Characterization of calcium sources and downstream signaling pathways following electrical field stimulation of rodent longitudinal esophageal muscle

Abstract

Aims To explore the mechanisms involved in esophageal body longitudinal muscle contraction induced by electrical field stimulation. Methods Isometric contractions of esophageal segments from wistar rats in an organ bath were induced by electrical field stimulation (duration 1 s, frequency 1-100 Hz, intensity 30-90 V) before and after application of pharmacological probes to test involvement of muscarinic receptors, Rho kinase, Ca2+ release, protein kinase C, calmodulin and L-type Ca2+ channels. Results Electrical field stimulation (EFS) induced contractions showed a frequency and intensity-dependent behavior. Based on the effect size, expressed by the Cohen’s d, they were insensitive to the muscarinic receptor blocker atropine (1 µM), the Rho kinase inhibitor Y-27632 (10 µM) as well as the inhibitors of Ca2+ release 2-aminoethoxydiphenylborane (2-APB, 100µM) and 1,1’-diheptyl-4,4’-bipyridinium (DHBP, 100 µM). In contrast, contractions were reduced by the protein kinase C inhibitor chelerythrine (10µM) and by the calmodulin antagonist N-[6-aminohexyl]-5-chloro-1-naphthalenesulfonamide hydrochloride (W-7, 100 µM). Verapamil (100 µM) abolished EFS-induced contractions. Conclusions Based on our findings with 2-APB, DHBP, and verapamil, extracellular Ca²+ appears to be the source of the increase in intracellular Ca²+ underlying EFS-induced EB contractions. Furthermore, the effects observed with chelerythrine and W-7 may suggest that this increase in intracellular Ca²+ may subsequently activate PKC- and calmodulin-dependent signaling pathways.