Effects of 5-hydroxytryptamine (serotonin) and forskolin on intracellular free calcium in isolated and fura-2 loaded smooth-muscle cells from the anterior byssur retractor (catch) muscle ofMytilus edulis

Abstract

Effects of 5-hydroxytryptamine (5-HT) and forskolin on intracellular free calcium concentration ([Ca²⁺]_i) were studied in suspensions of fura-2 loaded smooth-muscle cells from the anterior byssus retractor ‘catch’ muscle ofMytilus edulis. The successive addition of 5 mM carbachol (CCh) and 100 mM KCl to the suspension evoked a transient elevation of [Ca²⁺]_i from the resting value of 124±2.7 nM (mean ± SE,n=18) to 300–400 nM, which was associated with contraction. The change in [Ca²⁺]_i induced CCh was concentration-dependent with the EC₅₀ of 10⁻⁵ M. The resting [Ca²⁺]_i was unaffected by 10 μM 5-HT. The change in [Ca²⁺]_i induced by 5 mM CCh was suppressed by 5-HT from 167±14.0 (n=11) to 124±14.9 (n=8) nM whereas that induced by 100 mM KCl was enhanced from 321±31.9 to 405±17.6 nM (n=8). 5-HT applied during the decaying phase of the CCh response caused a rapid decline in [Ca²⁺]_i. In both the responses to CCh and KCl, the falling phase was accelerated by 5-HT. 10 μM forskolin, a potent activator of adenylate cyclase, mimicked the effects of 5-HT as did a membrane-permeant cyclic AMP analogue, 8-parachlorophenylthio cyclic AMP (cpt-cAMP). Application of 100 μM cpt-cAMP partially suppressed the Ca²⁺ _i response to CCh and enhanced that to KCl.d-Tubocurarine (500 μM) added during the decaying phase of the response induced by 100 μM CCh, caused a rapid decline in [Ca²⁺]_i similar to that caused by both 5-HT and forskolin. In essentially Ca²⁺-free sea water, or in the presence of 10 μM D600 in seawater containing 4 mM, Ca²⁺, the response to CCh was partially suppressed, whereas that to KCl was completely abolished, demonstrating a CCh-induced release of intracellularly stored Ca²⁺. The remaining component of the response to CCh, in either Ca²⁺-free sea water or in the presence of D600, was abolished by both 5-HT and forskolin. The results suggest that 5-HT has multiple effects on [Ca²⁺]_i in the ABRM, and implicate cyclic AMP in this effect, and that one of the mechanisms underlying these responses is the inhibition of an agonist-induced release of stored Ca²⁺. In addition, that Ca²⁺ _i is at, or close to resting values during the ‘catch state’.