SUMMARYThis study investigates the interaction between physiological doses of the synthetic gut hormones, cholecystokinin-octapeptide (CCK8) and secretin on pancreatic juice secretion in the anaesthetized rat and on amylase secretion and Ca2l and Mg2+ mobilization in isolated pancreatic segments and acinar cells. CCK8 (150 pmol kg-1 h-1) and secretin (100 pmol kg-1 h-1) evoked marked time course increases in pancreatic juice flow, total protein output and amylase secretion in the anaesthetized rat when administered separately compared to saline controls. Simultaneous intravenous infusion of CCK8 and secretin did not yield either an additive response or a potentiation but instead it caused a decrease in secretory responses. Administration of either polymyxin B (10-8 mol kg-1 h-1) or staurosporine (10-8 mol kg-1 h-1), two protein kinase C inhibitors, simultaneously with both CCK8 and secretin caused a further decrease in all secretory parameters. Superfusing pancreatic segments with either CCK8 (10-1 M) or secretin (10-1 M) elevated amylase output compared to the smaller response with a combination of CCK8 and secretin. Combining staurosporine (10-6 M) with CCK8 and secretin resulted in a further decrease in amylase output. CCK8 (10-1' M) evoked a large increase in radiolabelled Ca2+ influx into pancreatic segments and elevated cytosolic free Ca2+ concentration ([Ca2+]i) in acinar cells loaded with the fluorescent dye, Fura-2. Secretin (10-11 M) alone had no significant effect on Ca2+ mobilization but it markedly attenuated the increases in radiolabelled Ca2+ influx and [Ca2+]i elicited by CCK . In superfused pancreatic segments CCK8 (10-11 M) evoked a net efflux of Mg2+ whereas secretin (10-1' M) induced a net uptake of Mg2+. Combining secretin with CCK8 also resulted in a net uptake of Mg2+. The results indicate that both Ca2+ and Mg2+ mobilization may be associated with the interaction between CCK8 and secretin in the rat pancreas.

Application of either acetylcholine (ACh, 10(-5) M) or cholecystokinin-octapeptide (CCK-8, 10(-8) M) to the isolated rat pancreas elicited large increases in amylase secretion, radiolabelled 45Ca2+ influx and cytosolic free calcium [Ca2+]i levels in zero and normal (1.1 mM) extracellular magnesium [Mg2+]o. Elevated [Mg2+]o significantly (p < 0.001) reduced the secretagogue-evoked secretory responses and Ca2+ mobilisation. Stimulation of pancreatic segments with either ACh (10(-5) and 10(-6) M) or CCK-8 (10(-8) and 10(-10) M) resulted in marked elevation in Mg2+ concentration in effluent samples (net efflux). On removal of either ACh or CCK-8, Mg2+ concentration returned to resting level. In pancreatic acinar cells loaded the fluorescent dye magfura, ACh and CCK-8 evoked marked reduction in cytosolic free Mg2+ concentration [Mg2+]i compared to the resting value of 0.82 +/- 0.03 mM (n = 50) in normal medium in the absence of secretagogues. In elevated [Mg2+]o (10 mM) medium, [Mg2+]i rises to 0.98 +/- 0.04 mM (n = 6). Addition of CCK-8 led to only a small reduction in [Mg2+]i in elevated [Mg2+]o. In Mg2+ loaded pancreatic acinar cells, Mg2+ is released in a time dependent manner and this efflux of Mg2+ was sensitive to sodium, extracellular amiloride (1 mM), dinitrophenol (10 mM) and lidocaine (1 mM). The results indicate that Mg2+ is acting as an intracellular messenger to regulate the mobilisation of Ca2+ which in turn mediates enzyme secretion.

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+.

SUMMARYThis study investigates the effects of the gut peptide, secretin, on nerve-mediated and acetylcholine-evoked amylase secretion and calcium (Ca2") and magnesium (Mg2") mobilization in the in vitro rat pancreas. Both electrical field stimulation (EFS; 50 V, 20 Hz, 1 ms) and acetylcholine (ACh; 10-6 M) caused marked increases in amylase output in isolated pancreatic segments. These responses were inhibited by atropine (10-`M). Secretin (10-'°and 108 M) evoked only a small increase in amylase secretion, which was unaffected by atropine. Combining either EFS or ACh with secretin resulted in an attenuation in amylase output compared with the response obtained with either EFS or ACh alone. In a Mg2"-free medium, secretin failed to inhibit the amylase output evoked by EFS. When forskolin (10-5M) was combined with EFS there was a marked potentiation of amylase output. In fura-2-loaded acinar cells, ACh (10-6 M)

SUMMARYThis study investigates the mechanism of magnesium (Mg2") transport (efflux) from the exocrine rat pancreas. Permeabilized pancreatic acini were loaded with Mg2+ by employing a high-Mg2+ (12 mM) buffer containing A23 187 (6 /LM). Net Mg2+ efflux was measured using the technique of atomic absorbance spectrophotometry. Incubation of preloaded acini in a buffer deficient in Mg2+ resulted in a large and time-dependent release of Mg2+ with maximal efflux occurring within 40 min. Pretreatment of loaded acini with bumetanide, SITS or ouabain had no significant effect on Mg2+ efflux. In contrast, when acini were pretreated with 10 mM dinitrophenol, 10-4 M amiloride, 1 mm lidocaine or 1 mm quinidine there were significant (P < 0.001) decreases in net Mg2+ efflux. Replacement of extracellular sodium [Na']0 with either N-methyl-D-glucamine (NMDG), Tris or choline resulted in a significant (P < 0.001) inhibition of Mg2+ efflux. The results of this study indicate that Mg2+ transport (efflux) in pancreatic acinar cells may not be associated with the Na'-K+-ATPase, the Na'-K+-Cl-cotransporter or the anion exchanger, but with a Na'-sensitive Mg2+ transport system.