Cholecystokinin-induced residual stimulation of enzyme secretion from mouse pancreatic acini

When dispersed acini from mouse pancreas are first incubated with cholecystokinin octapeptide, washed and then reincubated with no additions there is significant stimulation of amylase secretion during the second incubation (residual stimulation of enzyme secretion). Cholecystokinin-induced residual stimulation of enzyme secretion is modified, but not abolished, by reducing the temperature of the first incubation from 37°C to 4°C. Measurement of binding of ¹²⁵I-labeled cholecystokinin octapeptide indicated that maximal cholecystokinin induced residual stimulation of enzyme secretion occurs when 12–20% of cholecystokinin receptors are occupied by cholecystokinin octapeptide. Moreover, maximal cholecystokinin-induced residual stimulation of amylase secretion is 25% greater than maximal cholecystokinin-induced direct stimulation of amylase secretion. Cholecystokinin tetrapeptide, which causes the same maximal direct stimulation of amylase secretion as does cholecystokinin octapeptide, causes a maximal residual stimulation of enzyme secretion that is only 30% of that caused by a maximally effective concentration of cholecystokinin octapeptide. Adding dibutyryl cyclic GMP to the second incubation can reverse the residual stimulation caused by adding cholecystokinin to the first incubation. The pattern and extent of the dibutyryl cyclic GMP-induced reversal of residual stimulation varies, depending on the temperature and concentration of cholecystokinin octapeptide in the first incubation. The present results are compatible with the hypothesis that mouse pancreatic acini possess two classes of cholecystokinin receptors. One class has a relatively high affinity for cholecystokinin and produces stimulation of enzyme secretion; the other class has a relatively low affinity for cholecystokinin and produces inhibition of enzyme secretion.     

Interaction of tryptophan-modified analogues of cholecystokinin-octapeptide with cholecystokinin receptors on pancreatic acini

None of six different tryptophan-modified analogues of the C-terminal octapeptide of cholecystokinin differed from the unaltered peptide in terms of their efficacies for stimulating amylase secretion from dispersed acini prepared from guinea-pig pancreas. Replacementof hydrogen with fluorine in position 5 or 6 on the indole ring of the tryptophan residue did not alter the potency with which the peptide stimulated amylase secretion; however, replacement of hydrogen by fluorine in positions 4, 5, 6, and 7 of the indole ring, of modifying or replacing the indole nitrogen caused a 30- to 300-fold decrease in potency. Changes in the ability of the peptide to stimulate amylase secretion were accompanied by corresponding changes in the ability of the peptide to inhibit binding of ¹²⁵I-labeled cholecystokinin. Our findings indicate that reducing the ability of the tryptophan residue to donate electrons produced a greater decrease in the affinity of the peptide for the cholecystokinin receptors than did abolishing the ability of tryptophan to form hydrogen bonds, and modifications that altered both abilities caused a greater decrease in affinity than did modification of only one ability. Finally, in the tryptophan residues of cholecystokinin octapeptide, tetrafluorination of the indole ring or replacing the indole nitrogen by oxygen reduced the ability of the peptide to cause residual stimulation of enzyme secretion, probably by accelerating the rate at which bound peptide dissociated from its receptors when the acini were washed and resuspended in fresh incubation solution.     

Phorbol ester attenuates cholecystokinin-stimulated amylase release in pancreatic acini of rats

12-O-tetradecanoylphorbol 13-acetate (TPA) and cholecystokinin octapeptide stimulate amylase secretion in dispersed pancreatic acini, presumably acting via the activation of protein kinase C. In this study, we examined TPA pretreatment on the subsequent response of rat pancreatic acini to secretagogues. Acini exposed to TPA (3 X 10(-7) M) at 37 degrees C reduced the subsequent amylase secretion as stimulated by cholecystokinin octapeptide and carbachol, but not by A23187 or VIP. The optimal effect was obtained after 5 min of preincubation with TPA. Longer incubation did not result in greater attenuation. The degree of attenuation was dependent on the concentration of TPA used in the pretreatment. Maximal effect was seen at TPA concentrations of 10(-7) M and higher. Preincubation with TPA resulted in alterations of the dose response of pancreatic acini to cholecystokinin octapeptide. A decrease in amylase secretion was obtained at optimal and suboptimal but not at supraoptimal concentrations of cholecystokinin octapeptide. The peak response to cholecystokinin octapeptide, furthermore, was shifted almost 1 log unit to the right, suggesting a decrease in cholecystokinin binding of the acini following TPA treatment. Binding studies demonstrated a reduction in the specific binding of 125I-labelled cholecystokinin octapeptide to acini following TPA treatment. Analysis of binding data revealed a decrease in affinity and binding capacity of the high-affinity component. No significant change in the binding capacity was detected with the low-affinity component, but a great increase in its affinity was observed. This suggests that the attenuation effect by TPA on the cholecystokinin octapeptide response in rat pancreatic acini in vitro is at the receptor level.     

Secretagogue-induced membrane alterations in dispersed acini from rat pancreas

Dispersed acini from rat pancreas were used to examine the effects of various pancreatic secretagogues on the fine structure of the acinar cell plasma membrane. With the C-terminal octapeptide of cholecystokinin, the C-terminal tetrapeptide of cholecystokinin, carbamylcholine, bombesin, A23187, vasoactive intestinal peptide or 8-bromo cyclic adenosine monophosphate, concentrations of the secretagogues that caused maximal stimulation of enzyme secretion did not produce alterations of the acinar cell plasma membrane. Supramaximal concentrations of the C-terminal octapeptide of cholecystokinin, the C-terminal tetrapeptide of cholecystokinin or carbamylcholine induced the formation of cytoplasmic protrusions at the basolateral plasma membrane of the pancreatic acinar cell, whereas supramaximal concentration of bombesin, A23187, vasoactive intestinal peptide or 8-bromo cyclic AMP did not alter the morphology of the acinar cell. Effects of the C-terminal octapeptide of cholecystokinin could be detected as early as after two minutes of incubation and these effects progressed for up to 30 minutes of incubation.     

Binding of cholecystokinin to high affinity receptors on isolated rat pancreatic acini

The binding of cholecystokinin (CCK) to its receptors on isolated rat pancreatic acini was investigated employing high specific activity, radioiodinated CCK (125I-BH-CCK), prepared by the conjugation of 125I-Bolton-Hunter reagent (125I-BH) to CCK. Binding was specific, time-dependent, reversible, and linearly related to the acinar protein concentration. After incubation for 30 min at 37 degrees C, the 125I-BH-CCK both in the incubation medium and bound to acini remained intact, as judged by gel filtration and trichloroacetic acid precipitation studies. Scatchard analysis was compatible with two classes of binding sites on acini: a very high affinity site (Kd, 64 pM) and a lower affinity site (Kd, 21 nM). 125I-BH-CCK binding to acini was competitively inhibited by CCK and four of its analogues in proportion to their biological potencies but not by unrelated hormones. Stimulation of amylase secretion by CCK and inhibition of 125I-BH-CCK binding by the same analogues carried out under identical conditions revealed a correlation (r = 0.99) between binding potency and amylase secretion. Stimulation of amylase secretion by CCK closely paralleled the occupancy of the high affinity CCK binding sites. It is concluded that the high affinity CCK binding sites most likely are the receptors mediating the stimulation of amylase secretion by CCK.     

Dissimilar effects of the protein kinase C inhibitors, staurosporine and H-7, on cholecystokinin-induced enzyme secretion from rabbit pancreatic acini.

The effects of two putative inhibitors of protein kinase C activity, staurosporine and H-7, on partially purified protein kinase C and amylase secretion from isolated rabbit pancreatic acini were investigated. Staurosporine dose-dependently inhibited amylase release stimulated by an optimal concentration of cholecystokinin C-terminal octapeptide. At a concentration of 100 nM, the drug inhibited the secretory response to the secretagogue by approximately 50%. At the same concentration, staurosporine inhibited 12-O-tetradecanoylphorbol 13-acetate-stimulated enzyme secretion by 90%. Moreover, the potentiating effect of this phorbol ester on cholecystokinin-induced amylase release was completely abolished in the presence of staurosporine. Interestingly, amylase release was decreased to the level observed with the combination of cholecystokinin and staurosporine. In contrast, H-7, potentiated rather than inhibited cholecystokinin-stimulated enzyme secretion, whereas the secretory response to 12-O-tetradecanoylphorbol 13-acetate was not affected by the drug. Both staurosporine and H-7, however, inhibited protein kinase C purified from exocrine pancreatic tissue. Kinetic analysis revealed that both compounds inhibited protein kinase C competitively with respect to ATP. The Ki value for staurosporine was 0.55 nM and for H-7 13.5 microM. Our results obtained with staurosporine are in line with a stimulatory role of protein kinase C in cholecystokinin-induced enzyme secretion from the exocrine pancreas. The results obtained with H-7 emphasize that care has to be taken in interpreting the biological effects of this drug.     

The actions of tetraethylammonium on dispersed acini from guinea pig pancreas

In dispersed acini from guinea pig pancreas, replacing extracellular sodium by tetraethylammonium (1) abolished carbamylcholine-stimulated amylase secretion but did not alter the increase in amylase secretion caused by the C-terminal octapeptide of cholecystokinin, bombesin, ionophore A23187, vasoactive intestinal peptide or 8-bromoadenosine 3':5' monophosphate, (2) caused a parallel rightward shift in the dose-response curve for carbamylcholine-stimulated amylase secretion and (3) inhibited binding of N-[3H]methyl scopolamine to muscarinic cholinergic receptors. Detectable inhibition of carbamylcholine-stimulated amylase secretion and binding of N-[3H]methyl scopolamine occurred with 300 microM tetraethylammonium, and half-maximal inhibition of these functions occurred with 1-2 mM tetraethylammonium. Replacing extracellular sodium by Tris did not alter the stimulation of enzyme secretion caused by any secretagogue tested. These results indicate that the tetraethylammonium is a muscarinic cholinergic receptor antagonist and that enzyme secretion from pancreatic acini does not depend on extracellular sodium.     

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.     

Cholecystokinin receptor occupation and cholecystokinin-induced calcium mobilization in the early phase in rat pancreatic acini.

We examined receptor occupation, calcium mobilization and amylase release for cholecystokinin octapeptide (CCK-8) within a 3-min incubation period at 37 degrees C using dispersed acini from rat pancreas. Analysis of competitive binding inhibition data obtained after a 3-min incubation revealed the presence of only a single class of CCK receptors, while two classes of CCK receptor, i.e., high-affinity and low-affinity CCK receptors, were detected when binding reached a steady-state after a 60-min incubation. The IC50 of CCK receptors calculated from the 3-min binding data was 19.0 +/- 0.5 nM (mean +/- S.D.), close to the Kd of the low-affinity CCK receptors determined by equilibrium binding studies. Exposure of fura-2-loaded acini to 10-1000 pM CCK-8 caused an immediate and dose-dependent increase in [Ca2+]i followed by a gradual decrease in [Ca2+]i. The CCK-stimulated amylase release after 3 min of incubation was biphasic; amylase release increased over the dose range of 3-300 pM CCK-8, peaked at 300 pM CCK-8 and decreased with supramaximal concentrations of CCK-8. Our data suggest that occupation of the low-affinity, but not the high-affinity, CCK receptors is more directly associated with calcium mobilization and subsequent stimulation of amylase release in rat pancreatic acini.     

Action of cholera toxin on dispersed acini from guinea pig pancreas.

In dispersed acini from guinea pig pancreas cholera toxin bound reversibly to specific membrane binding sites to increase cellular cyclic AMP and amylase secretion. Cholera toxin did not alter outflux of 45Ca or cellular cyclic AMP. Binding of 125I-labeled cholera toxin could be detected within 5 min; however, cholera toxin did not increase cyclic AMP or amylase release until after 40 min of incubation. There was a close correlation between the dose vs. response curve for inhibition of binding of 125I-labeled cholera toxin by native toxin and the action of native toxin on cellular cyclic AMP. With different concentrations of cholera toxin, maximal stimulation of amylase release occurred when the increase in cellular cyclic AMP was approximately 35% of maximal. Cholera toxin did not alter the increase in 45Ca outflux or cellular cyclic GMP caused by cholecystokinin or carbachol but significantly augmented the increase in cellular cyclic AMP caused by secretin or vasoactive intestinal peptide. The increase in amylase secretion caused by cholera toxin plus secretin or vasoactive intestinal peptide was the same as that with cholera toxin alone. On the other hand, the increase in amylase secretion caused by cholera toxin plus cholecystokinin or carbachol was significantly greater than the sum of the increases caused by each agent alone.     

Down-regulation and recycling of high affinity cholecystokinin receptors on pancreatic acinar cells

First incubating dispersed acini from rat pancreas with monensin, a cation ionophore that can inhibit recycling of receptors, inhibited binding of 125I-cholecystokinin 8 (125I-CCK-8) measured during a second incubation by as much as 50%. A maximal effect of monensin required 90 min of first incubation. Detectable inhibition of binding of 125I-CCK-8 occurred with 300 nM monensin, and inhibition increased progressively with concentrations of monensin up to 25 microM. Pancreatic acini possess two classes of receptors that bind 125I-CCK-8. One class has a high affinity (Kd = 461 pM) and a low capacity for CCK (512 fmol/mg DNA); the other class has a low affinity (Kd = 47 nM) and a high capacity for CCK (18 pmol/mg DNA). First incubating acini with monensin caused an 84% decrease in the number of high affinity CCK receptors with no change in the number of low affinity CCK receptors or the values of Kd for either class of receptors indicating that there is recycling of high affinity CCK receptors but not low affinity CCK receptors. First incubating acini with monensin did not alter CCK-stimulated amylase secretion indicating that in contrast to previous conclusions, occupation of low affinity CCK receptors mediates CCK-stimulated enzyme secretion. Moreover, the biphasic dose-response curve for CCK-stimulated enzyme secretion from monensin-treated acini suggests that pancreatic acini also possess a third, previously unrecognized class of very low affinity CCK receptors.     

Potentiating role of cyclic AMP in pancreatic enzyme secretion, demonstrated by means of forskolin

The role of cyclic AMP in the regulation of enzyme secretion by the rabbit pancreas has been investigated by means of forskolin, an activator of the catalytic subunit of adenylate cyclase. Forskolin increases the cyclic AMP level in isolated pancreatic acini in a dose-dependent way. Basal amylase release, however, remains unchanged. Forskolin potentiates the increase in amylase release induced by the C-terminal octapeptide of cholecystokinin (CCK-8). Potentiation is already apparent at hormone concentrations which are only marginally effective in stimulating amylase secretion. CCK-8 alone does not raise the cellular cAMP level, but it potentiates the forskolin-induced increase. In relative terms, potentiation is higher with decreasing concentration of forskolin. These results indicate that cAMP alone does not play a direct role in CCK-stimulated pancreatic enzyme secretion in the rabbit, but it potentiates enzyme secretion already stimulated through a cAMP-independent process.     

Increased lung uptake of liposomes coated with polysaccharides

The effect of amiloride on fluid and protein secretion in the isolated rabbit pancreas and on amylase secretion in rabbit pancreatic acini has been studied. Amiloride (1 mM) has no effect on the pancreatic fluid secretion either in a normal incubation medium (143 mM Na+), or in a medium containing only 25 mM Na+. The carbachol-induced enzyme secretion is inhibited by amiloride in both systems, whereas the enzyme secretion induced by the C-terminal octapeptide of cholecystokinin ( PzO ) is not affected. Amiloride also inhibits the carbachol-induced 45Ca efflux from rabbit pancreatic acini, but again not that induced by PzO . The amiloride concentrations for half-maximal inhibition of carbachol-induced amylase secretion and 45Ca efflux are 40 and 80 microM, respectively. Amiloride also competitively inhibits the specific binding of [3H]quinuclidinyl benzylate ( [3H]QNB) to rabbit pancreatic acini, suggesting that the amiloride effect is due to competition on the level of the muscarinic acetylcholine receptor.     

Action of cholera toxin on dispersed acini from guinea pig pancreas

In dispersed acini from guinea pig pancreas cholera toxin bound reversibly to specific membrane binding sites to increase cellular cyclic AMP and amylase secretion. Cholera toxin did not alter outflux of ⁴⁵Ca or cellular cyclic AMP. Binding of ¹²⁵I-labeled cholera toxin could be detected within 5 min; however, cholera toxin did not increase cyclic AMP or amylase release until after 40 min of incubation. There was a close correlation between the dose vs. response curve for inhibition of bindind of ¹²⁵I-labeled cholera toxin by native toxin and the action of native toxin on cellular cyclic AMP. With different concentrations of cholera toxin, maximal stimulation of amylase release occurred when the increase in cellular cyclic AMP was approximately 35% of maximal. Cholera toxin did not alter the increase in ⁴⁵Ca outflux or cellular cyclic GMP caused by cholecystokinin or carbachol but significantly augmented the increase in cellular cyclic AMP caused by secretion or vasoactive intestinal peptide. The increase in amylase secretion caused by cholera toxin plus secretin or vasoactive intestinal peptide was the same as that with cholera toxin alone. On the other hand, the increase in amylase secretion caused by cholera toxin plus cholecystokinin or carbachol was significantly greater than the sum of the increases caused by each agent alone.     

COOH-terminal fragments of cholecystokinin. A new class of cholecystokinin receptor antagonists

COOH-terminal fragments of cholecystokinin varying in length from 1 to 3 amino acids and their NH2-terminal butyloxycarbonyl derivatives were investigated for their ability to interact with the cholecystokinin receptor on dispersed acini from guinea pig pancreas. No fragment stimulated amylase secretion when present alone, but each of the butyloxycarbonyl derivatives and the COOH-terminal tripeptide amide inhibited the stimulation of enzyme secretion by cholecystokinin. In each case the inhibition was surmounted by increasing the concentration of cholecystokinin. Each fragment also inhibited binding of 125I-labeled cholecystokinin, with significant inhibition occurring with 30 microM butyloxycarbonyl tripeptide amide, 0.3 mM butyloxycarbonyl dipeptide amide, 10 mM butyloxycarbonyl phenylalanine amide and 3 mM tripeptide amide of cholecystokinin. In each case, there was a close correlation between the ability of the fragment to inhibit binding of 125I-labeled cholecystokinin and its ability to inhibit cholecystokinin-stimulated amylase release, cholecystokinin-stimulated 45Ca outflux and cholecystokinin-stimulated residual stimulation of amylase secretion. The inhibition of amylase secretion caused by the butyloxycarbonyl tripeptide of cholecystokinin was reversible and specific for those peptides which interact with the cholecystokinin receptor (i.e., cholecystokinin, caerulein, gastrin); it did not inhibit the actions of bombesin, carbachol, physalaemin, vasoactive intestinal peptide, secretin, PHI, ionophore A23187 or 8-bromo cyclic AMP. These results demonstrate that COOH-terminal fragments of cholecystokinin comprise a new class of cholecystokinin receptor antagonists.     

The effects of γ-hexachlorocyclohexane on amylase secretion and inositol phospholipid metabolism in mouse pancreatic acini

Dispersed mouse and guinea-pig pancreatic acini were used to examine the effects of the inositol analogue, γ-hexachlorocyclohexane (lindane) on agonist-stimulated amylase secretion. Secretion from mouse acini in response to carbachol and cholecystokinin octapeptide (CCK-8) was reduced by lindane. Similarly, amylase release from guinea-pig acini stimulated by carbachol was abolished by lindane. These acini, however, still remained responsive to dibutyryl-cAMP with only a slightly diminished secretion to this agent. Inositol phospholipid synthesis and hydrolysis was stimulated in mouse acini by both carbachol and CCK-8. Although hydrolysis of these lipids in response to CCK-8 was reduced by only 18%, stimulation of inositol phospholipid synthesis by either agonist was abolished by lindane. Dose-response curves for inositol phospholipid synthesis stimulated by carbachol and CCK-8 in mouse acini were biphasic and superimposable with those of amylase secretion. In contrast, the dose-response curve for phosphoinositide hydrolysis was sigmoid and clearly separable from that of synthesis. Reducing the external Ca²⁺ concentration caused the dose-response curves for carbachol- and CCK-8-induced inositol phospholipid synthesis to be displaced to the right, as has been observed for amylase secretion. A23187 was also found to induce amylase secretion and inositol phospholipid synthesis, and both of these responses were inhibited by lindane. Amylase secretion and inositol phospholipid synthesis may, therefore, be closely related events in the exocrine pancreas. Lindane may provide a valuable tool with which to determine the role of inositol phospholipid metabolism in stimulus-response coupling.     

An analogue of substance P with broad receptor antagonist activity

[DPro4,DTrp7,9,10]Substance P-4-11 functions as a substance P receptor antagonist in several different systems. Because some analogues of substance P can function as receptor antagonists for bombesin as well as substance P, we tested [DPro4,DTrp7,9,10]substance P-4-11 for its ability to modify the interaction of various pancreatic secretagogues with their receptors in dispersed acini from guinea pig pancreas. [DPro4,DTrp7,9,19]Substance P-4-11 did not stimulate amylase secretion and did not alter the stimulation of amylase secretion caused by secretin, vasoactive intestinal peptide, calcitonin gene-related peptide or carbachol, but did inhibit the stimulation of amylase secretion caused by substance P, bombesin or cholecystokinin. With substance P, bombesin and cholecystokinin, [DPro4,DTrp7,9,10]substance P-4-11 caused a parallel rightward shift in the dose-response curve for stimulation of amylase secretion with no change in the maximal response. Schild plots of these results gave straight lines with slopes that were not significantly different from unity. [DPro4,DTrp7,9,10]Substance P-4-11 inhibited binding of 125I-labeled substance P, 125I-[Tyr4]bombesin and 125I-cholecystokinin octapeptide over the same range of concentrations as that in which it inhibited biologic activity of each of these peptides. Half-maximal inhibition of binding of 125I-substance P occurred with 4 microM, of 125I-[Tyr4]bombesin with 17 microM and of 125I-cholecystokinin octapeptide with 5 microM. With each radiolabeled peptide the value of Ki for inhibition of binding by [DPro4,DTrp7,9,10]substance P-4-11 was not significantly different from the corresponding value of Ki calculated from the appropriate Schild plot. The present results indicate that [DPro4,DTrp7,9,10]substance P-4-11 is a competitive antagonist at receptors for substance P, for bombesin and for cholecystokinin. Thus, these receptors must share a common peptide recognition mechanism even though they interact with agonists that have no obvious structural similarity.     

Amiloride is a cholinergic antagonist in the rabbit pancreas

The effect of amiloride on fluid and protein secretion in the isolated rabbit pancreas and on amylase secretion in rabbit pancreatic acini has been studied. Amiloride (1 mM) has no effect on the pancreatic fluid secretion either in a normal incubation medium (143 mM Na⁺), or in a medium containing only 25 mM Na⁺. The carbachol-induced enzyme secretion is inhibited by amiloride in both systems, whereas the enzyme secretion induced by the C-terminal octapeptide of cholecystokinin (PzO) is not affected. Amiloride also inhibits the carbachol-induced ⁴⁵Ca efflux from rabbit pancreatic acini, but again not that induced by PzO. The amiloride concentrations for half-maximal inhibition of carbachol-induced amylase secretion and ⁴⁵Ca efflux are 40 and 80 μM, respectively. Amiloride also competitively inhibits the specific binding of [³H]quinuclidinyl benzylate ([³H]QNB) to rabbit pancreatic acini, suggesting that the amiloride effect is due to competition on the level of the muscarinic acetylcholine receptor.     

Action of cholecystokinin, cholinergic agents, and A-23187 on accumulation of guanosine 3':5'-monophosphate in dispersed guinea pig pancreatic acinar cells.

The COOH-terminal octapeptide of cholecystokinin (CCK-OP) and carbamylcholine each increased calcium outflux, cellular cyclic GMP and amylase secretion in dispersed guinea pig pancreatic acinar cells. Following addition of CCK-OP or carbamylcholine, cellular cyclic GMP increased as early as 15 s, became maximal after 1 to 2 min, and then decreased steadily during the subsequent incubation. For both CCK-OP and carbamylcholine there was close agreement between the dose-response curve for stimulation of calcium outflux and that for increase of cellular cyclic GMP. With CCK-OP an effect on both functions could be detected at 10(-10) M and maximal stimulation occurred at 3 X 10(-8) M. With carbamylcholine an effect on both functions could be detected at 10(-5) M and maximal stimulation occurred at 3 X 10(-3) M. Atropine inhibited stimulation of both cyclic GMP and calcium outflux by carbamylcholine but not by CCK-OP. Stimulation of calcium outflux or cellular cyclic GMP by CCK-OP or carbamylcholine did not require extracellular calcium since stimulation occurred in a calcium-free, ethylene glycol bis(beta, beta-aminoethyl ether) N,N'-tetraacetic acid (EGTA)-containing solution. The divalent cation ionophore A-23187 increased bidirectional fluxes of calcium, cellular cyclic GMP and secretion of amylase from dispersed pancreatic acinar cells. Like CCK-OP and carbamylcholine, the ionophore stimulated calcium outflux and cellular cyclic GMP in a calcium-free, EGTA-containing solution. These results suggest that in pancreatic acinar cells the initial step in the sequence of events mediating the action of ionophore as well as that of CCK-OP and carbamylcholine is stimulation of calcium outflux, and that this stimulation then increases cellular cyclic GMP.     

Stimulation of the glucose transport system in isolated mouse pancreatic acini by cholecystokinin and analogues.

Cholecystokinin and analogues increased the uptake of 2-deoxy-D-glucose and 3-O-methylglucose into isolated mouse pancreatic acini. This uptake was mediated by a facilitated glucose transport system that was saturable, stereospecific, and was inhibited by both phloretin and cytochalasin B. In agreement with previous studies of acinar function, caerulein was more potent and pentagastrin less potent than cholecystokinin in increasing sugar transport. The cholinergic analogue carbachol mimicked the effect of caerulein; atropine completely abolished the effects of carbachol but was without influence on the effects of the polypeptide hormones. In contrast, secretion, as well as dibutyryl cyclic AMP and dibutyryl cyclic GMP, had no effect on 2-deoxy-D-glucose uptake. Two lines of evidence suggested that hormonal stimulation of this sugar transport system was related to mobilization of cellular Ca2+. First, depletion of cellular Ca2+ by incubation of acini with ethylene glycol bis(beta-aminoethyl ether) N,N,N',N'-tetraacetic acid (EGTA) reduced the effect of caerulein. Second, the Ca2+ ionophore A23187 mimicked the effects of caerulein on 2-deoxy-D-glucose uptake when Ca2+ was present in the medium.