SOURCES OF CA2-INDUCED ENDOTHELIUM-DEPENDENT HYPERPOLARIZATION IN RATMESENTERIC-ARTERY( IN RELATION TO GENERATION OF ACETYLCHOLINE)

1 The aim of the present study was to identify the sources of Ca2+ con tributing to acetylcholine (ACh)-induced release of endothelium-derive d hyperpolarizing factor (EDHF) from endothelial cells of rat mesenter ic artery and to assess the pathway involved. The changes in membrane potentials of smooth muscles by ACh measured with the microelectrode t echnique were evaluated as a marker fur EDHF release. 2 ACh elicited m embrane hyperpolarization of smooth muscle cells in an endothelium-dep endent manner. The hyperpolarizing response was not affected by treatm ent with 10 mu M indomethacin, 300 mu M N-G-nitro-L-arginine or 10 mu M oxyhaemoglobin, thereby indicating that the hyperpolarization is not mediated by prostanoids or nitric oxide but is presumably by EDHF. 3 In the presence of extracellular Ca2+, 1 mu M ACh generated a hyperpol arization composed of the transient and sustained components. By contr ast, in Ca2+-free medium, ACh produced only transient hyperpolarizatio n. 4 Pretreatment with 100 nM thapsigargin and 3 mu M cyclopiazonic ac id, endoplasmic reticulum Ca2+-ATPase inhibitors, completely abolished ACh-induced hyperpolarization. Pretreatment with 20 mM caffeine also markedly attenuated ACh-induced hyperpolarization. However, the overal l pattern and peak amplitude of hyperpolarization were unaffected by p retreatment with 1 mu M ryanodine. 5 In the presence of 5 mM Ni2+ or 3 mM Mn2+, the hyperpolarizing response to ACh was transient, and the s ustained component of of hyperpolarization was not observed. On the ot her hand, 1 mu M nifedipine had no effect on ACh-induced hyperpolariza tion. 6 ACh-induced hyperpolarization was nearly completely eliminated by 500 nM U-73122 or 200 mu M 2-nitro-4-carboxyphenyl-N,N-diphenylcar bamate, inhibitors of phospholipase C, but was unchanged by 500 nM U-7 3343, an inactive form of U-73122. Pretreatment with 20 nM staurospori ne, an inhibitor of protein kinase C, did nor modify ACh-induced hyper polarization. 7 These results indicate that the ACh-induced release of EDHF from endothelial cells of rat mesenteric artery is possibly init iated by Ca2+ release from inositol 1,4,5-trisphosphate (IP3)-sensitiv e Ca2+ pool as a consequence of stimulation of phospholipid hydrolysis due to phospholipase C activation, and maintained by Ca2+ influx via a Ni2+- and Mn2+-sensitive pathway distinct from L-type Ca2+ channels. The Ca2+-influx mechanism seems to be activated following IP3-induced depletion of the pool.