Vasodilation Effects and Action Mechanisms of TMB-8 on Basilar Artery in Rabbits

Abstract

Background: 8-(N,N'-diethylamino)-n-octyl-3,4.5-trimethoxybenzoate (TMB-8) is a potent Ca2+-antagonist that can prevent/treat ischemic stroke and inhibit the contractility of smooth, skeletal. and cardiac muscles. Further studies are warranted to elucidate the efficacy of TMB-8 on rabbit basilar artery preparation and its action mechanisms on vascular smooth muscle cell cultures.

Methods and Results: Effects of TMB-8 on the contractility of rabbit's basilar artery in vitro and those on intracellular free Ca²⁺ concentrations. [Ca²⁺]_i, were studies with isolated organ bath and Fura-2 methods. Histamine-induced concentration-response curves were shifted by TMB-8 in a mixed manner whereas those of norepinephrine and KCI were shifted in a non-competitive manner. In the presence of nifedipine or in a Ca²⁺-free medium, 2,5-di(tert-butyl)-1,4-benzohydroquinone (BHQ) (10 μM) induced an immediate transient contraction in rabbit basilar artery, whereas ryanodine showed a slow, weak. sustained contraction, followed by a weak, sustained relaxation. TMB-8 (30 μM) significantly inhibited these contractions of BHQ and ryanodine. Further, aminophylline enhanced the inhibitory action of TMB-8 on vasocontractions. suggesting that TMB-8's inhibitory actions may be related to the increase of cAMP level. The muscle contraction induced by BHQ was enhanced by pretreatment of the artery ring with TMB-8 for 15 minutes and then TMB-8 was rinsed out. These results indicate that TMB-8 pretreatment can increase Ca²⁺ sequestration into sarcoplasmic reticulum. which leads to a larger subsequent Ca²⁺ release by BHQ. KCI-induced increase of [Ca²⁺]_i in vascular smooth muscle cells was reduced when the cells were bathed in the medium containing nifedipine. TMB-8 made further reduction on KCI-induced [Ca²⁺]_i increase in nifedipine-containing solution, which had already blocked the voltage-operated Ca²⁺ entry.

Conclusion: These results indicate that (a) TMB-8 can enhance Ca²⁺ sequestration into sarcoplasmic reticulum, which leads to a larger amount of Ca²⁺ that can be released by BHQ; (b) TMB-8 can inhibit KCI-induced muscle contraction caused by the reduction of [Ca²⁺]_i through saturation of Ca²⁺ inside the sarcoplasmic reticulum rather than a direct blockade of Ca²⁺-influx at cell membrane site; and (c) TMB-8 increases cAMP, which enhances Ca²⁺ uptake into the sarcoplasmic reticulum.