Guanine nucleotide-dependent inositol trisphosphate formation in chick heart cells.
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 chromotography 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) (GTP gamma S) stimulates the accumulation of [3H]InsP, [3H]InsP2, [3H]InsP3, and [3H]InsP4. The effect of GTP gamma S is half-maximal at 1 microM and maximal above 100 microM. In contrast, GTP gamma S 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.(ABSTRACT TRUNCATED AT 250 WORDS)
Jones
L G
LG
Department of Pharmacology, University of California, San Diego, La Jolla 92093.
Goldstein
D
D
Brown
J H
JH
D000818:Animals; D002217:Carbachol / Q000494:pharmacology; D002478:Cells, Cultured; D002642:Chick Embryo; D004789:Enzyme Activation; D019204:GTP-Binding Proteins / Q000502:physiology; D006150:Guanine Nucleotides / Q000494:pharmacology*; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D006160:Guanosine Triphosphate / Q000031:analogs & derivatives / Q000494:pharmacology; D015544:Inositol 1,4,5-Trisphosphate; D007295:Inositol Phosphates / Q000096:biosynthesis*; D009206:Myocardium / Q000378:metabolism*; D010716:Phosphatidylinositols / Q000378:metabolism; D013403:Sugar Phosphates / Q000096:biosynthesis*; D013873:Thionucleotides / Q000494:pharmacology; D010738:Type C Phospholipases / Q000032:analysis
D006150:Guanine Nucleotides; D007295:Inositol Phosphates; D010716:Phosphatidylinositols; D013403:Sugar Phosphates; D013873:Thionucleotides; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D015544:Inositol 1,4,5-Trisphosphate; D006160:Guanosine Triphosphate; D002217:Carbachol; D010738:Type C Phospholipases; D019204:GTP-Binding Proteins