Smooth muscle cell expression of type I cyclic GMP-dependent protein kinase is suppressed by continuous exposure to nitrovasodilators, theophylline, cyclic GMP, and cyclic AMP.

A key component of the nitric oxide-cyclic guanosine monophosphate (cGMP) pathway in smooth muscle cells (SMC) is the type I GMP-dependent protein kinase (PK-G I). Activation of PK-G I mediates the reduction of cytoplasmic calcium concentrations and vasorelaxation. In this manuscript, we demonstrate that continuous exposure of SMC in culture to the nitrovasodilators S-nitroso-N-acetylpenicillamine (SNAP) or sodium nitroprusside (SNP) results in approximately 75% suppression of PK-G I mRNA by 48 h. PK-G I mRNA and protein were also suppressed by continuous exposure to cGMP analogues 8-bromo- and 8-(4-chlorophenylthio) guanosine-3,5-monophosphate or the cAMP analogue dibutyryl cAMP. These results suggest that activation of one or both of the cyclic nucleotide-dependent protein kinases mediates PK-G I mRNA suppression. Using isoform-specific cDNA probes, only the PK-G I alpha was detected in SMC, either at baseline or after suppression, while PK-G I beta was not detected, indicating that isoform switch was not contributing to the gene regulation. Using the transcription inhibitor actinomycin D, the PK-G I mRNA half-life in bovine SMC was observed to be 5 h. The half-life was not affected by the addition of SNAP to actinomycin D, indicating no effect on PK-G I mRNA stability. Nuclear runoff studies indicated a suppression of PK-G I gene transcription by SNAP. PK-G I suppression was also observed in vivo in rats given isosorbide dinitrate in the drinking water, with a dose-dependent suppression of PK-G I protein in the aorta. PK-G I antigen in whole rat lung extract was also suppressed by administration of isosorbide or theophylline in the drinking water. These data may contribute to our understanding of nitrovasodilator resistance, a phenomenon resulting from continuous exposure to nitroglycerin or other nitrovasodilators.

Cyclic GMP-dependent protein kinase expression in coronary arterial smooth muscle in response to balloon catheter injury.

Arterial smooth muscle cells undergo phenotypic and proliferative changes in response to balloon catheter injury. Nitric oxide (NO) and cGMP have been implicated in the inhibition of vascular smooth muscle cell proliferation and phenotypic modulation in cultured-cell studies. We have examined the expression of the major cGMP receptor protein in smooth muscle, cGMP-dependent protein kinase I (PKG), in response to balloon catheter injury in the swine coronary artery. On injury, there was a transient decrease in the expression of PKG in neointimal smooth muscle cells when compared with medial smooth muscle cells. The decrease in PKG expression was observed in the population of proliferating cells expressing the extracellular matrix protein osteopontin but not in cells present in the uninjured portion of the media. Coincident with the suppression of PKG expression in neointimal cells after injury, there was a marked increase in the expression of type II NO synthase (inducible NOS [iNOS], NOS-II) in the neointimal cells. These results suggest that PKG expression is transiently reduced in response to injury in the population of coronary arterial smooth muscle cells that are actively proliferating and producing extracellular matrix proteins. The reduction in PKG expression is also correlated temporally with increases in inflammatory activity in the injured vessels as assessed by iNOS expression. Coupled with our current knowledge regarding the role of PKG in the regulation of cultured cell phenotypes, these results imply that PKG may also regulate phenotypic modulation of vascular smooth muscle cells in vivo as well.

Demonstration of Na+-dependent Ca2+ efflux using low concentrations of fluorometric Ca2+ probes.

The effects of different concentrations of the fluorometric Ca2+ probes, fura-2 and indo-1, on Ca2+ transients in cultured rat aortic smooth muscle cells were examined. When stimulated with the agonists, angiotensin II and arginine vasopressin, cells incubated with low concentrations of fura-2 or indo-1 (less than 1 microM) produced Ca2+ transients characterized by a small increase followed by a dramatic decrease in fluorescence below the original baseline. This effect of agonists was concentration-dependent, reversible, and blocked by receptor antagonists. In contrast to the agonists, stimulation of Ca2+ transients with depolarizing concentrations of K+ or with caffeine did not produce decreases in fluorescence and Ca2+ levels at any loading concentration of probe. The decrease in Ca2+ observed with agonists was dependent on the presence of extracellular Na+. These data suggest that under certain loading conditions, fluorescent Ca2+ indicators measure agonist-stimulated Ca2+ efflux mediated by a Na+/Ca2+ exchange mechanism.

Pleiotropic regulation of vascular smooth muscle tone by cyclic GMP-dependent protein kinase.

Cyclic GMP (cGMP) mediates vascular smooth muscle relaxation in response to nitric oxide and atrial natriuretic peptides. One mechanism by which cGMP decreases vascular tone is by lowering cytosolic Ca2+ levels in smooth muscle cells. Although mechanisms by which cGMP regulates cytosolic Ca2+ are unclear, an important role for the cGMP-dependent dependent protein kinase in regulating Ca2+ has been proposed. Cyclic GMP-dependent protein kinase has been shown to regulate several pathways that control cytosolic Ca2+ levels: inositol 1,4,5-trisphosphate production and action, Ca(2+)-ATPase ATPase activation, and activation of Ca(2+)-activated K+ channels. The pleiotropic action of cGMP-dependent protein kinase is proposed to occur through the phosphorylation of important proteins that control several signaling pathways in smooth muscle cells. One potential target for cGMP-dependent protein kinase is the class of okadaic acid-sensitive protein phosphatases that appears to regulate K+ channels among other potentially important events to reduce cytosolic Ca2+ and tone. In addition, cytoskeletal proteins are targets for cGMP-dependent protein phosphorylation, and it is now appreciated that the cytoskeleton may play a key role in signal transduction.

Reorganization of myofilament proteins and decreased cGMP-dependent protein kinase in the human uterus during pregnancy.

Excessive or premature contractions of uterine smooth muscle may contribute to preterm labor. Contractile stimuli induce myosin and actin filament interactions through calcium-dependent myosin phosphorylation. The mechanisms that maintain myometrial quiescence until term are not well established, but may include control of calcium levels by nitric oxide and cGMP signaling and thin filament (caldesmon and calponin) regulation. Previously, we reported that myometrial tissues from pregnant rats are not responsive to cGMP due to decreases in cGMP-dependent protein kinase. Considering the well documented differences in the endocrinology of parturition among species, this study was conducted to test the hypothesis that the levels and subcellular distribution of caldesmon, calponin, and cGMP-dependent protein kinase are regulated with the hormonal milieu of human pregnancy. Whereas cGMP-dependent protein kinase was significantly reduced in the human uterus during pregnancy, caldesmon expression was significantly increased, and both caldesmon and calponin were redistributed to a readily extractable subcellular pool. These data suggest that cGMP-dependent protein kinase does not mediate gestational quiescence. Redistribution of thin filament-associated proteins, however, may alter uterine smooth muscle tone or the cytoskeletal framework of myocytes to maintain gestation despite the substantial distention that accompanies all intrauterine pregnancies.

Nitric oxide synthase and cyclic GMP-dependent protein kinase concentrated at the neuromuscular endplate.

Nitric oxide mediates diverse functions in development and physiology of vertebrate skeletal muscle. Neuronal type nitric oxide synthase-mu is enriched in fast-twitch fibers and binds to syntrophin, a component of the sarcolemmal dystrophin glycoprotein complex. Here, we show that cyclic GMP-dependent protein kinase type I, a primary effector for nitric oxide, occurs selectively at the neuromuscular junction, in mice and rats, and both neuronal type nitric oxide synthase-mu and cyclic GMP-dependent protein kinase type I remain at skeletal muscle endplates at least two weeks following muscle denervation. Expression of neuronal type nitric oxide synthase-mu and cyclic GMP-dependent protein kinase type I are up-regulated following fusion of cultured primary myotubes. Interestingly, the highest levels of neuronal type nitric oxide synthase-mu in muscle are found complexed with dystrophin at the sarcolemma of intrafusal fibers in muscle spindles. Localization of neuronal type nitric oxide synthase-mu and cyclic GMP-dependent protein kinase type I at the neuromuscular junction suggests functions for nitric oxide and cyclic GMP in the regulation of synaptic actions of intra- and extrafusal muscle fibers.

Regulation of phosphorylase A formation and calcium content in aortic smooth muscle and smooth muscle cells: effects of atrial natriuretic peptide II.

Atrial natriuretic peptide II (ANP II) raises cyclic GMP and relaxes vascular smooth muscle in vitro. The manner in which ANP II relaxes vascular smooth muscle is unknown but may involve alterations in the concentration of free intracellular Ca++. To examine this possibility, changes in intracellular Ca++ were monitored in rat aortic strips using the Ca++-dependent conversion of phosphorylase b to a, while Ca++ levels and phosphorylase were measured in cultured rat aortic smooth muscle cells. ANP II produced time- and concentration-dependent decreases in phosphorylase a and tension in norepinephrine-contracted aortic strips. The decrease in the formation of phosphorylase a was accompanied by an increase in cyclic GMP content. ANP II also decreased phosphorylase a formation in K+-depolarized tissues but to a lesser extent. Agonists such as angiotensin II and arginine vasopressin, and depolarizing concentrations of K+ elevated Ca++ levels in cultured aortic cells. ANP II inhibited Ca++ accumulation to either agonists or K+, but was more effective against agonists. Phosphorylase a formation which was increased by agonists and K+ in cultured cells was also inhibited by ANP II. We conclude that phosphorylase a formation can be a useful indicator of intracellular Ca++ concentrations in smooth muscle preparations and that ANP II regulates Ca++ levels in agonist and depolarized smooth muscle, suggesting that ANP II affects mainly Ca++ removal from the cytoplasm.

Regulation of intracellular Ca2+ levels in cultured vascular smooth muscle cells. Reduction of Ca2+ by atriopeptin and 8-bromo-cyclic GMP is mediated by cyclic GMP-dependent protein kinase.

Primary rat aortic cells, when treated with arginine vasopressin or depolarizing concentrations of K+, responded to atriopeptin II and 8-bromo-cGMP (8-Br-cGMP) with decreases in intracellular Ca2+ levels. The effects of atriopeptin and 8-Br-cGMP were diminished in cells which had been passaged many times. Low levels of cGMP-dependent protein kinase were present in soluble extracts prepared from the unresponsive cells in later passage compared with extracts from responsive cells. Unresponsive cells, when induced to incorporate cGMP-dependent protein kinase into the cytoplasm using the osmotic lysis procedure of Okada and Rechsteiner (Okada, C. Y., and Rechsteiner, M. (1982) Cell 29, 33-41), responded to atriopeptin and 8-Br-cGMP with reductions in peak Ca2+ levels in response to vasopressin and depolarizing concentrations of K+. Cells which were furnished with affinity-purified antibody to the cGMP-dependent protein kinase after the introduction of the kinase remained unresponsive to the effects of atriopeptin. In addition, antibody furnished to responsive primary cultured cells inhibited the effects of atriopeptin and 8-Br-cGMP on Ca2+ levels. These data suggest that repetitively passaged cultured rat aortic smooth muscle cells lose their responsiveness to cGMP concurrently with the loss of cGMP-dependent protein kinase. Restoration of kinase to the cells results in the restoration of responsiveness to cGMP. Thus cGMP-dependent protein kinase appears to be the mediator of the reduction in Ca2+ levels upon elevation of intracellular cGMP.

Regulation of cGMP-induced relaxation and cGMP-dependent protein kinase in rat myometrium during pregnancy.

Increases in guanosine 3',5'-cyclic monophosphate (cGMP) induced by nitric oxide (NO), nitrovasodilators, and atrial peptides correlate with relaxation of vascular smooth muscle. Relaxation of myometrial smooth muscle by increases in cGMP, however, has required unusually high concentrations of the cyclic nucleotide. We tested the hypothesis that the sensitivity of myometrium to relaxation by cGMP is increased during pregnancy. Aortic smooth muscle was more sensitive to relaxation by cGMP than myometrial tissues, and, contrary to our hypothesis, myometrium from pregnant rats was least sensitive. Although levels of cGMP were elevated after treatment with the NO donor, S-nitroso-N-acetylpenicillamine, relaxation of myometrial tissues obtained from pregnant rats occurred only at extraordinarily high concentrations. The levels of cGMP-dependent protein kinase (PKG) were significantly decreased in myometrium from pregnant rats compared with myometrium from nonpregnant cycling animals or aortic smooth muscle. Administration of estradiol to ovariectomized rats increased myometrial PKG expression, and progesterone antagonized this response. We conclude that 1) myometrial tissues from pregnant rats are not sensitive to relaxation by cGMP and 2) this insensitivity to cGMP is accompanied by progesterone-mediated decreases in the level of PKG expression.

Intracellular cyclic GMP receptor proteins.

Cyclic GMP is recognized as an important intracellular mediator of extracellular signals such as nitric oxide and natriuretic peptides. Cyclic GMP interacts with three types of intracellular receptor proteins: cGMP-dependent protein kinases, cGMP-regulated ion channels, and cGMP-regulated cyclic nucleotide phosphodiesterases. This means that cGMP can alter cell function through protein phosphorylation or through mechanisms not directly related to protein phosphorylation. Cyclic GMP appears to regulate a number of intracellular processes, such as vascular smooth muscle relaxation and neutrophil activation, through these receptor proteins in the cell. It is also becoming clear that the localization of these cGMP receptor proteins in the cell is an important factor in the regulation of cell function by cGMP.

Towards an understanding of the mechanism of action of cyclic AMP and cyclic GMP in smooth muscle relaxation.

Cyclic GMP (cGMP) mediates the relaxing action of a variety of vasodilator drugs and endogenous vasodilator substances. Cyclic AMP (cAMP) mediates relaxation by beta-adrenergic agonists as well as other activators of adenylate cyclase. Both second messengers appear to reduce the concentration of intracellular Ca2+ in vascular smooth muscle cells, thus affecting relaxation. The presence of cGMP-dependent protein kinase in vascular smooth muscle cells is required for the reduction of Ca2+ by cAMP and cGMP, suggesting that this enzyme mediates the relaxing effects of both cyclic nucleotides. Although the specific substrate proteins for cGMP-dependent protein kinase are not well characterized in vascular smooth muscle, new evidence indicates that Ca2(+)-ATPase activation by phosphorylation of phospholamban by the kinase may underlie the mechanism of action of cyclic-nucleotide-dependent relaxation.

Purification and characterization of two forms of cyclic GMP-dependent protein kinase from bovine aorta.

Cyclic GMP-dependent protein kinase in extracts of bovine aortic tissue eluted from DEAE-cellulose ion-exchange resins as two distinct peaks of activity. This elution pattern was preserved when the peaks were combined, precipitated with ammonium sulfate, dialyzed, and rechromatographed. Proteolysis did not appear to account for the two forms of kinase because (i) aging of the extract did not cause interconversion of the two forms, and (ii) both forms retained cGMP sensitivity unlike the proteolytically formed monomer. In addition, treatment with saturating concentrations of cGMP (10 microM) did not cause interconversion of the two forms. The first peak of cGMP-dependent protein kinase eluting from DEAE-cellulose (form 1) had a slightly greater mobility on gradient sodium dodecyl sulfate-polyacrylamide gels than the second peak (form 2). On native, nondenaturing gradient polyacrylamide gels, however, form 2 displayed the greater electrophoretic mobility. Furthermore, form 1, when bound to cAMP-agarose, appeared to exchange more rapidly with cGMP than form 2 when subjected to affinity chromatography. Peptide maps generated from the two forms by protease treatment were very similar, although trypsin produced a unique peptide in form 1 and Streptomyces griseus protease gave rise to unique peptides in forms 1 and 2. Phosphorylation did not appear to account for the physical differences because both enzymes could be phosphorylated to similar extents and dephosphorylation using alkaline phosphatase did not result in the conversion of one form to the other. These results suggest that either differences in primary structure or post-translational modification, other than phosphorylation, are responsible for the presence of two forms of cGMP-dependent protein kinase in aortic tissue.

Effects of 8-bromo-cGMP on Ca2+ levels in vascular smooth muscle cells: possible regulation of Ca2+-ATPase by cGMP-dependent protein kinase.

The effects of 8-bromo-cGMP on intracellular calcium concentrations in cultured rat aortic smooth muscle cells were studied. Both angiotensin II and depolarizing concentrations of K+ stimulated Ca2+ accumulation in the cytoplasm. The increase in Ca2+ due to angiotensin II was associated with an increase in inositol phosphates, while that due to K+ was not. Preincubation of cells with 8-bromo-cGMP (100 microM) caused an inhibition of peak Ca2+ accumulation to either angiotensin II or K+. To probe the mechanism of action of cGMP in vascular smooth muscle, the effects of cGMP-dependent protein kinase on Ca2+-ATPase from the cultured cell particulate material were investigated. Ca2+-activated ATPase was stimulated approximately equal to 2-fold by exogenous calmodulin and up to 4-fold by low concentrations of purified cGMP-dependent protein kinase. The inclusion of both calmodulin and cGMP-dependent protein kinase resulted in an additive stimulation of Ca2+-ATPase. Stimulation of Ca2+-ATPase activity was observed at all Ca2+ concentrations tested (0.01-1.0 microM). cAMP-dependent protein kinase catalytic subunit and protein kinase C were either ineffective or less effective than cGMP-dependent protein kinase in stimulating the Ca2+-ATPase from rat aortic smooth muscle cells. These results suggest a possible mechanism of action for cGMP in mediating decreases in cytosolic Ca2+ through activation of a Ca2+-ATPase and the subsequent removal of Ca2+ from the cell.

cGMP-dependent protein kinase mediates the reduction of Ca2+ by cAMP in vascular smooth muscle cells.

The major action of forskolin, the diterpine activator of adenylate cyclase, in primary (unpassaged) rat aortic smooth muscle cells is to reduce vasopressin-stimulated Ca2+ concentrations. In repetitively passaged cells, however, forskolin by itself increased Ca2+ levels by apparently stimulating Ca2+ uptake into the cell and had much smaller effects on inhibiting vasopressin-stimulated Ca2+ elevations. Both primary and passaged smooth muscle cells contained adenosine 3',5'-cyclic monophosphate (cAMP)-dependent protein kinase. Guanosine 3',5'-cyclic monophosphate (cGMP)-dependent protein kinase was greatly reduced or absent in passaged smooth muscle cells. The introduction of purified cGMP-dependent protein kinase into the cytoplasm of passaged cells prevented forskolin from elevating intracellular Ca2+ and restored the capacity of forskolin to reduce vasopressin-stimulated Ca2+ mobilization. Similar effects were observed for isoproterenol in passaged smooth muscle cells. When introduced into cells, the active catalytic subunit of the cAMP-dependent protein kinase did not lead to reductions in Ca2+ levels. These results suggest that cAMP elevations lead to profound changes in Ca2+ metabolism through activation of both cAMP- and cGMP-dependent protein kinases. Activation of cGMP-dependent protein kinase by cAMP leads to the reduction in intracellular Ca2+, whereas activation of cAMP-dependent protein kinase may only mediate the uptake of Ca2+ from extracellular sources.

Demonstration of a direct role for myosin light chain kinase in fibroblast-populated collagen lattice contraction.

Mixing feed fibroblasts with soluble collagen and serum-supplemented culture medium at 37 degrees C results in the entrapment of cells within the polymerizing collagen matrix. This cellular-collagen complex is referred to as a fibroblast-populated collagen lattice (FPCL). In time, this FPCL undergoes a reduction in size called lattice contraction. The proposed mechanism for lattice contraction is cellular force produced by cytoplasmic microfilaments which organize collagen fibrils compacting the matrix. When the regulatory subunits of myosin, myosin light chains, are phosphorylated by myosin light chain kinase (MLCK), myosin ATPase activity is increased and actin-myosin dynamic filament sliding occurs. Elevated levels of myosin ATPase are required for maximal lattice contraction. Cholera toxin inhibits lattice contraction by increasing intracellular levels of cAMP. It is proposed that increased cytoplasmic concentrations of cAMP promote phosphorylation of MLCK, the enzyme important for maximizing myosin ATPase activity. Phosphorylating MLCK in vitro inhibits activity by decreasing its sensitivity to calcium-calmodulin complex. A decrease in MLCK activity would result in lower levels of myosin ATPase activity. MLCK, purified from turkey gizzard, was subjected to limited proteolytic digestion to produce calmodulin-independent-MLCK. The partially digested kinase does not require calcium-calmodulin for activation. Independent-MLCK is not subject to inhibition by phosphorylation. The electroporetic inoculation of independent-MLCK into fibroblasts before FPCL manufacture produced enhanced lattice contraction. Lattice contraction, in the presence of cholera toxin, was restored to normal levels by the prior electroporetic introduction of independent-MLCK. These findings support the hypothesis that increases in cAMP hinder lattice contraction by a mechanism involving inhibition of MLCK and myosin ATPase.

Regulation of sarcoplasmic reticulum protein phosphorylation by localized cyclic GMP-dependent protein kinase in vascular smooth muscle cells.

The role of cGMP-dependent protein kinase in the regulation of intracellular Ca2+ levels in vascular smooth muscle cells was examined by studying the effects of cGMP on the phosphorylation of the Ca(2+)-ATPase regulatory protein phospholamban. Cultured rat aortic smooth muscle cells incubated with atrial natriuretic peptide II or sodium nitroprusside responded with increased phosphorylation of the 6000-Da subunit of phospholamban. The identity of phospholamban was confirmed using immunoprecipitation methods. Phosphorylation was associated with an increase in the activation of membrane-associated ATPase by Ca2+. These results indicated that at least one site of action of cGMP in smooth muscle cells is the sarcoplasmic reticulum, where phosphorylation of proteins regulating Ca2+ fluxes occurs. Studies using confocal laser scanning microscopy to define the cellular distribution of cGMP-dependent protein kinase suggested that the enzyme was localized to the same cellular region(s) as was phospholamban. Phosphorylation of proteins by cGMP in broken cell fractions from rabbit aorta was also performed. Phospholamban and other proteins were phosphorylated in the presence of cGMP but not cAMP, suggesting that only cGMP-dependent protein kinase was associated with smooth muscle membrane fractions containing phospholamban. These results suggest that one mechanism of action of cGMP in the reduction of intracellular Ca2+ is the activation of sarcoplasmic reticulum Ca(2+)-ATPase via phosphorylation of phospholamban. The data also support the concept that compartmentalization of protein kinases with substrates in the intact cell is an important factor involved in protein phosphorylation.

Cyclic GMP-dependent protein kinase regulates vascular smooth muscle cell phenotype.

Nitric oxide (NO) and cyclic guanosine 3',5'-monophosphate (cGMP) have been reported to prevent vascular smooth muscle cell (VSMC) proliferation and have beneficial effects to reduce intimal thickening in response to arterial injury. The purpose of this study was to determine whether the downstream effector molecule of NO-cGMP signaling, cyclic GMP-dependent protein kinase (PKG), regulates phenotypic modulation and proliferation in cultured rat aortic VSMC. PKG-expressing VSMC lines were created by transfection of PKG-deficient cell lines and characterized. All forms of PKG, i.e. PKG-I alpha and PKG-I beta, as well as the constitutively active catalytic domain of PKG-I, transformed dedifferentiated 'synthetic' VSMC to a more contractile-like morphology. PKG expression resulted in an increased production of the contractile phenotype marker proteins, smooth muscle myosin heavy chain-2, calponin and alpha-actin and restored the capacity of cAMP and cGMP analogues to inhibit platelet-derived growth factor (PDGF)-induced cell migration. On the other hand, PKG expression had no significant effects on PDGF-induced cell proliferation. These results suggest that PKG expression contributes to the regulation of a contractile-like phenotypic expression in cultured VSMC, and the suppression of PKG expression during cultured growth in vitro may permit the modulation of cells to a more synthetic, dedifferentiated phenotype.

Regulation of the expression of cyclic GMP-dependent protein kinase by cell density in vascular smooth muscle cells.

Cyclic GMP-dependent protein kinase (cGMP kinase) is the major receptor protein for cGMP in vascular smooth muscle. Vascular smooth muscle cells (VSMC) isolated from the rat aorta express type I cGMP kinase at high levels, but expression decreases markedly upon passage of the cells. In primary or early passage, the expression of cGMP kinase is lowest when cells are plated at low density as assessed by immunological and Northern analyses. Expression increases at confluence and is maintained in postconfluent cultures. With repeated passaging, however, the levels of cGMP kinase decrease even in confluent and postconfluent cultures so that after several passages enzyme levels are undetectable. The decrease in expression in passaged cells is not due to exposure to serum-derived growth factors, but rather on the repeated exposure of cells to conditions in which cell density is reduced (i.e., subculturing). These results indicate that aortic VSMC grown at low density or those repetitively passaged have reduced expression of cGMP kinase, and thus may not represent appropriate cultures with which to investigate the role of nitric oxide and cGMP in VSMC function.