Reduction of insulin secretion in the insulinoma cell line INS-1 by overexpression of a Ca(v)2.3 (alpha1E) calcium channel antisense cassette

Abstract

Objective: Multiple types of voltage-activated Ca 2+ channels (T, L, N, P, Q and R type) coordinate a variety of Ca 2+ -dependent processes in neurons and neuroendocrine cells. In insulinoma cell lines as well as in endocrine tissues, the non-L-type a1E (Ca v 2.3) subunit is expressed as the tissue-specific splice variant a1Ee. Design and Methods: To understand the functional role of a1E-containing Ca 2+ channels, antisense a1E mRNA was overexpressed in INS-1 cells by stable transfection of an antisense a1E cassette cDNA. As controls, either a sense a1E cassette or a control vector containing enhanced green fluorescent protein as an unrelated gene was stably transfected. The overexpression of each transfected cassette cDNA was recorded by RT-PCR. Results: In three independent antisense a1E INS-1 clones, the glucose-induced insulin release was significantly reduced as compared with wild-type INS-1 cells and with a sense a1E INS-1 clone. However, in the antisense INS-1 clones, the KCl-induced insulin release was less impaired by overexpressing the antisense a1E cassette than the glucose-induced insulin release, leading to the assumption that glucose (15 mmol/l) and KCl (25 mmol/l) finally depolarize the membrane potential to a different extent. Conclusion: a1E is involved in glucose-induced insulin secretion probably by influencing the excitability of INS-1 cells.

CaV1.2 rather than CaV1.3 is coupled to glucose-stimulated insulin secretion in INS-1 832/13 cells

“…This is in accordance with our previous results from the experiments performed in INS-1 cells, where no effect of SNX-482 on exocytosis could be detected (MacDonald et al 2005). On the other hand, Pereverzev et al (2002) studied the effect of a Ca V 2.3 antisense expression cassette and reported a reduction in insulin secretion. In this study, the transfection procedure lowered the insulin content of the cells significantly.…”

Three-Dimensional Scaffolds for Tissue Engineering Applications: Role of Porosity and Pore Size

“…183 Cross-linking of the ECM is associated with local crosslinking of collagen and elastin by lysyl oxidase. [184][185][186] The increase in cross-linking also inevitably results in a reduction of the matrix porosity. Collagenases can increase the matrix porosity by disrupting the ECM network through uncoiling the triple-helical structure of collagen and exposing sites for proteolysis to occur.…”

Regulated Exocytosis and Kiss-and-Run of Synaptic-Like Microvesicles in INS-1 and Primary Rat β-Cells

“…It is well established that exocytosis of insulincontaining LDCVs is preferentially coupled to Ca 2ϩ entry through L-type channels (21,22). R-type channels mediate Ͼ60% of the non-L-type Ca 2ϩ current in mouse ␤-cells (22) and may regulate insulin secretion from INS-1 (23,24) and mouse ␤-cells (22,25). We therefore investigated the role of these channel types in exocytosis of single SLMVs and (Fig.…”