The cholecystokinin-1 receptor antagonist devazepide increases cholesterol cholelithogenesis in mice.

BACKGROUND: A defect in gallbladder contraction function plays a key role in the pathogenesis of gallstones. The cholecystokinin-1 receptor (CCK-1R) antagonists have been extensively investigated for their therapeutic effects on gastrointestinal and metabolic diseases in animal studies and clinical trials. However, it is still unknown whether they have a potential effect on gallstone formation. DESIGN: To study whether the CCK-1R antagonists enhance cholelithogenesis, we investigated cholesterol crystallization, gallstone formation, hepatic lipid secretion, gallbladder emptying function and intestinal cholesterol absorption in male C57BL/6J mice treated by gavage with devazepide (4 mg/day/kg) or vehicle (as controls) twice per day and fed the lithogenic diet for 21 days. RESULTS: During 21 days of feeding, oral administration of devazepide significantly accelerated cholesterol crystallization and crystal growth to microlithiasis, with 40% of mice forming gallstones, whereas only agglomerated cholesterol monohydrate crystals were found in mice receiving vehicle. Compared to the vehicle group, fasting and postprandial residual gallbladder volumes in response to the high-fat meal were significantly larger in the devazepide group during cholelithogenesis, showing reduced gallbladder emptying and bile stasis. Moreover, devazepide significantly increased hepatic secretion of biliary cholesterol, but not phospholipids or bile salts. The percentage of intestinal cholesterol absorption was higher in devazepide-treated mice, increasing the bioavailability of chylomicron-derived cholesterol in the liver for biliary hypersecretion into bile. These abnormalities induced supersaturated bile and rapid cholesterol crystallization. CONCLUSIONS: The potent CCK-1R antagonist devazepide increases susceptibility to gallstone formation by impairing gallbladder emptying function, disrupting biliary cholesterol metabolism and enhancing intestinal cholesterol absorption in mice.

Differential sensitivity of types 1 and 2 cholecystokinin receptors to membrane cholesterol.

Recent studies indicate that membrane cholesterol can associate with G protein-coupled receptors (GPCRs) and affect their function. Previously, we reported that manipulation of membrane cholesterol affects ligand binding and signal transduction of the type 1 cholecystokinin receptor (CCK1R), a Class A GPCR. We now demonstrate that the closely related type 2 cholecystokinin receptor (CCK2R) does not share this cholesterol sensitivity. The sequences of both receptors reveal almost identical cholesterol interaction motifs in analogous locations in transmembrane segments two, three, four, and five. The disparity in cholesterol sensitivity between these receptors, despite their close structural relationship, provides a unique opportunity to define the possible structural basis of cholesterol sensitivity of CCK1R. To evaluate the relative contributions of different regions of CCK1R to cholesterol sensitivity, we performed ligand binding studies and biological activity assays of wild-type and CCK2R/CCK1R chimeric receptor-bearing Chinese hamster ovary cells after manipulation of membrane cholesterol. We also extended these studies to site-directed mutations within the cholesterol interaction motifs. The results contribute to a better understanding of the structural requirements for cholesterol sensitivity in CCK1R and provides insight into the function of other cholesterol-sensitive Class A GPCRs.

Mechanisms mediating CCK-8S-induced contraction of proximal colon in guinea pigs.

AIM: To investigate the effects of sulfated cholecystokinin octapeptide (CCK-8S) on the contractile activity of guinea-pig proximal colon. METHODS: Proximal colonic smooth muscle (PCSM) strips were obtained from adult female guinea pigs and contractile response of PCSM strips was recorded using a polyphysiograph. PCSM cells were isolated by enzymatic digestion. Resting potential (RP), action potential (AP), large conductance potassium channel currents (I(BKCa)) and L-type calcium currents (I(Ca-L)) were recorded by patch-clamp techniques. RESULTS: (1) CCK-8S (10(-7) mol/L) enhanced the mean contractile amplitude of colonic circular muscle and longitudinal muscle (LM) strips by 56.53% + or - 11.92% (P = 0.038) and 65.93% + or - 12.98% (P = 0.019), respectively, as well as the mean frequency of LM by 31.69% + or - 13.58% (P = 0.023), which were significantly attenuated by pretreating strips with devazepide, nifedipine, iberiotoxin, thapsigargin (TG) and BAPTA-AM (BA) respectively; (2) CCK-8S (10(-7) mol/L) increased the AP amplitude by 38.6% + or - 3.2% (P = 0.015), decreased AP duration by 36.9% + or - 8.7% (P = 0.026), and depolarized the RP from -61.3 + or - 2.7 mV to -29.8 + or - 5.9 mV (P = 0.032); and (3) Compared with the normal control group, CCK-8S (10(-7) mol/L) enhanced the peak current of I(BKCa) by 18.7% + or - 2.1% (from 916 + or - 183 pA to 1088 + or - 226 pA; at +60 mV; P = 0.029), which was inhibited by respective pretreatment with iberiotoxin and devazepide. Additionally, CCK-8S (10(-7) mol/L) intensified the peak current of I(Ca-L) by 40% (from 60 + or - 8 pA to 84 + or - 11 pA; at +10 mV; P = 0.012), compared to the normal control group, which was apparently suppressed by respective pretreatment with nifedipine, devazepide, TG and BA. In the respective presence of heparin and staurosporine, CCK-8S did not significantly enhance I(BKCa) and I(Ca-L). CONCLUSION: The results suggest that CCK-8S promotes guinea-pig proximal colon contraction by CCK1 receptors, following activation of the inositol triphosphate-protein kinase C signal transduction pathway.

Therapeutic potential for novel drugs targeting the type 1 cholecystokinin receptor.

Cholecystokinin (CCK) is a physiologically important gastrointestinal and neuronal peptide hormone, with roles in stimulating gallbladder contraction, pancreatic secretion, gastrointestinal motility and satiety. CCK exerts its effects via interactions with two structurally related class I guanine nucleotide-binding protein (G protein)-coupled receptors (GPCRs), the CCK(1) receptor and the CCK(2) receptor. Here, we focus on the CCK(1) receptor, with particular relevance to the broad spectrum of signalling initiated by activation with the natural full agonist peptide ligand, CCK. Distinct ligand-binding pockets have been defined for the natural peptide ligand and for some non-peptidyl small molecule ligands. While many CCK(1) receptor ligands have been developed and have had their pharmacology well described, their clinical potential has not yet been fully explored. The case is built for the potential importance of developing more selective partial agonists and allosteric modulators of this receptor that could have important roles in the treatment of common clinical syndromes.

Gastrointestinal growth factors and hormones have divergent effects on Akt activation.

Akt is a central regulator of apoptosis, cell growth and survival. Growth factors and some G-protein-coupled receptors (GPCR) regulate Akt. Whereas growth-factor activation of Akt has been extensively studied, the regulation of Akt by GPCR's, especially gastrointestinal hormones/neurotransmitters, remains unclear. To address this area, in this study the effects of GI growth factors and hormones/neurotransmitters were investigated in rat pancreatic acinar cells which are high responsive to these agents. Pancreatic acini expressed Akt and 5 of 7 known pancreatic growth-factors stimulate Akt phosphorylation (T308, S473) and translocation. These effects are mediated by p85 phosphorylation and activation of PI3K. GI hormones increasing intracellular cAMP had similar effects. However, GI-hormones/neurotransmitters [CCK, bombesin, carbachol] activating phospholipase C (PLC) inhibited basal and growth-factor-stimulated Akt activation. Detailed studies with CCK, which has both physiological and pathophysiological effects on pancreatic acinar cells at different concentrations, demonstrated CCK has a biphasic effect: at low concentrations (pM) stimulating Akt by a Src-dependent mechanism and at higher concentrations (nM) inhibited basal and stimulated Akt translocation, phosphorylation and activation, by de-phosphorylating p85 resulting in decreasing PI3K activity. This effect required activation of both limbs of the PLC-pathway and a protein tyrosine phosphatase, but was not mediated by p44/42 MAPK, Src or activation of a serine phosphatase. Akt inhibition by CCK was also found in vivo and in Panc-1 cancer cells where it inhibited serum-mediated rescue from apoptosis. These results demonstrate that GI growth factors as well as gastrointestinal hormones/neurotransmitters with different cellular basis of action can all regulate Akt phosphorylation in pancreatic acinar cells. This regulation is complex with phospholipase C agents such as CCK, because both stimulatory and inhibitory effects can be seen, which are mediated by different mechanisms.

Activation of cholecystokinin (CCK 1) and serotonin (5-HT 3) receptors increases the discharge of pancreatic vagal afferents.

Cholecystokinin and serotonin are released from the gastrointestinal tract in response to the products of digestion and play critical roles in mediating pancreatic secretion via vago-vagal reflex pathways. This study was designed to investigate the effects of activation of cholecystokinin CCK(1) and serotonin (5-hydroxytryptamine, 5-HT) 5-HT(3) receptors on pancreatic vagal afferent discharge and to determine whether there is an interaction between these receptors. Male Sprague Dawley rats anaesthetised with isoflurane (1.5%/100% O(2)) were used in all experiments. The effects of systemic administration of cholecystokinin and the serotonin 5-HT(3) receptor agonist phenylbiguanide on pancreatic vagal afferent discharge were recorded before and after administration of cholecystokinin CCK(1) and serotonin 5-HT(3) receptor antagonists. Cholecystokinin (0.1-10 microg/kg, i.v.) and phenylbiguanide (1 and 10 microg/kg, i.v.) increased pancreatic vagal afferent discharge dose-dependently. Cholecystokinin CCK(1) receptor antagonists, lorglumide (10 mg/kg, i.v.) and devazepide (0.5 mg/kg, i.v.), reduced cholecystokinin- and phenylbiguanide-induced increases in pancreatic vagal afferent discharge significantly (n=5, P<0.05). On the other hand, serotonin 5-HT(3) receptor blockade with granisetron (1 mg/kg, i.v.) or MDL72222 ([(1S,5R)-8-methyl-8-azabicyclo[3.2.1]octan-3-yl] 3,5-dichlorobenzoate; 0.1 mg/kg, i.v.) inhibited the pancreatic vagal afferent discharge responses to phenylbiguanide but not those to cholecystokinin. This study has confirmed that cholecystokinin and phenylbiguanide activate pancreatic vagal afferent discharge via activation of cholecystokinin CCK(1) and serotonin 5-HT(3) receptors, respectively. In addition, it has demonstrated that (i) the serotonin 5-HT(3) agonist phenylbiguanide acts partly via an interaction with cholecystokinin CCK(1) receptors, and (ii) the actions of cholecystokinin are not dependent on serotonin 5-HT(3) receptor activation.

PEGylated cholecystokinin is more potent in inducing anorexia than conditioned taste aversion in rats.

BACKGROUND AND PURPOSE: The physiological involvement of endogenous cholecystokinin (CCK) in the termination of feeding has been challenged by evidence of aversive effects of exogenous CCK8. We previously prolonged the anorectic effect of CCK by conjugation to polyethylene glycol (PEGylation) to produce PEG-CCK9. In this study, we investigated the ability of different doses of PEG-CCK9 to induce conditioned taste aversion (CTA) and satiety and identified the receptors involved in CTA induction. EXPERIMENTAL APPROACH: Induction of CTA, measured by the saccharin preference ratio determined in a two-bottle CTA procedure, and of satiety in adult male Wistar rats after intraperitoneal (i.p.) injection of different doses of PEG-CCK9 (1, 2, 4, 8, 16 or 32 microg kg(-1)) was compared. Devazepide (100 microg kg(-1)) and 2-NAP (3 mg kg(-1)), two selective CCK1-receptor antagonists, were co-administered i.p. with PEG-CCK9 (8 microg kg(-1)) and the CTA effects monitored. KEY RESULTS: PEG-CCK9 dose-dependently induced CTA, with a minimal effective dose of 8 microg kg(-1), whereas the minimal effective dose to induce satiety was 1 microg kg(-1). The CTA effects of PEG-CCK9 were completely abolished by i.p. administration of devazepide prior to PEG-CCK9 treatment and only partially abolished by administration of 2-NAP. CONCLUSIONS AND IMPLICATIONS: Although PEG-CCK9-induced satiety and PEG-CCK9-induced CTA both increased with dose, the conjugate was more potent in inducing satiety, suggesting that the anorexia could not be completely attributed to the aversiveness of the drug. As observed with induction of satiety, PEG-CCK9-induced CTA was mediated by CCK1-receptors.

Control of gallbladder contractions by cholecystokinin through cholecystokinin-A receptors on gallbladder interstitial cells of Cajal.

AIM: To identify the cholecystokinin (CCK)-A receptors (CCK-AR) on the guniea pig gallbladder interstitial cells of cajal (ICC) and to study CCK-8 induced gallbladder muscle strip contractions through the CCK-AR. METHODS: The existence of CCK-AR was examined by immunohistofluorescence on sectioned tissue and cultured cells. In vitro contractile response of guinea pig gallbladder muscle strips and the strips with ICC removed were also studied with CCK-8 receptors added. RESULTS: In tissue sections, intensely CCKAR-immunoreactive interstitial cells were found mainly in the muscular layers. In cultured cell sections, distinctive double staining of C-kit and CCK-AR ICCs were found. When we removed the ICC of the gallbladder, CCK-8 induced muscle strip contraction dose response curve significantly shifted to the right. CONCLUSION: We proved that both the existence of CCK-AR on the guinea pig gallbladder ICC and CCK evoked contraction are mediated through direct action on CCK-AR on the gallbladder ICC.

The vagal afferent pathway does not play a major role in the induction of satiety by intestinal fatty acid in rats.

Intestinal infusion of long-chain fatty acids (LCFAs) strongly suppresses food intake and gut motility. Vagal afferents and cholecystokinin (CCK) signaling pathway are considered to play important roles in intestinal LCFA-induced satiety. Here, we first investigated the influence of vagus nerve on satiety following intestinal LCFA infusion in rats. Jejunal infusion of linoleic acid (LA) at 200 microL/h for 7 h suppressed food intake and the effect lasted for 24 h. The satiety induced by jejunal LA infusion occurred in a dose dependent manner. In contrast, the anorectic effect induced by octanoic acid, a medium-chain fatty acid, was weaker than that induced by LA. The reduction in food intake induced by jejunal LA infusion was not attenuated in rats treated with vagotomy, the ablation of bilateral subdiaphragmatic vagal trunks. Jejunal LA-induced satiety could also be observed in rats with bilateral midbrain transections, which ablates fibers between the hindbrain and hypothalamus. These findings suggest that the vagus nerve and fibers ascending from the hindbrain to the hypothalamus do not play a major role in intestinal LCFA-induced satiety. Jejunal LA infusion also reduced food intake in CCK-A receptor-deficient OLETF rats, suggesting that CCK signaling pathway is not critical for intestinal LCFA-induced anorexia. In conclusion, this study indicates that the vagus nerve and the CCK signaling pathway do not play major roles in conveying satiety signals induced by intestinal LCFA to the brain in rats.

Protein hydrolysates induce CCK release from enteroendocrine cells and act as partial agonists of the CCK1 receptor.

Protein has been reported to be the most satiating of all macronutrients. Upon gastrointestinal digestion, peptides are generated that stimulate the release of satiety hormones such as cholecystokinin (CCK) from enteroendocrine cells. As such, bioactive peptides could be the target of Functional Food ingredients with satiating effects. We set up an in vitro assay system to investigate if different protein hydrolysates exhibit varying CCK-releasing properties. Soy, pea, potato, casein, and whey protein hydrolysates were incubated with the enteric endocrine cell line STC-1 that endogenously expresses and secretes CCK. Release of CCK was measured by ELISA. All hydrolysates induced CCK release at low concentrations (>0.1 mg.L -1)); however, no significant differences in CCK-releasing properties between the different protein hydrolysates were found, suggesting a generic, nonspecific peptide-sensing mechanism in the STC-1 cells on hydrolyzed protein. As the ELISA exhibits sensitivity to all CCK isoforms possessing the C-terminal CCK octapeptide but varying in biological activity at the CCK 1 receptor (CCK 1R), a secondary module was added to the STC-1 cell assay. Intracellular calcium measurements were performed in CHO-CCK 1R cells. Following exposure of the STC-1 cells to the protein hydrolysates, the medium was tested on the CCK 1R assay. Released CCK was measured with higher sensitivity and lower variability than in the ELISA. Surprisingly, we found that some protein hydrolysates (soy > potato >> casein) also directly stimulated CCK 1R-expressing cells, while whey and pea protein hydrolysates were inactive. As CCK 1R is expressed in the GI tract, direct interaction of CCK 1R with dietary peptides may contribute to their satiety effects. Future experiments developing bioactive ingredients for Functional Foods for weight management could involve isolation of the active, CCK 1R-activating peptides in, for example, soy protein hydrolysates.

PEGylated cholecystokinin prolongs satiation in rats: dose dependency and receptor involvement.

BACKGROUND AND PURPOSE: Acute intraperitoneal (i.p.) administration of cholecystokinin (CCK) is known to induce a significant, but short-lasting, reduction in food intake, followed by recovery within hours. Therefore, we had covalently coupled CCK to a 10 kDa polyethylene glycol and showed that this conjugate, PEG-CCK(9), produced a significantly longer anorectic effect than unmodified CCK(9). The present study assessed the dose-dependency of this response and the effect of two selective CCK(1) receptor antagonists, with different abilities to cross the blood-brain barrier (BBB), on PEG-CCK(9)-induced anorexia. EXPERIMENTAL APPROACH: Food intake was measured, for up to 23 h, after i.p. administration of different doses (2, 4, 8, 16 and 32 microg kg(-1)) of CCK(9) or PEG-CCK(9) in male Wistar rats. Devazepide (100 microg kg(-1)), which penetrates the BBB or 2-NAP (3 mg kg(-1)), which does not cross the BBB, were coadministered i.p. with PEG-CCK(9) (6 microg kg(-1)) and food intake was monitored. KEY RESULTS: In PEG-CCK(9)-treated rats, a clear dose-dependency was seen for both the duration and initial intensity of the anorexia whereas, for CCK(9), only the initial intensity was dose-dependent. Intraperitoneal administration of devazepide or 2-NAP, injected immediately prior to PEG-CCK(9), completely abolished the anorectic effect of PEG-CCK(9). CONCLUSIONS AND IMPLICATIONS: The duration of the anorexia for PEG-CCK(9) was dose-dependent, suggesting that PEGylation of CCK(9) increases its circulation time. Both devazepide and 2-NAP completely abolished the anorectic effect of i.p. PEG-CCK(9) indicating that its anorectic effect was solely due to stimulation of peripheral CCK(1) receptors.

Vagally mediated, nonparacrine effects of cholecystokinin-8s on rat pancreatic exocrine secretion.

Cholecystokinin (CCK) has been proposed to act in a vagally dependent manner to increase pancreatic exocrine secretion via actions exclusively at peripheral vagal afferent fibers. Recent evidence, however, suggests the CCK-8s may also affect brain stem structures directly. We used an in vivo preparation with the aims of 1) investigating whether the actions of intraduodenal casein perfusion to increase pancreatic protein secretion also involved direct actions of CCK at the level of the brain stem and, if so, 2) determining whether, in the absence of vagal afferent inputs, CCK-8s applied to the dorsal vagal complex (DVC) can also modulate pancreatic exocrine secretion (PES). Sprague-Dawley rats (250-400 g) were anesthetized and the common bile-pancreatic duct was cannulated to collect PES. Both vagal deafferentation and pretreatment with the CCK-A antagonist lorglumide on the floor of the fourth ventricle decreased the casein-induced increase in PES output. CCK-8s microinjection (450 pmol) in the DVC significantly increased PES; the increase was larger when CCK-8s was injected in the left side of the DVC. Protein secretion returned to baseline levels within 30 min. Microinjection of CCK-8s increased PES (although to a lower extent) also in rats that underwent complete vagal deafferentation. These data indicate that, as well as activating peripheral vagal afferents, CCK-8s increases pancreatic exocrine secretion via an action in the DVC. Our data suggest that the CCK-8s-induced increases in PES are due mainly to a paracrine effect of CCK; however, a relevant portion of the effects of CCK is due also to an effect of the peptide on brain stem vagal circuits.

Cholecystokinin-8s excites identified rat pancreatic-projecting vagal motoneurons.

It is known that cholecystokinin (CCK) acts in a paracrine fashion to increase pancreatic exocrine secretion via vagal circuits. Recent evidence, however, suggests that CCK-8s actions are not restricted to afferent vagal fibers, but also affect brain stem structures directly. Within the brain stem, preganglionic neurons of the dorsal motor nucleus of the vagus (DMV) send efferent fibers to subdiaphragmatic viscera, including the pancreas. Our aims were to investigate whether DMV neurons responded to exogenously applied CCK-8s and, if so, the mechanism of action. Using whole cell patch-clamp recordings we show that perfusion with CCK-8s induced a concentration-dependent excitation in approximately 60% of identified pancreas-projecting DMV neurons. The depolarization was significantly reduced by tetrodotoxin, suggesting both direct (on the DMV membrane) and indirect (on local synaptic circuits) effects. Indeed, CCK-8s increased the frequency of miniature excitatory currents onto DMV neurons. The CCK-A antagonist, lorglumide, prevented the CCK-8s-mediated excitation whereas the CCK-B preferring agonist, CCK-nonsulfated, had no effect, suggesting the involvement of CCK-A receptors only. In voltage clamp, the CCK-8s-induced inward current reversed at -106 +/- 3 mV and the input resistance increased by 150 +/- 15%, suggesting an effect mediated by the closure of a potassium conductance. Indeed, CCK-8s reduced both the amplitude and the time constant of decay of a calcium-dependent potassium conductance. When tested with pancreatic polypeptide (which reduces pancreatic exocrine secretion), cells that responded to CCK-8s with an excitation were, instead, inhibited by pancreatic polypeptide. These data indicate that CCK-8s may control pancreas-exocrine secretion also via an effect on pancreas-projecting DMV neurons.

Cholecystokinin-induced satiety is mediated through interdependent cooperation of CCK-A and 5-HT3 receptors.

Recent evidence supports a role for the serotonin-3 (5-HT3) receptors in the modulation of cholecystokinin (CCK)-induced satiation. Likewise, 5-HT's anorectic response has been linked to recruitment of peripheral CCK-A receptors. Evidence to date, however, does not elucidate whether there is a concomitant interaction between CCK-A and 5-HT3 receptors or whether each receptor functions independently in the negative feedback control of food intake elicited by CCK. In the present study, we used selective receptor antagonists to investigate the roles of CCK-A and 5-HT3 receptors in CCK-induced satiation. Intraperitoneal administration of CCK-8 reduced 30-min 15% sucrose intake in a dose-responsive manner. Prior treatment with ondansetron (1.0 mg/kg ip), a highly selective 5-HT3 receptor antagonist, attenuated CCK-induced suppression of food intake in a dose-responsive manner. Pretreatment with lorglumide (1.0 mg/kg ip), a selective CCK-A receptor antagonist, reversed CCK-induced inhibition of sucrose intake. Finally, simultaneous blockade of CCK-A and 5-HT3 receptors by lorglumide and ondansetron, as well as concomitant administration of the two antagonists with CCK, produced a significant synergistic increase in sucrose intake compared with intakes after administration of saline, CCK, or either antagonist alone. These findings support evidence that CCK-A and 5-HT3 receptors cooperate interdependently in control of short-term food intake. Most likely, this interconnection exists through a feed-forward parallel model arising from CCK-A and 5-HT3 receptors, where activation of one system engages the other to intensify the overall satiety signal.

Anthranilic acid based CCK1 antagonists: the 2-indole moiety may represent a "needle" according to the recent homonymous concept.

Recently we described an innovative class of non-peptide CCK(1) antagonists keeping appropriate pharmacophoric groups on the anthranilic acid employed as a molecular scaffold. The lead compound obtained, VL-0395, characterized by the presence of Phe and the 2-indole moiety at the C- and N-termini of anthranilic acid, respectively, is endowed with submicromolar affinity towards CCK(1) receptors. Thus, we have prepared and tested on CCK receptors a library of VL-0395 analogues in order to investigate the precise topological and essential key interactions of the 2-indole group of the lead with the CCK(1) receptor. The obtained results confirm that this group establishes very specific interactions with this receptor sub-site and may be viewed as a "needle" group.

Differential bile-pancreatic secretory effects of CCK-58 and CCK-8.

In this work, we 1) synthesized rat CCK-58, 2) determined the amounts and forms of rat CCK in whole blood after stimulation of its release by casein, 3) determined the potency of CCK-8 and CCK-58 peptides to displace labeled CCK-8 from CCK(A) and CCK(B) receptors transfected into Chinese hamster ovary (CHO) cells, and 4) examined the biological actions of CCK-8 and rat CCK-58 in an anesthetized rat model. CCK-58 was the only detected endocrine form of CCK in rat blood. Synthetic rat CCK-58 was less potent than CCK-8 for displacing the label from CCK(A) and CCK(B) receptors in transfected CHO cells. However, rat CCK-58 was more potent than CCK-8 for stimulation of pancreatic protein secretion in the anesthetized rat. In addition, CCK-58 but not CCK-8 stimulated fluid secretion in this anesthetized rat model. These data suggest that regions outside the COOH terminus of rat CCK-58 influence the expression of CCK biological activity. The presence of only CCK-58 in the circulation and the fact that its biological activity differs from CCK-8 suggests that CCK-58 deserves scrutiny in other physiological models of CCK activity.

Dietary and hormonal stimulation of rat exocrine pancreatic function regulates CRHSP-28 phosphorylation in vivo.

Dietary regulation of digestive enzyme secretion from the pancreas is essential for the breakdown of macronutrients in the gastrointestinal tract. Ca(2+)-responsive heat stable protein (CRHSP)-28 is a regulatory protein that modulates the exocytosis of digestive enzymes from pancreatic acinar cells. In the present study, isoelectric focusing and immunoblotting were used to characterize CRHSP-28 phosphorylation in isolated rat acinar cells and also after hormonal and dietary stimulation of rat pancreas in vivo. CRHSP-28 was highly phosphorylated in isolated acini after stimulation with a physiologic range of concentrations of cholecystokinin-octapeptide (CCK-8). Activation of the high affinity state of the CCK-A receptor with the synthetic peptide JMV-180 confirmed the physiologic relevance of the response. CRHSP-28 phosphorylation was contingent on elevated cellular Ca2+ because it was maximally stimulated by Ca2+ ionophore, but unchanged after protein kinase C, cAMP or cyclic guanosine monophosphate activation. Intravenous infusion of rats with a secretory concentration of the CCK analog, caerulein, stimulated CRHSP-28 phosphorylation by 100% over control (P < 0.01) within 15 min of dosing. Moreover, CRHSP-28 phosphorylation was stimulated by 150% over control (P < 0.05) immediately after consumption of a semipurified AIN-93 diet. These data demonstrate that CRHSP-28 phosphorylation occurs in vivo and can be used as a functional indicator of nutrient-driven acinar cell activation.