TY - JOUR

T1 - A New Preparation of Pancreatic Ducts for Patch-Clamp Studies

AU - Novak, Ivana

AU - Hug, M.J.

N1 - Key Words Pancreas Ducts Secretin Acetylcholine ATP Patch clamp Fura-2 Calcium

PY - 1995

Y1 - 1995

N2 - Pancreatic HCO-3 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 °C and ducts were bathed with bicarbonate-containing solutions. Duct cells were found to have a resting membrane voltage, Vm, 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 Vm by 16 and 22 mV (n = 21; 7), respectively. Secretin (10-9 mol/l) and carbachol (10-5 mol/l) depolarized Vm by 12-18 mV (n = 7; 13). ATP in the bathing solution (10-4 mol/l) led to a 12-mV depolarization of Vm (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 at 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: Pflügers Arch 1988;411:58-68, 546-553], are comparable. Similar conclusion can be made about the calcium measurements [Hug M, et al: Pflügers 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.

AB - Pancreatic HCO-3 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 °C and ducts were bathed with bicarbonate-containing solutions. Duct cells were found to have a resting membrane voltage, Vm, 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 Vm by 16 and 22 mV (n = 21; 7), respectively. Secretin (10-9 mol/l) and carbachol (10-5 mol/l) depolarized Vm by 12-18 mV (n = 7; 13). ATP in the bathing solution (10-4 mol/l) led to a 12-mV depolarization of Vm (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 at 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: Pflügers Arch 1988;411:58-68, 546-553], are comparable. Similar conclusion can be made about the calcium measurements [Hug M, et al: Pflügers 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.

U2 - 10.1159/000154770

DO - 10.1159/000154770

M3 - Journal article

VL - 5

SP - 344

EP - 352

JO - Cellular Physiology and Biochemistry

JF - Cellular Physiology and Biochemistry

SN - 1015-8987

IS - 5

ER -