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

In mouse peritoneal macrophages, ₁-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 M) 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[S], 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[S]-induced current {I₀(GTP[S])} 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[S]) result from a rise in intracellular free Ca²⁺ concentration ([Ca²⁺]_i). Removal of extracellular Ca²⁺ did not abolish I₀(InsP₃) and I₀(GTP[S]). Both were blocked by bath-applied charybdotoxin. Intracellular D- myo-inositol 1,3,4,5-tetrakisphosphate (InsP₄, 50 M) did not evoke any responses, whereas D-myo-inositol 2,4,5-trisphosphate [InsP₃(2,4,5), 20 M] 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[S], 2 mM) or heparin (5 mg/ml) together with GTP[S] in the patch pipette solution completely blocked I₀(GTP[S]). These results indicate that intracellular injection of InsP₃ or GTP[S] mimic I₀(Adr). Furthermore, intracellular dialysis with heparin (3 mg/ ml) or GDP[S] (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[S] (0.2 mM). Therefore, it is concluded that I₀(Adr) results from stimulation of ₁-adrenoceptor and InsP₃ formation via a G protein.