Duck Pancreatic Acinar Cell as a Unique Model for Independent Cholinergic Stimulation–Secretion Coupling

This paper investigated the role of acetylcholine (ACh) in physiological regulation of amylase secretion in avian exocrine pancreas. In the isolated duck pancreatic acini, ACh dose dependently stimulated amylase secretion, with a maximal effective concentration at 10 μM. The cAMP-mobilizing compounds forskolin, vasoactive intestinal peptide (VIP)/pituitary adenylate cyclase activating peptide (PACAP) receptor (VPAC) agonists PACAP-38 and PACAP-27 had no effect on the dose–response curve. ACh dose dependently induced increases in cytosolic Ca²⁺ concentration ([Ca²⁺] _c ), with increasing concentrations transforming oscillations into plateau increases. Forskolin (10 μM), PACAP-38 (1 nM), PACAP-27 (1 nM), or VIP (10 nM) alone did not stimulate [Ca²⁺] _c increase; neither did they modulate ACh-induced oscillations, nor made ACh low concentration effective. These data indicate that ACh-stimulated zymogen secretion in duck pancreatic acinar cells is not subject to modulation from the cAMP signaling pathway; whereas it has been widely reported in the rodents that ACh-stimulated exocrine pancreatic secretion is significantly enhanced by cAMP-mobilizing agents. This makes the duck exocrine pancreas unique in that cholinergic stimulus-secretion coupling is not subject to cAMP regulation.     

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.     

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)     

Endothelin increases [Ca2+]i in rat pancreatic acinar cells by intracellular release but fails to increase amylase secretion.

In individual fura-2 loaded cells of rat pancreatic acini endothelin-1 (ET-1) (10-50 nM) induced sustained oscillations in [Ca2+]i. At higher concentrations a larger, but transient increase in [Ca2+]i was observed, which was largely unaffected by removal of extracellular Ca2+. ET-1 induced the release of Ca2+i from the same store as cholecystokinin (CCK), but with less potency. At concentrations of endothelin which transiently increased Ca2+, ET-1 increased the accumulation of inositol phosphates. Specific binding sites for 125I-endothelin were demonstrated on rat pancreatic acini. A single class of binding sites was identified with an apparent Kd 108 +/- 12 pM and Bmax of 171 +/- 17 fmol/mg for ET-1. The relative potency order for displacing [125I]ET was ET-1 greater than ET-2 greater than ET-3. In contrast to CCK and the non-phorbol ester tumour promoter Thapsigargin (TG) which induce both transient and sustained components of [Ca2+]i elevation, ET-1 failed to increase amylase release over the range 100 pM-1 microM.     

Calcium concentration and amylase secretion in guinea pig pancreatic acini: interactions between carbachol, cholecystokinin octapeptide and the phorbol ester, 12-O-tetradecanoylphorbol 13-acetate

The effects of the phorbol ester, 12-O-tetradecanoylphorbol 13-acetate (TPA) on amylase secretion and cytoplasmic free calcium concentration ([Ca2+]i) were investigated in dispersed guinea pig pancreatic acini. Carbachol evoked dose-dependent increases in amylase secretion and [Ca2+]i with half-maximal responses at 2.5 and 5 microM, respectively. Carbachol-induced calcium transients could be blocked by atropine. In the presence of a maximal effective dose of carbachol, cholecystokinin octapeptide caused no further increase in [Ca2+]i, suggesting that both agonists act on the same pool of trigger calcium. TPA (10(-9)-10(-6) M) stimulated amylase secretion with no change in [Ca2+]i. Maximum amylase secretion occurred at 0.5 microM TPA. Preincubation of acini in the presence of TPA resulted in a time- and dose-dependent inhibition (IC50 = 30 nM) of the carbachol-induced rise in [Ca2+]i, the maximal effect being observed within 3 min. The inactive phorbol ester, 4 alpha-phorbol 12,13-didecanoate was ineffective in inhibiting the carbachol-stimulated rise in [Ca2+]i. These findings suggest that, in addition to stimulating amylase secretion, probably through protein kinase C, TPA may also exert a negative feedback control over secretagogue-induced calcium transients.     

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.     

Nonparallel discharge of digestive enzymes from isolated pancreatic acini

The secretion of amylase, trypsinogen, chymotrypsinogen and proelastase from isolated rat dispersed pancreatic acini was investigated in the absence (basal) and presence of two concentrations of CCK8 (50 and 500 pM), carbachol (2.5 and 7.5 microM) and secretin (10 nM and 1 microM). The unstimulated (basal) rate of release of each of the digestive enzymes was essentially the same. However, whereas both doses of CCK8 and carbachol caused a preferential release of chymotrypsinogen over that of amylase and trypsinogen, the magnitude of stimulated release of amylase, trypsinogen and chymotrypsinogen by 1 microM secretin was found to be similar for each of the enzymes. Furthermore, none of the secretagogues caused a significant enhancement in proelastase release. The present data demonstrate that whereas CCK8 and carbachol induce a greater release of chymotrypsinogen over that of amylase or trypsinogen, release of all three enzymes was equally stimulated by secretin from isolated pancreatic acini.     

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.     

A synthetic peptide derivative that is a cholecystokinin receptor antagonist

So far, there are no known peptidic effective receptor antagonists of both peripheral and central effects of cholecystokinin (CCK). Here, we describe a synthetic peptide derivative of CCK, t-butyloxycarbonyl-Tyr(SO3-)-Met-Gly-D-Trp-Nle-Asp 2-phenylethyl ester 1 (where Nle is norleucine), which is a potent CCK receptor antagonist. In rat and guinea pig dispersed pancreatic acini, this peptide derivative did not alter amylase secretion, but was able to antagonize the stimulation caused by cholecystokinin-related agonists. It caused a parallel rightward shift in the dose-response curve for the stimulation of amylase secretion with half-maximal inhibition of CCK-8-stimulated amylase release at a concentration of about 0.1 microM. Compound 1 was able to inhibit the binding of labeled CCK-9 (the C-terminal nonapeptide of CCK) to rat and guinea pig pancreatic acini (IC50 = 5 X 10(-8) M) as well as to guinea pig cerebral cortical membranes (IC50 = 5 X 10(-7) M). These results indicate that Compound 1 is a potent competitive CCK receptor antagonist.     

Role of calcium and cAMP in the action of adrenocorticotropin on aldosterone secretion

When the dose-response curve of adrenocorticotropin (ACTH)-induced aldosterone secretion is compared to that of ACTH-induced intracellular cAMP, the ED50 for intracellular cAMP is more than 10 times as high as that for aldosterone production. In contrast, the dose-response curve of forskolin-induced aldosterone secretion correlates well with that for forskolin-induced intracellular cAMP. ACTH, but not forskolin, increases calcium influx into glomerulosa cells without inducing the mobilization of calcium from an intracellular pool. The effect of ACTH on calcium influx is dose-dependent and ED50 is 3.5 X 10(-11) M. In a perifusion system, the effect of 1 nM ACTH on aldosterone secretion is much greater than that of 1 microM forskolin, even though these two stimulators induce identical increases in the intracellular cAMP. Perifusion with combined A23187 (50 nM) and forskolin (1 microM) stimulates aldosterone secretion to a value comparable to that induced by 1 nM ACTH. Likewise, BAY K 8644 (1 nM), which induces a comparable increase in calcium influx, potentiates the effect of 1 microM forskolin. When the intracellular [Ca2+] is fixed at either 100 or 300 nM, forskolin-stimulated intracellular cAMP content is identical, but ACTH-stimulated intracellular cAMP content at 100 nM [Ca2+]i is 60% of that at 300 nM [Ca2+]i. Both the ACTH- and forskolin-induced aldosterone secretion rate is higher at 300 nM than at 100 nM [Ca2+]i. These results indicate that ACTH stimulates calcium influx, that calcium potentiates ACTH-induced but not forskolin-induced cAMP generation, and that Ca2+ and cAMP act as synarchic messengers in ACTH-mediated aldosterone secretion.     

Effects of gamma-aminobutyric acid on secretagogue-induced exocrine secretion of isolated, perfused rat pancreas

Because GABA and its related enzymes have been determined in beta-cells of pancreas islets, effects of GABA on pancreatic exocrine secretion were investigated in the isolated, perfused rat pancreas. GABA, given intra-arterially at concentrations of 3, 10, 30, and 100 microM, did not exert any influence on spontaneous or secretin (12 pM)-induced pancreatic exocrine secretion. However, GABA further elevated CCK (10 pM)-, gastrin-releasing peptide (100 pM)-, or electrical field stimulation-induced pancreatic secretions of fluid and amylase dose dependently. The GABA (30 microM)-enhanced CCK-induced pancreatic secretions were completely blocked by bicuculline (10 microM), a GABA(A) receptor antagonist, but were not affected by saclofen (10 microM), a GABA(B) receptor antagonist. The enhancing effects of GABA (30 microM) on CCK-induced pancreatic secretions were not changed by tetrodotoxin (1 microM) but were partially reduced by cyclo-(7-aminoheptanonyl-Phe-D-Trp-Lys-Thr[BZL]) (10 nM), a somatostatin antagonist. In conclusion, GABA enhances pancreatic exocrine secretion induced by secretagogues, which predominantly induce enzyme secretion, via GABA(A) receptors in the rat pancreas. The enhancing effect of GABA is partially mediated by inhibition of islet somatostatin release.     

Mode of inhibitory action of cholecystokinin in amylase release from isolated rat pancreatic acini--inhibition of secretory process post to protein kinase C-calcium ion systems

The incubation of isolated rat pancreatic acini with low doses (1 x 10(-11)-1 x 10(-10) M) of cholecystokinin-octapeptide (CCK8) induced amylase release. This CCK8-induced amylase release has been shown to be mediated through the protein kinase C activation and the Ca2+ mobilization which are linked to the phospholipase C-mediated hydrolysis of phosphoinositides. However, the incubation of the acini with high doses (1 x 10(-9)-1 x 10(-7) M) of CCK8 reduced amylase release to the level less than that induced by the maximally effective dose (1 x 10(-10) M) of this secretagogue. Under the same conditions, the high doses of this secretagogue did not inhibit the phospholipase C-mediated hydrolysis of phosphoinositides. The stimulatory action of the maximally effective dose of CCK8 in amylase release was mimicked by the simultaneous addition of protein kinase C-activating 12-O-tetradecanoylphorbol-13-acetate (TPA) and Ca2+ ionophore A23187. A high dose (1 x 10(-7) M) of CCK8 reduced the amylase release induced by the combination of TPA and A23187. These results suggest that the high doses of CCK8 inhibit the secretory process post to the protein kinase C-Ca2+ systems and thereby reduce the amylase release induced by the maximally effective dose of CCK8 in rat pancreatic acini.     

Cytosolic Ca2+ responses to sub-picomolar and nanomolar PACAP in pancreatic beta-cells are mediated by VPAC2 and PAC1 receptors

Pituitary adenylate cyclase-activating polypeptide (PACAP) potentiates glucose-induced insulin release and increases cytosolic Ca2+ concentration ([Ca2+]i) in islet beta-cells in a concentration-dependent manner with two peaks at 10(-13) and 10(-9) M. PAC1 receptor (PAC1-R) and VPAC2 receptor (VPAC2-R) are expressed in pancreatic beta-cells and thought to be involved in insulin release. We aimed to determine the receptor types involved in the [Ca2+]i responses to 10(-13) and 10(-9) M PACAP. We measured [Ca2+]i in beta-cells and examined comparative effects of PAC1-R-selective agonist maxadilan, its antagonist M65, VPAC2-R-selective agonist Ro25-1553, and native ligands of PACAP and VIP. In the presence of 8.3 mM glucose, maxadilan, Ro25-1553, PACAP, and VIP at 10(-13) and 10(-9) M all increased [Ca2+]i. PACAP and maxadilan elicited greater effects at 10(-9) M than at 10(-13) M both in the incidence and amplitude of [Ca2+]i responses. For VIP and Ro25-1553, in contrast, the effects at 10(-9) and 10(-13) M were comparable. Furthermore, the amplitude of [Ca2+]i responses to 10(-9) M PACAP, but not 10(-13) M PACAP, was suppressed by M65. The results suggest that VPAC2-R and PAC1-R contribute equally to [Ca2+]i responses to sub-picomolar concentrations of PACAP, while PAC1-R has greater contribution to [Ca2+]i responses to nanomolar concentrations of this peptide.     

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.     

PACAP-38, a novel peptide from ovine hypothalamus, is a potent modulator of amylase release from dispersed acini from rat pancreas.

Despite studies indicating the presence of specific pancreatic acinar receptors for PACAP-38, a peptide that was recently isolated from ovine hypothalamus, the actions of the new peptide on pancreatic enzyme secretion have not been examined. The present study demonstrates that in terms of cAMP production and amylase release from dispersed acini from rat pancreatic acini, PACAP-38 and an N-terminal fragment, PACAP-27, have the same potency and efficacy as vasoactive intestinal peptide (VIP). As with VIP, these actions are potentiated by adding an inhibitor of cyclic nucleotide phosphodiesterase, and combination of PACAP-38 with bombesin, CCK-8, carbachol or the calcium ionophore A23187 results in 2-fold augmentation of the secretory actions of these agents. Inhibition of PACAP-38-induced cAMP production and amylase release by two VIP-receptor antagonists indicates that the secretory effects of PACAP-38 are mediated by interaction with VIP receptors. PACAP-38, a new brain-gut peptide, may be a physiological modulator of pancreatic enzyme secretion.     

Effect of pituitary adenylate cyclase activating polypeptide on rat pancreatic exocrine secretion.

A novel neuropeptide, pituitary adenylate cyclase activating polypeptide (PACAP), which has been isolated from ovine hypothalami, shows 68% homology with vasoactive intestinal peptide (VIP). Since VIP stimulates amylase secretion from the pancreas, we investigated the effect of PACAP and VIP on rat pancreatic exocrine secretion after intravenous injections of PACAP-27, PACAP-38, or VIP at doses of 2.5, 5 or 10 nmol/kg. Results showed: 1) Bolus injection of PACAP stimulated pancreatic amylase and protein secretions in a dose-dependent manner; and 2) Stimulation of amylase secretion with 10 nmol/kg of PACAP-27 was greater than that induced with the same dose of VIP or PACAP-38 (p less than 0.05).     

Caerulein and carbamoylcholine stimulate pancreatic amylase release at resting cytosolic free Ca2+.

Cytosolic free calcium concentrations ([Ca2+]i) and amylase secretion were measured in isolated rat pancreatic acini loaded with the intracellularly trapped fluorescent indicator quin2. Both caerulein and carbamoylcholine caused a rapid increase in [Ca2+]i, with a maximal 3-fold increase at 10(-9) M-caerulein and 10(-4) M-carbamoylcholine. However, caerulein (10(-12) M and 10(-11) M) as well as carbamoylcholine (10(-7) M) caused a significant stimulation of amylase release, while not inducing any detectable rise in [Ca2+]i. Changes in [Ca2+]i after addition of either secretagogue were transient and did not last more than 2-3 min. By contrast, when amylase secretion was monitored as a function of time, two distinct secretory phases could be observed upon addition of either carbamoylcholine (10(-5) M) or caerulein (10(-10) M). An initial, rapid phase (0-5 min) which caused a 6-7-fold increase above basal, followed by a sustained (5-30 min), but less marked, secretory rate (2-3-fold above basal). Addition of atropine (10(-4) M) 5 min after carbamoylcholine (10(-5) M) (i.e. after termination of the rise in [Ca2+]i and of the first secretory phase) did not cause any significant change in [Ca2+]i, while significantly inhibiting amylase secretion from 5 to 30 min to the same rate observed in the absence of the secretagogue. These results show that caerulein and carbamoylcholine, two agents thought to activate secretion mainly through mobilization of Ca2+ from intracellular stores, are capable of eliciting amylase secretion independently of a concomitant rise in [Ca2+]i. Furthermore, with both secretagogues the rise in [Ca2+]i, when observed, was only transient, while the stimulation of amylase release was sustained.     

Secretin is not necessary for exocrine pancreatic development and growth in mice

Adaptive exocrine pancreatic growth is mediated primarily by dietary protein and the gastrointestinal hormone cholecystokinin (CCK). Feeding trypsin inhibitors such as camostat (FOY-305) is known to induce CCK release and stimulate pancreatic growth. However, camostat has also been reported to stimulate secretin release and, because secretin often potentiates the action of CCK, it could participate in the growth response. Our aim was to test the role of secretin in pancreatic development and adaptive growth through the use of C57BL/6 mice with genetic deletion of secretin or secretin receptor. The lack of secretin in the intestine or the secretin receptor in the pancreas was confirmed by RT-PCR. Other related components, such as vasoactive intestinal polypeptide (VIP) receptors (VPAC(1) and VPAC(2)), were not affected. Secretin increased cAMP levels in acini from wild-type (WT) mice but had no effect on acini from secretin receptor-deleted mice, whereas VIP and forskolin still induced a normal response. Secretin in vivo failed to induce fluid secretion in receptor-deficient mice. The pancreas of secretin or secretin receptor-deficient mice was of normal size and histology, indicating that secretin is not necessary for normal pancreatic differentiation or maintenance. When WT mice were fed 0.1% camostat in powdered chow, the pancreas doubled in size in 1 wk, accompanied by parallel increases in protein and DNA. Camostat-fed littermate secretin and secretin receptor-deficient mice had similar pancreatic mass to WT mice. These results indicate that secretin is not required for normal pancreatic development or adaptive growth mediated by CCK.     

Mechanism of action of bombesin on amylase secretion. Evidence for a Ca2+-independent pathway

The mode of action of bombesin on amylase secretion was investigated in rat pancreatic acini. Bombesin induced a dose-dependent increase in inositol 1,4,5-trisphosphate and cytosolic free Ca2+. The threshold concentration capable of inducing both effects was 0.1 nM and the half-maximal dose of the peptide for Ca2+ mobilization was approximately 0.6 nM. By contrast, amylase release was approximately 30 times more sensitive than inositol 1,4,5-trisphosphate production and Ca2+ mobilization to bombesin action, with 1 pM being the first stimulatory concentration and a half-maximal effect at approximately 20 pM. The ability of low bombesin doses to trigger enzyme secretion was unaffected by chelation of extracellular Ca2+ with EGTA. In order to test whether the stimulation of amylase release was truly a Ca2+-independent response, the intracellular Ca2+ stores were depleted by pretreating acini with EGTA plus ionomycin, the Ca2+ ionophore. Under these conditions bombesin was still capable of eliciting a significant twofold enhancement of the secretory activity. These results indicate that bombesin, an agonist thought to activate secretion mainly through mobilization of Ca2+ from intracellular stores, elicits amylase release at low concentrations, independently of a concomitant rise in cytosolic free Ca2+. The relevance of these findings to the physiological regulation of pancreatic exocrine secretion is discussed.     

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.