Inositol 1,4,5-trisphosphate mediates adrenaline activation of K+ conductance in mouse peritoneal macrophages

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Abstract
In mouse peritoneal macrophages, α1-adrenoceptor stimulation evokes a Ca2+-dependent K+ current [I0(Adr)] [Hara et al. (1991) Pflügers Arch 419:371–379]. The roles of D-myo-inositol 1,4,5-trisphosphate (InsP3) and a GTP-binding protein (G protein) in I0(Adr) were investigated with tight-seal whole-cell recordings and fura-2 fluorescence measurements. Intracellular injection of lnsP3 (5–50 μM) evoked transient outward currents [I0(InsP3)] with or without damped oscillations in membrane currents at -40 mV. Dialysis with 0.2 mM guanosine 5′-[3-thio]triphosphate (GTP[γS], a poorly hydrolysable GTP analogue) at -40 mV activated oscillatory outward currents or a slowly developing steady current on which such oscillations were superimposed after a delay of 10–90 s. I0(InsP3) and the GTP[γS]-induced current {I0(GTP[γS])} were accompanied by an increase in conductance. Reversal potentials of both responses closely depended on the extracellular K+ concentration. Fura-2 measurements revealed that I0(InsP3) and I0(GTP[γS]) result from a rise in intracellular free Ca2+ concentration ([Ca2+]i). Removal of extracellular Ca2+ did not abolish I0(InsP3) and I0(GTP[γS]). Both were blocked by bath-applied charybdotoxin. Intracellular D- myo-inositol 1,3,4,5-tetrakisphosphate (InsP4, 50 μM) did not evoke any responses, whereas D-myo-inositol 2,4,5-trisphosphate [InsP3(2,4,5), 20 μM] elicited an outward current at -40 mV. I0(InsP3) was completely blocked by prior dialysis with the InsP3 receptor antagonist heparin (5 mg/ml). Inclusion of guanosine 5′-[2-thio] diphosphate (GDP[βS], 2 mM) or heparin (5 mg/ml) together with GTP[γS] in the patch pipette solution completely blocked I0(GTP[γS]). These results indicate that intracellular injection of InsP3 or GTP[γS] mimic I0(Adr). Furthermore, intracellular dialysis with heparin (3 mg/ ml) or GDP[βS] (2 mM) greatly accelerated a run-down of I0(Adr). On the other hand, I0(Adr) was markedly prolonged in a cell dialysed with GTP[γS] (0.2 mM). Therefore, it is concluded that I0(Adr) results from stimulation of α1-adrenoceptor and InsP3 formation via a G protein.