Protein Kinase In Smooth Muscle Research Articles

Endogenous testosterone attenuates neointima formation after moderate coronary balloon injury in male swine.

Previous studies from our laboratory have demonstrated that testosterone increases coronary smooth muscle protein kinase C delta (PKC delta) both in vivo and in vitro and inhibits coronary smooth muscle proliferation by inducing G(0)/G(1) cell cycle arrest in a PKC delta-dependent manner. The purpose of the present study was to determine whether endogenous testosterone limits coronary neointima (NI) formation in a porcine model of post-angioplasty restenosis. Sexually mature, male Yucatan miniature swine were either left intact (IM), castrated (CM), or castrated with testosterone replacement (CMT; Androgel, 10 mg/day). Angioplasty was performed in both the left anterior descending and left circumflex coronary arteries with balloon catheter overinflation to induce either moderate (1.25-1.3 x diameter; 3 x 30 s) or severe (1.4x diameter; 3 x 30 s) injury, and animals were allowed to recover for either 10 or 28 days. Injured coronary sections were dissected, fixed, stained (Verheoff-Van Gieson, Ki67, PKC delta, p27), and analysed. Vessels without internal elastic laminal rupture were excluded. Following moderate injury, intimal area, intima-to-media ratio (I/M), and I/M normalized to rupture index (RI) were increased in CM compared with IM and CMT. RI, medial area, and intimal/medial thickness (IMT) were not different between groups. NI formation was inversely related to serum testosterone concentration. Conversely, following severe injury, there were no significant differences between the groups. Testosterone inhibited proliferation and stimulated PKC delta and p27(kip1) expression during NI formation (10 days post-injury). These findings demonstrate that endogenous testosterone limits coronary NI formation in male swine and provides support for a protective role for testosterone in coronary vasculoproliferative diseases, such as restenosis and atherosclerosis.

Signaling Mechanisms Contribute to the Vasodilatory Effects of HMC05

We investigated the signaling mechanisms contribute to the vasodilatory effects of a herbal extract, HMC05. Previous findings revealed that the action of BCT, a cardiovascular medicine the compositions are very similar to those of HMC05, was mediated by both nitric oxide (NO) release and a direct inhibition of smooth muscle protein kinase C (PKC). HMC05 also showed the vascular relaxing effects and lowered the phosphorylation level of the 20 kDa myosin light chains (LC20) in vascular strips, but it acted as endothelium-independent manner. To elucidate the fundamental mechanism of its vascular actions, we focused on the signaling molecules involved in actin-myosin filament regulation including Caldesmon (CaD), Rho-associated kinase (ROK), PKC and extracellular signal-regulated protein kinase (ERK) in the endothelium-denuded thoracic aorta or isolated smooth muscle cells (SMCs). The results showed ERK, ROK and PKC pathways played important roles in the effects, as confirmed by the observations with a specific inhibition or activation, and with the activity and the subcellular localization of these molecules. In particular, HMC05 dramatically inhibited the activity of ERK and the downstream signalings of ROK. It also changed the subcellular localization of the phophorylated PKC alpha as well as the amount of phosphorylation. Taken together, these data indicate that the vasorelaxation of HMC05 may be regulated by these signaling mechanisms. Acknowledgement This study was supported by a grant of the Oriental Medicine R&D Project (B050042), Ministry of Health and Welfare, Republic of Korea.

Modulation of smooth muscle contractility by CHASM, a novel member of the smoothelin family of proteins

Cyclic nucleotides acting through their associated protein kinases, the cGMP- and cAMP-dependent protein kinases, can relax smooth muscles without a change in free intracellular calcium concentration ([Ca 2+] i ), a phenomenon referred to as Ca 2+ desensitization. The molecular mechanisms by which these kinases bring about Ca 2+ desensitization are unknown and an understanding of this phenomenon may lead to better therapies for treating diseases involving defects in the contractile response of smooth muscles such as hypertension, bronchospasm, sexual dysfunction, gastrointestinal disorders and glaucoma. Utilizing a combination of real-time proteomics and smooth muscle physiology, we characterized a distinct subset of protein targets for cGMP-dependent protein kinase in smooth muscle. Among those phosphoproteins identified was calponin homology-associated smooth muscle (CHASM), a novel protein that contains a calponin homology domain and shares sequence similarity with the smoothelin family of smooth muscle specific proteins. Recombinant CHASM was found to evoke relaxation in a concentration dependent manner when added to permeabilized smooth muscle. A co-sedimentation assay with actin demonstrated that CHASM does not possess actin binding activity. Our findings indicate that CHASM is a novel member of the smoothelin protein family that elicits Ca 2+ desensitization in smooth muscle.

Selective phosphorylation of the IP3R-I in vivo by cGMP-dependent protein kinase in smooth muscle.

This study examined the expression of inositol 1,4,5-trisphosphate (IP(3)) receptor (IP(3)R) types and PKG isoforms in isolated gastric smooth muscle cells and determined the ability of PKG and PKA to phosphorylate IP(3)Rs and inhibit IP(3)-dependent Ca(2+) release, which mediates the initial phase of agonist-induced contraction. PKG-Ialpha and PKG-Ibeta were expressed in gastric smooth muscle cells, together with IP(3)-R-associated cG-kinase substrate, a protein that couples PKG-Ibeta to IP(3)R-I. IP(3)R-I and IP(3)R-III were also expressed, but only IP(3)R-I was phosphorylated by PKA and PKG in vitro and exclusively by PKG in vivo. Sequential phosphorylation by PKA and by PKG-Ialpha in vitro showed that PKA phosphorylated the same site as PKG (presumably S(1755)) and an additional PKA-specific site (S(1589)). In intact muscle cells, agents that activated PKG or both PKG and PKA induced IP(3)R-I phosphorylation that was reversed by the PKG inhibitor (8R,9S,11s)-(-)-9-methoxy-carbamyl-8-methyl-2,3,9,10-tetrahydro-8,11-epoxy-1H,8H,1H,-2,7b,11a-trizadizo-benzo9(a,g)cycloocta(c,d,e)-trinden-1-one. Agents that activated PKA induced IP(3)R-I phosphorylation in permeabilized but not intact muscle cells, implying that PKA does not gain access to IP(3)R-I in intact muscle cells. The pattern of IP(3)R-I phosphorylation in vivo and in vitro was more consistent with phosphorylation by PKG-Ialpha. Phosphorylation of IP(3)R-I in microsomes by PKG, PKA, or a combination of PKG and PKA inhibited IP(3)-induced Ca(2+) release to the same extent, implying that inhibition was mediated by phosphorylation of the PKG-specific site. We conclude that IP(3)R-I is selectively phosphorylated by PKG-I in intact smooth muscle resulting in inhibition of IP(3)-dependent Ca(2+) release.

Activation of phosphodiesterase 5 and inhibition of guanylate cyclase by cGMP-dependent protein kinase in smooth muscle.

The regulation of cGMP-specific phosphodiesterase (PDE) 5 and soluble guanylate cyclase (GC) by cGMP- and cAMP-dependent protein kinases (PKG and PKA respectively) was examined in gastric smooth muscle. The NO donor, sodium nitroprusside (SNP), stimulated PDE5 phosphorylation and activity, which was blocked by the selective PKG inhibitor, KT5823, resulting in an elevation of cGMP levels. Activation of PKA either directly by Sp-5,6-dichloro-1-beta-d-ribofuranosyl benzimidazole 3',5'-cyclic monophosphothioate, or via isoproterenol- and forskolin-dependent increase in cAMP, also caused an increase in PDE5 phosphorylation and activity, but only in the presence of cGMP; consistent with the dependence of PDE5 phosphorylation and activity on cGMP binding to allosteric sites in the regulatory domain of PDE5. The selective PKA inhibitors, myristoylated protein kinase inhibitor and H-89, blocked the increase in PDE5 phosphorylation and activity induced by PKA. SNP also stimulated soluble GC phosphorylation and activity. KT5823 abolished phosphorylation and augmented soluble GC activity, implying feedback inhibition of soluble GC by PKG-dependent phosphorylation. Phosphorylation by PKG was direct and could be induced in vitro. Activation of PKA had no effect on soluble GC. Thus cGMP levels are regulated by PKG- and PKA-dependent activation of PDE5 and PKG-specific inhibition of soluble GC.

Activation of phosphodiesterase 5 and inhibition of guanylate cyclase by cGMP-dependent protein kinase in smooth muscle

The regulation of cGMP-specific phosphodiesterase (PDE) 5 and soluble guanylate cyclase (GC) by cGMP- and cAMP-dependent protein kinases (PKG and PKA respectively) was examined in gastric smooth muscle. The NO donor, sodium nitroprusside (SNP), stimulated PDE5 phosphorylation and activity, which was blocked by the selective PKG inhibitor, KT5823, resulting in an elevation of cGMP levels. Activation of PKA either directly by Sp-5,6-dichloro-1-β-d-ribofuranosyl benzimidazole 3′,5′-cyclic monophosphothioate, or via isoproterenol- and forskolin-dependent increase in cAMP, also caused an increase in PDE5 phosphorylation and activity, but only in the presence of cGMP; consistent with the dependence of PDE5 phosphorylation and activity on cGMP binding to allosteric sites in the regulatory domain of PDE5. The selective PKA inhibitors, myristoylated protein kinase inhibitor and H-89, blocked the increase in PDE5 phosphorylation and activity induced by PKA. SNP also stimulated soluble GC phosphorylation and activity. KT5823 abolished phosphorylation and augmented soluble GC activity, implying feedback inhibition of soluble GC by PKG-dependent phosphorylation. Phosphorylation by PKG was direct and could be induced in vitro. Activation of PKA had no effect on soluble GC. Thus cGMP levels are regulated by PKG- and PKA-dependent activation of PDE5 and PKG-specific inhibition of soluble GC.

Role of phospholamban in the modulation of arterial Ca(2+) sparks and Ca(2+)-activated K(+) channels by cAMP.

Phospholamban (PLB) inhibits the sarcoplasmic reticulum (SR) Ca(2+)-ATPase, and this inhibition is relieved by cAMP-dependent protein kinase (PKA)-mediated phosphorylation. The role of PLB in regulating Ca(2+) release through ryanodine-sensitive Ca(2+) release channels, measured as Ca(2+) sparks, was examined using smooth muscle cells of cerebral arteries from PLB-deficient ("knockout") mice (PLB-KO). Ca(2+) sparks were monitored optically using the fluorescent Ca(2+) indicator fluo 3 or electrically by measuring transient large-conductance Ca(2+)-activated K(+) (BK) channel currents activated by Ca(2+) sparks. Basal Ca(2+) spark and transient BK current frequency were elevated in cerebral artery myocytes of PLB-KO mice. Forskolin, an activator of adenylyl cyclase, increased the frequency of Ca(2+) sparks and transient BK currents in cerebral arteries from control mice. However, forskolin had little effect on the frequency of Ca(2+) sparks and transient BK currents from PLB-KO cerebral arteries. Forskolin or PLB-KO increased SR Ca(2+) load, as measured by caffeine-induced Ca(2+) transients. This study provides the first evidence that PLB is critical for frequency modulation of Ca(2+) sparks and associated BK currents by PKA in smooth muscle.

Smooth muscle protein kinase C

Protein kinase C (PKC) was first implicated in the regulation of smooth muscle contraction with the observation that phorbol esters induce slowly developing, sustained contractions. In some vascular smooth muscles, e.g., ferret aorta, phorbol ester induced contractions occur without an increase in sarcoplasmic free-Ca2+ concentration ([Ca]i) or myosin light chain phosphorylation. This response appears to be mediated by a Ca(2+)-independent isoenzyme of PKC (probably PKC epsilon), since saponin-permeabilized single ferret aortic smooth muscle cells, which retain receptor coupling, developed force in response to phenylephrine at low free [Ca2+] (pCa 7.0-8.6) and the constitutively active proteolytic fragment of PKC (PKM) elicited a contraction at pCa 7 comparable with the phenylephrine-induced contraction. Both contractions were reversed by a pseudo-substrate peptide inhibitor of PKC. These observations suggest a mechanism whereby alpha-adrenergic agonists may elicit a contractile response without a Ca2+ signal: alpha-adrenergic stimulation of phosphatidylcholine-specific phospholipase C or D (the latter in conjunction with phosphatidate phosphohydrolase) generates diacylglycerol. In the absence of an increase in [Ca2+]i, diacylglycerol specifically activates so-called novel PKCs, of which epsilon is the only isoenzyme known to be expressed in vascular smooth muscle. Recent evidence suggests that PKC may trigger a cascade of phosphorylation reactions, resulting in activation of mitogen-activated protein kinase and phosphorylation of the thin filament associated protein caldesmon. Alternatively, or additionally, PKC may directly phosphorylate calponin, another thin filament associated protein. These phosphorylations are predicted to alleviate inhibition of the cross-bridge cycling rate by these thin-filament proteins.(ABSTRACT TRUNCATED AT 250 WORDS)

Physiology and Pathophysiology of the Heart

This comprehensive and updated Second edition is an impressive contribution to the fundamentals of basic science of the heart. The editor has assembled 48 chapters written by experts and wellknown researchers. Eight new chapters, a precious and timely addition to the first edition, include comprehensive discussions on contractile proteins of cardiac muscle, contractile proteins of smooth muscle, cyclic nucleotides and protein kinase in smooth muscle, calcium-activated ion currents in cardiac muscle, developmental changes in adrenergic modulation, endothelial cell interactions with vascular smooth-muscle cells, myocardial infarction and free radical effects on the heart, and extravascular coronary resistance. The basic factors affecting the function of the normal and diseased heart are evaluated by a comprehensive multidisciplinary approach including membrane biophysics, electrophysiology, physiology, pathophysiology, pharmacology, biochemistry, and ultrastructure. Adroitly written chapters, such as chapter 31 on hormonal cardiac performance, deal with topics of special interest to diabetologists and cardiologists alike. Diabetic cardiomyopathy and

Effect of Ca 2+ entry blockers on myosin light-chain kinase and protein kinase C

Thirteen Ca 2+ entry blockers were compared with respect to their inhibitory effects on the activity of smooth muscle myosin light-chain kinase and smooth muscle protein kinase C, and the Ca 2+-induced contraction of basilar artery rings. Comparison of the IC 50 values obtained for these compounds, suggests that with the exception of ferdiline inhibition of the enzymes does not contribute to vascular smooth muscle relaxation.

Phosphorylation of myosin light chain by Ca2+-activated, phospho-lipid-dependent protein kinase in smooth muscle

Myosin light chain phosphorylation is thought to play a central role in regulating the interaction of actin and myosin in smooth muscle, and even in non-muscle cells. Although myosin light chain phosphorylation is reportedly catalyzed mainly by Ca2+, calmodulin-dependent myosin light chain kinase (MLCK), myosin light chain was also phosphorylated by another species of Ca2+-dependent protein kinase (protein kinase C). Smooth muscle (chicken gizzard) myosin but not skeletal muscle was phosphorylated in vitro by protein kinase C from human platelets. The apparent Kms for ATP and 20,000-dalton light chain of chicken gizzard myosin were 7.5 uM and 93 uM, respectively. Two-dimensional peptide mapping of tryptic digests, of myosin light chain revealed that protein kinase C and MLCK catalyzed the incorporation of the same amount of phosphate into gizzard myosin light chain, at different sites. These results suggest that the protein kinase C phosphorylation of myosin light chain acts independently or in a complementary manner with the MLCK phosphorylation system, at least in smooth muscle.

Phosphoprotein phosphatase in bovine tracheal smooth muscle Multiple fractions and multiple substrates

Phosphoprotein phosphatase (phosphoprotein phosphohydrolase, EC 3.1.3.16) from bovine tracheal smooth muscle extracts was isolated and its activity determined using two [ 32P]phosphorylated proteins as substrates, i.e. phosphorylated histone (H-P) and a phosphorylated muscle specific substrate protein (MS-P) for the tracheal smooth muscle protein kinase. The enzyme was purified by the use of DEAE-cellulose followed by a two stage chromatography on a histone-Sepharose affinity column. Elution from the affinity column resolved the phosphoprotein phosphatase into four activity fractions. While fractions expressed phosphatase activity against both tested substrates the relative amounts of either activity varied. The ratio of activity towards H-P to activity towards MS-P changed from 11.5 to 0.12. The characterization of four phosphoprotein phosphatase fractions was based on the differences found in the following parameters: substrate specificity; sensitivity to NaF; influences of nucleotides (ATP, 5′-AMP, cyclic AMP, cyclic GMP) and the requirement of Mn 2+ for maximal activity. Mg 2+, Ba 2+ or Ca 2+ could not substitute for Mn 2+.

Phosphorylation of muscle proteins by cyclic adenosine 3′,5′-monophosphate-dependent protein kinase from muscle

Tissue-specific substrates for a cyclic AMP-dependent protein kinase from bovine tracheal smooth muscle have been partially characterized. The substrates are phosphorylated by purified smooth muscle kinase or by kinases found in crude extracts of bovine smooth, skeletal, and cardiac muscle. The substrates derived from and phosphorylated by smooth muscle protein kinase have a molecular weight of approximately 70 000, as determined by disc gel electrophoresis in the absence of sodium dodecyl sulfate and of approx. 20 000–25 000 when electrophoresed in the presence of sodium dodecyl sulfate. It appears that there are a liminated number of substrates for the smooth muscle cyclic AMP-dependent protein kinase.