Voltage-independent calcium channels mediate slow oscillations of cytosolic calcium that are glucose dependent in pancreatic beta-cells.

Abstract

Pancreatic beta-cells and HIT-T15 cells exhibit oscillations of cytosolic calcium ([Ca2+]i) that are dependent on glucose metabolism and appear to trigger pulsatile insulin secretion. Significantly, differences in the pattern of this [Ca2+]i oscillatory activity may have important implications for our understanding of how glucose homeostasis is achieved during the feeding and fasting states. When single beta-cells are exposed to a stepwise increase in glucose concentration that mimics the transition from fasting to feeding states, fast irregular oscillations of [Ca2+]i are observed. Alternatively, when single beta-cells are equilibrated in a steady state concentration of glucose that mimics the fasting state, slow periodic oscillations of [Ca2+]i are noted. Here we report a fundamental difference in the mechanism by which glucose induces these two types of [Ca2+]i oscillatory activity. In agreement with previous studies, we substantiate a role for L-type voltage-dependent Ca2+ channels as mediators of the fast oscillations of [Ca2+]i observed after a stepwise increase in glucose concentration. In marked contrast, we report that voltage-independent calcium channels (VICCs) mediate slow oscillations of [Ca2+]i that occur when beta-cells are equilibrated in steady state concentrations of glucose. Slow [Ca2+]i oscillations are mediated by VICCs which are pharmacologically and biophysically distinguishable from voltage-dependent Ca2+ channels that mediate fast oscillations. Specifically, slow [Ca2+]i oscillations are blocked by extracellular La3+, but not by nifedipine, and are independent of changes in membrane potential. Measurement of membrane conductance also indicate an important role for VICCs, as demonstrated by a steady state inward Ca2+ current that is blocked by La3+. The steady state Ca2+ current appears to generate slow [Ca2+]i oscillations by triggering Ca(2+)-induced Ca2+ release from intracellular Ca2+ stores, a process that is mimicked by extracellular application of caffeine, a sensitizer of the ryanodine receptor/Ca2+ release channel. Depletion of intracellular Ca2+ stores with thapsigargin stimulated Mn2+ influx, suggesting the presence of Ca(2+)-release-activated Ca2+ channels. Taken together, these observations are consistent with a role for VICCs (possibly G-type channels) and/or Ca(2+)-release-activated Ca2+ channels as mediators of slow [Ca2+]i oscillations in beta-cells. We propose that slow oscillations of [Ca2+]i probably serve as important initiators of insulin secretion under conditions in which tight control of glucose homeostasis is necessary, as is the case during the fasting normoglycemic state.

Insulinotropic Glucagon-like Peptide-1-mediated Activation of Non-selective Cation Currents in Insulinoma Cells Is Mimicked by Maitotoxin

“…3C, iv), consistent with a decrease in [Ca 2ϩ ] i . Similar activation of Mn 2ϩ quenching has been observed following stimulation of insulinoma cells with PACAP (6), cAMP (22), and thapsigargin (23,24).…”

“…3C) is similar to that observed following thapsigargin treatment of insulinoma cells (23,24). Thapsigargin-sensitive Ca 2ϩ pools can also be depleted by econazole, which inhibits Ca 2ϩ -ATPases (31) and thereby elevates [Ca 2ϩ ] i and also inhibits Mn 2ϩ quenching in HIT cells (23).…”

“…The presence of a Ca 2ϩ store depletion-activated current in pancreatic ␤-cells was proposed from studies showing: 1) that the state of filling of endoplasmic reticulum stores could regulate the membrane potential in mouse ␤-cells (39), 2) that thapsigargin can activate Mn 2ϩ quenching of intracellular fura-2 in RINm5F cells (24), and 3) that the Mn 2ϩ quenching pathway is inhibited by econazole in HIT-T15 cells (23). Activation of I Ca-NS by thapsigargin and its inhibition by both econazole and SKF 96365 are consistent with the suggestion that I Ca-NS may represent a Ca 2ϩ release-activated current (10,31).…”

Pituitary adenylate cyclase-activating polypeptide induces the voltage-independent activation of inward membrane currents and elevation of intracellular calcium in HIT-T15 insulinoma cells.

“…Cells were loaded in this solution for 1.5-2 h at room temperature (20-22 C). Loading HIT-T15 cells at room temperature, rather than at 37 C, substantially reduces the degree of intracellular compartmentalization of fura-2 (14). Glass coverslips with fura-2-loaded adherent cells formed the base of a recording chamber mounted on a Peltier temperature-controlled (32 C) stage of a Zeiss IM35 microscope (Zeiss, Thornwood, NY) equipped with a dual excitation video imaging system (IonOptix Corp., Milton, MA).…”

“…7B). Mn 2+ -permeant channels believed to be associated with capacitative Ca 2+ entry (I CRAC ) have been reported in HIT-T15 cells (14), RINm5F cells (19), and mouse β-cells (20). However, it remains to be determined whether the activation of an I CRAC explains the increased Mn 2+ permeability observed after treatment of HIT cells with PACAP.…”