Phorbol ester induces intracellular translocation of phospholipid/Ca2+-dependent protein kinase and stimulates amylase secretion in isolated pancreatic acini

Treatment of intact rat pancreatic acini with phorbol ester (12-O-tetradecanoyl-phorbol-13-acetate, TPA) resulted in a time- and concentration-dependent translocation of phospholipid/Ca2+-dependent protein kinase (PL/Ca-PK) from the soluble fraction. Redistribution of PL/Ca-PK was concurrent with stimulation of amylase secretion by TPA-treated acini. Polymyxin B, a potent and selective inhibitor of PL/Ca-PK completely inhibited TPA-induced amylase secretion. These findings are consistent with a role for PL/Ca-PK in the regulation of pancreatic exocrine secretion.     

Relationship of CCK/gastrin receptor binding to amylase release in dog pancreatic acini

Effects of synthetic peptides belonging to the CCK/gastrin family (CCK-39, CCK-8, G/CCK-4, G-17ns) on amylase release in dog pancreatic acini have been measured and correlated with binding of three radio-labelled CCK/gastrin peptides: ¹²⁵I-BH-(Thr,Nle)-CCK-9, ¹²⁵I-BH-(2–17)G-17ns and ¹²⁵I-BH-G/CCK-4 prepared by conjugation of the peptides to iodinated Bolton-Hunter reagent and purified by reverse-phase-HPLC. All the CCK/gastrin peptides produced the same maximal amylase release response. Half-maximal responses (D₅₀) were obtained with 2 · 10^?10 M CCK-8; 6 · 10^?10 M CCK-39; 10^?7 M G.17 ns and 2 · 10^?6 M G/CCK-4. Dose-response curves for G-17 ns and G/CCK-4 were similar in configuration but not parallel with those for CCK-8 and CCK-39.Binding studies with ¹²⁵I-BH(Thr,Nle)-CCK-9 demonstrated the presence of specific CCK receptors on dog pancreatic acini. There was a good correlation between receptor occupancy by CCK-8 and CCK-39 and amylase stimulation since maximal amylase stimulation was achieved when 40–50% of high affinity receptors were occupied. In contrast, a saturation of these receptors was required for maximal stimulation by G-17 ns and G/CCK-4 suggesting the existence of a fraction of receptors that can be occupied by G-17 ns and G/CCK-4 without stimulation of amylase release. Binding studies with labelled (2–17)-G-17 ns and G/CCK-4 confirmed the presence of high affinity sites for G-17 ns and G/CCK-4. These sites were not related to amylase release.This study points out a possible species specificity of biological action of gastrin/CCK peptides on pancreatic exocrine secretion in higher mammals.     

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.     

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.     

Photodynamic action of rose bengal on isolated rat pancreatic acini: stimulation of amylase release

The halogenated fluorescein derivative, rose bengal, upon photon activation, elicits amylase secretion from isolated, perifused pancreatic acini. This effect is due to production of highly reactive singlet delta oxygen which can permeabilize the cell membrane and may also react chemically with secretagogue receptors, or other functional components of the membrane such as the G-proteins. The profile of photodynamically induced amylase secretion is anion-dependent: it becomes biphasic when the chloride ion is substituted by the glutamate ion, an effect attributed to the action of glutamate on the ionic transport systems of the zymogen granule membrane.     

Effect of pancreatic polypeptide-antiserum on bombesin-stimulated pancreatic exocrine secretion in dogs

Bombesin is a potent stimulus of both pancreatic protein secretion and plasma pancreatic polypeptide (PP) release in dogs. Physiological plasma levels of PP have been shown to inhibit pancreatic exocrine secretion in dogs. We examined the question whether the concomitant release of PP exerts a suppressive action on the pancreatic exocrine response to bombesin in dogs by measuring pancreatic exocrine secretion with and without in vivo immunoneutralization of PP with a high affinity PP-antiserum. Bombesin was infused in a dose of 150 ng/kg·hr, resulting in a rise of plasma PP from 24±5 to 224±25 pM (p<0.01). Before this bombesin infusion, 7 ml of normal rabbit serum had been administered to the dogs (n=8). At a later stage, the study was repeated after administration of 7 ml of PP-antiserum to the same animals. The bombesin induced increase in pancreatic exocrine secretion during administration of PP-antiserum (flow rate 24±10 ml/hr, protein output 1.35±0.43 g/hr, and bicarbonate output 3.25±1.42 mmol/hr) was not significantly different from that during control rabbit serum (flow rate 21±7 ml/hr, protein output 1.26±0.38 g/hr, and bicarbonate output 3.18±1.10 mmol/hr). It is therefore concluded that the pancreatic exocrine response to bombesin is not affected by the concomitant secretion of PP.     

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.     

Effects of ionophore A23187 on calcium flux and amylase release in isolated mouse pancreatic acini

The divalent cation ionophore A23187 has been used extensively to demonstrate the importance of Ca²⁺ in the control of pancreatic enzyme secretion. The relative importance, however, of the ability of the ionophore to facilitate Ca²⁺ movement across plasma and intracellular membranes in the stimulation of amylase release is not clear. We therefore studied these relationships in isolated pancreatic acini, a preparation in which it is possible to precisely measure both ⁴⁵Ca²⁺ fluxes, Ca²⁺ content and amylase release. A23187 increased the initial rates of both ⁴⁵Ca²⁺ uptake and washout. In addition, the content of both exchangeable ⁴⁵Ca²⁺ and total Ca²⁺ were reduced. These results indicated, therefore, that A23187 increases Ca²⁺ fluxes across both plasma and intracellular membranes. Consistent with this observation, the initial stimulation of amylase release by A23187 was independent of extracellular Ca²⁺. In the absence of extracellular Ca²⁺, however, A23187 caused a rapid fall in acinar Ca²⁺ and subsequent amylase release was abolished. Depletion of intracellular Ca²⁺ by the ionophore also blocked the subsequent stimulation by cholecystokinin (CCK). The results indicate certain similarities in the actions of A23187 and CCK on pancreatic acini; both the agonists have striking effects on intracellular Ca²⁺ which in turn mediates their actions.     

Effect of AA861, a 5-lipoxygenase inhibitor,on amylase secretion from rat pancreatic acini

We have examined the effects of 2,3,5-trimethyl-6-(12-hydroxy-5,10-dodecadiynyl)-1,4-benzoquinone (AA861), a selective inhibitor of 5-lipoxygenase, on the action of cholecystokinin (CCK) and other secretagogues in the stimulation of amylase secretion from dispersed rat pancreatic acini. AA861 inhibited amylase secretion caused by CCK, carbamylcholine (carbachol), bombesin or calcium ionophore A23187 but failed to affect amylase secretion by vasoactive intestinal peptide or 12-O-tetradecanoyl-phorbol 13-acetate. Inhibition by AA861 of CCK or carbachol-induced amylase secretion was confined to the relatively lower concentrations of these secretagogues. AA861 did not inhibit receptor binding of CCK or alter the cellular calcium mobilization induced by CCK. In kinetic studies, AA861 was effective only on amylase secretion from pancreatic acini incubated with CCK for more than 5 min. Indomethacin, a known inhibitor of cyclooxygenase, did not affect the amylase secretion caused by all secretagogues used. These results indicate that the 5-lipoxygenase pathway of arachidonate metabolism may be involved in the actions of calcium-dependent secretagogues of amylase secretion in rat dispersed pancreatic acini, especially for sustaining stimulation of amylase secretion by CCK.     

Dominant negative Rab3D inhibits amylase release from mouse pancreatic acini

Rab3 proteins are believed to play an important role in regulated exocytosis and previous work has demonstrated the presence of Rab3D on pancreatic zymogen granules. To further understand the function of Rab3D in acinar cell exocytosis, adenoviral constructs were prepared encoding hemagglutinin-tagged wild type Rab3D and three mutant forms, N135I and T36N (both deficient in guanine nucleotide binding) and Q81L (deficient in GTP hydrolysis), which also expressed enhanced green fluorescent protein driven by a separate promoter. When isolated mouse pancreatic acini were cultured with 5 x 10(6) pfu/ml adenovirus, nearly 100% of acini were infected as visualized by expression of green fluorescent protein. Cultured acini showed a biphasic dose-response to cholecystokinin (CCK); basal amylase secretion was 1.8 +/- 0.3%/30 min, peak release was 7.3 +/- 0.2%/30 min at 30 pm CCK and reduced secretion was observed at higher CCK concentrations. Control beta-galactosidase virus infection had no effect on either basal or CCK-induced secretion in the titer range from 0.5 to 10 x 10(6) pfu/ml. While the expression of Rab3D and Rab3D Q81L had no effect on amylase secretion, Rab3D N135I and T36N functioned as dominant negative mutants and inhibited CCK-induced amylase release by 40-50% at all points on the CCK dose-response curve from 3 to 300 pm. Inhibition was stronger during the first 5 min (71 +/- 5%) than over 30 min (36%+/-5%). Similar inhibition was found using other agonists including bombesin, carbachol, and cAMP. Localization of adenoviral expressed Rab protein showed wild type Rab3D localized to zymogen granules. The two dominant negative mutants did not localize to granules and were primarily in the basolateral region of the cell. Since both dominant negative Rab3D mutants had no effect on intracellular calcium increase induced by CCK, it is unlikely that they acted at receptors or transmembrane signaling. These results suggest that Rab3D plays an important role in regulating the terminal steps of acinar exocytosis and that this effect is greatest on the early phase of amylase release.     

Electrophoretic and cytological evidence for heterogeneity of pancreatic acinar cell responsiveness to carbachol,caerulein and secretin

Summary Incubation of rat pancreatic lobules for 90 min with optimal concentrations of caerulein, carbachol or secretin caused the release of about 30% of the amylase content. Combination of secretin with carbachol or caerulein increased the amylase output to about 40%. With secretin, as with carbachol or caerulein, heterogeneity of cellular responsiveness was observed, some acini being partially or completely depleted of their zymogen granules, whereas others appeared to be resting. When secretin was combined with carbachol or caerulein, granule depletion, originally confined to small groups of neighbouring acini, spread to form large areas of degranulated cells, sometimes comprising a whole section of a lobule.In dispersed acini, under the same conditions, carbachol caused the release of about 60% of the amylase content, and secretin 40%. When both secretagogues were combined, a significant increase to 78% was observed. Under these conditions, there was some important cellular damage, as indicated by the release of 20% of the amylase content and between 6 and 12% of lactate dehydrogenase into the media, in the absence of stimulus. These results were corroborated by cytological observations. On the basis of their secretory response two groups of acini can be distinguished, those that respond to carbachol, caerulein or secretin and those that respond to the combination of secretin with carbachol or caerulein. Electrophoretic patterns of secretory proteins released by lobules stimulated by these different types of secretagogues were essentially similar. The pattern was quite different, however, in the absence of a stimulus. The most striking feature was the presence of a band at 63 Kd whereas a 73.5 Kd band was found only under conditions of stimulation. The latter results support the view that under resting and stimulated conditions secretory proteins are released from distinct compartments in the acinar cell.Abbreviations used PMSF phenylmethylsulfonyl fluoride - Carbachol carbamylcholine chloride - SBTI soybean trypsin inhibitor     

The importance of the amino acid in position 32 of cholecystokinin in determining its interaction with cholecystokinin receptors on pancreatic acini

In the C-terminal heptapeptide of cholecystokinin, replacement of the penultimate residue, aspartic acid, by β-alanine caused a 300-fold decrease in potency with which the peptide stimulated enzyme secretion, whereas replacement by glutamic acid caused a 1000-fold decrease in potency. The β-alanine-substituted peptide was approximately ten times more potent when the N terminus was blocked with t-butyloxycarbonyl than when it was blocked with benzyloxycarbonyl, and the glutamic acid-substituted peptide was approximately twice as potent when the N terminus was blocked with t-butyloxycarbonyl than when it was blocked with benzyloxycarbonyl. Changes in the ability of the peptide to stimulate amylase secretion were acompanied by corresponding changes in the ability of the peptide to inhibit binding of ¹²⁵I-labeled cholecystokinin. The magnitude of stimulation of enzyme secretion caused by a maximally effective peptide concentration was the same with each analogue as it was with the unaltered peptide. Rpelacing the aspartyl by β-alanine or glutamic acid or replacing of N-terminal t-butyloxycarbonyl moiety by benzyloxycarbonyl caused an equivalent decrease in the ability of the peptide to stimulate enzyme secretion and its ability to cause residual stimulation of enzyme secretion. In contrast, the N-terminal desamino analogue of cholecystokinin heptapeptide was ten times less potent than the unaltered peptide in stimulating amylase secretion, but 100 times less potent that the unaltered peptide in causing residual stimulation of enzyme secretion.     

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.     

Introduction of calcium chelators into isolated rat pancreatic acini inhibits amylase release in response to carbamylcholine

The Ca2+ chelators, EGTA and BAPTA, have been introduced into intact, isolated rat pancreatic acini using a hypotonic swelling method. This resulted in complete inhibition of amylase release, stimulated by carbamylcholine at a submaximal concentration and 82 - 85% inhibition at maximal concentrations. Acini swollen in the absence of Ca2+ chelators showed similar secretory responses to those of unswollen acini. Treatment of unswollen acini with chelators inhibited the maximum response to carbamylcholine by only 23%. The inhibitory effect of intracellular chelators was not due to ATP depletion or a lowering of the total cell Ca2+ content. Thus, these results provide the first direct demonstration that an increase in intracellular Ca2+ concentration is necessary for the stimulation of enzyme release from pancreatic acinar cells.     

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.     

Reversible acetaldehyde inhibition of A23187-stimulated amylase secretion from isolated rat pancreatic acini

The Ca2+ ionophore, A23187, stimulated amylase secretion from isolated rat pancreatic acini in a dose-dependent manner with a maximal effect at 6 microM. Acetaldehyde, a metabolite of ethanol, caused a reduction in the magnitude of ionophore-stimulated secretion with no evidence of competitive inhibition. Furthermore, 6 microM ionophore-stimulated amylase secretion was dose-dependently inhibited by acetaldehyde. This inhibitory effect of acetaldehyde, however, was reversible on washing and reincubating acetaldehyde-treated acini. These results suggest that acetaldehyde reversibly inhibits intracellular components mediating stimulated secretion and this inhibition requires a continuous chemical interaction between acetaldehyde and intracellular component(s) regulating stimulated enzyme secretion.     

Effect of an intracellular calcium antagonist (TMB-8) on carbamylcholine-induced amylase release from dispersed rat pancreatic acini

During 10-min incubation with increasing concentrations of carbamylcholine (carbachol), amylase release from dispersed rat pancreatic acini increased, became maximal at 2 X 10(-6)M and then decreased. In the concentration range of 10(-7)M to 10(-4)M, 8-(N,N-diethylamino)-octyl 3,4,5-trimethoxybenzoate hydrochloride (TMB-8) caused a dose-dependent inhibition of amylase release induced by a submaximal concentration of carbachol. No inhibitory effect was observed on basal and secretin-stimulated amylase release. TMB-8 showed a significantly greater ability of blocking the action of carbachol than verapamil and diltiazem. TMB-8 could reverse the submaximal stimulation of amylase release caused by supramaximal concentrations of carbachol to a maximal stimulation, while verapamil and diltiazem could not. These results confirm the hypothesis that mobilization of intracellular calcium is the primary step in the action of carbachol on pancreatin acinar cells and contributes to the submaximal secretory response of acinar cells induced by high concentrations of carbachol.     

Effects of an inhibitor of myosin light chain kinase on amylase secretion from rat pancreatic acini

Ca(2+)/calmodulin-dependent protein (CaM) kinases play an important role in Ca(2+)-mediated secretory mechanisms. Previously, we demonstrated that a CaM kinase II inhibitor KN-62 had a small inhibitory effect on amylase secretion stimulated by CCK. In the present study, we investigated the effects of a myosin light chain kinase (MLCK) inhibitor on amylase secretion and Ca(2+) signaling in rat pancreatic acini. A specific inhibitor of MLCK, wortmannin, inhibited amylase secretion stimulated by CCK-8 (30 pM) in a concentration-dependent manner. Wortmannin (10 microM) had no effects on basal secretion but reduced amylase secretion stimulated by CCK-8 (30 pM) by 67 +/- 3%. Wortmannin inhibited amylase secretion stimulated by calcium ionophore (A23187) and phorbol ester (TPA). Wortmannin also inhibited amylase response to thapsigargin by 76 +/- 8% and to both thapsigargin and TPA by 52 +/- 10%. Ca(2+) oscillations evoked by CCK-8 (10 pM) were inhibited by wortmannin (10 microM). Wortmannin had a little inhibitory effect on an initial rise in [Ca(2+)](i), and abolished a subsequent sustained elevation of [Ca(2+)](i) evoked by 1 nM CCK-8. In conclusion, MLCK plays a crucial role in amylase secretion from pancreatic acini and regulates Ca(2+) entry from the extracellular space.     

Interaction of secretin5–27 and its analogues with hormone receptors on pancreatic acini

The C-terminal tricosapeptide of secretin (S_5–27) and two analogues, one with asparagine replacing aspartic acid in position 15 (15-Asn-S_5–27) and one with lysine replacing aspartic acid in position 15 (15-Lys-S_5–27) were tested for their abilities to interact with hormone receptors on pancreatic acinar cells. In interacting with the receptors which prefer vasoactive intestinal peptide (vaso-active intestinal peptide-preferring receptors), the apparent affinity of 15-Asn-S_5–27 was equal to that of 15-Lys-S_5–27 and was greater than that of S_5–27. In interacting with secretin-preferring receptors, the apparent affinity of 15-Asn-S_5–27 was equal to that of S_5–27 and was greater than that of 15-Lys-S_5–27. In interacting with the secretin-preferring receptors each of the secretin fragments was approximately 2% as effective as secretin in causing an increase in cellular cyclic AMP. None of these fragments was able to cause a detectable increase in cyclic AMP mediated by the vasoactive intestinal peptide-preferring receptors. The dose vs. response curves for the action of secretin and vasoactive intestinal peptide on cellular cyclic AMP and on amylase secretion as well as the patetern of effects of secretin fragments on these actions indicated that the increase in amylase secretion caused by vasoactive intestinal peptide and secretin is mediated exclusively by the vasoactive intestinal peptide-preferring receptors. Furthermore, occupation of approximately 50% of the vasoactive intestinal peptide-preferring receptors is sufficient to cause maximal stimulation of amylase secretion.