InsP3 In Cells Research Articles

Effect of intracellular injection of inositol trisphosphate on cytosolic calcium and membrane currents in Aplysia neurons

Pacemaker cells of Aplysia californica display a regular bursting that results from a complex interplay of Ca(2+)-mediated conductances and a continuous influx and extrusion of Ca2+. The effect of the second messenger 1,4,5-inositol trisphosphate (InsP3) on intracellular free Ca2+ concentration (Cai) regulation and electrical properties was investigated in identified neurons of the abdominal ganglion (R15, L2-L4, L6). Double-barreled Ca-selective microelectrodes were used to pressure inject InsP3 and measure Cai at the same point. Brief injection of InsP3 resulted in an average increase of Cai of 9.2 +/- 10.0 microM (+/- SE; n = 14) that decayed in about 1 min. The InsP3-induced elevation of Cai increased in a dose-dependent manner and saturated when large amounts of InsP3 were injected. The InsP3-induced Cai increase was the result of mobilization from intracellular stores; Cai could be repeatedly mobilized by InsP3 in cells superfused with 0 Ca artificial seawater for more than 60 min. Following multiple injections of InsP3, there was no evidence of immediate inhibition or facilitation. the spatial nature of the InsP3-induced Cai increase was investigated by moving the double-barreled Ca-selective microelectrode tip in a stepwise manner relative to the membrane surface. The largest InsP3-induced Cai increases were measured in an area 0-80 microns from the membrane surface; some cells had their largest InsP3-induced Cai increase some 120-160 microns away from the membrane. Injection of InsP3 in a bursting neuron induced an immediate train of action potentials followed by membrane hyperpolarization and a decrease in the burst frequency. Injection of InsP3 in voltage-clamped cells induced a biphasic response: a rapid inward current followed by a more prolonged outward current; the temporal overlap of the currents was depth dependent. Injection of InsP3 or Ca2+ from a double-barreled injecting electrode induced currents that were different in waveform and time course, indicating that part of the conductance change induced by InsP3 is direct and not mediated by the mobilized Ca2+. In BAPTA [1,2-bis(2-aminophenoxy)ethane-N,N,N',N'tetra-acetic acid]-loaded cells, the InsP3-induced inward current was mostly unaffected while the Ca-induced outward current was largely attenuated. The results suggest that InsP3 mobilizes Ca2+ from discrete intracellular compartments and induces distinct changes in membrane currents that seem to be independent of the Cai increase.

Relationship between inositol polyphosphate production and the increase of cytosolic free Ca2+ induced by vasopressin in isolated hepatocytes.

Addition of vasopressin to rat hepatocytes prelabeled with myo-[2-3H]inositol resulted in a very rapid decrease [3H]phosphatidylinositol 4,5-bisphosphate (Ptd-Ins-4,5-P2) which was paralleled by increases of up to 3-fold in the levels of [3H]inositol trisphosphate (Ins-P3) and [3H]inositol bisphosphate (Ins-P2). Increases of [3H]inositol phosphate (Ins-P) were not detected until about 5 min after hormone addition. These data indicate that the major pathway for hormone-induced lipid breakdown in liver is through a phosphodiesterase for PtdIns-4,5-P2 and that decreases of phosphatidylinositol are a secondary result of increased PtdIns-4,5-P2 resynthesis. Using the fluorescent Ca2+ indicator Quin 2, cytosolic free Ca2+ increased from 160 nM to about 400 nM after vasopressin addition to hepatocytes and preceded the conversion of phosphorylase b to a. Half-maximal and maximal increases of cytosolic free Ca2+ and phosphorylase a activity were observed at 0.2 and 1 nM vasopressin, respectively. The dose-response curve for the initial rate of cytosolic free Ca2+ increase was very similar to those obtained for the initial rates of Ins-P3 production and PtdIns-4,5-P2 breakdown. Pretreatment of hepatocytes with Li+ caused a 3--4-fold potentiation of vasopressin-induced elevations of Ins-P, Ins-P2, and Ins-P3, with half-maximal effects at 0.5, 1, and 5 mM, respectively. The calculated maximal concentrations of Ins-P3 in cells treated with 20 nM vasopressin were 10 and 30 microM, respectively, without and with Li+. Lithium did not affect the initial rate of inositol polyphosphate production or Ca2+ mobilization. The increase of Ins-P3 which correlated with peak cytosolic free Ca2+ elevation was about 0.6 microM. In a saponin-permeabilized hepatocyte preparation, Ins-P3 (1 microM) caused Ca2+ release from a vesicular, ATP-dependent Ca2+ pool. The data presented here suggest that Ins-P3 may be a second messenger for the mobilization of intracellular Ca2+ by hormones in liver.