Calcium-independent activation of contractile apparatus in smooth muscle by calyculin-A

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 X 10(-8) to 1 X 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 X 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 induce 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 X 10(-3) M) and K-252a (2 X 10(-5) M). Low concentrations of Ca++ (approximately 1 X 10(-6) M) augmented the CL-A-induced contraction in the permeabilized taenia. In native actomyosin prepared from chicken gizzard CL-A induced phosphorylation of the 20 kDa myosin light chain (MLC) in the absence of Ca++.(ABSTRACT TRUNCATED AT 250 WORDS)