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(Circulation. 2003;108:2172.)
© 2003 American Heart Association, Inc.

Special Review

Physiology and Pathophysiology of Vascular Signaling Controlled by Cyclic Guanosine 3',5'-Cyclic Monophosphate–Dependent Protein Kinase

Thomas Münzel, MD; Robert Feil, PhD; Alexander Mülsch, PhD; Suzanne M. Lohmann, PhD, MD; Franz Hofmann, MD; Ulrich Walter, MD

From the Division of Cardiology, University Hospital Eppendorf, Hamburg, Germany (T.M.); Institut für Pharmakologie und Toxikologie, Technische Universität, München, Germany (R.F., F.H.); the Institute of Cardiovascular Physiology, J.W. Goethe–University Clinic, Frankfurt/Main, Germany (A.M.); and the Institute of Clinical Biochemistry and Pathobiochemistry, University of Würzburg, Würzburg, Germany (S.M.L., U.W.).

Correspondence to Prof Thomas Münzel, MD, Professor of Internal Medicine, Division of Cardiology, University Hospital Eppendorf, Martinistr 52, 20246 Hamburg, Germany. E-mail muenzel{at}uke.uni-hamburg.de

Key Words: nitric oxide • natriuretic peptides • endothelium • cardiovascular diseases • free radical

	Introduction

Top Introduction cGMP Synthesis References

 
Despite impressive medical advances¹ that have led to diminished cardiovascular death rates in some countries over the past 20 years, cardiovascular disease remains the leading cause of death in developed countries such as the United States. This promises to worsen as a result of aging populations; the incipient obesity and type II diabetes epidemic; sedentary lifestyle; and continued abuse of tobacco, alcohol, and other substances. Cardiovascular disease is clearly multifactorial, and the approach to its prevention necessarily likewise. Candidates for prevention include cyclic guanosine 3',5'-cyclic monophosphate (cGMP)–dependent signaling networks initiated by natriuretic peptides (NPs) and nitric oxide (NO), which demonstrate characteristics deemed worthy of diagnostic and therapeutic exploitation. cGMP signaling contributes to the function and interaction of several vascular cell types, and its dysfunction could be involved in major destructive processes such as atherosclerosis, hypertension, diabetic complications, (re)stenosis, and tissue infarction, as well as the undermining of clinical therapy like in the case of nitrate tolerance. This review takes a focused look at key elements of the cGMP signaling cascade in vascular tissue, particularly recent advances in our knowledge of cGMP-dependent protein kinase (cGK, also known as PKG) function. Finally, we discuss the potential of clinical monitoring of cGK activity for assessing the functional status of cGMP signaling and for guiding the design of therapeutic strategies to improve vascular function.

	cGMP Synthesis

Top Introduction cGMP Synthesis References

 
One of the 2 major synthetic pathways for cGMP generation from guanosine 5'-triphosphate (GTP) is directed by NPs (Figure 1), consisting of atrial (ANP), B-type (BNP), and C-type (CNP) natriuretic peptides, which act via the membrane receptor guanylate cyclases GC-A (highest affinity for ANP, BNP) and GC-B (highest affinity for CNP). ANP and BNP, released from the heart by mechanical stretch in response to increased atrial pressure/volume, and CNP, released from the endothelium, can all cause smooth muscle (SM) relaxation and vasodilation.^2,3 Particularly, ANP has multiple effects, which lead to vascular smooth muscle (VSM) relaxation, increased vascular permeability and glomerular filtration rate, inhibition of the sympathetic and renin-angiotensin-aldosterone systems, and natriuresis/diuresis.³ Germline deletion of ANP or its GC-A receptor in mice resulted in varying degrees of hypertension, cardiomyopathy, and heart failure (reviewed in reference ²), but also cardiac hypertrophy that is independent of hypertension.^4,5 ANP effects on VSM itself were analyzed in transgenic mice engineered to lack GC-A selectively in smooth muscle. These mice displayed resistance to vasodilation by acute ANP treatment or vascular volume expansion, but normal resting blood pressure,⁶ suggesting that the function of GC-A in VSM can be compensated for in chronic blood pressure regulation.

View larger version (25K): [in this window] [in a new window]   Figure 1. Mechanisms of cGKI inhibition of Ca2+ release (left) and Ca2+ sensitization (right) to inhibit myosin light chain (MLC) phosphorylation and SMC contraction. cGKI phosphorylation (P) of IRAG inhibits IP3/IRAG-mediated Ca2+ release from the endoplasmic reticulum (ER), thus inhibiting MLC kinase (MLCK) and vasoconstriction. cGKI phosphorylates Rho and interferes with Rho kinase inhibition of MLCP, thus MLCP dephosphorylates MLC and enhances SMC relaxation. Binding of cGKI to substrates (eg, IRAG and the myosin-binding subunit [MBS] of MLCP), which are also members of signaling complexes or scaffolds, could be an important aspect of achieving specificity of cGKI action. G indicates G-protein; IP3R, IP3 receptor; NO, nitric oxide; PLC, phospholipase C; and sGC, soluble guanylate cyclase.

The second major pathway for cGMP synthesis (Figure 1) entails stimulation of nitric oxide synthases (NOS) to produce NO, which activates soluble guanylate cyclases (sGC).² NO inhibits VSM constriction, proliferation, and migration, and reduces platelet adhesion and activation as well as vascular inflammation.⁷ Transgenic overexpression and deletion of endothelial NOS (eNOS, NOSIII) and inducible NOS (iNOS, NOSII) have substantiated the role of NO in reducing blood pressure and the consequences of vascular injury.^2,7 These results with experimental models have led to the suggestion of members of the NO pathway as promising candidates for gene therapy, and a phase I NOS gene transfer study has been announced.⁷ Recommended potential clinical applications of NOS gene transfer include inhibition of vascular proliferation, relevant, for example, for atherosclerosis and restenosis, and inhibition of pulmonary hypertension, vasospasm, vasculitis, oxidative and reperfusion injury, thrombosis, and (through antisense NOS) septicemia.⁷ Already idiopathic pulmonary hypertension has been shown to respond to inhaled NO in some patients with apparent defects in their endogenous NO system.⁸ Promising as this sounds, incipient signals generated by NO or NPs need to traverse long, divergent, and interacting chains of multiple mediators before reaching effector molecules, which trigger final effects. Also, defects, desensitization, or other aberrations in the pathways distal to NO/NPs must be recognized as capable of derailing the desired procession of events. The ensuing discussion illustrates that understanding the complex array of interactions beyond NO/NPs is a serious challenge and an integral aspect of achieving insights into successful strategies for opposing cardiovascular disease.

cGMP Effectors
The cGMP signal is diversified through cell type–specific collections of intracellular cGMP receptors. In the cardiovascular system, the major cGMP effectors are cGMP-regulated phosphodiesterases (PDEs)^9,10 and cGKs (see subsequently in this article).^11–14 Cyclic nucleotide PDEs hydrolyze cAMP and/or cGMP and thus terminate their action. They are presently classified as PDE families 1 through 11, encoded by 20 genes, generating more than 50 PDE isozymes.^14a cGMP-hydrolyzing PDEs include ones that specifically (PDE 5, 6, and 9) or preferentially (PDE 1A1) hydrolyze cGMP,¹⁵ as well as ones that hydrolyze both cGMP and cAMP (PDE 1 to 3 and 10 to 11).^9,10,16 cGMP may also be transported out of cells by the multidrug-resistant protein 5.¹⁷ The activity of certain PDEs can be stimulated (PDE 2) or inhibited (PDE 3) by cGMP. Also, cGKI phosphorylates and activates PDE 5 to rapidly terminate cGMP action.^18,19 Platelets contain at least PDE 2, 3, and 5^19,20; arterial VSM cells at least PDE 1 to 5; and venous VSM cells at least PDE 1, 4, and 5.²¹

There is limited information on other cGMP effectors, which include cyclic nucleotide-gated channels (CNG) and cAMP-dependent protein kinase (cAK). Endothelial cells can express CNG²²; however, the functional significance of these channels is unclear, because no cardiovascular phenotype has been reported for various CNG-knockout mice.^23,24 cGMP signals can be mediated by cAMP-dependent protein kinase²⁵ through either of 2 different mechanisms. cGMP can inhibit PDE 3 (also called cGI-PDE) at concentrations similar to those that activate cGK,^26,26a,26b and thus increase cAMP and its activation of cAK. In contrast to the native cGMP increased by physiological activators, membrane-permeable cGMP analogs inhibit PDE 3 with different efficiencies, 100 µmol/L 8-Br-cGMP^26b apparently reaching intracellular concentrations sufficient to inhibit PDE 3, whereas 100 µmol/L 8-pCPT-cGMP does not.^26a,27 Alternatively, cGMP has been proposed to directly activate cAK under certain circumstances. However, compared with cGK, much higher levels of cGMP are required to directly activate cAK.²⁶ Studies of SM and platelets from cGK I–deficient transgenic mice,^28,29 or cGK I-deficient platelets from chronic myelocytic leukemia patients,³⁰ suggest that cAK is not involved in NO/cGMP signaling under physiological conditions, although cAK could come into play in pathophysiological settings such as inflammatory conditions^30a and septic shock when high NO/cGMP levels are produced.³¹

cGMP-Dependent Protein Kinases
The mammalian cGK family consists of cGKI and Iß, splice forms derived from 1 gene, and cGKII, encoded by a second gene.^11,13 Several cardiovascular cells, and other cell types which regulate the cardiovascular system, contain cGKs. These include fibroblasts and certain types of endothelial cells (cGKI), but not HUVECs from human umbilical veins,^32,33 SM cells (cGKI and Iß),^34,35 platelets and T lymphocytes (cGKIß),^36,37 renin-secreting juxtaglomerular cells (cGKI and cGKII),³⁸ other renal cells (cGKI and cGKII),³⁹ and cardiac myocytes (cGKI).⁴⁰ It is important to note that cGKs are lost in many primary cell types on passaging in cell culture and are undetectable in many cell lines.^32,37,41 This and the use of selective but not totally specific agonists and inhibitors of cGK has compounded the problems associated with establishing cGK functions. A notable example is the cGKI inhibitor KT5823, which can stimulate cGKI in certain cell types.⁴²

cGKs can achieve specificity of function partly through their selective binding to and phosphorylation of various substrates. Both cGKI and Iß have been shown to associate with troponin T and phosphorylate troponin I in the heart⁴³; however, only cGKI interacted with and phosphorylated the myosin-binding subunit of myosin light chain (MLC) phosphatase in VSM.⁴⁴ Only cGKIß occurs in a complex with and phosphorylates the IP₃ receptor-associated cGKI substrate (IRAG) in VSM.⁴⁵

Insight into cGK functions has been obtained both from analyses of substrates phosphorylated by cGKs and from cGK knockout mice. cGKI substrates and functions (Table) have been reviewed^11,14,46,47 and are briefly discussed here. Except for CFTR,⁴⁸ there are almost no other well-studied substrates of cGKII. In conventional cGKI knockout mice, the major phenotype observed was decreased lifespan²⁸; impaired relaxation of vascular,²⁸ visceral,^28,49 and penile⁵⁰ SM; and disturbed platelet activation.²⁹ The phenotype of cGKII knockout mice included normal lifespan,⁵¹ decreased longitudinal bone growth,⁵¹ decreased intestinal chloride secretion,^51,52 and altered renin secretion.⁵³ Results obtained with knockout mice discussed here support an important role of cGKI in the cardiovascular system, particularly in VSM cells and platelets, as well as potential cGKII influences on hemodynamic parameters through regulation of renin release and ion transport in the kidney.

View this table: [in this window] [in a new window]   Substrates Phosphorylated by cGKI and Their Functions

cGKI—Endothelial Cells
Two lines of evidence exist for activation of endothelial NOSIII by cGKI. cGKI⁵⁴ as well as several other protein kinases (cAK, Akt kinase, AMP kinase, protein kinase C [PKC], and calmodulin-dependent kinase II)^54–57 regulate (eg, cGKI stimulates) the phosphorylation and function of NOSIII, and in some cases (including cGKI) reduce its Ca²⁺ dependence. Furthermore, cGKI and cGKII phosphorylate and activate 6-pyruvoyltetrahydropterin synthase to produce tetrahydrobiopterin (BH₄) needed by NOS for NO synthesis.⁵⁸

cGKI—Vascular Smooth Muscle Cells
NO/ANP/cGMP interfere with VSMC signaling initiated by vasoconstrictor receptors and G-proteins,⁵⁹ which elevate Ca²⁺ to promote activation of MLC kinase, phosphorylation of MLCs, interaction of myosin/actin filaments, and contraction (Figure 1). Evidence supports a dual role of cGKI in inhibition of both intracellular Ca²⁺ concentrations and Ca²⁺ sensitization to evoke SM relaxation.^14,46,60 Primary cells cultured from cGKI knockout mice have also been important in demonstrating differences in cGKI isoform involvement in SMC Ca²⁺ regulation. NO/cGMP inhibited noradrenaline-induced Ca²⁺-transients in wild-type VSMCs containing endogenous cGKI and cGKIß, but not in cGKI-deficient cells.^28,61 Interestingly, the defective Ca²⁺ regulation in cGKI-deficient VSMCs was rescued by transfection of only cGKI, not cGKIß,⁶¹ suggesting that cGKI relaxes VSM by decreasing the cytosolic Ca²⁺ level. The role of cGKIß is unclear at present, but it is perhaps more relevant to cGKI effects on SM proliferation, differentiation, and gene expression.^14,46

Contributing to reduction of intracellular Ca²⁺ concentrations could be a spectrum of SM cGKI substrates, including the IP₃ receptor and IRAG^14,45 (Figure 1). cGKI also phosphorylates a SM BK_Ca channel, which leads to K⁺ efflux from the cell, hyperpolarization, inhibition of Ca²⁺ entry through voltage-dependent ion channels, and relaxation.^14,46 cGKI phosphorylation of the sarcoplasmic reticulum (SR) protein phospholamban (PLB) was suggested to deinhibit the SR Ca²⁺-ATPase, thus increasing Ca²⁺ loading of SR for later release and activation of BK_Ca.⁴⁶ However, PLB deletion in mice has only a minor effect on cGMP-mediated VSM relaxation.⁶²

At constant Ca²⁺ concentration, SM relaxation can be promoted by cGKI phosphorylation of Rho A to inhibit Rho kinase and thus activate MLC phosphatase to dephosphorylate MLC (Figure 1).⁶³ cGKI binds to and phosphorylates the myosin-binding subunit of MLC phosphatase, which could be important for localization of cGKI near the enzyme it regulates.⁴⁴ Furthermore, cGKI inhibition of Rho reinforces insulin signaling and its stimulation of myosin-bound phosphatase activity.⁶⁴ The importance of the Rho pathway is illustrated by the fact that treatment of rats with the Rho kinase inhibitor Y-27632 corrected hypertension in several hypertensive rat models⁶⁵ and also suppressed neointimal formation in balloon-injured carotid arteries.⁶⁶

In agreement with a role of cGKI in SM relaxation, juvenile (4 to 5 week old) cGKI knockout mice had elevated blood pressure.²⁸ These and in vitro studies with small arteries from cGKI-deficient mice³¹ suggested that cGKI mediates at least part of the antihypertensive effects of NO and ANP. The release of renin from the kidney and subsequent generation of angiotensin II also affect blood pressure. Inactivation of the cGKII gene, but not the cGKI gene, abolished cGMP inhibition of renin secretion in vitro and in vivo.⁵³ Thus, both cGKI and cGKII could be involved in the regulation of blood pressure, although through different mechanisms and cell types.

Interestingly, 7-week-old and older cGKI knockout mice have normal, or only slightly elevated, blood pressure,²⁸ indicating that lack of cGKI can be bypassed in older animals. Similarly, the cardiac phenotype of young NOSIII null mutants⁶⁷ is not observed in older NOSIII knockout mice.^68,69 These results suggest that mice with defects in the NO/cGMP pathway develop mechanisms to compensate for lost gene functions or, alternatively, that the physiological role of components of this signaling pathway is age-dependent. However, cGKI null mutants develop multiple defects with increasing age, including infections and inflammation, which are known to induce massive NO synthesis. Therefore, it is tempting to speculate that the apparent normalization of blood pressure in older cGKI mutants could be the result of cross-activation of cAK by the high NO/cGMP levels potentially generated in these diseased mice.

Although conventional cGKI knockout mice have yielded important insights into cGKI functions in the cardiovascular system, the analysis of these mice has several limitations. These mice have low life expectancy (approximately 50% of these mice die before 5 to 6 weeks of age), precluding analysis of adult mice and performance of long-term experiments like telemetric blood pressure measurements. Furthermore, the interpretation of phenotypes of conventional null mutants (in which the cGKI gene is inactivated in the germline and thus in every cell) is complicated by the lack of temporal and regional specificity of gene disruption. cGKI null mutants show not only a cardiovascular phenotype, but also develop severe defects of the gastrointestinal²⁸ and immune systems (C. Werner and F. Hofmann, unpublished results, 2003). It is also difficult to distinguish whether the age-related hypertension of these mice reflects a function of cGKI in VSMCs, endothelial cells, or other cell types. To overcome these limitations, a mouse line has been generated that allows tissue-specific inactivation of the cGKI gene using the Cre/lox site-specific recombination system.⁷⁰ In the future, the combined use of conventional and conditional cGKI knockout mice and of cGKI-deficient primary cells will further advance our understanding of cGKI and cGKIß function in the vessel wall in health and disease, and provide insight needed to develop new strategies for the treatment of cardiovascular disorders.

Reductions in cGMP/cGKI signaling have also been associated with detrimental conditions such as nitrate tolerance (discussed in a later section) and neointimal formation. In some studies,⁴⁶ but not others,⁷¹ cGKI has been shown to be reduced in the synthetic or proliferative SMC phenotype found in neointima. Adenovirus-mediated gene transfer of cGKI increased the sensitivity of cultured VSMC to the antiproliferative and proapoptotic effects of NO and cGMP,⁷² and cGKI inhibited VSMC migration.^46,73,74 Adenovirus-mediated gene transfer of constitutively active cGKI reduced neointima formation after balloon injury in rat carotid arteries and reduced in-stent restenosis in a porcine model.⁷⁴

cGKI—Platelets
Antiplatelet therapy is presently standard in the secondary prevention of cardiovascular events.⁷⁵ Therapeutically used platelet inhibitors are aspirin (a cyclooxygenase inhibitor), glycoprotein IIb/IIIa inhibitors, ADP receptor P2Y12 inhibitors such as Clopidogrel, and adenosine uptake/PDE 5 inhibitors such as dipyridamole, which increase cGMP.^20,75,76 cGKI can mediate inhibitory effects on platelets through several of these same targets. Experiments with both cGK-deficient human and murine platelets demonstrated that cGKI mediates many aspects of NO/cGMP inhibition of platelet activation^29,30 (Figure 2). Importantly, a prominent role of cGKI in the inhibition of platelet adhesion/activation in vivo during ischemia/reperfusion of the microcirculation was conclusively demonstrated by intravital video microscopy analyses comparing cGKI-deficient versus wild-type murine platelets perfused back into mice.²⁹ These experiments clearly showed that platelet cGKI, but not endothelial or SM cGKI, is essential to prevent intravascular adhesion and aggregation of platelets after ischemia. Furthermore, NO/cGMP/cGKI causes phosphorylation of platelet VASP,⁷⁷ which closely correlates with inhibition of platelet activation both in vitro²⁰ and in vivo,⁷⁸ with fibrinogen receptor (integrin GPIIb/IIIa) inhibition, and with inhibition of both VASP binding to F-actin and VASP localization to focal adhesions/integrins.^20,77 Other platelet substrates phosphorylated by cGKI include the IP₃ receptor, heat shock protein 27,²⁰ the LIM and SH3 protein LASP,⁷⁹ the small GTPase Rap 1b,⁸⁰ and PDE 5.^18,19

View larger version (29K): [in this window] [in a new window]   Figure 2. Mechanisms of cGKI inhibition of platelet activation by phosphorylation (P) of substrate proteins (IP3 receptor, the small GTPase Rap 1b, vasodilator-stimulated phosphoprotein [VASP], heat shock protein [hsp]27, and the cGMP hydrolyzing phosphodiesterase, PDE5) by inhibition of G-protein-coupled (Gq/Gi) receptor complexes and by inhibition of cAMP hydrolysis by PDE3. ABP indicates actin-binding protein; AC, adenylate cyclase; cAK, cAMP-dependent protein kinase; EDRF, endothelium-derived relaxing factor; G, G-protein; GP, glycoprotein; IP3R, IP3 receptor; sGC, soluble guanylate cyclase; and TXA2, thromboxane A2.

NO/cGMP signaling through cGKI is also known to inhibit platelet Gq/Gi-coupled receptor responses,²⁰ and in particular, the platelet ADP receptor P2Y12.⁸¹ Thus, one consequence of the impaired NO/cGMP signaling found in cardiovascular diseases characterized by endothelial dysfunction could be enhanced signaling through purinergic Gq/Gi-coupled receptors (P2Y1 and P2Y12), leading to enhanced platelet activation. cGKI also contributes to negative feedback or to cycling in signaling systems it transduces. Recent results indicated that cGKI phosphorylation of PDE 5 and activation of cGMP degradation could also contribute to desensitization of the platelet NO/cGMP response and to the contraction–relaxation cycle in SM.^18,19 Furthermore, evidence indicates that cGKI mediates effects of dipyridamole, which, in combination with low-dose aspirin, is very effective in preventing recurrent stroke; dipyridamole inhibits cGMP hydrolysis by PDE 5 and enhances antiplatelet effects of NO, but not those of cAMP.⁷⁶ There is still lack of in vivo evidence in volunteers and/or patients of direct platelet inhibition by NO-generating drugs such as NTG, which activate sGC. However, recent preclinical studies with NO-independent sGC activators suggest that elevation of platelet cGMP in vivo is in fact associated with platelet inhibition.^82,83

cGKI—Fibroblasts
NO, ANP, and cGMP inhibit proliferative effects of norepinephrine on cardiac myocytes and fibroblasts.⁸⁴ Cardiac fibrosis, a hallmark of cardiac failure, is a major consequence of genetic deletion of BNP in mice.⁸⁵ Some data suggests that cGKI can mediate inhibitory effects on fibrosis. In NIH 3T3 fibroblasts, cGKI phosphorylates MAP/ERK kinase kinase (MEKK1), causing activation of JNK kinase, an inducer of apoptosis.⁸⁶ In adventitial fibroblasts, cGKI increased expression of p21 (Waf1/Cip1), a cyclin-dependent kinase inhibitor that can arrest cell proliferation during the cell cycle by preventing DNA replication in S-phase.⁸⁷ In 293T fibroblasts, cGKI stimulated p38 kinase phosphorylation and ATF-2-dependent gene expression.⁸⁸

cGKI—Cardiac Myocytes
In the heart, cGKI inhibits L-type Ca²⁺ channels,⁴⁰ has antihypertrophic effects,⁸⁹ and mediates the negative inotropic effect of cGMP.⁷⁰ Ca²⁺ signaling has been implicated in cardiac hypertrophy,^90–92 and cGKI inhibition of Ca²⁺ influx resulted in inhibition of the Ca²⁺-dependent calcineurin-NFAT signaling pathway and cell hypertrophy.⁹¹ cGKI also phosphorylates PLB,⁹³ releasing PLB inhibition of Ca²⁺-ATPase and enhancing Ca²⁺ reuptake into the SR, which would presumably enhance cardiac relaxation, availability of Ca²⁺ for contraction, and the maximal rate of contraction.^94,95 In a mouse model of dilated cardiomyopathy, ablation of PLB rescued phenotypes that resemble human heart failure,⁹⁶ suggesting that cGKI phosphorylation and inactivation of PLB might have a similar inhibitory effect with regard to heart failure.

Mice with cardiomyocyte-specific cGKI deletion are fully viable and can be studied throughout adulthood. The combined analysis of conventional and cardiomyocyte-specific cGKI knockout mice demonstrated that cGKI mediates the negative inotropic effect of cGMP in juvenile as well as adult murine heart, whereas the NO/cGMP/cGKI pathway seems not to be involved in the negative inotropic action of acetylcholine.^68–70 Potential mechanisms involved in the negative inotropic effects of cGKI might include cGKI inhibition of L-type Ca²⁺ channels,⁴⁰ and cGKI interaction with troponin T and phosphorylation of troponin I.⁴³

Endothelial Dysfunction, Oxidative Stress, and Defective NO/cGMP Signaling
The preceding sections provide considerable evidence that the NO/cGMP/cGK signaling axis is important in the control of normal cardiovascular function and should have important implications for our understanding and treatment of vascular disease. Endothelial dysfunction, manifested as thrombosis, inflammation, and vasoconstriction, is a common feature of atherosclerosis, hypercholesterolemia, hypertension, and nitrate tolerance. Quite often, endothelial dysfunction is characterized by impairment of endothelium-dependent, NO-mediated relaxation, in contrast to essentially normal vascular relaxation to endothelium-independent nitrovasodilators such as sodium nitroprusside (SNP) or NTG. In endothelial dysfunction, defects have been described in the coupling of stimuli to activation of NOSIII, in the activity and/or expression of NOSIII, and in NO bioavailability as a result of increased production of reactive oxygen species (ROS=oxidative stress). Impaired VSM relaxation can result from not only endothelial dysfunction, but also from defective activity and/or expression of components of the NO downstream signaling pathway (sGC, cGKI, PDEs).

Antioxidants are effective in combating the vascular oxidative stress, which reduces NO production or bioactivity. Acute administration of the antioxidant vitamin C improved endothelial dysfunction in patients with hypercholesterolemia, overt atherosclerosis, diabetes mellitus, hypertension, smoking, ischemia/reperfusion, and NTG therapy (reviewed in reference ⁹⁷) The major extent to which oxidative stress contributes to endothelial dysfunction is demonstrated by the fact that the degree to which vitamin C improves endothelial dysfunction is a strong and independent predictor of subsequent cardiovascular events.⁹⁸

In mammalian cells, potential enzymatic sources of reactive oxygen species (ROS) include mitochondrial respiration enzymes, lipoxygenases, cyclooxygenases, cytochrome P450 enzymes, NAD(P)H oxidases, xanthine oxidase, and the ceramide-activated protein kinase (reviewed in reference ⁹⁹). Another source of vascular ROS production that has received substantial attention is NOSIII itself. Circumstances in which NOSIII generates superoxide include inadequate intracellular concentrations of L-arginine (the NOSIII substrate) and BH₄ (a NOSIII cofactor), or interactions of ROS with the Zn thiolate complex of NOSIII itself.¹⁰⁰ Diminished intracellular L-arginine bioavailability can occur by diminished L-arginine recycling, increased L-arginine metabolism, or increased formation of asymmetric dimethyl-arginines, which inhibit NOSIII.¹⁰¹ BH₄ as well as the Zn thiolate complex are susceptible to oxidation by peroxynitrite (ONOO-), the reaction product of NO and superoxide (Figure 3). Consistent with this, recent animal¹⁰² and human studies^103,104 of endothelial dysfunction indicate that administration of either BH₄, or folic acid (which can restore endothelial BH₄ concentrations) improves endothelial dysfunction and simultaneously reduces oxidative stress.

View larger version (31K): [in this window] [in a new window]   Figure 3. Schematic representation of the NO/cGMP/cGKI pathway of vascular smooth muscle relaxation and mechanisms of its inactivation by endothelial dysfunction. Also illustrated is the analysis of NO/cGMP/cGKI action and endothelial function by monitoring cGKI phosphorylation of Ser239 of the substrate protein VASP. Under normal conditions, nitric oxide (NO), synthesized by nitric oxide synthase (NOS) III, stimulates soluble guanylate cyclase (sGC), increasing cGMP and thus stimulation of cGMP-dependent protein kinase I (cGKI) and vasorelaxation. This pathway can, however, be inhibited at several sites. Angiotensin II, hypertension, hypercholesterolemia, and nitrate tolerance enhance vascular superoxide (O2·-) production, which inactivates NO, diminishing cGKI action. Both O2·- and the NO/O2·- reaction product peroxynitrite (ONOO-) potently inhibit sGC. Peroxynitrite can also uncouple NOSIII by oxidizing the NOSIII cofactor tetrahydrobiopterin (BH4) to dihydrobiopterin (BH2) and/or by oxidizing Zn thiolate complexes within NOSIII. Angiotensin II and nitroglycerin (NTG) therapy stimulate the activity and expression of phosphodiesterase PDE1A1, thus decreasing cGKI action and endothelium-dependent and -independent vasodilation while inducing supersensitivity to vasoconstrictors. Interventions that reduce vascular oxidative stress such as therapy with statins, Ang II receptor (AT1 subtype) blockers, ACE inhibitors, or vitamins improves NO bioavailability by reducing vascular superoxide production. In all cases, P-VASP served as a reliable monitor of the NO/cGMP/cGKI pathway and endothelial function.

NO/cGMP Signaling Pathway as Monitor of Endothelial Dysfunction
In general, assessment of any individual component (NOSIII, sGC, PDE) of the NO/cGMP pathway has not been uniformly reliable as a predictor of endothelial dysfunction. However, because cGKI occupies a very distal position in this signaling chain, its activity could theoretically be used to reflect the sum of changes in activity and/or expression of upstream components as well as cGKI itself. Assessment of cGKI activity is facilitated by immunoblotting using antibodies that detect phosphorylation of the cGKI substrate, VASP, at Ser239 (P-VASP) (Figure 3). This assay has proven to reliably reflect NO/cGMP signaling activity in platelets, cultured endothelial and SM cells,^33,105 and even intact vascular tissues.¹⁰⁶ Immunoblot detection of P-VASP is even sufficiently sensitive to allow the assessment of basal NO release in endothelium-intact arteries. Indeed, either endothelial denudation or incubation with NOS inhibitors significantly attenuates the proportion of arterial P-VASP.¹⁰⁶ Conversely, exogenous NO donors such as SNP and NTG markedly increase P-VASP in vessels regardless of the presence or absence of intact endothelium.¹⁰⁶ P-VASP also proved to be a suitable monitor for reduced NO bioactivity as a result of oxidative stress in vascular tissue. For example, inhibition of Cu/Zn superoxide dismutase (SOD) increased vascular superoxide, reduced endothelial NO bioactivity, and resulted in marked reduction in the proportion of P-VASP.¹⁰⁶

Hypercholesterolemia
Hypercholesterolemia impairs endothelium-dependent vasodilation, and also arterial relaxation to authentic NO, but has little effect on responses to nitrovasodilators such as SNP and NTG. This hypercholesterolemia-dependent defect in NO bioactivity appears linked to oxidative stress, because animals rendered hyperlipidemic by cholesterol feeding,¹⁰⁷ LDL receptor deficiency,¹⁰⁶ or ApoE deletion¹⁰² all exhibit increased steady-state superoxide flux compared with control animals. Sources of superoxide that have been implicated in hypercholesterolemia include xanthine oxidase,¹⁰⁷ NAD(P)H oxidase,¹⁰⁸ and NOSIII.^102,106

The effects of hypercholesterolemia on sGC expression are inconsistent. Cholesterol feeding is associated with a 3- to 4-fold increase in the vascular expression of sGC subunits ₁ and ß₁ (mainly within neointimal lesions), whereas no change was observed in vessels from Watanabe heritable hyperlipidemic rabbits (WHHL).¹⁰⁹ The catalytic activity of sGC is unchanged but the specific activity reduced, perhaps as a result of increased smooth muscle-derived ROS production.¹¹⁰

The expression of cGKI appears to be normal, whereas cGK activity, assessed by the P-VASP/VASP ratio, is strongly decreased compared with controls, suggestive of a defect in the NO/cGMP pathway upstream of cGK.¹⁰⁶ Incubation of vessels from controls, as well as WHHL animals, with high concentrations of the exogenous NO donor, SNP, resulted in comparable maximal P-VASP levels, demonstrating that the cGMP signaling pathway downstream of sGC was, per se, not impaired.¹⁰⁶ Chronic treatment of WHHL with AT1 receptor blockers reduced oxidative stress within vascular tissue and improved endothelial function, most likely by inhibiting NADPH-oxidase and NOSIII-mediated superoxide production.^106,111 The improvement of endothelial function was associated with increased vascular NO concentrations detected with electron paramagnetic resonance, and a quite marked enhancement in vascular P-VASP levels, again demonstrating the usefulness of P-VASP as a reliable biomonitor of NO function in vascular tissue.

Hypertension
Endothelial dysfunction in different animal models of hypertension has been shown to be associated with increased expression of NOSIII^112,113 but decreased vascular NO bioavailability, as revealed by P-VASP analysis.¹¹² Uncoupled NOSIII¹¹² and NAD(P)H oxidase¹¹⁴ were identified as significant superoxide sources based on observations that (1) NOSIII inhibition decreased vascular superoxide production in hypertensive animals,¹¹² and (2) these animals exhibited increased NAD(P)H oxidase subunit (nox1, p22phox, p67phox, and gp91phox) expression and enzymatic activity.^112,115 Increased superoxide production involved all vessel layers^112,116 and was causally linked to both endothelial dysfunction and hypertension, because treatment with either antioxidants or SOD not only improved endothelial function, but also markedly reduced blood pressure.¹¹⁷

Blood vessels from hypertensive animals demonstrate a phenotype that is distinct from that observed with hypercholesterolemia. In particular, vessels from hypertensive animals displayed reduced vasodilation to both endothelium-dependent vasodilators and the endothelium-independent exogenous nitrovasodilators SNP and NTG.¹¹⁴ This finding can be explained in part by reduced expression of sGC because different hypertensive animal models typically demonstrate reduced levels of 1 or both sGC subunits (₁ and ß₁) as well as reduced NO-dependent sGC activity.^112,118,119 The specific mechanism(s) for reduced sGC expression in hypertension are not entirely clear. Changes in sGC expression seem not to be exclusively linked to increased superoxide formation. Reducing blood pressure normalizes or even enhances sGC expression,^112,113,120 but does not consistently alter vascular superoxide production. In the angiotensin II-infusion model, inhibition of PKC in vivo reduces both blood pressure and vascular superoxide formation¹¹² concomitant with normalization of sGC levels (M. Oelze, unpublished observation, 2003). In contrast, hydralazine treatment significantly lowers blood pressure but fails to normalize vascular superoxide formation in chronically, NOS-inhibited rats.¹¹⁹

In angiotensin II-induced hypertension, cGKI activity assessed by P-VASP was markedly reduced, indicative of a signaling defect upstream of cGKI.¹¹² Oxidative stress could be involved in this defect, because PKC inhibition with chelerythrine reduced oxidative stress, increased VASP phosphorylation, and improved endothelial dysfunction (M. Oelze, unpublished observation, 2003). Interpretation of these observations is not straightforward, however, because in vitro VSM cell experiments indicate that angiotensin II also increases the activity and expression of the cGMP-specific PDE1A1 isoform (Figure 3).¹⁵ Thus, increased cGMP hydrolysis could also contribute to the endothelial dysfunction and decreased cGKI activity observed in angiotensin II-induced hypertension.

Nitrate Tolerance
NTG produces potent vasodilatory and antiischemic effects that are rapidly lost on chronic treatment as a result of a phenomenon known as nitrate tolerance. Like in the case of hypertension and hypercholesterolemia, nitrate tolerance is associated with endothelial dysfunction (=cross-tolerance); increased expression of a dysfunctional, uncoupled NOSIII¹²¹; and activation of NAD(P)H oxidase.¹²² Treatment with antioxidants such as vitamin E¹²³ or C¹²⁴ ameliorates nitrate tolerance. Moreover, other substances that reduce oxidative stress such as ACE inhibitors and AT1 receptor blockers (both of which can reduce NADPH oxidase activation) or BH₄¹²⁵ and folic acid (which can prevent NOSIII uncoupling)¹²⁶ also reverse, in part, nitrate tolerance as well as cross-tolerance to endothelium-dependent vasodilators.

Baseline cGMP levels, and cGMP responses to acute NTG challenges, are lower in tolerant than control vessels.¹²⁷ Mechanisms involved can include decreased vascular NO bioavailability,¹²¹ increased production of SM endothelin-1,¹²⁸ and inhibition of NO-dependent sGC activity by NTG-stimulated ROS production. Posttranslational modifications of sGC such as heme oxidation can also be a cause of impaired sGC-specific activity in the setting of nitrate tolerance.¹²⁹ Changes in sGC expression do not explain the impaired cGMP response, because both mRNA and protein expression of sGC subunits ₁ and ß₁ appear increased rather than decreased in aortas from tolerant rats and rabbits.¹³⁰ cGKI expression does not appear to be changed in arterial tissue from either NTG-treated animals or from humans treated with therapeutic doses of NTG.^130,131 Only higher, clinically nonrelevant doses of ISDN and NTG appear to decrease cGKI expression by approximately 50%.^130–132 However, in vivo treatment with NTG consistently decreased the cGKI activity assessed by P-VASP in aortas from rats and rabbits,¹³⁰ and in mammary arteries from patients with coronary artery disease.¹³¹ Incubation of arterial tissue from NTG-treated animals with the antioxidant vitamin C restored P-VASP, improved tolerance, and reduced vascular superoxide levels, indicating a crucial role of ROS in inhibiting cGKI activity in the setting of nitrate tolerance.¹³⁰

In summary, in experimental and clinical examples of hypercholesterolemia, hypertension, and nitrate tolerance, ROS were shown to be a major cause of inhibition of the NO/cGMP/cGKI signaling pathway and induction of endothelial dysfunction. Analysis of distal pathway cGKI activity was a more reliable indicator of endothelial function/dysfunction than were the levels of individual components of the NO/cGMP/cGKI pathway. Monitoring of cGKI activity through P-VASP levels not only serves as an effective predictor of endothelial function, but also potentially represents a much needed biochemical assay for early recognition of disease states and for evaluation of therapeutic strategies.

Diagnostic and Therapeutic Potential of cGKI-Mediated Vascular Signaling
cGKI is clearly an important mediator of vasorelaxant, antithrombotic, and other cardiovascular protective effects of NO and NPs, and new avenues of diagnostic and therapeutic exploitation of NO/NP/cGMP/cGK pathways are emerging. Recently, measurement of plasma BNP has been demonstrated to be useful in the diagnosis of congestive heart failure.^133,134 Analysis of VASP phosphorylation as a reporter of cGKI activity in vessels is a reliable monitor of endothelial integrity in model systems of atherosclerosis, hypertension, and nitrate tolerance. Importantly, P-VASP analysis in vessels was found applicable for monitoring nitrate tolerance in humans. Analysis of P-VASP has proven useful for monitoring platelet inhibition by NO-independent sGC activators,^83,135 by PDE 5 inhibitors such as dipyridamole,⁷⁶ and by the ADP P2Y12-receptor inhibitor Clopidogrel.²⁰ Present studies focus on analyzing P-VASP in whole blood to expedite clinical monitoring of the antithrombotic/antiplatelet capacity of the endothelium as an indicator of cardiovascular disease status and the success of therapeutic regimens.

Components of the NO/NP/cGMP/cGK pathways also constitute promising targets for therapy of chronic vascular diseases. The large number of PDEs with different regulators and cellular distributions make them interesting candidate targets for specific intervention into cGMP signaling. The success of Sildenafil, a PDE 5 inhibitor for increasing cGMP and SM relaxation, used in clinical treatment of erectile dysfunction, is well known.¹³⁶ Other results have shown that PDE1C (hydrolyzes cAMP and cGMP with equal efficiency) is expressed only in proliferating aortic SMCs, and that PDE1C inhibition suppresses proliferation of SMC from normal and atherosclerotic human tissue, potentially useful in prevention of atherosclerotic lesions or restenosis.¹³⁷ Recently, short-term use of Nesiritide (human recombinant BNP) was shown to be beneficial in the management of decompensated congestive heart failure.¹³⁸ The longstanding clinical use of NO donors for the treatment of angina pectoris and coronary artery disease is of proven value, however, with the detraction that chronic treatment leads to tolerance and endothelial dysfunction. A promising strategy designed to circumvent this would be use of ROS inhibitors, NO-independent activators of sGC that do not invoke tolerance,^82,83 selective PDE 5 inhibitors, direct activators of cGKI, and combinations thereof. Currently, non-NO-based activators of sGC used in in vivo animal studies have caused long-term reduction in blood pressure and thrombosis,⁸³ as well as cardiorenal effects, which would be beneficial for treatment of diseases such as congestive heart failure.¹³⁹ By definition, these activators would also avoid any undesirable cGMP-independent effects of NO. Further downstream, activators of cGKI or cGKII would have even more focused effects by excluding cGMP actions on other mediators.

	References

Top Introduction cGMP Synthesis References

 

1. Lefkowitz RJ, Willerson JT. Prospects for cardiovascular research. JAMA. 2001; 285: 581–587.[Abstract/Free Full Text]
   
2. Wedel B, Garbers D. The guanylyl cyclase family at Y2K. Annu Rev Physiol. 2001; 63: 215–233.[CrossRef][Medline] [Order article via Infotrieve]
   
3. Venugopal J. Cardiac natriuretic peptides—hope or hype? J Clin Pharm Ther. 2001; 26: 15–31.[CrossRef][Medline] [Order article via Infotrieve]
   
4. Knowles JW, Esposito G, Mao L, et al. Pressure-independent enhancement of cardiac hypertrophy in natriuretic peptide receptor A-deficient mice. J Clin Invest. 2001; 107: 975–984.[Medline] [Order article via Infotrieve]
   
5. Holtwick R, van Eickels M, Skryabin BV, et al. Pressure-independent cardiac hypertrophy in mice with cardiomyocyte-restricted inactivation of the atrial natriuretic peptide receptor guanylyl cyclase-A. J Clin Invest. 2003; 111: 1399–1407.[CrossRef][Medline] [Order article via Infotrieve]
   
6. Holtwick R, Gotthardt M, Skryabin B, et al. Smooth muscle-selective deletion of guanylyl cyclase-A prevents the acute but not chronic effects of ANP on blood pressure. Proc Natl Acad Sci U S A. 2002; 99: 7142–7147.[Abstract/Free Full Text]
   
7. von der Leyen HE, Dzau VJ. Therapeutic potential of nitric oxide synthase gene manipulation. Circulation. 2001; 103: 2760–2765.[Free Full Text]
   
8. Beckman JS. OONO: rebounding from nitric oxide. Circ Res. 2001; 89: 295–297.[Free Full Text]
   
9. Beavo JA. Cyclic nucleotide phosphodiesterases: functional implications of multiple isoforms. Physiol Rev. 1995; 75: 725–748.[Abstract/Free Full Text]
   
10. Hetman JM, Robas N, Baxendale R, et al. Cloning and characterization of two splice variants of human phosphodiesterase 11A. Proc Natl Acad Sci U S A. 2000; 97: 12891–12895.[Abstract/Free Full Text]
    
11. Lohmann SM, Vaandrager AB, Smolenski A, et al. Distinct and specific functions of cGMP-dependent protein kinases. Trends Biochem Sci. 1997; 22: 307–312.[CrossRef][Medline] [Order article via Infotrieve]
    
12. Eigenthaler M, Lohmann SM, Walter U, et al. Signal transduction by cGMP-dependent protein kinases and their emerging roles in the regulation of cell adhesion and gene expression. Rev Physiol Biochem Pharmacol. 1999; 135: 173–209.[Medline] [Order article via Infotrieve]
    
13. Pfeifer A, Ruth P, Dostmann W, et al. Structure and function of cGMP-dependent protein kinases. Rev Physiol Biochem Pharmacol. 1999; 135: 105–149.[Medline] [Order article via Infotrieve]
    
14. Hofmann F, Ammendola A, Schlossmann J. Rising behind NO: cGMP-dependent protein kinases. J Cell Sci. 2000; 113: 1671–1676.[Abstract]
    
14. Rybalkin SD, Rybalkina I, Beauo JA, et al. Cyclic nucleotide phosphodiesterase 1C promotes human arterial smooth muscle cell proliferation. Circ Res. 2002; 90: 151–157.[Abstract/Free Full Text]
    
15. Kim D, Rybalkin SD, Pi X, et al. Upregulation of phosphodiesterase 1A1 expression is associated with the development of nitrate tolerance. Circulation. 2001; 104: 2338–2343.[Abstract/Free Full Text]
    
16. Soderling SH, Beavo JA. Regulation of cAMP and cGMP signaling: new phosphodiesterases and new functions. Curr Opin Cell Biol. 2000; 12: 174–179.[CrossRef][Medline] [Order article via Infotrieve]
    
17. Jedlitschky G, Burchell B, Keppler D. The multidrug resistance protein 5 functions as an ATP-dependent export pump for cyclic nucleotides. J Biol Chem. 2000; 275: 30069–30074.[Abstract/Free Full Text]
    
18. Rybalkin SD, Rybalkina IG, Feil R, et al. Regulation of cGMP-specific phosphodiesterase (PDE5) phosphorylation in smooth muscle cells. J Biol Chem. 2002; 277: 3310–3317.[Abstract/Free Full Text]
    
19. Mullershausen F, Friebe A, Feil R, et al. Direct activation of PDE5 by cGMP: long-term effects within NO/cGMP signaling. J Cell Biol. 2003; 160: 719–727.[Abstract/Free Full Text]
    
20. Schwarz UR, Walter U, Eigenthaler M. Taming platelets with cyclic nucleotides. Biochem Pharmacol. 2001; 62: 1153–1161.[CrossRef][Medline] [Order article via Infotrieve]
    
21. Wallis RM, Corbin JD, Francis SH, et al. Tissue distribution of phosphodiesterase families and the effects of sildenafil on tissue cyclic nucleotides, platelet function, and the contractile responses of trabeculae carneae and aortic rings in vitro. Am J Cardiol. 1999; 83: 3C–12C.[Medline] [Order article via Infotrieve]
    
22. Zhang J, Xia SL, Block ER, et al. NO upregulation of a cyclic nucleotide-gated channel contributes to calcium elevation in endothelial cells. Am J Physiol Cell Physiol. 2002; 283: C1080–C1089.[Abstract/Free Full Text]
    
23. Baker H, Cummings DM, Munger SD, et al. Targeted deletion of a cyclic nucleotide-gated channel subunit (OCNC1): biochemical and morphological consequences in adult mice. J Neurosci. 1999; 19: 9313–9321.[Abstract/Free Full Text]
    
24. Biel M, Seeliger M, Pfeifer A, et al. Selective loss of cone function in mice lacking the cyclic nucleotide-gated channel CNG3. Proc Natl Acad Sci U S A. 1999; 96: 7553–7557.[Abstract/Free Full Text]
    
25. Lincoln TM, Komalavilas P, Boerth NJ, et al. cGMP signaling through cAMP- and cGMP-dependent protein kinases. Adv Pharmacol. 1995; 34: 305–322.[Medline] [Order article via Infotrieve]
    
26. Butt E, Nolte C, Schulz S, et al. Analysis of the functional role of cGMP-dependent protein kinase in intact human platelets using a specific activator 8-para-chlorophenylthio-cGMP. Biochem Pharmacol. 1992; 43: 2591–2600.[CrossRef][Medline] [Order article via Infotrieve]
    
26. Nolte C, Eigenthaler M, Horstrup R, et al. Synergistic phosphorylation of the focal adhesion-associated vasodilator-stimulated phosphoprotein in intact human platelets in response to cGMP- and cAMP-elevating platelet inhibitors. Biochem Pharmacol. 1994; 48: 1569–1575.[CrossRef][Medline] [Order article via Infotrieve]
    
26. Vandecasteele G, Verde I, Rucker-Martin C, et al. Cyclic GMP regulation of the L-type Ca(2+) channel current in human atrial myocytes. J Physiol. 2001; 533: 329–340.[Abstract/Free Full Text]
    
27. Osinski MT, Rauch BH, Schror K. Antimitogenic actions of organic nitrates are potentiated by sildenafil and mediated via activation of protein kinase A. Mol Pharmacol. 2001; 59: 1044–1050.[Abstract/Free Full Text]
    
28. Pfeifer A, Klatt P, Massberg S, et al. Defective smooth muscle regulation in cGMP kinase I-deficient mice. Embo J. 1998; 17: 3045–3051.[CrossRef][Medline] [Order article via Infotrieve]
    
29. Massberg S, Sausbier M, Klatt P, et al. Increased adhesion and aggregation of platelets lacking cyclic guanosine 3', 5'-monophosphate kinase I. J Exp Med. 1999; 189: 1255–1264.[Abstract/Free Full Text]
    
30. Eigenthaler M, Ullrich H, Geiger J, et al. Defective nitrovasodilator-stimulated protein phosphorylation and calcium regulation in cGMP-dependent protein kinase-deficient human platelets of chronic myelocytic leukemia. J Biol Chem. 1993; 268: 13526–13531.[Abstract/Free Full Text]
    
30. Cornwell TL, Arnold E, Boerth NJ, et al. Inhibition of smooth muscle cell growth by nitric oxide and activation of cAMP-dependent protein kinase by cGMP. Am J Physiol. 1994; 267: C1405–C1413.[Medline] [Order article via Infotrieve]
    
31. Sausbier M, Schubert R, Voigt V, et al. Mechanisms of NO/cGMP-dependent vasorelaxation. Circ Res. 2000; 87: 825–830.[Abstract/Free Full Text]
    
32. Draijer R, Vaandrager AB, Nolte C, et al. Expression of cGMP-dependent protein kinase I and phosphorylation of its substrate, vasodilator-stimulated phosphoprotein, in human endothelial cells of different origin. Circ Res. 1995; 77: 897–905.[Abstract/Free Full Text]
    
33. Smolenski A, Poller W, Walter U, et al. Regulation of human endothelial cell focal adhesion sites and migration by cGMP-dependent protein kinase I. J Biol Chem. 2000; 275: 25723–25732.[Abstract/Free Full Text]
    
34. Sekhar KR, Hatchett RJ, Shabb JB, et al. Relaxation of pig coronary arteries by new and potent cGMP analogs that selectively activate type I alpha, compared with type I beta, cGMP-dependent protein kinase. Mol Pharmacol. 1992; 42: 103–108.[Abstract]
    
35. Keilbach A, Ruth P, Hofmann F. Detection of cGMP dependent protein kinase isozymes by specific antibodies. Eur J Biochem. 1992; 208: 467–473.[Medline] [Order article via Infotrieve]
    
36. Bader B, Butt E, Palmetshofer A, et al. A cGMP-dependent protein kinase assay for high throughput screening based on time-resolved fluorescence resonance energy transfer. J Biomol Screen. 2001; 6: 255–264.[Abstract]
    
37. Fischer TA, Palmetshofer A, Gambaryan S, et al. Activation of cGMP-dependent protein kinase Ibeta inhibits interleukin 2 release and proliferation of T cell receptor-stimulated human peripheral T cells. J Biol Chem. 2001; 276: 5967–5974.[Abstract/Free Full Text]
    
38. Gambaryan S, Wagner C, Smolenski A, et al. Endogenous or overexpressed cGMP-dependent protein kinases inhibit cAMP-dependent renin release from rat isolated perfused kidney, microdissected glomeruli, and isolated juxtaglomerular cells. Proc Natl Acad Sci U S A. 1998; 95: 9003–9008.[Abstract/Free Full Text]
    
39. Gambaryan S, Hausler C, Markert T, et al. Expression of type II cGMP-dependent protein kinase in rat kidney is regulated by dehydration and correlated with renin gene expression. J Clin Invest. 1996; 98: 662–670.[Medline] [Order article via Infotrieve]
    
40. Mery PF, Lohmann SM, Walter U, et al. Ca2+ current is regulated by cyclic GMP-dependent protein kinase in mammalian cardiac myocytes. Proc Natl Acad Sci U S A. 1991; 88: 1197–1201.[Abstract/Free Full Text]
    
41. Cornwell TL, Lincoln TM. 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. J Biol Chem. 1989; 264: 1146–1155.[Abstract/Free Full Text]
    
42. Burkhardt M, Glazova M, Gambaryan S, et al. KT5823 inhibits cGMP-dependent protein kinase activity in vitro but not in intact human platelets and rat mesangial cells. J Biol Chem. 2000; 275: 33536–33541.[Abstract/Free Full Text]
    
43. Yuasa K, Michibata H, Omori K, et al. A novel interaction of cGMP-dependent protein kinase I with troponin T. J Biol Chem. 1999; 274: 37429–37434.[Abstract/Free Full Text]
    
44. Surks HK, Mochizuki N, Kasai Y, et al. Regulation of myosin phosphatase by a specific interaction with cGMP-dependent protein kinase Ialpha. Science. 1999; 286: 1583–1587.[Abstract/Free Full Text]
    
45. Schlossmann J, Ammendola A, Ashman K, et al. Regulation of intracellular calcium by a signalling complex of IRAG, IP3 receptor and cGMP kinase Ibeta. Nature. 2000; 404: 197–201.[CrossRef][Medline] [Order article via Infotrieve]
    
46. Lincoln TM, Dey N, Sellak H. Invited review: cGMP-dependent protein kinase signaling mechanisms in smooth muscle: from the regulation of tone to gene expression. J Appl Physiol. 2001; 91: 1421–1430.[Abstract/Free Full Text]
    
47. Smolenski A, Lohmann SM. Cyclic GMP dependent protein kinase (cGK, PKG). In: Creighton TE, ed. Wiley Encyclopedia of Molecular Medicine. New York: John Wiley & Sons, Inc; 2002: 954–957.
    
48. Vaandrager AB, Tilly BC, Smolenski A, et al. cGMP stimulation of cystic fibrosis transmembrane conductance regulator Cl-channels co-expressed with cGMP-dependent protein kinase type II but not type Ibeta. J Biol Chem. 1997; 272: 4195–4200.[Abstract/Free Full Text]
    
49. Ny L, Pfeifer A, Aszodi A, et al. Impaired relaxation of stomach smooth muscle in mice lacking cyclic GMP-dependent protein kinase I. Br J Pharmacol. 2000; 129: 395–401.[CrossRef][Medline] [Order article via Infotrieve]
    
50. Hedlund P, Aszodi A, Pfeifer A, et al. Erectile dysfunction in cyclic GMP-dependent kinase I-deficient mice. Proc Natl Acad Sci U S A. 2000; 97: 2349–2354.[Abstract/Free Full Text]
    
51. Pfeifer A, Aszodi A, Seidler U, et al. Intestinal secretory defects and dwarfism in mice lacking cGMP-dependent protein kinase II. Science. 1996; 274: 2082–2086.[Abstract/Free Full Text]
    
52. Vaandrager AB, Bot AG, Ruth P, et al. Differential role of cyclic GMP-dependent protein kinase II in ion transport in murine small intestine and colon. Gastroenterology. 2000; 118: 108–114.[CrossRef][Medline] [Order article via Infotrieve]
    
53. Wagner C, Pfeifer A, Ruth P, et al. Role of cGMP-kinase II in the control of renin secretion and renin expression. J Clin Invest. 1998; 102: 1576–1582.[Medline] [Order article via Infotrieve]
    
54. Butt E, Bernhardt M, Smolenski A, et al. Endothelial nitric-oxide synthase (type III) is activated and becomes calcium independent upon phosphorylation by cyclic nucleotide-dependent protein kinases. J Biol Chem. 2000; 275: 5179–5187.[Abstract/Free Full Text]
    
55. Fleming I, Fisslthaler B, Dimmeler S, et al. Phosphorylation of Thr(495) regulates Ca(2+)/calmodulin-dependent endothelial nitric oxide synthase activity. Circ Res. 2001; 88: E68–E75.[CrossRef][Medline] [Order article via Infotrieve]
    
56. Boo YC, Hwang J, Sykes M, et al. Shear stress stimulates phosphorylation of eNOS at Ser(635) by a protein kinase A-dependent mechanism. Am J Physiol Heart Circ Physiol. 2002; 283: H1819–H1828.[Abstract/Free Full Text]
    
57. Boo YC, Sorescu G, Boyd N, et al. Shear stress stimulates phosphorylation of endothelial nitric-oxide synthase at Ser1179 by Akt-independent mechanisms: role of protein kinase A. J Biol Chem. 2002; 277: 3388–3396.[Abstract/Free Full Text]
    
58. Scherer-Oppliger T, Leimbacher W, Blau N, et al. Serine 19 of human 6-pyruvoyltetrahydropterin synthase is phosphorylated by cGMP protein kinase II. J Biol Chem. 1999; 274: 31341–31348.[Abstract/Free Full Text]
    
59. Gohla A, Schultz G, Offermanns S. Role for G(12)/G(13) in agonist-induced vascular smooth muscle cell contraction. Circ Res. 2000; 87: 221–227.[Abstract/Free Full Text]
    
60. Pfitzer G. Invited review: regulation of myosin phosphorylation in smooth muscle. J Appl Physiol. 2001; 91: 497–503.[Abstract/Free Full Text]
    
61. Feil R, Gappa N, Rutz M, et al. Functional reconstitution of vascular smooth muscle cells with cGMP-dependent protein kinase I isoforms. Circ Res. 2002; 90: 1080–1086.[Abstract/Free Full Text]
    
62. Lalli MJ, Shimizu S, Sutliff RL, et al. [Ca2+]i homeostasis and cyclic nucleotide relaxation in aorta of phospholamban-deficient mice. Am J Physiol. 1999; 277: H963–H970.[Medline] [Order article via Infotrieve]
    
63. Sauzeau V, Le Jeune H, Cario-Toumaniantz C, et al. Cyclic GMP-dependent protein kinase signaling pathway inhibits RhoA-induced Ca2+ sensitization of contraction in vascular smooth muscle. J Biol Chem. 2000; 275: 21722–21729.[Abstract/Free Full Text]
    
64. Begum N, Sandu OA, Ito M, et al. Active Rho kinase (ROK-alpha) associates with insulin receptor substrate-1 and inhibits insulin signaling in vascular smooth muscle cells. J Biol Chem. 2002; 277: 6214–6222.[Abstract/Free Full Text]
    
65. Uehata M, Ishizaki T, Satoh H, et al. Calcium sensitization of smooth muscle mediated by a Rho-associated protein kinase in hypertension. Nature. 1997; 389: 990–994.[CrossRef][Medline] [Order article via Infotrieve]
    
66. Sawada N, Itoh H, Ueyama K, et al. Inhibition of rho-associated kinase results in suppression of neointimal formation of balloon-injured arteries. Circulation. 2000; 101: 2030–2033.[Abstract/Free Full Text]
    
67. Han X, Kubota I, Feron O, et al. Muscarinic cholinergic regulation of cardiac myocyte ICa-L is absent in mice with targeted disruption of endothelial nitric oxide synthase. Proc Natl Acad Sci U S A. 1998; 95: 6510–6515.[Abstract/Free Full Text]
    
68. Vandecasteele G, Eschenhagen T, Scholz H, et al. Muscarinic and beta-adrenergic regulation of heart rate, force of contraction and calcium current is preserved in mice lacking endothelial nitric oxide synthase. Nat Med. 1999; 5: 331–334.[CrossRef][Medline] [Order article via Infotrieve]
    
69. Godecke A, Heinicke T, Kamkin A, et al. Inotropic response to beta-adrenergic receptor stimulation and anti-adrenergic effect of ACh in endothelial NO synthase-deficient mouse hearts. J Physiol. 2001; 532: 195–204.[Abstract/Free Full Text]
    
70. Wegener JW, Nawrath H, Wolfsgruber W, et al. cGMP-dependent protein kinase I mediates the negative inotropic effect of cGMP in the murine myocardium. Circ Res. 2002; 90: 18–20.[Abstract/Free Full Text]
    
71. Monks D, Lange V, Silber RE, et al. Expression of cGMP-dependent protein kinase I and its substrate VASP in neointimal cells of the injured rat carotid artery. Eur J Clin Invest. 1998; 28: 416–423.[CrossRef][Medline] [Order article via Infotrieve]
    
72. Chiche JD, Schlutsmeyer SM, Bloch DB, et al. Adenovirus-mediated gene transfer of cGMP-dependent protein kinase increases the sensitivity of cultured vascular smooth muscle cells to the antiproliferative and pro-apoptotic effects of nitric oxide/cGMP. J Biol Chem. 1998; 273: 34263–34271.[Abstract/Free Full Text]
    
73. Jacob A, Molkentin JD, Smolenski A, et al. Insulin inhibits PDGF-directed VSMC migration via NO/cGMP increase of MKP-1 and its inactivation of MAPKs. Am J Physiol Cell Physiol. 2002; 283: C704–C713.[Abstract/Free Full Text]
    
74. Sinnaeve P, Chiche JD, Gillijns H, et al. Overexpr