Cyclic GMP Causes Ca2+ Desensitization in Vascular Smooth Muscle by Activating the Myosin Light Chain Phosphatase

doi:10.1074/jbc.272.8.5063

Cyclic GMP Causes Ca²⁺ Desensitization in Vascular Smooth Muscle by Activating the Myosin Light Chain Phosphatase*

From the Department of Physiology and Biophysics, Georgetown University Medical Center, Washington, D. C. 20007

^‡ To whom correspondence should be addressed:
Dept. of Physiology and Biophysics, Georgetown University Medical Center, 3900 Reservoir Rd, NW, Washington, D. C. 20007.
Tel.: 202-687-4298; Fax: 202-687-7407; E-mail: tkitaz01{at}medlib.georgetown.edu

Abstract

Using permeabilized, arterial smooth muscle strips where membrane-associated pathways remain intact but intracellular Ca²⁺ stores are depleted, we investigated mechanism(s) for the Ca²⁺ desensitization of contractile force by cGMP. The nonhydrolyzable analog 8-bromo-cGMP, when applied to these strips with submaximal Ca²⁺ levels clamped, dramatically and reversibly reduced the steady state levels of phosphorylation at 20-kDa myosin light chain and contractile force, with a nanomolar concentration required to obtain 50% reduction. Supramaximal concentrations of 8-bromo-cGMP (10 μM), however, did not change the steady state relationship between phosphorylation and force. When light chain phosphatase activity was blocked at pCa 6.7, 10 μM 8-bromo-cGMP did not affect the rates of rise of light chain phosphorylation and contractile force. When light chain kinase activity was blocked, 10 μM 8-bromo-cGMP significantly accelerated light chain dephosphorylation and force relaxation from the maximal contraction steady state. The light chain phosphorylation time course of a pCa 6.0-induced contraction in the presence of 8-bromo-cGMP exhibited kinetics that are predictable from a mathematical model in which only light chain phosphatase activity is increased. The results of this study strongly suggest that cGMP indirectly activates light chain phosphatase, the first proposed mechanism for cGMP-induced Ca²⁺ desensitization in vasodilatation.

Footnotes

↵* This work was supported by National Institute of Health Grant HL51824. The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵¹ The abbreviations used are:

MLC₂₀

20-kDa myosin light chain

8Br-cGMP

8-bromo guanosine 3′,5′-cyclic monophosphate

MLCK

myosin light chain kinase

MLCP

myosin light chain phosphatase

MC-LR

microcystin-LR

ML-9

1-(5-chloronaphthalene-1-sulfonyl)-¹H-hexahydro-1,4-diazepine

PKG

protein kinase G or cGMP-activated kinase

GTPγS

guanosine 5′-3-O-(thio)triphosphate.
- Received July 10, 1996.
- Revision received December 16, 1996.