Calcium modulates the generation of inositol 1,3,4-trisphosphate in human platelets by the activation of inositol 1,4,5-trisphosphate 3-kinase.

Histamine (0.5 mM) stimulated the cyclic AMP content of cell suspensions containing greater than 80% parietal cells. Epidermal growth factor (EGF) inhibited this stimulatory effect of histamine, but had no effect on basal cyclic AMP content. The half-maximally effective concentration of EGF for inhibition of histamine-stimulated cyclic AMP was 3.9 nM. The equivalent measurement for the inhibition of histamine-stimulated aminopyrine accumulation was 3.0 nM. Aminopyrine accumulation was measured because it provides an index of the secretory activity of the cell. The cyclic AMP phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX) prevented the inhibitory effect of EGF on cyclic AMP content. This effect of IBMX was not caused by its ability to raise cellular cyclic AMP content in the presence of histamine. Prevention by IBMX of the inhibitory action of EGF on histamine-stimulated aminopyrine accumulation had been shown previously [Shaw, Hatt, Anderson & Hanson (1987) Biochem. J. 244, 699-704]. EGF stimulated prostaglandin E2 (PGE2) production in the cell fraction containing greater than 80% parietal cells, with the half-maximally effective concentration being 7.5 nM. EGF was ineffective in stimulating PGE2 production if the cell fraction was depleted of parietal cells (12%), or if 0.5 mM-histamine was added to the enriched parietal-cell fraction. In conclusion, EGF may inhibit histamine-stimulated acid secretion by decreasing the cyclic AMP content of parietal cells. This effect could be mediated by an increase in cyclic AMP phosphodiesterase activity, but it is unlikely to involve an effect of EGF on parietal-cell prostaglandin production.     

Pertussis toxin reverses prostaglandin E2- and somatostatin-induced inhibition of rat parietal cell H(+)-production.

In enzymatically dispersed enriched rat parietal cells we studied the effect of pertussis toxin on prostaglandin E2 (PGE2)- or somatostatin-induced inhibition of H(+)-production. Parietal cells were incubated in parallel in the absence (control cells) and presence of pertussis toxin (250 ng/ml; 4 h). [14C]Aminopyrine accumulation by both pertussis toxin-treated and control cells was used as an indirect measure of H(+)-production after stimulation with either histamine, forskolin or dibutyryl adenosine 3',5'-cyclic monophosphate (dbcAMP) alone and in the presence of PGE2 (10(-9)-10(-7) M) or somatostatin (10(-9)-10(-6) M). PGE2 inhibited histamine- and forskolin-stimulated [14C]aminopyrine accumulation but failed to alter the response to dbcAMP. Somatostatin was less effective and less potent than PGE2 in inhibiting stimulation by histamine or forskolin and reduced the response to dbcAMP. Pertussis toxin completely reversed inhibition by both PGE2 and somatostatin on histamine- and forskolin-stimulated H(+)-production but failed to affect inhibition by somatostatin of the response to dbcAMP. After incubation of crude control cell membranes with [32P]NAD+, pertussis toxin catalysed the incorporation of [32P]adenosine diphosphate (ADP)-ribose into a membrane protein of molecular weight of 41,000, the known molecular weight of the inhibitory subunit of adenylate cyclase (Gi alpha). Pertussis toxin treatment of parietal cells prior to the preparation of crude membranes almost completely prevented subsequent pertussis toxin-catalysed [32P]ADP ribosylation of the 41,000 molecular weight protein.(ABSTRACT TRUNCATED AT 250 WORDS)     

Multiple effects of phorbol ester on secretory activity in rabbit gastric glands and parietal cells

Acid secretory activity and respiration in rabbit gastric glands are stimulated by cAMP-dependent and -independent agonists. Potentiation between agonists suggests interaction of the activation pathways. Regulation of secretory response by protein kinase C was investigated with 12-0-tetradecanoyl phorbol-13-acetate (TPA). TPA elevated basal respiration, pepsin release, and acid secretion but inhibited histamine and carbachol stimulation of acid secretion by gastric glands, as measured by [dimethylamino-14C]aminopyrine accumulation. The inhibition of histamine response was specific for protein kinase C activators, occurred after a 20-min lag, and was not reversed by removal of TPA after 3 min of preincubation. TPA pretreatment inhibited acid secretory responses to cholera toxin and forskolin but enhanced the response to cAMP analogues. Cholera toxin and pertussis toxin simulated ADP-ribosylation of 45 and 41 kDa proteins, respectively, in parietal cell membranes. Therefore, both stimulatory (Gs) and inhibitory (Gi) GTP binding proteins of adenylyl cyclase appear to be present in parietal cells. Pretreatment with pertussis toxin attenuated PGE2 but not TPA inhibition of histamine stimulation of aminopyrine accumulation. Thus, the inhibitory effect of TPA does not appear to be associated with an action on Gi. The results with histamine and carbachol suggest that protein kinase C may regulate both cAMP-dependent and -independent stimulation of parietal cell acid secretion.     

Mechanisms for direct inhibition of canine gastric parietal cells by somatostatin

To examine the potential mechanisms by which somatostatin inhibits gastric acid secretion we studied its effects on isolated canine gastric parietal cells. Using 125I-[Leu8-D-Trp22-Tyr25]somatostatin-28 as ligand, we identified somatostatin-binding sites in parietal cell-enriched fractions of fundic mucosa. Two binding sites with respective dissociation constants of 3.2 X 10(-9) and 2.1 X 10(-7) M were identified. Somatostatin-14 and -28 were equally potent both in displacing bound ligand and in inhibiting parietal cell activity as measured by [14C]aminopyrine uptake. Pertussis toxin reversed the ability of somatostatin to inhibit the uptake of [14C]aminopyrine and production of cAMP by parietal cells stimulated with histamine and forskolin but not with dibutyryl cAMP or pentagastrin. Furthermore, somatostatin had no effect on parietal cell membrane inositol phospholipid turnover or changes in protein kinase C (Ca2+/phospholipid-dependent enzyme) activity induced by carbachol or pentagastrin. These data indicate that somatostatin directly inhibits parietal cell activity via mechanisms both dependent on and independent of the pertussis toxin-sensitive inhibitory guanine nucleotide-binding protein.     

Inhibition of Ca(2+)-induced calcitonin secretion by somatostatin: roles of voltage dependent Ca2+ channels and G-proteins.

Somatostatin has recently been applied therapeutically for hypercalcitonemia in patients with calcitonin-producing tumours. Using calcitonin-secreting cells (C-cells) of the medullary thyroid carcinoma cell line rMTC 44-2, we investigated the inhibitory action of somatostatin on calcitonin release, cytosolic Ca2+ and Ca2+ channel currents. The Ca(2+)-induced rises of the cytosolic Ca2+ and calcitonin secretion were greatly inhibited by somatostatin or its stable analogue octreotide. The effects of somatostatin were pertussis toxin-sensitive. Under voltage clamp conditions, C-cells exhibited slowly inactivating Ca2+ channel currents. Bath application of 100 nM somatostatin reversibly reduced the Ca2+ channel current by about 30%. The Ca2+ channel current and its inhibition by somatostatin were not affected by intracellularly applied cyclic AMP. Moreover, pretreating the cells with pertussis toxin had no effect on the control Ca2+ channel currents but greatly abolished its inhibition by somatostatin. The data show that somatostatin suppresses the Ca(2+)-stimulated calcitonin secretion by inhibiting voltage-dependent Ca2+ channel currents and by lowering cytosolic Ca2+. These actions of somatostatin involve pertussis toxin-sensitive G-proteins and occur independently of changes in the cyclic AMP concentration.     

Cyclic AMP-independent, dual regulation of voltage-dependent Ca2+ currents by LHRH and somatostatin in a pituitary cell line.

Voltage-dependent Ca2+ currents appear to be involved in the actions of hormones that regulate pituitary secretion. In order to investigate modulation of Ca2+ currents by release-inducing and release-inhibiting hormones, we performed whole-cell clamp experiments in the pituitary cell line GH3. The resting potential was approximately -40 mV; spontaneous action potentials were observed in the majority of cells. Superfusion of cells with the stimulatory hormone, LHRH, depolarized the plasma membrane to approximately -10 mV, whereas the inhibitory hormone, somatostatin, caused hyperpolarization to approximately -60 mV; both hormones suppressed spontaneous action potentials. Under voltage clamp conditions, GH3 cells exhibited slowly and fast inactivating Ca2+ currents. LHRH increased whereas somatostatin decreased the slowly inactivating currents; fast inactivating currents were not affected by these hormones. The stimulatory effect of LHRH was not mimicked by intracellularly applied cAMP. In contrast to vasoactive intestinal peptide and forskolin, LHRH did not activate adenylate cyclase in membranes of GH3 cells, but rather appeared to cause inhibition of the enzyme. Hormonal stimulation and inhibition of inward currents were abolished by pretreatment of the cells with pertussis toxin. In membranes of GH3 cells, we identified a pertussis toxin-sensitive G-protein of the Gi-type and Go. We conclude that LHRH and somatostatin modulate voltage-dependent Ca2+ currents via cAMP-independent mechanisms involving pertussis toxin-sensitive G-proteins. The occurrence of both pertussis toxin-sensitive hormonal stimulation and inhibition of voltage-dependent Ca2+ currents in one cell type suggest that these opposite regulations are mediated by distinct G-proteins.     

Pancreastatin: a novel peptide inhibitor of parietal cell signal transduction

The direct inhibition of secretion by pancreastatin was investigated in rabbit isolated parietal cells. Pancreastatin exerted no influence on basal aminopyrine uptake. Pancreastatin inhibited histamine stimulated aminopyrine uptake through a decrease in intracellular cAMP. Pancreastatin inhibition of histamine stimulated uptake was blocked in the presence of pertussis toxin. Pancreastatin also inhibited the carbachol stimulated increase in aminopyrine accumulation. However, the effects of pancreastatin on carbachol stimulation were not reversed by pertussis toxin. Pancreastatin did not alter the carbachol induced increase in cytosolic free calcium. Thus, pancreastatin appears to inhibit parietal cell signal transduction at multiple points along the second messenger pathways.     

Secretion-stimulating and secretion-inhibiting hormones stimulate high-affinity pertussis-toxin-sensitive GTPases in membranes of a pituitary cell line

Different peptide hormones influence hormone secretion in pituitary cells by diverse second messenger systems. Recent data indicate that luteinizing-hormone-releasing hormone (LHRH) stimulates and somatostatin inhibits voltage-dependent Ca2+ channels of GH3 cells via pertussis-toxin-sensitive mechanisms [Rosenthal et al. (1988) EMBO J. 7, 1627-1633]. In other pituitary cell lines, somatostatin has been shown to cause a pertussis-toxin-sensitive decrease in adenylate cyclase activity, and LHRH and thyrotropin-releasing hormone (TRH) stimulate phosphoinositol lipid hydrolysis in a pertussis-toxin-independent manner. Whether stimulation of Ca2+ influx by TRH is affected by pertussis toxin is not known. In order to elucidate which of the hormone receptors interact with pertussis-toxin-sensitive and -insensitive G-proteins, we measured the effects of LHRH, somatostatin and TRH on high-affinity GTPases in membranes of GH3 cells. In control membranes, both LHRH and TRH stimulated the high-affinity GTPase by 20%, somatostatin by 25%. Maximal hormone effects were observed at a concentration of about 1 microM. Pretreatment of cells with pertussis toxin abolished pertussis-toxin-catalyzed [32P]ADP-ribosylation of 39-40-kDa proteins in subsequently prepared membranes and reduced basal GTPase activity. The toxin also reduced by more than half the increases in GTPase activity induced by LHRH and TRH; stimulation of GTPase by somatostatin was completely suppressed. Stimulation of adenylate cyclase by vasoactive intestinal peptide (VIP) was not impaired by pretreatment of cells with pertussis toxin. Somatostatin but not LHRH and TRH decreased forskolin-stimulated adenylate cyclase activity. The results suggest that the activated receptors for LHRH and TRH act via pertussis-toxin-sensitive and -insensitive G-proteins, whereas effects of somatostatin are exclusively mediated by pertussis-toxin-sensitive G-proteins.     

Prostaglandin E receptors in bovine adrenal medulla are coupled to adenylate cyclase via Gi and to phosphoinositide metabolism in a pertussis toxin-insensitive manner

Prostaglandin E (PGE) receptor is coupled to a pertussis toxin-insensitive GTP-binding protein in bovine adrenal medulla, but PGE receptor partially purified from bovine adrenal medulla was functionally reconstituted with Gi into phospholipid vesicles (Negishi, M., Ito, S., Yokohama, H., Hayashi, H., Katada, T., Ui, M., and Hayaishi, O. (1988) J. Biol. Chem. 263, 6893-6900). We demonstrate here that PGE2 inhibited forskolin-induced accumulation of cAMP in cultured bovine chromaffin cells. In plasma membranes prepared from bovine adrenal medulla, PGE2 inhibited forskolin-stimulated adenylate cyclase activity in a GTP-dependent manner. This inhibitory action of PGE2 was abolished by treatment of the membrane with pertussis toxin. Reconstitution of the membranes ADP-ribosylated by pertussis toxin with Gi purified from bovine brain restored the potency of PGE2 to inhibit the adenylate cyclase activity. Inhibition of forskolin-induced cAMP accumulation by PGE2 was also abolished by exposure to the toxin in the cells, indicating that PGE receptors are coupled to Gi. In contrast, PGE2 stimulated the formation of inositol phosphates in chromaffin cells, but this effect was not affected by treatment of the cells with pertussis toxin, suggesting that the PGE receptors are coupled to phosphoinositide metabolism via a pertussis toxin-insensitive G-protein. Both the inhibitory action of cAMP accumulation and stimulation of phosphoinositide metabolism were specific for PGE1 and PGE2, and the Scatchard plot analysis of PGE2 binding to the membrane showed a single high-affinity binding site (Kd = 2 nM). In bovine adrenal chromaffin cells PGE2 enhanced catecholamine release in the presence of ouabain by stimulation of phosphoinositide metabolism (Yokohama, H., Tanaka, T., Ito, S., Negishi, M., Hayashi, H., and Hayaishi, O. (1988) J. Biol. Chem. 263, 1119-1122). We further examined the modulation of catecholamine release by PGE2 through its inhibitory coupling to the adenylate cyclase system. Prior exposure of chromaffin cells to forskolin or dibutyryl-cAMP reduced nicotine-stimulated catecholamine release, and PGE2 attenuated forskolin-induced inhibition of catecholamine release stimulated by nicotine, but not dibutyryl-cAMP-induced inhibition. In the absence of evidence that PGE receptor subtypes exist, these results suggest that the PGE receptor is coupled to two signal transduction systems leading to inhibition of cAMP accumulation via Gi and to production of inositol phosphates via a pertussis toxin-insensitive G-protein, both of which may modulate catecholamine release from bovine chromaffin cells.     

Pertussis toxin blocks somatostatin's inhibition of stimulated cyclic AMP accumulation in anterior pituitary tumor cells

Somatostatin inhibits both forskolin and (-) isoproterenol-stimulated cyclic AMP accumulation in AtT-20 cells. Pretreatment of these cells with pertussis toxin prevents somatostatin's inhibitory effects on cyclic AMP production. This pretreatment also enhances the cyclic AMP response to forskolin and (-) isoproterenol without affecting basal cyclic AMP levels. The blockade of somatostatin's inhibitory effect was dependent both on the time of preincubation and concentration of pertussis toxin used. The rise in forskolin-stimulated cyclic AMP formation following pertussis toxin treatment preceded the blockade of somatostatin's inhibitory actions. The results suggest that somatostatin acts through an inhibitory guanine nucleotide regulatory protein to affect adenylate cyclase activity.     

Somatostatin, misoprostol and galanin inhibit gastrin- and PACAP-stimulated secretion of histamine and pancreastatin from ECL cells by blocking specific Ca2+ channels

The oxyntic mucosa is rich in ECL cells. They secrete histamine and chromogranin A-derived peptides, such as pancreastatin, in response to gastrin and pituitary adenylate cyclase-activating peptide (PACAP). Secretion is initiated by Ca2+ entry. While gastrin stimulates secretion by opening L-type and N-type Ca2+ channels, PACAP stimulates secretion by activating L-type and receptor-operated Ca2+ channels. Somatostatin, galanin and prostaglandin E2 (PGE2) inhibit gastrin- and PACAP-stimulated secretion from the ECL cells. In the present study, somatostatin and the PGE2 congener misoprostol inhibited gastrin- and PACAP-stimulated secretion 100%, while galanin inhibited at most 60-65%. Bay K 8644, a specific activator of L-type Ca2+ channels, stimulated ECL-cell secretion, an effect that was inhibited equally effectively by somatostatin, misoprostol and galanin (75-80% inhibition). Pretreatment with pertussis toxin, that inactivates inhibitory G-proteins, prevented all three agents from inhibiting stimulated secretion (regardless of the stimulus). Pretreatment with nifedipine (10 microM), an L-type Ca2+ channel blocker, reduced PACAP-evoked pancreastatin secretion by 50-60%, gastrin-evoked secretion by approximately 80% and abolished the response to Bay K 8644. The nifedipine-resistant response to PACAP was abolished by somatostatin and misoprostol but not by galanin. Gastrin and PACAP raised the intracellular Ca2+ concentration in a biphasic manner, believed to reflect mobilization of internal Ca2+ followed by Ca2+ entry. Somatostatin and misoprostol blocked Ca2+ entry (and histamine and pancreastatin secretion) but not mobilization of internal Ca2+. The present observations on isolated ECL cells suggest that Ca2+ entry rather than mobilization of internal Ca2+ triggers exocytosis, that gastrin and PACAP activate different (but over-lapping) Ca2+ channels, that somatostatin, misoprostol and galanin interact with inhibitory G-proteins to block Ca2+ entry via L-type Ca2+ channels, and that somatostatin and misoprostol (but not galanin) in addition block N-type and/or receptor-operated Ca2+ channels.     

The epidermal growth factor receptor is coupled to a phospholipase A2-specific pertussis toxin-inhibitable guanine nucleotide-binding regulatory protein in cultured rat inner medullary collecting tubule cells

Studies were performed to examine a potential role for a guanine nucleotide-binding protein in epidermal growth factor (EGF)-stimulated phospholipase A2 (PLA2) activity. EGF increased prostaglandin E2 (PGE2) production in intact or saponin-permeabilized rat inner medullary collecting tubule (RIMCT) cells. Incubation of permeabilized cells with guanosine 5'-O-(thiotriphosphate) (GTP gamma S) enhanced and with guanosine 5'-O-(2-thiodiphosphate) (GDP beta S) inhibited the response to EGF. GDP beta S had no effect on ionomycin-stimulated PGE2 production. Exposure of intact cells to 25 mM NaF + 10 microM AlCl3 enhanced both basal and EGF-stimulated PGE2 production. Pertussis toxin ADP-ribosylated a 41-kDa protein in RIMCT cell membranes. Pretreatment of cells with pertussis toxin (100 ng/ml for 16 h) eliminated the response to EGF in intact cells and the response to EGF + GTP gamma S in permeabilized cells. Pertussis toxin had no effect on the response to ionomycin. The effect of pertussis toxin was not due to alterations in cAMP as cellular cAMP levels were unaffected by pertussis toxin both in the basal state and in the presence of EGF. PGE2 production in response to EGF was not transduced by a G protein coupled to phospholipase C (PLC) as neomycin, which inhibited PLC, did not decrease EGF-stimulated PGE2 production. Also, PGE2 production was not increased by inositol trisphosphate and did not require the presence of extracellular Ca2+. In contrast to EGF-stimulated PLC activity, stimulation of PLA2 by EGF was not susceptible to inhibition by phorbol 12-myristate 13-acetate. These results clearly demonstrate the existence of a PLA2-specific pertussis toxin-inhibitable guanine nucleotide-binding protein coupled to the EGF receptor in RIMCT cells.     

Pertussis toxin blocks the inhibitory effects of somatostatin on cAMP-dependent vasoactive intestinal peptide and cAMP-independent thyrotropin releasing hormone-stimulated prolactin secretion of GH3 cells

It was shown that somatostatin (SRIF) inhibited cAMP-dependent vasoactive intestinal peptide (VIP)-stimulated prolactin (PRL) release by a GH3 clonal strain of rat pituitary tumor cells and decreased basal PRL secretion and inhibited PRL release in response to thyrotropin releasing hormone (TRH) whose action was independent of prior synthesis of cAMP. Pretreatment of these cells with pertussis toxin prevented SRIF's inhibitory effects on basal and TRH-stimulated hormone secretion as well as its VIP-stimulated responses. The blockade of SRIF's inhibitory effect on the actions of TRH or VIP was dependent on both the duration of preincubation and concentration of the toxin and was correlated with the ability of the toxin to catalyze the ADP-ribosylation of the 39,000-Da membrane protein. It is likely that this pertussis toxin substrate is involved in signal transduction of SRIF on cAMP-dependent actions of VIP and cAMP-independent action of TRH. However, the mechanism of SRIF's action on TRH is not clear, since SRIF did not affect the intracellular responses by TRH, neither intracellular Ca2+ mobilization nor the increase of 1,2-diacylglycerol formation following the breakdown of polyphosphoinositides.     

Pertussis toxin blocks an inhibition of hormone-stimulated glycogenolysis by prostaglandin E2 and its analogue in cultured hepatocytes

Prostaglandin E2 (PGE2) and 16,16-dimethyl PGE2 were found to inhibit a hepatic glycogenolysis stimulated by epinephrine in the presence of propranolol (alpha 1-adrenergic response), isoproterenol (beta-adrenergic response) and glucagon in primary cultures of rat hepatocytes. The inhibitory effects to these stimulations were maximally increased (60-100%) in the cultures on day 2 or 3. Pretreatment of the cultured hepatocytes with pertussis toxin (islet-activating protein) resulted in a complete blockage of the prostaglandin-induced inhibition of glycogenolysis in a dose-dependent manner. Pertussis toxin had no significant effect on the glycogenolysis stimulated by these compounds in the absence of prostaglandin. The data suggest that the hepatic glycogenolysis stimulated by alpha 1- and beta-adrenergic responses and glucagon are modulated by the E series of prostaglandins via pertussis toxin-sensitive guanine nucleotide regulatory protein.     

Functional reconstitution of prostaglandin E receptor from bovine adrenal medulla with guanine nucleotide binding proteins

Prostaglandin E2 (PGE2) was found to bind specifically to a 100,000 x g pellet prepared from bovine adrenal medulla. The PGE receptor was associated with a GTP-binding protein (G-protein) and could be covalently cross-linked with this G-protein by dithiobis(succinimidyl propionate) in the 100,000 x g pellet (Negishi, M., Ito, S., Tanaka, T., Yokohama, H., Hayashi, H., Katada, T., Ui, M., and Hayaishi, O. (1987) J. Biol. Chem. 262, 12077-12084). In order to characterize the G-protein associated with the PGE receptor and reconstitute these proteins in phospholipid vesicles, we purified the G-protein to apparent homogeneity from the 100,000 x g pellet. The G-protein served as a substrate of pertussis toxin but differed in its alpha subunit from two known pertussis toxin substrate G-proteins (Gi and Go) purified from bovine brain. The molecular weight of the alpha subunit was 40,000, which is between those of Gi and Go. The purified protein was also distinguished immunologically from Gi and Go and was referred to as Gam. PGE receptor was solubilized by 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid and freed from G-proteins by wheat germ agglutinin column chromatography. Reconstitution of the PGE receptor with pure Gam, Gi, or Go in phospholipid vesicles resulted in a remarkable restoration of [3H]PGE2 binding activity in a GTP-dependent manner. The efficiency of these three G-proteins in this capacity was roughly equal. When pertussis toxin- or N-ethylmaleimide-treated G-proteins, instead of the native ones, were reconstituted into vesicles, the restoration of binding activity was no longer observed. The displacement of [3H]PGE2 binding was specific for PGE1 and PGE2. Furthermore, addition of PGE2 stimulated the GTPase activity of the G-proteins in reconstituted vesicles. These results indicate that the PGE receptor can couple functionally with Gam, Gi, or Go in phospholipid vesicles and suggest that Gam may be involved in signal transduction of the PGE receptor in bovine adrenal medulla.     

Functional role of bone morphogenetic protein-4 in isolated canine parietal cells

Bone morphogenetic protein (BMP)-4 is an important regulator of cellular growth and differentiation. Expression of BMP-4 has been documented in the gastric mucosa. We reported that incubation of canine parietal cells with EGF for 72 h induced both parietal cell morphological transformation and inhibition of H(+)/K(+)-ATPase gene expression through MAPK-dependent mechanisms. We explored the role of BMP-4 in parietal cell maturation and differentiation. Moreover, we investigated if BMP-4 modulates the actions of EGF in parietal cells. H(+)/K(+)-ATPase gene expression was examined by Northern blots and quantitative RT-PCR. Acid production was assessed by measuring the uptake of [(14)C]aminopyrine. Parietal cell apoptosis was quantitated by Western blots with anti-cleaved caspase 3 antibodies and by counting the numbers of fragmented, propidium iodide-stained nuclei. MAPK activation and Smad1 phosphorylation were measured by Western blots with anti-phospho-MAPK and anti-phospho-Smad1 antibodies. Parietal cell morphology was examined by immunohistochemical staining of cells with anti-H(+)/K(+)-ATPase alpha-subunit antibodies. BMP-4 stimulated Smad1 phosphorylation and induced H(+)/K(+)-ATPase gene expression. BMP-4 attenuated EGF-mediated inhibition of H(+)/K(+)-ATPase gene expression and blocked EGF induction of both parietal cell morphological transformation and MAPK activation. Incubation of cells with BMP-4 enhanced histamine-stimulated [(14)C]aminopyrine uptake. BMP-4 had no effect on parietal cell apoptosis, whereas TGF-beta stimulated caspase-3 activation and nuclear fragmentation. In conclusion, BMP-4 promotes the induction and maintenance of a differentiated parietal cell phenotype. These findings may provide new clues for a better understanding of the mechanisms that regulate gastric epithelial cell growth and differentiation.     

Protein acylation in the inhibition of insulin secretion by norepinephrine,somatostatin, galanin,and PGE2

The major physiological inhibitors of insulin secretion, norepinephrine, somatostatin, galanin, and prostaglandin E2, act via specific receptors that activate pertussis toxin (PTX)-sensitive G proteins. Four inhibitory mechanisms are known: 1) activation of ATP-sensitive K channels and repolarization of the beta-cell; 2) inhibition of L-type Ca2+ channels; 3) decreased activity of adenylyl cyclase; and 4) inhibition of exocytosis at a "distal" site in stimulus-secretion coupling. We have examined the underlying mechanisms of inhibition at this distal site. In rat pancreatic islets, 2-bromopalmitate, cerulenin, and polyunsaturated fatty acids, all of which suppress protein acyltransferase activity, blocked the distal inhibitory effects of norepinephrine in a concentration-dependent manner. In contrast, control compounds such as palmitate, 16-hydroxypalmitate, and etomoxir, which do not block protein acylation, had no effect. Furthermore, 2-bromopalmitate also blocked the distal inhibitory actions of somatostatin, galanin, and prostaglandin E2. Importantly, neither 2-bromopalmitate nor cerulenin affected the action of norepinephrine to decrease cAMP production. We also examined the effects of norepinephrine, 2-bromopalmitate, and cerulenin on palmitate metabolism. Palmitate oxidation and its incorporation into lipids seemed not to contribute to the effects of 2-bromopalmitate and cerulenin on norepinephrine action. These data suggest that protein acylation mediates the distal inhibitory effect on insulin secretion. We propose that the inhibitors of insulin secretion, acting via PTX-sensitive G proteins, activate a specific protein acyltransferase, causing the acylation of a protein or proteins critical to exocytosis. This particular acylation and subsequent disruption of the essential and precise interactions involved in core complex formation would block exocytosis.     

Pertussis toxin abolishes the inhibitory effects of prostaglandins E1, E2, I2 and F2 alpha on hormone-induced cAMP accumulation in cultured hepatocytes

Several prostaglandins inhibit the cAMP response to glucagon and beta-adrenergic stimulation in hepatocytes. To probe the mechanism of this inhibition, we have examined in primary hepatocyte cultures how pretreatment with pertussis toxin (islet-activating protein) influences the ability of the cells to respond to hormones and prostaglandins. Pertussis toxin augmented the effects of glucagon, epinephrine and isoproterenol, and also markedly enhanced the cAMP response to prostaglandin E1 (PGE1). Furthermore, whereas PGE1, PGE2, PGI2 and PGF2 alpha attenuated the cAMP responses to glucagon in control cultures, this inhibition was abolished in cells pretreated with pertussis toxin. A more detailed comparison was made of the effects of PGE1 and PGF2 alpha. In cells not treated with pertussis toxin, both these prostaglandins at high concentrations reduced the cAMP response to glucagon and isoproterenol by approximately 50%, but dose-effect curves showed that PGE1 was about 100-fold more potent as an inhibitor than PGF2 alpha. Pertussis toxin abolished the inhibitory effects of PGE1 and PGF2 alpha with almost identical time and dose requirements. The results obtained with PGE1, PGE2, PGI2 and PGF2 alpha suggest that prostaglandins of different series attenuate hormone-activable adenylate cyclase in hepatocytes through a common mechanism, dependent on the inhibitory GTP-binding protein.     

Modulation of responsiveness to cAMP stimulating agonists by phorbol ester in fetal rat osteoblasts.

We studied the effect of activation of protein kinase C (PKC) by a phorbol ester on cAMP accumulation in fetal rat osteoblasts. Activation of PKC by phorbol 12-myristate 13-acetate (PMA) caused a potentiation of cAMP accumulation induced by parathyroid hormone (PTH), forskolin, and cholera toxin. The results suggest that the potentiating effect of PMA on PTH-induced cAMP accumulation was not due to an effect on the PTH-receptor nor to an effect on cAMP degradation, as the effect of PMA persisted in the presence of a phosphodiesterase inhibitor. Pretreatment of the cells with pertussis toxin did not prevent the action of PMA, indicating that PMA does not act via the inhibitory G-protein. PMA had a biphasic effect on prostaglandin E2 (PGE2)-induced cAMP accumulation; i.e., at concentrations greater than or equal to 10(-6) M, PMA potentiated the PGE2-induced cAMP response but PMA attenuated cAMP accumulation induced by concentrations of PGE2 less than or equal to 5.10(77) M. From our data we conclude that PKC can interact with a stimulated cAMP pathway in a stimulatory and inhibitory manner. Potentiation of cAMP accumulation is probably due to modification of the adenylate cyclase complex, whereas attenuation of stimulated cAMP accumulation appears to be due to an effect on a different site of the cAMP generating pathway, which may be specific to PGE2-induced cAMP accumulation.