Phosphatidylinositol hydrolysis and phosphatidylinositol 4′,5′-diphosphate hydrolysis are separable responses during secretagogue action in the rat pancreas

Rat pancreatic fragments and acinar preparations were incubated in vitro to characterize further the changes in phosphoinositide metabolism that occur during secretagogue action. Two distinct responses were discernible. The first response, most notably involving a decrease in phosphatidylinositol content, was (a) observed at lower carbachol concentrations in dose-response studies, (b) inhibited by incubation in Ca²⁺-free media containing 1 mM EGTA, (c) associated with increases in inositol monophosphate production, and (d) provoked by all tissue secretagogues (carbachol, cholecystokinin, secretin, insulin, dibutyryl cAMP and the ionophore A23187), regardless of whether their mechanism of action primarily involved Ca²⁺ mobilization or cAMP generation. This decrease in phosphatidylinositol content was at least partly due to phospholipase C (and/or D) activation, as evidenced by the increase in inositol monophosphate. The second response, most notably involving markedly increased incorporation of ³²PO₄ into phosphatidic acid and phosphatidylinositol, was (a) observed at higher carbachol concentrations, (b) not influenced by incubation in Ca²⁺-free media containing 1 mM EGTA, and (c) associated with increases in inositol triphosphate production. This ³²PO₄ turnover response was probably largely the result of phospholipase C-mediated hydrolysis of phosphatidylinositol 4′,5′-diphosphate, which, as shown previously, also occurs at higher carbachol concentrations and is insensitive to comparable EGTA-induced Ca²⁺ deficiency. This phosphatidylinositol 4′,5′-diphosphate hydrolysis response was only observed in the action of agents (carbachol and cholecystokinin) which mobilize Ca²⁺ via activation of cell surface receptors. The present results indicate that phosphatidylinositol and phosphatidylinositol 4′,5′-diphosphate hydrolysis are truly separable responses to secretagogues acting in the rat pancreas. Furthermore, phosphatidylinositol 4′,5′-diphosphate, rather than phosphatidylinositol hydrolysis is more likely to be associated with receptor activation and Ca²⁺ mobilization.     

U73122 inhibits Ca2+ oscillations in response to cholecystokinin and carbachol but not to JMV-180 in rat pancreatic acinar cells.

Stimulation of rat pancreatic acinar cells with low concentrations of phosphatidylinositol (PI)-linked secretagogues induces [Ca2+]i oscillations, without measurable changes in the formation of inositol 1,4,5-trisphosphate. Therefore, we tested U73122 a new phospholipase C inhibitor to determine if PI turnover is necessary for the generation of [Ca2+]i oscillations. In acini prelabeled with [3H]inositol, PI hydrolysis on stimulation with either cholecystokinin or carbachol was inhibited dose-dependently by U73122, with a maximal effect seen at 10 microM; the formation of inositol 1,4,5-trisphosphate, measured using a radioreceptor assay, was also similarly inhibited. By contrast secretin- or vasoactive intestinal peptide-stimulated production of cAMP was unaffected by 10 microM U73122. These studies indicate that U73122 is a relatively specific inhibitor of G-protein-mediated phospholipase C activation in pancreatic acini. In fura-2-loaded acini, U73122 inhibited the increases in [Ca2+]i stimulated by these high concentrations of secretagogues which can be demonstrated to elicit PI turnover. The [Ca2+]i signal generated by directly stimulating G-proteins with sodium fluoride was also inhibited by U73122; however, the [Ca2+]i rise induced by thapsigargin was unaffected. These data indicate that the mechanism of inhibition was distal to the occupation of cell surface receptors but did not involve an interference of Ca2+ metabolism in general. When [Ca2+]i oscillations were elicited by low concentrations of cholecystokinin or carbachol, U73122 rapidly inhibited the oscillating [Ca2+]i signal. In contrast, oscillations induced by an analogue of cholecystokinin, JMV-180, which does not stimulate changes in PI metabolism at any concentration, were unaffected. This indicates that cholecystokinin- and carbachol-induced oscillations are probably initiated by small, localized changes in PI metabolism, which are not readily detectable. However, the inability of U73122 to inhibit JMV-180-induced oscillations indicates that PI metabolism may not necessarily be a prerequisite for the generation of [Ca2+]i oscillations.     

Ca2+-Independent Stimulation of Adenylate Cyclase Activity by Muscarinic Receptors in Rat Olfactory Bulb

In rat olfactory bulb homogenate, carbachol stimulated adenylate cyclase activity in a concentration-dependent manner (EC50 = 1.1 microM). The carbachol stimulation occurred fully in membranes that had been prepared in the presence of 1 mM EGTA and incubated in a Ca2(+)-free enzyme reaction medium. Under these conditions, exogenous calmodulin (1 microM) failed to stimulate adenylate cyclase activity. In miniprisms of olfactory bulb, carbachol (1 mM) increased accumulation of inositol phosphates, but this response was markedly reduced in a Ca2(+)-free medium. Moreover, the carbachol stimulation of adenylate cyclase activity was not affected by staurosporine at a concentration (1 microM) that completely blocked the stimulatory effect of phorbol 12-myristate 13-acetate, an activator of Ca2+/phospholipid-dependent protein kinase. Quinacrine, a nonselective phospholipase A2 inhibitor, reduced the carbachol stimulation of adenylate cyclase activity, but this inhibition appeared to be competitive with a Ki of 0.2 microM. Nordihydroguaiaretic acid and indomethacin, two inhibitors of arachidonic acid metabolism, failed to affect the carbachol response. These results indicate that in rat olfactory bulb, muscarinic receptors stimulate adenylate cyclase activity through a mechanism that is independent of Ca2+ and phospholipid hydrolysis.     

Inositol Phospholipid Hydrolysis by Rat Sciatic Nerve Phospholipase C

Rat sciatic nerve cytosol contains a phosphodiesterase of the phospholipase C type that catalyzes the hydrolysis of inositol phospholipids, with preferences of phosphatidylinositol 4'-phosphate (PIP) greater than phosphatidylinositol (PI) much greater than phosphatidylinositol 4',5'-bisphosphate (PIP2), at a pH optimum of 5.5-6.0 and at maximum rates of 55, 13, and 0.7 nmol/min/mg protein, respectively. Analysis of reaction products by TLC and formate exchange chromatography shows that inositol 1,2-cyclic phosphate (83%) and diacylglycerol are the major products of PI hydrolysis. [32P]-PIP hydrolysis yields inositol bisphosphate, inositol phosphate, and inorganic phosphate, indicating the presence of phosphodiesterase, phosphomonoesterase, and/or inositol phosphate phosphatase activities in nerve cytosol. Phosphodiesterase activity is Ca2+-dependent and completely inhibited by EGTA, but phosphomonoesterase activity is independent of divalent cations or chelating agents. Phosphatidylcholine (PC) and lysophosphatidylcholine (lysoPC) inhibit PI hydrolysis. They stimulate PIP and PIP2 hydrolysis up to equimolar concentrations, but are inhibitory at higher concentrations. Both diacylglycerols and free fatty acids stimulate PI hydrolysis and counteract its inhibition by PC and lysoPC. PIP2 is a poor substrate for the cytosolic phospholipase C and strongly inhibits hydrolysis of PI. However, it enhances PIP hydrolysis up to an equimolar concentration.     

Carbachol induces a rapid and sustained hydrolysis of polyphosphoinositide in bovine tracheal smooth muscle measurements of the mass of polyphosphoinositides, 1,2-diacylglycerol, and phosphatidic acid

The effects of carbachol on polyphosphoinositides and 1,2-diacylglycerol metabolism were investigated in bovine tracheal smooth muscle by measuring both lipid mass and the turnover of [3H]inositol-labeled phosphoinositides. Carbachol induces a rapid reduction in the mass of phosphatidylinositol 4,5-bisphosphate and phosphatidylinositol 4-monophosphate and a rapid increase in the mass of 1,2-diacylglycerol and phosphatidic acid. These changes in lipid mass are sustained for at least 60 min. The level of phosphatidylinositol shows a delayed and progressive decrease during a 60-min period of carbachol stimulation. The addition of atropine reverses these responses completely. Carbachol stimulates a rapid loss in [3H]inositol radioactivity from phosphatidylinositol 4,5-bisphosphate and phosphatidylinositol 4-monophosphate associated with production of [3H]inositol trisphosphate. The carbachol-induced change in the mass of phosphoinositides and phosphatidic acid is not affected by removal of extracellular Ca2+ and does not appear to be secondary to an increase in intracellular Ca2+. These results indicate that carbachol causes phospholipase C-mediated polyphosphoinositide breakdown, resulting in the production of inositol trisphosphate and a sustained increase in the actual content of 1,2-diacylglycerol. These results strongly suggest that carbachol-induced contraction is mediated by the hydrolysis of polyphosphoinositides with the resulting generation of two messengers: inositol 1,4,5-trisphosphate and 1,2-diacylglycerol.     

Two distinct pathways of platelet-activating factor-induced hydrolysis of phosphoinositides in primary cultures of rat Kupffer cells

Stimulation of rat Kupffer cells in primary culture with platelet-activating factor (PAF) caused a rapid hydrolysis of phosphatidylinositol 4,5-bisphosphate and phosphatidylinositol 4-phosphate with a concomitant increase in the levels of myo-inositol 1,4,5-trisphosphate and myo-inositol 1,4-bisphosphate. This phospholipase C-mediated hydrolysis of polyphosphoinositides was independent of extracellular Ca2+ but was inhibited by the intracellular Ca2+ antagonist TMB-8. A second slower response to PAF was characterized by deacylation of PI leading to the accumulation of glycerophosphoinositol (GPI). PAF-induced GPI synthesis was not inhibited by TMB-8. These effects of PAF were accompanied by initial transient mobilization of Ca2+ from intracellular stores followed by a rather slow influx of Ca2+ from the extracellular medium. PAF-stimulated deacylation and phosphodiesteric hydrolysis of inositol lipids were differentially affected by cholera toxin and pertussis toxin. Pretreatment of the Kupffer cells with either of these toxins caused inhibition of phospholipase C activity. Pertussis toxin also inhibited PAF-stimulated deacylation. However, cholera toxin itself stimulated GPI release and addition of PAF to the cholera toxin-treated cells caused a further increase in GPI release. Phorbol ester inhibited PAF-induced phosphodiesteric hydrolysis of phosphoinositides, but not deacylation. PAF-induced metabolism of phosphoinositides was inhibited by the PAF antagonist, U66985. These results suggest that PAF-induced phosphodiesteric hydrolysis and deacylation of inositol phospholipids are regulated via distinct mechanisms involving activation of separate G-proteins in rat Kupffer cells. Also the regulation of phosphoinositide metabolism by Ca2+ mobilization from two separate Ca2+ pools is indicated by this study.     

Metabolism and functions of inositol phosphates

Activation of Ca2+-mobilizing receptors rapidly increases the cytoplasmic Ca2+ concentration both by releasing Ca2+ stored in endoplasmic reticulum and by stimulating Ca2+ entry into the cells. The mechanism by which Ca2+ release occurs has recently been elucidated. Receptor activation of phospholipase C results in the hydrolysis of the plasma membrane lipid, phosphatidylinositol 4,5-bisphosphate (PIP2), to yield two intracellular messengers, diacylglycerol (DAG) and (1,4,5)inositol trisphosphate [(1,4,5)IP3]. DAG remains in the plasma membrane where it stimulates protein phosphorylation via the phospholipid-dependent protein kinase C. (1,4,5)IP3 diffuses to and interacts with specific sites on the endoplasmic reticulum to release stored Ca2+. Receptor stimulation of phospholipase C appears to be mediated by one or more guanine nucleotide-dependent regulatory proteins by a mechanism analogous to hormonal activation of adenylyl cyclase. The actions of (1,4,5)IP3 on Ca2+ mobilization are terminated by two metabolic pathways, sequential dephosphorylation to inositol bisphosphate (IP2), inositol monophosphate (IP) and inositol or by phosphorylation to inositol tetrakisphosphate (IP4) and sequential dephosphorylation to different inositol phosphates. A sustained cellular response also requires Ca2+ entry into the cell from the extracellular space. The mechanism by which hormones increase Ca2+ entry is not known; a recent proposal involving movement of Ca2+ through the endoplasmic reticulum, possibly regulated by IP4, will be considered here.     

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

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

Deoxycholate induces the preferential hydrolysis of polyphosphoinositides by human platelet and rat corneal phospholipase C

Deoxycholate promotes phospholipase C degradation of endogenous phosphatidyl[3H]inositol (Pl), phosphatidyl[3H]inositol monophosphate (PIP) and phosphatidyl[3H]inositol bisphosphate (PIP2) in rat cornea and human platelets. Hydrolysis of phosphatidyl[3H]inositol significantly lags polyphospho[3H]inositide degradation. Concomitantly, formation of [3H]inositol monophosphate (IP1) lags behind [3H]inositol bisphosphate (IP2) and [3H]inositol trisphosphate (IP3) production. These results demonstrate that rat cornea and human platelet phospholipase C cause a preferential hydrolysis of the endogenous polyphosphoinositides rather than phosphatidylinositol.     

Activation of muscarinic receptors in PC12 cells. Correlation between cytosolic Ca2+ rise and phosphoinositide hydrolysis.

The intracellular signals generated by carbachol activation of the muscarinic receptor [release of inositol phosphates as a consequence of phosphoinositide hydrolysis and rise of the cytosolic Ca2+ concentration ([Ca2+]i, measured by quin2)] were studied in intact PC12 pheochromocytoma cells that had been differentiated by treatment with nerve growth factor. When measured in parallel samples of the same cell preparation 30 s after receptor activation, the release of inositol trisphosphate and of its possible metabolites, inositol bis- and mono-phosphate, and the [Ca2+]i rise were found to occur with almost superimposable carbachol concentration curves. At the same time carbachol caused a decrease in the radioactivity of preloaded phosphatidylinositol 4,5-bisphosphate, the precursor of inositol trisphosphate. Neither the inositol phosphate nor the [Ca2+]i signal was modified by preincubation of the cells with either purified Bordetella pertussis toxin or forskolin, the direct activator of adenylate cyclase. Both signals were partially inhibited by dibutyryl cyclic AMP, especially when the nucleotide analogue was applied in combination with the phosphodiesterase inhibitors RO 201724 and theophylline. The latter drug alone profoundly inhibited the carbachol-induced [Ca2+]i rise, with only minimal effect on phosphoinositide hydrolysis. Because of the diverging results obtained with forskolin on the one hand, dibutyryl cyclic AMP and phosphodiesterase inhibitors on the other, the effects of the latter drugs are considered to be pharmacological, independent of the intracellular cyclic AMP concentration. Two further drugs tested, mepacrine and MY5445, inhibited phosphoinositide hydrolysis at the same time as the 45Ca2+ influx stimulated by carbachol. Taken together, our results concur with previous evidence obtained with permeabilized cells and cell fractions to indicate phosphatidylinositol 4,5-bisphosphate hydrolysis and [Ca2+]i rise as two successive events in the intracellular transduction cascade initiated by receptor activation. The strict correlation between the carbachol concentration curves for inositol trisphosphate generation and [Ca2+]i rise, and the inhibition by theophylline of the Ca2$ signal without major effects on inositol phosphate generation, satisfy important requirements of the abovementioned interpretation.     

Modulation of carbachol-stimulated inositol phospholipid hydrolysis in rat cerebral cortex

Cortical slices from rat brain were used to study carbachol-stimulated inositol phospholipid hydrolysis. Omission of calcium during incubation of slices with [³H]inositol increased its incorporation into receptor-coupled phospholipids. Carbachol-stimulated hydrolysis of [³H]inositol phospholipids in slices was dose-dependent, was affected by the concentrations of calcium and lithium present and resulted in the accumulation of mostly [³H]inositol-l-phosphate. Incubation of slices withN-ethylmaleimide or a phorbol ester reduced the response to carbachol. Membranes prepared from cortical slices labeled with [³H]inositol retained the receptor-stimulated inositol phospholipid hydrolysis reaction. The basal rate of inositol phospholipid hydrolysis was higher than in slices and addition of carbachol further stimulated the process. Addition of GTP stimulated inositol phospholipid hydrolysis, suggesting the presence of a guanine nucleotide-binding protein coupled to phospholipase C. Carbachol and GTP-stimulated inositol phospholipid hydrolysis in membranes was detectable following a 3 min assay period. In contrast to slices, increased levels of inositol bisphosphate and inositol trisphosphate were detected following incubation of membranes with carbachol. These results demonstrate that agonist-responsive receptors are present in cortical membranes, that the receptors may be coupled to phosphatidylinositol 4,5-bisphosphate, rather than phosphatidylinositol, hydrolysis and that a guanine nucleotide-binding protein may mediate the coupling of receptor activation to inositol phospholipid hydrolysis in brain.     

Effects of angiotensin II and dibutyryl cyclic adenosine monophosphate on phosphatidylinositol metabolism, 45Ca2+ fluxes, and aldosterone synthesis in bovine adrenal glomerulosa cells

Angiotensin II (AII) and N6,O2'-dibutyryladenosine 3':5'-cyclic monophosphate (dibutyryl cyclic AMP) both stimulated aldosterone synthesis in bovine adrenal glomerulosa cells. AII altered 45Ca2+ fluxes and increased 32PO4 incorporation into phosphatidylinositol in these cells, whereas dibutyryl cyclic AMP did not affect either process. Neither AII nor dibutyryl cyclic AMP increased the mass of phosphatidylinositol. Both agents are known to stimulate pregnenolone synthesis. Thus, although dibutyryl cyclic AMP and AII may increase aldosterone synthesis at a common site (pregnenolone synthesis), they do so by different mechanisms. AII stimulation of phosphatidylinositol labeling by 32PO4 (the "PI effect") was blocked when cells were incubated in a medium containing both EGTA and the calcium antagonist, 8-(N,N-diethylamino)-octyl 3,4,5-trimethoxy-benzoate hydrochloride (TMB-8), suggesting a calcium requirement for the PI effect.     

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.     

Inositol Phospholipid Hydrolysis in Rat Cerebral Cortical Slices: II. Calcium Requirement

The calcium requirement for agonist-dependent breakdown of phosphatidylinositol and polyphosphoinositides has been examined in rat cerebral cortex. The omission of added Ca2+ from the incubation medium abolished [3H]inositol phosphate accumulation from prelabelled phospholipid induced by histamine, reduced that due to noradrenaline and 5-hydroxytryptamine, but did not affect carbachol-stimulated breakdown. EC50 values for agonists were unaltered in the absence of Ca2+. Removal of Ca2+ by preincubation with EGTA (0.5 mM) abolished all responses, but complete restoration was achieved by replacement of Ca2+. The EC50 for Ca2+ for histamine-stimulated [3H]inositol phosphate accumulation was 80 microM. Noradrenaline-stimulated breakdown was antagonised by manganese (IC50 1.7 mM), but not by the calcium channel blockers nitrendipine or nimodipine (30 microM). The calcium ionophore A23187 stimulated phosphatidylinositol/polyphosphoinositide hydrolysis with an EC50 of 2 microM, and this response was blocked by EGTA. Omission of Ca2+ or preincubation with EGTA or Mn2+ (EC50 = 230 microM) greatly enhanced the incorporation of [3H]inositol into phospholipids. The IC50 for Ca2+ in inhibiting incorporation was 25 microM. The results show that different receptors mediating phosphatidylinositol/polyphosphoinositide breakdown in rat cortex have quantitatively different Ca2+ requirements, and it is suggested that rigid opinions regarding phosphatidylinositol/polyphosphoinositide breakdown as either cause or effect of calcium mobilisation in rat cortex are inappropriate.     

Effects of carbachol and pancreozymin (cholecystokinin-octapeptide) on polyphosphoinositide metabolism in the rat pancreas in vitro.

We studied the possibility that hydrolysis of phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] may be the initiating event for the increase in [32P]Pi incorporation into phosphatidic acid (PtdA) and phosphatidylinositol (PtdIns) during carbachol and pancreozymin (cholecystokinin-octapeptide) action in the rat pancreas. After prelabelling acini for 2h, [32P]Pi incorporation into PtdA, PtdIns(4,5)P2 and phosphatidylinositol 4-phosphate (PtdIns4P) had reached equilibrium. Subsequent addition of carbachol or pancreozymin caused 32P in PtdIns(4,5)P2 to decrease by 30-50% within 10-15 s, and this was followed by sequential increases in [32P]Pi incorporation into PtdA and PtdIns. Similar changes in 32P-labelling of PtdIns4P were not consistently observed. Confirmation that the decrease in 32P in chromatographically-purified PtdIns(4,5)P2 reflected an actual decrease in this substance was provided by the fact that similar results were obtained (a) when PtdIns(4,5)P2 was prelabelled with [2-3H]inositol, and (b) when PtdIns(4,5)P2 was measured as its specific product (glycerophosphoinositol bisphosphate) after methanolic alkaline hydrolysis and ion-exchange chromatography. The secretogogue-induced breakdown of PtdIns(4,5)P2 was not inhibited by Ca2+ deficiency (severe enough to inhibit amylase secretion and Ca2+-dependent hydrolysis of PtdIns), and ionophore A23187 treatment did not provoke PtdIns(4,5)P2 hydrolysis. The increase in the hydrolysis of PtdIns(4,5)P2 and the increase in [32P]Pi incorporation into PtdA commenced at the same concentration of carbachol in dose-response studies. Our findings suggest that the hydrolysis of PtdIns(4,5)P2 is an early event in the action of pancreatic secretogogues that mobilize Ca2+, and it is possible that this hydrolysis may initiate the Ca2+-independent labelling of PtdA and PtdIns. Ca2+ mobilization may follow these responses, and subsequently cause Ca2+-dependent hydrolysis of PtdIns and exocytosis.     

Release of intracellular Ca2+ and elevation of inositol trisphosphate by secretagogues in parietal and chief cells isolated from rabbit gastric mucosa

The fluorescent intracellular Ca2+ indicator, fura2/AM, was used to determine the effects of carbachol, cholecystokinin octapeptide (CCK-8), gastrin and histamine on intracellular Ca2+ ([Ca2+]i) in parietal cells from rabbit gastric mucosa enriched to more than 95% purity by a new Nycodenz gradient/centrifugal elutriation technique. Changes in [Ca2+]i in response to the same agonists were also measured in enriched chief cells. Carbachol, histamine, gastrin and CCK-8 increased parietal cell [Ca2+]i with the response to carbachol greater than CCK -8 = histamine = gastrin. Prestimulation with msximal doses of carbachol blocked histamine-induced increases in [Ca2+]i. In chief cells, carbachol increased [Ca2+]i but to a lesser degree than CCK-8, while histamine had no significant effect on [Ca2+]i. Neither removal of extracellular Ca2+ coupled with acute addition of 1 mM EGTA nor addition of the Ca2+-channel blocker nicardipine prevented agonist-induced changes in [Ca2+]i in either cell type. In the presence and absence of 10 mM LiCl2, carbachol and CCK-8 were found to increase inositol trisphosphate (IP3) content in both parietal and chief cells while histamine had no significant effect on this phosphoinositide hydrolysis product. From these results and previous observations with gastric glands (Chew, C.S. (1986) Am. J. Physiol. 13, G814-G823) we conclude that: carbachol, CCK-8, gastrin and histamine increase parietal cell [Ca2+]i initially by release of Ca2+ from the same intracellular store(s); the release of [Ca2+]i in response to carbachol and CCK-8 in both chief and parietal cells appear to be mediated by IP3; however, other mechanisms may be involved in histamine-induced release of parietal cell Ca2+.     

Thyrotropin-releasing hormone stimulation of polyphosphoinositide hydrolysis in GH3 cell membranes is GTP dependent but insensitive to cholera or pertussis toxin

Thyrotropin-releasing hormone (TRH), like numerous other Ca2+-mobilizing agonists, has been found to stimulate polyphosphoinositide hydrolysis in responsive cells. The present studies further clarify the mechanism of action of this peptide hormone by demonstrating direct in vitro effects of TRH on polyphosphoinositide hydrolysis in GH3 pituitary cell membranes. Membranes from [3H]myoinositol-labeled cells were found to generate inositol bis- and tris- but not monophosphate upon incubation. Inositol polyphosphate generation was stimulated 2-3-fold by nanomolar concentrations of TRH in a reaction which was potentiated by micromolar concentrations of GTP; hormone-stimulated hydrolysis observed in the absence of GTP was fully antagonized by guanosine 5'-O-(2-thiodiphosphate). Guanosine 5'-O-(3-thiotriphosphate), Ca2+, and sodium fluoride also activated phosphoinositide hydrolysis in vitro. Stimulated inositol polyphosphate generation was accompanied by stimulated 1,2-diacylglycerol formation. Evidence that both phosphatidylinositol 4,5-bisphosphate as well as phosphatidylinositol 4-phosphate served as substrates for the activated phosphoinositide phosphodiesterase is presented. Pretreatment of GH3 cells with cholera or pertussis toxin did not influence stimulated hydrolysis in membranes. It is concluded that the TRH receptor directly regulates polyphosphoinositide hydrolysis in GH3 cell plasma membranes by a GTP-dependent process. The GTP dependence does not appear to be mediated through a cholera or pertussis toxin substrate and may involve a novel GTP-binding protein (NP).     

Costimulation of T cell receptor/CD3-mediated activation of resting human CD4+ T cells by leukocyte function-associated antigen-1 ligand intercellular cell adhesion molecule-1 involves prolonged inositol phospholipid hydrolysis and sustained increase of intracellular Ca2+ levels.

Activation of resting human CD4+ T cells mediated by mAb ligation of the TCR/CD3 complex requires costimulatory signals to result in proliferation; these can be provided by intercellular cell adhesion molecule-1 (ICAM-1, CD54) a natural ligand of leukocyte function-associated Ag-1 (LFA-1, CD11a/CD18). We analyzed early signaling events involved in T cell activation to determine the contribution by the LFA-1/ICAM-1 interaction. We studied in detail the hydrolysis of phosphatidylinositol(4,5)bisphosphate and intracellular levels of free Ca2+ during stimulation with beads coated with the CD3 mAb OKT3 alone or in combination with purified ICAM-1 protein. Our investigations show no response to LFA-1/ICAM-1 alone, but that costimulation by LFA-1/CAM-1 interaction induces prolonged inositol phospholipid hydrolysis (up to 4 h), resulting in generation of both inositol(1,4,5)phosphate3 and inositol(1,3,4,5)phosphate4 and their derivatives. Based on studies with cycloheximide, this costimulatory effect of prolonged inositol phospholipid hydrolysis appears dependent in part on de novo protein synthesis. A sustained increase in intracellular levels of free Ca2+ level is also observed after LFA-1/ICAM-1 costimulation, which is at least partly dependent on extracellular sources of Ca2+. Kinetic studies indicate that costimulation requires a minimal period of 4 h of LFA-1/ICAM-1 interaction to provide maximal costimulation for OKT3-mediated T cell proliferation. Thus, the necessary costimulation required for OKT3-mediated proliferation in this model system may be provided by an extended LFA-1/ICAM-1 interaction that in combination with OKT3 mAb leads to signal-transducing events, resulting in prolonged phospholipase C activation and phosphatidylinositol(4,5)bisphosphate hydrolysis, and a sustained increase in intracellular levels of free Ca2+.     

Differential effects of extracellular calcium removal and nonspecific effects of Ca2+ antagonists on acid secretory activity in isolated gastric glands

The role of extracellular calcium in the action of the secretagogues, carbachol, histamine and forskolin, on parietal cell HCl secretion was investigated using glands isolated from rabbit gastric mucosa. Omission of calcium from the cellular incubation medium and chelation of a major portion of contaminating calcium with EGTA resulted in a disappearance of the initial transient response to carbachol (as measured by uptake of the weak base, amino[14C]pyrine), but the sustained response to carbachol persisted. Neither histamine nor forskolin-stimulated increase in amino[14C]pyrine uptake were affected by omission of extracellular calcium. Furthermore, the potentiating interactions between histamine and carbachol and between forskolin and carbachol appeared to occur independent of extracellular calcium. Attempts to assess the contribution of intracellular calcium to secretory activity using the Ca2+ antagonists, verapamil, nifedipine, nicardipine and lanthanum, and the putative intracellular Ca2+ antogonist, TMB-8 (3,4,5-trimethyloxybenzoic acid 8-(diethyl-amino)-octyl ester) were unsuccessful. Nifedipine had no effect on secretagogue stimulated amino[14C]pyrine accumulation even at concentration well above the pA2 reported for excitable tissues. Verapamil, nicardipine, lanthanum and TMB-8 all appeared to have nonspecific inhibitory effects on amino [14C]pyrine uptake. From these results we conclude that: (1) parietal cell HCl secretion can occur independent of extracellular Ca2+; (2) influx of extracellular Ca2+ enhances the response to carbachol but has little influence on the secretory response initiated by cAMP-dependent secretagogues; and (3) parietal cell Ca2+ channels have a different molecular configuration than Ca2+ channels in excitable cells.     

Receptor-mediated metabolism of the phosphoinositides and phosphatidic acid in rat lacrimal acinar cells.

The metabolism of the inositol lipids and phosphatidic acid in rat lacrimal acinar cells was investigated. The muscarinic cholinergic agonist methacholine caused a rapid loss of 15% of [32P]phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] and a rapid increase in [32P]phosphatidic acid (PtdA). Chemical measurements indicated that the changes in 32P labelling of these lipids closely resembled changes in their total cellular content. Chelation of extracellular Ca2+ with excess EGTA caused a significant decrease in the PtdA labelling and an apparent loss of PtdIns(4,5)P2 breakdown. The calcium ionophores A23187 and ionomycin provoked a substantial breakdown of [32P]PtdIns(4,5)P2 and phosphatidylinositol 4-phosphate (PtdIns4P); however, a decrease in [32P]PtdA was also observed. Increases in inositol phosphate, inositol bisphosphate and inositol trisphosphate were observed in methacholine-stimulated cells, and this increase was greatly amplified in the presence of 10 mM-LiCl; alpha-adrenergic stimulation also caused a substantial increase in inositol phosphates. A23187 provoked a much smaller increase in the formation of inositol phosphates than did either methacholine or adrenaline. Experiments with excess extracellular EGTA and with a protocol that eliminates intracellular Ca2+ release indicated that the labelling of inositol phosphates was partially dependent on the presence of extracellular Ca2+ and independent of intracellular Ca2+ mobilization. Thus, in the rat lacrimal gland, there appears to be a rapid phospholipase C-mediated breakdown of PtdIns(4,5)P2 and a synthesis of PtdA, in response to activation of receptors that bring about an increase in intracellular Ca2+. The results are consistent with a role for these lipids early in the stimulus-response pathway of the lacrimal acinar cell.