Stimulation of glucose production from glycogen by glucagon, noradrenaline and non-degradable adenosine analogues is counteracted by adenosine and ATP in cultured rat hepatocytes.

We have shown previously that exposure of a non-transformed continuous line of rat liver epithelial (WB) cells to epidermal growth factor (EGF), adrenaline, angiotensin II or [Arg8]vasopressin results in an accumulation of the inositol phosphates InsP1, InsP2 and InsP3 [Hepler, Earp & Harden (1988) J. Biol. Chem. 263, 7610-7619]. Studies were carried out with WB cells to determine whether the EGF receptor and other, non-tyrosine kinase, hormone receptors stimulate phosphoinositide hydrolysis by common, overlapping or separate pathways. The time courses for accumulation of inositol phosphates in response to angiotensin II and EGF were markedly different. Whereas angiotensin II stimulated a very rapid accumulation of inositol phosphates (maximal by 30 s), increases in the levels of inositol phosphates in response to EGF were measurable only following a 30 s lag period; maximal levels were attained by 7-8 min. Chelation of extracellular Ca2+ with EGTA did not modify this relative difference between angiotensin II and EGF in the time required to attain maximal phospholipase C activation. Under experimental conditions in which agonist-induced desensitization no longer occurred in these cells, the inositol phosphate responses to EGF and angiotensin II were additive, whereas those to angiotensin II and [Arg8]vasopressin were not additive. In crude WB lysates, angiotensin II, [Arg8]vasopressin and adrenaline each stimulated inositol phosphate formation in a guanine-nucleotide-dependent manner. In contrast, EGF failed to stimulate inositol phosphate formation in WB lysates in the presence or absence of guanosine 5'-[gamma-thio]triphosphate (GTP[S]), even though EGF retained the capacity to bind to and stimulate tyrosine phosphorylation of its own receptor. Pertussis toxin, at concentrations that fully ADP-ribosylate and functionally inactivate the inhibitory guanine-nucleotide regulatory protein of adenylate cyclase (Gi), had no effect on the capacity of EGF or hormones to stimulate inositol phosphate accumulation. In intact WB cells, the capacity of EGF, but not angiotensin II, to stimulate inositol phosphate accumulation was correlated with its capacity to stimulate tyrosine phosphorylation of the 148 kDa isoenzyme of phospholipase C. Taken together, these findings suggest that, whereas angiotensin II, [Arg8]vasopressin and alpha 1-adrenergic receptors are linked to activation of one or more phospholipase(s) C by an unidentified G-protein(s), the EGF receptor stimulates phosphoinositide hydrolysis by a different pathway, perhaps as a result of its capacity to stimulate tyrosine phosphorylation of phospholipase C-gamma.     

Neural regulation of parvalbumin expression in mammalian skeletal muscle.

The receptor mechanisms underlying vasopressin-induced human platelet activation were investigated with respect to stimulation of phosphoinositide metabolism and changes in the cytosolic free Ca2+ concentration ([Ca2+]i). Vasopressin stimulated phosphoinositide metabolism, as indicated by the early formation of [32P]phosphatidic acid ([32P]PtdA) and later accumulation of [32P]phosphatidylinositol ([32P]PtdIns). In addition, vasopressin elicited a transient depletion of [glycerol-3H]PtdIns and accumulation of [glycerol-3H]PtdA. The effects of vasopressin on phosphoinositide metabolism were concentration-dependent, with half maximal [32P]PtdA formation occurring at 30 +/- 15 nM-vasopressin. In the presence of 1 mM extracellular free Ca2+, vasopressin induced a rapid, concentration-dependent elevation of [Ca2+]i in quin2-loaded platelets: half-maximal stimulation was observed at 53 +/- 20 nM-vasopressin. The V1-receptor antagonist [1-(beta-mercapto-beta, beta-cyclopentamethylenepropionic acid),2-(O-methyl)tyrosine,8-arginine]-vasopressin selectively inhibited vasopressin (100 nM)-induced [32P]PtdA formation [I50 (concn. giving 50% inhibition) = 5.7 +/- 2.4 nM] and elevation of [Ca2+]i (I50 = 3 +/- 1.5 nM). Prior exposure of platelets to vasopressin rendered them unresponsive, in terms of [32P]PtdA formation and elevation of [Ca2+]i, to a subsequent challenge with vasopressin, but responsive to a subsequent challenge with U44069, a thromboxane-A2 mimetic. These results indicate that vasopressin-induced human platelet activation is initiated by combination with specific V1 receptors on the platelet, and that the sequelae of receptor occupancy (stimulation of phosphoinositide metabolism and elevation of [Ca2+]i) are equally susceptible to inhibition by receptor antagonists and by receptor desensitization.     

Dissociation of Ca2+ entry and Ca2+ mobilization responses to angiotensin II in bovine adrenal chromaffin cells

In fura-2-loaded bovine adrenal chromaffin cells, 0.5 microM angiotensin II (AII) stimulated a 185 +/- 19 nM increase of intracellular-free calcium [( Ca2+]i) approximately 3 s after addition. The time from the onset of the response until achieving 50% recovery (t 1/2) was 67 +/- 10 s. Concomitantly, AII stimulated both the release of 45Ca2+ from prelabeled cells, and a 4-5-fold increase of [3H]inositol 1,4,5-trisphosphate [( 3H]Ins(1,4,5)P3) levels. In the presence of 50 microM LaCl3, or when extracellular-free Ca2+ [( Ca2+]o) was less than 100 nM, AII still rapidly increased [Ca2+]i by 95-135 nM, but the t 1/2 for recovery was then only 23-27 s. In medium with 1 mM MnCl2 present, AII also stimulated a small amount of Mn2+ influx, as judged by quenching of the fura-2 signal. When [Ca2+]o was normal (1.1 mM) or low (less than 60 nM), 1-2 microM ionomycin caused [Ca2+]i to increase 204 +/- 26 nM, while also releasing 45-55% of bound 45Ca2+. With low [Ca2+]o, ionomycin pretreatment abolished both the [Ca2+]i increase and 45Ca2+ release stimulated by AII. However, after ionomycin pretreatment in normal medium, AII produced a La3+-inhibitable increase of [Ca2+]i (103 +/- 13 nM) with a t 1/2 of 89 +/- 8 s, but no 45Ca2+ release. No pretreatment condition altered AII-induced formation of [3H]Ins(1,4,5)P3. We conclude that AII increased [Ca2+]i via rapid and transient Ca2+ mobilization from Ins(1,4,5)P3- and ionomycin-sensitive stores, accompanied (and/or followed) by Ca2+ entry through a La3+-inhibitable divalent cation pathway. Furthermore, the ability of AII to activate Ca2+ entry in the absence of Ca2+ mobilization (i.e. after ionomycin pretreatment) suggests a receptor-linked stimulus other than Ca2+ mobilization initiates Ca2+ entry.     

Inhibition by nicardipine of endothelin-mediated inositol phosphate formation and Ca2+ mobilization in smooth muscle cell

We have investigated the effects of endothelin on phosphoinositide metabolism and Ca2+ mobilization in cultured A10 cells. Endothelin stimulated a significant increase in inositol phosphate formation in a time- and dose-dependent manner. IP3 was significantly elevated by 30 sec and reached a 2.0-fold above control at 1 min. The EC50 for endothelin was 0.5 nM. The initiation of inositol phosphate formation was independent of extracellular Ca2+, and the Ca2+ ionophore, A23187, did not stimulate IP3 formation. However, the sustained elevation of inositol phosphates was partially inhibited by incubating cells in buffer lacking Ca2+ or in buffer containing nicardipine. Endothelin mobilized both intracellular and extracellular Ca2+ reaching a peak intracellular concentration of 350 +/- 11 nM by 1 min when cells were bathed with Ca2+-complete buffer. Intracellular Ca2+ remained 2-fold above baseline for at least 15 min. In contrast, when cells were exposed to endothelin in Ca2+-free buffer, the peak value of [Ca2+]i was 195 +/- 20 nM and returned to baseline by 2 min. Nicardipine completely blocked the influx of extracellular Ca2+ but did not interfere with the mobilization of intracellular stores. We conclude that endothelin produces a rapid and sustained elevation in inositol phosphate formation. The rapid production of IP3 is consistent with the time course for mobilization of intracellular Ca2+. Elevated cytosolic Ca2+ levels are maintained by the influx of extracellular Ca2+ through a nicardipine-sensitive Ca2+ channel and are involved in the sustained formation of inositol phosphates. These data provide an explanation for the sustained, nicardipine-inhibitable contraction of coronary artery strips induced by endothelin.     

Temporal relationship between inositol polyphosphate formation and increases in cytosolic Ca2+ in quiescent 3T3 cells stimulated by platelet-derived growth factor, bombesin and vasopressin.

We determined the temporal relationship between the formation of inositol phosphates and increase in cytosolic [Ca2+] elicited by bombesin, vasopressin and platelet-derived growth factor (PDGF) in quiescent Swiss 3T3 cells. These responses were measured under identical conditions. Bombesin caused a rapid increase in inositol 1,4,5-trisphosphate which coincided with the increase in cytosolic [Ca2+]. This was followed by a slower but marked increase in inositol 1,3,4-trisphosphate and inositol-bisphosphate. Vasopressin elicited a similar sequence of events. In sharp contrast, highly purified porcine PDGF induced increases in cytosolic [Ca2+] and inositol 1,4,5-trisphosphate that were temporally uncoupled: detectable inositol polyphosphate formation occurred after Ca2+ mobilization from intracellular stores. The same temporal dissociation was observed when a recombinant v-sis product was used instead of porcine PDGF. However, PDGF was as effective as bombesin in stimulating the formation of inositol phosphates after 5-10 min of incubation. The data suggest that PDGF increases cytosolic [Ca2+] via a different signal transduction pathway from that utilized by bombesin and vasopressin. These findings have important implications for understanding the signal transduction pathway activated by PDGF.     

A novel human plasma factor(s) capable of mobilizing intracellular Ca2+ in cultured rat vascular smooth muscle cells

Using fura-2-loaded rat vascular smooth muscle cells (VSMCs) in culture, we have attempted to partially purify and characterize yet unidentified factor(s) from normal human plasma that stimulates cytoplasmic free Ca2+ concentration ([Ca2+]i). The plasma extract caused an immediate and transient increase of [Ca2+]i in a dose-dependent manner, of which effect was not prevented by pretreatment with either any of receptor antagonists for -adrenergic agonist, angiotensin II, arginine vasopressin, serotonin, thromboxane A2, or with EGTA and nifedipine. This novel plasma factor(s) was heat-stable and completely inactivated by pronase E, suggesting its protein nature. Furthermore, plasma extracts dose-dependently stimulated the accumulation of [3H] inositol phosphates in rat VSMCs. Sephadex G-50 gel chromatography of plasma extracts resolved one major component (mol wt 13,000) and two minor components with larger (greater than 30,000) and smaller (3,000) mol wt. Present study demonstrates the presence of hitherto unidentified plasma factor(s) with size heterogeneity capable of stimulating both mobilization of [Ca2+]i and breakdown of phosphatidylinositol-4,5-biphosphates in rat VSMCs.     

Long-term phorbol ester treatment down-regulates protein kinase C and sensitizes the phosphoinositide signaling pathway to hormone and growth factor stimulation. Evidence for a role of protein kinase C in agonist-induced desensitization

Exposure of a nontransformed, continuous line of epithelial cells derived from rat liver (WB cells) to epidermal growth factor, angiotensin II, [Arg8]vasopressin, and epinephrine resulted in rapid accumulation of the inositol phosphates (InsP) InsP1, InsP2, and InsP3. Although short-term (5-60 min) pretreatment of WB cells with the phorbol ester 4 beta-phorbol 12 beta-myristate 13 alpha-acetate (PMA) markedly attenuated InsP accumulation in response to all agonists, the inhibitory effects on the InsP response were lost after 2 h incubation with PMA; and, with extended (6-24 h) preincubation, a time-dependent potentiation of the InsP response to angiotensin II, epidermal growth factor and [Arg8]vasopressin was observed. The InsP response during a 15-min challenge with angiotensin II in cells pretreated for 18 h with 600 nM and 10 microM PMA was increased by 2-3-fold and 4-6-fold, respectively. Long-term (18 h) treatment with 600 nM and 10 microM PMA caused a similar 90-100% loss of measurable Ca2+/phospholipid-dependent enzyme (protein kinase C) activity in cytosolic and soluble particulate fractions. The effects of long-term PMA pretreatment do not represent a general enhancement of hormone responsiveness since the InsP response to epinephrine was not affected. In control cells, the InsP response to angiotensin II and epinephrine desensitized very rapidly. Long-term pretreatment with PMA greatly reduced the contribution of agonist-induced desensitization to the angiotensin II response; in contrast, the extent of desensitization occurring during incubation of WB cells with epinephrine was unaltered by long-term treatment with PMA suggesting that an additional mechanism may be involved in alpha 1-adrenergic receptor desensitization. No PMA-induced change in resting levels of [3H]phosphoinositides or the metabolism of exogenous [3H]inositol 1,4,5-trisphosphate by WB homogenates occurred. Stimulation of InsP formation in intact cells by NaF and activation of phospholipase C by GTP gamma S in membranes both were unaltered by short-term or long-term PMA pretreatment. These data are consistent with the idea that following long-term treatment of WB cells with PMA, the occurrence of agonist-induced desensitization of receptors linked to the phosphoinositide/Ca2+ signaling system is reduced, apparently at least in part due to the loss of contribution of a negative feedback regulatory role of protein kinase C.     

Insulin-stimulated phosphoinositide metabolism in isolated fat cells

Treatment of isolated fat cells with insulin produced increases of up to 4.8-fold in the incorporation of [3H]inositol into phosphatidylinositol. This effect of insulin was both time- and dose-dependent with half-maximal stimulation at 30 microunits/ml of insulin. Insulin increased the labeling of phosphatidylinositol and phosphatidylinositol 4,5-bisphosphate but not phosphatidylinositol 4-monophosphate in cells which had been preincubated with [3H]inositol for 90 min. Incubation of the cells in a Ca2+-free buffer increased the basal level of phosphatidylinositol labeling and enhanced the effect of insulin. Glucagon and isoprenaline, both of which stimulate lipolysis, had no effect on phosphatidylinositol labeling but did potentiate insulin-stimulated incorporation of [3H]inositol into phosphatidylinositol. Phosphoinositide breakdown was measured by the accumulation of inositol phosphates. Insulin did not increase the level of the inositol phosphates at all concentrations of the hormone tested. By comparison, phenylephrine and vasopressin were able to stimulate phosphoinositide breakdown. Pretreatment of the cells with insulin enhanced the effect of phenylephrine on inositol phosphates' accumulation, suggesting that insulin may potentiate phenylephrine-mediated phosphoinositide turnover. From these data we conclude that insulin stimulates the de novo synthesis of phosphatidylinositol and phosphatidylinositol 4,5-biphosphate, but has no effect on phosphoinositide breakdown.     

Studies on molecular regulation of phagocytosis in neutrophils. Con A-mediated ingestion and associated respiratory burst independent of phosphoinositide turnover, rise in [Ca2+]i, and arachidonic acid release

The role of the activation of phosphoinositide turnover and of the increase in cytosolic free calcium, [Ca2+]i, in the phagocytosis and associated activation of the respiratory burst was investigated. We report the results obtained on the phagocytosis of yeast cells mediated by Con A in normal and in Ca2+-depleted human neutrophils. In normal neutrophils the phagocytosis was associated with a respiratory burst, a stimulation in the formation of [3H] inositol phosphates and [32P]phosphatidic acid, the release of [3H]arachidonic acid, and a rise in [Ca2+]i. Ca2+-depleted neutrophils are able to perform the phagocytosis of yeast cells mediated by Con A and to activate the respiratory burst without stimulation of [3H]inositol phosphates and [32P]phosphatidic acid formation, [3H]arachidonic acid release, and rise in [Ca2+]i. In both normal and Ca2+-depleted neutrophils the phagocytosis and the associated respiratory burst, 1) were inhibited by cytochalasin B; 2) were insensitive to H-7, an inhibitor of protein kinase C; and 3) did not involve GTP-binding protein sensitive to pertussis toxin. These findings indicate that the activation of phosphoinositide turnover, the liberation of arachidonic acid, the rise in [Ca2+]i, and the activity of protein kinase C are not necessarily required for ingestion of Con A-opsonized particles and for associated activation of the NADPH oxidase, the enzyme responsible for the respiratory burst. The molecular mechanisms of these phosphoinositide and Ca2+-independent responses are discussed.     

Okadaic acid inhibits angiotensin II stimulation of Ins(1,4,5)P3 and calcium signalling in rat hepatocytes.

OKA2 and CL-A significantly inhibit the ability of angiotensin II, ATP and vasopressin to raise [Ca2+]i in rat hepatocytes, with a partial inhibition of the initial spike, and a complete inhibition of the following plateau. In contrast, the [Ca2+]i response to thapsigargin, which releases intracellular calcium stores through a mechanism independent of inositol phosphates, is much less affected. The ability of angiotensin II to stimulate Ins(1,4,5)P3 production is also reduced by OKA, with kinetics consistent with the inhibited [Ca2+]i response. Since OKA and CL-A are potent and selective inhibitors of phosphoprotein phosphatases, these results provide further evidence that agonist-stimulated Ins(1,4,5)P3 signalling can be inhibited by protein phosphorylation.     

Localization and heterogeneity of agonist-induced changes in cytosolic calcium concentration in single bovine adrenal chromaffin cells from video imaging of fura-2.

Temporal and spatial changes in the concentration of cytosolic free calcium ([Ca2+]i) in response to a variety of secretagogues have been examined in adrenal chromaffin cells using digital video imaging of fura-2-loaded cells. Depolarization of the cells with high K+ or challenge with nicotine resulted in a rapid and transient elevation of [Ca2+]i beneath the plasma membrane consistent with Ca2+ entry through channels. This was followed by a late phase in which [Ca2+]i rose within the cell interior. Agonists that act through mobilization of inositol phosphates produced an elevation in [Ca2+]i that was most marked in an internal region of the cell presumed to be the site of IP3-sensitive stores. When the same cells were challenged with nicotine or high K+, to trigger Ca2+ entry through voltage-dependent channels, the rise in [Ca2+]i was most prominent in the same localized region of the cells. These results suggest that Ca2+ entry through voltage-dependent channels results in release of Ca2+ from internal stores and that the bulk of the measured rise in [Ca2+]i is not close to the exocytotic sites on the plasma membrane. Analysis of the time courses of changes in [Ca2+]i in response to bradykinin, angiotensin II and muscarinic agonists showed that these agonists produced highly heterogeneous responses in the cell population. This heterogeneity was most marked with muscarinic agonists which in some cells elicited oscillatory changes in [Ca2+]i. Such heterogeneous changes in [Ca2+]i were relatively ineffective in eliciting catecholamine secretion from chromaffin cells. A single large Ca2+ transient, with a component of the rise in [Ca2+]i occurring beneath the plasma membrane, may be the most potent signal for secretion.     

Effects of caffeine on phosphatidylinositide turnover and calcium mobilization in human neuroblastoma SK-N-SH cells

The effects of caffeine on receptor-controlled Ca2+ mobilization and turnover of inositol phosphates in human neuroblastoma SK-N-SH cells were studied. Caffeine inhibited both the rise in cytosolic Ca2+ concentration ([Ca2+]i) evoked by muscarinic receptor agonists and the total production of inositol phosphates in a dose-dependent manner, but to different extents. At 10 mM, caffeine inhibited agonist-evoked generation of inositol phosphates almost completely, whereas the agonist-evoked [Ca2+]i rise remained observable after caffeine treatment, in the absence or presence of extracellular Ca2+. Raising the cytosolic cAMP concentration increased the carbachol-induced [Ca2+]i rise, and this effect was abolished in the presence of caffeine. Our data suggested that caffeine may exert two effects on receptor-controlled Ca2+ mobilization: 1) inhibition of inositol phosphate production, 2) augmentation of the size of the releasable Ca2+ pool by elevating cytosolic cAMP concentration.     

Gonadotropin-releasing hormone activates a rapid Ca2+-independent phosphodiester hydrolysis of polyphosphoinositides in pituitary gonadotrophs

Addition of gonadotropin releasing hormone (GnRH) to pituitary cells prelabeled with [32P]Pi or with myo-[2-3H]inositol, resulted in a rapid decrease in the level of [32P]phosphatidylinositol 4,5-bisphosphate (approximately 10 s), and in [32P]phosphatidylinositol 4-phosphate (approximately 1 min), followed by increased labeling of [32P]phosphatidylinositol and [32P]phosphatidic acid (1 min). GnRH stimulated the appearance of [3H]myo-inositol 1,4,5-trisphosphate (10 s), [3H]myo-inositol 1,4-bisphosphate (15 s), and [3H]myo-inositol 1-phosphate (1 min) in the presence of Li+ (10 mM). Li+ alone stimulated the accumulation of [3H]myo-inositol 1-phosphate and [3H]myo-inositol 1,4-bisphosphate but not [3H]myo-inositol 1,4,5-trisphosphate, but had no effect on luteinizing hormone release. The effect of GnRH on inositol phosphates (Ins-P) production was dose-related (ED50 = 1-5 nM), and was blocked by a potent antagonist [D-pGlu,pClPhe,D-Trp]GnRH. Elevation of cytosolic free Ca2+ levels ([Ca2+]i), by ionomycin and A23187 from intracellular or extracellular Ca2+ pools, respectively, had no significant effect on [3H]Ins-P production. GnRH-induced [3H]Ins-P production was not dependent on extracellular Ca2+ and was noticed also after extracellular or intracellular Ca2+ mobilization by A23187 or ionomycin, respectively. The effect of GnRH on [3H]Ins-P accumulation was not affected by prior treatment of the cells with the tumor promoter phorbol ester 12-O-tetradecanoylphorbol-13-acetate or with islet-activating protein pertussis toxin. These results indicate that GnRH stimulates a rapid phosphodiester hydrolysis of polyphosphoinositides. The stimulatory effect is not mediated via an islet-activating protein-substrate, is not dependent on elevation of [Ca2+]i, neither is it negatively regulated by 12-O-tetradecanoylphorbol-13-acetate which activates Ca2+/phospholipid-dependent protein C kinase. The results are consistent with the hypothesis that GnRH-induced phosphoinositide turnover is responsible for Ca2+ mobilization followed by gonadotropin release.     

Inhibition of prostaglandin E2-induced phosphoinositide metabolism by phorbol ester in bovine adrenal chromaffin cells.

In bovine adrenal chromaffin cells, prostaglandin E2 (PGE2) stimulates the formation of inositol phosphates and Ca2+ mobilization through its specific receptor [Yokohama, Tanaka, Ito, Negishi, Hayashi & Hayaishi (1988) J. Biol. Chem. 263, 1119-1122]. Here we show that PGE2-induced phosphoinositide metabolism was blocked by pretreatment with 12-O-tetradecanoylphorbol 13-acetate (TPA). Using intact cells, we also examined the inhibitory effect of TPA on the individual steps of the activation process of phosphoinositide metabolism. The inhibition was observed within 1 min and complete by 10 min after addition of 1 microM-TPA, and half-maximal inhibition by TPA occurred at 20 nM. TPA prevented Ca2+ mobilization induced by PGE2, but not by the Ca2+ ionophore ionomycin. The inactive phorbol ester 4 alpha-phorbol 12,13-didecanoate did not inhibit the formation of inositol phosphates and Ca2+ mobilization by PGE2. TPA treatment affected neither the high-affinity binding of [3H]PGE2 to intact cells and membrane fractions nor the ability of guanosine 5'-[gamma-thio]triphosphate to decrease the binding in membrane fractions. TPA also abolished phosphoinositide metabolism induced by muscarinic-receptor activation. NaF plus AlCl3 and ionomycin caused the accumulation of inositol phosphates, probably by directly activating a GTP-binding protein(s) and phospholipase C respectively; neither accumulation was inhibited by TPA treatment. These results suggest that protein kinase C serves as a feedback regulator for PGE2-induced phosphoinositide metabolism. The site of action of TPA appears to be distal to the coupling of the receptor to GTP-binding protein, but on a component(s) specific to the agonist-induced phosphoinositide metabolism.     

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.     

Glucagon and vasopressin interactions on Ca2+ movements in isolated hepatocytes.

The effects of glucagon and vasopressin, singly or together, on cytosolic free Ca2+ concentration [( Ca2+]i) and on the 45Ca2+ efflux were studied in isolated rat liver cells. In the presence of 1 mM external Ca2+, glucagon and vasopressin added singly induced sustained increases in [Ca2+]i. The rate of the initial fast phase of the [Ca2+]i increase and the magnitude of the final plateau were dependent on the concentrations (50 pm-0.1 microM) of glucagon and vasopressin. Preincubating the cells with a low concentration of glucagon (0.1 nM) for 2 min markedly accelerated the fast phase and elevated the plateau of the [Ca2+]i increase caused by vasopressin. In the absence of external free Ca2+, glucagon and vasopressin transiently increased [Ca2+]i and stimulated the 45Ca2+ efflux from the cells, indicating mobilization of Ca2+ from internal store(s). Preincubating the cells with 0.1 nM-glucagon accelerated the rate of the fast phase of the [Ca2+]i rise caused by the subsequent addition of vasopressin. However, unlike what was observed in the presence of 1 mM-Ca2+, glucagon no longer enhanced the maximal [Ca2+]i response to vasopressin. In the absence of external free Ca2+, higher concentrations (1 nM-0.1 microM) of glucagon, which initiated larger increases in [Ca2+]i, drastically decreased the subsequent Ca2+ response to vasopressin (10 nM). At these concentrations, glucagon also decreased the vasopressin-stimulated 45Ca2+ efflux from the cells. It is suggested that, in the liver, glucagon accelerates the fast phase and elevates the plateau of the vasopressin-mediated [Ca2+]i increase respectively by releasing Ca2+ from the same internal store as that permeabilized by vasopressin, probably the endoplasmic reticulum, and potentiating the influx of extracellular Ca2+ caused by this hormone.     

Effect of thapsigargin on cytosolic Ca2+ level and amylase release in rat parotid acinar cells.

At concentrations greater than 0.01 microM, thapsigargin (ThG) dose-dependently caused an increase in cytosolic free Ca2+ concentration ([Ca2+]i) in rat parotid acinar cells, as measured by the fluorescent Ca(2+)-indicator fura-2. In the absence of extracellular Ca2+, a transient increase in [Ca2+]i by ThG was observed, and subsequent addition of carbachol (CCh) did not produce a further [Ca2+]i response, suggesting that ThG released Ca2+ from the CCh-sensitive intracellular Ca2+ pool. Since ThG did not stimulate formation of inositol phosphates, the ThG-induced Ca2+ mobilization is independent of phosphoinositide breakdown. High concentrations (greater than 0.1 microM) of ThG induced amylase release from rat parotide acini, but the effect was very poor as compared with that of CCh or the protein kinase C activator, PMA (phorbol 12-myristate 13-acetate). Combined addition of ThG and PMA modestly potentiated amylase release induced by PMA alone. These results support the view that amylase release by muscarinic stimulation is mediated mainly by activation of protein kinase C rather than a rise in [Ca2+]i, although Ca2+ may modulate the secretory response.     

Changes in free cytosolic calcium and accumulation of inositol phosphates in isolated hepatocytes by [Leu]enkephalin.

Isolated hepatocytes from fed rats were used to study the effects of the opioid peptide [Leu]enkephalin on intracellular free cytosolic Ca2+ ([Ca2+]i) and inositol phosphate production. By measuring the fluorescence of the intracellular Ca2+-selective indicator quin-2, [Leu]enkephalin was found to increase [Ca2+]i rapidly from a resting value of 0.219 microM to 0.55 microM. The magnitude of this response was comparable with that produced by maximally stimulating concentrations of either vasopressin (100 nM) or phenylephrine (10 microM). The opioid-peptide-mediated increase in [Ca2+]i showed a dose-dependency comparable with the activation of phosphorylase, but it preceded the increase in phosphorylase alpha activity. Addition of [Leu]enkephalin to hepatocytes prelabelled with myo-[2-3H(n)]inositol resulted in a significant stimulation of inositol phosphate production. At 10 min after hormone addition, there were increases in the concentrations of inositol mono-, bis- and tris-phosphate fractions of 12-, 9- and 14-fold respectively. No effect was apparent on the glycerophosphoinositol fraction. The effect of 10 microM-[Leu]enkephalin on inositol phosphate production was significantly greater than that obtained with 10 microM-phenylephrine, but marginally smaller than that induced by 100 nM-vasopressin. However, at these concentrations all three agonists gave a comparable increase in [Ca2+]i and activation of phosphorylase a. These data provide evidence for [Leu]enkephalin acting via a mechanism involving a mobilization of Ca2+ as a result of increased phosphatidylinositol turnover.     

Ca2(+)-mobilizing hormones stimulate Ca2+ efflux from hepatocytes

Treatment of hepatocytes with 2,5-di-(tert-butyl)-1,4-benzohydroquinone (tBuBHQ), a novel mobilizer of the inositol 1,4,5-trisphosphate-sensitive Ca2+ pool, produces a sustained elevation of [Ca2+]i (Kass, G. E. N., Duddy, S. K., and Orrenius, S. (1989) J. Biol. Chem. 264, 15192-15198). Exposure of hepatocytes to the Ca2(+)-mobilizing hormones, vasopressin, angiotensin II, or ATP following [Ca2+]i elevation by tBuBHQ produced a rapid return of [Ca2+]i to basal or near basal levels. Release of the inositol 1,4,5-trisphosphate-sensitive Ca2+ pool by tBuBHQ following pretreatment with vasopressin or angiotensin II resulted in a [Ca2+]i transient and not the sustained [Ca2+]i elevation observed in the absence of the Ca2(+)-mobilizing hormones. The G-protein activator, NaF plus AlCl3, mimicked both effects of the Ca2(+)-mobilizing hormones on [Ca2+]i. The mechanism for Ca2+ removal from the cytosol by Ca2(+)-mobilizing hormones did not involve cyclic nucleotides nor did it require protein kinase C activation or cyclo- and lipoxygenase-dependent metabolites of arachidonic acid. Furthermore, the hormone-mediated decrease in [Ca2+]i did not involve the pertussis toxin-sensitive Gi-protein. Removal of the tBuBHQ-mobilized Ca2+ from the cytosol of hepatocytes by Ca2(+)-mobilizing hormones was mediated by stimulation of a Ca2+ efflux pathway. Thus, in addition to initiating [Ca2+]i transients by releasing Ca2+ from the inositol 1,4,5-trisphosphate-sensitive Ca2+ store and stimulating Ca2+ influx, Ca2(+)-mobilizing hormones also regulate the termination of the [Ca2+]i transient by stimulating a Ca2+ efflux pathway.     

Inositol tetrakisphosphate isomers and elevation of cytosolic Ca2+ in vasopressin-stimulated insulin-secreting RINm5F cells.

Signal generation during the stimulation of insulin secretion by arginine vasopressin (AVP) was investigated in RINm5F cells. AVP (0.1 microM) caused a biphasic cytosolic Ca2+ ([Ca2+]i) rise, namely a rapid transient marked elevation after stimulation followed by a series of oscillations. In the absence of extracellular Ca2+, the sustained oscillations were abolished, while the initial [Ca2+]i transient was only partly decreased, indicating that the former are due to Ca2+ influx and the latter due mainly to mobilization from internal Ca2+ stores. AVP also evoked a transient depolarization of the average membrane potential. AVP-induced Ca2+ influx during the sustained phase, which was strictly dependent on receptor occupancy, was attenuated by membrane hyperpolarization with diazoxide. However, blockade of Ca2+ channels of the L- or T-type was ineffective. AVP stimulated production of diacylglycerol and inositol phosphates; for the latter both [3H] inositol labeling and mass determinations were performed. A transient increase in Ins(1,4,5)P3 was followed by a marked enhancement of Ins(1,3,4,5)P4 (8-fold) peaking at 15 s and gradually returning to basal values. Ins(1,3,4,6)P4 and Ins(3,4,5,6)P4 exhibited the most long-lasting augmentation (4- and 1.7-fold, respectively), and therefore correlated best with the period of sustained [Ca2+]i oscillations. InsP5 and InsP6 were not elevated. The effects of AVP, including the stimulation of insulin secretion from perifused cells, were obliterated by a V1 receptor antagonist. In conclusion, AVP induces protracted [Ca2+]i elevation in RINm5F cells which is associated with long-lasting increases in InsP4 isomers. The accumulation of InsP4 isomers reflects receptor occupancy and accelerated metabolism of the inositol phosphates. Activation of second messenger-operated Ca2+ channels is not necessarily implicated because of the attenuating effect of membrane hyperpolarization.