sincalide and fura-2-am

Cholecystokinin (CCK) stimulates insulin secretion. It is, however, not established whether CCK receptors are expressed in insulin-producing cells. We therefore investigated, by in situ hybridization, whether CCK-A or CCK-B receptor mRNA could be detected in normal rat pancreatic islets and in the rat insulinoma cell line, RINm5F. The CCK-A, but not the CCK-B, receptor transcript was detected in both islets and RINm5F cells. Islet CCK-A receptors were mostly confined to the center of the islets corresponding to the distribution of the B cells. In RINm5F cells, insulin release was not significantly affected by cholecystokinin (CCK)-8-S (10(-13) to 10(-7) M), which is in contrast to the insulinotropic effect of CCK-8-S in normal rat islets. Similarly, in FURA-2AM-loaded cells, CCK-8-S (10(-11) to 10(-7) M) was without effect on the intracellular Ca2+ concentration ([Ca2+]ic) in RINm5F cells, whereas CCK-8-S (10(-7) M) markedly increased [Ca2+]ic (by 366+/-2 nM (P < 0.001) in normal rat islet cells. We conclude that the CCK-A, but not the CCK-B, receptor subtype is expressed in both normal rat islets and in the rat insulinoma-derived cell line RINmS5F. There is, however, a functional difference between normal islets and the RINm5F cells with respect to effects of CCK-8-S on insulin release and [Ca2+]ic.

Topics: Animals; Calcium; Carbachol; Fura-2; Gastric Mucosa; Immunohistochemistry; In Situ Hybridization; Insulin; Insulinoma; Intestinal Mucosa; Islets of Langerhans; Male; Pancreas; Rats; Rats, Sprague-Dawley; Receptors, Cholecystokinin; RNA, Messenger; Sincalide; Tumor Cells, Cultured

This study investigates the effect of magnesium (Mg2+) on the secretory responses and the mobilization of calcium (Ca2+) and Mg2+ evoked by cholecystokinin-octapeptide (CCK-8) in the exocrine rat pancreas. In the isolated intact perfused pancreas CCK-8 (10(-10) M) produced marked increases in juice flow and total protein output in zero and normal (1.1 mM) extracellular Mg2+ [Mg2+]o compared to a much reduced secretory response in elevated (5 mM and 10 mM) [Mg2+]o. Similar effects of perturbation of [Mg2+]o on amylase secretion and 45 Ca2+ uptake (influx) were obtained in isolated pancreatic segments. In pancreatic acinar cells loaded with the fluorescent bioprobe fura-2 acetomethylester (AM), CCK-8 evoked marked increases in cytosolic free Ca2+ concentration [Ca2+]i in zero and normal [Mg2+]o compared to a much reduced response in elevated [Mg2+]o. Pretreatment of acinar cells with either dibutyryl cyclic AMP (DB2 cAMP) or forskolin had no effect on the CCK-8 induced changes in [Ca2+]i. In magfura-2-loaded acinar cells CCK-8 (10(-8) M) stimulated an initial transient rise in intracellular free Mg2+ concentration [Mg2+]i followed by a more prolonged and sustained decrease. This response was abolished when sodium (Na+) was replaced with N-methyl-D-glucamine (NMDG). Incubation of acinar cells with 10 mM Mg2+ resulted in an elevation in [Mg2+]i. Upon stimulation with CCK-8, [Mg2+]i decreased only slightly compared with the response obtained in normal [Mg2+]o. CCK-8 caused a net efflux of Mg2+ in pancreatic segments; this effect was abolished when extracellular sodium [Na+]o was replaced with either NMDG or choline. The results indicate that Mg2+ can regulate CCK-8-evoked secretory responses in the exocrine pancreas possibly via Ca2+ mobilization. Moreover, the movement of Mg2+ in pancreatic acinar cells is dependent upon extracellular Na+.

Topics: Amylases; Analysis of Variance; Animals; Bucladesine; Calcium; Cytosol; Dose-Response Relationship, Drug; Fluorescent Dyes; Fura-2; In Vitro Techniques; Kinetics; Magnesium; Meglumine; Pancreas; Pancreatic Juice; Rats; Rats, Wistar; Sincalide; Sodium; Spectrophotometry, Atomic; Time Factors

The effects of the partial muscarinic agonist pilocarpine on physiological responses were investigated in rat pancreatic acinar cells and compared with carbachol, a full muscarinic agonist, together with previous results using JMV-180, a partial agonist of CCK-A receptors. Pilocarpine was found to stimulate amylase release from isolated pancreatic acini in a concentration-dependent manner. At a maximal concentration (10 microM), pilocarpine was only capable of stimulating 63% of the secretion stimulated by a maximal concentration of carbachol. Moreover pilocarpine did not induce a decrease in secretion at supramaximal concentrations as does carbachol. In acini loaded with fura-2, superfusion of pilocarpine resulted exclusively in generation of intracellular Ca2+ concentration ([Ca2+]i) oscillations at all concentrations tested (0.3 microM-1 mM), in marked contrast to high concentrations of full agonists, which result in a biphasic sustained increase in [Ca2+]i. In common with low concentrations of other secretagogues that stimulate [Ca2+]i oscillations, pilocarpine at all concentrations was only able to stimulate a very small increase in phosphoinositide (PI) hydrolysis. In acini previously incubated with [3H]inositol, pilocarpine was shown to stimulate PI hydrolysis 27% above basal, compared with 872% for carbachol. To ascertain if this small degree of PI hydrolysis seen with pilocarpine is responsible for the generation of [Ca2+]i oscillations, an inhibitor of phospholipase C-linked processes, U-73122, which has been shown to inhibit Ca2+ oscillations induced by carbachol and CCK but not JMV-180 was tested. This agent rapidly inhibited pilocarpine-stimulated oscillations, indicating that in contrast to JMV-180, oscillations induced by pilocarpine are the result of PI hydrolysis.

Topics: Amylases; Animals; Calcium; Carbachol; Dose-Response Relationship, Drug; Fluorescent Dyes; Fura-2; In Vitro Techniques; Inositol; Inositol Phosphates; Male; Pancreas; Phosphatidylinositols; Pilocarpine; Rats; Rats, Sprague-Dawley; Signal Transduction; Sincalide

1. Procaine (0.03-10 mM) inhibited carbachol (CCh)-induced amylase release from rat isolated pancreatic acini in a competitive manner. Kinetic analysis of the relation between CCh concentrations and the amount of amylase released in the presence of various procaine concentrations indicated that procaine caused competitive inhibition with the affinity constant (pA2) value of 5.00 +/- 0.08. 2. Receptor binding assay confirmed that procaine (0.01-10 mM) competitively inhibited [N-methyl-3H]-scopolamine chloride ([3H]-NMS) binding to its receptor with binding affinity (pKi) of 4.63 +/- 0.10. 3. Procaine transformed CCh-evoked [Ca2+]i dynamics: the initial rise in [Ca2+]i followed by a gradual decay during continuous stimulation with 3 microM CCh was transformed by 0.3 mM procaine to the oscillatory [Ca2+]i dynamics, which resembled the response to 0.3 microM CCh in the absence of procaine. The initial phase of [Ca2+]i oscillation corresponded to the initial phase of CCh-induced amylase release in isolated perfused acini. 4. Procaine (0.3-3 mM) did not inhibit the secretory response to cholecystokinin octapeptide (CCK-8) in isolated incubated acini. A higher concentration of procaine (10 mM) caused weak but significant inhibition of the response to only limited concentrations of CCK-8, 30 and 100 pM. Procaine lower than 10 mM was ineffective on [125I]-BH-CCK-8 binding, although procaine (10 mM) caused weak but significant inhibition of the binding.

Topics: Amylases; Animals; Binding, Competitive; Calcium; Carbachol; Fluorescent Dyes; Fura-2; Image Processing, Computer-Assisted; In Vitro Techniques; Iodine Radioisotopes; Kinetics; Male; N-Methylscopolamine; Pancreas; Parasympatholytics; Perfusion; Procaine; Rats; Rats, Sprague-Dawley; Receptors, Cholecystokinin; Receptors, Muscarinic; Scopolamine Derivatives; Sincalide

CCK-8 stimulates insulin secretion by an effect involving phosphoinositide (PI) hydrolysis and release of Ca2+ from intracellular stores. In this study, we examined the dependence of Na+ for the effects of CCK-8. CCK-8 (100 nM) stimulated insulin secretion from isolated rat islets. A first phase, which lasted 10 min, was not affected by removal of external Na+, whereas a second phase was abolished. CCK-8-stimulated 45Ca2+ and 3H efflux in 45Ca(2+)- and myo-[2-3H]-inositol-prelabelled islets were not affected by removal of external Na+. In a second series of experiments, pancreatic rat islet cells were loaded with the Ca2+ fluorophore FURA 2-AM. CCK-8 (100 nM) induced a rapid and transient increase in the cytoplasmic free Ca2+ concentration ([Ca2+]IC), followed by a subsequent reduction of [Ca2+]IC below the prestimulatory levels. The CCK-8-induced increase in [Ca2+]IC was not dependent on extracellular Na+, whereas the decline in [Ca2+]IC after the initial peak was slower in the absence than in the presence of Na+. Thus, the present study shows that both the second phase of CCK-8-stimulated insulin secretion and the CCK-8-stimulated postpeak reduction in [Ca2+]IC are dependent on extracellular Na+.

Topics: Animals; Calcium; Cells, Cultured; Fluorescent Dyes; Fura-2; Glucose; Inositol; Insulin; Insulin Secretion; Islets of Langerhans; Kinetics; Male; Rats; Rats, Sprague-Dawley; Sincalide; Sodium; Spectrometry, Fluorescence; Time Factors

The intracellular free Ca2+ ion concentration [( Ca2+]i) was measured in individual rat pancreatic B-cells loaded with fura-2. The cells were prepared by enzymatic digestion and fluorescence-activated cell sorting. The resting concentration of [Ca2+]i in B-cells was 126.3 +/- 3.1 nM in the presence of 4.4 mM glucose. Addition of cholecystokinin-8 (CCK-8) resulted in rapid and transient rises in [Ca2+]i. Perifusion of B-cells with galanin attenuated the amplitude and duration of CCK-8-induced [Ca2+]i changes and this inhibitory effect was concentration-dependent and reversible. Perifusion of B-cells with nifedipine, a voltage-sensitive Ca2+ channel blocker, reduced the duration of the [Ca2+]i increase induced by CCK-8, indicating that the Ca2+ entry from the extracellular space was, at least in part, involved in CCK-8-induced increases in [Ca2+]i.

Topics: Animals; Calcium; Cells, Cultured; Culture Techniques; Fluorescent Dyes; Fura-2; Galanin; Islets of Langerhans; Kinetics; Male; Nifedipine; Peptides; Rats; Rats, Inbred Strains; Sincalide; Spectrometry, Fluorescence

The C-terminal octapeptide of cholecystokinin (CCK-8) is known to stimulate insulin secretion. We examined its effects on the cytoplasmic free calcium concentration ([Ca2+]IC) in isolated rat pancreatic islet cells. At 8.3 mM glucose and 1.28 mM Ca2+, CCK-8 (100 nM) rapidly increased [Ca2+]IC to a short-lived peak, whereafter the [Ca2+]IC, within 1.5 minutes, fell to values below baseline. CCK-8 also rapidly increased the [Ca2+]IC at 3.3 mM glucose and in a calcium deficient medium. However, either at low glucose or in the absence of extracellular Ca2+, the post-peak [Ca2+]IC did not fall below baseline levels. The CCKA receptor antagonist, L-364,718 (20 nM), inhibited the effects of CCK-8 on [Ca2+]IC. The results suggest that CCK-8 in islet cells liberates calcium from intracellular stores by activating CCKA receptors.

Topics: Animals; Benzodiazepinones; Calcium; Carbachol; Cells, Cultured; Cholecystokinin; Cytoplasm; Devazepide; Egtazic Acid; Fluorescent Dyes; Fura-2; Glucose; Islets of Langerhans; Kinetics; Male; Rats; Rats, Inbred Strains; Sincalide