| 71JUNG et al.

Although the actin-binding and actomyosin adenosinetriphosphatase (ATPase) inhibitory properties of calponin are well documented in vitro, its function in the smooth muscle cell has not been elucidated. To address this question, we utilized the ferret aortic smooth muscle cell, which shows a protein kinase C-dependent contraction even at pCa (-log [Ca2+]) 9.0 in the absence of a change in myosin light chain phosphorylation. Force was recorded from single, briefly permeabilized cells stimulated via a Ca(2+)-independent pathway by either phenylephrine or the epsilon isoenzyme of protein kinase C. Treatment of stimulated cells with wild-type recombinant calponin reduced steady-state contractile force by 45-60%. When calponin application preceded protein kinase C epsilon treatment, contraction was completely suppressed. On the other hand, calponin phosphorylated at Ser175 or mutant calponin with a Ser175 --> Ala replacement had no effect on contractile force. A peptide corresponding to Leu166-Gly194 of calponin, which included an actin-binding domain but excluded the actomyosin ATPase inhibitory region, was synthesized. Treatment of aortic smooth muscle cells with this peptide triggered a concentration-dependent contraction, presumably by alleviating the inhibitory effect of endogenous calponin. A control peptide with a scrambled sequence of the same residues produced no detectable contractile response. Although other interpretations are possible, these results are consistent with the view that calponin participates in thin filament-mediated regulation of smooth muscle contraction and that it may be part of a Ca(2+)-independent pathway downstream of protein kinase C epsilon.

No abstract

To study the essential features of acetylcholine (ACh)- and caffeine-sensitive cellular Ca2+ storage sites in single vascular smooth muscle cells of the porcine coronary artery, the effects of ryanodine on both ACh- and caffeine-induced Ca2+ mobilization were investigated by measuring intracellular Ca2+ concentration ([Ca2+]i) using Fura 2 in Ca(2+)-containing or Ca(2+)-free solution. The resting [Ca2+]i of the cells was 122 nM in normal physiological solution and no spontaneous activity was observed. In a solution containing 2.6 mM Ca2+, 10 microM ACh or 128 mM K+ produced a phasic, followed by a tonic, increase in [Ca2+]i but 20 mM caffeine produced only a phasic increase. In Ca(2+)-free solution containing 0.5 mM ethylenebis(oxonitrilo)tetraacetate (EGTA), the resting [Ca2+]i rapidly decreased to 102 nM within 5 min, and 10 microM ACh or 20 mM caffeine (but not 128 mM K+) transiently increased [Ca2+]i. Ryanodine (50 microM) greatly inhibited the phasic increase in [Ca2+]i induced by 10 microM ACh or 5 mM caffeine and increased the time to peak and to the half decay after the peak in the presence or absence of extracellular Ca2+. By contrast, ryanodine (50 microM) enhanced the tonic increase in [Ca2+]i induced by 128 mM K+ and also by 10 microM ACh in Ca(2+)-containing solution. In Ca(2+)-free solution containing 0.5 mM EGTA, ACh (10 microM) failed to increase [Ca2+]i following application of 20 mM caffeine. The level of [Ca2+]i induced by 20 mM caffeine was greatly reduced, but not abolished, following application of 10 microM ACh in Ca(2+)-free solution.(ABSTRACT TRUNCATED AT 250 WORDS)