Nancy J. Boerth

cGMP signaling through cAMP- and cGMP-dependent protein kinases.

Publisher Summary The signaling pathways by which nitric oxide (NO) affects cell function, are by no means limited to the stimulation of guanylate cyclase. Concentrations of NO that activate guanylate cyclase may also have other effects on cells. This is because, at least in part, of the fact that NO binds with high affinity to heme moieties in proteins—guanylate cyclase being the only example of a heme-containing enzyme. At high concentrations of NO, that is, those that might be realized as a consequence of the induction on NO synthase by cytokines and other biological modifier molecules, enzymes containing iron-sulfur groups bind NO. One of the recently described mechanisms of NO signaling, at least in terms of its pathophysiological effects on cells, is the formation of peroxynitrite. NO reacts with superoxide generated in response to cellular responses to oxidative injury to form the free radical peroxynitrite. Peroxynitrite, in turn, may have a variety of effects on cells, including orthonitration of tyrosine residues on proteins. The significance of this effect of NO is not clear at this time, but peroxynitrite production and protein “nitration” have been correlated with tissue injury and pathological responses of the tissues to insult.

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.

Cyclic GMP–Dependent Protein Kinase Inhibits Osteopontin and Thrombospondin Production in Rat Aortic Smooth Muscle Cells

Vascular lesions resulting from injury are characterized by a thickening of the intima brought about in part through the production of increased amounts of extracellular matrix proteins by the vascular smooth muscle cells (VSMCs). In this study, we tested the hypothesis that cGMP-dependent protein kinase (PKG), an important mediator of NO and cGMP signaling in VSMCs, inhibits the production of two extracellular matrix proteins, osteopontin and thrombospondin, which are involved in the formation of the neointima. VSMCs deficient in PKG were stably transfected with cDNAs encoding either the holoenzyme PKG-Ialpha or the constitutively active catalytic domain of PKG-I in order to directly examine the effects of PKG on osteopontin and thrombospondin production. Cells expressing either of the PKG constructs had dramatically reduced levels of osteopontin and thrombospondin-1 protein compared with control-transfected PKG-deficient cells. PKG transfection also altered the morphology of the VSMCs. These results indicate that PKG may be involved in suppressing extracellular matrix protein expression, which is one important characteristic of synthetic secretory VSMCs. Suppression of these matrix proteins may underlie the effects of NO-cGMP signaling to inhibit VSMC migration and phenotypic modulation.

Cyclic GMP-dependent protein kinase in nitric oxide signaling.

Abstract Cyclic GMP-dependent protein kinase is now implicated in a number of important cellular signaling events. The role of PKG in processes as diverse as the regulation of intracellular Ca 2+ levels in smooth muscle tissues to its potential role in gene expression has been the subject of investigations over the past few years. Despite the importance of this enzyme in cellular regulation, few details of the molecular mechanisms of action of PKG are available. There are a number of important issues to consider, however, when studying the role of NO, cGMP, and PKG in cellular function. In the first case, it is important to acknowledge the diversity of effects of NO on cellular processes. At submicromolar concentrations of NO-generating drugs such as nitroprusside, NO is known to activate soluble guanylate cyclase. Predictably, this leads to the activation of PKG and the phosphorylation of proteins relevant to the signaling cascade under investigation. At higher concentrations of NO-generating drugs, however, other effects of NO occur that may be unrelated to PKG activation. These include cross-activation of PKA by cGMP, and the modification of proteins by the NO radical. Another important consideration when investigating the role of cGMP and PKG in cell regulation is the nonspecific actions of cyclic nucleotide analogs (e.g., 8-Br-cyclic nucleotides) and drugs used to inhibit protein kinase activity. For example, high concentrations of cyclic nucleotide analogs may cross-activate both cyclic nucleotide-dependent protein kinases when incubated with cultured cells at high concentrations for prolonged periods of time. And finally, the specificity of protein phosphorylation catalyzed by protein kinases must be considered. Both PKA and PKG, for example, catalyze the phosphorylation of identical residues in protein substrates in vitro . In the intact cell, the pattern of protein phosphorylation may be affected by the localization of the kinases or the presence of adaptor or anchoring proteins. Many of these experimental problems may be addressed with appropriate pharmacological protocols (dose-response curves and time courses), and there are now available specific cDNAs for expressing catalytic domains or subunits of protein kinases. In this way, the specific role of PKG may be addressed through transfection studies.

Expression of the catalytic domain of cyclic GMP-dependent protein kinase in a baculovirus system

The Type I cGMP‐dependent protein kinase catalytic domain (residues 336‐671 from the Iα isofonn) has been expressed as a cGMP independent kinase in a baculovirus system. Using peptide substrates, the protein retains similar substrate specificity as the native holoenzyme. The recombinant catalytic domain catalyzes the phosphorylation of histone, but does not display the inhibition using non‐substrate histones which has been described for the holoenzyme. The catalytic domain is an active kinase in mammalian cells also since vascular smooth muscle cells transfected with the cDNA encoding the catalytic domain display altered morphology. The catalytic domain of G‐kinase may be a useful tool for delineating the role of cGMP‐mediated protein phosphorylation in cell systems.

Cyclic nucleotides in smooth muscle

Publisher Summary Studies on the role of cyclic nucleotides, that is cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP) in smooth muscle relaxation began in the mid 1960s. If the role of cAMP (cyclic nucleotides) in smooth muscle relaxation appears to be relatively straight-forward, the role of cGMP (cyclic nucleotides) was less clear. The initial studies suggested a role for cGMP in mediating smooth muscle contraction. These conclusions were based on correlations between cGMP content and contraction in response to contractile agonists such as acetylcholine. A more thorough analysis, however, revealed that the elevations in cGMP observed with acetylcholine actually occurred after the onset of contraction, suggesting that cGMP was produced in response to intracellular Ca2+ elevations. It was not until the late 1970s that several investigators demonstrated that nitrogen oxide containing vasodilators such as nitroprusside and nitroglycerine elevated cGMP in smooth muscle tissues. These findings suggested that cGMP mediated relaxation of smooth muscle, a finding that was eventually supported by the discovery of the potent smooth muscle relaxing effects of cGMP analogs. Nevertheless, there have been dissociations reported between cyclic nucleotide elevating compounds or analogs on one hand, and the relaxation of different smooth muscle preparations on the other. Uterine smooth muscle relaxation, for example, is unaffected by elevations in cGMP. Vascular and bronchial smooth muscle, however, are highly responsive to the relaxing effects of cGMP. The diversity of responses to cyclic nucleotides in the various smooth muscle types may reflect different mechanisms that lead to the decrease in tone in the different types of smooth muscle.