Cytosolic heparin inhibits muscarinic and alpha-adrenergic Ca2+ release in smooth muscle. Physiological role of inositol 1,4,5-trisphosphate in pharmacomechanical coupling.
In order to test the physiological significance of inositol 1,4,5-trisphosphate (InsP3) in pharmacomechanical coupling, we have utilized two near-physiological systems, in which relatively high molecular weight solutes can be applied intracellularly and receptor coupling is retained: beta-escin permeabilization and reversible permeabilization. We showed that in smooth muscle permeabilized with beta-escin, one of the saponin esters, alpha 1-adrenergic (phenylephrine) and muscarinic (carbachol) agonists, as well as caffeine and InsP3, cause contractions mediated by Ca2+ release. These contractions were calmodulin-dependent and blocked by depletion of Ca2+ stored in the sarcoplasmic reticulum. Intracellular heparin (Mr = about 5000), a blocker of InsP3 binding to its receptor and a specific inhibitor of InsP3-induced Ca2+ release in smooth muscles, inhibited the responses to the agonists and to InsP3, but not those to caffeine, nor did it block the enhanced contractile response to cytoplasmic Ca2+ induced by agonists and by GTP gamma S. Neomycin blocked Ca2+ release induced by carbachol, but not by caffeine. In reversibly permeabilized ileum smooth muscle cells, loaded with Fura-2 acid and heparin, the intracellular heparin inhibited Ca2+ release and contractions induced by carbachol in Ca2+-free, high K+ solution. Heparin did not inhibit the high K+ contractions (with 1.2 mM Ca2+) and had no significant inhibitory effects on carbachol-induced responses in the presence of extracellular Ca2+. These results, obtained under near-physiological conditions, support the conclusion that InsP3 is the major physiological messenger of the Ca2+ release component of pharmacomechanical coupling, but not of the components mediated by Ca2+ influx or by potentiation of the contractile response to Ca2+.
Kobayashi
S
S
Department of Physiology, University of Virginia School of Medicine, Charlottesville 22908.
Kitazawa
T
T
Somlyo
A V
AV
Somlyo
A P
AP
D000818:Animals; D002110:Caffeine / Q000494:pharmacology; D002217:Carbachol / Q000494:pharmacology*; D003600:Cytosol / Q000378:metabolism; D004533:Egtazic Acid / Q000494:pharmacology; D006168:Guinea Pigs; D006493:Heparin / Q000494:pharmacology*; D007082:Ileum / Q000187:drug effects / Q000502:physiology; D066298:In Vitro Techniques; D015544:Inositol 1,4,5-Trisphosphate / Q000494:pharmacology / Q000502:physiology*; D007537:Isometric Contraction / Q000187:drug effects*; D007700:Kinetics; D009119:Muscle Contraction; D009130:Muscle, Smooth / Q000187:drug effects / Q000502:physiology*; D009131:Muscle, Smooth, Vascular / Q000187:drug effects / Q000502:physiology*; D010656:Phenylephrine / Q000494:pharmacology*; D011188:Potassium / Q000494:pharmacology; D011651:Pulmonary Artery / Q000187:drug effects / Q000502:physiology; D012433:Ryanodine / Q000494:pharmacology
D012433:Ryanodine; D010656:Phenylephrine; D002110:Caffeine; D004533:Egtazic Acid; D015544:Inositol 1,4,5-Trisphosphate; D002217:Carbachol; D006493:Heparin; D011188:Potassium