Endothelin and a Ca2+ ionophore raise cyclic GMP levels in a neuronal cell line via formation of nitric oxide

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Abstract
1 The vasoconstrictor peptide endothelin-1 caused a fast, transient rise in guanosine 3′:5′-cyclic monophosphate (cyclic GMP) levels in a neuronal cell line (mouse neuroblastoma x rat glioma hybrid cells 108CC15). The mechanism of activation of guanylate cyclase by endothelin-1 was investigated. The endothelin-1-induced rise depended on the release of internal Ca2+. 2 The stimulation of cyclic GMP synthesis induced by endothelin-1 was suppressed after preincubating the cells in medium containing haemoglobin (IC50 3 μm). Similarly, pretreatment of the cells with the l-arginine analogues, l-canavanine (IC50 60 μm) or NG-monomethyl-l-arginine (IC50 2.5 μm), inhibited the cyclic GMP response to endothelin-1. Therefore, endothelin-1 activates guanylate cyclase most probably via formation of nitric oxide, which is released from l-arginine. 3 The Ca2+ ionophore ionomycin induced a transient rise in cyclic GMP levels, which was also suppressed by preincubation in the presence of either haemoglobin or the l-arginine analogues l-canavanine or NG-monomethyl-l-arginine. Therefore, we conclude that ionomycin can activate guanylate cyclase by a mechanism involving nitric oxide formation, similar to that induced by endothelin-1. 4 The alkaloid veratridine, which activates Na+ channels and also causes influx of Ca2+ induced a transient rise of cyclic GMP levels in the neuronal cell line. This stimulation was blocked by pretreating the cells with l-canavanine, NG-monomethyl-l-arginine or haemoglobin. 5 Loading the cells with the Ca2+ chelator BAPTA suppresed the cyclic GMP response to application of endothelin-1, ionomycin, or veratridine. Thus, in the neuronal cell line a rise in cytosolic Ca2+ activity seems to be sufficient to stimulate the nitric oxide forming enzyme which synthesizes the activator of soluble guanylate cyclase.