Stimulation of plasma membrane Ca2+ -pump ATPase of vascular smooth muscle by cGMP-dependent protein kinase: functional reconstitution with purified proteins.

A 240-kDa protein isolated from porcine aortic smooth muscle as a substrate for cGMP-dependent protein kinase (cGMP kinase) whose phosphorylation was in a close association with stimulation of partially purified plasma membrane Ca2+ -pump ATPase by the kinase was later shown to represent splicing variants of type 1 inositol 1,4,5-trisphosphate (IP3) receptor. To further clarify the role played by this protein in the stimulation of Ca2+ -pumpATPase, it was attempted in thepresent study to specifically remove the protein by immunoprecipitation with an antibody specific to type 1 IP3 receptor. Contrary to expectation, stimulation of the ATPase by cGMP kinase was still observed after removal of the IP3 receptor. Furthermore, cGMP kinase stimulated a highly purified preparation of Ca2+ -pump ATPase deprived of IP3 receptor when the concentrations of the ATPase were low enough (10-20 nM) to make it retain a monomeric form, while it did not produce stimulation when the concentration of the enzyme was increased to 40 nM at which the enzyme is known to take an oligomeric, fully activated form insensitive to activation by calmodulin. Heat-inactivated cGMP kinase and cGMP kinase without cGMP failed to stimulate the highly purified Ca2+ -pumpATPase. In addition, type Ialpha but not type Ibeta cGMP kinase was found to stimulate the ATPase. The stimulation of Ca2+ -pump ATPase by cGMP kinase occurs without any detectable phosphorylation of the ATPase. In conclusion, cGMP kinase can stimulate the plasma membrane Ca2+ -pump ATPase when it is in a monomeric form without phosphorylating the Ca2+ -pump ATPase and that of the two cGMP kinase isozymes found in the vascular smooth muscle, only type Ialpha cGMP kinase participates in the stimulation.

cGMP-kinase mediates cGMP- and cAMP-induced Ca2+ desensitization of skinned rat artery.

(Rp)-8-Bromo-guanosine 3',5'-cyclic monophosphorothioate (Rp-8-Br-cGMPS) inhibited competitively both isozymes of type I alpha and I beta cGMP-dependent protein kinase (cGMP-kinase) purified from porcine aorta with apparent Ki values (microM) of 3.7 for I alpha and 1.8 for I beta. The compound also inhibited bovine heart type II cAMP-dependent protein kinase (cAMP-kinase), but with a Ki of 25 microM. Thus, it is a selective inhibitor of cGMP-kinase. In alpha-toxin-skinned smooth muscle preparations from rat mesenteric artery, 8-Br-cGMP (10(-7) M) and 8-Br-cAMP (10(-6) M) produced a rightward shift of the concentration-contraction curves for Ca2+, denoting a decrease in Ca2+ sensitivity of the contractile elements. The shift by 8-Br-cAMP as well as by 8-Br-cGMP was completely reversed by Rp-8-Br-cGMPS, while a selective inhibitor of activation of cAMP-kinase, (Rp)-adenosine-3',5'-cyclic monophosphorothioate (Rp-cAMPS), was without effects on the shift produced by these two compounds. These findings indicate the pivotal role that the activation of cGMP-kinase plays in the production of a decrease in Ca2+ sensitivity of contractile elements.

Expression of inducible nitric oxide synthase in rat experimental autoimmune myocarditis with special reference to changes in cardiac hemodynamics.

Excessive NO produced by an inducible NO synthase (iNOS) has been implicated in many types of immune-associated disorders of the cardiovascular system, but it remains to be determined whether NO plays a role in myocarditis. Thus, the significance of iNOS expression in the development of experimental autoimmune myocarditis (EAM), an animal model of human giant cell myocarditis, was investigated. Lewis rats were immunized with cardiac myosin and were killed 7, 14, 21, 28, and 49 days after immunization. The development of severe myocarditis was observed on days 14, 21, and 28 in association with significant deterioration of hemodynamics determined by cardiac catheterization, which peaked on day 21. In parallel with histological severity of myocarditis and deterioration of cardiac performance, iNOS activity in the heart measured by [14C]L-citrulline formation was markedly increased on days 14, 21, and 28. The expression of iNOS was confirmed by immunoblotting and was localized to the infiltrating inflammatory cells found in the vicinity of necrotic myocytes by immunohistochemical analysis. Aminoguanidine, a selective inhibitor of iNOS, significantly decreased the iNOS activity (1.04 +/- 0.37 compared with 29.1 +/- 8.62 pmol.min-1.mg protein-1 in untreated myosin-immunized rats, P < .01) and effectively attenuated histopathological changes of EAM on day 21. Hemodynamic parameters were also improved from 64 +/- 3 to 89 +/- 3 mm Hg for mean blood pressure, from 80 +/- 2 to 113 +/- 4 mm Hg for left ventricular systolic pressure, from 7.8 +/- 0.3 to 3.2 +/- 0.3 mm Hg for left ventricular end-diastolic pressure, from 2867 +/- 137 to 4180 +/- 102 mm Hg/s for +dP/dt, and from 2717 +/- 132 to 4180 +/- 184 mm Hg/s for -dP/dt (P < .01). The values after aminoguanidine treatment were not significantly different from the control values. These results suggest an important role for NO in mediating pathophysiological changes in myocarditis of autoimmune origin.

Isolation and characterization of vascular smooth muscle inositol 1,4,5-trisphosphate receptor.

myo-Inositol 1,4,5-trisphosphate (InsP3) receptor of porcine aorta was purified to near homogeneity and its biochemical properties were compared with those of cerebellar InsP3 receptor of the same animal species. The aortic InsP3 receptor consisted of equal amounts of two polypeptides with slightly differing molecular masses of around 240 kDa and was found to possess a single population of InsP3-binding site (Kd of 1.2 nM). The InsP3 receptor purified from porcine cerebellum was also comprised of two polypeptides. However, the molecular mass was slightly but definitely larger, being 250 kDa, and the amounts of the two polypeptides were not equal. The aortic InsP3 receptor cross-reacted with polyclonal antibody specific to type 1 InsP3 receptor as did the cerebellar InsP3 receptor. The aortic InsP3 receptor bound to calmodulin-Sepharose in a Ca(2+)-dependent manner, while the cerebellar InsP3 receptor did not. Reverse transcriptase-PCR analysis revealed two splicing variants of the type 1 InsP3 receptor in porcine aortic smooth muscle distinct from those of the type 1 InsP3 receptor of porcine cerebellum. The possible relevance of this difference to difference in calmodulin-binding property was discussed.

Purification and characterization of 240-kDa cGMP-dependent protein kinase substrate of vascular smooth muscle. Close resemblance to inositol 1,4,5-trisphosphate receptor.

The 240-kDa, cGMP-dependent protein kinase substrate protein obtained from porcine aortic smooth muscle, whose phosphorylation was closely associated with stimulation of plasma membrane Ca(2+)-pump ATPase (Yoshida, Y., Sun, H.-T., Cai, J.-Q., and Imai, S. (1991) J. Biol. Chem. 266, 19819-19825), was purified to near homogeneity by three successive chromatographic runs with calmodulin-, concanavalin A-, and heparin-Sepharose columns from microsomes solubilized with Triton X-100. The purified protein was found to bind inositol 1,4,5-trisphosphate (InsP3) in a specific, heparin-inhibitable manner with a Kd of 2.0 nM and Bmax of 450 pmol/mg protein (the binding of inositol 1,3,4,5-tetrakisphosphate was much weaker). In sedimentation experiments on a linear sucrose density gradient the InsP3 binding activity was always with the 240-kDa protein. Protein kinase G phosphorylated the InsP3 receptor purified from the rat cerebellum as well as the 240-kDa protein. Sialic acid content of the protein measured with Limulus polyphemus agglutinin was not significantly different from that of the cerebellar InsP3 receptor. Thus, 240-kDa protein closely resembles InsP3 receptor and may be a type of InsP3 receptor. The only difference was the behavior on SDS-polyacrylamide gel electrophoresis. The 240-kDa protein presented itself as two polypeptides with similar but slightly differing M(r) values, both of which were phosphorylated by protein kinase G.