Long-Term Culture of Neonatal Rat Pancreatic Endocrine Cells as Model for Insulin-Secretion and Ion-Channel Studies

Abstract

So far, only freshly isolated cells or short-term cultures have been used to study ion-channel activity in pancreatic nontumor beta-cells. We report a procedure for the long-term cultivation of pancreatic endocrine cells to study the relationship between ion channels and insulin secretion. Using thimerosal to suppress fibroblastoid cell proliferation and a preliminary 2-day cell exposure to alternating normal (5.6 mM) and high (16.7 mM) glucose levels, we observed a significant secretory responsiveness of the cells to a glucose challenge for at least 4 wk in culture. Cells also responded to glucose or other secretagogues, such as quinine and the sulfonylurea glyburide, with membrane voltage oscillations. In the cell-attached configuration of the patch-clamp technique, a 65-pS-conductance K+ channel was observed, which was inhibited by glucose, quinine, and glyburide. In the inside-out configuration, the activity of this channel was suppressed by ATP applied to the cytoplasmic side of the membrane. A K+ channel with a conductance of 200 pS was also observed, which was activated by intracellular Ca2+. A 13-pS-conductance glucose-insensitive K+ channel was present in both cell-attached and inside-out patch recordings. Even after 3 wk, the characteristics of these currents and channels were comparable to those reported by other investigators with freshly dissociated or short-term-cultured beta-cells from neonatal and adult rats and adult mice. Therefore, the neonatal rat endocrine cell culture characterized herein provides an improved model for long-term investigations combining secretion and electrophysiological studies.