A NEW PREPARATION OF PANCREATIC DUCTS FOR PATCH-CLAMP STUDIES

Pancreatic HCO3- secretion is ascribed to small intra- and interlobular ducts. Although the general transport mechanisms for ducts obtained from the rat pancreas are known, the ion channels and their regulation are not extensively studied in the normal fresh tissue. This paper presents a method for isolation of pancreatic ducts from normal rat pancreas by collagenase digestion and a study of their electrical parameters with the whole-cell nystatin method. In addition, intracellular Ca2+ activity was estimated. All experiments were carried out at 37 degrees C and ducts were bathed with bicarbonate-containing solutions. Duct cells were found to have a resting membrane voltage, V-m, of -55 +/- 2 mV (n = 63). An increase in the bath K+ concentration (to 19 mmol/l) or an introduction of Ba2+ (3-5 mmol/l) into the bathing solution depolarized V-m by 16 and 22 mV (n = 21; 7), respectively. Secretin (10(-9) mol/l) and carbachol(10(-5) mol/l) depolarized V, by 12-18 mV (n = 7; 13). ATP in the bathing solution (10(-4) mol/l) led to a 12-mV depolarization of V-m (n = 17), often preceded by 3-4 mV hyperpolarization. In a parallel series of experiments, ducts were loaded with fura-2 and the fluorescence ratio al 340/380 nm was measured as an indication of the intracellular Ca2+ activity, [Ca2+](i). Carbachol and ATP evoked characteristic calcium transients, the peak changes in [Ca2+](i) were about 370 and 410 nmol/l, respectively (n = 16; 11). Taken together, responses of the present preparation of pancreatic ducts in the electrical parameters, monitored with the whole-cell patch-clamp method, and those in isolated perfused ducts, monitored with the conventional microelectrodes [Novak I, Greger R: Pflugers Arch 1988;411:58-68, 546-553], are comparable. Similar conclusion can be made about the calcium measurements [Hug M, et al: Pflugers Arch 1994;426:412-418]. Hence, the present paper offers a method for preparation of pancreatic duct suitable to study whole-cell conductances, as well as single-ion channels, and their regulation.