Attenuation of inositol trisphosphate generation and cytosolic Ca2+ elevation in dispersed rat parotid acini stimulated simultaneously at muscarinic and alpha 1-adrenergic receptors

We have examined intracellular signalling events, peak cytosolic [Ca2+] and inositol trisphosphate levels, in rat parotid acini simultaneously stimulated with two Ca2+ mobilizing agonists, carbachol (muscarinic-cholinergic) and epinephrine (alpha 1-adrenergic). When the agonists were added together, either at sub-maximal (200 nM each, i.e. 400 nM total agonist concentration) or maximal (10 uM each, i.e. 20 uM total) stimulatory concentrations, the resulting elevations in both cytosolic [Ca2+] and inositol trisphosphate levels were not greater than those achieved when each agonist was added individually. However, with 400 nM carbachol these responses were significantly greater than those seen with either 200 nM carbachol or 200 nM carbachol + 200 nM epinephrine. The data indicate that when muscarinic and alpha 1-adrenergic receptors of rat parotid acini are simultaneously stimulated a novel regulatory mechanism is induced, which attenuates inositol trisphosphate generation and, consequently, intracellular Ca2+ release.     

Effect of secretin on protein phosphorylation in dispersed acini from rat pancreas

Dispersed acini from rat pancreas, incubated in the presence of KH2(32)PO4 to steady state 32P incorporation into cellular proteins, were exposed to secretin. 32P incorporated into selected proteins, separated by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis, reached a plateau by 150 min. Effect of secretin on amylase release, cellular cyclic AMP levels and protein phosphorylation was then examined. Stimulation of amylase release was apparent with 10(-10)M and was maximal with 10(-7)M by 10 min incubation. Almost maximal increase in cellular cyclic AMP levels and 32P incorporation into selected proteins was also observed with 10(-7)M secretin by 10 min in the presence of 10 mM theophyllin. Both secretin (10(-8)M) and dibutyryl cyclic AMP (10(-3)M) induced the phosphorylation of similar proteins analyzed by counting 32P content in each peptide band after SDS gel electrophoresis. Addition of cyclic AMP (10(-6)M) to homogenates of acini also augmented 32P incorporation from [gamma-32P]ATP into similar proteins. These results indicate that secretin enhances protein phosphorylation in pancreatic acinar cells and cyclic AMP may mediate the action of secretin on protein phosphorylation.     

Stimulatory effects of human pancreatic polypeptide on rat pancreatic acini

The effect of human pancreatic polypeptide (HPP) on rat pancreatic acini has been studied. It was found that HPP stimulated amylase and lipase release from the acini. The secretory response of acini to HPP was dose-dependent in a sigmoidal fashion. Between 10(-9) M and 10(-8) M concentration of HPP there was a slow increase of enzyme release to about 40-60% over basal release. At concentrations of HPP above 10(-8) M there was a rapid increase of enzyme release, amounting to 4-6 times over basal release at 10(-6) M concentration of HPP. The potency of HPP compared to other secretagogues at 10(-7) M concentration was 45% of CCK, 60% of carbachol and 75% of secretin. HPP did not inhibit the effect of CCK, secretin and carbachol on amylase release. The amylase release stimulated by HPP was accompanied by an increase in 45Ca2+ efflux. Atropine or dibutyryl cyclic GMP did not influence the effect of HPP. It is concluded that HPP stimulates the release of enzymes from rat pancreatic acini and that Ca2+ may be a mediator for this secretion.     

C-type natriuretic peptide (CNP) effects on intracellular calcium [Ca2+]i in mouse gonadotrope-derived alphaT3-1 cell line.

C-type natriuretic peptide (CNP), the third member of the atrial natriuretic peptide family, acts via guanylyl cyclase containing GC-B receptors to stimulate cyclic guanosine 3',5' monophosphate (cGMP) accumulation in the gonadotrope-derived alphaT3-1 cell line and rat pituitary cells. This effect is inhibited by concomitant activation of the phospholipase C (PLC)-coupled gonadotrophin hormone-releasing hormone (GnRH) receptors in these cells. Since GnRH stimulates gonadotrophin secretion from gonadotropes by increasing the cytosolic Ca2+ concentration ([Ca2+]i) and natriuretic peptides have been found to influence PLC/Ca2+ signalling in other systems, we have investigated whether CNP can alter basal or GnRH-stimulated changes in [Ca2+]i in alphaT3-1 cells. In Ca 2+-containing medium, 10(-7) M CNP modestly, but significantly increased [Ca2+]i over several min, but subsequently inhibited the elevation of [Ca2+]i in response to 10(-7) M GnRH in both Ca2+-containing and Ca2+-free medium. This inhibitory effect was mimicked by 10(-6) M 8-Br-cGMP, but not by ANP, indicating mediation by cyclic GMP and the CNP-specific GC-B receptor. However, basal and GnRH-stimulated inositol (1,4,5) trisphosphate (Ins(1,4,5)P3) generation were not measurably affected by CNP, and CNP failed to affect thapsigargin-induced capacitative Ca2+ entry. Thus, it appears that the cross-talk between CNP and GnRH in these cells is reciprocal in that GnRH modulates CNP effects on cGMP generation, whereas, CNP modulates GnRH effects on Ca2+ mobilisation.     

Stoichiometry of GTP hydrolysis in a poly(U)-dependent cell-free translation system. Determination of GTP/peptide bond ratios during codon-specific elongation and misreading

Pancreatic secretory factor (PSF), a 17.5-kDa protein purified from the venom of Gila monster (Heloderma suspectum), stimulated amylase secretion from dispersed rat pancreatic acini more efficiently than CCK-8, bombesin, carbachol and secretin, and without increasing 45Ca2+ efflux and cyclic AMP levels. The secretory action was dependent on the presence of extracellular calcium and was additive to the secretion induced by agents acting via cyclic AMP or via Ca2+ efflux.     

Two functionally distinct cholecystokinin receptors show different modes of action on Ca2+ mobilization and phospholipid hydrolysis in isolated rat pancreatic acini. Studies using a new cholecystokinin analog, JMV-180.

A new hepatapeptide cholecystokinin (CCK) analog, JMV-180 (Boc-Tyr(SO3-)-Nle-Gly-Trp-Nle-Asp-2-phenylethylester), acts as an agonist at high affinity CCK receptors on rat pancreatic acini to stimulate amylase release but unlike cholecystokinin octapeptide (CCK8) does not act on low affinity CCK receptors to inhibit amylase release (Galas, M. D., Lignon, M. F., Rodriguez, M., Mendre, C., Fulcrand, P., Laur, J., and Martinez, J. (1988) Am. J. Physiol. 254, G176-G188). To investigate the biochemical mechanisms initiated by CCK acting on each class of CCK receptor, the effects of JMV-180 and CCK8 on amylase release, Ca2+ mobilization, and phospholipid hydrolysis were studied in isolated rat pancreatic acini. When acini were loaded with the intracellular Ca2+ chelator BAPTA, amylase release stimulated by both JMV-180 and CCK8 was reduced. Measurement of 45Ca2+ efflux and cytosolic free calcium concentration ([Ca2+]i) by the fluorescence of fura-2-loaded acini in a stirred cuvette showed that JMV-180 induced a concentration-dependent increase but with a maximal response only two-thirds that induced by CCK8. When [Ca2+]i of individual fura-2-loaded acinar cells was measured by microspectrofluorometry, all concentrations of JMV-180 (1 nM-10 microM) induced repetitive transient [Ca2+]i spikes (Ca2+ oscillations). By contrast, stimulation with a high concentration of CCK8 (1 nM) caused a large increase in [CA2+]i followed by a small sustained elevation of [Ca2+]i. The measurement of inositol trisphosphate (IP3) production by both [3H]inositol labeling and 1,4,5-IP3 radioreceptor assay showed that JMV-180 had only minimal effects at 10 microM in contrast to the large increase induced by high concentrations of CCK8 (more than 1 nM). JMV-180 blocked the effect of a high concentration of CCK8 on both [Ca2+]i and 1,4,5-IP3 productions but did not affect the response to carbamylcholine. JMV-180 caused a delayed monophasic stimulation of 1,2-diacylglycerol (DAG) sustained to 60 min without the early increase in DAG observed in response to CCK8. Furthermore, JMV-180 stimulated the release of [3H]choline metabolites, primarily phosphorylated choline, from [3H]choline-labeled acini at low concentrations and to the same extent as CCK8. Since JMV-180 interacts not only with high affinity CCK receptors as an agonist but also with low affinity CCK receptors as a functional antagonist, the present results indicate that the occupancy of high affinity state receptors by CCK induces Ca2+ oscillations, DAG formation from phosphatidylcholine hydrolysis, and amylase release with minimal phosphatidylinositol 4,5-bisphosphate hydrolysis.(ABSTRACT TRUNCATED AT 400 WORDS)     

Calcium rather than cyclic AMP is an intracellular messenger of parathyroid hormone action on glycogen metabolism in isolated rat hepatocytes.

The synthetic 1-34 fragment of human parathyroid hormone (1-34hPTH) stimulated glucose production in isolated rat hepatocytes. The effect of 1-34hPTH was dose-dependent and 10(10) M-1-34 hPTH elicited the maximum glucose output, which was approx. 80% of that by glucagon. Although 1-34hPTH induced a small increase in cyclic AMP production at concentrations higher than 10(-9) M, 10(-10) M-1-34hPTH induced the maximum glucose output without significant elevation of cyclic AMP. This is in contrast to the action of forskolin, which increased glucose output to the same extent as 10(-10) M-1-34hPTH by causing a 2-fold elevation of cyclic AMP. In addition to increasing cyclic AMP, 1-34hPTH caused an increase in cytoplasmic free calcium concentration ([Ca2+]c). When the effect of 1-34hPTH on [Ca2+]c was studied in aequorin-loaded cells, low concentrations of 1-34hPTH increased [Ca2+]c: the 1-34hPTH effect on [Ca2+]c was detected at as low as 10(-12) M and increased in a dose-dependent manner. 1-34hPTH increased [Ca2+]c even in the presence of 1 microM extracellular calcium, suggesting that PTH mobilizes calcium from an intracellular pool. In line with these observations, 1-34hPTH increased the production of inositol trisphosphate. These results suggest that: (1) PTH activates both cyclic AMP and calcium messenger systems and (2) PTH stimulates glycogenolysis mainly via the calcium messenger system.     

Luteinizing hormone stimulates the formation of inositol trisphosphate and cyclic AMP in rat granulosa cells. Evidence for phospholipase C generated second messengers in the action of luteinizing hormone.

The following studies were conducted to determine whether luteinizing hormone (LH), a hormone which increases cellular levels of cyclic AMP, also provokes increases in 'second messengers' derived from inositol lipid metabolism (i.e. inositol phosphates and diacylglycerol). Rat granulosa cells isolated from mature Graafian follicles were prelabelled for 3 h with myo-[2-3H]inositol. LH provoked rapid (5 min) and sustained (up to 60 min) increases in the levels of inositol mono-, bis, and trisphosphates (IP, IP2 and IP3, respectively). Time course studies revealed that IP3 was formed more rapidly than IP2 and IP following LH treatment. The response to LH was concentration-dependent with maximal increases at LH concentrations of 1 microgram/ml. LiCl (2-40 mM) enhanced the LH-provoked accumulation of all [3H]inositol phosphates, presumably by inhibiting the action of inositol phosphate phosphatases. The effectiveness of LH, however, was dependent on the concentration of lithium employed; maximal increases in IP were observed at 10 mM-LiCl, whereas maximal increases in IP2 and IP3 were observed at 20 mM- and 40 mM-LiCl, respectively. The stimulatory effects of LH on inositol phosphate and progesterone accumulation were also compared with changes in cyclic nucleotide levels. LH rapidly increased levels of inositol phosphates, progesterone and cyclic AMP, but transiently reduced levels of cyclic GMP. These results demonstrate that LH increases both cyclic AMP and inositol trisphosphate (and presumably diacylglycerol) in rat granulosa cells. Our findings suggest that two messenger systems exist to mediate the action of LH in granulosa cells.     

Phosphorylation of 3 particulate proteins in rat pancreatic acini in response to vasoactive intestinal peptide (VIP), secretin and cholecystokinin (CCK-8)

Rat pancreatic acini were preincubated with 0.4 mM 32Pi for 45 min at 37 degrees C, then exposed for 15 min to VIP, secretin or CCK-8. The incubation was terminated with a stop solution and a fraction rich in mitochondria and zymogen granules was separated from a microsome-rich fraction by differential centrifugation. After heating in the presence of SDS, beta-mercaptoethanol was added and the pattern of equivalent amounts of 32P-labelled proteins was examined by autoradiography of SDS-PAGE gels. VIP, secretin, and CCK-8 stimulated the phosphorylation of a Mr=33 K microsomal protein and that of two proteins of Mr=21 K and Mr=25 K mostly present in a fraction rich in mitochondria and zymogen granules. Stimulations were dose-dependent, the highest stimulant concentrations tested allowing 2- to 3-fold increases of phosphorylation over basal. When 1 nM CCK-8 was used simultaneously with 1 microM VIP, the cyclic AMP levels attained and the pattern of protein phosphorylation were similar to those obtained with VIP alone, and there was a potentiation of amylase secretion; when a supra-maximal 0.1 microM CCK-8 concentration was added, the VIP-induced elevation in cyclic AMP levels and the phosphorylation of the Mr=21 K and Mr=25 K proteins were partially antagonized, and no potentiation any more of secretion occurred. To conclude the in vitro phosphorylation of three particulate proteins (Mr=33 K, 25 K, and 21 K) was similarly increased in rat pancreatic acini in response to secretin and VIP (acting through cyclic AMP) and to CCK-8 (acting mostly through Ca2+).     

Ouabain- and potassium-induced stimulation of amylase release in fragments and acini of rabbit pancreas

Ouabain increases the enzyme secretion from the isolated rabbit pancreas and pancreatic fragments, but not from isolated pancreatic acini. The increase occurs after a delay of 45-60 min and is not accompanied by an increase in lactate dehydrogenase release. The stimulatory effect of ouabain (10(-5) M) is dependent on the presence of extracellular calcium, and is not antagonized by 10(-4) M atropin, 10(-4) M propranolol, 10(-5) M phentolamine, 10(-3) M dibutyryl-cyclic GMP, 10(-6) M tetrodotoxin, 10(-4) M verapamil or 10(-4) M D-600. Elevation of the extracellular potassium concentration to 120 mM in the presence of 10(-4) M atropin also increases the enzyme secretion from rabbit pancreatic fragments. The increase is again dependent on the presence of extracellular calcium and is resistant to adrenergic blockade and to tetrodotoxin, verapamil or D-600. Forskolin also stimulates a Ca2+-dependent release of amylase from pancreatic fragments but not from pancreatic acini. In the presence of the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IMX), ouabain (10(-5) M) and K+ (120 mM) cause an immediate increase in the cyclic AMP content of pancreatic fragments which does not occur in the absence of extracellular calcium. In pancreatic acini, the cAMP production is only slightly increased by ouabain. In the absence of IMX, the cAMP levels in fragments or acini are not detectably altered by ouabain or K+. The results suggest that the stimulation of enzyme secretion by ouabain and high K+ is an indirect effect, mediated by the release of an endogenous transmitter from non-cholinergic, non-adrenergic nerves in the intact preparations. The release and/or the effect of the transmitter appears to be mediated primarily by Ca2+ and secondarily by cyclic AMP.     

Dopamine-stimulated cyclic AMP and binding of [3H] dopamine in acini from dog pancreas

Dispersed acini from dog pancreas were used to examine the ability of dopamine to increase cyclic AMP cellular content and the binding of [3H] dopamine. Cyclic AMP accumulation caused by dopamine was detected at 1.10(-8) M and was half-maximal at 7.9 +/- 3.4 10(-7) M. The increase at 1.10(-5) M, (7.5-fold) was equal to the half-maximal increase caused by secretin at 1.10(-9) M. Haloperidol, a dopaminergic receptor antagonist inhibited cyclic AMP accumulation caused by dopamine. The IC50 value for haloperidol, calculated from the inhibition of cyclic AMP increase caused by 1.10(-5) M dopamine was 2.3 +/- 0.9.10(-6) M. Haloperidol did not alter basal or secretin-stimulated cyclic AMP content. [3H] Dopamine binding was studied on the same batch of cells as cyclic AMP accumulation. At 37 degrees C, it was rapid, reversible, saturable and stereospecific. The Kd value for high affinity binding sites was 0.43 +/- 0.1.10(-7) M and 4.7 +/- 1.6.10(-7) M for low affinity binding sites. The concentration of drugs necessary to inhibit specific binding of dopamine by 50% was 1.2 +/- 0.4.10(-7) M epinine, 4.1 +/- 1.8.10(-6) M fluphenazine, 8.0 +/- 1.6.10(-6) M haloperidol, 4.2 +/- 1.2.10(-6) M cis-flupenthixol, 2.7 +/- 4.0.10(-5) M trans-flupenthixol, less than 1.10(-5) M apomorphine, sulpiride, naloxone and isoproterenol.     

Pertussis toxin stimulates cholecystokinin-induced cyclic AMP formation but is without effect on secretagogue-induced calcium mobilization in exocrine pancreas

The role of a pertussis toxin sensitive GTP-binding protein in mediating between cholecystokinin receptors and phosphatidylinositol 4,5-bisphosphate phosphodiesterase as well as in preventing cholecystokinin from increasing cellular cyclic AMP has been investigated using dispersed acini from rabbit pancreas. Pertussis toxin pretreatment (500 ng/ml, 2 h) did not affect cholecystokinin(octapeptide) (CCK-8)-induced increases in cytosolic free Ca2+ as judged from changes in fluorescence obtained from quin2-loaded acini. Although pretreatment with pertussis toxin was also without effect on resting acinar cell cyclic AMP levels, adenylate cyclase activity was increased, since inhibition of cyclic AMP phosphodiesterase activity by isobutylmethylxanthine (IBMX) resulted in an additional increase in cyclic AMP levels in toxin-treated acini, indicating that acinar cell adenylate cyclase activity is under some tonic inhibitory control by the pertussis toxin-sensitive inhibitory GTP-binding protein (Gi) of the adenylate cyclase system. CCK-8 gave an increase in cyclic AMP levels in both control (1.6-fold) and toxin-treated (2.3-fold) acini, leading to cyclic AMP levels in the toxin-treated acini 2-times as high as those in control acini. In the presence of IBMX, the cyclic AMP response to CCK-8 was again markedly enhanced in acini pretreated with the toxin (3.2- vs. 1.8-fold), resulting in cAMP levels in the toxin-treated acini 3.7-times those in the absence of IBMX, 2.5-times those in control acini in the presence of IBMX and 7.0-times those in control acini in the absence of IBMX. Neither the pretreatment with pertussis toxin, nor the presence of IBMX alone, nor the combination had an effect on basal amylase secretion. However, all three treatments potentiated the stimulatory effect of CCK-8 on amylase secretion and the amount of potentiation was proportional to the cyclic AMP levels reached. Our findings suggest that in the intact pancreatic acinar cell Gi inhibition of the catalytic subunit of the adenylate cyclase may largely be responsible for preventing cholecystokinin from increasing cellular cyclic AMP. They moreover show that cyclic AMP is a modulatory agent in rabbit pancreatic enzyme secretion, not able to stimulate secretion itself, but potentiating effects mediated by the phosphatidylinositol-calcium pathway.     

An in vitro evaluation of adrenergic action on rat pancreatic amylase secretion: lack of stimulatory effect.

To assess direct evidence of adrenergic stimulation in pancreatic amylase secretion, effects of catecholamines on amylase release and intracellular cyclic AMP accumulation were examined with rat dispersed pancreatic acini. We first carried out control studies with CCK-8 and carbamylcholine to evaluate the usefulness of the material for the examination of amylase secretion, and examined VIP-induced cyclic AMP accumulation to assess the agonist evoked intracellular response. As a result, significant effects of CCK-8, carbamylcholine and VIP were observed, which confirmed that dispersed pancreatic acini used in this study were useful in examining exocrine pancreatic secretion. However, catecholamines failed to stimulate amylase release from pancreatic acini, although a significant increase in intracellular cyclic AMP accumulation was observed. Thus the present study strongly suggests that direct involvement of catecholamine is unlikely in pancreatic amylase secretion, in contrast to results reported previously.     

Rat pancreatic adenylate cyclase. IV. Effect of hormones and other agents on cyclic AMP level and enzyme release.

1. The effects of secretin and pancreozymin-C-octapeptide and phosphodiesterase inhibitors on the concentration of adenosine 3',5'-cyclic monophosphate (cyclic AMP) and on the release of enzymes from rat pancreas have been studied. 2. In determininging cyclic AMP by means of the saturation assay of Brown et al. ((1971) Biochem. J. 121, 561-563) it is found essential to purify the pancreatic tissue extract by ion-exchange chromatography prior to the assay. 3. Injection of synthetic secretin or pancreozymin-C-octapeptide in anaesthetized rats in a secretory active dose (0.1 nmol) has no effect on the pancreatic cyclic AMP level. 4. Incubation for up to 10 min of pancreatic slices in Krebs-Ringer bicarbonate glucose medium containing 10(-2) M theophylline as phosphodiesterase inhibitor does not result in an increase of the cyclic AMP level. With 10(-2) M 1-methyl-3-isobutylxanthine as phosphodiesterase inhibitor the level is more than doubled after the first min of incubation and remains constant thereafter. 5. Addition of 3-10(-7) M secretin to slices incubated in the presence of 10(-2) M theophylline causes 84% increase of the cyclic AMP level above control, whereas the addition of 3-10(-7) M pancreozymin-C-octapeptide has no significant effect. In the presence of 10(-2) M 1-methyl-3-isobutylxanthine the latter hormone causes significant increases of up to 34% above control during 10 min of incubation. Secretin in this condition augments the cyclic AMP level by up to 296% above control during a 10 min incubation period. Addition of secretin and pancreozymin-C-octapeptide together has no greater effect than of secretin alone. 6. A broken cell fraction of rat pancreas contains adenylate cyclase activity which can be stimulated to 457 and 600% above the basal activity by 3-10(-7) M pancreozymin-C-octapeptide and secretin, respectively. Incubation of pancreatic slices with either hormone has no effect on the cyclic AMP phosphodiesterase activity in the homogenate of these slices. 7. Pancreozymin-C-octapeptide, dibutyryl cyclic AMP, 1-methyl-3-isobutylxanthine and carbamylcholine cause an elevated release of chymotrypsin from pancreatic slices incubated for 2 h in Krebs-Ringer bicarbonate medium, containing 10 mM glucose, while secretin, cyclic AMP and butyric acid have no significant effect. The release of the cytoplasmic enzyme lactate dehydrogenase is also elevated by dibutyryl cyclic AMP, 1-methyl-3-isobutylxanthine and carbamylcholine, but not significantly by pancreozymin-C-octapeptide. 8. The results support the role of cyclic AMP in the action of secretin, and do not exclude a mediating function of this nucleotide in the actions of pancreozymin in rat pancreas.     

The interaction of glucagon, gastric inhibitory peptide and somatostatin with cyclic AMP production systems present in rat gastric glands

The effects of glucagon, gastric inhibitory peptide (GIP) and somatostatin on the generation of cyclic AMP have been studied under basal and histamine- or secretin-stimulated conditions in tubular gastric glands isolated by means of EDTA from the rat fundus and antrum. Four types of cell could be identified by electron microscopy; namely, parietal, mucous, peptic and some endocrine cells with a good morphological preservation of the cellular topography as seen in the intact mucosa. Immunoreactive somatostatin was found in antral glands (210 +/- 16 ng/g cell, wet wt., n = 9) as well as in fundic glands, but in smaller concentration (50 +/- 8 ng/g cell, wet wt., n = 9). (1) In rat fundic glands, glucagon, in supraphysiologic doses (3 . 10(-9) -5 . 10(-7) M), raised cyclic AMP levels 46 times above the basal. At maximally effective doses, combination of glucagon plus histamine was not additive whereas glucagon and secretin stimulations resulted in an additive response. Somatostatin (10(-10) -10(-7) M) inhibited both glucagon- and histamine-induced cyclic AMP production, whereas cimetidine specifically blocked the histaminergic stimulation. (2) In the same conditions, 10(-6)M glucagon produced a marginal effect (4-fold increase) in rat antrum, whereas GIP (10(-9) -10(-6)M) was unable to induce a significant rise of cyclic AMP production in either fundic or antral glands, or to prevent cyclic AMP production stimulated by histamine. (3) The present data do not support the view that circulating glucagon or GIP may regulate gastric secretion directly by a cyclic AMP-dependent mechanism in rat gastric glands and raise the possibility that gastric somatostatin may be the final mediator of the inhibitory actions of these hormones on acid secretion. (4) It is proposed that pancreatic glucagon acts through a receptor-cyclic AMP system which is specific for the bioactive peptide enteroglucagon ('oxyntomodulin'), probably in rat parietal cells.     

Inositol trisphosphate independent increase of intracellular free calcium and amylase secretion in pancreatic acini

It is generally believed that the activation of various cell surface receptors results in the phospholipase C-catalyzed production of inositol trisphosphate which, in turn, increases the intracellular concentration of free Ca2+ by stimulating its release from nonmitochondrial sources. We have investigated both the production of inositol trisphosphate and changes in intracellular Ca2+ concentration in rat pancreatic acini in response to caerulein and CCK-JMV-180, two analogs of cholecystokinin. Both of these analogs cause comparable increases in the rate of amylase secretion and in intracellular Ca2+ concentration but their effects on inositol phosphate generation are dramatically different; caerulein stimulates significant production of inositol phosphates within 1 min of its addition, whereas no detectable levels of inositol phosphates were generated within the same time after addition of CCK-JMV-180. These results suggest that the CCK-JMV-180 stimulated release of intracellular Ca2+ is not mediated by inositol trisphosphate but some other as yet unidentified messenger.     

Evidence for a cyclic AMP system highly sensitive to secretin in gastric glands isolated from the rat fundus and antrum

The effects of secretin and vasointestinal peptide (VIP) on the production of cyclic AMP have been studied in gastric glands isolated by means of EDTA from rat fundic and antral mucosa. (1) In gastric fundus, secretin and VIP caused a time- and temperature-dependent stimulation of cyclic AMP production that was maximal when the test agents were incubated for 60 min at 20 degrees C in the presence of 0.5 mM 3-isobutyl-1-methylxanthine as a phosphodiesterase inhibitor. The dose-response curve was monophasic for both peptides, the production of cyclic AMP being sensitive to 10(-10) M secretin and to 5 . 10(-8) M VIP. Half-maximal stimulation was obtained with 2.9 10(-9) M secretin or 2 . 10(-7) M VIP and the maximal stimulation represented a 21-fold and a 19-fold increase above control for secretin and VIP, respectively. Histamine also stimulated cyclic AMP production, with a Km of about 5 . 10(-4) M. No additive effect on cyclic AMP production was oberved when secretin and VIP were simultaneously added at maximally active concentrations, while an additive effect was observed when secretin and histamine were added together. (2) In gastric antrum, the characteristics of the secretin- and VIP-stimulated cyclic AMP production were similar to those observed in gastric fundus. Histamine nevertheless failed to stimulate the formation of cyclic AMP in antral mucosa. (3) These data demonstrate the existence of a cyclic AMP system highly sensitive to secretin in gastric glands isolated from the rat fundus and antrum and suggest that VIP operates through this system. (4) It is proposed that the pepsinogen- and/or mucous-secreting cells are implicated in the regulation of cyclic AMP production by secretin in gastric glands of the rat.     

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.     

Noncyclooxygenase metabolites of arachidonic acid amplify the vasopressin-induced Ca2+ signal in glomerular mesangial cells by releasing Ca2+ from intracellular stores

Noncyclooxygenase metabolites of arachidonic acid may be potent modulators of the mitogenic response of renal mesangial cells to the mitogenic vasoactive peptide arginine vasopressin (AVP). Since Ca2+ is a critical second messenger in the response of mesangial cells to AVP, and Ca2+ has been implicated in the regulation of growth, we determined whether noncyclooxygenase metabolites altered the phospholipase C-Ca2+ signalling cascade which is activated by AVP. Pretreatment of mesangial cells for 10 min with lipoxygenase and cytochrome P450 monooxygenase inhibitors, nordihydroguaiaretic acid (NDGA, 10(-5) M) or SKF-525A (2.5 x 10(-5) M), but not the cyclooxygenase inhibitor indomethacin (2 x 10(-5) M), reduced the magnitude of the AVP (10(-8) and 10(-7) M)-induced increase in cytosolic free Ca2+ concentration ([Ca2+]i) without affecting inositol trisphosphate production. With 10(-8) M AVP, [Ca2+]i increased to 250 +/- 47 nM in NDGA-treated cells versus 401 +/- 59 nM in control cells (p less than 0.01). [Ca2+]i, measured 2 min after exposure to AVP, was also lower with NDGA (152 +/- 21 nM) when compared with AVP alone (220 +/- 22 nM, p less than 0.01). 14,15-epoxyeicosatrienoic acid (EET) (10(-8) M), which had no effect on inositol trisphosphate production, completely reversed the NDGA-induced inhibition of the [Ca2+]i transient, whereas 5-hydroperoxyeicosatetraenoic acid (HPETE) (5 x 10(-7) M) did not. Pretreatment with higher concentrations of 14,15-EET (10(-7)-10(-6) M) markedly potentiated the AVP-induced increase in [Ca2+]i. NDGA-induced inhibition of the AVP-generated [Ca2+]i transient was also observed when cells were incubated in low Ca2+ media ([Ca2+] less than 5 x 10(-8) M), suggesting that NDGA pretreatment impaired intracellular release of Ca2+. Since NDGA had no direct effect on inositol 1,4,5-trisphosphate-induced Ca2+ release, we postulated that NDGA blocked production of a metabolite that releases Ca2+ from intracellular stores. 14,15-EET and 15-HPETE, but not 15-hydroxyeicosatetraenoic acid (each at 3 x 10(-7) M), raised [Ca2+]i when added directly to cells in low Ca2+ media. In permeabilized cells 14,15-EET and 15-HPETE (10(-7) M) potently released Ca2+ from intracellular stores. In summary, noncyclooxygenase metabolites of arachidonic acid, and in particular P450 metabolites, are potent endogenous amplifiers of the AVP-induced [Ca2+]i signal by mechanisms not directly involving phospholipase C activation. This effect is mediated, at least in part, by enhanced release of Ca2+ from intracellular storage sites by an inositol 1,4,5-trisphosphate-independent mechanism.     

Effects of prostaglandin I2 and forskolin on the secretion from platelets evoked at basal concentrations of cytoplasmic free calcium by thrombin, collagen, phorbol ester and exogenous diacylglycerol.

Cytoplasmic free calcium ([Ca2+]i) and secretion of ATP were measured in quin2-loaded human platelets. In certain conditions thrombin and collagen cause secretion while [Ca2+]i remains at basal concentrations, a response attributed to activation of protein kinase by diacylglycerol formed by hydrolysis of inositol lipids. This secretion evoked by thrombin could be totally suppressed by prostaglandin I2 or forskolin, as expected from the known ability of cyclic AMP to inhibit phospholipase C. The secretory response evoked by collagen at basal [Ca2+]i and that evoked by exogenous diacylglycerol or phorbol ester, direct activators of protein kinase-C, were much less affected by these inhibitors, suggesting that thrombin and collagen may promote formation of diacylglycerol by different mechanisms.