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Departments of Anesthesiology and Pharmacology, Tulane University Health Science Center, New Orleans, Louisiana 70112
Submitted 16 December 2002 ; accepted in final form 20 August 2003
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ABSTRACT |
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vasodilator responses; vasoconstrictor responses; oxidant; cyclooxygenase product; hypoxia
) first reported by Blough and Zafiriou in 1985 (5). Beckman and colleagues (1, 2) were the first to postulate that ONOO- and the resulting acid were a biologically relevant source of the cytotoxic hydroxyl radical that could induce endothelial cell injury. In acute lung injury, overproduction of NO by inducible NO synthase and of superoxide by activated neutrophils leads to the formation of ONOO- and tissue injury (1, 2, 10, 21-24). It has been reported that ONOO- is formed during endotoxin- and ischemia-induced pulmonary injury and exposure to CO and that ONOO- may be responsible for the observed vascular dysfunction (10, 18, 24, 27). However, the role of ONOO- in lung injury is controversial (11, 18). It has been reported that ONOO- inhibits leukocyte-endothelial cell interactions, has a protective effect in ischemia-reperfusion injury in the heart in vivo, and produces relaxation in isolated dog and human coronary arteries and in bovine pulmonary arteries, and it has been postulated that ONOO- modulates Ca2+ signaling in endothelial cells (7, 8, 16, 17, 31). However, in other studies, tolerance to the vasorelaxant response to ONOO- was observed, ONOO- and its product 3-nitro-L-tyrosine have been shown to impair responses to vasoactive agonists, and ONOO- contributes to ischemia-reperfusion injury in the isolated rat heart (3, 10, 14, 29, 32). The mechanism by which ONOO- impairs vascular responsiveness is uncertain, although it has been reported that sulfhydryl oxidation, lipid peroxidation enzyme, and DNA damage are involved (22, 23, 25, 33). It has also been reported that activation of the cyclooxygenase pathway and formation of NO donors may be involved in mediating responses to ONOO- (7, 15, 19, 26, 31). Despite many investigations into the biological activity of ONOO-, very little is known about the actions of ONOO- on the pulmonary and peripheral vascular beds of the rat. We, therefore, sought to investigate and compare responses to ONOO- in the pulmonary and systemic vascular beds of the rat and to examine the hypothesis that repeated short-term exposure to ONOO- alters responses to vasoactive agonists and hypoxia. |
METHODS |
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9-12 mmHg and was not changed during an experiment. The preparation was allowed to stabilize for 30 min before an experiment. In some experiments, responses to the vasodilator agents were investigated when pulmonary arterial perfusion pressure was raised to a high steady level by addition of U-46619 (10-8 to 10-9 M) to the perfusion reservoir. In experiments with hypoxia, the lung was ventilated with a 3% O2-5% CO2-balance N2 mixture to elicit a hypoxic pulmonary vasoconstrictor response. Arterial blood-gas tensions and pH were measured (Corning model 178, Corning Instruments), and blood pH was maintained between 7.35 and 7.45 by adding small amounts of NaHCO3 solution. The modified Krebs-Henseleit solution used in the isolated rat lung studies had the following composition (in g/l): 66.37 NaCl, 3.58 KCl, 3.68 CaCl2·2 H2O, 1.63 KH2PO4, 1.45 MgSO4·7 H2O, 1.6 NaHCO3, 1.0 dextrose (pH 7.4) and was prepared daily in double-distilled water.
Systemic arterial pressure and hindquarters preparation. Fifty-five Sprague-Dawley rats of either sex weighing 240-360 g were anesthetized with pentobarbital sodium (50 mg/kg iv), and supplemental doses were given during the course of the experiment to ensure a uniform level of anesthesia. The trachea was cannulated, and the rats breathed spontaneously or were ventilated with a Harvard model 683 rodent ventilator at a tidal volume of 2-4 ml and at a rate of 30 breaths/min with room air enriched with 95% O2-5% CO2. Catheters were inserted into the external jugular vein for the iv administration of agonists and antagonists and into the carotid artery for the measurement of systemic arterial pressure. In 20 rats, responses to iv injections of ONOO- and to the vasoactive agonists on systemic arterial pressure were investigated. For constant-flow perfusion of the hindquarters vascular bed (n = 34), a 1.0- to 1.5-cm segment of the distal aorta was exposed through a ventral midline incision and cleared of surrounding connective tissue. After administration of heparin sodium (1,000 U/kg iv), the aorta was ligated, and catheters were inserted into the aorta both proximal and distal to the ligature. Blood was withdrawn from the proximal catheter and pumped (Masterflex Pump, Cole-Parmer) at a constant flow rate into the distal aortic catheter. Perfusion pressure was measured from a lateral tap in the perfusion circuit located between the pump and the distal catheter. Agonists were injected directly into the hindquarters perfusion circuit distal to the pump in small volumes in a random sequence. Hindquarters blood flow was set to achieve a baseline perfusion pressure of 125 mmHg and was not changed during an experiment. The flow rate was determined by time collection and ranged from 5-7 ml/min. The lumbar sympathetic chain ganglia between L2 and L4 were ligated and cut to denervate the hindquarters vascular bed. Blood gases were measured and were within physiological range.
Vascular pressures were measured with Viggo-Spectramed transducers. Mean pressures were obtained by electronic averaging and were recorded on a Grass model 7 recorder (Grass Instruments) and, in some experiments, on a MacLab data-acquisition system (AD Instruments, Castle Hill, Australia) or a Biopac MP100 data-acquisition system.
Materials. Sodium peroxynitrite (Cayman Chemical) supplied as a solution in sodium hydroxide is stable for 1 mo when stored at -80°C. In the hindquarters experiments, ONOO- was diluted in the lowest doses used with saline and rapidly injected into the hindquarters perfusion circuit. In the other experiments, the ONOO- from Cayman was injected undiluted and responses were consistent with a number of Cayman ONOO- preparations. The concentration of ONOO- was determined by diluting an aliquot of the stock solution 40-fold with cold 0.3 M NaOH and measuring the absorbance at 302 nm with 0.3 M NaOH as a blank in a Beckman DU spectrophotometer. The concentration of the stock solution can be calculated using the extinction coefficient for ONOO- (1,670 M-1·cm-1). Doses of ONOO- administered ranged from 37 nmol to 10 µmol. U-46619 (11
,9-epoxymethano-9,11-dideoxyprostaglandin F2; Cayman Chemical), a thromboxane A2 mimic, was dissolved in ethanol 100% at a concentration of 10 mg/ml, and further dilutions were made in 0.9% NaCl. Angiotensin II, bradykinin, norepinephrine hydrochloride, adenosine triphosphate, serotonin creatinine sulfate, nitroglycerin, sodium nitroprusside, isoproterenol (Sigma Chemical), and the NO donor Proli/NO (supplied by Dr. Larry K. Keefer) (12) were dissolved in 0.9% NaCl. The adrenomedullin analog (ADM15-52) and sodium meclofenamate (Parke-Davis) were also dissolved in 0.9% NaCl. Working solutions of the vasoactive agonists were prepared on a frequent basis, stored in brown stoppered bottles, and kept on crushed ice during experiments. All injections were made in small volumes, and sufficient time was permitted between agonist injections for pressures to return to baseline values. Injections of the saline vehicle for the ONOO- solution or equivalent amounts of NaOH in saline or sodium nitrate or nitrite had no significant effect on pulmonary, systemic, or hindquarters perfusion pressure or on responses to the vasoactive agonists. In addition to controlling for the pH of the ONOO- solution and the presence of excess nitrite and nitrate, experiments were carried out with ONOO- that had been allowed to decompose for several hours or overnight at room temperature. In these experiments the decomposed ONOO- had very little effect on vascular pressure in the rat as seen in Fig. 1A.
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Responses to ONOO- were investigated in the pulmonary vascular bed under low- and high-tone conditions. Tone was increased with U-46619 (10-8 to 10-9 M), and in some experiments tone was increased by ventilating the lung with a 3% O2-5% CO2-balance N2 mixture. Ventilation with the hypoxic gas mixture decreased PO2 but did not change PCO2 or pH (pH: control 7.35 ± 0.1, hypoxia 7.43 ± 0.1; arterial PO2: control 116 ± 5 Torr, hypoxia 44 ± 3 Torr; arterial PCO2: control 28 ± 3 Torr, hypoxia 27 ± 2 Torr).
In experiments carried out to determine whether ONOO- altered pulmonary vascular responses, responses to nitroglycerin, nitroprusside, the NO donor Proli/NO, isoproterenol, the adrenomedullin analog ADM15-52, angiotensin II, bradykinin, norepinephrine, and serotonin were obtained before and beginning 10-30 min after exposure to ONOO-.
The role of cyclooxygenase product release in mediating or modulating responses to ONOO- was studied by use of the cyclooxygenase inhibitor sodium meclofenamate, and responses to ONOO- were compared before and beginning 10-30 min after administration of meclofenamate. The effect of meclofenamate on responses to arachidonic acid was used to assess the extent of cyclooxygenase blockade.
Statistics. The hemodynamic data are expressed (in mmHg) as means ± SE. The data were analyzed by using analysis of variance with post hoc Scheffé's F test (StatView, Abacus Concepts) on a Power Macintosh 7200/75, and a P value of <0.05 was used as the criterion for statistical significance.
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RESULTS |
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Effects in the hindquarters vascular bed. Responses to injections of ONOO- were investigated in the hindquarters vascular bed, and these data are summarized in Fig. 2. Under constant-flow conditions, injections (37-3,700 nmol) of ONOO- into the hindquarters perfusion circuit produced dose-related decreases in perfusion pressure (Fig. 2A). The time course of the decrease in hindquarters perfusion pressure in response to injection of a midrange dose of ONOO- (1,110 nmol) is shown in Fig. 2B. The vasodilator response was rapid in onset, and perfusion pressure returned to control value within 240 s. Hindquarters vasodilator responses to repeated injections of a midrange dose (1,110 nmol) of ONOO- were assessed over time, and responses were not different from control at time periods of up to 4 h (Fig. 3A). Hindquarters vasodilator responses to acetylcholine and ATP or vasoconstrictor responses to angiotensin II were not different from control after 3-6 injections of 370-3,700 nmol ONOO- (Fig. 3B).
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Effect of ONOO- on pulmonary arterial perfusion pressure. Pulmonary vascular responses to ONOO- were investigated in the isolated blood perfused rat lung, and these results are summarized in Fig. 4. Under baseline-tone conditions when pulmonary arterial perfusion pressure averaged 10 ± 2 mmHg, injections of ONOO- (1.23 and 4.10 µmol) into the perfusion circuit increased perfusion pressure in a dose-related manner (Fig. 4A). Pulmonary pressor responses to ONOO- were rapid in onset and short in duration. The recovery half-times of the responses to ONOO- in 1.23 and 4.10 µmol injections were 25 ± 5 and 85 ± 17 s, respectively.
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Influence of ONOO- under elevated-tone conditions. Responses to ONOO- were also investigated under elevated-tone conditions in the isolated, blood-perfused rat lung, and these data are summarized in Fig. 4B. When pulmonary arterial pressure had been increased to a high steady value of 33 ± 5 mmHg with addition of U-46619 to the perfusion reservoir, injections of ONOO- of 1.23 and 4.10 µmol increased pulmonary arterial perfusion pressure in a dose-dependent manner. Responses were rapid in onset and short in duration, and perfusion pressure returned to baseline value and did not fall below baseline value (Fig. 4B). The recovery half-time of the response to ONOO- injections under elevated-tone conditions was 22 ± 1 and 30 ± 3 s, respectively. The effect of the passage of time on the pulmonary pressor response to injections of ONOO- in 1.23 and 4.10 µmol injections was investigated, and responses were not different when compared during the control period and after a 30- to 60-min period (Fig. 5A).
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Influence of ONOO- on pulmonary vascular responses. The effects of repeated injections of ONOO- on pulmonary vascular responses to ventilatory hypoxia and vasoactive agonists were investigated, and these data are summarized in Figs. 5B and 6. Pulmonary pressor responses to three sequential trials of ventilation with a 3% O2-5% CO2-balance N2 gas mixture for 5-8 min before and after 6-10 injections of ONOO- (1.23 and 4.10 µmol) were compared. Ventilation with the hypoxic gas mixture produced a consistent increase in pulmonary arterial perfusion pressure in the three hypoxic trials (Fig. 5B). Repeated injections of ONOO- in doses of 1.23 and 4.10 µmol (total 6-10 injections) had no significant effect on the hypoxic pulmonary vasoconstrictor response (Fig. 5B).
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Short-term exposure to ONOO- (5-10 injections of 1.23 or 4.10 µmol) did not decrease pulmonary vasodilator responses to nitroglycerin, nitroprusside, the NO donor Proli/NO, isoproterenol, or to the adrenomedullin analog ADM15-22 when pulmonary vascular tone was increased with U-46619 (Fig. 6).
Short-term exposure to ONOO- (5-10 injections of 1.23 or 4.10 µmol) did not alter pulmonary pressor responses to injections of angiotensin II, norepinephrine, or serotonin when responses were studied under baseline-tone conditions (data not shown).
Effects of NaNO2 and NaNO3 and decomposed ONOO-. Responses to sodium nitrate and sodium nitrite were studied under baseline and high-tone conditions in the pulmonary vascular bed and in the hindquarters vascular bed under constant-flow conditions. Injections of sodium nitrate or sodium nitrite in doses up to 300 µg had little if any effect on pulmonary arterial pressure under baseline-tone conditions or under elevated-tone conditions (data not shown). In the hindquarters vascular bed, injections of sodium nitrate or sodium nitrite in doses of 10-300 µg, amounts greater than could be present in the ONOO- solution, had little if any effect on hindquarters perfusion pressure (data not shown). The injections of ONOO- that had been allowed to decompose for several hours or overnight at room temperature were investigated, and injection of the decomposed ONOO- in doses up to 4.8 µmol had very little effect on hindquarters or pulmonary arterial pressures (data not shown) and in doses of 4.8 and 10 µmol had little effect on systemic arterial pressure (Fig. 1A).
Role of cyclooxygenase product release. The role of cyclooxygenase product release in mediating or in modulating responses to ONOO- in the hindquarters and pulmonary vascular bed was investigated, and these data are summarized in Fig. 7. In the hindquarters vascular bed, the decreases in hindquarters perfusion pressure in response to injections of ONOO- (111-1,110 nmol) were not altered at a time when vasodilator responses to the prostaglandin precursor arachidonic acid were significantly decreased after administration of sodium meclofenamate (Fig. 7A). The increases in pulmonary arterial perfusion pressure in response to injections of ONOO- (1.23 and 4.10 µmol) were not altered after addition of sodium meclofenamate (2.5 mg/kg) to the perfusion reservoir (Fig. 7B). The response to arachidonic acid was decreased significantly in the isolated perfused rat lung after administration of the cyclooxygenase inhibitor (data not shown).
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DISCUSSION |
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ONOO- is a potent oxidant that, after protonation, is highly unstable and forms species with the reactivity of hydroxyl radical (1, 2, 10, 22, 23). In addition to generating nitrogen dioxide and hydroxyl radical, ONOO- reacts with thiols and other substituents to form NO donors (1, 7, 19, 21, 31). Injections of ONOO- in the nanomole and low micromole range caused dose-related decreases in systemic arterial pressure and hindlimb vascular resistance, and responses to ONOO- were rapid in onset and did not exhibit tachyphylaxis. The observation that decomposed ONOO- had little effect on vascular pressures in the present study suggests that responses were not mediated by breakdown products, such as nitrogen dioxide, or the alkaline pH of the solution or by H2O2 (19). In addition, sodium nitrite and nitrate, which are present in the ONOO- preparation and can be formed from the breakdown of an S-nitrosothiol, had little if any effect on vascular pressures in the present study. The observation that ONOO- can react with plasma proteins, such as albumin, that inactivate it and form NO donors may be the explanation for the decrease in arterial pressure and hindlimb vascular resistance observed in response to injected ONOO- in the rat and is similar to responses to NO donors, such as sodium nitroprusside (19).
Repeated injections of ONOO- produced reproducible responses, but the tachyphylaxis, increased arterial pressure, and elevated hindquarters vascular resistance observed in other studies, suggesting that ONOO- may be involved in the pathogenesis of hypertension (3, 4, 6, 13, 29), were not observed in the present study. Repeated short-term ONOO- administration did not affect the hypoxic pulmonary vasoconstrictor response or responses to a variety of vasoactive agonists. These results are in contrast to the reported selective impairment of adrenoreceptor-mediated responses in the rat but are in general agreement with the concept that ONOO- did not affect responses to acetylcholine in pulmonary arterial rings or alter responses to vasopressin in the hindquarters vascular bed of the rat (3, 4, 6, 13). It has also been reported that repeated injections of ONOO- result in attenuated responses to acetylcholine and PGI2 in the rat but that responses to bradykinin and an NO donor were preserved (3, 4, 6).
A number of studies in the literature indicate that ONOO- is a cytotoxic molecule capable of causing sulfhydryl oxidation, lipid peroxidation, and enzyme and DNA damage (1, 2, 21-23, 25, 33). It has been reported that ONOO- produces vascular dysfunction and impairs responses to vasodilator agents and that ONOO- generation contributes to ischemia-reperfusion injury in the isolated rat heart (3, 4, 29, 32). However, in other studies, administration of ONOO- protects against ischemiare-perfusion injury in vivo in both rats and cats when injected into the blood (16, 20). The difference in results in the literature may depend on the organ system studied, the experimental conditions, the presence of plasma proteins in the organ perfusate, the time of exposure, and amount of ONOO- administered, and whether ONOO- is endogenously formed or exogenously administered (3, 4, 6, 13, 16, 19, 20, 32).
It has been reported that ONOO- relaxes vascular smooth muscle and that formation of S-nitrosothiols and NO donors are involved (7, 19, 31). It has, however, been reported that ONOO--mediated vasorelaxation does not involve the formation of S-nitrosothiols and may be mediated by the activation of poly adenosine 5'-diphosphoribose synthase (6, 9). The results of the present study showing that ONOO- has substantial vasodilator activity in the systemic vascular bed of the rat are consistent with previous studies (16, 17, 19, 20). The observation that responses to ONOO- were reproducible and did not exhibit tachyphylaxis or cause vascular dysfunction, as measured by responses to endothelium-dependent vasodilator agents, argues against a role for the formation of large amounts of a chemical species cytotoxic to the endothelium. The vasodilator responses observed in the systemic vascular bed in the present study are consistent with the hypothesis that exogenous ONOO- can be rapidly converted to an NO donor in the presence of plasma proteins (19). However, the observation that ONOO- causes vasoconstriction in the isolated rat lung perfused with blood suggests that chemical species other than or in addition to an NO donor are formed, although it has been reported that NO can induce vasoconstriction in the rat lung (30). The identity of the vasoconstrictor substance formed when ONOO- is injected into the isolated blood perfused rat lung is unknown but is not a product in the cyclooxygenase pathway, because responses were not altered by a cyclooxygenase inhibitor (15, 26).
In summary, the present results show that exogenous ONOO- in nanomole and low micromole doses decreases systemic arterial pressure and hindlimb vascular resistance in the rat and induces vasoconstriction in the isolated, blood-perfused rat lung. Responses to ONOO- were reproducible, did not exhibit tachyphylaxis, and were not altered by a cyclooxygenase inhibitor. Repeated administration of ONOO- did not impair responses to vasoactive agonists, including acetylcholine and bradykinin, and did not alter hypoxic pulmonary vasoconstriction. These results show that ONOO- produces opposite cyclooxygenase-independent responses in the pulmonary and systemic vascular beds and that repeated short exposure to exogenous ONOO- does not impair vascular responsiveness in the rat.
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GRANTS |
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ACKNOWLEDGMENTS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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REFERENCES |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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