CALCIUM-INDEPENDENT ACTIVATION OF CONTRACTILE APPARATUS IN SMOOTH-MUSCLE BY CALYCULIN-A

  • 1 July 1989
    • journal article
    • research article
    • Vol. 250  (1) , 388-396
Abstract
Calyculin-A (CL-A), a novel marine toxin isolated from Discodermia calyx, caused contraction in the smooth muscle of guinea pig taenia ceci and rat aorta in the presence or absence (with 1 mM ethylene glycol bis(.beta.-aminoethyl ether)-N,N''tetraacetic acid) of external Ca++ at concentrations ranging from 1 .times. 10-8 to 1 .times. 10-6 M. In the presence of external Ca++, the contraction induced by CL-A was accompanied by an increase in the cytosolic free Ca++ concentration ([Ca++[cyt) as measured by the fluorescence indicator fura-2. Verapamil (3 .times. 10-6 M) inhibited the increase in [Ca++]cyt, but not tension development caused by CL-A. In the absence of external Ca++, CL-A still caused contraction without changing [Ca++]cyt. Thus, from studies with intact smooth muscle it was demonstrated that, in the absence of external Ca++,CL-A can induced a contraction that was not accompanied by an increase in [Ca++]cyt. In permeabilized taenia, CL-A caused contraction in the absence of Ca++ (with 2 mM ethylene glycol bis(.beta.-aminoethyl ether)N,N''-tetraacetic acid) at concentrations similar to those required to contract intact tissue. This contraction was inhibited by the nonselective kinase inhibitors such as amiloride (1 .times. 10-3 M) and K-252a (2 .times. 10-5 M). Low concentrations of Ca++ (approximately 1 .times. 10-6 M) augmented the CL-A-induced contraction in the permeabilized taenia. In the native actomyosin prepared from chicken gizzard CL-A induced phosphorylation of the 20 kDa myosin light chain (MLC) in the absence of Ca++. This phosphorylation was inhibited by amiloride and K-252a, but not by calmodulin (CaM) inhibitor, trifluoperazine (1 .times. 10-4 M), or protein kinase C inhibitor, polymyin B (1 .times. 10-4 g/ml). CL-A was not effective as a CaM antagonist, or did not influence the activity of the MLC kinase measured under a variety of conditions. CL-A inhibited the dephosphorylation of MLC upon removal of Ca++ and ATP from the native actomyosin, indicating that CL-A inhibits myosin phosphatase. These data suggest that CL-A induced contraction through the direct activation of contractile elements which attributable to the phosphorylation of MLC. This may be due either to the direct activation of Ca++ CaM independent MLC kinase or the unmasking of Ca++/CaM-independent MLC kinase activity via the inhibition of phosphatase activity. In addition, CL-A activates voltage-dependent Ca++ channels, but this effect appears to be independent of the CL-A induced contraction.