8-bromocyclic-gmp and Myocardial-Ischemia

Nitric oxide (NO) inhibits myocardial glucose transport and metabolism, although the underlying mechanism(s) and functional consequences of this effect are not clearly understood. We tested the hypothesis that NO inhibits the activation of AMP-activated protein kinase (AMPK) and translocation of cardiac glucose transporters (GLUTs; GLUT-4) and reduces lactate production. Ischemia was induced in open-chest dogs by a 66% flow reduction in the left anterior descending coronary artery (LAD). During ischemia, dogs were untreated (control) or treated by direct LAD infusion of (i) nitroglycerin (NTG) (0.5 microg.kg(-1).min(-1)); (ii) 8-Br-cGMP (50 microg.kg(-1).min(-1)); or (iii) NO synthase inhibitor L-nitro-argininemethylester (40 microg.kg(-1).min(-1); n = 9 per group). Cardiac substrate oxidation was measured with isotopic tracers. There were no differences in myocardial blood flow or oxygen delivery among groups; however, at 45 min of ischemia, the activation of AMPK was significantly less in NTG (77 +/- 12% vs. nonischemic myocardium) and 8-Br-cGMP (104 +/- 13%), compared with control (167 +/- 17%). Similarly, GLUT-4 translocation was significantly reduced in NTG (74 +/- 7%) and 8-Br-cGMP (120 +/- 11%), compared with control (165 +/- 17%). Glucose uptake and lactate output were 30% and 60% lower in NTG compared with control. Inhibition of NO synthesis stimulated glucose oxidation (67% increase compared with control) but did not affect AMPK phosphorylation, GLUT-4 translocation and glucose uptake. Contractile function in the ischemic region was significantly improved by NTG and L-nitro-argininemethylester. In conclusion, in ischemic myocardium an NO donor inhibits glucose uptake and lactate production via a reduction in AMPK stimulation of GLUT-4 translocation, revealing a mechanism of metabolic modulation and myocardial protection activated by NO donors.

Topics: AMP-Activated Protein Kinases; Animals; Biological Transport; Biopsy; Cell Membrane; Coronary Vessels; Cyclic GMP; Dogs; Glucose; Heart Ventricles; Lactates; Male; Monosaccharide Transport Proteins; Multienzyme Complexes; Myocardial Ischemia; Myocardium; NG-Nitroarginine Methyl Ester; Nitric Oxide; Nitric Oxide Synthase; Oxygen; Perfusion; Phosphorylation; Protein Serine-Threonine Kinases; Protein Transport; Protons; Subcellular Fractions; Time Factors

We tested the hypothesis that the second messenger activated by nitric oxide, cyclic GMP, would reduce the effects of myocyte stunning following simulated ischemia-reperfusion and that this was related to cyclic GMP protein kinase. Ventricular cardiac myocytes were isolated from New Zealand White rabbits (n = 8). Cell shortening was measured by a video edge detector and protein phosphorylation was determined autoradiographically after SDS gel electrophoresis. Cell shortening data were acquired at: (i) baseline followed by 8-Bromo-cGMP 10(-6) M (8-Br-cGMP) and then KT 5823 10(-6) M (cyclic GMP protein kinase inhibitor) and (ii) simulated ischemia (20 min of 95% N(2)-5% CO(2) at 37 degrees C) followed by simulated reperfusion (reoxygenation) with addition of 8-Br-cGMP 10(-6) M followed by KT 5823 10(-6) M, (iii) addition of 8-Br-cGMP prior to ischemia followed by the addition of KT 5823 10(-6) M after 30 min of reoxygenation. In the control group, 8-Br-cGMP 10(-6) M decreased percentage shortening (%short) (5.0 +/- 0.6 vs 3.8 +/- 0. 4) and the maximum velocity (V(max), microm/s) (48.6 +/- 6.9 vs 40.2 +/- 6.4). KT 5823 10(-6) M added after 8-Br-cGMP partially restored %short (4.6 +/- 0.5) and V(max) (46.6 +/- 8.0). After stunning, baseline myocytes had decreased %short (3.4 +/- 0.2) and V(max) (36. 0 +/- 4.2). After the addition of 8-Br-cGMP, the %short (2.7 +/- 0. 2) and V(max) (27.6 +/- 2.5) decreased further. The addition of KT 5823 did not change either the %short or the V(max). The myocytes with 8-Br-cGMP during ischemia had increased %short (4.2 +/- 0.2) and V(max) (37.2 +/- 3.4) when compared to the stunned group. The addition of KT 5823 did not significantly alter %short (3.3 +/- 0.4) or V(max) (29.2 +/- 5.0) in the myocytes pretreated with 8-Br-cGMP. Protein phosphorylation was increased by 8-Br-cGMP in control and stunned myocytes. KT 5823 blocked this effect in control but not stunned myocytes, suggesting some change in the cyclic GMP protein kinase. Ischemia-reperfusion produced myocyte stunning that was reduced when 8-Br-cGMP was added prior to but not after ischemia.

Topics: Alkaloids; Animals; Carbazoles; Cell Size; Cells, Cultured; Cyclic GMP; Enzyme Inhibitors; Heart; Heart Ventricles; Indoles; Myocardial Ischemia; Myocardial Reperfusion; Myocardial Stunning; Myocardium; Phosphoproteins; Phosphorylation; Rabbits

Previous studies in isolated cardiac myocytes suggest that impaired relaxation during reoxygenation after brief hypoxia results from abnormal Ca(2+)-myofilament interaction. Recent studies indicate that guanosine 3',5'-cyclic monophosphate (cGMP)-elevating interventions selectively enhance myocardial relaxation. We investigated the effect of 8-bromoguanosine 3',5'-cyclic monophosphate (8-BrcGMP) on posthypoxic relaxation in single rat myocytes, with simultaneous measurement of contraction and intracellular Ca2+ (indo 1 fluorescence). In control myocytes (n = 11), reoxygenation after 10 min of hypoxia markedly prolonged time to peak shortening (+36.5 +/- 4.2%) and half-relaxation time (+75.7 +/- 11.3% cf. normoxic values; both P < 0.001) and reduced diastolic length but did not change cytosolic Ca2+. Under normoxic conditions, 50 microM 8-BrcGMP slightly reduced time to peak shortening and half-relaxation time and increased diastolic length but did not alter cytosolic Ca2+. In the presence of 8-BrcGMP, there was no posthypoxic delay in twitch relaxation nor was there a decrease in diastolic length (half-relaxation time -5.8 +/- 3.3% cf. normoxic values; P < 0.05 cf. control group; n = 11). Cytosolic Ca2+ remained unaltered. Thus, 8-BrcGMP fully prevents impaired posthypoxic relaxation in isolated cardiac myocytes, probably by altering Ca(2+)-myofilament interaction.

Topics: Animals; Calcium; Cell Hypoxia; Cells, Cultured; Cyclic GMP; Fluorescent Dyes; Heart; Indoles; Myocardial Contraction; Myocardial Ischemia; Myocardium; Oxygen; Rats; Rats, Wistar; Time Factors

The cardioprotective effect of angiotensin-converting enzyme (ACE) inhibitors in cardiac ischemia/reperfusion damage is assumed to result largely from inhibition of the enzymatic breakdown of endogenous bradykinin (BK). We assessed the role of nitric oxide (NO) in mediating the beneficial actions of BK and the possible mechanism of the effect of NO. We experimentally infringed myocardial function in a working guinea pig heart preparation by ischemia (15 min) and reperfusion. The parameter external heart work (EHW), determined before and after ischemia, served as criterion for quantitation of recovery. We assessed oxidative stress during reperfusion by measuring glutathione release in coronary venous effluent; lactate release was used as a measure of ischemic challenge. The principal ability of NO to scavenge oxygen radicals was separately investigated in a chemiluminescence (CL) assay with the NO-donor sodium nitroprusside (SNP) and lucigenin. The ACE inhibitor ramiprilat (RT 25 microM) improved postischemic function significantly (55% recovery of EHW vs. 29% for controls). BK 1 nM was even more cardioprotective (71% recovery). The NO-synthase inhibitor Ng-nitro-L-arginine (NOLAG 10 microM) inhibited the effects of RT and BK (18% recovery each). SNP (0.3 microM) improved recovery to 57%, the prostacyclin analogue iloprost (ILO, 0.1 and 3 nM) had no beneficial effect (21 and 20% recovery, respectively). With 8-bromo-cyclicGMP, a membrane-permeable cGMP analogue, function was not better than control (30% recovery). Release of glutathione during reperfusion was decreased by the three compounds known to increase NO concentration in the heart; lactate release was the same in all groups.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Acridines; Angiotensin-Converting Enzyme Inhibitors; Animals; Cricetinae; Cyclic GMP; Free Radical Scavengers; Glutathione; Guinea Pigs; Heart; Hemodynamics; In Vitro Techniques; Lactates; Lactic Acid; Luminescent Measurements; Male; Myocardial Ischemia; Myocardium; Nitric Oxide; Nitroprusside; Purines; Reactive Oxygen Species