Differential effects of cGMP produced by soluble and particulate guanylyl cyclase on mouse ventricular myocytes

Particulate guanylyl cyclase (pGC) and soluble guanylyl cyclase (sGC) are cGMP-generation systems distributed in different intracellular locations. Our aim was to test the hypothesis that the functional effects of cGMP produced by pGC and sGC on contraction and Ca2+ transients would differ in ventricular myocytes. We measured myocyte shortening from adult mice using a video edge-detector and investigated the functional changes after stimulating pGC with C-type natriuretic peptide (CNP; 10(-8) M and 10(-7) M) or sGC with S-nitroso-N-acetyl-penicillamine (SNAP; nitric oxide donor; 10(-6) M and 10(-5) M). Significant concentration-dependent decreases in percentage shortening (PCS), maximal rate of shortening (RSmax), and relaxation (RRmax) were produced by CNP. To a similar degree, SNAP concentration-dependently reduced PCS, RSmax, and RRmax. The addition of Rp-8-[(4-chlorophenyl)thio]-cGMPS triethylamine (cGMP-dependent protein kinase inhibitor; 5 x 10(-6) M) or erythro-9-(2-hydroxy-3-nonyl) adenine (cGMP-stimulated cAMP phosphodiesterase inhibitor; 10(-5) M) reduced the responses induced by CNP or SNAP, suggesting that their actions were through cGMP-mediated pathways. While SNAP significantly increased intracellular cGMP concentration by 57%, CNP had little effect on cGMP production. We also found that CNP markedly decreased the amplitude of Ca2+ transients while SNAP had little effect, suggesting the cGMP generated by sGC may decrease myofilament Ca2+ sensitivity. The small amount of cGMP generated by pGC had a major effect in reducing Ca2+ level. This study suggested the existence of compartmentalization for cGMP in ventricular myocytes.     

Alterations in nitric oxide-cGMP pathway in ventricular myocytes from obese leptin-deficient mice

Leptin is a regulator of body weight and affects nitric oxide (NO) production. This study was designed to determine whether the myocardial NO-cGMP signal transduction system was altered in leptin-deficient obese mice. Contractile function, guanylyl cyclase activity, and cGMP-dependent protein phosphorylation were assessed in ventricular myocytes isolated from genetically obese (B6.V-Lepob) and age-matched lean (C57BL/6J) mice. There were no differences in baseline contraction between the lean and obese groups. After stimulation with the NO donor S-nitroso-N-acetyl-penicillamine (SNAP, 10-6 and 10-5 M) or a membrane-permeable cGMP analog 8-bromo-cGMP (8-Br-cGMP, 10(-6) and 10(-5) M), cell contractility was depressed. However, 8-Br-cGMP had significantly greater effects in obese mice than in lean controls with percent shortening reduced by 47 vs. 39% and maximal rate of shortening decreased by 46 vs. 36%. The negative effects of SNAP were similar between the two groups. Soluble guanylyl cyclase activity was not attenuated. This suggests that the activity of the cGMP-independent NO pathway may be enhanced in obesity. The phosphorylated protein profile of cGMP-dependent protein kinase showed that four proteins were more intensively phosphorylated in obese mice, which suggests an explanation for the enhanced effect of cGMP. These results indicate that the NO-cGMP signaling pathway was significantly altered in ventricular myocytes from the leptin-deficient obese mouse model.     

Role of phospholamban in cyclic GMP mediated signaling in cardiac myocytes.

We tested the hypothesis that the negative functional effects of cyclic GMP on cardiac myocytes were mediated through phospholamban (PLB) and activation of sarcoplasmic reticulum Ca(2+)-ATPase. Using ventricular myocytes from wild type (WT, n=10) and PLB knockout (PLB-KO, n=10) mouse hearts, functional changes were measured using a video edge detector at baseline and after 10(-6), 10(-5)M 8-bromo-cyclic GMP (cGMP), 10(-8), 10(-7)M C-type natriuretic peptide (CNP), or 10(-6), 10(-5)M S-nitroso-N-acetyl-penicillamine (SNAP, nitric oxide donor). Changes in cytosolic Ca(2+) concentration were assessed in fura 2-loaded WT and PLB-KO myocytes. Cyclic GMP dependent phosphorylation analysis was also performed in WT and PLB-KO myocytes. 8-bromo-cGMP 10(-5)M caused a significant decrease in %shortening (3.6+/-0.2% to 2.3+/-0.1%) in WT, but little change in PLB-KO myocytes (3.4+/-0.1% to 3.2+/-0.2%). Similarly, CNP and SNAP reduced %shortening of WT, but not PLB-KO myocyte. Changes in other contractile parameters such as maximum rate of shortening and relaxation were consistent with the changes in % shortening. Intracellular Ca(2+) transients changed similarly to cell contractility in WT and PLB-KO myocytes treated with cGMP and CNP; i.e. Ca(2+) transients decreased with cGMP or CNP in WT myocytes, but were unchanged in PLB-KO myocytes. cGMP dependent phosphorylation analysis showed that some proteins were phosphorylated by cGMP to a lesser extent in PLB-KO compared with WT myocytes, suggesting impaired cGMP-kinase function in PLB-KO cardiac myocytes. These results indicated that cGMP-induced reductions in cardiac myocyte function were at least partially mediated through the action of phospholamban.     

Nitric oxide attenuates endothelin-1-induced activation of ERK1/2, PKB, and Pyk2 in vascular smooth muscle cells by a cGMP-dependent pathway

Nitric oxide (NO), in addition to its vasodilator action, has also been shown to antagonize the mitogenic and hypertrophic responses of growth factors and vasoactive peptides such as endothelin-1 (ET-1) in vascular smooth muscle cells (VSMCs). However, the mechanism by which NO exerts its antimitogenic and antihypertrophic effect remains unknown. Therefore, the aim of this study was to determine whether NO generation would modify ET-1-induced signaling pathways involved in cellular growth, proliferation, and hypertrophy in A-10 VSMCs. Treatment of A-10 VSMCs with S-nitroso-N-acetylpenicillamine (SNAP) or sodium nitroprusside (SNP), two NO donors, attenuated the ET-1-enhanced phosphorylation of several key components of growth-promoting and hypertrophic signaling pathways such as ERK1/2, PKB, and Pyk2. On the other hand, inhibition of the endogenous NO generation with N(G)-nitro-L-arginine methyl ester, a nitric oxide synthase inhibitor, increased the ET-1-induced phosphorylation of these signaling components. Since NO mediates its effect principally through a cGMP-soluble guanylyl cyclase (sGC) pathway, we investigated the role of these molecules in NO action. 8-Bromoguanosine 3',5'-cyclic monophosphate, a nonmetabolizable and cell-permeant analog of cGMP, exhibited a effect similar to that of SNAP and SNP. Furthermore, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), an inhibitor of sGC, reversed the inhibitory effect of NO on ET-1-induced responses. SNAP treatment also decreased the protein synthesis induced by ET-1. Together, these data demonstrate that NO, in a cGMP-dependent manner, attenuated ET-1-induced phosphorylation of ERK1/2, PKB, and Pyk2 and also antagonized the hypertrophic effects of ET-1. It may be suggested that NO-induced generation of cGMP contributes to the inhibition of ET-1-induced mitogenic and hypertrophic responses in VSMCs.     

Nitric-oxide-dependent activation of pig oocytes: the role of the cGMP-signalling pathway

Pig oocytes matured in vitro were parthenogenetically activated (78%) after treatment with 2 mM nitric oxide-donor (+/-)-S-nitroso-N-acetylpenicillamine (SNAP) for 24 h. Inhibition of soluble guanylyl cyclase with the specific inhibitors 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) or 6-anilino-5,8-quinolinequinone (LY83583) suppressed the SNAP-induced activation in a dose-dependent manner (23% of activated oocytes after treatment with 400 microM ODQ; 12% of activated oocytes after treatment with 40 microM LY83583). 8-Bromo-cyclic guanosine monophosphate (8-Br-cGMP), a phosphodiesterase-resistant analogue of cGMP, enhances the effect of suboptimal doses (0.1 or 0.5 mM) of the NO donor SNAP. DT3, a specific inhibitor of cGMP-dependent protein kinase (PKG, PKG), is also able to inhibit the activation of pig oocytes after NO donor treatment. Involvement of the cGMP-dependent signalling pathway is specific for NO-induced oocyte activation, because both the guanylyl cyclase inhibitor ODQ and the PKG inhibitor DT3 are unable to inhibit activation in oocytes treated with the calcium ionophore A23187. These data indicate that the activation of pig oocytes with an NO donor is cGMP-dependent and that PKG plays an important role in this mode of oocyte activation.     

Reduction in interaction between cGMP and cAMP in dog ventricular myocytes with hypertrophic failure

Baseline function and signal transduction are depressed in hearts with hypertrophic failure. We tested the hypothesis that the effects of cGMP and its interaction with cAMP would be reduced in cardiac myocytes from hypertrophic failing hearts. Ventricular myocytes were isolated from control dogs, dogs with aortic valve stenosis hypertrophy, and dogs with pacing hypertrophic failure. Myocyte function was measured using a video edge detector. Cell contraction data were obtained at baseline, with 8-bromo-cGMP (10(-7), 10(-6), and 10(-5) M), with erythro-9-(2-hydroxy-3-nonyl)adenine [EHNA; a cAMP phosphodiesterase (PDE(2)) inhibitor] plus 8-bromo-cGMP, or milrinone (a PDE(3) inhibitor) plus 8-bromo-cGMP. Baseline percent shortening and maximal rates of shortening (R(max)) and relaxation were slightly reduced in hypertrophic myocytes and were significantly lower in failing myocytes (R(max): control dogs, 95.3 +/- 17.3; hypertrophy dogs, 88.2 +/- 5.5; failure dogs, 53.2 +/- 6.4 mum/s). 8-Bromo-cGMP dose dependently reduced myocyte function in all groups. However, EHNA (10(-6) M) and milrinone (10(-6) M) significantly reduced the negative effects of cGMP on cell contractility in control and hypertrophy but not in failing myocytes (R(max) for control dogs: cGMP, -46%; +EHNA, -21%; +milrinone, -19%; for hypertrophy dogs: cGMP, -40%; +EHNA, -13%; +milrinone, -20%; for failure dogs: cGMP, -40%; +EHNA, -29%; +milrinone, -32%). Both combinations of EHNA-cGMP and milrinone-cGMP significantly increased intracellular cAMP in control, hypertrophic, and failing myocytes. These data indicated that the cGMP signaling pathway was preserved in hypertrophic failing cardiac myocytes. However, the interaction of cGMP with the cAMP signaling pathway was impaired in these failing myocytes.     

Chronic nitric oxide synthase blockade desensitizes the heart to the negative metabolic effects of nitric oxide

The consequences of chronic nitric oxide synthase (NOS) blockade on the myocardial metabolic and guanylyl cyclase stimulatory effects of exogenous nitric oxide (NO) were determined. Thirty-three anesthetized open-chest rabbits were randomized into four groups: control, NO donor S-nitroso-N-acetyl-penicillamine (SNAP, 10(-4 )M), NOS blocking agent N(G)-nitro-L-arginine methyl ester (L-NAME, 20 mg/kg/day) for 10 days followed by a 24 hour washout and L-NAME for 10 days followed by a 24 hour washout plus SNAP. Myocardial O(2) consumption was determined from coronary flow (microspheres) and O(2) extraction (microspectrophotometry). Cyclic GMP and guanylyl cyclase activity were determined by radioimmunoassay. There were no baseline metabolic, functional or hemodynamic differences between control and L-NAME treated rabbits. SNAP in controls caused a reduction in O(2) consumption (SNAP 5.9+/-0.6 vs. control 8.4+/-0.8 ml O(2)/min/100 g) and a rise in cyclic GMP (SNAP 18.3+/-3.8 vs. control 10.4+/-0.9 pmol/g). After chronic L-NAME treatment, SNAP caused no significant changes in O(2) consumption (SNAP 7.1+/-0.8 vs. control 6.4+/-0.7) or cyclic GMP (SNAP 14.2+/-1.8 vs. control 12.1+/-1.3). In controls, guanylyl cyclase activity was significantly stimulated by SNAP (216.7+/-20.0 SNAP vs. 34.4+/-2.5 pmol/mg/min base), while this increase was blunted after L-NAME (115.9+/-24.5 SNAP vs. 24.9+/-4.7 base). These results demonstrated that chronic NOS blockade followed by washout blunts the response to exogenous NO, with little effect on cyclic GMP or myocardial O(2) consumption. This was related to reduced guanylyl cyclase activity after chronic L-NAME. These results suggest that, unlike many receptor systems, the NO-cyclic GMP signal transduction system becomes downregulated upon chronic inhibition.     

Differential relaxing responses to particulate or soluble guanylyl cyclase activation on endothelial cells: a mechanism dependent on PKG-I alpha activation by NO/cGMP

cGMP is generated in endothelial cells after stimulation of soluble guanylyl cyclase (sGC) by nitric oxide (NO) or of particulate guanylyl cyclase (pGC) by natriuretic peptides (NP). We examined whether localized increases in cytosolic cGMP have distinct regulatory roles on the contraction induced by H₂O₂ treatment in human umbilical vein endothelial cells. cGMP concentrations and temporal dynamics were different upon NO stimulation of sGC or C-type NP (CNP) activation of pGC and did not correlate with their relaxing effects measured as planar cell surface area after H₂O₂ challenge. cGMP production due to sGC stimulation was always smaller and more brief than that induced by pGC stimulation with CNP, which was greater and remained elevated longer. The NO effects on cell relaxation were cGMP dependent because they were blocked by sGC inhibition with 1H-(1,2,4)Oxadiazolo(4,3-a)quinoxaline-1-one and mimicked by 8-Br-cGMP. An antagonist of the cGMP-dependent protein kinase type-I (PKG-I) also inhibited the NO-induced effects. The cell contraction induced by H₂O₂ produces myosin light chain (MLC) phosphorylation and NO prevented it completely, whereas CNP only produced a partial inhibition. Transfection with a dominant negative form of PKG type-I completely reversed the NO-induced effects on MLC phosphorylation, whereas it only partially inhibited the effects due to CNP. Taken together, these results demonstrate that the NO/sGC/cGMP pathway induces endothelial cell relaxation in a more efficient manner than does CNP/pGC/cGMP pathway, an effect that might be related to a selective stimulation of PKG-1 by NO-derived cGMP. Consequently, stimulated PKG-I may phosphorylate important protein targets that are necessary to inhibit the endothelial contractile machinery activated by oxidative stress. nitric oxide; C-type natriuretic peptide; myosin light chain; cGMP-dependent protein kinase type I; endothelial cell barrier dysfunction     

The alpha2beta1 isoform of guanylyl cyclase mediates plasma membrane localized nitric oxide signalling

Nitric oxide (NO) is a mediator of copious biological processes, in many cases through the production of cGMP from the enzyme nitric oxide-sensitive guanylyl cyclase. Natriuretic peptides also elevate cGMP, often with distinct biological effects, raising the issue of how specificity is achieved. Here we show that a recently described alpha(2)beta(1) isoform of guanylyl cyclase is expressed in a number of epithelia, where it is localized to the apical plasma membrane. We measured the functional properties of the alpha(2)beta(1) isoform by utilizing the NO-dependent activation of the ion channel cystic fibrosis transmembrane conductance regulator (CFTR), which occurs by phosphorylation via the membrane-bound type II isoform of cGMP-dependent protein kinase. We found that cGMP generated by NO activation of the alpha(2)beta(1) isoform of guanylyl cyclase is an exceptionally efficient mediator of nitric oxide action on membrane targets, activating CFTR far more effectively than the cytoplasmically located alpha(1)beta(1) guanylyl cyclase isoform. Targeting the alpha(1)beta(1) isoform of guanylyl cyclase to the membrane also dramatically enhanced the effects of nitric oxide on CFTR within the membrane. This was not due to increased enzymatic activity of guanylyl cyclase in a membrane location, but to production of a localised membrane pool of cGMP by membrane-localized NO-dependent guanylyl cyclase that was resistant to degradation by phosphodiesterases. Selective effects of cGMP produced from this enzyme in response to NO are directed at membrane targets and suggest that drugs selectively activating or inhibiting this alpha(2)beta(1) isoform of guanylyl cyclase may have unique pharmacological properties.     

Nitric oxide inhibits gastric acid secretion by increasing intraparietal cell levels of cGMP in isolated human gastric glands

We have previously identified cells containing the enzyme nitric oxide (NO) synthase (NOS) in the human gastric mucosa. Moreover, we have demonstrated that endogenous and exogenous NO has been shown to decrease histamine-stimulated acid secretion in isolated human gastric glands. The present investigation aimed to further determine whether this action of NO was mediated by the activation of guanylyl cyclase (GC) and subsequent production of cGMP. Isolated gastric glands were obtained after enzymatic digestion of biopsies taken from the oxyntic mucosa of healthy volunteers. Acid secretion was assessed by measuring [(14)C]aminopyrine accumulation, and the concentration of cGMP was determined by radioimmunoassay. In addition, immunohistochemistry was used to examine the localization of cGMP in mucosal preparations after stimulation with the NO donor S-nitroso-N-acetylpenicillamine (SNAP). SNAP (0.1 mM) was shown to decrease acid secretion stimulated by histamine (50 microM); this effect was accompanied by an increase in cGMP production, which was histologically localized to parietal cells. The membrane-permeable cGMP analog dibuturyl-cGMP (db-cGMP; 0.1-1 mM) dose dependently inhibited acid secretion. Additionally, the effect of SNAP was prevented by preincubating the glands with the GC inhibitor 4H-8-bromo-1,2,4-oxadiazolo[3,4-d]benz[b][1,4]oxazin-1-one (10 microM). We therefore suggest that NO in the human gastric mucosa is of physiological importance in regulating acid secretion. Furthermore, the results show that NO-induced inhibition of gastric acid secretion is a cGMP-dependent mechanism in the parietal cell involving the activation of GC.     

Exogenous NO triggers preconditioning via a cGMP- and mitoKATP-dependent mechanism

Exogenous nitric oxide (NO) triggers a preconditioning-like effect in heart via a pathway that is dependent on reactive oxygen species. This study examined the signaling pathway by which the NO donor S-nitroso-N-acetylpenicillamine (SNAP, 2 microM) triggers its anti-infarct effect. Isolated rabbit hearts experienced 30 min of regional ischemia and 120 min of subsequent reperfusion. Infarct size was determined by triphenyltetrazolium chloride staining. Infarct size was reduced from 30.5 +/- 3.0% of the risk zone in control hearts to 10.2 +/- 2.0% in SNAP-treated hearts. Bracketing the SNAP infusion with either the guanylyl cyclase blocker 1H-[1,2,4]oxadiazole[4,3-a]quinoxalin-1-one (2 microM) or the mitochondrial ATP-sensitive K(+) (mitoK(ATP)) channel blocker 5-hydroxydecanoate (200 microM) completely blocked the infarct-sparing effect of SNAP (34.3 +/- 3.8 and 32.2 +/- 1.6% infarction, respectively). Pretreatment of hearts with 8-(4-chlorophenylthio)-guanosine 3',5'-cyclic monophosphate (10 microM), which is a cell-permeable cGMP analog that activates protein kinase G, mimicked the preconditioning effect of SNAP by reducing infarct size to 7.5 +/- 1.1% of the risk zone. This salutary effect was abolished by either the free radical scavenger N-(2-mercaptopropionyl)glycine (1 mM) or 5-hydroxydecanoate (100 microM; 28.9 +/- 2.7 and 33.6 +/- 5.0% infarction of the risk zone, respectively). To confirm these functional data and the effect of SNAP on the guanylyl cyclase-protein kinase G signaling pathway, cGMP levels were measured. SNAP increased the level from 0.18 +/- 0.04 to 0.61 +/- 0.14 pmol/mg of protein (P < 0.05). These data suggest that exogenous NO triggers the preconditioning effect by initiating a cascade of events including stimulation of guanylyl cyclase to make cGMP, activation of protein kinase G, opening of mitoK(ATP) channels, and, finally, production of reactive oxygen species.     

Natriuretic peptides and nitric oxide stimulate cGMP synthesis in different cellular compartments

Cyclic nucleotide-gated (CNG) channels are a family of ion channels activated by the binding of cyclic nucleotides. Endogenous channels have been used to measure cyclic nucleotide signals in photoreceptor outer segments and olfactory cilia for decades. Here we have investigated the subcellular localization of cGMP signals by monitoring CNG channel activity in response to agonists that activate either particulate or soluble guanylyl cyclase. CNG channels were heterologously expressed in either human embryonic kidney (HEK)-293 cells that stably overexpress a particulate guanylyl cyclase (HEK-NPRA cells), or cultured vascular smooth muscle cells (VSMCs). Atrial natriuretic peptide (ANP) was used to activate the particulate guanylyl cyclase and the nitric oxide donor S-nitroso-n-acetylpenicillamine (SNAP) was used to activate the soluble guanylyl cyclase. CNG channel activity was monitored by measuring Ca2+ or Mn2+ influx through the channels using the fluorescent dye, fura-2. We found that in HEK-NPRA cells, ANP-induced increases in cGMP levels activated CNG channels in a dose-dependent manner (0.05-10 nM), whereas SNAP (0.01-100 microM) induced increases in cGMP levels triggered little or no activation of CNG channels (P < 0.01). After pretreatment with 100 microM 3-isobutyl-1-methylxanthine (IBMX), a nonspecific phosphodiesterase inhibitor, ANP-induced Mn2+ influx through CNG channels was significantly enhanced, while SNAP-induced Mn2+ influx remained small. In contrast, we found that in the presence of IBMX, both 1 nM ANP and 100 microM SNAP triggered similar increases in total cGMP levels. We next sought to determine if cGMP signals are compartmentalized in VSMCs, which endogenously express particulate and soluble guanylyl cyclase. We found that 10 nM ANP induced activation of CNG channels more readily than 100 muM SNAP; whereas 100 microM SNAP triggered higher levels of total cellular cGMP accumulation. These results suggest that cGMP signals are spatially segregated within cells, and that the functional compartmentalization of cGMP signals may underlie the unique actions of ANP and nitric oxide.     

T4-induced cardiac hypertrophy disrupts cyclic GMP mediated responses to brain natriuretic peptide in rabbit myocardium

Brain natriuretic peptide (BNP) affects the regulation of myocardial metabolism through the production of cGMP and these effects may be altered by cardiac hypertrophy. We tested the hypothesis that BNP would cause decreased metabolism and function in the heart and cardiac myocytes by increasing cGMP and that these effects would be disrupted after thyroxine-induced cardiac hypertrophy (T4). Open-chest control and T4 rabbits were instrumented to determine local effects of epicardial BNP (10(-3) M). Function of isolated cardiac myocytes was examined with BNP (10(-8)-10(-7) M) with or without KT5823 (10(-6) M, cGMP protein kinase inhibitor). Cyclic GMP levels were measured in myocytes. In open-chest controls, O2 consumption was reduced in the BNP area of the subepicardium (6.6+/-1.3 ml O2/min/100 g versus 8.9+/-1.4 ml O2/min/100 g) and subendocardium (9.4+/-1.3 versus 11.3+/-0.99). In T4 animals, functional and metabolic rates were higher than controls, but there was no difference between BNP-treated and untreated areas. In isolated control myocytes, BNP (10(-7) M) reduced percent shortening (PSH) from 6.5+/-0.6 to 4.3+/-0.4%. With KT5823 there was no effect of BNP on PSH. In T4 myocytes, BNP had no effect on PSH. In control myocytes, BNP caused cGMP levels to rise from 279+/-8 to 584+/-14 fmol/10(5) cells. In T4 myocytes, baseline cGMP levels were lower (117+/-2 l) and were not significantly increased by BNP. Thus, BNP caused decreased metabolism and function while increasing cGMP in control. These effects were lost after T4 due to lack of cGMP production. These data indicated that the effects of BNP on heart function operated through a cGMP-dependent mechanism, and that this mechanism was disrupted in T4-induced cardiac hypertrophy.     

Altered beta-adrenergic signal transduction in nonfailing hypertrophied myocytes from Dahl salt-sensitive rats

Desensitization of the beta-adrenergic receptor (beta-AR) response is well documented in hypertrophied hearts. We investigated whether beta-AR desensitization is also present at the cellular level in hypertrophied myocardium, as well as the physiological role of inhibitory G (G(i)) proteins and the L-type Ca(2+) channel in mediating beta-AR desensitization. Left ventricular (LV) myocytes were isolated from hypertrophied hearts of hypertensive Dahl salt-sensitive (DS) rats and nonhypertrophied hearts of normotensive salt-resistant (DR) rats. Cells were paced at a rate of 300 beats/min at 37 degrees C, and myocyte contractility and intracellular Ca(2+) concentration ([Ca(2+)](i)) were simultaneously measured. In response to increasing concentrations of isoproterenol, DR myocytes displayed a dose-dependent augmentation of cell shortening and the [Ca(2+)](i) transient amplitude, whereas hypertrophied DS myocytes had a blunted response of both cell shortening and the [Ca(2+)](i) transient amplitude. Interestingly, inhibition of G(i) proteins did not restore beta-AR desensitization in DS myocytes. The responses to increases in extracellular Ca(2+) and an L-type Ca(2+) channel agonist were also similar in both DS and DR myocytes. Isoproterenol-stimulated adenylyl cyclase activity, however, was blunted in hypertrophied myocytes. We concluded that compensated ventricular hypertrophy results in a blunted contractile response to beta-AR stimulation, which is present at the cellular level and independent of alterations in inhibitory G proteins and the L-type Ca(2+) channel.     

Nitric Oxide Regulates Cyclic GMP-Dependent Protein Kinase Phosphorylation in Rat Brain

Abstract: Nitric oxide (NO) acts via soluble guanylyl cyclase to increase cyclic GMP (cGMP), which can regulate various targets including protein kinases. Western blotting showed that type II cGMP-dependent protein kinase (cGK II) is widely expressed in various brain regions, especially in the thalamus. In thalamic extracts, the phosphorylation of several proteins, including cGK II, was increased by exogenous NO or cGMP. In vivo pretreatment with a NO synthase inhibitor reduced the phosphorylation of cGK II, and this could be reversed by exogenous NO or cGMP. Conversely, brainstem electrical stimulation, which enhances thalamic NO release, caused a NO synthase-dependent increase in the phosphorylation of thalamic cGK II. These results indicate that endogenous NO regulates cGMP-dependent protein phosphorylation in the thalamus. The activation of cGKII by NO may play a role in thalamic mechanisms underlying arousal.     

Restoration of calcium handling properties of adult cardiac myocytes from hypertrophied hearts

Reductions in cardiac sarcoplasmic reticulum calcium-ATPase (Serca2a) levels are thought to underlie the prolonged calcium (Ca(2+)) transients and consequent reduced contractile performance seen in human cardiac hypertrophy and heart failure. In freshly isolated cardiac myocytes from rats with monocrotaline-induced right ventricular hypertrophy we found reduced sarcoplasmic reticulum Serca2a expression and prolonged Ca(2+)transients, characteristic of hypertrophic cardiac disease.Modulation of intracellular Ca(2+)levels, Ca(2+) kinetics or Ca(2+)sensitivity is the focus of many current therapeutic approaches to improve contractile performance in the hypertrophic or failing heart. However, the functional effects of increasing Serca2a expression on Ca(2+) handling properties in myocytes from an animal model of cardiac hypertrophy are largely unknown. Here, we describe enhancement of the deficient Ca(2+) handling properties evident in myocytes from hypertrophied hearts following adenoviral-mediated transfer of the human Serca2a gene to these myocytes.These results highlight the importance of Serca2a deficiencies in the hypertrophic phenotype of cardiac muscle and suggest a simple, effective approach for manipulation of normal cardiac function.     

Implication of microRNAs in atrial natriuretic peptide and nitric oxide signaling in vascular smooth muscle cells

MicroRNAs (miRs) are endogenous small RNA molecules that suppress gene expression by binding to complementary sequences in the 3' untranslated regions of their target genes. miRs have been implicated in many diseases, including heart failure, ischemic heart disease, hypertension, cardiac hypertrophy, and cancers. Nitric oxide (NO) and atrial natriuretic peptide (ANP) are potent vasorelaxants whose actions are mediated through receptor guanylyl cyclases and cGMP-dependent protein kinase. The present study examines miRs in signaling by ANP and NO in vascular smooth muscle cells. miR microarray analysis was performed on human vascular smooth muscle cells (HVSMC) treated with ANP (10 nM, 4 h) and S-nitroso-N-acetylpenicillamine (SNAP) (100 μM, 4 h), a NO donor. Twenty-two shared miRs were upregulated, and 21 shared miRs were downregulated, by both ANP and SNAP (P < 0.05). Expression levels of four miRs (miRs-21, -26b, -98, and -1826), which had the greatest change in expression, as determined by microarray analysis, were confirmed by quantitative RT-PCR. Rp-8-Br-PET-cGMPS, a cGMP-dependent protein kinase-specific inhibitor, blocked the regulation of these miRs by ANP and SNAP. 8-bromo-cGMP mimicked the effect of ANP and SNAP on their expression. miR-21 was shown to inhibit HVSMC contraction in collagen gel lattice contraction assays. We also identified by computational algorithms and confirmed by Western blot analysis new intracellular targets of miR-21, i.e., cofilin-2 and myosin phosphatase and Rho interacting protein. Transfection with pre-miR-21 contracted cells and ANP and SNAP blocked miR-21-induced HVSMC contraction. Transfection with anti-miR-21 inhibitor reduced contractility of HVSMC (P < 0.05). The present results implicate miRs in NO and ANP signaling in general and miR-21 in particular in cGMP signaling and vascular smooth muscle cell relaxation.     

Atrial natriuretic peptide provokes a dramatic increase in cyclic GMP formation and markedly inhibits muscarinic-stimulated Ca2+ mobilisation in SV-40 transformed cat iris sphincter smooth muscle (SV-CISM-2) cells

We investigated the effects of cGMP-elevating agents, including atrial natriuretic peptide (ANP), C-type natriuretic peptide (CNP) and sodium nitroprusside (SNP), on cGMP accumulation and on carbachol (CCh)-stimulated intracellular calcium ([Ca2+]i) mobilisation in SV-40 transformed cat iris sphincter smooth muscle (SV-CISM-2) cells and in primary cultured cat iris sphincter smooth muscle (CISM) cells. The stimulatory effects of the natriuretic peptides on cGMP production correlated well with their inhibitory effects on CCh-induced [Ca+1]i mobilisation, and these effects were significantly more pronounced in the SV-CISM-2 cells than in the CISM cells. Thus, ANP (1 microM) increased cGMP production in the SV-CISM-2 cells and CISM cells by 487- and 1.7-fold, respectively, and inhibited CCh-induced [Ca2+]i mobilisation by 95 and 3%, respectively. In the SV-CISM-2 cells, ANP and CNP dose dependently inhibited CCh-induced [Ca2+]i mobilisation with IC50 values of 156 and 412 nM, respectively, and dose dependently stimulated cGMP formation with EC50 values of 24 and 88 nM, respectively, suggesting that the inhibitory actions of the peptides are mediated through cGMP. Both ANP and CNP stimulated cGMP accumulation in a time-dependent manner. The potency of the cGMP-elevating agents were in the following order: ANP>CNP>SNP; these agents had no effect on cAMP accumulation. The inhibitory effects of the natriuretic peptides were mimicked by 8-Br-cGMP, a selective activator of cGMP-dependent protein kinase. LY83583, a soluble guanylyl cyclase inhibitor, significantly inhibited SNP-induced cGMP formation but had no effect on those of ANP and CNP. The basal activities of the guanylyl cyclase and the dissociation constant (Kd) and total receptor density (Bmax) values of the natriuretic peptide receptor for [125I]ANP binding were not significantly different between the two cell types. The cGMP system, as with the cAMP system, has a major inhibitory influence on the muscarinic responses in the iris sphincter smooth muscle cells, and SV-CISM-2 cells can serve as an excellent model for investigating the cross talk between cGMP and the Ca2+ signalling system.     

NO and cGMP mediate angiotensin AT2 receptor-induced renal renin inhibition in young rats

We hypothesized that angiotensin subtype-2 receptor (AT(2)R) inhibits renal renin biosynthesis in young rats via nitric oxide (NO). We monitored changes in renal NO, cGMP, renal renin content (RRC), and ANG II in 4-wk-old rats in response to low sodium (LNa(+)) intake alone and combined with 8-h direct renal cortical administration of AT(1) receptor blocker valsartan (VAL), AT(2)R blocker PD123319 (PD), NO synthase inhibitor N(G)-nitro-l-arginine methyl ester (l-NAME), NO donor S-nitroso-N-acetyl penicillamine (SNAP), or guanylyl cyclase inhibitor 1H-[1,2,4] oxadiazolo[4,2-alpha] quinoxaline-1-one (ODQ). In addition, we monitored renal endothelial nitric oxide synthase (eNOS) and neuronal nitric oxide synthase (nNOS) in response to VAL or PD. LNa(+), VAL, PD, l-NAME, and ODQ increased RRC, ANG II, and renin mRNA. PD and l-NAME decreased NO and cGMP, while SNAP reduced RRC, ANG II, renin mRNA, and reversed the effects of PD. PD also reduced eNOS and nNOS protein and mRNA. Combined treatment with PD, l-NAME, or ODQ and VAL reversed the effects of VAL and caused further increase in RRC, ANG II, renin mRNA, and protein. ODQ reversed the effects of SNAP. These data demonstrate that the renal AT(2) receptor decreases renal renin biosynthesis and ANG II production in young rats. Reversal of the PD effects by SNAP and SNAP effects by ODQ confirms that NO and cGMP mediate the AT(2) receptor inhibition of renal renin production.