Effects of imidazoline compounds on intracellular CA2+ and apoptosis

Abstract

Cytoplasmic free Ca2+ concentration ([Ca2+]i) plays a pivotal role in the regulation of insulin release from the pancreatic beta-cell. [Ca2+]i is also important in the control of apoptosis. The extracellular Ca2+ and intracellular Ca2' pools are responsible for the changes in [Ca2+]i in the pancreatic beta-cell subsequent to stimulation with nutrients, hormones and neuropeptides. The metabolism of glucose leads to an increase in the ATP/ADP ratio and subsequent closure of ATP-dependent K' channels (KATP channels), membrane depolarization, opening of voltage-gated L-type Ca2+ channels and, finally, an influx of extracellular Ca2+.

A great number of pharmacological agents affect [Ca2+]i and thus regulate the insulin secretory machinery. Among these are the imidazolines, compounds with the characteristic imidazoline moiety. Importantly, imidazoline compound RX871024 (1-phenyl-2-(imidazolin-2-yl)benzimidazole) increased glucose- and arginine-induced insulin and somatostatin release and decreased arginine-stimulated glucagon release. We have therefore explored the mechanism of the insulinotropic action of imidazoline compounds in pancreatic beta-cells. Furthermore, we have investigated the effect of RX871024 on apoptosis of the P-cell.

RX871024 increased [Ca2+]i by closure of KATP channels, leading to membrane depolarization and opening of voltage-gated L-type Ca2+ channels and, finally, influx of extracellular Ca2+. This effect was observed in single islets and in single beta-cells isolated from Wistar rats, oblob mice, and insulin-secreting HIT-T15 cells. Moreover, RX871024 increased intracellular [Ca2+]i in human pancreatic islets. In addition to the increase in [Ca2+]i due to activation of Ca2+ influx into the cell, the imidazoline compounds RX871024 and phentolamine induce release of Ca2+ from non-mitochondrial thapsigargin-sensitive intracellular stores.

The insulinotropic activity of the imidazoline compound RX871024 was studied in diabetic GK (Goto-Kakizaki) rats. The spontaneously diabetic non-obese GK rat exhibited high basal plasma glucose and insulin levels and poor glucose-induced insulin secretion, which makes it a suitable model for type 2 diabetes. The insulinotropic activity of RX871024 was higher than that of the sulfonylurea glibenclamide in islets from both control and GK rats. This difference was more pronounced in GK rat islets. Glucose induced a delayed [Ca2+]i response in the GK rat pancreatic islet. This delay in [Ca2+]i increase is likely to result from the defective metabolism of glucose in the diabetic islet. RX871024 induced a faster increase in [Ca 2+]i and a faster inhibition of KATP channel activity in GK rat islets compared to control islets. Thus, RX871024 substantially improved glucose sensitivity in diabetic beta-cells.

In many cases elevated levels of [Ca2+]i over physiological concentrations have been demonstrated to trigger cell death. In the present studies, the role of Ca2+ in beta-cell apoptosis has been investigated. High glucose concentrations, as well as the absence of glucose in the medium, interleukin 1-0 (IL-1beta) and the sulfonylurea tolbutamide induced apoptosis of pancreatic beta-cells. by a process which was Ca2+-dependent. IL-10 triggered apoptosis in ob/ob mouse beta-cells. by activating nitric oxide synthase and increasing nitric oxide (NO) production. Protein kinase G and the Ca/calmodulin-dependent protein phosphatases calcineurin are involved in the apoptotic action of IL-1beta.

In contrast to the sulfonylurea tolbutamide, the imidazoline compounds RX871024 and efaroxan promoted survival of beta-cells. by decreasing beta-cell apoptosis. RX871024 suppressed IL-1beta-induced apoptosis by the inhibition of inducible nitric oxide synthase and a subsequent decrease in NO production. In conclusion, the imidazoline compound RX871024 enhanced insulin and somatostatin release and inhibited glucagon release. The increase in [Ca2+]i is the important mechanism behind the insulinotropic effect of RX871024. This compound substantially improved glucose sensitivity in diabetic beta-cells. Moreover, the imidazoline compounds RX871024 and efaroxan exerted an antiapoptotic action. These studies may help in design of new drugs for the management of diabetes mellitus.