Effects of an Endogenous Cyclic AMP-Dependent Protein Kinase Catalytic Subunit on Ca-Uptake by Plasma Membrane Vesicles from Rat Mesenteric Artery

Abstract

A plasma membrane-enriched fraction of vesicles from rat mesenteric artery possessed ATP-dependent calcium properties similar to those which have been reported previously. The catalytic subunit from a soluble, cyclic AMP-dependent protein kinase was prepared from the same tissue, in partially purified form. Calcium uptake by membrane vesicles increased 30% in the presence of this kinase. This effect was proportional to the kinase concentration, and was blocked by either catalytic subunit inhibitor or by prior boiling of the subunit preparation. Stimulation of calcium uptake by the kinase occurred maximally between 1 and 10 µMfree calcium. These findings suggest that cyclic AMP and its dependent protein kinase may participate in the regulation of smooth muscle tension at high free myoplasmic calcium concentrations.

Purification and properties of d-myo-inositol 1,4,5-trisphosphate 3-kinase from bovine iris sphincter smooth muscle: effects of protein phosphorylation in vitro and in intact muscle

“…To determine the specificity of the purified InsP3 3-kinase, the reaction products of the enzyme assay were analysed by HPLC. As shown in Figure 3, when 3H-labelled InsP3 was incubated with the purified enzyme in the presence of 5 Substrate-velocity relationships InsP., 3-kinase activity increased as a function of substrate concentration ( Figure 5). In the presence of 50 nM CaM, the apparent Km for InsP3 was 0.56 ,uM and the Vmax 2.5 4umol/min per mg of the purified enzyme protein.…”

“…Ins (1,4,5)P3 (InsP!3), a second messenger molecule generated by receptor-mediated hydrolysis of Ptdlns(4,5)P2 (PtdlnsP2), has been shown to mobilize intracellular Ca2+ in a wide variety of tissues [1]. In smooth muscle, such as that of the iris sphincter, activation of Ca2+-mobilizing receptors by appropriate agonists results in a rapid increase in InsP3 leading to rapid contraction of the muscle (see [2,3] for reviews).…”

“…These findings on a cross-talk between the two second messenger systems suggest that phosphorylation of specific protein(s) in the InsP3-Ca2+ signal transduction pathway, via cAMP-dependent protein kinase A (PKA), are probably involved in the mechanism underlying relaxation in smooth muscle. While the precise loci for cAMP inhibition are still unclear, potential sites include phosphorylation of cell surface receptors, G-proteins, phospholipase C, InsP3 receptor and myosin light chain kinase (see [3] for review; [4][5][6]). The activities of InsP!3 metabolizing enzymes may also be modulated by PKA.…”

Activation of cAMP-Dependent Protein Kinase in Rat Aorta by cAMP Analogs Is Not Correlated with Relaxation

“…It is generally held that relaxation of smooth muscle induced by cAMP-elevating agents such as (3-adrenergic agonists is mediated via activation of cAMP-dependent protein kinase (PICA) and subsequent phosphorylation of specific proteins [for reviews, see 1, 2], The identities of these proteins are unknown, and the precise mechanism by which PKA activation can produce smooth muscle relaxation has not been fully elucidated. It has been var iously ascribed to increased calcium uptake by the sarco plasmic reticulum [3], increased calcium extrusion across the plasma membrane [4,5] or direct inhibition of myosin light chain kinase activity [6]. The activation state of PKA in intact vascular smooth muscle preparations during relaxation by cAMP-elevating agents has not been exten sively explored.…”