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Endothelial nitric-oxide synthase (eNOS) is an important regulator of endothelial function and vascular tone in biological tissues. While endothelial dysfunction occurs following ischemia and has been attributed to altered NO⋅ formation, the biochemical basis for this dysfunction is unknown. Therefore, studies were performed to determine the effects of myocardial ischemia and reperfusion on eNOS in isolated rat hearts subjected to periods of global ischemia or ischemia followed by reperfusion. eNOS activity was assayed by l-14Carginine tol-14Ccitrulline conversion and alterations in the amount and distribution of eNOS determined by Western blotting and immunohistochemistry. While activity was preserved after 30 min of ischemia with a value of 1.1 ± 0.1 pmol × min−1 × mg of protein−1, it decreased by 77% after 60 min and became nearly undetectable after 120 min. Reperfusion resulted in only a partial restoration of activity. The decline in activity with ischemia was due, in part, to a loss of eNOS protein. Hemodynamic studies showed that the onset of impaired vascular reactivity paralleled the loss of functional eNOS. Subjecting isolated eNOS to conditions of acidosis, which occur during ischemia, followed by restoration of pH as occurs on reperfusion, caused a combination of reversible and irreversible loss of activity similar to that seen in ischemic and reperfused hearts. Thus, loss of endothelial function following ischemia is paralleled by a loss of eNOS activity due to a combination of pH-dependent denaturation and proteolysis. Endothelial nitric-oxide synthase (eNOS) is an important regulator of endothelial function and vascular tone in biological tissues. While endothelial dysfunction occurs following ischemia and has been attributed to altered NO⋅ formation, the biochemical basis for this dysfunction is unknown. Therefore, studies were performed to determine the effects of myocardial ischemia and reperfusion on eNOS in isolated rat hearts subjected to periods of global ischemia or ischemia followed by reperfusion. eNOS activity was assayed by l-14Carginine tol-14Ccitrulline conversion and alterations in the amount and distribution of eNOS determined by Western blotting and immunohistochemistry. While activity was preserved after 30 min of ischemia with a value of 1.1 ± 0.1 pmol × min−1 × mg of protein−1, it decreased by 77% after 60 min and became nearly undetectable after 120 min. Reperfusion resulted in only a partial restoration of activity. The decline in activity with ischemia was due, in part, to a loss of eNOS protein. Hemodynamic studies showed that the onset of impaired vascular reactivity paralleled the loss of functional eNOS. Subjecting isolated eNOS to conditions of acidosis, which occur during ischemia, followed by restoration of pH as occurs on reperfusion, caused a combination of reversible and irreversible loss of activity similar to that seen in ischemic and reperfused hearts. Thus, loss of endothelial function following ischemia is paralleled by a loss of eNOS activity due to a combination of pH-dependent denaturation and proteolysis. Over the last decade it has been shown that the endothelium plays a critical role in the control of vascular tone (1Furchgott R.F. Zawadzki J.V. Nature. 1980; 288: 373-376Crossref PubMed Scopus (10026) Google Scholar). A labile vasodilating substance termed endothelium-derived relaxing factor was identified as nitric oxide (NO⋅), 1The abbreviations used are: NO⋅, nitric oxide; eNOS, endothelial nitric-oxide synthase; NOS, nitric-oxide synthase; l-NAME, N-nitro-l-arginine methyl ester. which is synthesized by a calcium dependent nitric-oxide synthase (NOS) in endothelial cells (2Palmer R.M.J. Ferrige A.G. Moncada S. Nature. 1987; 327: 524-526Crossref PubMed Scopus (9366) Google Scholar, 3Huang P.L. Huang Z. Mashimo H. Bloch K.D. Moskowitz M.A. Bevan J.A. Fishman M.C. Nature. 1995; 327: 239-242Crossref Scopus (1789) Google Scholar, 4Pollock J.S. Forstermann U. Mitchell J.A. Warner T.D. Schmidt H.H.H.W. Nakane M. Murad F. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 10480-10484Crossref PubMed Scopus (900) Google Scholar). Subsequently, it was observed that ischemia causes impaired endothelial reactivity (5Johns R.A. Linden J.M. Peach M.J. Circ. Res. 1989; 65: 1508-1515Crossref PubMed Scopus (158) Google Scholar). In the heart it was observed that coronary artery occlusion, as occurs in heart attack, results in endothelial dysfunction. In both isolated vascular ring andin vivo models, endothelium-dependent vasodilation is markedly decreased after myocardial ischemia and reperfusion (6Van Benthuysen K.M. McMurtry I.F. Horwitz L.D. J. Clin. Invest. 1987; 79: 265-274Crossref PubMed Scopus (331) Google Scholar, 7Tsao P.S. Lefer A.M. Am. J. Physiol. 1990; 259: H1660-H1666PubMed Google Scholar, 8Mehta J.L. Nichols W.W. Donnelly W.H. Lawson D.L. Saldeen T.G.P. Circ. Res. 1989; 64: 43-54Crossref PubMed Scopus (153) Google Scholar). Studies in humans have identified a similar decline in acetylcholine-induced vasodilation or even paradoxical vasoconstriction in areas adjacent to atherosclerotic plaques present in coronary arteries (9Ludmer P.L. Selwyn A.P. Shook T.L. Wayne R.R. Mudge G.H. Alexander R.W. N. Engl. J. Med. 1986; 315: 1046-1051Crossref PubMed Scopus (2032) Google Scholar). Likewise, a diffuse vasomotor impairment in hypercholesterolemic and diabetic patients was seen showing that vascular dysfunction might occur in different pathological conditions (10Zeiher A.M. Drexler H. Saurier B. Just H. J. Clin. Invest. 1993; 92: 652-662Crossref PubMed Scopus (629) Google Scholar, 11Johnstone M.T. Creager S.J. Scales K.M. Cusco J.A. Lee B.K. Creager M.A. Circulation. 1993; 88: 2510-2516Crossref PubMed Scopus (1009) Google Scholar). Because of the major pathophysiological significance of impaired endothelial reactivity following ischemia, there has been great interest in determining its underlying mechanisms. Earlier studies verified that while receptor mediated endothelial-dependent responses to acetylcholine as well as receptor-independent responses to calcium ionophore A23187 were lost, endothelium-independent agents such as the NO⋅ donor nitroprusside could elicit normal vasodilation in coronary rings of reperfused hearts (5Johns R.A. Linden J.M. Peach M.J. Circ. Res. 1989; 65: 1508-1515Crossref PubMed Scopus (158) Google Scholar, 6Van Benthuysen K.M. McMurtry I.F. Horwitz L.D. J. Clin. Invest. 1987; 79: 265-274Crossref PubMed Scopus (331) Google Scholar). These observations implied the site of inhibition of endothelium-dependent dilation to be distal to receptor-mediated events and proximal to the activation of the vascular smooth muscle, suggesting that impaired enzymatic synthesis of NO⋅ could be one of the mechanisms causing endothelial dysfunction. However, questions remain regarding the contribution of a dysfunctional NO⋅ synthetic pathway to the endothelial impairment of postischemic hearts. The exact alterations in eNOS activity and expression which occur during ischemia or following reperfusion are unknown. Furthermore, it is not known what processes during ischemia cause these alterations. In the heart, the endothelial isoform of nitric-oxide synthase (eNOS or NOS III) present in the endothelium of coronary vessels and myocardium, normally accounts for most NO⋅ production (12Ursell P.C. Mayes M. Cardiovasc. Res. 1993; 27: 1920-1924Crossref PubMed Scopus (46) Google Scholar, 13Balligand J.L. Kobzik L. Han X. Kaye D.M. Belhassen L. O'Hara D.S. Kelly R.A. Smith T.W. Michel T. J. Biol. Chem. 1995; 270: 14582-14586Abstract Full Text Full Text PDF PubMed Scopus (362) Google Scholar). Catalysis by eNOS involves the oxidation of one of the terminal guanidino nitrogens of l-arginine to yield NO⋅ plusl-citrulline (14Lowenstein C.J. Dinerman J.L. Snyder S.H. Ann. Intern. Med. 1994; 120: 227-237Crossref PubMed Scopus (853) Google Scholar). Since nitric-oxide synthases are relatively labile enzymes (15Bredt D.S. Snyder S.H. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 682-685Crossref PubMed Scopus (3128) Google Scholar) whose function can be impaired by a variety of conditions (16Marletta M.A. J. Biol. Chem. 1993; 268: 12231-12234Abstract Full Text PDF PubMed Google Scholar), we hypothesized that the metabolic disorders associated with ischemia and reperfusion might also impair eNOS activity. Therefore, the purpose of this study was to characterize the alterations in eNOS that occur following myocardial ischemia, as well as the mechanisms that trigger this process and their significance in causing endothelial dysfunction. Female Sprague Dawley rats (250–300 g) were heparinized and anesthetized with intraperitoneal pentobarbital. The hearts were excised, the aorta cannulated, and retrograde perfusion initiated at a pressure of 80 mm Hg using Krebs bicarbonate buffer (17 mm glucose, 120 mmsodium chloride, 25 mm sodium bicarbonate, 2.5 mm calcium chloride, 0.5 mm EDTA, 5.9 mm potassium chloride, and 1.2 mm magnesium chloride) bubbled with 95% O2 and 5% CO2 gas at 37 °C, as described previously (17Thompson-Gorman S.L. Zweier J.L. J. Biol. Chem. 1990; 265: 6656-6663Abstract Full Text PDF PubMed Google Scholar). Hearts were perfused for 15 min to allow functional stabilization and then subjected to the desired duration of 37 °C global ischemia or ischemia followed by reflow. For studies of endothelial reactivity, coronary flow was measured after 15 min of perfusion. The vasodilatory effect of histamine (10−5m) was then tested to measure base-line endothelium-dependent relaxation. Hearts were then subjected to control perfusion or 30, 60, or 90 min of ischemia followed by 45 min of reperfusion, and the effect of histamine on coronary flow was reassessed followed by measurement of the effect of the eNOS inhibitor N-nitro-l-arginine methyl ester (l-NAME, 1 mm). Hearts were immediately frozen in liquid nitrogen, finely ground, and suspended in 3 ml of ice-cold buffer consisting of 50 mm Tris, pH 7.4, containing 0.1 mm EDTA, 0.1 mm EGTA, 12 mm mercaptoethanol, and the protease inhibitors 2 mm phenylmethylsulfonyl fluoride and 4 μmleupeptin. The suspension was homogenized and centrifuged at 100,000 × g for 60 min at 5 °C. The particulate fraction was subsequently washed in 3 ml of ice-cold buffer containing 1 m KCl for 5 min and centrifuged at 100,000 ×g for 30 min at 5 °C. The supernatant was discarded, and the pellet was rinsed several times with buffer to remove excess KCl. Finally, the pellet was resuspended in buffer containing calmodulin (330 nm) and tetrahydrobiopterin (10 μm) which are essential cofactors removed from the pellet by the KCl wash (18Forstermann U. Pollock J.S. Schmidt H.H.H.W. Heller M. Murad F. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 1788-1792Crossref PubMed Scopus (551) Google Scholar). eNOS activity was measured from the conversion rate of l-14Carginine tol-14Ccitrulline (19Bredt D.S. Snyder S.H. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 9030-9033Crossref PubMed Scopus (1750) Google Scholar) in heart tissue subcellular preparations. The reaction mixture contained 3.0 mm NADPH, 200 μm CaCl2, 30 μm EDTA, 30 μm EGTA, 100 nmcalmodulin, and 3 μm tetrahydrobiopterin in Tris buffer. The reaction was initiated by the addition of purifiedl-14Carginine (317 mCi/mmol) to produce a 10 μm final concentration and carried out for 8 min at 37 °C. The reaction was quenched with 3 ml of ice-cold stop buffer (20 mm HEPES and 2 mm EDTA, pH 5.5). Experiments were also performed in the presence of either EGTA (5.0 mm) or l-NAME (250 μm).l-14CCitrulline content was determined by liquid scintillation counting after separation from the reaction mixture by passage through a column of the cation exchange resin Dowex AG 50WX-8 of the of buffer containing l-14Carginine in the of heart tissue were to the Dowex resin column to determine which were from eNOS activity was determined by and for content by the and conversion activity was and by EGTA, that it was from eNOS. For studies of the pH of particulate of heart were with of potassium buffer different final pH and eNOS activity was pH that occur in ischemic and reperfused were with buffer at pH for or 10 min or followed by restoration of pH to with eNOS activity was measured at either immediately after of pH or after 10 min. and reperfused heart were in pH containing and 5% followed by denaturation at °C for 5 min. were a and was carried out for 30 min at 200 were to a and with 5% in with the of eNOS were with the at 4 °C. were with the for 2 at followed by with the was for 10 min using a of heart tissue were in for 10 min and on The were rinsed with and for min in a with 0.1 followed by and then quenched with were with and normal for 10 min. was by of a the of the eNOS and The was used for were with in of with and heart were with and in the of the and were from was from and and were from and eNOS were from are as ± and significance of was by A value of or was to eNOS activity was in hearts subjected to 30 min of ischemia ± pmol × mg of with ± pmol of × mg of However, ischemic of 30 min caused a decline in eNOS which became undetectable after 120 min of global heart ischemia ± pmol × mg of These while in the of ischemia, normal activity after ischemia of 60 min or eNOS activity is markedly impaired and a factor for the synthesis of activity in these was contribution of the isoform of the nitric-oxide synthase to the global activity reperfusion, a partial of eNOS activity was observed 60 min of ischemia followed by 45 min of reperfusion, eNOS activity with ischemic from a to of control In this to be in hearts subjected to periods of ischemia, suggesting that metabolic alterations that occur during ischemia cause the loss of eNOS determine alterations in the amount of eNOS after ischemia and reperfusion for the in Western of eNOS were performed on the heart A of with eNOS was by the of and of the eNOS was at 60 and 90 min of myocardial ischemia with of and 3 A and 4 However, alterations were seen after reperfusion 3 and 4 These that of the loss in activity that occurs is due to process is flow is that it is ischemia reperfusion which results in of eNOS in Western of and reperfused heart to the value of the from and are A that 60 min of ischemia a in the eNOS that to of control after 90 min. In results are shown after 60 or 90 min of ischemia or ischemia followed by 45 min of reperfusion. is seen after In control hearts a of endothelial was seen with in in and The in eNOS with ischemia paralleled seen in the Western periods of ischemia of 30 min or of eNOS in endothelial cells or was In ischemic of 60 min a loss of in endothelium and with a loss of in These observations that loss of eNOS occurs in endothelial cells and followed by loss in the loss of eNOS with the onset of endothelial studies were performed to measure endothelial-dependent and endothelial-dependent of the endothelium-dependent histamine (10−5m) in the coronary of rat hearts at conditions a in coronary the hearts were to 30 min of ischemia followed by 45 min flow was only ± with the control ± flow mm) was preserved 30 ± control ± However, periods of ischemia were associated with a decline of the endothelial These results that endothelium-dependent is impaired after ischemic periods markedly impaired after The of the onset of this endothelial dysfunction that of the loss of eNOS activity. Because pH markedly in ischemic hearts and cause a loss of the effect of pH on eNOS activity was particulate that eNOS were the pH from to and activity was measured and as a function of pH eNOS function was observed at pH markedly decreased at pH and at pH function was The observed of activity pH was by the with a value of Therefore, eNOS activity is in the and activity is as the pH is with the of a functional with of determine the effect of periods of similar to that in the ischemic heart after 30 min of ischemia pH to as well as the effects of pH as occurs on reperfusion, of the eNOS was performed in buffer at pH followed by to pH and in this buffer caused a loss of enzymatic as in the for 1 min to this pH resulted in a in eNOS as as 10 min a decline of of the enzymatic activity. However, were at pH for 10 min after to pH and then assayed for a partial the in vivo was subjected to periods of of only 1 or 5 of activity was seen 10 min following of pH and with to for periods of 10 min restoration of activity was seen with a in eNOS activity from the value observed immediately after pH These results that the pH associated with ischemia might a role in the loss of eNOS activity the of pH to during reperfusion to the partial restoration of endothelium-dependent occurs in hearts subjected to ischemia or ischemia followed by reperfusion (6Van Benthuysen K.M. McMurtry I.F. Horwitz L.D. J. Clin. Invest. 1987; 79: 265-274Crossref PubMed Scopus (331) Google Scholar, 7Tsao P.S. Lefer A.M. Am. J. Physiol. 1990; 259: H1660-H1666PubMed Google Scholar, 8Mehta J.L. Nichols W.W. Donnelly W.H. Lawson D.L. Saldeen T.G.P. Circ. Res. 1989; 64: 43-54Crossref PubMed Scopus (153) Google X. Lefer Lefer A.M. Circ. Res. 1993; PubMed Google Scholar). receptor-mediated and endothelial responses are while endothelium-independent responses to which NO⋅ are (5Johns R.A. Linden J.M. Peach M.J. Circ. Res. 1989; 65: 1508-1515Crossref PubMed Scopus (158) Google Scholar, 6Van Benthuysen K.M. McMurtry I.F. Horwitz L.D. J. Clin. Invest. 1987; 79: 265-274Crossref PubMed Scopus (331) Google Scholar). Therefore, it has been that impaired NO⋅ synthesis from eNOS is the cause of postischemic endothelial the basis for this was not While it was hypothesized that alterations in eNOS might the and of these alterations were not previously The the of the alterations in eNOS and activity during myocardial ischemia and the role of these alterations in endothelial dysfunction. The of NO⋅ from NOS can be by a of the and activity of the the concentration of the NADPH, and and the of the tetrahydrobiopterin (16Marletta M.A. J. Biol. Chem. 1993; 268: 12231-12234Abstract Full Text PDF PubMed Google Scholar, Snyder S.H. Zweier J.L. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). While of these NO⋅ the presence and activity of the is of Therefore, the present study on the alterations in and function which occur during myocardial ischemia and reperfusion. was observed that ischemic of 30 min or not activity as measured by l-arginine measured by Western blotting were also and on in the amount or of the was However, with ischemic of 60 min or a loss of activity was seen with 77% loss after 60 loss after 90 and loss after 120 min. loss of activity was after reperfusion, even after 45 min of eNOS activity was markedly decreased from with and seen in hearts which were subjected to 60, or 120 min of ischemia, Thus, ischemia resulted in a loss of activity with only partial restoration during reperfusion. to the of these isolated heart there was for these observations that ischemia results in a combination of irreversible and reversible denaturation of eNOS. loss of activity could be due to of eNOS protein. Western blotting that while ischemic times of 30 min caused in ischemic times of 60 or 90 caused of and has been previously that ischemia results in activation of J.M. N. Engl. J. Med. PubMed Scopus Google Scholar). Therefore, it is that the loss of eNOS on Western blotting is due to loss of eNOS was not after reperfusion that the partial restoration of eNOS activity seen after reperfusion was not due to results in which can in denaturation with a loss of activity. studies have previously that after 30 min of global ischemia pH to a value of J.L. A. Med. 1995; PubMed Scopus Google Scholar). has been previously that eNOS its function the pH is to this M. Circ. Res. 1994; PubMed Scopus Google Scholar). Experiments were performed to determine the effect of on the activity of eNOS. observed that loss of activity to the of a functional with of Since this value most to that for of the of and the critical of eNOS an it is that the loss of activity seen to of the it could of on the or be due to effects on These that the loss of eNOS activity observed during ischemia could be by the following in the of a critical in this to altered with a protein. Finally, in the then be to to the irreversible loss of activity. the loss of eNOS activity seen during ischemia that from reversible loss of activity to irreversible loss of activity by of the also for the loss of activity and that occurs as a function of ischemic The with in could be of pH as occurs after the loss of activity be The of 1 and 2 a partial restoration of activity can be seen following reperfusion, while 3 the irreversible loss of which The loss of activity with the loss of endothelial vascular reactivity seen in these hearts. While only in reactivity were seen after 30 min of ischemia, after 60 loss of vascular reactivity of the NOS inhibitor l-NAME decreased flow is normally after 60 min or 90 min of ischemia, this was decreased by 45 or the loss of eNOS activity A loss of vasodilation was seen with a after 60 min of ischemia and a 95% seen after 90 min of The loss of vasodilation at 30 min in addition to loss of alterations also occur that impair the vasodilatory vasodilation by calcium which in eNOS, a calcium dependent and following ischemia calcium are J. Clin. Invest. 1990; PubMed Scopus Google Scholar). in calcium in the postischemic heart could in base-line activation of eNOS that subsequently the of vasodilation to of NO⋅ in postischemic hearts after 30 min of ischemia that NO⋅ are after reperfusion with J.L. J. Biol. Chem. 1995; 270: Full Text Full Text PDF PubMed Scopus Google Scholar). Furthermore, it has been shown that after periods of ischemia NO⋅ occurs due to from J.L. A. Med. 1995; PubMed Scopus Google Scholar). Thus, while the loss in eNOS activity observed in the present study paralleled the observed loss of endothelial reactivity mechanisms also to this of alterations in eNOS eNOS and vascular of activity and are as of control while either in flow due to l-NAME mm) or due to histamine in a eNOS activity and are as of control while either in flow due to l-NAME mm) or due to histamine studies in endothelial have mechanisms that could also in enzymatic of has been that or of or could be has been shown that endothelial is by inhibitors of M.J. Cardiovasc. Res. 1986; PubMed Scopus Google Scholar) which in decreased and to l-arginine by endothelial cells can be causing the l-arginine to M. J.A. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: PubMed Scopus Google Scholar). tetrahydrobiopterin also endothelial function F. Circulation. 1995; PubMed Scopus Google Scholar). The that eNOS has been described A. A. J. Moncada S. J. Res. Scholar). While these mechanisms could to the alterations in vascular reactivity which occur during ischemia and reperfusion, the presence of functional eNOS, that loss of be of critical even alterations also these mechanisms could be important in the of altered vascular reactivity with relatively ischemic eNOS activity is has been that reperfusion also in of NO⋅ Am. J. Physiol. 1986; PubMed Google Scholar, Zweier J.L. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). Studies have that can postischemic P.S. Lefer A.M. Am. J. Physiol. 1990; 259: H1660-H1666PubMed Google Scholar, P.S. N. Lefer Lefer A.M. Circulation. 1990; PubMed Scopus Google Scholar, J.L. Lawson D.L. Nichols W.W. Am. J. Physiol. 1989; Google Scholar). it has been that and NO⋅ during reperfusion to which in causes Zweier J.L. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). Since is only during the of reperfusion this could be in of NO⋅ during the of reperfusion. However, the endothelial impairment in well after the of it is that the observed alterations in endothelial function be by of NO⋅ by has also been hypothesized that eNOS could be by during Am. J. Physiol. 1987; Scholar). However, we that function was impaired during ischemia and after reperfusion. Thus, we observed that a loss of eNOS activity during ischemia with only partial restoration reperfusion. loss of activity paralleled the loss of vascular reactivity that endothelial dysfunction due to decreased functional eNOS. The loss of activity and was by which was was followed by proteolysis. These that loss of eNOS activity and is a critical factor that results in endothelial dysfunction in the postischemic the of in the
Giraldez et al. (Fri,) studied this question.