Regulation of reactivated elongation in lysed cell models of teleost retinal cones by cAMP and calcium.

Abstract

Teleost retinal cones elongate in the dark and contract in the light. In isolated retinas of the green sunfish Lepomis cyanellus, cone myoids undergo microtubule-dependent elongation from 5 to 45 micron. We have previously shown that cone contraction can be reactivated in motile models of cones lysed with Brij-58. Reactivated contraction is both actin and ATP dependent, activated by calcium, and inhibited by cAMP. We report here that we have obtained reactivated cone elongation in lysed models prepared by the same procedures. Reactivated elongation is ATP dependent, activated by cAMP, and inhibited by calcium. The rate of reactivated elongation is proportional to the cAMP concentration between 10 microM and 0.5 mM, but is constant between 10 microM and 1.0 mM Mg-ATP. No elongation occurs if cAMP or Mg-ATP concentration is less than or equal to 5 microM. Mg-ATP is required for both cAMP-dependent and cAMP-independent processes, suggesting that Mg-ATP is required both for a regulatory process entailing cAMP-dependent phosphorylation and for a force-producing process. Free calcium concentrations greater than or equal to 10(-7) reduce the elongation rate by 78% or more, completely inhibiting elongation at 10(-5) M. This inhibition is not due to competition from calcium-activated contraction. Cytochalasin D blocks reactivated contraction, but does not abolish calcium inhibition of reactivated elongation. Thus calcium directly affects the elongation mechanism. Calcium inhibition is calmodulin dependent. The calmodulin inhibitor trifluoperazine abolishes calcium inhibition of elongation. Furthermore, calcium blocks elongation only if present during the lysis step; subsequent calcium addition has no effect. However, if calcium plus exogenous calmodulin are subsequently added, elongation is again inhibited. Thus calcium inhibition appears to require a soluble calmodulin which is lost shortly after lysis.