Regulation of vascular smooth muscle tone by N-terminal region of caldesmon - Possible role of tethering actin to myosin

To assess the functional significance of tethering actin to myosin by calde smon in the regulation of smooth muscle contraction, we investigated the ef fects of synthetic peptides, containing the myosin-binding sequences in the N-terminal region of caldesmon, on force directly recorded from single per meabilized smooth muscle cells of ferret portal vein. Two peptides were use d, IK29C and MY27C, containing residues from Ile(25) to Lys(53) and from Me t(1) to Tyr(27) Of the human and chicken caldesmon sequence, respectively, plus an added cysteine at the C terminus. In cells clamped at pCa 6.7, both peptides increased basal tone. Pretreatment of cells at pCa 6.7 with IK29C or MY27C decreased the amplitude of subsequent phenylephrine-induced contr actions but not microcystin-racemic mixture-induced contractions. In all ca ses the effects of the peptides were concentration-dependent, and IK29C was more potent than MY27C, in agreement with their relative affinity toward m yosin, The peptides were ineffective after the phenylephrine contraction wa s established. MY27C did not further increase the magnitude of contraction caused by a maximally effective concentration of IK29C, consistent with the two peptides having the same mechanism of action. Neither polylysine nor t wo control peptides containing scrambled sequences of IK29C, which do not b ind myosin, had any effect on basal or phenylephrine-induced force. Our res ults suggest that IK29C and MY27C induce contraction by competing with the myosin-binding domain of endogenous caldesmon, Digital imaging of fluoroiso thiocyanate-tagged IK29C confirmed the association of the peptide with intr acellular filamentous structures. The results are consistent with a model w hereby tethering of actin to myosin by caldesmon may play a role in regulat ing vascular tone by positioning the C-terminal domain of caldesmon so that it is capable of blocking the actomyosin interaction.