Cytosolic heparin inhibits muscarinic and alpha-adrenergic Ca2+ release in smooth muscle. Physiological role of inositol 1,4,5-trisphosphate in pharmacomechanical coupling

• Published in: The Journal of biological chemistry 1989-10, Vol.264 (30), p.17997-18004
• Main Authors: Kobayashi, S, Kitazawa, T, Somlyo, A V, Somlyo, A P
• Format: Article
• Language: English
• Subjects: Animals; Caffeine - pharmacology; Carbachol - pharmacology; Cytosol - metabolism; Egtazic Acid - pharmacology; Guinea Pigs; Heparin - pharmacology; Ileum - drug effects; Ileum - physiology; In Vitro Techniques; Inositol 1,4,5-Trisphosphate - pharmacology; Inositol 1,4,5-Trisphosphate - physiology; Isometric Contraction - drug effects; Kinetics; Muscle Contraction; Muscle, Smooth - drug effects; Muscle, Smooth - physiology; Muscle, Smooth, Vascular - drug effects; Muscle, Smooth, Vascular - physiology; Phenylephrine - pharmacology; Potassium - pharmacology; Pulmonary Artery - drug effects; Pulmonary Artery - physiology; Ryanodine - pharmacology
• ISSN: 0021-9258; 1083-351X

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+.