Guanine nucleotide-dependent inositol trisphosphate formation in chick hearts cells

Abstract

Stimulation of muscarinic receptors in dissociated embryonic chick heart cells promotes the hydrolysis of the phosphoinositides resulting in accumulation of the breakdown products inositol trisphosphate, bisphosphate, and monophosphate (InsP3, InsP2, and InsP, respectively). [3H]InsP3 and [3H]InsP2 are significantly elevated within 10 seconds of carbachol addition, while there is a lag in the accumulation of [3H]InsP. The time courses of the formation of the inositol phosphates suggest that carbachol activates a polyphosphoinositide-specific phospholipase C resulting in the formation of InsP3, which is subsequently metabolized to InsP2 and InsP. High-performance liquid chromatography analysis demonstrates the formation of both naturally occurring InsP3 isomers (Ins-1,4,5-P3 and Ins-1,3,4-P3) and of inositol tetrakisphosphate (InsP4) as well. To investigate whether a guanine nucleotide-binding protein couples receptor stimulation to phosphoinositide (PI) hydrolysis in the heart, we developed a saponin-permeabilized cell preparation that would allow external manipulation of the intracellular guanosine triphosphate (GTP) concentration. In the permeabilized cell preparation, guanosine-5'-O-(3-thiotriphosphate) (GTPγS) stimulates the accumulation of [3H]InsP, [3H]InsP2, [3H]InsP3, and [3H]InsP4. The effect of GTPγS is half-maximal at 1 μM and maximal above 100 μM. In contrast, GTPγS is ineffective in promoting PI hydrolysis in the nonpermeabilized cell except at high concentrations. Other guanine nucleotides also lead to the accumulation of [3H]InsP in the permeabilized cell, while 5'-adenylylimidodiphosphate does not. Carbachol also stimulates PI hydrolysis in the permeabilized cell preparation although it is less effective than in the intact cell. The response to carbachol is at least additive with the response to GTPγS. The observation that GTPγS stimulates the accumulation of [3H]InsP3 provides evidence that a guanine nucleotide-binding protein is involved in the regulation of the polyphosphoinositide-specific phospholipase C in the heart. The data also demonstrate the formation of the biologically active isomer of InsP3 and suggest that InsP3 may serve a physiological role in the chick heart.