Effects of calcium and chelating agents on the ability of various agonists to increase cyclic GMP in pancreatic acinar cells

175

Cholecystokinin (CCK) increases pancreatic enzyme secretion by virtue of its ability to mobilize cellular calcium (for review see refs. 1 and 2), and this calcium mobilization is accompanied by increased cellular cyclic GMP (cGMP) (3-6) and changes in the electrical properties of the acinar cell plasma membrane (7,8). Previous attempts to examine the interaction of CCK with its cell-surface receptors have been hampered by the low specific activity of the tritium-labeled ligand (9-11). As a result, correlations between changes in binding of the labeled peptide and changes in cell function have been poor (10,11). In the present study we have prepared an 125I-labeled derivative of CCK with a specific activity sufficiently high (900-1300 Ci/ mmol; 1 Ci = 3.7 X 1010 becquerels) to be used to examine the interaction of CCK and structurally related peptides with their receptors on dispersed pancreatic acini. MATERIALS AND METHODSUnless stated otherwise the standard incubation solution contained 24.5 mM Hepes (pH 7.4), 98 mM NaCl, 6 mM KCI, 2.5 mM KH2PO4, 1.2 mM MgCI2, 11.5 mM glucose, 5 mM sodium fumarate, 5 mM sodium glutamate, 5 mM sodium pyruvate, 0.5 mM CaCl2, 2 mM glutamine, 1% (wt/vol) albumin, 0.01% trypsin inhibitor, 1% (vol/vol) amino acid mixture, 1% (vol/vol) essential vitamin mixture, and 5 mM theophylline.125I-Labeled natural porcine CCK (125I-CCK-33) was prepared by using the modification of the procedure of Bolton and Hunter (12) described by Rehfeld (13). CCK-33 (1.3 nmol) was dissolved in 4 ,l of 2.5 mM acetic acid and then mixed with 20 ,ul of 50 mM sodium borate, pH 10.0. ['25I]Iodinated N-succinimidyl-3-(4-hydroxyphenyl)propionate (1.0 mCi; 1500 Ci/mmol; New England Nuclear) was dried under a gentle stream of N2 and combined with the CCK-33-containing solution. The mixture was incubated at 0°C for 30 min, after which 250 ,ul of 0.2 M glycine in 50 mM sodium borate, pH 10.0, was added and the incubation was continued at 0°C for 5 min. At the end of the incubation, 500 ,gl of 6 M guanidine hydrochloride and then 250 ,l of 0.1% gelatin in 0.1 M acetic acid, pH 4.5, were added to the reaction tube. The radiolabeled peptide was isolated by applying the reaction mixture to a column of Sephadex G-50 superfine (1.5 X 50 cm) that had been equilibrated with 0.1% gelatin in 0.1 M acetic acid (pH 4.5). The column was eluted with the gelatin/acetic acid solution at a flow rate of 7.5 ml/hr, and the elution profile of 125I was essentially the same as that illustrated in figure 1 of ref. 13. The fractions containing 125I-CCK-33 (those corresponding to fractions 69-74 in figure 1 in ref. 13) were combined. The specific activities of the various preparations of 125I-CCK-33 used for the present studies were 900-1300 Ci/mmol and their biological activities, determined from their abilities to increase amylase release from pancreatic acini (14), were at least 75% of the ability of native CCK-33.Dispersed acini from guinea pig pancreas were prepared by using the procedure described previously (14) and were suspended in s...

“…Binding of 125I-CCK-33 (15), amylase release (14), outflux of 45Ca (16), and cellular cGMP (17) were measured with the procedures described in detail previously.…”

Section: Methodsmentioning

confidence: 99%

Interaction of cholecystokinin with specific membrane receptors on pancreatic acinar cells

Jensen

Lemp

Gardner

1980

175

“…Cyclic GMP was measured by using a modification (12) of the procedure of Harper and Brooker (13). In each experiment each value was determined in quadruplicate and the coefficient of variation was always <7%.…”

mentioning

confidence: 99%

Interaction of physalaemin, substance P, and eledoisin with specific membrane receptors on pancreatic acinar cells.

Jensen¹,

Gardner²

1979

We have prepared '251-labeled physalaemin and have examined the kinetics, stoichiometry, and chemical specificity with which the labeled peptide binds to dispersed acini from guinea pig pancreas. Binding of '25I-labeled physalaemin was saturable, temperature-ependent, and reversible and reflected interaction of the labeled peptide with We (1, 2) and others (3, 4) have found that peptides isolated from amphibian skin (caerulein, bombesin, litorin, and physalaemin) as well as eledoisin, a peptide isolated from the salivary gland of a Mediterranean octopod,t produce changes in the function of pancreatic acinar cells similar to those caused by muscarinic cholinergic agents and cholecystokinin (CCK). In pancreatic fragments as well as dispersed acinar cells, each of these amphibian peptides increases amylase secretion, cellular cyclic GMP, and outflux of exchangeable cellular calcium but does not alter cellular cyclic AMP (1). These effects of amphibian peptides are not inhibited by atropine (1, 4), and therefore are not mediated by muscarinic cholinergic receptors. The effects of caerulein appear to be mediated by receptors that also interact with CCK because the actions of caerulein and CCK, but not those of other pancreatic secretagogues, can be inhibited competitively by dibutyryl cyclic GMP (5). The effects of bombesin and litorin on pancreatic acinar cells appear to be mediated by a common receptor that does not interact with other secretagogues. Binding of 125I-labeled [Tyr4] bombesin (125I-[Tyr4]bombesin) to pancreatic acini in vitro can be inhibited by bombesin and litorin but not by other secretagogues (6) and there is a close correlation between the abilities of bombesin and litorin to inhibit binding of 1251-[Tyr4]bombesin and their abilities to alter the function of pancreatic acini (6). Because physalaemin and eledoisin have similar COOHterminal amino acid sequences and do not interact with receptors for other pancreatic secretagogues, we have proposed (1) that these peptides might interact with a common class of receptors.To explore further the interaction of physalaemin and structurally similar peptides with pancreatic acini, we have prepared 125I-labeled physalaemin (125I-physalaemin) and have examined its ability to bind to dispersed pancreatic acini. We have also explored the abilities of various peptides to inhibit this binding, and the relationship between a peptide's ability to inhibit binding of 125I-physalaemin and its ability to alter acinar cell function. Our results indicate that pancreatic acini possess a single class of receptors that interact reversibly with physalaemin, eledoisin, and substance P and that occupation of approximately 45% of these receptors is sufficient to cause a maximal change in acinar cell function.MATERIALS AND METHODS Materials. Male guinea pigs (175-225 g) were obtained from the Small Animals Section, Veterinary Resources Branch, National Institutes of Health. Hepes and cyclic somatostatin were obtained from Calbiochem; synthetic human gastrin was from ...

“…Receptors for peptide hormones such as angiotensin II (with no known adenylate cyclase linkage) also manifest specific regulation by guanine nucleotides (31). CCK enhancement of amylase secretion in the pancreas is associated with calcium outflux and cyclic GMP accumulation (32,33). Conceivably the GTP effect is associated with calcium regulatory sites.…”

mentioning

confidence: 99%

Distinct cholecystokinin receptors in brain and pancreas.

Innis¹,

Snyder²

1980

428

187