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.     

Glucocorticoids increase cholecystokinin receptors and amylase secretion in pancreatic acinar AR42J cells

We recently reported in AR42J pancreatic acinar cells that glucocorticoids increased the synthesis, cell content, and mRNA levels for amylase (Logsdon, C.D., Moessner, A., Williams, J.A., and Goldfine, I.D. (1985) J. Cell Biol. 100, 1200-1208). In addition, in these cells glucocorticoids increased the volume density of secretory granules and rough endoplasmic reticulum. In the present study we investigate the effects of glucocorticoids on the receptor binding and biological effects of cholecystokinin (CCK) on AR42J cells. Treatment with 10 nM dexamethasone for 48 h increased the specific binding of 125I-CCK. This increase in binding was time-dependent, with maximal effects occurring after 48 h, and dose-dependent, with a one-half maximal effect elicited by 1 nM dexamethasone. Other steroid analogs were also effective and their potencies paralleled their relative effectiveness as glucocorticoids. Analyses of competitive binding experiments conducted at 4 degrees C to minimize hormone internalization and degradation revealed the presence of a single class of CCK binding sites with a Kd of approximately 6 nM and indicated that dexamethasone treatment nearly tripled the number of CCK receptors/cell with little change in receptor affinity. Treatment with 10 nM dexamethasone increased both basal amylase secretion and the amylase released in response to CCK stimulation. In addition, dexamethasone increased the sensitivity of the cells to CCK. The glucocorticoid decreased the concentration of CCK required for one half-maximal stimulation of amylase secretion from 35 +/- 6 to 8 +/- 1 pM. These data indicate, therefore, that glucocorticoids induce an increase in the number of CCK receptors in AR42J cells, and this increase leads to enhanced sensitivity to CCK.     

Role of cholecystokinin in the intestinal phase of pancreatic circulation in dogs

The regulatory mechanisms of postprandial pancreatic hyperemia are not well characterized. The aim of this study is to clarify the role of cholecystokinin (CCK) in the intestinal phase of pancreatic circulation. Pancreatic, gastric, and intestinal blood flows were measured by ultrasound transit-time blood flowmeters in five conscious dogs. Pancreatic and gastric secretion and blood pressure were also monitored. Synthetic CCK octapeptide (CCK-8) or gastrin heptadecapeptide (gastrin-17) was infused intravenously, and milk was infused into the duodenum with or without loxiglumide, a specific CCK-A receptor antagonist. CCK-8 induced dose-related increases of pancreatic, but not gastric or intestinal, blood flow and protein secretion without affecting systemic blood pressure. Gastrin-17 did not affect pancreatic blood flow. An intraduodenal infusion of milk increased pancreatic and intestinal blood flows and pancreatic protein secretion. Loxiglumide completely inhibited pancreatic blood flow and protein responses to CCK-8 and milk but not the intestinal blood flow response. CCK is a potent and specific pancreatic vasodilator, with its effect mediated by CCK-A receptors. CCK plays an important role in the regulation of the intestinal phase of the pancreatic circulation in dogs.     

Glucagon-like peptide-1 does not mediate amylase release from AR42J cells.

In this study, AR42J pancreatic acinar cells were used to investigate if glucagon-like peptide-1 (GLP-1) or glucagon might influence amylase release and acinar cell function. We first confirmed the presence of GLP-1 receptors on AR42J cells by reverse trasncriptase-polymerase chain reaction (RT-PCR), Western blotting, and partial sequencing analysis. While cholecystokinin (CCK) increased amylase release from AR42J cells, GLP-1, alone or in the presence of CCK, had no effect on amylase release but both CCK and GLP-1 increased intracellular calcium. Similar to GLP-1, glucagon increased both cyclic adenosine monophosphate (cAMP) and intracellular calcium in AR42J cells but it actually decreased CCK-mediated amylase release (n = 20, P < 0.01). CCK stimulation resulted in an increase in tyrosine phosphorylation of several cellular proteins, unlike GLP-1 treatment, where no such increased phosphorylation was seen. Instead, GLP-1 decreased such protein phosphorylations. Genestein blocked CCK-induced phosphorylation events and amylase secretion while vanadate increased amylase secretion. These results provide evidence that tyrosine phosphorylation is necessary for amylase release and that signaling through GLP-1 receptors does not mediate amylase release in AR42J cells. J. Cell. Physiol. 181:470-478, 1999. Published 1999 Wiley-Liss, Inc.     

Characterization of interactions between CCK-33 and CCK receptors in isolated dispersed pancreatic acini.

In isolated dispersed pancreatic acini, we have characterized the interactions between cholecystokinin (CCK) and CCK receptors by simultaneously measuring CCK-33 immunoreactivity and CCK bioactivity. Incubation of acinar cells with CCK-33 at cell density of 0.2-0.3 mg acinar protein per ml resulted in stimulation of amylase release concomitant with significant and time-dependent decrease of the immunoreactive CCK. With L-364,718 (0.1 microM), a specific CCK receptor antagonist, immunoreactive CCK levels in the media were not significantly altered during incubation; however, CCK-stimulated amylase release was almost completely abolished (94% inhibition). Vasoactive intestinal peptide (1 nM) significantly potentiated CCK stimulated amylase release without affecting immunoreactive CCK in the media. Insulin (167 nM) did not affect the CCK stimulated amylase release or immunoreactive CCK in the media. Incubation of acinar cells with CCK-33 at 4 degrees C did not affect the levels of immunoreactive CCK; however, a significant change in levels of immunoreactive CCK were found at 37 degrees C at 90 min. Incubation of cell free medium with CCK-33 in the presence or absence of secreted enzymes revealed no changes in CCK immunoreactivity in the medium at 90 min. Addition of bacitracin in the incubation media did not affect the CCK immunoreactivity or bioactivity. These findings indicate that in isolated rat pancreatic acini, CCK-33 stimulates amylase release through a receptor that is specifically blocked by L-364,718. Specificity of the interactions of CCK-33 with acinar cells in the media appears to be receptor-mediated and time- and temperature-dependent.     

Importance of sulfation of gastrin or cholecystokinin (CCK) on affinity for gastrin and CCK receptors

We investigated the importance of sulfation of gastrin or cholecystokinin (CCK) on influencing their affinity for gastrin or CCK receptors by comparing the abilities of sulfated gastrin-17 (gastrin-17-II), desulfated gastrin-17 (gastrin-17-I), CCK-8 and desulfated CCK-8 [des(SO3)CCK-8] to interact with CCK or gastrin receptors on guinea pig pancreatic acini. For inhibiting binding of 125I-gastrin to gastrin receptors, gastrin-17-II (Kd 0.08 nM) greater than CCK-8 (Kd 0.4 nM) greater than gastrin-17-I (Kd 1.5 nM) greater than des(SO3)CCK-8 (Kd 28 nM). For inhibiting binding of 125I-Bolton Hunter-labeled CCK-8 to CCK receptors the relative potencies were: CCK-8 much greater than des(SO3)CCK-8 = gastrin-17-II greater than gastrin-17-I. Each peptide interacted with both high and low affinity CCK binding sites. The relative abilities of each peptide to interact with high affinity CCK receptors showed a close correlation with their abilities to cause half-maximal stimulation of enzyme secretion. These results demonstrate that, in contrast to older studies, sulfation of both CCK and gastrin increase their affinities for both gastrin and CCK receptors. Moreover, the gastrin receptor is relatively insensitive to the position of the sulfate moiety, whereas the CCK receptor is extremely sensitive to both the presence and exact position of the sulfate moiety.     

Characterization of cholecystokinin receptors in bullfrog (Rana catesbeiana) brain and pancreas

The binding of biologically active 125I-Bolton-Hunter-CCK-33 to bullfrog brain and pancreatic membrane particles was characterized. Both tissues exhibited time-dependent, saturable, reversible, and high affinity binding without evidence for cooperative interaction. Both bullfrog CCK receptors resembled their mammalian counterparts in having acidic pH optima for tracer binding and a Kd of about 0.5 nM. However, the receptors differed from their mammalian counterparts in that (1) the bullfrog brain membranes bound more tracer per mg protein than did the pancreatic membranes, (2) both bullfrog CCK receptors were relatively insensitive to dibutyryl cGMP, and (3) both bullfrog brain and pancreatic CCK receptors exhibited the same general specificity toward a variety of CCK and gastrin peptides. For both tissues, the relative order of receptor binding potency was CCK-8 greater than caerulein = CCK-33 greater than gastrin-17-II greater than CCK-8-ns = gastrin-17-I greater than caerulein-ns greater than gastrin-4 with the sulfated CCK peptides being 1000-fold more potent than their nonsulfated analogs. Sulfated gastrin was also relatively potent, being only 10-fold weaker than CCK-8. Gastrin-4 was 20 000-fold weaker than CCK-8 in interacting with the brain CCK receptor. The latter finding is in sharp contrast to the mammalian brain CCK receptor. We conclude that the bullfrog brain and pancreas contain similar CCK receptors of probable physiological significance and may represent an ancestral condition from which the two distinct CCK receptors present in mammalian brain and pancreas have evolved.     

Structure activity of C-terminal modified analogs of Ac-CCK-7.

Previous work indicates that both the C-terminal phenylalanine amide and the tryptophan moieties of cholecystokinin (CCK) are critical pharmacophores for interaction with either the A or B receptor subtypes. We have examined a series of analogs of Ac-CCK-7 [Ac-Tyr(SO3H)-Met-Gly-Trp-Met-Asp-Phe33-NH2] (2) in which the phenyl ring of the C-terminal Phe-NH2 has been modified. Compounds were assessed in binding assays using homogenated rat pancreatic membranes and bovine striatum as the source of CCK-A and CCK-B receptors respectively and for anorectic activity after intraperitoneal administration to rats. Substitution of a number of cycloalkyl or bicyclic aryl moieties for the phenyl ring of phenylalanine33 including cyclopentyl (20), cyclohexyl (21), cyclooctyl (23), 2-(5,6,7,8-tetrahydro)naphthyl (26), 2-naphthyl (27), and 1-naphthyl (29) led to analogs with 10-70 times the anorectic potency of 2. The anorectic activity of 21 was blocked by the specific CCK-A receptor antagonist MK-329. Other bulky aliphatic groups in place of the phenylalanine33 aromatic ring such as isopropyl, 2-adamantyl and cyclohexylmethyl gave derivatives similar to 2 in potency. While most of the new compounds were comparable to CCK in binding assays, 23, 26, 27 and 29 were exceptionally potent with IC50s 10(-11)-10(-14) M in the pancreas. Compounds 23 and 29 were further evaluated for their ability to stimulate amylase secretion and found to have potencies similar to that of CCK. The dissociation between potency in the binding and amylase secretion assays suggests that they may interact with a high affinity binding site which is not coupled to amylase secretion. We conclude that CCK receptors possess a generous hydrophobic pocket capable of accommodating large alkyl groups in place of the side chain of phenylalanine33 and that the pharmacological profile of CCK analogs can be tailored by appropriate exploitation of this finding.     

Effects of GP2 expression on secretion and endocytosis in pancreatic AR4-2J cells

GP2 is the major membrane protein present in secretory granules of the exocrine pancreas. GP2's function is unknown, but a role in digestive enzyme packaging or secretion from secretory granules has been proposed. In addition, GP2 has been proposed to influence endocytosis and membrane recycling following stimulated secretion. Adenovirus-mediated GP2 overexpression in the rat pancreatic cell line AR4-2J was used to study its impact on digestive enzyme secretion and membrane recycling. Immunoelectron microscopy showed that GP2 and amylase co-localized in secretory granules in infected AR4-2J cells. CCK-8 stimulation resulted in a fourfold increase in amylase secretion with or without GP2 expression. GP2 expression also did not influence endocytosis following CCK-8 stimulation. Thus, GP2 expression in AR4-2J cells does not affect amylase packaging in secretory granules or stimulated secretion. GP2 expression also does not influence membrane recycling in response to stimulated stimulation in AR4-2J cells.     

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.     

Exocrine pancreatic secretion: effect of subarachnoidal application of cholecystokinin octapeptide in rats

Administration of cholecystokinin octapeptide (CCK-8) intravenously, or in the subarachnoidal surface of the olfactory lobe in rats, caused an increase in pancreatic protein and amylase secretion. It was observed that for subarachnoidal administration of CCK-8 both protein and amylase outputs were higher than that seen after i.v. injection. This result is consistent with the presence of central CCK receptors which when activated can enhance pancreatic exocrine secretion. The blockade of the effect of CCK by administration of CCK-8-specific antisera proves the specificity of the subarachnoidal CCK-8 stimulation.     

In vitro analysis of the effects of cholecystokinin on rat brain stem motoneurons

Using whole cell patch clamp in thin brain stem slices, we tested the effects of cholecystokinin (CCK) on identified gastric-projecting neurons of the rat dorsal motor nucleus of the vagus (DMV). Perfusion with the sulfated form of CCK octapeptide (CCK8s, 30 pM-300 nM, EC50 approximately 4 nM) induced a concentration-dependent inward current in 35 and 41% of corpus- and antrum/pylorus-projecting DMV neurons, respectively. Conversely, none of the fundus-projecting DMV neurons responded to perfusion with CCK8s. The CCK8s-induced inward current was accompanied by a 65 +/- 17% increase in membrane input resistance and reversed at 90 +/- 4 mV, indicating that the excitatory effects of CCK8s were mediated by the closure of a potassium conductance. Pretreatment with the synaptic blocker TTX (0.3-1 microM) reduced the CCK8s-induced current, suggesting that a portion of the CCK8s-induced current was mediated indirectly via an action on presynaptic neurons apposing the DMV membrane. Pretreatment with the selective CCK-A receptor antagonist lorglumide (0.3-3 microM) attenuated the CCK8s-induced inward current in a concentration-dependent manner, with a maximum inhibition of 69 +/- 12% obtained with 3 microM lorglumide. Conversely, pretreatment with the selective CCK-B antagonist triglumide did not attenuate the CCK8s-induced inward current; pretreatment with triglumide (3 microM) and lorglumide (1 microM) attenuated the CCK8s-induced current to the same extent as pretreatment with lorglumide alone. Immunohistochemical experiments showed that CCK-A receptors were localized on the membrane of 34, 65, and 60% of fundus-, corpus-, and antrum/pylorus-projecting DMV neurons, respectively. Our data indicate that CCK-A receptors are present on a subpopulation of gastric-projecting neurons and that their activation leads to excitation of the DMV membrane.     

Phosphatidic acid production,required for cholecystokinin octapeptide-stimulated amylase secretion from pancreatic acinar AR42J cells,is regulated by a wortmannin-sensitive process

To investigate the role of phospholipids in exocytotic secretory events, we utilized rat pancreatic acinar AR42J cells that secreted amylase in response to cholecystokinin octapeptide (CCK-8). Wortmannin, an inhibitor of phosphoinositide 3-kinase (PI3K), was found to inhibit the secretion in a dose-dependent manner. When changes in cell membrane phospholipids were investigated before and after CCK-8 stimulation using [32P]orthophosphoric acid-labeled AR42J cells, we observed a rapid increase in phosphatidic acid (PtdOH) levels right after stimulation, which was not observed in non-stimulated cells. The increase, however, was suppressed by wortmannin pre-treatment, which also inhibited amylase secretion. Changes in other major phospholipids were not significant. These results indicate that CCK-8 induces amylase secretion through PI3K-regulated production of PtdOH in cell membranes.     

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.     

Functional CCK-A and Y2 receptors in guinea pig esophagus

Effects of cholecystokinin octapeptide (CCK-8), peptide YY (PPY), neuropeptide Y (NPY) and their analogs on muscle contractions of esophageal strips were investigated. CCK-8 induced a tetrodotoxin and atropine-sensitive contraction. The relative potencies for CCK related peptides to induce contractions were CCK-8 > desulfated CCK-8 > gastrin-17-I. The CCK-A receptor antagonist L-364,718 was 300-fold more potent than the CCK-B receptor antagonist L-365,260 at inhibiting CCK-8-induced contraction. These indicate that neural CCK-A receptors mediate this contraction. PYY or NPY did not cause muscle contraction or inhibit muscle contraction induced by carbachol, endothelin-1 or KCl. However, both PYY and NPY concentration-dependently inhibited contraction induced by CCK-8. This inhibition was not affected by nitric oxide (NO) synthase inhibitors L-NMMA or L-NAME. The relative potencies of PYY related peptides to inhibit CCK-8 induced contraction were PYY > NPY > NPY13-36 > [Leu(31), Pro(34)]NPY > pancreatic polypeptide (PP). We conclude that CCK interacts with neural CCK-A receptors to cause esophageal muscle contraction. PYY and NPY interact with Y2 receptors to inhibit this CCK-induced muscle contraction by an effect not related to NO.     

Mutual dependence of VIP/PACAP and CCK receptor signaling for a physiological role in duck exocrine pancreatic secretion

Unlike in rodents, CCK has not been established as a physiological regulator in avian exocrine pancreatic secretion. In the isolated duck pancreatic acini, 1 nM CCK was required for stimulation of amylase secretion, maximal effect being achieved at 10 nM; picomolar CCK was without effect. Vasoactive intestinal peptide (VIP)/pituitary adenylate cyclase activating peptide (PACAP) receptor (VPAC) agonists PACAP-38 and PACAP-27 (10(-12)-10(-7) M) alone had no effect, but made picomolar CCK effective. VPAC agonist VIP 10(-10)-10(-7) M stimulated amylase secretion marginally, but made CCK 10(-12)-10(-10) M effective also. PACAP-27 and VIP both shifted the maximal CCK concentration from 10(-8) to 10(-9) M. This sensitizing effect was mimicked by forskolin. CCK dose dependently induced intracellular Ca2+ concentration ([Ca2+]i) oscillations. PACAP-38 (1 nM), PACAP-27 (1 nM), VIP (10 nM), or forskolin (10 microM) alone did not stimulate [Ca2+]i increase, neither did they modulate CCK (1 nM)-induced oscillations; but when they were added to cells simultaneously exposed to subthreshold CCK (10 pM), calcium spikes emerged. Amylase secretion induced by the simultaneous presence of 10 pM CCK and VPAC agonists was completely blocked by removing extracellular calcium, but the protein kinase C inhibitor staurosporine (1 microM) was without effect. CCK (10 nM)-induced secretion was inhibited by CCK1 receptor antagonist FK480 (1 microM). Gastrin from 10(-12) to 10(-6) M did not stimulate amylase secretion nor did it (100 nM) induce [Ca2+]i increase. The above data suggest that duck pancreatic acini possess both CCK1 and VPAC receptors; simultaneous activation of both is required for each to play a physiological role.     

Cholecystokinin- and gastrin-induced protein and amylase secretion from the parotid gland of the anaesthetized rat

I.V. infusion of pentagastrin (20 microg/kg/h) or cholecystokinin (CCK)-8 (1 microg/kg/h) for 10 min caused secretion of salivary proteins from the parotid gland in the anaesthetized rat without any accompanying overt fluid secretion. This "occult" response was revealed by a subsequent wash-out injection of methacholine (5 microg/kg, I.V.) 10 min after the end of the infusion period (aiming at avoiding synergistic interactions). While the fluid response to methacholine was unaffected by the preceding infusion of pentagastrin and CCK-8, the output of protein increased by 147% (pentagastrin) and 74% (CCK-8) and that of amylase by 45% (CCK-8) compared to the responses to methacholine upon saline infusion. Those increases were abolished by the CCK-A receptor blocker (lorglumide), but not by the CCK-B receptor blocker (itriglumide). Evisceration, combined sympathetic and parasympathetic denervation of the glands and assay under adrenoceptor blockade excluded contribution from the gastro-intestinal tract, central or ganglionic mechanisms and circulating catecholamines to the increase in protein/amylase. Furthermore, Western blot demonstrated CCK receptors for both A and B subtypes in normal and chronically denervated glands. In the submandibular gland, both pentagastrin and CCK-8 evoked a trace secretion of saliva but, under the present experimental set-up, no statistically significant increase in protein output. Thus, in addition to the autonomic nervous system, gastrointestinal hormones may, in some types of glands, be involved in the secretion of salivary gland proteins.     

Role of cholecystokinin in the intestinal fat- and acid-induced inhibition of gastric secretion.

This study was designed to determine the role of cholecystokinin (CCK) in the inhibition of gastric HCl secretion by duodenal peptone, fat and acid in dogs with chronic gastric and pancreatic fistulas. Intraduodenal instillation of 5% peptone stimulated both gastric HCl secretion and pancreatic protein secretion and caused significant increments in plasma gastrin and CCK levels. L-364,718, a selective antagonist of CCK-A receptors, caused further increase in gastric HCl and plasma gastrin responses to duodenal peptone but reduced the pancreatic protein outputs in these tests by about 75%. L-365,260, an antagonist of type B receptors, reduced gastric acid by about 25% but failed to influence pancreatic response to duodenal peptone. Addition of 10% oleate or acidification of peptone to pH 3.0 profoundly inhibited acid secretion while significantly increasing the pancreatic protein secretion and plasma CCK levels. Administration of L-364,718 reversed the fall in gastric HCl secretion and significantly attenuated pancreatic protein secretion in tests with both peptone plus oleate and peptone plus acid. Exogenous CCK infused i.v. in a dose (25 pmol/kg per h) that raised plasma CCK to the level similar to that achieved by peptone meal plus fat resulted in similar inhibition of gastric acid response to that attained with fat and this effect was completely abolished by the pretreatment with L-364,718. We conclude that CCK released by intestinal peptone meal, containing fat or acid, exerts a tonic inhibitory influence on gastric acid secretion and gastrin release through the CCK-A receptors.     

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.     

Cholecystokinin activates Gi1-, Gi2-, Gi3- and several Gs-proteins in rat pancreatic acinar cells.

On separation of rat pancreatic plasma membrane proteins by two-dimensional gel electrophoresis, 15 GTP-binding protein (G-protein) alpha-subunits could be detected immunochemically using an alpha common antibody. These consisted of five 48 kDa proteins (pI 5.70, 5.80, 5.90, 6.10 and 6.25) and five 45 kDa proteins (pI 5.90, 6.05, 6.25, 6.30 and 6.70), presumably corresponding to low- and high-molecular mass forms of the Gs-protein, as well as three 40/41 kDa proteins (pI 5.50, 5.70 and 6.00) and two 39 kDa proteins (pI 5.50 and 6.00). All of these proteins except for the more acidic 39 kDa protein were ADP-ribosylated by cholera toxin (CT). In addition, the three 40/41 kDa proteins and the more alkaline 39 kDa protein were also ADP-ribosylated by pertussis toxin (PT). CT- and PT-induced ADP-ribosylation changed the pI values of G-protein alpha-subunits by 0.2 pI units to more acidic values. Preincubation of isolated pancreatic membranes with cholecystokinin octapeptide (CCK-OP), which stimulates phospholipase C in acinar cells, decreased CT-induced as well as PT-induced ADP-ribosylation of the three 40/41 kDa proteins, whereas CT-induced ADP-ribosylation of one 45 kDa (pI 5.80) and all 48 kDa proteins was enhanced in the presence of CCK. Carbachol, another stimulant of phospholipase C, had no effect. The three 40/41 kDa proteins and one 48 kDa protein could be labelled with the GTP analogue [alpha-32P]GTP-gamma-azidoanilide. CCK, but not carbachol, stimulated incorporation of the GTP analogue into all of these four proteins. Using different anti-peptide antisera specific for alpha-subunits of G-proteins we identified the three 40/41 kDa Gi-proteins as Gi1 (pI 6.00), Gi2 (pI 5.50) and Gi3 (pI 5.70). The Gi3-protein was found to be the major Gi-protein of pancreatic plasma membranes. One of the 39 kDa proteins (pI 6.0) was identified as Go. These results indicate that CCK receptors functionally interact with six Gs-proteins and with Gi1, Gi2 and Gi3-proteins. Since evidence suggests that a 40/41 kDa CT substrate is involved in the stimulation of phospholipase C in pancreatic acinar cells, it is likely that one, two or all three 40/41 kDa Gi-proteins are involved in the coupling of CCK receptors with phospholipase C.