates PKCepsilon and cross-desensitizes CCR5, the data further suggest that signal duration leading to activation of novel PKC may modulate receptor-mediated cross-inhibitory signals.     

P2Y receptors activate neuroprotective mechanisms in astrocytic cells

Mechanical or ischemic trauma to the CNS causes the release of nucleotides and other neurotransmitters into the extracellular space. Nucleotides can activate nucleotide receptors that modulate the expression of genes implicated in cellular adaptive responses. In this investigation, we used human 1321N1 astrocytoma cells expressing a recombinant P2Y2 receptor to assess the role of this receptor in the regulation of anti-apoptotic (bcl-2 and bcl-xl) and pro-apoptotic (bax) gene expression. Acute treatment with the P2Y2 receptor agonist UTP up-regulated bcl-2 and bcl-xl, and down-regulated bax, gene expression. Activation of P2Y2 receptors was also coupled to the phosphorylation of cyclic AMP responsive element binding protein that positively regulates bcl-2 and bcl-xl gene expression. Cyclic AMP responsive element decoy oligonucleotides markedly attenuated the UTP-induced increase in bcl-2 and bcl-xl mRNA levels. Activation of P2Y2 receptors induced the phosphorylation of the pro-apoptotic factor Bad and caused a reduction in bax/bcl-2 mRNA expression ratio. All these signaling pathways are known to be involved in cell survival mechanisms. Using cDNA microarray analysis and RT-PCR, P2Y2 receptors were found to up-regulate the expression of genes for neurotrophins, neuropeptides and growth factors including nerve growth factor 2; neurotrophin 3; glia-derived neurite-promoting factor, as well as extracellular matrix proteins CD44 and fibronectin precursor--genes known to regulate neuroprotection. Consistent with this observation, conditioned media from UTP-treated 1321N1 cells expressing P2Y2 receptors stimulated the outgrowth of neurites in PC-12 cells. Taken together, our results suggest an important novel role for the P2Y2 receptor in survival and neuroprotective mechanisms under pathological conditions.     

Expression and localization of the thromboxane A2 receptor in human nasal mucosa

Thromboxane A2 (TXA2) has been thought a potent mediator involved in allergic rhinitis, because TXA2 was recovered from the nasal lavage fluid of allergic rhinitis patients after allergen provocation and TXA2 receptor antagonists relief nasal allergic symptoms. In order to clarify the expression of TXA2 receptor in human nasal mucosa, we investigated TXA2 receptor mRNA expression and its protein localization by polymerase chain reaction (PCR) and immunohistochemistry, respectively. Human turbinates were obtained after turbinectomy from 10 patients with nasal obstruction refractory to medical therapy. RT-PCR analysis of total RNA from nasal mucosa demonstrated the expression of TXA2 receptor alpha mRNA. The immunohistochemical studies revealed that anti-TXA2 receptor alpha antibody labeled vascular smooth muscle cells, vascular endothelial cells, epithelial cells and submucosal glands in the nasal mucosa. The results may have an important clinical implication for understanding the role of TXA2 receptor on upper airway diseases such as allergic rhinitis and non-allergic rhinitis.     

Expression of Corticosteroid-Binding Globulin in Human Astrocytoma Cell Line

Glial tumor cells are known to be sensitive to glucocorticoids (GC) in vivo and in vitro. Here we studied the expression of corticosteroid-binding globulin (CBG) in the low-grade malignant human astrocytoma cell line 1321N1. CBG was observed in cytoplasm of most of these cells with immunocytochemistry. RT-PCR revealed the presence of the respective mRNA. Only scattered cells contained nuclear immunoreactivity for glucocorticoid receptor as visualized by double immunostaining. Immunoreactive CBG could be recovered from the supernatant of cultures that had been exposed to 10⁻⁵ M cortisol. Our observations indicate the endogenous expression of CBG in 1321N1 cells which may occur independently from classical glucocorticoid receptor pathways. Cortisol seems to facilitate liberation of CBG in a paracrine manner, perhaps through membrane action of the steroid. Effects of adrenal steroids on proliferation and apoptosis of certain glial tumors may in part depend on these mechanisms.     

Purification, characterization and proteinase-inhibitory activity of a Clara-cell secretory protein from the pulmonary extracellular lining of rabbits.

Although activation of muscarinic cholinergic receptors on 1321N1 human astrocytoma cells results in a linear accumulation of inositol phosphates for up to 60 min in the presence of LiCl [Masters, Quinn & Brown (1985) Mol. Pharmacol. 27, 325-332], activation of H1-histamine receptors resulted in an increase in total inositol phosphate formation that was maintained for less than 5 min. The effects of stimulation of these two receptors on accumulation of inositol 1,4,5-trisphosphate [Ins(1,4,5)P3], inositol 1,3,4-trisphosphate [Ins(1,3,4)P3] and inositol 1,3,4,5-tetrakisphosphate [Ins(1,3,4,5)P4] were also examined. Incubation of 1321N1 cells with carbachol resulted in a rapid accumulation of all three inositol phosphates, reaching a maximum within 30 s; this elevated value was maintained for up to 60 min. The rate of disappearance of Ins(1,3,4)P3 from carbachol-treated cells after the addition of atropine paralleled or exceeded the rate of disappearance of Ins(1,4,5)P3. Although the initial rates of accumulation of Ins(1,4,5)P3, Ins(1,3,4)P3 and Ins(1,3,4,5)P4 in the presence of histamine were similar to that observed with carbachol, the amounts of these inositol phosphates had returned to control values within 5 min after the addition of histamine. The results indicate that, although the acute effects of muscarinic receptor and H1-histamine receptor stimulation on phosphoinositide hydrolysis are very similar, the histamine receptor is desensitized rapidly, whereas the muscarinic receptor is not. This effect on histamine-receptor function is apparently homologous, since preincubation of 1321N1 cells with histamine did not decrease the subsequent response to carbachol.