cGMP Regulation Research Articles

Microarray Transfection Analysis of cAMP and cGMP Regulation of Gene Expression

Transcription factor activation by phosphorylation is a common mechanism for both developmental and hormonal control of gene expression. Over 2000 transcription factors and 600 protein kinases are predicted from the human genome sequence but only a small fraction of the potential regulatory interactions have been experimentally tested. We have defined transfection conditions to functionally assess these interactions in a microarray transfection format. The transfection method employed is termed Surface Transfection and Expression Protocol (STEP) and relies on the production of recombinant polyfunctional proteins to mediate the high efficiency in situ transfection of different cell types. Cis-acting transcriptional regulatory sequences driving the expression of GFP and other reporters were used to probe the activity of specific transcription factor families in the presence or absence of transfected protein kinases. We find that regulation of native promoters such as the c-fos promoter by cAMP- and cGMP-dependent protein kinases is controlled by conserved non-genic (CNG) sequences surrounding the c-fos gene. In particular, an evolutionarily conserved sequence within the first intron of the c-fos gene plays a key role in transcriptional regulation by these two protein kinases. The regulation by the CNG sequences was promoter specific in most cases. This microarray transfection approach should be useful in defining novel pathways by which protein phosphorylation regulates transcription factor activity as well as other functional interactions in vivo. This work was supported by NIH grants HG002367 and DK063340.

The impact of cGMP compliance on consumer confidence in dietary supplement products

The FDA estimates that US citizens spend more than $ 8.5 billion a year on dietary supplements and world wide the market is estimated at more than $ 60 billion. However, although a majority of consumers express confidence in the safety of these products, 74% believe the government should be more involved in ensuring that these products are safe and efficacious. Recent regulatory initiatives such as the imminent adoption of cGMPs for dietary supplements in the US, implementation of cGMPs in Canada and the recent EU dietary supplement initiative represent legislative and industry response to public clamor for more comprehensive oversight of dietary supplements. Regardless of mandated practices, the majority of dietary supplement manufacturers have done an excellent job of protecting the safety and quality of their products. The promulgation of these cGMPs will help ensure consumers that equal standards are followed throughout the industry. For some companies with established processes based on existing food or pharmaceutical cGMP regulations, the transition will be relatively painless while, for many, it will represent a significant increase in the level of documentation and testing. However, consumers deserve and demand that products meet standards for safety and quality and the implementation of cGMPs for these products are an important first step. Although the cGMPs are designed to ensure products are safe from a standpoint of identity, purity, quality, strength and composition, they do not address preclinical or clinical testing of ingredients for safety or efficacy. This would involve ingredients meeting the requirements of Generally Recognized as Safe (GRAS) status or going through the New Dietary Ingredient (NDI) process.

Cyclic GMP-dependent Protein Kinase Regulates CCAAT Enhancer-binding Protein β Functions through Inhibition of Glycogen Synthase Kinase-3

The CCAAT enhancer-binding protein (C/EBPbeta) plays an important role in the regulation of gene expression during cell proliferation, differentiation, and apoptosis. We previously showed that C/EBPbeta participates in cGMP-regulated transcription of c-fos in osteoblasts (Chen, Y., Zhuang, S., Cassenaer, S., Casteel, D. E., Gudi, T., Boss, G. R., and Pilz, R. B. (2003) Mol. Cell. Biol. 23, 4066-4082). In the present work, we show that cGMP/cGMP-dependent protein kinase (PKG) induced dephosphorylation and activation of C/EBPbeta by inhibiting glycogen synthase kinase-3beta (GSK-3beta). Phosphorylation of GSK-3beta on Ser9 negatively regulates the enzyme activity, and we found that PKG phosphorylated this site both in vitro and in vivo; the in vivo phosphorylation occurred rapidly and preceded C/EBPbeta dephosphorylation. Previous studies with GSK-3 inhibitors suggest that GSK-3beta is a C/EBPbeta kinase in resting cells. We determined that GSK-3beta phosphorylated C/EBPbeta in vitro on Thr189, Ser185, Ser181, and Ser177; C/EBPbeta was phosphorylated on these same sites in intact, unstimulated osteoblasts, and phosphorylation was decreased in cGMP-treated cells. Mutation of the GSK-3 phosphorylation sites in C/EBPbeta prevented C/EBPbeta phosphorylation in resting cells, enhanced C/EBPbeta DNA binding, and led to increased target gene transactivation, mimicking the stimulatory effects of cGMP on C/EBPbeta. cGMP regulation of C/EBPbeta was disrupted by a mutant GSK-3beta(Ala9) resistant to cGMP/PKG phosphorylation and inhibition. We conclude that cGMP increases the DNA binding potential of C/EBPbeta by preventing the negative effects of GSK-3 phosphorylation.

NO responsiveness in pulmonary artery and airway smooth muscle: the role of cGMP regulation

The purpose of this study was to assess intrinsic smooth muscle mechanisms contributing to greater nitric oxide (NO) responsiveness in pulmonary vascular vs. airway smooth muscle. Canine pulmonary artery smooth muscle (PASM) and tracheal smooth muscle (TSM) strips were used to perform concentration response studies to an NO donor, (Z)-1-[N-(2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1-ium-1,2-diolate (DETA-NO). PASM exhibited a greater NO responsiveness whether PASM and TSM were contracted with receptor agonists, phenylephrine and acetylcholine, respectively, or with KCl. The >10-fold difference in NO sensitivity in PASM was observed with both submaximal and maximal contractions. This difference in NO responsiveness was not due to differences in endothelial or epithelial barriers, since these were removed, nor was it due to the presence of cGMP-independent NO-mediated relaxation in either tissue. At equal concentrations of NO, the intracellular cGMP concentration ([cGMP]i) was also greater in PASM than in TSM. Phosphodiesterase (PDE) inhibition using isobutylmethylxanthine indicated that the greater [cGMP]i in PASM was not due to greater PDE activity in TSM. Expression of soluble guanylate cyclase (sGC) subunit mRNA (2 +/- 0.2 and 1.3 +/- 0.2 attomol/microg total RNA, respectively) and protein (47.4 +/- 2 and 27.8 +/- 3.9 ng/mg soluble homogenate protein, respectively) was greater in PASM than in TSM. sGCalpha1 and sGCbeta1 mRNA expression was equal in PASM but was significantly different in TSM, suggesting independent regulation of their expression. An intrinsic smooth muscle mechanism accounting for greater NO responsiveness in PASM vs. TSM is greater sGC activity.

Structural and Functional Features in Human PDE5A1 Regulatory Domain That Provide for Allosteric cGMP Binding, Dimerization, and Regulation

The cGMP-binding cGMP-specific phosphodiesterase (PDE5) contains a catalytic domain that hydrolyzes cGMP and a regulatory (R) domain that contains two GAFs (a and b; GAF is derived from the proteins mammalian cGMP-binding PDEs, Anabaena adenylyl cyclases, and Escherichia coli (FhlA)). The R domain binds cGMP allosterically, provides for dimerization, and is phosphorylated at a site regulated by allosteric cGMP binding. Quaternary structures and cGMP-binding properties of 10 human PDE5A1 constructs containing one or both GAFs were characterized. Results reveal that: 1) high affinity homo-dimerization occurs between GAF a modules (K(D) < 30 nM) and between GAF b modules (K(D) = 1-20 pM), and the sequence between the GAFs (Thr322-Asp403) contributes to dimer stability; 2) 176 amino acids (Val156-Gln331) in GAF a are adequate for cGMP binding; 3) GAF a has higher affinity for cGMP (K(D) < 40 nM) than does the isolated R domain (K(D) = 110 nM) or holoenzyme (K(D) = 200 nM), suggesting that the sequence containing GAF b and its flanking amino acids autoinhibits GAF a cGMP-binding affinity in intact R domain; 4) a mutant (Met1-Glu321) containing only GAF a has high affinity, biphasic cGMP-binding kinetics consistent with structural heterogeneity of GAF a, suggesting that the presence of GAF b is not required for biphasic cGMP-dissociation kinetics observed in holoenzyme or isolated R domain; 5) significant cGMP binding by GAF b was not detected; and 6) the sequence containing GAF b and its flanking amino acids is critical for cGMP stimulation of Ser102 phosphorylation by cyclic nucleotide-dependent protein kinases. Results yield new insights into PDE5 functions, further define boundaries that provide for allosteric cGMP binding, and identify regions that contribute to dimerization.

Role of cyclic GMP in gene regulation

Cyclic GMP is produced in response to nitric oxide and natriuretic peptides; cGMP is a key regulator of cell proliferation, differentiation, and apoptosis, and plays an important role in many (patho)physiological processes such as synaptic plasticity, angiogenesis, inflammation, and cardiac hypertrophy. The regulation of gene expression by cGMP has been recognized relatively recently, but cGMP-mediated increases or decreases in the mRNA expression of >60 different genes have been described, and gene expression profiling is just beginning to contribute to the growing list of cGMP-regulated genes. Deletion or over-expression experiments in mice involving components of the cGMP signaling pathway have contributed to our understanding of long-term effects of altered cGMP signaling, including the regulation of gene expression. We will discuss transcriptional and post-transcriptional mechanisms of gene regulation by cGMP, and review specific transcription factors and RNA binding proteins targeted by cGMP. Some of the effects of cGMP on gene expression are indirect, through cGMP modulation of other signaling pathways, e. g. mitogen-activated protein kinase pathways. However, some effects of cGMP can be directly attributed to cGMP regulation of specific transcription factors such as CREB, TFII-I or c-Fos, and are mediated by cGMP-dependent protein kinases. We will discuss specific genes regulated by cGMP in the context of their contribution to particular (patho)physiologic processes regulated by cGMP.

CD86 and CD54 Co-Expression on VSV-G Pseudotyped HIV-1 Based Vectors Improves Transduction and Activation of Human Primary CD4+ T Lymphocytes.

We established the first clinical ex vivo HIV-based vector gene therapy trial in humans with HIV+ CD4+ T-cells. Briefly, this therapy involves modifying patient CD4+ T-cells with our modified lentiviral vector carrying an anti-HIV payload. These cells are then activated and expanded, and re-infused back into the patient. However, cGMP regulations require the use of costly clinical grade reagents (i.e. Retronectin™, CD3/CD28 stimulating paramagnetic beads). In an attempt to reduce ex-vivo processing costs, but not at the expense of transduction levels, we sought to determine a way to directly activate CD4+ T-cells with modified lentiviral vectors. 293FT HEK cell lines, used for producing our lentiviral vectors, were modified to co-express the natural CD28 stimulatory ligand B7.2 (CD86) and ICAM-1 (CD54) proteins on their membrane for co-stimulation and anchoring purposes. When CliniMACS purified normal donor CD4+ T cells were co-cultured with CD54/CD86-expressing cells, in the presence of soluble OKT3 CD3 antibody, CD25 and CD69 activation markers were upregulated, indicating that functional proteins were being expressed at the cell membrane. These CD54 and/or CD86 expressing cells could subsequently be transfected with lentiviral vector plasmid constructs in order to produce host-derived CD54 and/or CD86 bearing HIV-based vectors. EGFP-expressing lentiviral vectors, VRX494, with CD54/CD86-modified envelopes were produced both in these cell lines and by transient transfection of all relevant plasmids, and titers were assayed on Hela-Tat cells by FACS. CD54 modified lentiviral vectors showed increased binding to CD4+ T-cells, as evidenced by significant cell clumping. CD86 (as well as CD54 plus CD86) modified lentiviral vector, with soluble OKT3 CD3 antibody, was shown to activate T-cells, above the levels seen with unmodified lentiviral vectors, as evidenced by the increase in cell surface CD25 and CD69 expression and also the increase in cell size. Cellular expansion of modified lentiviral vector transduced CD4+ T cells reached levels close to CD3:CD28 bead stimulated CD4+ T cell controls over a period of 2 to 3 weeks. The CD3/TCR repertoire was assessed by flow cytometry and, compared to the well-established CD3/CD28 coated M450 Dynabeads stimulatory system as a control, no skewing of the repertoire was observed. CD86 was shown to improve levels of transduction in pre-activated lymphocytes with CD3/CD28 coated M450 Dynabeads. However, CD86 co-expression was crucial for transducing minimally activated CD4+ T cells with only soluble OKT3 CD3 antibody. Levels of transduction and activation were on average 2 to 3 times higher with the modified lentiviral vectors. To our knowledge, we are reporting the first generation of lentiviral particles exhibiting an adhesion property with stimulatory abilities. The development of such a lentiviral vector has valuable implications for clinical application by reducing the number of exogenous reagents in large scale cell processing.

SO MANY INSECTS, SO MANY SIGNALS

Edited by P. D. Evans London: Academic Press (2002) 428p. ISBN 012024229X £89.95 hardback. ![Figure][1] Reviews published in Advances in Insect Physiology are typically comprehensive treatments of an area by renowned experts in the field, and volume 29 is no

Guanylyl cyclases, nitric oxide, natriuretic peptides, and airway smooth muscle function.

Airway smooth muscle (ASM) plays an important role in asthma pathophysiology through its contractile and proliferative functions. The cyclic nucleotides adenosine 3',5'-cyclic monophosphate (cAMP) and guanosine 3',5'-cyclic monophosphate (cGMP) are second messengers capable of mediating the effects of a variety of drugs and hormones. There is a large body of evidence to support the hypothesis that cAMP is a mediator of the ASM's relaxant effects of drugs, such as beta2-adrenoceptor agonists, in human airways. Although most attention has been paid to this second messenger and the signal transduction pathways it activates, recent evidence suggests that cGMP is also an important second messenger in ASM with important relaxant and antiproliferative effects. Here, we review the regulation and function of cGMP in ASM and discuss the implications for asthma pathophysiology and therapeutics. Recent studies suggest that activators of soluble and particulate guanylyl cyclases, such as nitric oxide donors and natriuretic peptides, have both relaxant and antiproliferative effects that are mediated through cGMP-dependent and cGMP-independent pathways. Abnormalities in these pathways may contribute to asthma pathophysiology, and therapeutic manipulation may complement the effects of beta2-adrenoceptor agonists.

Capsaicin-sensitive sensory neurons regulate myocardial nitric oxide and cGMP signaling

We studied whether tissue levels of nitric oxide (NO) and cGMP are regulated by sensory nerves in normoxic and ischemic hearts. Wistar rats were treated with capsaicin to deplete neurotransmitters from capsaicin-sensitive sensory nerves. In separate experiments, capsaicin was applied perineurally to both vagus nerves for selective chemodenervation of vagal cardiac afferent nerves. Systemic capsaicin administration significantly decreased basal myocardial NO content assessed by electron spin resonance (ESR) spectroscopy, whereas, local treatment of vagus nerves did not change it. Both systemic and local capsaicin treatment decreased cardiac cGMP content measured by radioimmunoassay. In separate experiments, isolated hearts from control and systemic capsaicin-treated rats were subjected to 30-min global ischemia. NO signal intensity increased 10-fold after ischemia, whereas, cardiac cGMP decreased. Capsaicin pretreatment did not influence ischemic NO or cGMP content. These results suggest a major role for capsaicin-sensitive sensory neurons in the maintenance of basal but not ischemic myocardial NO and cGMP content. Vagal sensory nerves may be involved in the regulation of basal myocardial cGMP but not basal NO level. Consequently, basal NO content in the heart is regulated primarily by spinal afferent nerves.

Expression and regulation of CGMP degrading phosphodiesterases in the kidney of DAHL rats

Expression and regulation of CGMP degrading phosphodiesterases in the kidney of DAHL rats

CGMP regulation of phosphodiesterases: structure and function

cGMP regulation of phosphodiesterases: structure and function

Inhibition of cyclic GMP hydrolysis with zaprinast reduces basal and cyclic AMP-elevated L-type calcium current in guinea-pig ventricular myocytes.

(1) Cyclic GMP (cGMP) has been shown to be an important modulator of cardiac contractile function. A major component of cGMP regulation of contractility is cGMP-mediated inhibition of the cardiac calcium current (I(Ca)). An under-appreciated aspect of cyclic nucleotide signalling is hydrolysis of the cyclic nucleotide (i.e., breakdown by phosphodiesterases (PDEs)). The role of cGMP hydrolysis in regulating I(Ca) has not been studied. Thus the purpose of this study was to investigate if inhibition of cGMP hydrolysis can modulate I(Ca) in isolated guinea-pig ventricular myocytes. (2) Zaprinast, a selective inhibitor of cGMP-specific PDE (PDE5), caused a significant increase in cGMP levels in myocytes, but was without affect on basal or beta-adrenergic stimulated cAMP levels (consistent with its actions as a specific inhibitor of PDE5). (3) Zaprinast inhibited I(Ca) that was pre-stimulated with cAMP elevating agents (isoproterenol, a beta-adrenergic agonist; or forskolin, a direct activator of adenylate cyclase). The effect of zaprinast was greatly reduced by KT5823, an inhibitor of cGMP-dependent protein kinase (PKG). (4) Zaprinast also significantly inhibited basal I(Ca) when perforated-patch or whole-cell recording with physiological pipette calcium concentration (10(-7) M) was used. However, this effect was not observed when using standard calcium-free whole-cell recording conditions. (5) These results indicate that inhibition of cGMP hydrolysis can decrease both basal and cAMP-stimulated I(Ca). Thus, cGMP hydrolysis may likely be an important step for physiological modulation of I(Ca). This regulation may also be important in disease states in which cGMP production is increased and PDE5 expression is altered, such as heart failure.

SCHERING-PLOUGH PAYS RECORD FINE

SCHERING-PLOUGH WILL PAY a $500 million fine—the largest penalty ever—to the Food & Drug Administration for violating current Good Manufacturing Practice (cGMP) regulations. The company has signed ...

Impact of current good manufacturing practices and emission regulations and guidances on the discharge of pharmaceutical chemicals into the environment from manufacturing, use, and disposal.

The current Good Manufacturing Practice (cGMP) and effluent emission (use and disposal) regulations of the U.S. Food and Drug Administration (FDA) and manufacturing effluent discharge and emission regulations of the U.S. Environmental Protection Agency (U.S. EPA) require contained manufacture, use, and disposal of pharmaceuticals with the goal of minimizing the release of pharmaceutical chemicals into the environment. However, debate has recently arisen in several scientific forums over whether these regulations adequately protect human and environmental health from the new pharmaceutical drugs introduced each year into the marketplace and the multitude of existing products, each with many distinct biochemical modes of actions. To address this issue, it is important to understand the relevance of current cGMP regulations and emission regulations that have a direct bearing on the releases of pharmaceutical chemicals into the environment during the manufacture, use, and disposal of active pharmaceutical ingredients (drug substances) and drug products. This knowledge may help us assess the quantity of residues that may be released into the environment. Additionally, the information on physical, chemical, and degradation and sorption properties of the pharmaceutical chemicals may help determine the net residue levels that could persist in the environment to evaluate if such residues have any bearing on human and environmental health. The scientific and regulatory aspects of issues related to the manufacture, use, and disposal of pharmaceutical chemicals are discussed in this article, with special emphasis on potential environmental exposure pathways during the life cycle of an active pharmaceutical ingredient or drug product. The mechanisms of degradation (transformation or depletion) and dilution of pharmaceutical residues that may be released into aquatic or terrestrial environmental compartments are described. Such degradation and dilution of pharmaceutical chemicals in the environment may significantly reduce the residues. It is important to evaluate whether such residue levels have any measurable impact on human and/or environmental health.

Cardiac phosphodiesterase 5 (cGMP-specific) modulates beta-adrenergic signaling in vivo and is down-regulated in heart failure.

Recent studies implicate increased cGMP synthesis as a postreceptor contributor to reduced cardiac sympathetic responsiveness. Here we provide the first evidence that modulation of this interaction by cGMP-specific phosphodiesterase PDE5A is also diminished in failing hearts, providing a novel mechanism for blunted beta-adrenergic signaling in this disorder. In normal conscious dogs chronically instrumented for left ventricular pressure-dimension analysis, PDE5A inhibition by EMD82639 had modest basal effects but markedly blunted dobutamine-enhanced systolic and diastolic function. In failing hearts (tachypacing model), however, EMD82639 had negligible effects on either basal or dobutamine-stimulated function. Whole myocardium from failing hearts had 50% lower PDE5A protein expression and 30% less total and EMD92639-inhibitable cGMP-PDE activity. Although corresponding myocyte protein and enzyme activity was similar among groups, the proportion of EMD82639-inhibitable activity was significantly lower in failure cells. Immunohistochemistry confirmed PDE5A expression in both the vasculature and myocytes of normal and failing hearts, but there was loss of z-band localization in failing myocytes that suggested altered intracellular localization. Thus, PDE5A regulation of cGMP in the heart can potently modulate beta-adrenergic stimulation, and alterations in enzyme localization and reduced synthesis may blunt this pathway in cardiac failure, contributing to dampening of the beta-adrenergic response.

E3KARP (NHE3 kinase a anchoring protein) is necessary for cGMP regulation of NHE3: Demonstration of a plasma membrane signaling complex containing NHE3, E3KARP, and cGMP kinase II

E3KARP (NHE3 kinase a anchoring protein) is necessary for cGMP regulation of NHE3: Demonstration of a plasma membrane signaling complex containing NHE3, E3KARP, and cGMP kinase II

E3KARP (NHE3 kinase a anchoring protein) is necessary for cGMP regulation of NHE3: Demonstration of a plasma membrane signaling complex containing NHE3, E3KARP, and cGMP kinase II

E3KARP (NHE3 kinase a anchoring protein) is necessary for cGMP regulation of NHE3: Demonstration of a plasma membrane signaling complex containing NHE3, E3KARP, and cGMP kinase II

CGMP-dependent protein kinase mediates stimulation of L-type calcium current by cGMP in rabbit atrial cells.

cGMP has been shown to exert both stimulatory and inhibitory effects on cardiac L-type calcium current (I(Ca)). The physiological role of cGMP in regulation of cardiac activity is still controversial. cGMP may be of importance in regulation of I(Ca) in atrial cells. The present study was focused on the role of cGMP in the modulation of I(Ca) in rabbit atrial cells. Enzymatically isolated adult rabbit atrial cells were used to measure I(Ca) using whole cell voltage clamp. Expressed levels of cGMP-dependent protein kinase (PKG) were determined by Western blotting using PKG specific antibody in homogenates from atrial and ventricular cells. Nitrosoglutathione (GSNO), a nitric oxide donor that stimulates soluble guanylyl-cyclase to elevate cGMP levels increased I(Ca) while soluble G-cyclase inhibitors, ODQ or methylene blue inhibited I(Ca). Intracellular application of 8BrcGMP increased I(Ca) and blocked the inhibitory effect of methylene blue. KT-5823, an inhibitor of PKG inhibited I(Ca) and the stimulatory effect of GSNO was completely blocked ODQ or KT-5823. Inhibition of cAMP dependent protein kinase (PKA) by the 6-22 peptide completely blocked the stimulation of I(Ca) by the beta-agonist isoproterenol but not by GSNO. The potency of isoproterenol to stimulate I(Ca) was very high for atrial cells (EC(50) 2.4+/-0.6 nM) and only 100 nM isoproterenol was required to stimulate I(Ca) maximally (21.4+/-0.7 pA/pF) to a level (23.8+/-1.6 pA/pF) achieved with the inclusion of 100 microM cAMP in the pipette solution. GSNO produced an additive effect on I(Ca) already stimulated by either 10 microM isobutylmethylxanthine (phosphodiesterase inhibitor) or a low concentration (1 nM) isoproterenol but failed to produce any effect on I(Ca) maximally stimulated by 100 nM isoproterenol. Inhibition of PKG by KT-5823 significantly decreased the efficacy of isoproterenol and the maximal I(Ca) achieved with 100 nM isoproterenol was decreased to 8.2+/-0.6 pA/pF in the presence of KT-5823. Western blot analysis showed much higher expression of PKG in atrial cells compared to ventricular cells. These findings suggest that stimulatory effects of cGMP on I(Ca) in rabbit atrial cells are likely to be mediated via PKG dependent phosphorylation of calcium channels or associated proteins and that the effects of cGMP are not antagonistic to cAMP. PKG is highly expressed in atrial cells and PKG dependent phosphorylation may be necessary for maintaining basal I(Ca) and fully stimulating I(Ca) by beta-adrenergic activation in atrial cells.

Modulation of mouse cardiac function in vivo by eNOS and ANP.

To study the role of endothelial nitric oxide synthase (eNOS) in cardiac function, we compared eNOS expression, contractility, and relaxation in the left ventricles of wild-type and eNOS-deficient mice. eNOS immunostaining is localized to the macro- and microvascular endothelium throughout the myocardium in wild-type mice and is absent in eNOS-/- mice. Whereas blood pressure is elevated in eNOS-/- mice, baseline cardiac contractility (dP/dt(max)) is similar in wild-type and eNOS-/- mice (9,673 +/- 2, 447 and 9,928 +/- 1,566 mmHg/s, respectively). The beta-adrenergic agonist isoproterenol (Iso) at doses of >/=1 ng causes enhanced increases in dP/dt(max) in eNOS-/- mice compared with wild-type controls in vivo (P < 0.01) as well as in Langendorff isolated heart preparations (P < 0.02). beta-Adrenergic receptor binding (B(max)) is not significantly different in the two groups of animals (B(max) = 41.4 +/- 9.4 and 36.1 +/- 5.1 fmol/mg for wild-type and eNOS-/-). Iso-stimulated ventricular relaxation is also enhanced in the eNOS-/- mice, as measured by dP/dt(min) in the isolated heart. However, baseline ventricular relaxation is normal in eNOS-/- mice (tau = 5.2 +/- 1.0 and 5.6 +/- 1.5 ms for wild-type and eNOS-/-, respectively), whereas it is impaired in wild-type mice after NOS inhibition (tau = 8.3 +/- 2.4 ms). cGMP levels in the left ventricle are unaffected by eNOS gene deletion (wild-type: 3.1 +/- 0.8 pmol/mg, eNOS-/-: 3.1 +/- 0.6 pmol/mg), leading us to examine the level of another physiological regulator of cGMP. Atrial natriuretic peptide (ANP) expression is markedly upregulated in the eNOS-/- mice, and exogenous ANP restores ventricular relaxation in wild-type mice treated with NOS inhibitors. These results suggest that eNOS attenuates both inotropic and lusitropic responses to beta-adrenergic stimulation, and it also appears to regulate baseline ventricular relaxation in conjunction with ANP.