Endothelin (ET) may have both detrimental (reduced coronary flow) and beneficial effects (positive inotrope, reduced arrhythmogenesis) following ischaemia. We examined the effects of ET on cardiac function during reperfusion following prolonged hypothermic cardioplegic arrest in a protocol mimicking cardiac transplantation. Isolated working rat hearts were perfused with Krebs buffer to which increasing concentrations of ET-1 or sarafotoxin S6c had been added. Identical experiments were performed after 4 h of cardioplegic arrest at 4 degrees C. Under pre-ischaemic conditions ET-1 caused a dose-dependent decrease in cardiac function compared with controls. In contrast, following ischaemia low doses of ET-1 (10(-10) M) caused a significant and beneficial increase in cardiac output (109.1% versus 81.3%), dP/dt i.e. the rate of change of pressure with time (94.7% versus 75.6%) and stroke volume (100.3% versus 77.5%) compared with controls (P<0.05). At higher doses of ET-1 there was a detrimental effect on cardiac output, dP/dt and stroke volume similar to that seen prior to ischaemia. Sarafotoxin S6c had no significant effect pre or post ischaemia on any of the parameters measured compared with controls (P=not significant). ET-1 at low concentrations during reperfusion can improve the recovery of cardiac function mediated via ET(A) receptors. ET may play an important physiological role in the recovery of cardiac function following prolonged ischaemia.

BackgroundHeat shock protein 72 (HSP72) is known to provide myocardial protection against ischemia-reperfusion injury by its chaperoning function. Target molecules of this effect are presumed to include not only structural proteins but also other self-preservation proteins. The details, however, remain unknown. Manganese superoxide dismutase (Mn-SOD) is an enzyme that preserves mitochondria, a key organelle for cellular respiration, from reperfusion injury and limits mitochondria-related apoptosis. We hypothesized that Mn-SOD would play a role in HSP72-mediated cardioprotection. Methods and ResultsRat hearts were transfected with human HSP72 by intra-coronary infusion of Hemagglutinating Virus of Japan-liposome, resulting in global myocardial overexpression of HSP72. After ischemia-reperfusion injury, cardiac function (left ventricular systolic pressure, maximum dP/dt, minimum dP/dt, and coronary flow) was improved in the HSP72-transfected hearts compared with control-transfected ones, corresponding with less leakage of creatine kinase and mitochondrial aspartate aminotransferase. Postischemic Mn-SOD content and activity in the HSP72-transfected hearts were enhanced in comparison with the controls (content: 96.9±4.1 versus 85.5±2.5% to the preischemic level, P =0.038; activity: 93.9±2.2 versus 82.2±3.7%, P =0.022), associated with improved mitochondrial respiratory function (postischemic percent respiratory control index; NAD+-linked: 81.3±3.8 versus 18.5±4.4%; FAD-linked: 71.8±5.5 versus 20.7±5.3%, P <0.001). In addition, incidence of postischemic cardiomyocyte apoptosis was attenuated in the HSP72-transfected hearts (4.0±1.1 versus 10.3±3.3%, P =0.036), correlating with an increased Bcl-2 level and reduced up-regulation of caspase-3. ConclusionsThese data suggest that the enhanced Mn-SOD activity during ischemia-reperfusion injury, which is associated with mitochondrial protection and apoptosis reduction, is a possible mechanism of HSP72-induced cardioprotection.

Hyperthermic stress is known to protect against myocardial dysfunction after ischemia-reperfusion injury. It is unclear however, what energetic mechanisms are affected by the molecular adaptation to heat stress. We hypothesized that mild hyperthermic stress can increase mitochondrial respiratory enzyme activity, affording protection to mitochondrial energetics during prolonged cardiac preservation for transplantation. Rat hearts were excised after heat-stress or sham treatment and subjected to cold cardioplegic arrest and ischemia followed by reperfusion in an ex vivo perfusion system. Cardiac function, mitochondrial respiratory, and complex activities were assessed before and after ischemia. Heat shock protein (Hsp 32, 60, and 72) expression was increased in heat-stressed hearts. This was associated with increased mitochondrial complex activities in heat-stress versus sham-treated groups for complex I-V. During reperfusion, higher complex activities and respiratory control ratios were observed in heat-stressed versus sham-treated groups. Recovery of ventricular function was improved in heat-stressed hearts. Furthermore, mitochondria in reperfused heat-stressed myocardium exhibited intact membranes with packed, parallel, lamellar cristae, whereas in sham-treated myocardium, mitochondria were severely disrupted. This study provides the first evidence of heat-stress-mediated enhancement of mitochondrial energetic capacity. This is associated with increased tolerance to ischemia-reperfusion injury. Protection by heat stress against myocardial dysfunction may be partially due to enhancement of mitochondrial energetics.

Background Upregulation of heat shock protein 70 (HSP70) is beneficial in cardioprotection against ischemia-reperfusion injury, but the mechanism of action is unclear. We studied the role of HSP70 overexpression through gene therapy on mitochondrial function and ventricular recovery in a protocol that mimics clinical donor heart preservation. Methods and Results Hemagglutinating virus of Japan (HVJ)-liposome technique was used to transfect isolated rat hearts via intracoronary infusion of either the HSP70 gene (HSP group, n=16) or no gene (CON group, n=16), which was heterotopically transplanted into recipient rats. Four days after surgery, hearts were either perfused on a Langendorff apparatus for 30 minutes at 37°C (preischemia studies [n=8/group]) or perfused for 30 minutes at 37°C, cardioplegically arrested for 4 hours at 4°C, and reperfused for 30 minutes at 37°C (postischemia studies [n=8/group]). Western blotting and immunohistochemistry confirmed HSP70 upregulation in the HSP group. Postischemic mitochondrial respiratory control indices (RCIs) were significantly better preserved in HSP than in CON hearts: NAD + -linked RCI values were 9.54±1.1 versus 10.62±0.46 before ischemia (NS) but 7.98±0.69 versus 1.28±0.15 after ischemia ( P <0.05), and FAD-linked RCI values were 6.87±0.88 versus 6.73±0.93 before ischemia (NS) but 4.26±0.41 versus 1.34±0.13 after ischemia ( P <0.05). Postischemic recovery of mechanical function was greater in HSP than in CON hearts: left ventricular developed pressure recovery was 72.4±6.4% versus 59.7±5.3% ( P <0.05), maximum dP/dt recovery was 77.9±6.6% versus 52.3±5.2% ( P <0.05), and minimum dP/dt recovery was 72.4±7.2% versus 54.8±6.9% ( P <0.05). Creatine kinase release in coronary effluent after reperfusion was 0.20±0.04 versus 0.34±0.06 IU · min −1 · g wet wt −1 ( P <0.05) in HSP versus in CON hearts. Conclusions HSP70 upregulation protects mitochondrial function after ischemia-reperfusion injury; this was associated with improved preservation of ventricular function. Protection of mitochondrial function may be important in the development of future cardioprotective strategies.

OBJECTIVEEndothelin plays a role in the regulation of basal coronary tone. We hypothesized that low coronary reflow and reduced cardiac function after prolonged ischemia may be due to increased release of endogenous endothelin.METHODSUsing an isolated perfused rat heart, we examined the effect of the addition of various endothelin antagonists during reperfusion after 4 hours of cardioplegic arrest at 4 degrees C. Hearts were freeze-clamped at the end of reperfusion for analysis of high-energy phosphate levels. Results are expressed as the percentages of preischemic values.RESULTSThe addition of bosentan or Ro61-0612 (nonselective endothelin antagonists) resulted in a significant increase in the recovery of coronary flow after 30 minutes of reperfusion (100.9% vs 85.3% [P =.03] and 122.4% vs 83.7% [P <.001], respectively, versus controls). The addition of PD155080 (endothelin A antagonist) had a similar effect (129.5% vs 91.4%, P =.008). BQ788 (endothelin B antagonist) and phosphoramidon (endothelin-converting enzyme inhibitor) had no effect. Myocardial adenosine triphosphate levels were significantly (12.1%) higher after reperfusion with Ro61-0612 (18.1 +/- 0.4 micromol/g vs 16.2 +/- 0.5 micromol/g, P =.01). There was no difference in the recovery of cardiac mechanical function with any of the antagonists studied.CONCLUSIONThese results suggest that endogenous endothelin plays a role in low coronary reflow after prolonged cardioplegic arrest but does not impair recovery of myocardial function.

Objectives: Cardioplegic arrest during cardiac surgery induces severe abnormalities of the pyruvate metabolism, which may affect functional recovery of the heart. We aimed to evaluate the effect of pyruvate and dichloroacetate administration during reperfusion on recovery of mechanical function and energy metabolism in the heart subjected to prolonged cardioplegic arrest. Methods: Four groups of rat hearts perfused in working mode were subjected to cardioplegic arrest (St. Thomas' No. 1), 4 h of ischaemia at 8 degreesC and reperfusion with either Krebs buffer alone (C) or with 2.8 mM pyruvate (P), with 1 mM dichloroacecate (D), or with a combination of both (PD). Mechanical function was recorded before cardioplegic arrest and at the end of experiments. In groups C and PD, additional experiments were performed using; P-31 nuclear magnetic resonance spectroscopy in non-working Langendorff mode to evaluate cardiac high-energy phosphate concentration changes throughout the experiment. Results: Improved recovery of cardiac output (% of the preischaemic value +/- SEM, n = 9-12) was observed in all three treated groups (65.7 +/- 4.3, 59.5 +/- 5.2 and 59.5 +/- 5.3% in PD, P and D, respectively) as compared with C (42.2 +/- 4.6%; P < 0.05). Recovery of coronary flow was improved from 66.4 +/- 3.8 in C to 94.9 +/- 8.6%, in PD (P < 0.05). The phosphocreatine recovery rate in the first minutes of reperfusion was increased from 9.9 +/- 1.5 in C to 31.5 +/- 4.3 mu mol/min per g dry wt in PD (P < 0.001). No differences were observed in ATP or phosphocreatine concentrations at the end of experiment. Conclusions: The administration of pyruvate and dichloroacetate improves the recovery of mechanical function following hypothermic ischaemia. Accelerated restoration of the energy equilibrium in the initial phase of reperfusion map underlie the metabolic mechanism of this effect. (C) 2001 Elsevier Science B.V. All rights reserved.

The inhibition of adenosine deaminase with erythro-9 (2-hydroxy-3-nonyl)-adenine (EHNA) and the es-ENT1 transporter with p-nitro-benzylthioinosine (NBMPR), entraps myocardial intracellular adenosine during on-pump warm aortic crossclamping, leading to a complete recovery of cardiac function and adenosine triphosphate (ATP) during reperfusion. The differential role of entrapped intracellular and circulating adenosine in EHNA/NBMPR-mediated protection is unknown. Selective (8-cyclopentyl-1,3-dipropyl-xanthine) or nonselective [8-(p-sulfophenyl)theophyline] A1 receptor antagonists were used to block adenosine A1-receptor contribution in EHNA/NBMPR-mediated cardiac recovery.Anesthetized dogs (n = 45), instrumented to measure heart performance using sonomicrometry, were subjected to 30 minutes of warm aortic crossclamping and 60 minutes of reperfusion. Three boluses of the vehicle (series A) or 100 μM EHNA and 25 μM NBMPR (series B) were infused into the pump at baseline, before ischemia and before reperfusion. 8-Cyclopentyl-1,3-dipropyl-xanthine (10 μM) or 8-(p-sulfophenyl)theophyline (100 μM) was intra-aortically infused immediately after aortic crossclamping distal to the clamp in series A and series B. The ATP pool and nicotinamide adenine dinucleotide was determined using high-performance liquid chromatography.Ischemia depleted ATP in all groups by 50%. The adenosine/inosine ratios were more than 10-fold greater in series B than in series A (P < .001). ATP and function recovered in the EHNA/NBMPR-treated group (P < .05 vs control group). 8-Cyclopentyl-1,3-dipropyl-xanthine and 8-(p-sulfophenyl)theophyline partially reduced cardiac function in series A and B to the same degree but did not abolish the EHNA/NBMPR-mediated protection in series B.In addition to the cardioprotection mediated by activation of the adenosine receptors by extracellular adenosine, EHNA/NBMPR entrapment of intracellular adenosine provided a significant component of myocardial protection despite adenosine A1 receptor blockade.

OBJECTIVE:Both superoxide dismutase (SOD), a free radical scavenger, and nitric oxide (NO), a vasodilator with anti-inflammatory properties, have been shown to protect the myocardium from reperfusion injury. They are known to interact in vivo, the influence of which on myocardial protection has not been studied. METHODS:Four groups of rats (n=7, per group) were subjected to experimental infarction following injections into the anterior wall of the left ventricle with adenoviral vector encoding beta-galactosidase (group A), eNOS (group B), Mn-SOD (group C) and both eNOS and MnSOD (group D). Hearts were assessed for protein expression and size of infarction. RESULTS:Efficiency of gene up regulation was confirmed by immunostaining for eNOS and Mn-SOD, and X-gal staining for beta-gal respectively. In B and D, overexpression of eNOS was demonstrated in cardiac myocytes in addition to that in the endothelium, while in C and D, Mn-SOD was overexpressed in mainly cardiomyocytes. Infarct size was 49.7+/-4.8% in A, and was significantly reduced in the other groups (29.8+/-2.7%, 21.8+/-2.5% and 24.9+/-2.4% in B, C and D respectively). CONCLUSION:Adenoviral gene transfer of Mn-SOD was superior to eNOS in reducing the extent of in vivo ischemia-reperfusion injury in the rat heart in our model. The effect of combined application of Mn-SOD and eNOS was not different from their individual effect.

Background Adenosine (Ado) triggers numerous protective mechanisms in the heart that may attenuate ischemia-reperfusion injury in cardiac grafts. We aimed to establish whether sustained increase in endogenous Ado production by the combined application of Ado metabolism inhibitors and nucleotide precursors attenuates reperfusion injury in transplanted hearts. Methods and Results Rat hearts were collected after the infusion of St Thomas’ Hospital cardioplegic solution, stored at 4°C for 4 hours, and heterotopically transplanted into the abdomen of recipient rats. A solution containing Ado deaminase inhibitor erythro-9(2-hydroxy-3-nonyl)adenine, Ado kinase inhibitor 5′-aminoadenosine, and nucleotide precursors adenine and ribose was administered at the time of reperfusion in the treated group, whereas saline was administered to control animals. After 1 or 24 hours, mechanical function of the transplanted hearts was evaluated in an ex vivo perfusion system followed by the determination of myocardial ATP with related metabolites and measurement of the activity of neutrophil-specific enzyme myeloperoxidase in cardiac homogenates. After 24 hours of reperfusion, maximum left ventricular developed pressure increased from 87.0±6.8 mm Hg (mean±SEM) in controls to 118.1±8.2 mm Hg in the treated group ( P <0.05), ATP increased from 11.0±0.8 μmol/g dry wt in controls to 15.1±1.2 μmol/g dry wt in the treated group ( P <0.01), and myeloperoxidase activity decreased from 2.23±0.60 U/g wet wt in controls to 0.58±0.12 U/g wet wt in the treated group ( P <0.001). No differences in cardiac function, ATP, or myeloperoxidase activity were observed between the treated group and controls after 1 hour of reperfusion. Conclusions The administration of Ado metabolism inhibitors with nucleotide precursors causes a sustained increase in endogenous Ado production and exerts a potent protective effect against reperfusion injury in transplanted hearts. Improved cardiac function and elevated ATP concentration were accompanied by complete amelioration of neutrophil infiltration in treated hearts, suggesting that reduction in postischemic inflammation could be an important mechanism of this protective effect.

Background —Cell transplantation is a promising strategy to treat end-stage heart failure. At present, a popular method to deliver cells into the heart is direct intramuscular injection. This method, however, may not be efficient in spreading cells globally into the myocardium. We have developed a novel method for cell transplantation using intracoronary infusion. Methods and Results —An L6 rat skeletal muscle cell line expressing β-galactosidase (β-gal) was generated by gene transfection and clonal selection. These cells (10 6 in 1 mL medium) were infused into explanted rat hearts through the coronary artery, followed by heterotopic heart transplantation into the abdomen of recipients. Control hearts were infused with cell-free medium. According to β-gal activity measurements, ≈5×10 5 grafted cells per heart existed on day 3, increasing to 5×10 6 on day 28 in the cell-transplanted hearts. At day 28, discrete loci positively stained for β-gal were observed throughout the cardiac layers of both left and right coronary territories. Some of them differentiated into β-gal–positive multinucleated myotubes that aligned with the cardiac fiber axis and integrated into the native myocardium, whereas others formed colonies consisting of undifferentiated myoblasts. Connexin 43, a cardiac gap junction protein, was expressed between grafted cells and native cardiomyocytes. No reduction in cardiac function was observed in a Langendorff perfusion system. Conclusions —We have developed a unique method for efficient cell transplantation based on intracoronary infusion. This method, potentially applicable in the clinical setting during cardiac surgery, could be useful to globally supply cells to the heart.

Background-Heat shock protein 70 (HSP70) gene transfection has been shown to enhance myocardial tolerance after normothermic ischemia-reperfusion. We investigated the effect of HSP70 gene transfection on mechanical and endothelial function in a protocol mimicking clinical heart preservation.Methods and Results-Rat hearts were infused ex vivo with Hemagglutinating Virus of Japan-liposome complex containing HSP70 gene (HSP, n=8) or no gene (CON, n=8), and heterotopically transplanted into recipient rats. Four days after surgery, transfected hearts were perfused on a Langendorff apparatus for 45 minutes, arrested with St Thomas' No, 1 cardioplegia for 4 hours at 4 degreesC, and reperfused for 1 hour. Mechanical and endothelial function was studied before and after ischemia. Creatine kinase was measured in reperfusion effluent. Hearts underwent Western blotting and immunohistochemistry to confirm HSP70 overexpression. Postischemic recovery of mechanical function (% preischemic+/-SEM) was greater in HSP versus CON: Left ventricular developed pressure recovery was 76.7+/-3.9% versus 60.5+/-3.1% (P<0.05); dP/dtmax recovery was 79.4+/-4.9% versus 56.2+/-3.2% (P<0.05); dP/dtmin recovery was 74.8+/-4.6% versus 57.3+/-3.6% (P<0.05), Creatine kinase release was attenuated in HSP versus CON: 0.22+/-0.02 versus 0.32+/-0.04 IU/min/g wet wt. (P<0.05). Recovery of coronary flow was greater in HSP versus CON: 76.5+/-3.8% versus 59.2+/-3.2% (P<0.05). Recovery of coronary response to 5-hydroxytryptamine (5x10(-5) mol/L) was 55.6+/-4.7% versus 23.9+/-3.2% (P<0.05); recovery of coronary response to glyceryltrinitrate (15 mg/L) was not different between HSP and CON: 87.4+/-6.9% versus 84.3+/-5.8% (NS).Conclusions-In a clinically relevant donor heart preservation protocol, HSP70 gene transfection protects both mechanical and endothelial function.

Inhibition of adenosine metabolism and supply of nucleotide precursors resulted in marked improvement in cardiac function and increase in cardiac ATP concentration following cardioplegic arrest and hypothermic ischemia. Stimulation of endogenous adenosine production could be thus an effective treatment following cardiac transplantation.

Background —Graft survival after skeletal myoblast transplantation is affected by various pathological processes caused by environmental stress. Heat shock is known to afford protection of several aspects of cell metabolism and function. We hypothesized that prior heat shock treatment of graft cells would improve their survival after cell transplantation. Methods and Results —L6 rat skeletal myoblasts expressing β-galactosidase (β-gal) were subjected to heat shock (42°C, 1 hour). Increased expression of heat shock protein 72 was detected 24 hours later in the heat-shocked cells. After hypoxia-reoxygenation in vitro, lactate dehydrogenase leakage was significantly attenuated in the heat-shocked cells; in addition, the percentage of early apoptosis was lower in this group measured by flow cytometry with annexin V staining. For the in vivo study, 1×10 6 heat-shocked (hsCTx) or normal-cultured (CTx) myoblasts were infused into the explanted rat hearts through the coronary artery followed by heterotopic heart transplantation. β-gal activity was significantly higher in the hsCTx group after cell transplantation, with an estimated 8×10 6 surviving cells per heart in the hsCTx group and 5×10 6 cells in the CTx group on day 28. Discrete loci of grafted cells were globally observed in the myocardium of the hsCTx and CTx groups, with a higher frequency in the hsCTx group. Surviving myoblasts occasionally differentiated into myotubes and had integrated with the native cardiomyocytes. Conclusions —Heat-shocked skeletal myoblasts demonstrated improved tolerance to hypoxia-reoxygenation insult in vitro and enhanced survival when grafted into the heart. Heat shock treatment could be useful in improving graft cell survival in cell transplantation.

BACKGROUND:Alterations in metabolic pathways may contribute to the cardioprotective effects of heat stress (HS). We investigated the effects of HS on ATP and phosphocreatine (PCr) levels in the ischemic rat myocardium, after both normothermic and hypothermic ischemia.METHODS:Two protocols were used: (1) normothermic ischemia (20 min at 37 degrees C) with no myocardial protection (n=6 HS; n=6 control); (2) hypothermic ischemia (4 hrs at 4 degrees C) after cardioplegic arrest (n=6 HS; n=6 control). ATP and PCr levels in the heart were measured using 31P nuclear magnetic resonance spectroscopy.RESULTS:At the end of normothermic ischemia, ATP levels were better maintained in HS hearts (C vs HS: 4.51+/-0.66 vs 7.81+/-1.06 micromol/g dry wt+/-SEM, p=0.04). A trend for higher ATP content in HS hearts was observed after 40 min of reperfusion (C vs HS: 11.7+/-1.5 vs 16.9+/-2.0 micromol/g dry wt+/-SEM, p=0.09). PCr content was also higher at the end of 40 minutes of reperfusion in HS hearts (C vs HS: 46.4+/-2.9 vs 56.9+/-3.0 micromol/g dry wt+/-SEM, p=0.03). After prolonged hypothermic ischemia under cardioplegic arrest, heat stress again led to better preservation of ATP levels at the end of ischemia (C vs HS: 5.71+/-0.88 vs 9.23+/-1.38 micromol/g dry wt+/-SEM, p=0.05) and after 40 minutes of reperfusion (C vs HS: 16.8+/-1.4 vs 24.6+/-2.8 micromol/g dry wt+/-SEM, p=0.03). PCr levels were also better maintained at the end of ischemia (C vs HS: 4.87+/-0.77 vs 12.4+/-3.0 micromol/g dry wt+/-SEM, p=0.03) and after 40 minutes of reperfusion in HS hearts (C vs HS: 55.1+/-7.0.vs 79.8+/-7.3 micromol/g dry wt+/-SEM, p=0.03).CONCLUSIONS:Heat stress induces changes in the energy profile of the heart which results in better preservation of ATP and phosphocreatine levels. These changes could be observed after brief normothermic ischemia and also after prolonged hypothermic ischemia under cardioplegic arrest, mimicking conditions of preservation for cardiac transplantation.

Objective: Ageing is known to be associated with changes within the heart. We investigated whether the coronary response to endothelin-1 (ET) and sarafotoxin S6c (S6c) is altered with increasing age, before and after cardioplegic arrest. Methods: Using an isolated rat heart model, increasing concentrations of ETT and S6c were administered to rats of different ages (group I=one month; group II-five months; group III=21 months). An identical series of experiments was performed following the addition of indomethacin and N-G-nitro-L-arginine methyl ester (L-NAME) to the Krebs perfusion fluid. In a third series of experiments, increasing doses of ET-I were added to hearts following 4 h of cardioplegic arrest at 4 degrees C. Results: Coronary hows are expressed as a percentage of initial coronary flow+/-SEM. There was a greater decrease in coronary flow in the older rats for all doses of ET-1. ET-I (10(-9) M) reduced coronary flows to 72.8+/-3.7, 53.2+/-6.7 and 56.5+/-10.7% for groups I-Ill respectively (P=0.01 I vs. Ii; P=0.1 I vs. III). A similar response to ET-I was seen in hearts perfused with indomethacin and L-NAME when compared to those perfused without (P=NS) Perfusion with ET-I (10(-9) M) following 4 h of cardioplegic arrest reduced coronary flows to 40.5+/-4.9, 26.8+/-4.8 and 24.1+/-3.9%, respectively (P=0.08 I vs. II; P=0.03 I vs. III). Perfusion with S6c (10(-10) NI) produced coronary flows of 93.3+/-5.5, 77.0+/-3.5 and 73.9+/-3.9% for groups I-Ill, respectively (P=0.03 I vs. II; P=0.01 I vs. III). Perfusion with S6c (10(-9) M) in the presence of L-NAME and indomethacin reduced coronary hows to 85.7+/-3.0, 81.6+/-2.2 and 74.6+/-3.6% (P=NS I vs. Il; P=0.03 I vs. III). Conclusions: The coronary vasoconstrictor response to ET-I and S6c increases with age. The increased vasoconstriction in response to ET-I is independent of the decrease in NO release seen with ageing. (C) 1999 Published by Elsevier Science B.V. All rights reserved.