Involvement of Ca2+ influx in F(-)-stimulated pepsinogen release from guinea pig gastric chief cells

In isolated guinea pig gastric chief cells, pepsinogen release was stimulated by NaF in a dose-dependent manner. Cholecystokinin (CCK) and Ca2+ ionophore A23187 had no additional effect on NaF-stimulated pepsinogen release. CCK caused a rapid increase in intracellular free Ca2+ concentration ([Ca2+]i) monitored by Quin-2 and markedly stimulated inositol phosphate accumulation in chief cells. By contrast, NaF did not cause any change in [Ca2+]i. NaF, even at a maximal concentration for pepsinogen release, appeared to be relatively ineffective on inositol phosphate accumulation. On the other hand, NaF markedly stimulated Ca2+ influx into chief cells. These results suggest that F- stimulates pepsinogen release probably by increasing Ca2+ influx into chief cells. Since F- is a well known activator of guanine nucleotide regulatory proteins (G proteins), it is proposed that there may exist a G protein regulating the opening of Ca2+ channel in gastric chief cells.     

Cellular distribution of gastric chief cell protein kinase C activity: differential effects of diacylglycerol, phorbol esters, carbachol, and cholecystokinin.

Stimulation of chief cells with carbachol or cholecystokinin (CCK) results in the production of inositol trisphosphate (IP3) and diacylglycerol (DAG). Although IP3 increases cell calcium concentration, thereby stimulating pepsinogen secretion, the role of DAG and its target, protein kinase C (PKC), is less clear. To examine the relation between the cellular distribution of PKC activity and pepsinogen secretion, we determined PKC activity in cytosolic and membrane fractions from dispersed chief cells from guinea pig stomach. To validate our assay, we studied the actions of the phorbol ester PMA. PMA caused a rapid, dose-dependent, 6-fold increase in pepsinogen secretion and membrane-associated PKC activity. Similarly, dose-response curves for pepsinogen secretion and the increase in membrane-associated PKC activity induced by a membrane-permeant DAG (1-oleoyl-2-acetylglycerol) were superimposable. In contrast, CCK (0.1 nM to 1.0 microM) and carbachol (0.1 microM to 1.0 mM) caused a 4-fold increase in pepsinogen secretion, but did not alter the distribution of PKC activity. These results indicate that in gastric chief cells, PMA- and DAG-induced pepsinogen secretion is accompanied by increased membrane-associated PKC activity. However, the cellular distribution of PKC activity is not altered by CCK or carbachol.     

Difference in effects of sodium fluoride and cholecystokinin on diacylglycerol accumulation and calcium increase in guinea pig gastric chief cells

In isolated guinea pig gastric chief cells, sodium fluoride (NaF) stimulated a monophasic increase in diacylglycerol accumulation, while cholecystokinin (CCK) strongly stimulated its biphasic accumulation. NaF evoked an increase in initial Ca2+ influx rate with a slow increase in intracellular free Ca2+ concentration [( Ca2+]i), while CCK stimulated a rapid increase in [Ca2+]i followed by a late sustained phase of the [Ca2+]i increase. Lanthanum chloride (La3+) effectively blocked NaF-stimulated increase in [Ca2+]i, but it blocked only CCK-stimulated late sustained phase of [Ca2+]i increase. The effect of NaF on pepsinogen secretion was enhanced in the presence of 100 microM AlCl3. Furthermore, pertussis toxin did not affect NaF-evoked diacylglycerol accumulation at all. These results suggest that NaF may activate a pertussis-toxin insensitive guanine nucleotide regulatory protein (G protein) coupled to a signal transducing mechanism which seems to be distinct from that activated by CCK, thereby inducing increases in diacylglycerol accumulation, Ca2+ influx and pepsinogen secretion in guinea pig gastric chief cells.     

Cholinergic agonist-induced pepsinogen secretion from murine gastric chief cells is mediated by M1 and M3 muscarinic receptors

Muscarinic cholinergic mechanisms play a key role in stimulating gastric pepsinogen secretion. Studies using antagonists suggested that the M3 receptor subtype (M3R) plays a prominent role in mediating pepsinogen secretion, but in situ hybridization indicated expression of M1 receptor (M1R) in rat chief cells. We used mice that were deficient in either the M1 (M1R-/-) or M3 (M3R-/-) receptor or that lacked both receptors (M(1/3)R-/-) to determine the role of M1R and M3R in mediating cholinergic agonist-induced pepsinogen secretion. Pepsinogen secretion from murine gastric glands was determined by adapting methods used for rabbit and rat stomach. In wild-type (WT) mice, maximal concentrations of carbachol and CCK caused a 3.0- and 2.5-fold increase in pepsinogen secretion, respectively. Maximal carbachol-induced secretion from M1R-/- mouse gastric glands was decreased by 25%. In contrast, there was only a slight decrease in carbachol potency and no change in efficacy when comparing M3R-/- with WT glands. To explore the possibility that both M1R and M3R are involved in carbachol-mediated pepsinogen secretion, we examined secretion from glands prepared from M(1/3)R-/- double-knockout mice. Strikingly, carbachol-induced pepsinogen secretion was nearly abolished in glands from M(1/3)R-/- mice, whereas CCK-induced secretion was not altered. In situ hybridization for murine M1R and M3R mRNA in gastric mucosa from WT mice revealed abundant signals for both receptor subtypes in the cytoplasm of chief cells. These data clearly indicate that, in gastric chief cells, a mixture of M1 and M3 receptors mediates cholinergic stimulation of pepsinogen secretion and that no other muscarinic receptor subtypes are involved in this activity. The development of a murine secretory model facilitates use of transgenic mice to investigate the regulation of pepsinogen secretion.     

PAR-2 modulates pepsinogen secretion from gastric-isolated chief cells

In the present study, we investigated whether activation of protease-activated receptor type 2 (PAR-2) with SLIGRL (SL)NH2, a short mimetic agonistic peptide, directly stimulates pepsinogen secretion from gastric-isolated, pepsinogen-secreting (chief) cells. Immunostaining of gastric-dispersed chief cells with a specific anti-PAR-2 antibody demonstrated expression of PAR-2 receptors on membrane and cytoplasm. SL-NH2 and trypsin potently stimulated pepsinogen secretion (EC50 = 0.3 nM) and caused Ca2+ mobilization (EC50 = 0.6 nM). In contrast to SL-NH2, the scramble peptide LSIGRL-NH2 failed to stimulate pepsinogen release. Exposure to SL-NH2 also resulted in ERK1/2 phosphorylation and activation. Exposure of chief cells to phosphotyrosine kinase inhibitors and 2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one, a selective MEK inhibitor, significantly reduced secretion induced by SL-NH2. Pepsinogen secretion induced by SL-NH2 was desensitized by pretreating the cells with the mimetic peptide and trypsin, and exposure to SL-NH2 abrogates pepsinogen secretion induced by carbachol and CCK-8, but not secretion induced by secretin and vasointestinal peptide. Exposure to Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH2 (substance P) but not to calcitonin gene-related peptide increased pepsinogen release. The neurokinin-1 receptor antagonist, N-acetyl-l-tryptophan 3,5-bis(trifluoromethyl)benzyl ester, inhibited substance P-stimulated pepsinogen secretion, whereas it did not affect secretion induced by SL-NH2. Collectively, these data indicate that PAR-2 is expressed on gastric chief cells and that its activation causes a Ca2+-ERK-dependent stimulation of pepsinogen secretion.     

Cholecystokinin-stimulated pepsinogen secretion and cholecystokinin receptors on gastric chief cells in guinea pigs

We investigated cholecystokinin (CCK) receptors on isolated gastric chief cells from guinea pig. CCK stimulated pepsinogen secretion from chief cells at the same efficacy as that induced by carbamylcholine. Binding of 125I-labeled CCK-33 (125I-CCK) to chief cells was temperature-dependent, and was saturable and reversible at 37 degrees C. Hofstee plots of the ability of CCK-8 to inhibit binding of 125I-CCK showed a linear regression line, suggesting that CCK receptors possessed one binding site. The dissociation constant of the binding site was calculated to be 3.8 x 10(-10) M. The dose-response curve of CCK for pepsinogen secretion was superimposed on that for the binding to its receptors. These results indicated that gastric chief cells from the guinea pig possess CCK receptors that relate closely to the action of CCK involved in pepsinogen secretion.     

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

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

Difference in effects of pirenzepine and atropine on carbachol-induced pepsinogen secretion from isolated gastric glands

The effect of pirenzepine on carbamylcholine (carbachol)-stimulated pepsinogen secretion was compared with that of atropine in the isolated guinea pig gastric glands. Pirenzepine and atropine caused a dose dependent inhibition of carbachol-stimulated pepsinogen secretion. Moreover, pirenzepine as well as atropine produced a rightward shift in the dose response curve of carbachol-stimulated pepsinogen secretion but did not alter the maximum increase in pepsinogen secretion. Results therefore demonstrate that pirenzepine acts as a specific receptor antagonist in the interaction of carbachol with its receptor on gastric chief cells. However, pirenzepine was 50 times less potent than atropine in inhibiting pepsinogen secretion. Half maximal inhibitory concentration of pirenzepine was 2 X 10(-5) M when a maximally effective concentration of carbachol was used, while that of atropine was 4 X 10(-7) M. Results, therefore, suggest that muscarinic receptor on gastric chief cells to which pirenzepine binds may be an intermediate affinity type.     

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

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

Porcine ileal polypeptide causes an increase in cytoplasmic Ca2+ in both parietal and chief cells resulting in acid and pepsinogen secretion

Porcine ileal polypeptide, an enterooxyntin isolated from distal small intestinal mucosal epithelium, has been observed to stimulate gastric acid secretion in vivo as well as in vitro (Wider, M.D. et al. (1984) Endocrinology 115, 1484-1491, Wider M.D. et al. (1986) Endocrinology 118, 1546-1550). We report here that porcine ileal polypeptide stimulates both acid (aminopyrine accumulation) and pepsinogen secretion in isolated, enriched populations of guinea pig parietal and chief cells in a dose-dependent manner. Further, 10(-9) M porcine ileal polypeptide caused an increase in cytoplasmic Ca2+ concentration in both parietal and chief cells similar in magnitude to that observed with gastrin-17 (10(-8) M) (as measured by both fura-2 and aequorin) and cholecystokinin octapeptide (CCK-OP) (10(-8) M), respectively. Porcine ileal polypeptide has been observed to cause no stimulation of cAMP production in gastric glands from guinea pigs (Gespach, C., personal communication) nor is there any effect of medium Ca2+ depletion on acid production observed with guinea pig gastric mucosal sections. It is concluded that porcine ileal polypeptide, at concentrations similar to circulating levels observed in plasma of normal pigs (5 x 10(-9) M), acts directly on the parietal and chief cells to cause the mobilization of intracellular Ca2+ from the stores resulting in acid and pepsinogen secretion. These experiments demonstrate that this peptide is a potent enterooxyntin and chief cell secretagogue which acts via the same signal transduction mechanisms as gastrin and cholecystokinin.     

Combined effect of phorbol ester and, A23187 or dibutyryl cyclic AMP on pepsinogen secretion from isolated gastric glands

In isolated guinea pig gastric glands, pepsinogen secretion was stimulated by the phorbol ester, 12-0-tetradecanoyl-phorbol-13-acetate (TPA) in a dose dependent manner. Calcium-deprivation from the medium resulted in the decrease in TPA-induced pepsinogen secretion. The combination of 0.4 microM Ca2+ionophore A23187 and TPA stimulated pepsinogen secretion slightly higher than the calculated additive value for each agent. This synergistic effect of the agents supports a role of calcium-activated, phospholipid-dependent protein Kinase (protein Kinase C) in gastric pepsinogen secretion. Furthermore, pepsinogen secretion was also stimulated by dibutyryl cyclic AMP (dbc AMP) and dbc AMP slightly enhanced TPA-induced pepsinogen secretion. Results suggest that gastric chief cells possess at least two different secretory pathways for pepsinogen which are probably dependent on protein kinase C and cyclic AMP, respectively.     

Influence of thyroid status on Ca2+ mobilization and amylase secretion in rat pancreatic acini

Thyroid hormones influence Ca2+ homeostasis in both skeletal and cardiac muscle. Since secretory cells, like muscle cells, store and use Ca2+ in stimulus-response coupling, we have studied the effects of thyroid status on Ca2+ mobilization and secretion in a model secretory tissue, the pancreatic acinar cell. Hyperthyroidism was induced by rats by daily, subcutaneous injections of triiodothyronine for 8 days and hypothyroidism by adding 6-n-propyl-2-thiouracil to the drinking water for 14 days. Pancreatic acini were prepared by collagenase digestion of pancreatic tissue from hyper- and hypo-thyroid animals and from euthyroid controls. Ca2(+)-mobilization was assessed using Quin-2 fluorescence and secretion by assaying amylase release. The data indicate that the amount of Ca2+ mobilized by the muscarinic agonist carbachol or by cholecystokinin octapeptide increases with increasing thyroid hormone concentrations. Only in hypothyroidism was this change in Ca2+ homeostasis reflected by a parallel change in amylase secretion. This implies the existence of some compensatory mechanism which stabilizes secretory rate in the face of stimulus-evoked increases in intracellular Ca2+ concentration.     

The cholecystokinin-induced Ca2+ shuttle from the inositol trisphosphate-sensitive and ATP-dependent pool, and initial pepsinogen release connected with cytoskeleton of the chief cell

In guinea pig chief cells, inositol 1,4,5-trisphosphate (IP3) caused release of Ca2+, which was accumulated by ATP, from an endoplasmic reticulum-enriched fraction in both the permeable system and the cell-free system. This was mimicked with the Ca2+ ionophores A23187 and ionomycin on a large scale since an IP3-sensitive Ca2+ pool might be a subset of the Ca2+ ionophore-sensitive Ca2+ pool. The permeable chief cells, but not the cell-free system, retained the ability to react to synthetic cholecystokinin octapeptide (CCK-OP) with Ca2+ release from an IP3-sensitive pool due to of the non-additive but constant effect in exerting Ca2+ release from the store(s) induced by the combination with IP3 and CCK-OP. The increase in the cytosolic free Ca2+ concentration of intact chief cells responding to CCK-OP or the Ca2+ ionophore, ionomycin, comprised two components, namely, that by the Ca2+ entry from the extracellular space, and that by the Ca2+ release from the intracellular space(s) (as measured by fura-2). When CCK-OP or ionomycin was added, there was a biphasic response of pepsinogen secretion. An initial but transient response reaching a peak in 5 min was followed by a sustained response reaching a peak in 30 min. The initial pepsinogen release was independent of medium Ca2+, whereas the sustained one was dependent on medium Ca2+. The results suggest that the intracellular Ca2+ release from the store(s), presumably endoplasmic reticulum, may trigger the initial pepsinogen release, whereas the sustained pepsinogen secretion may be caused by acting in concert with the initial response and external Ca2+ entry. On the other hand, the disruption of the microtubular-microfilamentous system by colchicine or cytochalasin D failed to cause the Ca2+ release evoked by either IP3, CCK-OP or Ca2+ ionophores and to cause the CCK-OP- or ionomycin-induced initial pepsinogen release. These findings suggest that the IP3-sensitive pool is the same Ca2+ store which is completely or partially sensitive to CCK-OP and Ca2+ ionophores, respectively, and that the assembly of the cytoskeletal system is involved in initial intracellular Ca2+ metabolism and the following initial pepsinogen release. The assembly of the cytoskeletal system may be an early event in mediating the CCK-OP-induced initial pepsinogen release, perhaps by causing the Ca2+ release from an IP3-sensitive pool of the chief cell. The translocation or attachment of the IP3-sensitive pool brought about by cytoskeletal system might be necessary to cause Ca2+ release after the cell stimulation with CCK-OP.     

Interaction between the calcium and adenylate cyclase messenger systems in dispersed chief cells from guinea pig stomach. Possible cellular mechanism for potentiation of pepsinogen secretion

To determine the role of the adenylate cyclase system in potentiation of enzyme secretion, we used cholera toxin to activate adenylate cyclase before examining the effects of agents on chief cell cAMP and pepsinogen secretion. Dispersed chief cells were obtained from guinea pig stomach by fractionation of mucosal cells on a Percoll gradient. Incubation of cells with 100 nM cholera toxin for 90 min and subsequent incubation with carbachol or cholecystokinin resulted in augmentation of cellular cAMP and potentiation of pepsinogen secretion. The rate of increase in cAMP with carbachol or cholecystokinin was similar to that for the potentiated secretory response. To determine the role of changes in cell calcium on these effects, we examined the actions of the ionophore A23187. In cells preincubated with cholera toxin, A23187 augmented cAMP and caused potentiation of pepsinogen secretion. The effects of A23187, carbachol, and cholecystokinin on cells preincubated with cholera toxin were abolished by removing extracellular calcium or by adding the calmodulin inhibitor trifluoperazine. These data indicate that in chief cells preincubated with cholera toxin, secretagogue-induced increases in cell calcium concentration activate calmodulin thereby augmenting levels of cAMP and causing potentiation of pepsinogen secretion. Modulation of adenylate cyclase by changes in chief cell calcium concentration appears to be one mechanism whereby secretagogue interaction can result in potentiation of pepsinogen secretion.     

Changes in free cytosolic Ca2+ in hepatocytes following alpha 1-adrenergic stimulation. Studies on Quin-2-loaded hepatocytes

The Ca2+ selective fluorescent indicator, Quin-2, was employed to monitor continuously the concentration of free cytosolic Ca2+ [ Ca2+ ]i in isolated rat hepatocytes. Epinephrine (10(-6) M) and phenylephrine (10(-5) M), acting via alpha 1-adrenergic receptors, increases [ Ca2+ ]i from a basal concentration of approximately 0.2 microM to approximately 0.6 microM. This increase in [ Ca2+ ]i is evident as early as 1 to 1.5 s, the earliest time so far reported for any hepatic alpha 1-adrenergic event. Vasopressin (10(-8) M), after a lag which is 2 to 3 s longer, increases [ Ca2+ ]i to the same extent and at the same rate as the alpha 1-adrenergic agonists. Glucagon (10(-8) M) also increases [ Ca2+ ]i but at a significantly slower rate and only after a lag of about 10 s. All of these agents also induce an increase in the fluorescence of control cells. This Quin-2 independent fluorescence, which is due to an increased reduction of pyridine nucleotides, must be corrected for before the maximum change in [ Ca2+ ]i can be calculated but is sufficiently slow so as not to contribute to the initial rate of increase in the Quin-2-dependent fluorescence.     

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.     

Secretagogue-induced Ca2+ oscillations in isolated canine gastric chief cells.

Agonist-induced changes in cytoplasmic free Ca2+ concentration [( Ca2+]i) of isolated canine gastric chief cells were evaluated by microspectrofluorometry of superfused fura-2 loaded cells. Application of high concentrations of carbachol (CCh, 10(-5) M) or cholecystokinin octapeptide (10(-8) M) resulted in biphasic Ca2+ mobilization comprising an initial large transient followed by a small sustained elevation above the prestimulation level. Submaximal concentrations of CCh (10(-6) M) or cholecystokinin (10(-9) M) led to either a transient series of large amplitude Ca2+ spike(s) or a higher frequency of sustained Ca2+ oscillations of smaller amplitude. Cholecystokinin at 10(-10) M induced only sustained Ca2+ oscillations. Elimination of Ca2+ from the medium had no immediate effect on oscillations indicating an intracellular source of Ca2+. Thus the Ca2+ signalling mode in chief cells is dependent on agonist concentrations.     

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

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

Thyroidal and neural control of myosin transitions during development of rat fast and slow muscles

The uptake of 125I-labeled epidermal growth factor (125I-EGF) by mouse pancreatic acini was inhibited (40-50%) by the secretagogue cholecystokinin octapeptide (CCK8). Analysis of competitive binding data showed that the apparent Kd of EGF binding increased 135% while the binding capacity was only slightly altered (30% increase). That the effect of CCK8 on acini was mediated by intracellular Ca2+ was indicated by the following: (i) Inhibition of 125I-EGF binding to acini was dose-dependent and paralleled the known abilities of CCK8, its analogs, and the cholinergic secretagogue carbachol to induce Ca2+ efflux from acini; and (ii) addition of the Ca2+ ionophore A23187 also inhibited 125I-EGF binding. In addition, EGF association with A431 cells was also inhibited by A23187 in the presence but not the absence of Ca2+.     

Studies on the role of inositol trisphosphate in the regulation of insulin secretion from isolated rat islets of Langerhans.

Glucose (20 mM) and carbachol (1 mM) produced a rapid increase in [3H]inositol trisphosphate (InsP3) formation in isolated rat islets of Langerhans prelabelled with myo-[3H]inositol. The magnitude of the increase in InsP3 formation was similar when either agent was used alone and was additive when they were used together. In islets prelabelled with 45Ca2+ and treated with carbachol (1 mM), the rise in InsP3 correlated with a rapid, transient, release of 45Ca2+ from the cells, consistent with mobilization of 45Ca2+ from an intracellular pool. Under these conditions, however, insulin secretion was not increased. In contrast, islets prelabelled with 45Ca2+ and exposed to 20mM-glucose exhibited a delayed and decreased 45Ca2+ efflux, but released 7-8-fold more insulin than did those exposed to carbachol. Depletion of extracellular Ca2+ failed to modify the increase in InsP3 elicited by either glucose or carbachol, whereas it selectively inhibited the efflux of 45Ca2+ induced by glucose in preloaded islets. Under these conditions, however, glucose was still able to induce a small stimulation of the first phase of insulin secretion. These results demonstrate that polyphosphoinositide metabolism, Ca2+ mobilization and insulin release can all be dissociated in islet cells, and suggest that glucose and carbachol regulate these parameters by different mechanisms.