Effect of methylene blue and N-ethylmaleimide on internal anal sphincter relaxation

Abstract

The present studies were performed in vitro to define the participation of regulatory cyclic nucleotides in the relaxation of internal anal sphincter (IAS) smooth muscle in response to neural stimulation by electrical field stimulation (EFS) vs. exogenous vasoactive intestinal peptide (VIP). EFS and VIP both caused relaxation of the resting tone in the opossum-isolated IAS smooth muscle strips. The addition of permeant cyclic nucleotide derivatives, the guanylate cyclase stimulant sodium nitroprusside (SNP), and the adenylate cyclase stimulant forskolin caused a dose-dependent fall in the resting tension of IAS smooth muscle. The inhibitory effect of the agonists on the IAS smooth muscle was not modified by tetrodotoxin (TTX), a neurotoxin. TTX almost abolished the IAS responses to EFS. The effects of SNP and forskolin were selectively blocked by the putative inhibitors of corresponding enzyme systems, i.e., methylene blue (MB) (3 X 10(-5) M) for guanylate cyclase and N-ethylmaleimide (NEM) (10(-4) M) in the case of adenylate cyclase. NEM and not MB caused significant antagonism of the fall in IAS tension in response to both EFS and VIP during the control experiments. Such data suggest a common biochemical link (adenosine 3',5'-cyclic monophosphate as second messenger system) between the IAS smooth muscle relaxations with neural stimulation and VIP. In addition, a part of the IAS smooth muscle relaxation in response to EFS also involves the mediation of guanosine 5'-cyclic monophosphate.

Role of Adenylate and Guanylate Cyclases in β1-, β2-, and β3-Adrenoceptor-Mediated Relaxation of Internal Anal Sphincter Smooth Muscle

“…This suggests that ␤ 1 -AR activates the cGMP pathway. Because cGMP is more potent than cAMP in the IAS (Moummi and Rattan, 1988), one could predict that xamoterol would induce relaxation with E max values higher than obtained with procaterol and close to that with CL 316243 because the ␤ 2 agonist activates the cAMP pathway and the ␤ 3 agonist activates the cGMP. However, on actual experimentation, xamoterol was found to be least efficacious (E max ϭ 35.15 Ϯ 5.99%; p Ͻ 0.05).…”

“…The procedure to describe details of tissue preparation and isometric tension recording in the IAS smooth muscle has been published in a number of our earlier publications (Moummi and Rattan, 1988;Banwait and Rattan, 2003;Rathi et al, 2003;Sarma et al, 2003).…”

Role of nitric oxide in β3-adrenoceptor activation on basal tone of internal anal sphincter

“…Smooth muscle strips from the IAS of opossums were prepared for recording of isometric tension as described previously (32). Briefly, the animals were anesthetized with pentobarbital sodium (40-50 mg/kg ip), and the entire anal canal was isolated and transferred to oxygenated (95% O 2-5% CO2) Krebs physiological solution (in mM: 118.07 NaCl, 4.69 KCl, 2.52 CaCl2, 1.16 MgSO4, 1.01 NaH2PO4, 25 NaHCO3, and 11.10 glucose.…”

“…During this period, the muscle strips developed steady-state tone. Baseline and optimal length (L o) of the smooth muscle strips were determined as described previously (32). Only smooth muscle strips that developed steady tone and relaxed in response to electrical field stimulation (EFS) were included in the study.…”

Lack of nitrate tolerance in isosorbid dinitrate- and sodium nitroprusside-induced relaxation of rabbit internal anal sphincter

“…Prominent nonadrenergic noncholinergic (NANC) innervations of IAS have been demonstrated [1][2][3] . In vivo and in vitro studies showed that the NANC neurons caused relaxation of the IAS [4][5][6] . The tonic resting pressure of IAS is called "anal resting pressure".…”