Guanylate cyclase inhibitors: Effect on tone, relaxation, and cGMP content of lower esophageal sphincter

Relaxation of the lower esophageal sphincter (LES) results from activation of its intrinsic innervation. This relaxation is associated temporally with an increase in the guanosine 3',5'-cyclic monophosphate (cGMP) content of the muscle. This study tests the hypothesis that variations in the production of cGMP mediate resting LES tone and nerve-induced relaxation. We examined the effects of guanylate cyclase inhibitors, such as cystamine and methylene blue (MB), on the resting tone, resting membrane potential, electrical field stimulation (EFS)-induced relaxation, and cGMP content of circular smooth muscle from the LES of the opossum. Strips of sphincter muscle were placed in a tissue bath and stretched to 125% resting length. Both cystamine and MB increased the resting tone of LES muscle in a concentration-dependent manner (EC₅₀ = 1.1 ± 0.2, n = 12, and 1.6 ± 0.4 mM, n = 10, respectively). The increase in tone by cystamine was not blocked by tetrodotoxin, atropine, or propranolol. Cystamine (1 mM) did not alter the resting membrane potential of circular muscle cells of the LES. The removal of extracellular Ca²⁺ by the addition of ethylene glycol-bis(β-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA, 4 mM) and nifedipine (1 μM) shortened the duration but not the amplitude of the response to cystamine. Pretreatment with caffeine (5 mM) in the presence of EGTA and nifedipine to deplete intracellular Ca²⁺ stores blocked the increase in tone by cystamine. Cystamine (1 mM) failed to inhibit LES relaxation induced by EFS. Carbachol, at a concentration that induced a similar increase in baseline tone, attenuated the nerve-mediated relaxation. Cystamine did not alter basal cGMP levels, but inhibited the rise in cGMP induced by EFS. The data indicate that cystamine increases LES tone but does not inhibit EFS-induced relaxation, even though it inhibits EFS-induced increases in cGMP content. The increase in tone is dependent on the presence of intracellular Ca²⁺ stores.