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Methacholine-induced cGMP production is regulated by nitric oxide generation in rabbit submandibular gland cells
Toshihiko Sakai1, Hiromi Michikawa, Shunsuke Furuyama
1Department of Physiology, Nihon University School of Dentistry at Matsudo, Chiba 271-8587, Matsudo, Japan.
Abstract:
Guanosine 3',5'-monophosphate (cGMP) is an intracellular messenger in various kinds of cell. We investigated the regulation of cGMP production by nitric oxide (NO) in rabbit submandibular gland cells. Methacholine, a muscarinic cholinergic agonist, stimulated cGMP production in a dose- and time-dependent manner, but the alpha-agonist phenylephrine, substance P and the beta-agonist isoproterenol failed to evoke cGMP production. In fura-2-loaded cells, methacholine induced an increase in intracellular Ca2+ ([Ca2+]i) in a concentration-dependent manner, which was similar to that for cGMP production. When the external Ca2+ was chelated with EGTA, methacholine failed to induce cGMP production. Ca2+ ionophore A23187 and thapsigargin, which induce the increase in [Ca2+]i without activation of Ca2+-mobilizing receptors, mimicked the effect of methacholine. cGMP production induced by methacholine, A23187 and thapsigargin was clearly inhibited by NG-nitro-L-arginine methylester (L-NAME), a specific inhibitor of nitric oxide synthase (NOS). S-Nitroso-N-acetyl-DL-penicillamine (SNAP), a NO donor, induced cGMP formation. In the lysate of rabbit submandibular gland cells, Ca2+-regulated nitric oxide synthase activity was detected. These findings suggest that cGMP production induced by the activation of muscarinic cholinergic receptors is regulated by NO generation via the increase in [Ca2+]i.
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