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Relaxant Effects of Sodium Nitroprusside and NONOates in Goat Middle Cerebral Artery: Delayed Impairment by Global Ischemia–Reperfusion
NITRIC OXIDE: Biology and Chemistry Vol. 3, No. 1, pp. 85–93 (1999) Article ID niox.1999.0212, available online at http://www.idealibrary.com on
Relaxant Effects of Sodium Nitroprusside and NONOates in Goat Middle Cerebral Artery: Delayed Impairment by Global Ischemia–Reperfusion Juan B. Salom,* ,† ,1 Marı´a D. Barbera´,‡ Jose´ M. Centeno,* ,‡ Marta Ortı´,* ,‡ Germa´n Torregrosa,* ,† and Enrique Alborch* ,‡ *Centro de Investigacio´n, Hospital Universitario “La Fe,” †Departamento de Biologı´a Animal, and ‡Departamento de Fisiologı´a, Universidad de Valencia, Valencia, Spain
Received October 6, 1998, and in revised form February 1, 1999
Global cerebral ischemia and subsequent reperfusion induce early impairment of the vasodilator responses to hypercapnia and vasoactive substances. Nitric oxide (NO) is involved in the regulation of cerebral blood flow (CBF) in both health and disease. The present study was designed to assess possible changes in the cerebrovascular reactivity to NO donors induced by cerebral ischemia– reperfusion in goats. Female goats (n 5 9) were subjected to 20 min global cerebral ischemia under halothane/N 2O anesthesia. Sixteen additional goats were sham-operated as a control group. One week later the effects of ischemia–reperfusion on relaxations to NO donors sodium nitroprusside (SNP), diethylamine/NO (DEA/NO), diethylenetriamine/NO (DETA/NO), and spermine/NO (SPER/NO) were studied in rings of middle cerebral artery (MCA) isolated in an organ bath for isometric tension recording. SNP, DEA/NO, DETA/NO, and SPER/NO induced concentration-dependent relaxations of MCA precontracted with KCl (DEA/NO > SPER/NO > SNP > DETA/NO) or with endothelin-1 (DEA/NO > SNP > SPER/NO > DETA/NO). Relaxations were always higher in endothelin-1-precontracted arteries. One week after cerebral ischemia concentration– response curves to SNP and DEA/NO were displaced 1
To whom correspondence should be addressed at Centro de Investigacio´n, Hospital Universitario “La Fe,” Ave. Campanar 21, E46009 Valencia, Spain. Fax: 34 96 3868718. E-mail: jbsalom@ san.gva.es. 1089-8603/99 $30.00 Copyright © 1999 by Academic Press All rights of reproduction in any form reserved.
to the right, indicating a reduction in relaxant potency of NO donors. The classical nitrovasodilator SNP and NONOates induce relaxation of isolated goat MCA which is partially inhibited by arterial depolarization. Global cerebral ischemia followed by reperfusion induces delayed impairment of the relaxant effects of NO on cerebrovascular smooth muscle, which results in reduced vasodilatory potency of NO donors in large cerebral arteries. © 1999 Academic Press Key Words: nitric oxide; cerebral arteries; vasodilation; ischemia–reperfusion; goat.
Cerebral ischemia and subsequent reperfusion induce changes in the regulation of cerebral blood flow by CO 2 (1). Abolition or significant attenuation in the ability of hypercapnia to increase cerebral blood flow after global cerebral ischemia has been reported in rats (2, 3), cats (4, 5), dogs (6 – 8), and pigs (9, 10). Piglets have been widely used to study the effects of global cerebral ischemia induced by increasing intracranial pressure on the reactivity of cerebral arterioles to specific activation of membrane receptors and channels. Global ischemia reduces vasodilator responses to N-methyl-D-aspartate (NMDA) (11), calcitonin gene related peptide (CGRP) (12), oxytocin (13), and activation of ATP-sensitive K 1 channels (14), while a similar ischemic insult does not modify vasodilator responses to kainate (15) and 85
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activation of Ca 21-activated K 1 channels (16). In cats, global cerebral ischemia induces abolition of pial arteriolar dilation to acetylcholine (17–19), adenosine (17), and the Ca 21 ionophore A23187 (18), while vasoconstriction induced by serotonin and angiotensin are well preserved (17). Few studies have been made on the effects of cerebral ischemia on cerebrovascular reactivity assessed in vitro in the absence of influences from the cerebral parenchyma. Ischemia–reperfusion does not affect reactivity of isolated canine basilar artery (20), while reperfusion decreases myogenic reactivity and alters middle cerebral artery function after focal cerebral ischemia in rats (21). In the present study we have examined the effects of global cerebral ischemia on the vasodilator responses of isolated goat middle cerebral artery to NO donors, which are considered to have a potential role in therapeutic strategies related to NO (22). We have previously reported that sodium nitroprusside (SNP) 2 and other NO donors of the NONOate type induce relaxation of isolated rabbit basilar artery (23). Since there is evidence suggesting that NO is involved in the regulation of cerebral circulation both in health and disease (24), the effects of NO donors are being studied in models of cerebral ischemia (25, 26) and thrombus formation (27). We have investigated vasodilator responses of goat middle cerebral artery to SNP, diethylamine/NO complex (DEA/NO), diethylenetriamine/NO complex (DETA/ NO), and spermine/NO complex (SPER/NO), and we have looked for changes in the vasodilator responses to SNP and DEA/NO after transient global cerebral ischemia followed by 1 week of reperfusion. After ischemia, we used SNP as a representative of classic nitrovasodilators that releases NO through metabolic activation (28) and compared it with DEA/NO, a representative of NONOates that releases NO spontaneously on dissolution (29). EXPERIMENTAL PROCEDURES
Induction of transient global cerebral ischemia. Experiments were performed in accordance with the “Principles of Laboratory Animal Care” (NIH Publi2
Abbreviations used: SNP, sodium nitroprusside; DEA, diethylamine; DETA, diethyenetriamine; SPER, spermine; ET-1, endothelin-1; SIN-1, sydnonimine-1.
cation No. 86-23, revised 1985), as well as the guidelines from the Council of the European Economic Community (86/609/EEC, Article 5, Appendix II), promulgated by the Spanish legislation on March 14, 1988 (R.D. 223/1988). The experiments were carried out in 25 anesthetized (1.5% halothane in a mixture of 80% N 2 O and 20% O 2 ) female goats weighing 38 –54 kg. We have recently reported the details on the dynamic anatomy of the blood supply to the goat’s brain and surgical procedures to induce transient global cerebral ischemia (30). Briefly, the external carotid arteries were exposed bilaterally by means of an incision made along each mandible, and snare-type occluders were placed around them cephalad to the mandibular and occipital branches and tunneled subcutaneously to be exteriorized on the back of the horns. The animals were instrumented to continuously monitor the following parameters: cortical perfusion (laser-Doppler probe on the left dorsolateral cortex), intracranial pressure (16-G cannula advanced into the right lateral ventricle), monopolar electrocorticogram (needle electrode inserted into the right dorsolateral cortex and the reference electrode fastened to the goat’s ear), mean arterial blood pressure (femoral artery catheter), and heart rate (obtained from the blood pressure pulse by means of a tachymeter). After a period of stabilization following surgery, an episode of 20 min transient global cerebral ischemia was achieved in 9 goats by occlusion of the two external carotid arteries and simultaneous external compression of the jugular veins by means of a neck tourniquet. The reperfusion period started when the various occlusions were released, and it was monitored for 2 h. At the end of that time, the animals were recovered from anesthesia and returned to the pen. The animals were allowed to reperfuse for 7 days. A group of 16 sham-operated goats (the surgical procedures were followed but ischemia was not induced) served as the control group. Isometric tension recording in isolated arteries. The goats, previously sedated with 10 mg/kg im ketamine (Ketolar; Parke Davis, Barcelona, Spain) and anesthetized with iv 2% sodium thiopental (Tiobarbital; Braun Medical, Jae´n, Spain), were killed by iv injection of 30 mEq of KCl. The whole brain was rapidly removed, and the middle cerebral arter-
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ISCHEMIA–REPERFUSION AND RELAXATION TO NO DONORS
ies were dissected free and cut into cylindrical segments measuring 4 mm in length and about 1 mm in outer diameter. Each segment was prepared for isometric tension recording in an organ bath. Two stainless steel L-shaped pins (diameter of 125 mm) were introduced through the arterial lumen. One pin was fixed to the organ bath wall, while the other was connected to a strain gauge (Universal Transducing Cell, UC3; Gould Statham, Oxnard, CA) for isometric tension recording (Omniscribe D 5237-5; Houston Instrument, Gistel, Belgium). The organ bath contained 5 ml of Ringer–Locke solution, which was bubbled continuously with 95% O 2 and 5% CO 2 to give a pH of 7.3–7.4. The temperature was kept at 37°C. A resting tension of 1 g was applied to the arterial segments and they were allowed to equilibrate for 60 –90 min. Tension was readjusted when necessary and the bath fluid was changed every 15 min before starting the experiments. The contractile capacity of all the arterial segments was examined by exposure to a high-K 1 (50 mmol/L) Ringer–Locke solution. The relaxant effects of SNP, DEA/NO, DETA/NO, and SPER/NO were assessed by cumulative addition (10 29 –3 3 10 24 mol/L) to arterial segments previously contracted either with KCl (50 mmol/L) or with endothelin-1 (ET-1, 10 29 mol/L) in order to study whether arterial depolarization affected the relaxant effects of NO donors. Only one concentration–response curve was carried out in each arterial segment. The relaxant responses elicited by SNP, DEA/NO, DETA/NO, and SPER/NO were expressed as percentages of the active tone achieved with high-K 1 solution or ET-1. Maximal relaxant effects (E max) and concentrations of NO donors producing 50% of the E max (EC 50) were calculated for each concentration–response curve. The pEC 50 was calculated as the negative logarithm to base 10 of the EC 50 for statistical analysis. Student’s t test for grouped data or the Newman–Keuls test (for multiple comparisons) was used for statistical analysis, and P , 0.05 was considered statistically significant. SNP, DEA/NO, DETA/NO, and SPER/NO were from Research Biochemicals International (Natick, MA). ET-1 was from Peptide Institute (Osaka, Japan). Care was taken to protect SNP solutions from light due to its light-sensitivity (31). DEA/NO,
TABLE I
Hemodynamic Parameters [Cortical Perfusion (CP), Intracranial Pressure (ICP), Mean Arterial Blood Pressure (MABP), and Heart Rate (HR)] in Goats Subjected to Transient Global Cerebral Ischemia
CP (%) ICP (mm Hg) MABP (mm Hg) HR (beats/min)
Control
Ischemia
Reperfusion a
100 561 116 6 4 96 6 2
10 6 1** 10 6 1* 121 6 3 96 6 3
150 6 4**/74 6 4** 761 111 6 3 104 6 3
Note. Values are means 6 SE. a Two values of CP are given during reperfusion: first for early hyperemic reperfusion and second for delayed hypoperfusion. * Significantly different from control (P , 0.05). ** Significantly different from control (P , 0.01).
DETA/NO, and SPER/NO were dissolved and diluted in ice-cold 10 mmol/L NaOH to prevent NO release until addition to the organ bath (32). Final NaOH concentration in the organ bath did not have effects on the arterial isometric tension. The Ringer–Locke solution had the following composition (mmol/L): NaCl, 120; KCl, 5.4; CaCl 2 , 2.2; MgCl 2, 1.0; NaHCO 3, 25; and glucose, 5.6. In KCldepolarizing solution, NaCl was replaced by an equimolar amount of KCl. RESULTS
Bilateral occlusion of external carotid arteries and compression of jugular veins produced significant (P , 0.01) reduction in cortical perfusion to 10% of the control value, accompanied by a significant (P , 0.05) increase in intracranial pressure, which doubled the control value. No significant changes were induced in mean arterial blood pressure and heart rate. After 20 min ischemia, release of the occlusions induced a peak of hyperemic reperfusion for 64 6 17 min (cortical perfusion values significantly above control, P , 0.01), followed by delayed hypoperfusion which lasted until the end of the 2-h recording period (cortical perfusion values under control, P , 0.01). During reperfusion intracranial pressure decreased, and mean arterial blood pressure and heart rate were in the normal range (Table I). The four NO donors, SNP (10 29–3 3 10 24 mol/L), DEA/NO (10 29–3 3 10 24 mol/L), DETA/NO (10 27–3
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FIG. 1. Concentration–response curves to sodium nitroprusside (SNP), diethylamine/NO (DEA/NO), diethylenetriamine/NO (DETA/NO), and spermine/NO (SPER/NO) in MCA rings from sham-operated goats. (Top) Arteries precontracted with KCl (50 mmol/L). (Bottom) Arteries precontracted with endothelin-1 (ET-1, 10 29 mol/L). Values are means 6 SE from 16 rings with KCl and SNP, 16 rings with KCl and DEA/NO, 15 rings with KCl and DETA/NO, 15 rings with KCl and SPER/NO, 16 rings with ET-1 and SNP, 15 rings with ET-1 and DEA/NO, 14 rings with ET-1 and DETA/NO, and 15 rings with ET-1 and SPER/NO.
3 10 24 mol/L), and SPER/NO (10 28–3 3 10 24 mol/L) induced concentration-dependent relaxations of goat middle cerebral arteries obtained from shamoperated goats and precontracted with 50 mmol/L KCl (active tone, 2083 6 119 mg) or with 10 29 mol/L ET-1 (active tone, 1544 6 74 mg). In all cases arterial segments were fully relaxed (Fig. 1). According to the pEC 50 values for relaxation induced by each
NO donor, in KCl-precontracted arteries the order of potency was DEA/NO . SPER/NO .SNP . DETA/ NO, and in ET-1-precontracted arteries the order of potency was DEA/NO . SNP . SPER/NO . DETA/ NO. Values of pEC 50 and E max for the four NO donors and the two precontractile agents are summarized in Table II. The nature of the precontractile agent conditioned the relaxant response to NO donors. According to pEC 50 values, NO donors were always significantly (P , 0.01) more potent in ET-1-precontracted arteries than in KCl-precontracted arteries. Moreover, except for DEA/NO, E max values were always significantly higher in ET-1-precontracted arteries than in KCl-precontracted arteries (P , 0.01 for SNP and DETA/NO and P , 0.05 for SPER/NO; Table II). Active tone induced by KCl (50 mmol/L) in arteries obtained from goats subjected to transient global ischemia (1941 6 109 mg) did not differ from that induced in control arteries (sham-operated goats). Active tone induced by ET-1 (10 29 mol/L) was significantly (P , 0.05) higher in arteries from ischemic goats (1801 6 111 mg) when compared to control arteries. In arteries from ischemic goats, the relaxant responses elicited by NO donors differed from the responses obtained in arteries from shamoperated goats. With regard to SNP (Fig. 2), and according to pEC 50 values, the relaxant potency was significantly (P , 0.01) reduced in KClprecontracted and, to a higher extent, in ET-1precontracted arteries from goats subjected to ischemia. The E max for relaxant effects of SNP in KClprecontracted arteries was significantly (P , 0.01) higher after ischemia (Table II). As for DEA/NO (Fig. 3), a highly significant (P , 0.01) reduction in relaxant potency was observed in KCl-precontracted arteries obtained after ischemia and, to a lower extent, in ET-1-precontracted arteries. The E max for relaxant effects of DEA/NO in KCl-precontracted arteries was significantly (P , 0.01) higher after ischemia (Table II). DISCUSSION
This study shows that DEA/NO, DETA/NO, and SPER/NO, three NO/nucleophile complexes, induce relaxation of isolated goat middle cerebral arteries. Although the three NO donors showed the same
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ISCHEMIA–REPERFUSION AND RELAXATION TO NO DONORS TABLE II
Concentration–Response Curve Parameters for NO Donors in MCA from Sham-Operated (Control) and Ischemic Goats Control
SNP KCl ET-1 DEA/NO KCl ET-1 DETA/NO KCl ET-1 SPER/NO KCl ET-1
Transient global ischemia
pEC 50
E max (%)
n
pEC 50
E max (%)
n
5.4 6 0.1 6.7 6 0.1**
90 6 5 112 6 2**
16 16
5.0 6 0.1*** 6.2 6 0.1** ,***
105 6 2*** 109 6 5
16 13
6.5 6 0.1 7.2 6 0.1**
87 6 6 101 6 4
16 15
5.2 6 0.1*** 6.8 6 0.1** ,***
103 6 1*** 103 6 1
24 15
4.8 6 0.1 5.3 6 0.1**
88 6 3 106 6 3**
15 14
5.7 6 0.1 6.2 6 0.1**
88 6 4 100 6 2*
15 15
Note. Order of potency of NO donors: DEA/NO . SPER/NO . SNP . DETA/NO in KCl-precontracted arteries and DEA/NO . SNP . SPER/NO . DETA/NO in ET-1-precontracted arteries. Values are means 6 SE. * Significantly different from KCl-precontracted arteries (P , 0.05). ** Significantly different from KCl-precontracted arteries (P , 0.01). *** Significantly different from control arteries (P , 0.01).
efficacy by fully relaxing the arteries, significant differences were shown in their relaxant potencies. Independently of the precontractile agent used (KCl or ET-1), the order of potency was DEA/NO . SPER/ NO . DETA/NO, which coincides with what we have previously reported for rabbit basilar artery (23). The rate at which NO is released by NONOates on dissolution depends on the carrier nucleophile, determines half-life (DEA/NO, t 1/2 5 2.1 min; SPER/ NO, t 1/2 5 39 min; and DETA/NO, t 1/2 5 20 h) and correlates highly with the actual vasodilatory potency of each NONOate (29). Our results in this and previous studies confirm that the relaxant potencies of NO/nucleophile complexes in cerebral arteries can be predicted from data on the rates at which NO is generated in simple aqueous buffers: The lower the t 1/2 value, the higher the relaxant potency. The invariability of the order of potency of NONOates is in agreement with their mechanism of action. Since they are not drugs acting on membrane receptors but prodrugs that release NO, the order of potency does not depend on the receptor subtype present in the particular tissue studied. The classical nitrovasodilator SNP also relaxed goat middle cerebral arteries, but its relaxant potency is more variable than that of NONOates. The
vasodilatory potency was lower than that of SPER/NO in KCl-precontracted arteries, while it was higher that that of SPER/NO in ET-1precontracted arteries. In both cases the potency of SNP was lower than that of DEA/NO and higher than that of DETA/NO. In contrast, in rabbit basilar artery the relaxant potency of SNP is higher than that of DEA/NO (23). Vasodilatory effects of SNP are mediated by metabolic activation of the drug to NO by a SNP-directed, membrane-associated enzymatic activity present in vascular smooth muscle (28). This enzymatic activity may differ between species and may be affected by KCl-induced depolarization, and this could explain the variability in SNPinduced relaxation. Regardless of the NO donor, SNP, DEA/NO, DETA/NO, or SPER/NO, in our study relaxations were much more potent and effective in ET-1precontracted arteries than in KCl-precontracted arteries. SNP-induced relaxations of rabbit ear and femoral arteries are also reduced by high K 1 concentration with respect to ET-1-precontracted arteries (33). Endothelium-dependent relaxations induced by acetylcholine in rabbit mesenteric (34), ear, and femoral arteries (33) are also lower in KClprecontracted arteries than in agonist-precon-
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inhibition of arterial relaxation also applies to NONOates like DEA/NO, DETA/NO, and SPER/NO in goat cerebral arteries and is in agreement with the involvement of guanylyl cyclase activation to produce cGMP, which mediates NONOate-induced relaxation, as previously reported in rabbit basilar artery (23). In this study, 20 min global cerebral ischemia followed by 7 days of reperfusion affected reactivity of goat middle cerebral artery to NO donors. Both
FIG. 2. Concentration–response curves to SNP in MCA rings from sham-operated goats (control) and from goats subjected to 20 min of global cerebral ischemia and 7 days of reperfusion. (Top) Arteries precontracted with KCl (50 mmol/L). (Bottom) Arteries precontracted with ET-1 (10 29 mol/L). Values are means 6 SE from 16 control rings with KCl, 16 ischemic rings with KCl, 16 control rings with ET-1, and 13 ischemic rings with ET-1.
tracted arteries. It has been shown that this nonspecific inhibitory effect of depolarization on smooth muscle relaxation is due to an inhibition of cGMP formation by high KCl concentrations (35). It has been reported that CAS1609, a NO donor of the furoxan class, relaxes guinea pig pulmonary arteries precontracted with phenylephrine with a higher potency than KCl-precontracted arteries (36). Therefore, our results show that depolarization-induced
FIG. 3. Concentration–response curves to diethylamine/NO (DEA/NO) in MCA rings from sham-operated goats (control) and from goats subjected to 20 min of global cerebral ischemia and 7 days of reperfusion. (Top) Arteries precontracted with KCl (50 mmol/L). (Bottom) Arteries precontracted with ET-1 (10 29 mol/L). Values are means 6 SE from 16 control rings with KCl, 24 ischemic rings with KCl, 15 control rings with ET-1, and 15 ischemic rings with ET-1.
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ISCHEMIA–REPERFUSION AND RELAXATION TO NO DONORS
SNP-induced relaxations and DEA/NO-induced relaxations were attenuated in arteries subjected to ischemia–reperfusion when compared to relaxations induced in arteries from sham-operated goats. Since SNP releases NO through metabolic activation (28) and DEA/NO releases NO spontaneously (29), it can be concluded that the reactivity of large cerebral arteries in response to NO is reduced by ischemia– reperfusion. Previous in vivo studies have demonstrated that SNP-induced vasodilation of pial arterioles is not modified shortly (from 10 min to 4 h) after global cerebral ischemia in mice (37), cats (18), and piglets (11). In an in vitro study, reactivity of canine basilar artery to SNP and SIN-1, another NO donor, is not modified after 100 min of reperfusion following global cerebral ischemia (20). It must be noted that after these relatively short reperfusion periods the cerebrovascular responses to hypercapnia (4, 7, 9) and to vasodilator substances like acetylcholine (18, 37) adenosine (17), and CGRP (12) are impaired. Our results show that prolonged reperfusion periods (1 week) lead to changes in the reactivity of large cerebral arteries to NO donors, which do not appear early after ischemia. It recently has been reported that a significant portion of relaxation induced in cerebral arteries by the NO donors SNP and SIN-1 is mediated by largeconductance Ca 21-activated K 1 channels, through guanylate cyclase activation but also in part by direct activation independently of cGMP production (38). Our present results do not allow any conclusion regarding the mechanism impaired in goat cerebral arteries after ischemia–reperfusion, but they do show that arterial reactivity is not altered in a nonspecific way since KCl-induced contractions remain unchanged. Further research would be necessary to elucidate if reduced relaxation induced by SNP and DEA/NO through NO release is due to a reduction in cGMP production or to an impairment in Ca 21 activated K 1 channel function. In a previous study, we have shown that an ET1–NO interaction exists in goat cerebral arteries in such a way that endothelial and nonendothelial NO partially counteract the contractile response to ET-1 (39). Our present results show that ET-1-induced contraction is higher in arteries from ischemic goats when compared to sham-operated goats. This find-
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ing, although not conclusive, is compatible with an impairment of the role of NO in counteracting ET1-induced contraction. We have previously reported that such an impairment occurs in goat cerebral arteries after subarachnoid hemorrhage (39). In conclusion, the classical nitrovasodilator SNP and more recently discovered NO donors like NONOates DEA/NO, DETA/NO, and SPER/NO induce relaxation of isolated goat middle cerebral artery, which is partially inhibited by arterial depolarization. In the case of NONOates, the relaxant potency depends directly on the rate of NO release determined by the nucleophile. Global cerebral ischemia followed by reperfusion induces delayed impairment of the relaxant effects of NO on cerebrovascular smooth muscle, which results in reduced vasodilatory potency of NO donors in large cerebral arteries. This partial loss of vasodilatory potency should be taken into account when the therapeutic potential of NO donors is considered in the field of cerebrovascular disorders. ACKNOWLEDGMENTS This work was supported in part by Fondo de Investigacio´n Sanitaria Grant 95/1668. J. M. Centeno and M. Ortı´ hold research fellowships from Ministerio de Educacio´n y Cultura and Generalitat Valenciana, respectively. The authors are grateful to M. C. Tirados and M. C. Ma´n˜ez for technical assistance.
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middle cerebral artery function after focal cerebral ischemia in rats. Stroke 28, 176 –180. 22. Moncada, S., and Higgs, E. A. (1995). Molecular mechanisms and therapeutic strategies related to nitric oxide. FASEB J. 9, 1319 –1330. 23. Salom, J. B., Barbera´, M. D., Centeno, J. M., Ortı´, M., Torregrosa, G., and Alborch, E. (1998). Relaxant effects of sodium nitroprusside and NONOates in rabbit basilar artery. Pharmacology 57, 79 – 87. 24. Watkins, L. D. (1995). Nitric oxide and cerebral blood flow: An update. Cerebrovasc. Brain Metab. Rev. 7, 324 –337. 25. Zhang, F., White, J. G., and Iadecola, C. (1994). Nitric oxide donors increase blood flow and reduce brain damage in focal ischemia: Evidence that nitric oxide is beneficial in the early stages of cerebral ischemia. J. Cereb. Blood Flow Metab. 14, 217–226. 26. Chi, O. Z., Wei, H. M., and Weiss, H. R. (1995). Effects of CAS 754, a new nitric oxide donor, on regional cerebral blood flow in focal cerebral ischemia. Anesth. Analg. 80, 703–708. 27. Sasaki, Y., Seki, J., Giddings, J. C., and Yamamoto, J. (1996). Effects of NO-donors on thrombus formation and microcirculation in cerebral vessels of the rat. Thromb. Haemost. 76, 111–117. 28. Kowaluk, E. A., Seth, P., and Fung, H. L. (1992). Metabolic activation of sodium nitroprusside to nitric oxide in vascular smooth muscle. J. Pharmacol. Exp. Ther. 262, 916 –922. 29. Morley, D., and Keefer, L. K. (1993). Nitric oxide/nucleophile complexes: A unique class of nitric oxide-based vasodilators. J. Cardiovasc. Pharmacol. 22 (Suppl 7), S3-S9. 30. Torregrosa, G., Barbera´, M. D., Centeno, J. M., Ortı´, M., Salom, J. B., Jover, T., and Alborch, E. (1998). Characterization of the cortical laser-Doppler flow and hippocampal degenerative patterns after global cerebral ischemia in the goat. Pflu¨ger’s Arch. (Eur. J. Physiol.) 435, 662– 669. 31. Feelisch, M. (1991). The biochemical pathways of nitric oxide formation from nitrovasodilators: Appropriate choice of exogenous NO donors and aspects of preparation and handling of aqueous NO solutions. J. Cardiovasc. Pharmacol. 17(Suppl. 3), S25–S33. 32. Maragos, C. M., Morley, D., Wink, D. A., Dunams, T. M., Saavedra, J. E., Hoffman, A., Bove, A. A., Isaac, L., Hrabie, J. A., and Keefer, L. K. (1991). Complexes of NO with nucleophiles as agents for the controlled biological release of nitric oxide: Vasorelaxant effects. J. Med. Chem. 34, 3242–3247. 33. Garcı´a-Villalo´n, A. L., Ferna´ndez, N., Monge, L., Garcı´a, J. L., Go´mez, B., and Die´guez, G. (1995). Role of nitric oxide and potassium channels in the cholinergic relaxation of rabbit ear and femoral arteries: effects of cooling. J. Vasc. Res. 32, 387–397.
20. Katusˇic, Z. S., Michenfelder, J. D., and Milde, J. H. (1991). Ischemia–reperfusion does not affect reactivity of isolated canine basilar artery. J. Cereb. Blood Flow Metab. 11, 824 – 828.
34. Ferrer, M., Encabo, A., Conde, M. V., Marı´n, J., and Balfago´n, G. (1995). Heterogeneity of endothelium-dependent mechanisms in different rabbit arteries. J. Vasc. Res. 32, 339 –346.
21. Cipolla, M. J., McCall, A. L., Lessov, N., and Porter, J. M. (1997). Reperfusion decreases myogenic reactivity and alters
35. Rapoport, R. M., Schwartz, K., and Murad, F. (1985). Effect of sodium–potassium pump inhibitors and membrane-
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depolarizing agents on sodium nitroprusside-induced relaxation and cyclic guanosine monophosphate accumulation in rat aorta. Circ. Res. 57, 164 –170.
38. Onoue, H., and Katusˇic, Z. S. (1997). Role of potassium channels in relaxations of canine middle cerebral arteries induced by nitric oxide donors. Stroke 28, 1264 –1271.
36. Bohn, H., Brendel, J., Martorana, P. A., and Scho¨nafinger, K. (1995). Cardiovascular actions of the furoxan CAS 1609, a novel nitric oxide donor. Br. J. Pharmacol. 114, 1605–1612.
39. Alabadı´, J. A., Torregrosa, G., Miranda, F. J., Salom, J. B., Centeno, J. M., and Alborch, E. (1997). Impairment of the modulatory role of nitric oxide on the endothelin-1-elicited contraction of cerebral arteries: A pathogenetic factor in cerebral vasospasm after subarachnoid hemorrhage? Neurosurgery 41, 245–253.
37. Rosenblum, W. I. (1997). Selective impairment of the response to acetylcholine after ischemia/reperfusion in mice. Stroke 28, 448 – 452.
Copyright © 1999 by Academic Press. All rights of reproduction in any form reserved.
Relaxant Effects of Sodium Nitroprusside and NONOates in Goat Middle Cerebral Artery: Delayed Impairment by Global Ischemia–Reperfusion Juan B. Salom,* ,† ,1 Marı´a D. Barbera´,‡ Jose´ M. Centeno,* ,‡ Marta Ortı´,* ,‡ Germa´n Torregrosa,* ,† and Enrique Alborch* ,‡ *Centro de Investigacio´n, Hospital Universitario “La Fe,” †Departamento de Biologı´a Animal, and ‡Departamento de Fisiologı´a, Universidad de Valencia, Valencia, Spain
Received October 6, 1998, and in revised form February 1, 1999
Global cerebral ischemia and subsequent reperfusion induce early impairment of the vasodilator responses to hypercapnia and vasoactive substances. Nitric oxide (NO) is involved in the regulation of cerebral blood flow (CBF) in both health and disease. The present study was designed to assess possible changes in the cerebrovascular reactivity to NO donors induced by cerebral ischemia– reperfusion in goats. Female goats (n 5 9) were subjected to 20 min global cerebral ischemia under halothane/N 2O anesthesia. Sixteen additional goats were sham-operated as a control group. One week later the effects of ischemia–reperfusion on relaxations to NO donors sodium nitroprusside (SNP), diethylamine/NO (DEA/NO), diethylenetriamine/NO (DETA/NO), and spermine/NO (SPER/NO) were studied in rings of middle cerebral artery (MCA) isolated in an organ bath for isometric tension recording. SNP, DEA/NO, DETA/NO, and SPER/NO induced concentration-dependent relaxations of MCA precontracted with KCl (DEA/NO > SPER/NO > SNP > DETA/NO) or with endothelin-1 (DEA/NO > SNP > SPER/NO > DETA/NO). Relaxations were always higher in endothelin-1-precontracted arteries. One week after cerebral ischemia concentration– response curves to SNP and DEA/NO were displaced 1
To whom correspondence should be addressed at Centro de Investigacio´n, Hospital Universitario “La Fe,” Ave. Campanar 21, E46009 Valencia, Spain. Fax: 34 96 3868718. E-mail: jbsalom@ san.gva.es. 1089-8603/99 $30.00 Copyright © 1999 by Academic Press All rights of reproduction in any form reserved.
to the right, indicating a reduction in relaxant potency of NO donors. The classical nitrovasodilator SNP and NONOates induce relaxation of isolated goat MCA which is partially inhibited by arterial depolarization. Global cerebral ischemia followed by reperfusion induces delayed impairment of the relaxant effects of NO on cerebrovascular smooth muscle, which results in reduced vasodilatory potency of NO donors in large cerebral arteries. © 1999 Academic Press Key Words: nitric oxide; cerebral arteries; vasodilation; ischemia–reperfusion; goat.
Cerebral ischemia and subsequent reperfusion induce changes in the regulation of cerebral blood flow by CO 2 (1). Abolition or significant attenuation in the ability of hypercapnia to increase cerebral blood flow after global cerebral ischemia has been reported in rats (2, 3), cats (4, 5), dogs (6 – 8), and pigs (9, 10). Piglets have been widely used to study the effects of global cerebral ischemia induced by increasing intracranial pressure on the reactivity of cerebral arterioles to specific activation of membrane receptors and channels. Global ischemia reduces vasodilator responses to N-methyl-D-aspartate (NMDA) (11), calcitonin gene related peptide (CGRP) (12), oxytocin (13), and activation of ATP-sensitive K 1 channels (14), while a similar ischemic insult does not modify vasodilator responses to kainate (15) and 85
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activation of Ca 21-activated K 1 channels (16). In cats, global cerebral ischemia induces abolition of pial arteriolar dilation to acetylcholine (17–19), adenosine (17), and the Ca 21 ionophore A23187 (18), while vasoconstriction induced by serotonin and angiotensin are well preserved (17). Few studies have been made on the effects of cerebral ischemia on cerebrovascular reactivity assessed in vitro in the absence of influences from the cerebral parenchyma. Ischemia–reperfusion does not affect reactivity of isolated canine basilar artery (20), while reperfusion decreases myogenic reactivity and alters middle cerebral artery function after focal cerebral ischemia in rats (21). In the present study we have examined the effects of global cerebral ischemia on the vasodilator responses of isolated goat middle cerebral artery to NO donors, which are considered to have a potential role in therapeutic strategies related to NO (22). We have previously reported that sodium nitroprusside (SNP) 2 and other NO donors of the NONOate type induce relaxation of isolated rabbit basilar artery (23). Since there is evidence suggesting that NO is involved in the regulation of cerebral circulation both in health and disease (24), the effects of NO donors are being studied in models of cerebral ischemia (25, 26) and thrombus formation (27). We have investigated vasodilator responses of goat middle cerebral artery to SNP, diethylamine/NO complex (DEA/NO), diethylenetriamine/NO complex (DETA/ NO), and spermine/NO complex (SPER/NO), and we have looked for changes in the vasodilator responses to SNP and DEA/NO after transient global cerebral ischemia followed by 1 week of reperfusion. After ischemia, we used SNP as a representative of classic nitrovasodilators that releases NO through metabolic activation (28) and compared it with DEA/NO, a representative of NONOates that releases NO spontaneously on dissolution (29). EXPERIMENTAL PROCEDURES
Induction of transient global cerebral ischemia. Experiments were performed in accordance with the “Principles of Laboratory Animal Care” (NIH Publi2
Abbreviations used: SNP, sodium nitroprusside; DEA, diethylamine; DETA, diethyenetriamine; SPER, spermine; ET-1, endothelin-1; SIN-1, sydnonimine-1.
cation No. 86-23, revised 1985), as well as the guidelines from the Council of the European Economic Community (86/609/EEC, Article 5, Appendix II), promulgated by the Spanish legislation on March 14, 1988 (R.D. 223/1988). The experiments were carried out in 25 anesthetized (1.5% halothane in a mixture of 80% N 2 O and 20% O 2 ) female goats weighing 38 –54 kg. We have recently reported the details on the dynamic anatomy of the blood supply to the goat’s brain and surgical procedures to induce transient global cerebral ischemia (30). Briefly, the external carotid arteries were exposed bilaterally by means of an incision made along each mandible, and snare-type occluders were placed around them cephalad to the mandibular and occipital branches and tunneled subcutaneously to be exteriorized on the back of the horns. The animals were instrumented to continuously monitor the following parameters: cortical perfusion (laser-Doppler probe on the left dorsolateral cortex), intracranial pressure (16-G cannula advanced into the right lateral ventricle), monopolar electrocorticogram (needle electrode inserted into the right dorsolateral cortex and the reference electrode fastened to the goat’s ear), mean arterial blood pressure (femoral artery catheter), and heart rate (obtained from the blood pressure pulse by means of a tachymeter). After a period of stabilization following surgery, an episode of 20 min transient global cerebral ischemia was achieved in 9 goats by occlusion of the two external carotid arteries and simultaneous external compression of the jugular veins by means of a neck tourniquet. The reperfusion period started when the various occlusions were released, and it was monitored for 2 h. At the end of that time, the animals were recovered from anesthesia and returned to the pen. The animals were allowed to reperfuse for 7 days. A group of 16 sham-operated goats (the surgical procedures were followed but ischemia was not induced) served as the control group. Isometric tension recording in isolated arteries. The goats, previously sedated with 10 mg/kg im ketamine (Ketolar; Parke Davis, Barcelona, Spain) and anesthetized with iv 2% sodium thiopental (Tiobarbital; Braun Medical, Jae´n, Spain), were killed by iv injection of 30 mEq of KCl. The whole brain was rapidly removed, and the middle cerebral arter-
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ISCHEMIA–REPERFUSION AND RELAXATION TO NO DONORS
ies were dissected free and cut into cylindrical segments measuring 4 mm in length and about 1 mm in outer diameter. Each segment was prepared for isometric tension recording in an organ bath. Two stainless steel L-shaped pins (diameter of 125 mm) were introduced through the arterial lumen. One pin was fixed to the organ bath wall, while the other was connected to a strain gauge (Universal Transducing Cell, UC3; Gould Statham, Oxnard, CA) for isometric tension recording (Omniscribe D 5237-5; Houston Instrument, Gistel, Belgium). The organ bath contained 5 ml of Ringer–Locke solution, which was bubbled continuously with 95% O 2 and 5% CO 2 to give a pH of 7.3–7.4. The temperature was kept at 37°C. A resting tension of 1 g was applied to the arterial segments and they were allowed to equilibrate for 60 –90 min. Tension was readjusted when necessary and the bath fluid was changed every 15 min before starting the experiments. The contractile capacity of all the arterial segments was examined by exposure to a high-K 1 (50 mmol/L) Ringer–Locke solution. The relaxant effects of SNP, DEA/NO, DETA/NO, and SPER/NO were assessed by cumulative addition (10 29 –3 3 10 24 mol/L) to arterial segments previously contracted either with KCl (50 mmol/L) or with endothelin-1 (ET-1, 10 29 mol/L) in order to study whether arterial depolarization affected the relaxant effects of NO donors. Only one concentration–response curve was carried out in each arterial segment. The relaxant responses elicited by SNP, DEA/NO, DETA/NO, and SPER/NO were expressed as percentages of the active tone achieved with high-K 1 solution or ET-1. Maximal relaxant effects (E max) and concentrations of NO donors producing 50% of the E max (EC 50) were calculated for each concentration–response curve. The pEC 50 was calculated as the negative logarithm to base 10 of the EC 50 for statistical analysis. Student’s t test for grouped data or the Newman–Keuls test (for multiple comparisons) was used for statistical analysis, and P , 0.05 was considered statistically significant. SNP, DEA/NO, DETA/NO, and SPER/NO were from Research Biochemicals International (Natick, MA). ET-1 was from Peptide Institute (Osaka, Japan). Care was taken to protect SNP solutions from light due to its light-sensitivity (31). DEA/NO,
TABLE I
Hemodynamic Parameters [Cortical Perfusion (CP), Intracranial Pressure (ICP), Mean Arterial Blood Pressure (MABP), and Heart Rate (HR)] in Goats Subjected to Transient Global Cerebral Ischemia
CP (%) ICP (mm Hg) MABP (mm Hg) HR (beats/min)
Control
Ischemia
Reperfusion a
100 561 116 6 4 96 6 2
10 6 1** 10 6 1* 121 6 3 96 6 3
150 6 4**/74 6 4** 761 111 6 3 104 6 3
Note. Values are means 6 SE. a Two values of CP are given during reperfusion: first for early hyperemic reperfusion and second for delayed hypoperfusion. * Significantly different from control (P , 0.05). ** Significantly different from control (P , 0.01).
DETA/NO, and SPER/NO were dissolved and diluted in ice-cold 10 mmol/L NaOH to prevent NO release until addition to the organ bath (32). Final NaOH concentration in the organ bath did not have effects on the arterial isometric tension. The Ringer–Locke solution had the following composition (mmol/L): NaCl, 120; KCl, 5.4; CaCl 2 , 2.2; MgCl 2, 1.0; NaHCO 3, 25; and glucose, 5.6. In KCldepolarizing solution, NaCl was replaced by an equimolar amount of KCl. RESULTS
Bilateral occlusion of external carotid arteries and compression of jugular veins produced significant (P , 0.01) reduction in cortical perfusion to 10% of the control value, accompanied by a significant (P , 0.05) increase in intracranial pressure, which doubled the control value. No significant changes were induced in mean arterial blood pressure and heart rate. After 20 min ischemia, release of the occlusions induced a peak of hyperemic reperfusion for 64 6 17 min (cortical perfusion values significantly above control, P , 0.01), followed by delayed hypoperfusion which lasted until the end of the 2-h recording period (cortical perfusion values under control, P , 0.01). During reperfusion intracranial pressure decreased, and mean arterial blood pressure and heart rate were in the normal range (Table I). The four NO donors, SNP (10 29–3 3 10 24 mol/L), DEA/NO (10 29–3 3 10 24 mol/L), DETA/NO (10 27–3
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FIG. 1. Concentration–response curves to sodium nitroprusside (SNP), diethylamine/NO (DEA/NO), diethylenetriamine/NO (DETA/NO), and spermine/NO (SPER/NO) in MCA rings from sham-operated goats. (Top) Arteries precontracted with KCl (50 mmol/L). (Bottom) Arteries precontracted with endothelin-1 (ET-1, 10 29 mol/L). Values are means 6 SE from 16 rings with KCl and SNP, 16 rings with KCl and DEA/NO, 15 rings with KCl and DETA/NO, 15 rings with KCl and SPER/NO, 16 rings with ET-1 and SNP, 15 rings with ET-1 and DEA/NO, 14 rings with ET-1 and DETA/NO, and 15 rings with ET-1 and SPER/NO.
3 10 24 mol/L), and SPER/NO (10 28–3 3 10 24 mol/L) induced concentration-dependent relaxations of goat middle cerebral arteries obtained from shamoperated goats and precontracted with 50 mmol/L KCl (active tone, 2083 6 119 mg) or with 10 29 mol/L ET-1 (active tone, 1544 6 74 mg). In all cases arterial segments were fully relaxed (Fig. 1). According to the pEC 50 values for relaxation induced by each
NO donor, in KCl-precontracted arteries the order of potency was DEA/NO . SPER/NO .SNP . DETA/ NO, and in ET-1-precontracted arteries the order of potency was DEA/NO . SNP . SPER/NO . DETA/ NO. Values of pEC 50 and E max for the four NO donors and the two precontractile agents are summarized in Table II. The nature of the precontractile agent conditioned the relaxant response to NO donors. According to pEC 50 values, NO donors were always significantly (P , 0.01) more potent in ET-1-precontracted arteries than in KCl-precontracted arteries. Moreover, except for DEA/NO, E max values were always significantly higher in ET-1-precontracted arteries than in KCl-precontracted arteries (P , 0.01 for SNP and DETA/NO and P , 0.05 for SPER/NO; Table II). Active tone induced by KCl (50 mmol/L) in arteries obtained from goats subjected to transient global ischemia (1941 6 109 mg) did not differ from that induced in control arteries (sham-operated goats). Active tone induced by ET-1 (10 29 mol/L) was significantly (P , 0.05) higher in arteries from ischemic goats (1801 6 111 mg) when compared to control arteries. In arteries from ischemic goats, the relaxant responses elicited by NO donors differed from the responses obtained in arteries from shamoperated goats. With regard to SNP (Fig. 2), and according to pEC 50 values, the relaxant potency was significantly (P , 0.01) reduced in KClprecontracted and, to a higher extent, in ET-1precontracted arteries from goats subjected to ischemia. The E max for relaxant effects of SNP in KClprecontracted arteries was significantly (P , 0.01) higher after ischemia (Table II). As for DEA/NO (Fig. 3), a highly significant (P , 0.01) reduction in relaxant potency was observed in KCl-precontracted arteries obtained after ischemia and, to a lower extent, in ET-1-precontracted arteries. The E max for relaxant effects of DEA/NO in KCl-precontracted arteries was significantly (P , 0.01) higher after ischemia (Table II). DISCUSSION
This study shows that DEA/NO, DETA/NO, and SPER/NO, three NO/nucleophile complexes, induce relaxation of isolated goat middle cerebral arteries. Although the three NO donors showed the same
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ISCHEMIA–REPERFUSION AND RELAXATION TO NO DONORS TABLE II
Concentration–Response Curve Parameters for NO Donors in MCA from Sham-Operated (Control) and Ischemic Goats Control
SNP KCl ET-1 DEA/NO KCl ET-1 DETA/NO KCl ET-1 SPER/NO KCl ET-1
Transient global ischemia
pEC 50
E max (%)
n
pEC 50
E max (%)
n
5.4 6 0.1 6.7 6 0.1**
90 6 5 112 6 2**
16 16
5.0 6 0.1*** 6.2 6 0.1** ,***
105 6 2*** 109 6 5
16 13
6.5 6 0.1 7.2 6 0.1**
87 6 6 101 6 4
16 15
5.2 6 0.1*** 6.8 6 0.1** ,***
103 6 1*** 103 6 1
24 15
4.8 6 0.1 5.3 6 0.1**
88 6 3 106 6 3**
15 14
5.7 6 0.1 6.2 6 0.1**
88 6 4 100 6 2*
15 15
Note. Order of potency of NO donors: DEA/NO . SPER/NO . SNP . DETA/NO in KCl-precontracted arteries and DEA/NO . SNP . SPER/NO . DETA/NO in ET-1-precontracted arteries. Values are means 6 SE. * Significantly different from KCl-precontracted arteries (P , 0.05). ** Significantly different from KCl-precontracted arteries (P , 0.01). *** Significantly different from control arteries (P , 0.01).
efficacy by fully relaxing the arteries, significant differences were shown in their relaxant potencies. Independently of the precontractile agent used (KCl or ET-1), the order of potency was DEA/NO . SPER/ NO . DETA/NO, which coincides with what we have previously reported for rabbit basilar artery (23). The rate at which NO is released by NONOates on dissolution depends on the carrier nucleophile, determines half-life (DEA/NO, t 1/2 5 2.1 min; SPER/ NO, t 1/2 5 39 min; and DETA/NO, t 1/2 5 20 h) and correlates highly with the actual vasodilatory potency of each NONOate (29). Our results in this and previous studies confirm that the relaxant potencies of NO/nucleophile complexes in cerebral arteries can be predicted from data on the rates at which NO is generated in simple aqueous buffers: The lower the t 1/2 value, the higher the relaxant potency. The invariability of the order of potency of NONOates is in agreement with their mechanism of action. Since they are not drugs acting on membrane receptors but prodrugs that release NO, the order of potency does not depend on the receptor subtype present in the particular tissue studied. The classical nitrovasodilator SNP also relaxed goat middle cerebral arteries, but its relaxant potency is more variable than that of NONOates. The
vasodilatory potency was lower than that of SPER/NO in KCl-precontracted arteries, while it was higher that that of SPER/NO in ET-1precontracted arteries. In both cases the potency of SNP was lower than that of DEA/NO and higher than that of DETA/NO. In contrast, in rabbit basilar artery the relaxant potency of SNP is higher than that of DEA/NO (23). Vasodilatory effects of SNP are mediated by metabolic activation of the drug to NO by a SNP-directed, membrane-associated enzymatic activity present in vascular smooth muscle (28). This enzymatic activity may differ between species and may be affected by KCl-induced depolarization, and this could explain the variability in SNPinduced relaxation. Regardless of the NO donor, SNP, DEA/NO, DETA/NO, or SPER/NO, in our study relaxations were much more potent and effective in ET-1precontracted arteries than in KCl-precontracted arteries. SNP-induced relaxations of rabbit ear and femoral arteries are also reduced by high K 1 concentration with respect to ET-1-precontracted arteries (33). Endothelium-dependent relaxations induced by acetylcholine in rabbit mesenteric (34), ear, and femoral arteries (33) are also lower in KClprecontracted arteries than in agonist-precon-
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inhibition of arterial relaxation also applies to NONOates like DEA/NO, DETA/NO, and SPER/NO in goat cerebral arteries and is in agreement with the involvement of guanylyl cyclase activation to produce cGMP, which mediates NONOate-induced relaxation, as previously reported in rabbit basilar artery (23). In this study, 20 min global cerebral ischemia followed by 7 days of reperfusion affected reactivity of goat middle cerebral artery to NO donors. Both
FIG. 2. Concentration–response curves to SNP in MCA rings from sham-operated goats (control) and from goats subjected to 20 min of global cerebral ischemia and 7 days of reperfusion. (Top) Arteries precontracted with KCl (50 mmol/L). (Bottom) Arteries precontracted with ET-1 (10 29 mol/L). Values are means 6 SE from 16 control rings with KCl, 16 ischemic rings with KCl, 16 control rings with ET-1, and 13 ischemic rings with ET-1.
tracted arteries. It has been shown that this nonspecific inhibitory effect of depolarization on smooth muscle relaxation is due to an inhibition of cGMP formation by high KCl concentrations (35). It has been reported that CAS1609, a NO donor of the furoxan class, relaxes guinea pig pulmonary arteries precontracted with phenylephrine with a higher potency than KCl-precontracted arteries (36). Therefore, our results show that depolarization-induced
FIG. 3. Concentration–response curves to diethylamine/NO (DEA/NO) in MCA rings from sham-operated goats (control) and from goats subjected to 20 min of global cerebral ischemia and 7 days of reperfusion. (Top) Arteries precontracted with KCl (50 mmol/L). (Bottom) Arteries precontracted with ET-1 (10 29 mol/L). Values are means 6 SE from 16 control rings with KCl, 24 ischemic rings with KCl, 15 control rings with ET-1, and 15 ischemic rings with ET-1.
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ISCHEMIA–REPERFUSION AND RELAXATION TO NO DONORS
SNP-induced relaxations and DEA/NO-induced relaxations were attenuated in arteries subjected to ischemia–reperfusion when compared to relaxations induced in arteries from sham-operated goats. Since SNP releases NO through metabolic activation (28) and DEA/NO releases NO spontaneously (29), it can be concluded that the reactivity of large cerebral arteries in response to NO is reduced by ischemia– reperfusion. Previous in vivo studies have demonstrated that SNP-induced vasodilation of pial arterioles is not modified shortly (from 10 min to 4 h) after global cerebral ischemia in mice (37), cats (18), and piglets (11). In an in vitro study, reactivity of canine basilar artery to SNP and SIN-1, another NO donor, is not modified after 100 min of reperfusion following global cerebral ischemia (20). It must be noted that after these relatively short reperfusion periods the cerebrovascular responses to hypercapnia (4, 7, 9) and to vasodilator substances like acetylcholine (18, 37) adenosine (17), and CGRP (12) are impaired. Our results show that prolonged reperfusion periods (1 week) lead to changes in the reactivity of large cerebral arteries to NO donors, which do not appear early after ischemia. It recently has been reported that a significant portion of relaxation induced in cerebral arteries by the NO donors SNP and SIN-1 is mediated by largeconductance Ca 21-activated K 1 channels, through guanylate cyclase activation but also in part by direct activation independently of cGMP production (38). Our present results do not allow any conclusion regarding the mechanism impaired in goat cerebral arteries after ischemia–reperfusion, but they do show that arterial reactivity is not altered in a nonspecific way since KCl-induced contractions remain unchanged. Further research would be necessary to elucidate if reduced relaxation induced by SNP and DEA/NO through NO release is due to a reduction in cGMP production or to an impairment in Ca 21 activated K 1 channel function. In a previous study, we have shown that an ET1–NO interaction exists in goat cerebral arteries in such a way that endothelial and nonendothelial NO partially counteract the contractile response to ET-1 (39). Our present results show that ET-1-induced contraction is higher in arteries from ischemic goats when compared to sham-operated goats. This find-
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ing, although not conclusive, is compatible with an impairment of the role of NO in counteracting ET1-induced contraction. We have previously reported that such an impairment occurs in goat cerebral arteries after subarachnoid hemorrhage (39). In conclusion, the classical nitrovasodilator SNP and more recently discovered NO donors like NONOates DEA/NO, DETA/NO, and SPER/NO induce relaxation of isolated goat middle cerebral artery, which is partially inhibited by arterial depolarization. In the case of NONOates, the relaxant potency depends directly on the rate of NO release determined by the nucleophile. Global cerebral ischemia followed by reperfusion induces delayed impairment of the relaxant effects of NO on cerebrovascular smooth muscle, which results in reduced vasodilatory potency of NO donors in large cerebral arteries. This partial loss of vasodilatory potency should be taken into account when the therapeutic potential of NO donors is considered in the field of cerebrovascular disorders. ACKNOWLEDGMENTS This work was supported in part by Fondo de Investigacio´n Sanitaria Grant 95/1668. J. M. Centeno and M. Ortı´ hold research fellowships from Ministerio de Educacio´n y Cultura and Generalitat Valenciana, respectively. The authors are grateful to M. C. Tirados and M. C. Ma´n˜ez for technical assistance.
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Copyright © 1999 by Academic Press. All rights of reproduction in any form reserved.