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Aims: Both natriuretic peptides and nitric oxide may be protective in cardiac hypertrophy, although their functional effects are diminished in hypertrophy. Hypoxia inducible factor-1 (HIF-1) may also protect in cardiac hypertrophy. We hypothesized that upregulation of HIF-1 would protect the functional effects of cyclic GMP (cGMP) signaling in hypertrophied ventricular myocytes., Main Methods: A cardiac hypertrophy model was created in mice by transverse aorta constriction. HIF-1 was increased by deferoxamine (150 mg/kg for 2 days). HIF-1alpha protein levels were examined. Functional parameters were measured (edge detector) on freshly isolated myocytes at baseline and after BNP (brain natriuretic peptide, 10(-8)-10(-7)M) or CNP (C-type natriuretic peptide, 10(-8)-10(-7)M) or SNAP (S-nitroso-N-acetyl-penicillamine, a nitric oxide donor, 10(-6)-10(-5)M) followed by KT5823 (a cyclic GMP-dependent protein kinase (PKG) inhibitor, 10(-6)M). We also determined PKG expression levels and kinase activity., Key Findings: We found that under control conditions, BNP (-24%), CNP (-22%) and SNAP (-23%) reduced myocyte shortening, while KT5823 partially restored function. Deferoxamine treated control myocytes responded similarly. Baseline function was reduced in the myocytes from hypertrophied heart. BNP, CNP, SNAP and KT5823 also had no significant effects on function in these myocytes. Deferoxamine restored the negative functional effects of BNP (-22%), CNP (-18%) and SNAP (-19%) in hypertrophic cardiac myocytes and KT5823 partially reversed this effect. Additionally, deferoxamine maintained PKG expression levels and activity in hypertrophied heart., Significance: Our results indicated that the HIF-1 protected the functional effects of cGMP signaling in cardiac hypertrophy through preservation of PKG., (Copyright (c) 2010 Elsevier Inc. All rights reserved.)

Ischemia-reperfusion reduces the negative functional effects of cyclic GMP in cardiac myocytes. In this study, we tested the hypothesis that upregulation of hypoxic inducible factor-1 (HIF-1) would improve the actions of cyclic GMP signaling following simulated ischemia-reperfusion. HIF-1 alpha was increased with deferoxamine (150 mg/kg for 2 days). Rabbit cardiac myocytes were subjected to simulated ischemia [15 min 95% N(2)-5% CO(2)] and reperfusion [reoxygenation] to produce myocyte stunning. Cell function was measured utilizing a video-edge detector. Shortening was examined at baseline and after brain natriuretic peptide (BNP, 10(-8), 10(-7)M) or S-nitroso-N-acetyl-penicillamine (SNAP, 10(-6), 10(-5)M) followed by KT5823 (cyclic GMP protein kinase inhibitor, 10(-6)M). Kinase activity was measured via a protein phosphorylation assay. Under control conditions, BNP (-30%) and SNAP (-41%) reduced percent shortening, while KT5823 partially restored function (+18%). Deferoxamine treated control myocytes responded similarly. In stunned myocytes, BNP (-21%) and SNAP (-25%) reduced shortening less and KT5823 did not increase function (+2%). Deferoxamine increased the effects of BNP (-38%) and SNAP (-41%) in stunning and restored the effects of KT5823 (+12%). The cyclic GMP protein kinase increased phosphorylation of several proteins in control HIF-1 +/- cells. Phosphorylation was reduced in stunned cells and was restored in deferoxamine treated stunned cells. This study demonstrated that simulated ischemia-reperfusion reduced the negative functional effects of increasing cyclic GMP and this was related to reduced effects of the cyclic GMP protein kinase. Increased HIF-1 alpha protects the functional effects of cyclic GMP thorough maintenance of cyclic GMP protein kinase activity after ischemic-reperfusion., ((c) 2008 S. Karger AG, Basel.)

Exposure to nitrates causes tachyphylaxis to nitric oxide (NO), which reduces the effects of the second messenger cyclic guanosine-3',-5'-monophosphate (cyclic GMP). We tested the hypothesis that prolonged exposure to NO would also blunt the effects of natriuretic peptides. Cardiac myocytes were isolated from control (N=7) and chronic nitroglycerin (patched, N=7) rabbits. Patched animals received a transdermal nitroglycerin patch (0.3mg/h for 5 days). Myocyte function was determined at baseline, after C-type natriuretic peptide (CNP, 10(-8) and 10(-7)M) or brain natriuretic peptide (BNP, 10(-8) and 10(-7)M) or S-nitroso-N-acetyl-penicilliamine (SNAP, a NO donor, 10(-6) and 10(-5)M) followed by KT5823 (a cyclic GMP protein kinase inhibitor, 10(-6)M). Soluble and particulate guanylyl cyclase activities were measured in vitro and phosphoprotein analysis was performed. In control animals, CNP 10(-8)M (5.14+/-0.5%) and 10(-7)M (4.4+/-0.7%) significantly reduced percentage shortening from baseline (6.1+/-1.6%). KT5823 restored percentage shortening to 4.9+/-0.8%. Similar data were obtained with BNP and SNAP. In patched animals, CNP, BNP, SNAP had no significant effects on percentage shortening. The data on maximal rate of shortening and relaxation were consistent with these results. Guanylyl cyclase activities were not different in the control and patched animals. The myocytes from control and patched animals had similar protein phosphorylation patterns. Our data suggested that in addition to NO, the responses to both natriuretic peptides were downregulated after chronic exposure to nitroglycerin, but these effects were not due to changes in either guanylyl cyclase or cyclic GMP protein kinase, suggesting an altered downstream pathway.

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.

Lack of endothelial nitric oxide synthase (eNOS) may affect the sensitivity of cyclic GMP signaling through soluble guanylyl cyclase (sGC). We hypothesized that in eNOS knockout (eNOS-/-) mice, stimulation of guanylyl cyclase would have enhanced effects inhibiting cardiac contraction. We measured cell shortening and calcium transients in isolated ventricular myocytes from adult eNOS-/- and wild-type (WT) mice after stimulating particulate guanylyl cyclase (pGC) with C-type natriuretic peptide (CNP, 10(-8) and 10(-7) M) or sGC with S-nitroso-N-acetyl-penicillamine (SNAP, NO donor, 10(-6) and 10(-5) M). Although sGC activity was increased by +71% in eNOS-/-, SNAP had similar effects in the two groups (%shortening -39% control vs. -37% eNOS-/-), suggesting that the cyclic GMP pathway was desensitized in eNOS-/- myocytes. CNP had significantly smaller effects on cell contraction (%shortening -34% control vs. -14% eNOS-/-) and pGC activity was not changed in eNOS-/- myocytes. Similar effects were also produced by guanylin and carbon monoxide, stimulators of pGC and sGC. CNP's effects on Ca(2+) transients were also attenuated in eNOS-/- myocytes. SNAP did not alter Ca(2+) transients in eNOS-/- or control cells. In the eNOS-/- mice, cyclic GMP-dependent protein kinase and cyclic AMP phosphodiesterase activity were reduced. This study demonstrated that the downstream cyclic GMP pathway was attenuated in eNOS-/- mice and this was partially compensated for by increased sGC, but not pGC activity in ventricular myocytes.

Increases in the myocardial level of cGMP usually exert negative inotropic effects in the mammalian hearts. We tested the hypothesis that the negative functional effects caused by nitric oxide (NO) or C-type natriuretic peptide (CNP) through cGMP would be blunted in hypertrophied cardiac myocytes. Contractile function, guanylyl cyclase activity, cGMP-dependent protein phosphorylation, and calcium transients were assessed in ventricular myocytes from aortic stenosis-induced hypertrophic and age-matched control mice. Basal percentage shortening was similar in control and hypertrophic myocytes. S-nitroso-N-acetyl-penicillamine (SNAP, an NO donor, 10(-6) and 10(-5) M) or CNP (10(-8) and 10(-7) M) reduced percentage shortening in both groups, but their effects were blunted in hypertrophic myocytes. Maximal rates of shortening and relaxation were depressed at the basal level, and both reagents had attenuated effects in hypertrophy. Similar results were also found after treatment with guanylin and carbon monoxide, other stimulators of particulate, and soluble guanylyl cyclase, respectively. Guanylyl cyclase activity was not significantly changed in hypertrophy. Addition of Rp-8-[(4-chlorophenyl)thio]-cGMPS triethylamine (an inhibitor of cGMP-dependent protein kinase, 5 x 10(-6) M) blocked SNAP or the effect of CNP in control mice but not in hypertrophy, indicating the cGMP-dependent kinase (PKG) may not mediate the actions of cGMP induced by NO or CNP in the hypertrophic state. Calcium transients after SNAP or CNP were not significantly changed in hypertrophy. These results suggest that in hypertrophied mice, diminished effects of NO or CNP on ventricular myocyte contraction are not due to changes in guanylyl cyclase activity. The data also indicated that PKG-mediated pathways were diminished in hypertrophied myocardium, contributing to blunted effects.

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.

Increasing stimulation rate increases function in cardiac myocytes and nitric oxide and cyclic GMP inhibit this effect. We tested the hypothesis that myocyte stunning would blunt both the effects of increases in rate and of nitric oxide and cyclic GMP. Ventricular myocytes from 11 rabbits were used to determine maximum rate of shortening (R(max), microm/s) and % shortening during control and after simulated ischemia [15 min 95% N(2)- 5% CO(2)] and reperfusion [reoxygenation]. Measurements were obtained at 1-4 Hz with vehicle, 1H[1,2,4]oxadiazolo[4,3,alpha] quinoxaline-1-one (ODQ) 10(-6) M, soluble guanylyl cyclase inhibitor, or N(G)-nitro-L-arginine methyl ester, nitric oxide synthase inhibitor (L-NAME) 10(-5) M. In control, increases in rate increased R(max) from 69 +/- 3 to 254+/-12 and % shortening from 5.3 +/- 0.3 to 8.7 +/- 0.5. Both ODQ and L-NAME shifted values higher. With stunning, the effects of pacing on Rmax and % shortening were blunted and ODQ and L-NAME failed to alter these values. Cyclic GMP was 322+/-37 pmol/10(5) myocytes at baseline and these values were lowered by ODQ (244 +/- 31) and LNAME (207 +/- 23), and similar changes were observed in stunned myocytes. Increasing frequency increased function, and reducing nitric oxide/cyclic GMP enhanced this relationship. The effect of nitric oxide was diminished by stunning, but this was not related to altered cyclic GMP levels. This suggested changes in effects of cyclic GMP downstream to its production during myocardial stunning.

1. In the present study, we tested the hypothesis that the negative myocardial metabolic effects of nitric oxide (NO) were reduced in old hearts. 2. Studies were conducted in 17 young (approximately 6 months) and 18 old (> 36 months) New Zealand anaesthetized open-chest rabbits. Either vehicle or s-nitroso-N-acetylpenicillamine (SNAP; 10-4 mol/L; a NO donor) was applied to the epicardial surface of the left ventricle. Coronary blood flow (microspheres) and artero-venous (a-v) O2 difference (microspectrophotometry) were used to determine subepicardial (EPI) and subendocardial (ENDO) O2 consumption. Wall thickening was determined ultrasonically. Cyclic GMP and guanylyl cyclase activity were also determined. Myocardial a-v O2 difference, flow, O2 consumption and wall thickening were comparable in young and old hearts. 3. The EPI and ENDO O2 consumption of SNAP-treated young hearts decreased significantly (> 25%) compared with vehicle (saline). However, SNAP had no significant effects on the O2 consumption of old hearts. In addition, SNAP decreased the percentage wall thickening in young (from 18.0 +/- 1.7 to 13.4 +/- 1.6%), but not old (from 14.5 +/- 0.9 to 11.4 +/- 1.6%), hearts. Basal cGMP levels in old hearts were greater (approximately 70%) than those in young hearts (15.7 +/- 2.0 vs 9.0 +/- 0.8 pmol/g, EPI). s-Nitroso-N-acetylpenicillamine increased cGMP in EPI (13.7 +/- 1.8 pmol/g) and ENDO of young, but not old (18.7 +/- 2.3 pmol/g, EPI), hearts. Similar results were also obtained using another NO donor, namely sodium nitroprusside (SNP; 10-4 mol/L). Guanylyl cyclase activity was elevated in old rabbit hearts with 0.5 mmol/L SNP (131 +/- 12 vs 80 +/- 12 pmol/min per mg protein for old and young rabbits, respectively). 4. Thus, while older hearts had similar O2 consumption and wall thickening compared with young hearts, they responded less well to NO and had significantly elevated basal levels of myocardial cGMP.

This investigation tested the hypothesis that the effects of nitric oxide synthase on myocardial capillary perfusion were reduced during myocardial stunning. Anaesthetized open-chest rabbits were assigned to either a control group or a group treated with a nitric oxide synthase inhibitor. To induce myocardial stunning, a coronary artery was occluded (for 15 min) and then reperfused (for 15 min) twice. During reperfusion, rabbits were given either saline or N(G)-nitro-L-arginine methyl ester (L-NAME, 30 mg kg(-1)) followed by i.v. injection of 150 mg kg(-1) fluorescein isothiocyanate (FITC)-labelled dextran (molecular weight, 150,000) for 14 s. Fluorescence microscopy was used to identify the perfused vessels and an alkaline phosphatase stain was used to locate the total microvasculature. The 'closest-individual' method was used to estimate the geometric distribution of capillaries. No significant differences were observed in the total volume fraction (mm(3) of capillaries per mm(3) of tissue) between the control and stunned regions in either the saline- (0.045 +/- 0.008 and 0.042 +/- 0.009, respectively) or the L-NAME-treated hearts (0.060 +/- 0.010 and 0.049 +/- 0.005, respectively). There were no significant differences in the percentage volume fraction of perfused capillaries between the control and the stunned regions (49 +/- 4 % and 54 +/- 4 %, respectively) in saline-treated hearts. In hearts treated with L-NAME, the percentage of perfused capillaries was significantly reduced. The reduction was significantly greater in the control region (approximately 27 %) than the stunned region (approximately 17 %). Closest-individual analysis of the perfused capillary distribution in both groups demonstrated a similar unchanged distribution. Thus, nitric oxide synthase is an important regulator of basal coronary capillary perfusion, and its effects are significantly reduced by myocardial stunning.

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 Ca2+-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, 10-5M 8-bromo-cyclic GMP (cGMP), 10-8, 10-7M C-type natriuretic peptide (CNP), or 10-6, 10-5M S-nitroso-N-acetyl-penicillamine (SNAP, nitric oxide donor). Changes in cytosolic Ca2+ 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 10-5M 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 Ca2+ transients changed similarly to cell contractility in WT and PLB-KO myocytes treated with cGMP and CNP; i.e. Ca2+ 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. Copyright © 2007 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Increases in the myocardial level of cGMP usually exert negative inotropic effects in the mammalian hearts. We tested the hypothesis that the negative functional effects caused by nitric oxide (NO) or C-type natriuretic peptide (CNP) through cGMP would be blunted in hypertrophied cardiac myocytes. Contractile function, guanylyl cyclase activity, cGMP-dependent protein phosphorylation, and calcium transients were assessed in ventricular myocytes from aortic stenosis-induced hypertrophic and age-matched control mice. Basal percentage shortening was similar in control and hypertrophic myocytes. S-nitroso-N-acetyl-penicillamine (SNAP, an NO donor, 106 and 10-5 M) or CNP (10-8 and 10-7 M) reduced percentage shortening in both groups, but their effects were blunted in hypertrophic myocytes. Maximal rates of shortening and relaxation were depressed at the basal level, and both reagents had attenuated effects in hypertrophy. Similar results were also found after treatment with guanylin and carbon monoxide, other stimulators of particulate, and soluble guanylyl cyclase, respectively. Guanylyl cyclase activity was not significantly changed in hypertrophy. Addition of Rp-8-[(4-chlorophenyl)thio]-cGMPS triethylamine (an inhibitor of cGMP-dependent protein kinase, 5 × 10-6 M) blocked SNAP or the effect of CNP in control mice but not in hypertrophy, indicating the cGMP-dependent kinase (PKG) may not mediate the actions of cGMP induced by NO or CNP in the hypertrophic state. Calcium transients after SNAP or CNP were not significantly changed in hypertrophy. These results suggest that in hypertrophied mice, diminished effects of NO or CNP on ventricular myocyte contraction are not due to changes in guanylyl cyclase activity. The data also indicated that PKG-mediated pathways were diminished in hypertrophied myocardium, contributing to blunted effects. [ABSTRACT FROM AUTHOR]

Deals with a study which determined whether the negative effects of nitric oxide on cardiac myocytes would be reduced by aging. Methods; Results; Discussion.

We tested the hypothesis that lowering the level of cyclic GMP would have positive functional effects on isolated rabbit ventricular myocytes regardless of the basal cyclic GMP level. Cell shortening data were collected with a video detector; O[sub 2] consumption data were obtained with a Clark electrode; intracellular cyclic GMP levels were obtained by radioimmunoassay. Data were obtained: (1) at baseline; (2) after the addition of 1H-[1,2,4]oxadiazolo[4,3-α]quinoxaline-1-one (ODQ) 10[sup –6] and 10[sup –4] mol/l, a selective soluble guanylyl cyclase inhibitor, and (3) after zaprinast 10[sup –6] mol/l, a cyclic GMP phosphodiesterase inhibitor, followed by ODQ 10[sup –6] and 10[sup –4] mol/l. We found that ODQ 10[sup –4] mol/l significantly decreased the cyclic GMP level from 493 ± 75 to 301 ± 78 (fmol/100,000 myocytes) and increased percent shortening (Pcs, %; 4.9 ± 0.3 vs. 5.8 ± 0.6) and maximum rate of shortening (Rs, μm/s; 58.7 ± 5.7 vs. 73.6 ± 4.9). Zaprinast significantly increased the cyclic GMP level from 419 ± 140 to 599 ± 241 and decreased Pcs (6.2 ± 0.5 vs. 4.4 ± 0.4) and Rs (65.5 ± 5.3 vs. 49.6 ± 4.3). After zaprinast, ODQ 10[sup –4] mol/l decreased the cyclic GMP level to 439 ± 139 and increased percent shortening and rate of shortening by a similar percentage compared to the non-zaprinast treated myocytes. We conclude that in rabbit ventricular myocytes, a reduction in the level of myocyte cyclic GMP increases myocyte function independent of the initial cyclic GMP level.Copyright © 2000 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Chronic exposure to nitrates causes tachyphylaxis to nitric oxide (NO), which reduces the effects of cyclic GMP. We tested the hypothesis that prolonged exposure to NO would not only blunt the effects of NO, but also natriuretic peptides. Cardiac myocytes were isolated from control (N=7) and chronic nitroglycerin (patched, N=7) rabbits. Patched animals received a transdermal nitroglycerin patch (0.3mg/hr for 5 days). Myocyte function was determined at baseline, CNP (C-type natriuretic peptide) or BNP (Brain natriuretic peptide) 10-8M, 10-7M, followed by KT5823 (cyclic GMP protein kinase inhibitor) 10-6M; baseline, SNAP (S-nitroso-N-acetyl-penicilliamine, NO donor) 10-6M, 10-5M, followed by KT5823. Soluble and particulate guanylyl cyclase activities were measured and in vitro phosphoprotein analysis was performed. In control animals, CNP 10-8M, 10-7M significantly reduced percentage shortening from baseline 6.1±1.6% to 5.14±0.5 % and 4.4±0.7%. KT5823 restored percentage shortening to 4.9±0.8%. Similar data were obtained with BNP and SNAP. In patched animals, CNP, BNP, SNAP had no significant effects on percentage shortening. The data on maximal rate of shortening and relaxation were consistent with these results. Guanylyl cyclase activities were not different in the control and patched animals. The myocytes from control and patched animals had similar protein phosphorylation patterns. Our data suggested that in addition to NO; the responses to both natriuretic peptides were downregulated after chronic exposure to nitroglycerin, but these effects were not due to changes in either guanylyl cyclase or cyclic GMP protein kinase, suggesting altered downstream effects. [ABSTRACT FROM AUTHOR]

Summary 1. In the present study, we tested the hypothesis that the negative myocardial metabolic effects of nitric oxide (NO) were reduced in old hearts. 2. Studies were conducted in 17 young (approximately 6 months) and 18 old (> 36 months) New Zealand anaesthetized open-chest rabbits. Either vehicle or s-nitroso-N -acetylpenicillamine (SNAP; 10-4 mol/L; a NO donor) was applied to the epicardial surface of the left ventricle. Coronary blood flow (microspheres) and artero–venous (a-v) O2 difference (microspectrophotometry) were used to determine subepicardial (EPI) and subendocardial (ENDO) O2 consumption. Wall thickening was determined ultrasonically. Cyclic GMP and guanylyl cyclase activity were also determined. Myocardial a-v O2 difference, flow, O2 consumption and wall thickening were comparable in young and old hearts. 3. The EPI and ENDO O2 consumption of SNAP-treated young hearts decreased significantly (> 25%) compared with vehicle (saline). However, SNAP had no significant effects on the O2 consumption of old hearts. In addition, SNAP decreased the percentage wall thickening in young (from 18.0 ± 1.7 to 13.4 ± 1.6%), but not old (from 14.5 ± 0.9 to 11.4 ± 1.6%), hearts. Basal cGMP levels in old hearts were greater (approximately 70%) than those in young hearts (15.7 ± 2.0 vs 9.0 ± 0.8 pmol/g, EPI). s-Nitroso-N -acetylpenicillamine increased cGMP in EPI (13.7 ± 1.8 pmol/g) and ENDO of young, but not old (18.7 ± 2.3 pmol/g, EPI), hearts. Similar results were also obtained using another NO donor, namely sodium nitroprusside (SNP; 10-4 mol/L). Guanylyl cyclase activity was elevated in old rabbit hearts with 0.5 mmol/L SNP (131 ± 12 vs 80 ± 12 pmol/min per mg protein for old and young rabbits, respectively). 4. Thus, while older hearts had similar O2 consumption and wall thickening compared... [ABSTRACT FROM AUTHOR]