Antinociceptive effect of spinal cholinergic stimulation: interaction with substance P

Activation of central muscarinic receptors results in an antinociceptive response in experimental animals. Employing intrathecal (i.t.) injection and radiant heat applied to a rat's tail as the experimental paradigm, a spinally-mediated antinociceptive response was obtained following injection of cholinergic agonists. Since "cholinergic' analgesia is mediated independently of the opiate system, the possibility was considered that this response was mediated through inhibition of the local release of substance P. Rats were prepared with indwelling i.t. catheters which terminated in the L2-L3 region of the spinal cord. I.t. injection of carbachol (0.05-5 micrograms) or neostigmine (1-10 micrograms), but not nicotine (0.5-10 micrograms) produced dose-related increases in tail flick latencies. Pretreatment with i.t. injection of atropine or hemicholinium-3 significantly inhibited the antinociceptive response to neostigmine. Spinal substance P levels were measured 30 min following 0.5 micrograms carbachol. Levels in the dorsal horn were reduced by 30% compared with saline controls. Levels in the ventral horn were unchanged by carbachol. These results support the role of endogenous spinal acetylcholine in pain modification and suggest an interaction with substance P neurons of the dorsal spinal cord.     

Cholinomimetic teratogens. II. Interaction with inorganic ions.

Treatment of chicken embryos after 96 h of incubation showed that the teratogenic activity of the cholinomimetic compounds carbachol, neostigmine, decamethonium, and tetramethylammonium is reduced in the presence of calcium acetate. Similarly, supplementation with potassium acetate reduced the teratogenicity of carbachol and tetramethylammonium.     

Cholinomimetic teratogens: studies with chicken embryos.

The cholinomimetic compounds carbachol, decamethonium, neostigmine, succinylcholine, trimethylphenylammonium, and others were tested for their interference with normal chick development. All these compounds led to abnormalities of the cervical vertebrae; at higher dosage interference with normal morphogenesis involved the whole vertebral column. Hypoplasia of the leg muscles occurred with lower incidence. Responses, tested with carbachol, rose from 24 to 72 and 96 h, then declined to 120 h of incubation. Two of the cholinometic compounds used in combined treatment produced a high degree of synergism. Gallamine, benzoquinomium, butyrylcholine, and bethanechol had protective effects. Acetylcholine, at high dosage, caused defects different from the above. It is suggested that the cholinomimetic teratogens interfere with normal development by displacing acetylcholine from its receptors or by forming complexes with it.     

Regulation of Muscarinic Agonist-Induced Activation of Phosphoinositidase C in Electrically Permeabilized SH-SY5Y Human Neuroblastoma Cells by Guanine Nucleotides

myo-[3H]Inositol-labelled SH-SY5Y cells were permeabilized with electrical discharges. 3H-Inositol phosphate formation in cells shown to be fully permeable was stimulated by the muscarinic agonist carbachol, by guanosine 5'-(gamma-thio)triphosphate [GTP(S)], and by guanosine 5'-(beta gamma-imido)diphosphate (GppNHp). Synergism was observed on coincubation of these GTP analogues with carbachol. GTP was also stimulatory and guanosine 5'-(beta-thio)diphosphate was inhibitory in the presence of agonist. Atropine blocked the effects of carbachol. Stimulation by GTP(S) (0.1 mM) occurred after a 1-2-min lag, whereas Ca2+ (0.5 mM), carbachol (1 mM), and carbachol plus GTP(S) stimulated without delay. The effects of carbachol plus GTP(S) but not those of Ca2+ were inhibited by spermine (4 mM). Accumulation of 3H-inositol phosphates was enhanced by Li+ (4 mM) only in intact cells. In intact or permeabilized cells, the "partial" agonist arecoline was maximally 40-50% as efficacious as carbachol. In permeabilized cells, the maximal effects of carbachol and arecoline were enhanced 2.8- and 5.3-fold, respectively, by 0.1 mM GTP(S), but only the EC50 for carbachol was substantially reduced. The binding affinity of carbachol but not that of arecoline in permeabilized cells was significantly reduced by 0.1 mM GppNHp. These data indicate that a guanine nucleotide-binding regulatory protein is involved in coupling muscarinic receptors to phosphoinositidase C in SH-SY5Y cells and that the activity of this protein influences the relationship between receptor occupation and phosphoinositide response.     

Dual effect of fluoride on phosphoinositide metabolism in rat brain cortex. Stimulation of phospholipase C and inhibition of polyphosphoinositide synthesis.

We have studied the effects of fluoride, guanosine 5'-[gamma-thio]triphosphate (GTP[S]) and carbachol on phospholipase C and polyphosphoinositide synthesis. The experimental system consisted of membranes from rat brain cortex, with exogenous [3H]phosphatidylinositol ([3H]PtdIns) as substrate. In such systems, we have not found evidence to support carbachol and/or GTP[S] stimulation of PtdIns phosphorylation. Fluoride inhibited synthesis of PtdIns4P and PtdIns(4,5)P2 from PtdIns. Consequently, under conditions where breakdown of polyphosphoinositides by phospholipase C was dependent on PtdIns kinase activity, fluoride inhibited activation by GTP[S] plus carbachol of phospholipase C. When conditions allowed direct breakdown of PtdIns and precluded PtdIns kinase activity, the stimulatory effects of fluoride and GTP[S] plus carbachol on phospholipase C activity were additive.     

Bicarbonate stimulatory action of nizatidine, a histamine H(2)-receptor antagonist, in rat duodenums.

Nizatidine, a histamine H(2)-antagonist, is known to inhibit acetylcholinesterase (AChE) activity and is used clinically as a gastroprokinetic agent as well as the anti-ulcer agent. We examined whether or not nizatidine stimulates duodenal HCO(3)(-) secretion in rats through vagal-cholinergic mechanisms by inhibiting AChE activity. Under pentobarbital anesthesia, a proximal duodenal loop was perfused with saline, and the HCO(3)(-) secretion was measured at pH 7.0 using a pH-stat method and by adding 10 mM HCl. Nizatidine, neostigmine, carbachol, famotidine or ranitidine was administered i.v. as a single injection. Intravenous administration of nizatidine (3-30 mg/kg) dose-dependently increased the HCO(3)(-) secretion, and the effect at 10 mg/kg was equivalent to that obtained by carbachol at 0.01 mg/kg. The HCO(3)(-) stimulatory action of nizatidine was observed at the doses that inhibited the histamine-induced acid secretion and enhanced gastric motility. This effect was mimicked by neostigmine (0.03 mg/kg) and significantly attenuated by bilateral vagotomy and pretreatment with atropine but not indomethacin. The IC(50) of nizatidine for AChE of rat erythrocytes was 1.4 x 10(-6) M, about 12 times higher than that of neostigmine. Ranitidine showed the anti-AchE activity and increased duodenal HCO(3)(-) secretion, similar to nizatidine, whereas famotidine had any influence on neither AChE activity nor the HCO(3)(-) secretion. On the other hand, duodenal damage induced by acid perfusion (100 mM HCl for 4 h) in the presence of indomethacin was significantly prevented by nizatidine and neostigmine, at the doses that increased the HCO(3)(-) secretion. These results suggest that nizatidine increases HCO(3)(-) secretion in the rat duodenum, mediated by vagal-cholinergic mechanism, the action being associated with the anti-AChE activity of this agent.     

The interaction of cyproheptadine with agents affecting neuromuscular transmission.

Using the rat phrenic nerve diaphragm, cyproheptadine at concentrations of 1 to 8 mug/ml did not affect or slightly augmented indirect muscle twitches, but potentiated blockade by tubocurarine, decamethonium and succinylcholine, and antagonized the augmentation of twitches by neostigmine. Ketamine, choline and tetraethylammonium at concentrations causing no blockade produced, when given after cyproheptadine (6 mug/ml), a high degree of blockade. At concentrations of 9 to 20 mug/ml, cyproheptadine induced neuromuscular blockade which was slow in onset, more apparent at higher rate of stimulation and was not reversed by neostigmine, choline or tetraethylammonium. In the cat tibialis anterior muscle, it potentiated blockade by tubocurarine, decamethonium and succinylcholine, and blocked acetylcholine twitches. In the chick biventer cervicis muscle, the durg was more effective in blocking indirect twitches than responses to carbachol.     

The Incidence of Enamel Hypoplasia among the Krapina Neandertals

Developmental defects of teeth often appear as a record of metabolic disturbances during growth. One defect in particular, enamel hypoplasia, has been described among recent prehistoric human populations, but few analyses have been made of fossil hominids. This study describes the enamel quality of the dental remains of the Krapina Neandertals. Of the 18 individuals represented by dental arch fragments, 13 showed evidence of one or more teeth with enamel defects described as hypoplasia. This was a high frequency of occurrence when compared to Bronze and Copper Age populations in Central Europe or Woodland and Mississippian populations in North America. The reason for the higher incidence among the Neandertals is not fully explainable given the data available. However, there is a suggestion that fluorosis could be a factor that reduced the expression of enamel hypoplasia in the European populations.     

Activation of phospholipase C in rabbit brain membranes by carbachol in the presence of GTP gamma S; effects of biological detergents

Rabbit brain cortical membranes incubated with carbachol in the presence of GTP gamma S show a marked increase in the degradation of exogenous phosphatidylinositol 4,5-bisphosphate. This activation of phospholipase C is dependent on the presence of deoxycholate and maximal at 0.8-1 mM deoxycholate. There is negligible activation by carbachol alone but in the presence of GTP gamma S a carbachol effect can be readily demonstrated. Optimal activation of phospholipase C by carbachol was seen at 10 to 100 nM free Ca2+. Washing cortical membranes with hypertonic buffer extracted 60% of the membrane protein yet the carbachol and GTP gamma S coupling remained intact. Incubation of the membranes with lysophosphatidylcholine, Nonidet P-40, sodium deoxycholate or digitonin at concentrations considerably less than those frequently used to solubilize membrane proteins abolished the carbachol response. Octyl glucoside and sodium cholate also uncoupled receptor regulation of phospholipase C but only at concentrations where solubilization of membrane proteins occurred. Prior exposure of membranes to carbachol did not prevent the uncoupling observed as a result of detergent treatment. Incubation of the membranes with carbachol and GTP gamma S did not appear to be accompanied by specific release of either active phospholipase C or inhibitors of phospholipase C activity.     

G regulatory proteins and muscarinic receptor signal transduction in mucous acini of rat submandibular gland

The involvement of G regulatory proteins in muscarinic receptor signal transduction was examined in electrically permeabilized rat submandibular acinar cells. The guanine nucleotide analog, GTP gamma S, caused the dose dependent hydrolysis of membrane phosphatidylinositol 4,5-bisphosphate to release IP3. This response was insensitive to pertussis toxin treatment and was duplicated by NaF but not by GDP beta S. Enhanced IP3 synthesis was observed with a combination of GTP gamma S and carbachol. Exogenous IP3, as well as carbachol and GTP gamma S, provoked the release of sequestered 45Ca2+ from non-mitochondrial stores. In intact cells, carbachol significantly reduced the level of cyclic AMP induced by the beta-adrenergic agonist, isoproterenol, to 69% of its normal value. Pertussis toxin abolished this inhibitory action of carbachol on cyclic nucleotide levels. These results suggest that muscarinic receptors are coupled to two separate G regulatory proteins in submandibular mucous acini-the pertussis toxin-insensitive Gp of the phosphoinositide transduction pathway associated with elevated cytosolic calcium levels, and the pertussis toxin-sensitive Gi inhibitory protein of the adenylate cyclase complex.     

Agonist-induced phosphorylation and desensitization of human m2 muscarinic cholinergic receptors in Sf9 insect cells.

The human m1 (hm1) and m2 (hm2) muscarinic cholinergic receptors (mAChR) expressed in Sf9 insect cells using recombinant baculovirus were tested for their ability to undergo agonist-dependent phosphorylation and desensitization. The muscarinic agonist carbachol induced phosphorylation of the hm2 mAChR in the Sf9 cells incubated with 32P(i) to an extent of 4-5 mol of phosphate/mol of receptor. In contrast, no phosphorylation of the hm1 mAChR was observed. The hm2 mAChR stimulated [35S]GTP gamma S binding to, and GTPase activity of, the insect cell G-proteins. These receptor-mediated activities were reduced by 50% in membranes prepared from agonist-treated cells compared to control, suggesting that the agonist-induced phosphorylation of the hm2 mAChR resulted in desensitization of the receptors. No role for protein kinase C or cyclic nucleotide-dependent kinases in receptor phosphorylation and desensitization was suggested from studies using agents known to modulate the activity of these enzymes. However, pertussis toxin was found to completely eliminate the interaction of the hm2 receptors with the insect cell G-proteins, but did not perturb the ability of carbachol to induce agonist-dependent phosphorylation of the receptors. These results suggested that G-proteins and/or G-protein-activated signalling were not necessary for the agonist-induced phosphorylation of the receptors. Overall, the data indicated that the human m2 (but not the human m1) mAChR expressed in Sf9 insect cells undergo phosphorylation and desensitization in an agonist-dependent, G-protein-independent fashion by an endogenous insect cell kinase. The results demonstrated that a human G-protein-linked receptor is regulated in insect cells in a manner that is similar to that involving members of the G-protein receptor-kinase family.     

Involvement of pertussis-toxin-sensitive G protein in muscarinic-receptor-mediated inhibition of K(+)-activated 4-nitrophenylphosphatase activity of cardiac sarcolemma

The effects of the cholinergic agonist carbachol on ouabain-sensitive K(+)-activated 4-nitrophenylphosphatase (K(+)-O2NPhPase) activity of rabbit and pig ventricular sarcolemma were examined. Carbachol (0.01-1000 microM) alone had no effect on K(+)-O2NPase. However, in the presence of GTP (100 microM) or its analog guanosine 5'-[gamma-thio]triphosphate (GTP[S], 1 microM) the agonist reduced this enzymatic activity (IC50 = 0.3 microM) by about 45% in a concentration-dependent manner. The GTP[S]-dependent effect of carbachol was blocked by 10 microM atropine, an antagonist of muscarinic acetylcholine receptor (mAcChoR). In the presence of micromolar concentrations of ATP or the GDP analog guanosine 5'-[beta-thio]diphosphate, carbachol did not change sarcolemmal K(+)-O2NPhPase activity. GTP[S] alone reduced this activity (IC50 = 2 microM) by about 40% in a concentration-dependent manner with a lag period of about 3 min. This lag disappeared in the presence of carbachol. Treatment of sarcolemmal membranes with 20 micrograms/ml pertussis toxin, which catalyzed ADP-ribosylation of the 40-41-kDa alpha-subunits of inhibitory GTP-binding protein (Gi), abolished the GTP[S]-promoted inhibitory effect of carbachol. Immunochemically, these alpha-subunits were identified as alpha 12- and alpha i3-subunits. It is suggested that the carbachol-induced inhibition of ouabain-sensitive K(+)-O2NPhPase activity of mammalian myocardial sarcolemma is a result of a negative coupling between mAcChoR and Na+/K(+)-ATPase via Gi protein.     

Extracellular ATP increases free cytosolic calcium in rat parotid acinar cells. Differences from phospholipase C-linked receptor agonists.

The effects of extracellular ATP on intracellular free calcium concentration [( Ca2+]i), phosphatidylinositol (PtdIns) turnover, amylase release and Ca2+-activated membrane currents were examined in isolated rat parotid acinar cells and contrasted with the effects of receptor agonists known to activate phospholipase C. ATP was more effective than muscarinic and alpha-adrenergic agonists and substance P as a stimulus for elevating [Ca2+]i (as measured with quin2). The ATP effect was selectively antagonized by pretreating parotid cells with the impermeant anion-exchange blocker 4,4'-di-isothiocyano-2,2'-stilbenedisulphonate (DIDS), which also inhibited binding of [alpha-32P]ATP to parotid cells. By elevating [Ca2+]i, ATP and the muscarinic agonist carbachol both activated Ca2+-sensitive membrane currents, which were measured by whole-cell and cell-attached patch-clamp recordings. However, there were marked contrasts between the effects of ATP and the receptor agonists linked to phospholipase C, as follows. (1) Although the combination of maximally effective concentrations of carbachol, substance P and phenylephrine had no greater effect on [Ca2+]i than did carbachol alone, there was some additivity between maximal ATP and carbachol effects. (2) Intracellular dialysis with guanosine 5'-[beta-thio]diphosphate did not block activation of ion channels by ATP, but did block channel activation by the muscarinic agonist carbachol. This suggests that a G-protein is involved in the muscarinic response, but not in the response to ATP. (3) Despite its pronounced effect on [Ca2+]i, ATP had little effect on PtdIns turnover in these cells, in contrast with the effects of carbachol and other Ca2+-mobilizing agents. (4) Although ATP was able to stimulate amylase release from parotid acinar cells, the stimulation was only 33 +/- 9% of that obtained with phospholipase C-linked receptor agonists. These differences suggest that ATP increases [Ca2+]i through specific activation of a pathway which is distinct from that shared by the classical phospholipase C-linked receptor agonists.     

A new enzymatic method of nitrile synthesis by Rhodococcus sp. strain YH3-3

The substrate specificity of a novel aldoxime dehydratase from E-pyridine-3-aldoxime assimilating bacterium, Rhodococcus sp. strain YH3-3, was examined. The enzyme catalyzed a dehydration reaction of various aryl- and alkyl-aldoximes to form the corresponding nitriles, but did not act on arylalkyl- and substituted alkyl-aldoximes. Of various aldoximes tested, E-pyridine-3-aldoxime was the most suitable substrate for the enzyme. E-Pyridine-3-aldoxime analogs such as O-acetyl-E-pyridine-3-aldoxime, Z-pyridine-3-aldoxime, and E/Z-pyridine-3-aldehyde-hydrazone also acted as substrates and were converted to 3-cyanopyridine. Heat-treatment of the cells increased the accumulation of 3-cyanopyridine from E-pyridine-3-aldoxime because the nitrile degrading enzyme, nitrile hydratase was inactivated. Under the optimized reaction conditions (pH 7.0, 30°C), various nitriles were synthesized from the corresponding aldoximes in preparative scales with heat-treated cells of the strain. This is the first report on the microbial synthesis of nitriles from aldoximes.     

Alterations in capsaicin-evoked electrolyte transport during the evolution of guinea pig TNBS ileitis

The efferent secretomotor activity of capsaicin-sensitive nerves was monitored during the evolution of 2,4,6-trinitrobenzenesulfonic acid (TNBS)-induced ileitis in the guinea pig by recording changes in short-circuit current (DeltaI(sc)) in response to capsaicin, substance P (SP), and carbachol. Submucosal-mucosal preparations mounted in standard Ussing chambers were studied at time 0, at 8 h, and 1, 3, 5, 7, 14, and 30 days following the intraluminal instillation of TNBS or saline. Maximal DeltaI(sc) responses to capsaicin were dramatically attenuated (54%) by 24 h. By day 7, SP- and TTX-insensitive carbachol-stimulated DeltaI(sc) were also significantly reduced. Similar attenuation in capsaicin and carbachol responses was observed in jejunal tissue 20 cm proximal to the inflamed site at day 7. These studies demonstrate that efferent secretomotor function of capsaicin-sensitive nerves is maintained early in TNBS ileitis but significantly reduced by 24 h. By day 7, defects in enterocyte secretory function at inflamed and noninflamed sites also occurred, an effect that may be mediated by circulating cytokines.     

Cholinergic desensitization of pepsinogen secretion and calcium mobilization of dispersed guinea pig chief cells

When dispersed chief cells from guinea pig stomach were first incubated with carbachol, washed, and then reincubated with carbachol in fresh incubation solution, the stimulation of pepsinogen secretion and the rise in intracellular calcium concentration during the second incubation were reduced. Carbachol did not cause residual enzyme secretion, but the same range of concentrations that causes enzyme secretion caused desensitization that was rapid, temperature dependent, and reversible with time. Preincubation with carbachol caused approximately a 65% reduction in enzyme secretion stimulated during a subsequent incubation with this agonist, but the potency of carbachol was unaffected. Prior exposure to carbachol also reduced subsequent stimulation caused by cholecystokinin (CCK-8), gastrin I, ionophore A23187, or 12-O-tetradecanoylphorbol 13-acetate but did not alter stimulation by any agonist that increases cellular cAMP. Carbachol pretreatment of Fura-loaded chief cells caused a threefold increase in the EC50 for carbachol-stimulated [Ca2+]i and approximately a 30% reduction in the maximal rise in [Ca2+]i in response to carbachol or CCK-8. Inhibition of [N-methyl-3H] scopolamine binding by carbachol following carbachol pretreatment indicated that modulation of receptor affinity or number did not account for functional desensitization. These data indicate that carbachol causes heterologous desensitization of pepsinogen secretion stimulated by agonists that mobilize cellular Ca2+ or activate protein kinase C through a postreceptor action and suggest that an attenuated rise in chief cell calcium is one mechanism mediating the desensitization of enzyme secretion.     

Mitochondrial calcium oscillations in C2C12 myotubes

Mitochondrial Ca(2+) concentration ([Ca(2+)](m)) was monitored in C2C12 skeletal muscle cells stably expressing the Ca(2+)-sensitive photoprotein aequorin targeted to mitochondria. In myotubes, KCl-induced depolarization caused a peak of 3.03 +/- 0.14 micrometer [Ca(2+)](m) followed by an oscillatory second phase (5.1 +/- 0.1 per min). Chelation of extracellular Ca(2+) or blockade of the voltage-operated Ca(2+) channel attenuated both phases of the KCl response. The inhibitor of the sarcoplasmic reticulum Ca(2+)-ATPase, cyclopiazonic acid, reduced the amplitude of the KCl-induced [Ca(2+)](m) peak and prevented the oscillations, suggesting that these were generated intracellularly. No such [Ca(2+)](m) oscillations occurred with the nicotinic agonist carbachol, cyclopiazonic acid alone, or the purinergic agonist ATP. In contrast, caffeine produced an oscillatory behavior, indicating a role of ryanodine receptors as mediators of the oscillations. The [Ca(2+)](m) response was desensitized when cells were exposed to two consecutive challenges with KCl separated by a 5-min wash, whereas a second pulse of carbachol potentiated [Ca(2+)](m), indicating differences in intracellular Ca(2+) redistribution. Cross-desensitization between KCl and carbachol and cross-potentiation between carbachol and KCl were observed. These results suggest that close contacts between mitochondria and sarcoplasmic reticulum exist permitting Ca(2+) exchanges during KCl depolarization. These newly demonstrated dynamic changes in [Ca(2+)](m) in stimulated skeletal muscle cells might contribute to the understanding of physiological and pathological processes in muscular disorders.     

Formation of inositol polyphosphates in cultured human sweat duct cells in response to cholinergic stimulation

Inositol phosphate formation in response to cholinergic stimulation was studied in cultured human sweat duct cells, prelabelled with myo-[2-3H]inositol. Formation of inositol mono-, bis-, tris- and tetrakisphosphates was increased after 15 min stimulation by 30 microM carbachol. Formation of inositol 1,3,4-trisphosphate and inositol tetrakisphosphate was significantly increased within 1 min at carbachol concentrations between 10 microM and 100 microM. No detectable increase in inositol 1,4,5-trisphosphate formation was observed at 15 s or 1 min, but an increase was observed after 15 min at a carbachol concentration of 30-100 microM. The data are consistent with an involvement of inositol polyphosphates in the biphasic response of ion transport, to cholinergic stimulation in these cells (see Pederson, P.S. (1986) 6th Professional Conference "Broken Arrow 1986". Genetic and Eptihelial Dysfunction in Cystic Fibrosis (Riordan, J.R. and Buchwalds, M., eds.), Alan Liss, New York and Pedersen, P.S. (1987) Med. Sci. Res. 15, 769-770) and suggest a different pattern of metabolism from exocrine acinar cells.     

In vitro study of rat uterus after chronic formaldehyde exposure.

To measure cholinergic, adrenergic and tryptaminergic receptor activity of formaldehyde (HCHO) in rat uterus, albino rats were treated with 5 and 10 mg/kg, ip HCHO for 30 days. Acetylcholine (ACh) in doses 1.33, 2 and 3 micrograms/ml produced mild to moderate contraction of isolated rat uterus in control group. HCHO had no effect on isolated rat uterus per se, however it reduced ACh and carbachol induced contraction and presence of adrenaline influences in respect of ACh and carbachol activity. Adrenaline per se had no effect in control preparations, but reduced carbachol induced contraction. Propranolol had no effect on rat uterus; but its presence in the bathing medium increased activity of adrenaline. 5-Hydroxytryptamine (5-HT) had no effect of its own on isolated rat uterus but its presence in the bathing medium enhanced contractions of carbachol and oxytocin.     

Short-term desensitization of phosphatidylinositol turnover via muscarinic acetylcholine receptors and histamine H1-receptors in smooth muscle

Smooth muscle of guinea-pig taenia caecum was desensitized by treatment with 10(-4)M carbachol or 10(-4)M histamine for 30 min in Ca-free solution containing 2mM EGTA. Phosphatidylinositol turnover stimulated by carbachol was not reduced by desensitization with either carbachol or histamine, while the turnover stimulated by histamine was reduced by desensitization with histamine, but not with carbachol. These results are consistent with our previous report (1) that heterologous desensitization induced by carbachol occurs at intracellular Ca stores and homologous desensitization by histamine occurs at H1 receptors.