Methacholine-induced cGMP production is regulated by nitric oxide generation in rabbit submandibular gland cells

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 α-agonist phenylephrine, substance P and the β-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 N^G-nitro--arginine methylester (L-NAME), a specific inhibitor of nitric oxide synthase (NOS). S-Nitroso-N-acetyl--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.