CALCIUM-ACTIVATED CHLORIDE CHANNEL AND MYOSIN LIGHT CHAIN KINASE INHIBITORS ABOLISH NANC SLOW IJP IN CIRCULAR SMOOTH MUSCLE OF OPOSSUM ESOPHAGUS

Y Zhang, WG Paterson

Gastrointestinal Diseases Research Unit, Queen’s University, Kingston, Ontario

The effects of 9-anthroic acid (9-AC) and niflumic acid (NA), Ca²⁺-activated Cl- channel (I_Ca(Cl)) blockers, and wortmannin and 1-(5-chloronaphthalene-1-sulfonyl)-1-H-hexahydro-1,4-diazepine (ML-9), inhibitors of myosin light chain kinase (MLCK), on slow IJP (sIJP) were studied using conventional intracellular microelectrode recordings in circular smooth muscle of opossum esophageal body perfused with Kreb’s solution which was preheated at 35°C and gased with 5% CO2 + 95% O2 in the presence of atropine (3 µM) plus guanethidine (3 µM) and substance P (1 µM). Resting membrane potential (MP) was –51.9 ± 0.9 mV (n = 57) with MP fluctuations of 1 - 3 mV. A single square wave nerve stimulation of 0.5 ms duration and 80 V induced a sIJP with amplitude of 6.2 ± 0.3 mV, half amplitude duration of 633 ± 25 ms and rebound depolarization amplitude of 2.6 ± 0.2 mV (n = 57). 9-AC and NA hyperpolarized MP and abolished MP fluctuations, sIJP and rebound depolarization in a concentration-dependent manner. IC₅₀ of NA for MP hyperpolarization and suppression of sIJP and rebound depolarization were 25.7 ± 3.5 µM, 76.5 ± 18.3 µM and 38.8 ± 5.4 µM (n = 6), respectively. Wortmannin and ML-9 did not significantly change the MP, but concentration-dependently inhibited sIJP and sIJP rebound with IC₅₀ of 10.7 ± 0.8 µM and 4.4 ± 1.9 µM, 26.7 ± 3.3 µM and 17.3 ± 0.1 µM (n = 5). The maximal effective concentrations of 9-AC, NA, wortmannin and ML-9 were 1 mM, 300 µM, 30 µM and 100 µM. These data suggest that in the tissues studied, activation of I_Ca(Cl), which requires MLCK, contributes to resting MP, and that closing of I_Ca(Cl) is responsible for sIJP.

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