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Vasoconstriction to endothelin-1 in the goat middle cerebral artery after transient global cerebral ischemia
Vasoconstriction to Endothelin-1 in the Goat Middle Cerebral Artery After Transient Global Cerebral Ischemia Juan B. Salom, PhD,Jos6 M. Centeno, PhD,German Torregrosa, PhD, Marta Orti, PhD,Maria D. Barber& PhD,and Enrique Alborch, MD, PhD
We have examined the effects of global ischemia and subsequent reperfusion on the reactivity of goat middle cerebral artery to endothelin-1. The participation of extracellular Ca 2+ in endothelin-l-induced contractions and the ability of the Ca 2+ entry blocker, nicardipine, to counteract them after ischemia were also assessed. Concentration-response curves to endothelin-1 (10-12 - 3 • 10 s tool/L) obtained in arteries from goats subjected to either 5 or 10 minutes of ischemia and 7-day reperfusion did not significantly differ from curves obtained in arteries from sham-operated goats, Concentration-response curves to endothelin-1 in arteries from goats subjected to 20 minutes of ischemia and 7-day reperfusion, however, showed significantly higher pECs0 (negative logarithm to base 10 of the concentration of endothelin-1 producing 50% of the maximal effects) value (P < .01) than in arteries from sham-operated goats. The effect of 20 minutes of ischemia was assessed at various reperfusion times. The maximal effects (Ema• values of the concentration-response curves to endothelin-1 were significantly reduced (P < .01) 1 and 3 days after ischemia when compared with sham-operated animals. The concentration-response curve to endothelin-1 was displaced to the left 7 days after the ischemia. Two weeks after the ischemia, the Ernaxvalue was again significantly reduced (P < .01). When arteries obtained from goats 7 days after 20 minutes of ischemia were incubated in Ca2+-free medium or nicardipine (10-l~ and 10 s mol/L), the concentration-response curve to endothelin-1 was significantly inhibited (P < .01) both in terms of pECs0 and of Ema• In conclusion, there was an increase in the endothelin-1 potency 1 week after 20 minutes of ischemia. At this time, the involvement of dihydropyridine-sensitive L-type Ca2+ channels in Ca2+ entry induced by endothelin-1 to develop contraction is reduced when compared with normal arteries. We suggest that the increased reactivity to endothelin-1 could have pathophysiological relevance, and the reduced effect of nicardipine could have therapeutical relevance in cerebral ischemia. Key Words: Endothelin-l~Ca2+ chann e l - M i d d l e cerebral artery--Cerebral ischemia--Goat.
From the Centro de Investigaci6n, Hospital Universitario La Fe, Departamento de BiologiaAnimal, and Departamento de Fisiologia, Universidad de Valencia,Valencia,Spain. Received March 8,1999; accepted July 1, 1999. Partially supported by a grant from Fondo de Investigaci6n Sanitaria (FIS 95/1668). J.M.C. and M.O. hold research fellowships from Ministeriode Educaci6n y Cultura and Conselleriade Cultura, Educaci6 i Ci6ncia,respectively. Address reprint requests to Juan B. Salom, PhD, Centro de Investigaci6n, Hospital Universitario La Fe, Ave Campanar 21, E46009, Valencia,Spain. Copyright 9 2000by National Stroke Association 1052-3057/00/0901-000353.00/0
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Endothelins are potent constrictors of cerebral arteries that are isolated from various species, including humans. 1 Abluminal local application of endothelin-1 to the middle cerebral artery (MCA) in rats results in profound and long-lasting reductions in local cerebral blood flow (CBF), focal cerebral ischemia, and development of cerebral infarction.2,3 Intrastriatal injection of endothelin-1 in rats produces ischemic lesions caused by vasoconstriction of cerebral microvessels.4 Endothelins are believed to be involved in the pathophysiology of most c o m m o n cerebrovascular diseases, such as subarachnoid hemorrhage, cerebral ischemia, and
Journal of Stroke and Cerebrovascular Diseases, Vol, 9, No. 1 (January-February), 2000: pp 16-21
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CEREBROVASCULAR REACTIVITY AFTER ISCHEMIA
migraine. -~In regard to the involvement of endothelin-1 in the neuropathological consequences of cerebral ischemia, immunoreactive endothelin-1 is increased in rat brain, 6,7 and in rabbit brain and plasma s after focal ischemia. After global ischemia, concentration of endothelin-1 increases in gerbil forebrain, 9,1~ rat striatum, 6 and rat hippocampus, u,12 in which selective neuronal death takes place. 13In the clinical setting, plasma endothelin-1 is increased in patients with acute ischemic stroke 14-16 and in patients resuscitated from cardiac arrest. 17The use of endothelin-1 receptor antagonists has added to the evidence that endothelin-I has a role in the pathophysiology of cerebral ischemia. Intravenous administration of PD156707 (a nonselective receptor antagonist) increases cerebral perfusion and reduces ischemic damage in feline focal ischemia. ~8 Oral administration of SB217242 (a nonselective receptor antagonist) also reduces cerebral focal ischemic injury in the rat. 19 Finally, BQ-123 (selective endothelin A receptor antagonist) reverses late postischemic hypoperfusion 2~ and increases hippocampal CA1 neuron survival 21 in gerbils subjected to transient global ischemia. We have previously shown that endothelin-1 reduces CBF in goats by causing contraction of the cerebral arteries, which depends partially on the activation of Ca 2+ influx through dihydropyridine-sensitive channels. 22 After experimental subarachnoid hemorrhage, the cerebral vasoconstrictor effects of endothelin-I are increased, and dependence on extracellular Ca 2+ is reduced. 23 To gain insight into the role of endothelin-1 in the pathophysiology of cerebral ischemia, we have examined, in this study, the effects of global ischemia and subsequent reperfusion on the reactivity of goat MCA to endothelin-1. Participation of extracellular Ca 2+ in endothelin-l-induced contractions and the ability of the Ca 2+ entry blocker, nicardipine, to counteract them after ischemia were also assessed.
Materials a n d M e t h o d s Experiments were performed in accordance with the guidelines from the Council of the European Economic Community (86 / 609 / EEC, Article 5, Appendix II), promulgated by the Spanish legislature on March 14, 1988 (R.D. 223/1988).
and surgical procedures have been reported previously. 24 Briefly, the external carotid arteries were exposed bilaterally by means of an incision made along each mandible. Snare-type occluders were placed around the external carotid arteries, cephalad to the mandibular and occipital branches, and tunneled subcutaneously to be exteriorized on the back of the horns. The following parameters were continuously monitored: cortical perfusion (laser-Doppler probe on the left dorsolateral cortex); intracranial pressure (16-gauge 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 (the blood pressure pulse by means of a tachymeter). After a period of stabilization after surgery, transient global cerebral ischemia (5, 10, or 20 minutes) was achieved under anesthesia by occlusion of the 2 external carotid arteries and simultaneous external compression of the jugular veins by means of a neck tourniquet. Successful ischemia was assessed by a reduction of cortical perfusion by more than 80% and flattening of brain waves seen on the electrocorticogram. The reperfusion period began when the occlusions were released and was monitored for 2 hours. The animals were then allowed to recover from anesthesia. The animals were allowed to reperfuse for 1, 3, 7, or 14 days. Sham-operated goats, in which the surgical procedures were followed but ischemia was not induced and the different reperfusion times were allowed, served as the control. The number of animals used in each experimental group was, as follows: 7 sham-operated goats (included in the 4 different reperfusion groups), 4 goats subjected to 5 minutes of ischemia, 4 goats subjected to 10 minutes of ischemia, and 18 goats subjected to 20 minutes of ischemia (which were included in the 4 different reperfusion groups). The 7 sham-operated goats were distributed, as follows: 1 goat in the 1-day reperfusion group, 1 goat in 3-day reperfusion, 4 goats in 7-day reperfusion, and 1 goat in the 14-day reperfusion group. Finally, the 18 goats subjected to 20 minutes of ischemia were distributed, as follows: 4 goats in the 1-day reperfusion group, 4 goats in 3-day reperfusion, 7 goats in 7-day reperfusion, and 3 goats in the 14-day reperfusion group.
Induction of Transient Global Cerebral Ischemia Thirty-three female goats (30 to 35 kg) were sedated with 10 m g / k g ketamine (Ketolar; Parke-Davis, Barcelona, Spain) administered intramuscularly (i.m.) and anesthetized with 2% sodium thiopental (Tiobarbital; Palex, Ja6n, Spain) administered intravenously (i.v.). After gastric and tracheal intubation, anesthesia was maintained by means of a ventilator (Themel VT/3, Valencia, Spain), supplying 1.5% halothane (Fluothane; Zeneca Farma, Pontevedra, Spain) in 80% N20 and 20% 02. Details on the dynamic anatomy of the blood supply to the goat's brain
Isometric Tension Recording in Isolated Arteries The goats, sedated and anesthetized as described previously, were killed by i.v. injection of 30 mEq KC1 (UCB Pharma, Barcelona, Spain). The whole brain was rapidly removed. The 2 MCAs were dissected free and cut into a total of 8 to 10 cylindrical segments, measuring 4 m m in length and about 1 m m in outer diameter. Each segment was prepared for isometric tension recording in an organ bath. Two stainless-steel L-shaped pins (125 ~m in diameter) were introduced through the arterial lumen. One pin
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J.B. S A L O M E T AL.
was fixed to the organ bath wall; 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% o2 and 5% co2 to give a pH of 7.3 to 7.4. The temperature was kept at 37~ A resting tension of 1 g was applied to the arterial segments, and they were allowed to equilibrate for 60 to 90 minutes. Tension was readjusted when necessary, and the bath fluid was changed every 15 minutes before beginning the experiments. The contractile capacity of the arterial segments was examined by exposure to a high-K + (50 mmol/L) RingerLocke solution. The effects of endothelin-1 (10 12 3 • 10 8 mol/L) were studied by cumulative addition to the organ bath. The influx of extracellular Ca 2+, on stimulation with endothelin-1, was studied by obtaining concentration-response curves to the peptide after incubation in Ca2+-free medium for 20 minutes. The involvement of dihydropyridine-sensitive Ca 2+ channels in endothelin-1induced responses was assessed by obtaining concentration-response curves to the peptide after incubation with the Ca 2+ entry blocker, nicardipine (10 10or 10 8 mol/L), for 20 minutes. Only 1 concentration-response curve was performed in each arterial segment. _
Statistical Analysis The contractile responses to endothelin-1 were expressed as a percentage of the previous contraction induced by 50 mmol/L of KC1. Maximal effects (Ema• and the concentration of endothelin-1 producing 50% of the Er~ax (EC50) were calculated for each concentrationresponse curve. The pEC50 was calculated as the negative logarithm to base 10 of the EC50 for statistical analysis. The Dunnett test was used for comparisons with the control. Probability values of less than 5% were considered statistically significant.
Drugs and Solutions Endothelin-1 (Peptide Institute Inc, Osaka, Japan) was dissolved in 0.1% aqueous acetic acid and diluted in a phosphate-buffered saline solution with 0.05% bovine serum albumin, fraction V (Sigma Chemicals Co, St Louis, MO). Nicardipine (Ferrer Int, Barcelona, Spain) was dissolved in twice-distilled water and diluted in saline solution. The Ringer-Locke solution had the following composition: 120 mmol/L of NaC1, 5.4 mmol/L of KC1, 2.2 mmol/L of CaC12, 10 mmol/L of MgC12, 25 mmol/L of NaHCO3, and 5.6 mmol/L of glucose. In KCl-depolarizing solution, NaC1 was replaced by an equimolar amount of KC1. The Ca2+-free solution was prepared by omitting CaC12. The phosphate-buffered saline solution had the
following composition: 150 mmol/L of NaC1, and 10 mmol/L of NaH2PO4.
Results Endothelin-1 (10-12 - 3 x 10-s tool/L) induced concentration-dependent contractions of goat MCAs were obtained from sham-operated goats, as well as from goats subjected to different times of global cerebral ischemia and reperfusion. Reactivity to endothelin-1 was not significantly different in MCAs from sham-operated goats killed at 1, 3, 7, and 14 days after surgery. The effects of ischemia duration were assessed after 1 week of reperfusion. Concentration-response curves to endothelin-1 obtained in arteries from goats subjected to either 5 or 10 minutes of ischemia did not significantly differ, either in terms of pECs0 or in terms of E.... from curves obtained in arteries from sham-operated goats (Fig 1 and Table 1). Concentration-dependent contractions induced by endothelin-1 in arteries from goats subjected to 20 minutes of ischemia showed significantly higher pECs0 value (P < .01) than in arteries from sham-operated goats. The Emax value, however, remained unchanged (Table 1). Therefore, the concentration-response curve to endothelin-1 was slightly displaced to the left in arteries from goats subjected to 20 minutes of ischemia and 1 week of reperfusion with respect to arteries from shamoperated animals (Fig 1). The effect of 20 minutes of ischemia was assessed at various reperfusion times. The Emaxvalues of the concen--0---I---rn---II--
180 160 O ~" o z E O E F- o On o z
Sham-0perated 5 min 10 min 20 min
140 120
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100 80
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40
~
20
q
I
i
i
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I
i
I
12
11
10
9
8
7
ENDOTHELIN-1 (-log mol/L)
Figure1. Concentration-response curves to endothelin-1 in MCA isolated from sham-operated goats (n = 37) and from goats subjected to transient global cerebral ischemia of 5 (n = 32), 10 (n = 36), or 20 (n = 23) minutes duration. All goats were allowed 7 days of reperfusion. The symbols represent mean values of contraction expressed as a percentage of previous response to 50 mmol/L KCI. The vertical bars show SD.
19
CEREBROVASCULAR REACTIVITY AFTER ISCHEMIA
Table 1. Effects o f ischemia duration on
Table 2. Effects o f repeffusion duration on
concentration-response curve parameters f o r endothelin- 1
concentration-response curve parameters f o r endothelin- 1
in M C A f r o m goats qfter 1 week o f reperfusion
in M C A from goats subjected to 20 minutes o f ischemia
pECso Sham-operated 5-min ischemia 10-rain ischemia 20-rain ischemia
9.6 9.6 9.8 10.2
-+ 0.3 + 0.5 -+ 0.6 _+ 0.5*
Emax (%)
n
125 107 135 116
37 32 36 23
_+ 38 + 18 _+ 36 _+ 38
pECso Sham-operated 1-day reperfusion 3-day reperfusion 7-day reperfusion 14-day reperfusion
NOTE: Values are expressed as mean _+ SD. *Significantly different from sham-operated goats (P < .01).
-6 E E o ~o
- - O - - Control
- - O - - Sham-operated - - t - - 1 day - - r n - - 3 days
160
O
140
z
40-
~
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E
O E t- o O Lo o
80-
O
--13-- Nicardipine 10 -1~ mol/L --I--
Nicardipine 10 -8 mol/L
T
T
140 120 -
O
100-
60-
Ca2+-free medium
v
-
gr O o Z
60 29 29 23 30
_+ 34 + 24* _+ 16" -4- 38 -+ 15"
Our results show that transient global cerebral ischemia in the goat induces changes in the reactivity of the MCA to endothelin-1, which d e p e n d both on the duration of the ischemic insult and the subsequent reperfusion. Up to 10 minutes of ischemia did not induce changes in the reactivity of MCA to endothelin-1. A 20-minute ischemic insult was necessary to produce an increase in the reactivity to endothelin-1, consisting of a higher contractile potency of the peptide on the MCA. In regard to the reperfusion time, the increase in reactivity to endothelin-1 appeared 7 days after ischemia, whereas, after shorter (1 and 3 days) and longer (14 days) reperfusion periods,
120 z O Io
121 103 97 116 93
Discussion
--I--
O
_+ 0.3 + 0.3 _+ 0.3 -4- 0.5* -+ 0.3
n
NOTE: Values are expressed as mean _+ SD. *Significantly different from sham-operated goats (P < .01).
tration-response curves to endothelin-1 were significantly reduced (P < .01) 1 and 3 days after ischemia when compared with sham-operated animals (Fig 2 and Table 2). Then, as mentioned previously, the concentrationresponse curve to endothelin-1 was displaced to the left 1 week after the ischemic episode. Two weeks after the ischemia, the Emax value was again significantly reduced (P < .01) with respect to sham-operated goats (Fig 2 and Table 2). When arteries obtained from goats 7 days after 20 minutes of ischemia were incubated in Ca2+-free medium, the concentration-response curve to endothelin-1 was significantly inhibited (P < .01; Fig 3), both in terms of pECs0 and of Emax (Table 3). Incubation with the Ca 2+ entry blocker, nicardipine (10 ]0, 10 8 mol/L), also induced significant inhibition (P < .01) of the concentration-response curves to endothelin-1 (Fig 3 and Table 3).
160
9.6 9.7 9.7 10.2 9.7
E ..... (%)
Iz
O O
t-
100 8060-
O
40-
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2o-
20 O0 i
12
1
i
i
i
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10
9
8
7
t
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12
11
10
9
8
ENDOTHELIN-1 (-log mol/L) ENDOTHELIN-1 (-log mol/L) Figure 2. Concentration-response curves to endothelin-1 in MCA isolated from sham-operated goats (n - 60) and from goats subjected to 20 minutes of transient global cerebral ischemia, followed by 1 (n = 29), 3 (n = 29), 7 (n = 23), or 14 (n = 30) days of reperfusion. The symbols represent mean values of contraction expressed as a percentage of previous response to 50 mmol/L KC1. The vertical bars show SD.
Figure 3. Concentration-response curves to endothelin-1 in MCA isolated from goats subjected to 20 minutes of transient global cerebral ischemia, followed by 7 days of reperfusion. Curves were obtained in control conditions (n - 23), in Ca2+-free medium (n - 7), or during incubation with the Ca2~ entry blocker, nicardipine 10 7o mol/L (n = 8), or 10 8 mol/L (n - 8). The symbols represent mean values of contraction expressed as a percentage of previous response to 50 mmol/L KCI. The vertical bars show SD.
20
J.B. SALOM ET AL.
Table 3. Effects of Ca2+ removal and nicardipine on concentration-response curve parameters for endothelin-1 in MCA from goats subjected to 20 minutes of ischemia and 1-week of reperfusion
Control Ca2+-free medium Nicardipine 10-1~mol/L Nicardipine 10 8 mol/L
pECs0
Emax(%)
n
10.2 • 0.5 9.3 -+ 0.3* 9.1 _+ 0.4* 9.2 --_ 0.3*
116 --+ 38 48 • 17" 7.4 • 20* 65 • 21"
23 7 8 8
NOTE: Values are expressed as mean _+SD. *Significantly different from control (P < .01). the reactivity of MCA to endothelin-1 was reduced in terms of efficacy (maximum effect) of the peptide. Therefore, the increase in reactivity induced by ischemia showed up acutely I week after the ischemic insult. We have previously shown that there is a functional interaction between endothelin-1 and nitric oxide (NO) in goat MCA, consisting of a partial counteraction of the contractile response to endothelin-1 by NO. 25 Moreover, we have recently found that the relaxant effects of NO are impaired in MCA from goats subjected to 20 minutes of global cerebral ischemia and 7-day reperfusion. 26 Therefore, impairment of the counteracting effect of NO is in line with the potentiation of endothelin-l-induced contractions observed in this study. Other investigators have observed that cerebral ischemia induces changes in the presence and function of brain endothelin receptors. In the gerbil, transient forebrain ischemia results in a 15% reduction in endothelin receptor density 60 minutes after reperfusion. 1~In the rat brain, the endothelin-binding sites are remarkably increased in the hippocampal CA1 field and dentate gyrus, ventral thalamic nucleus, and cortical vessels 4 and 7 days after transient forebrain ischemia. 27 Finally, endothelin B receptor-mediated dilatation, elicited by the direct adventitial application of BQ-3020 onto cat pial arterioles, is lost within 30 minutes of focal ischemia. 28 Therefore, our results and those from other investigators indicate that early and delayed changes in endothelin receptors of brain tissue and blood vessels, together with increases in the synthesis and availability of endothelin-1, 6,8,9,16could contribute to the pathophysiology of cerebral ischemia. In a previous study, 24 we have shown that delayed neuronal death in the hippocampal CA1 subfield of the goat appears 7 days after 20 minutes of ischemia but not after shorter ischemia insults (5 or 10 minutes). Therefore, there is a temporal correlation (7 days) between the appearance of hyperreactivity to endothelin-1 and delayed neuronal death induced by transient global ischemia in the goat. Moreover, there is a coincidence in the severity of ischemia (20 minutes) causing hyperreactivity and delayed neuronal death. A direct causal relationship, however, between maintained cerebral hypoperfusion
eventually induced by hyperreactivity to endothelin-1 and delayed neuronal death can not be inferred. Additional studies using selective blockers of endothelin-1 receptors in vivo to counteract hyperreactivity to endothelin-1 could help to clarify this point. A postischemic increase in the contractile response to endothelin-1 seems not to be restricted to the cerebrovascular bed. Augmented reactivity to endothelin-1 has been reported in dog isolated left anterior descending coronary artery after myocardial ischemia and reperfusion. 29Moreover, decreases in coronary flow induced by endothelin-1 are enhanced in isolated perfused rat hearts after ischemia and reperfusion. 3~ Therefore, endothelin-1 seems to be involved in the pathophysiology of ischemic insults both in heart and brain. Our results show that enhanced endothelin-l-induced contractions in the MCAs of goats subjected to ischemia and reperfusion depend on extracellular Ca2+ to a higher extent than in normal arteries. Whereas incubation in Ca2+-free medium reduces contractions to 58% of control in normal arteries, 22the response was reduced to a 41% in this study. The inhibitory effects, however, of nicardipine on endothelin-l-induced contractions were lower in this study in reperfused arteries than in normal arteries. 22 Therefore, involvement of dihydropyridine-sensitive Ltype Ca R+channels in Ca 2+ entry induced by endothelin-1 is reduced by ischemia and reperfusion. It has been shown that Ca2+-permeable nonselective cation channels can contribute to endothelin-l-induced contraction of rabbit aorta. 31 Such a compensatory mechanism of Ca 2+ entry could be increased after ischemia and reperfusion in goat MCAs. We have previously reported that the ability of nicardipine to inhibit the cerebrovascular effects of endothelin-1 is impaired after experimental subarachnoid hemorrhage in the goat, caused by reduced dependence of the cerebral arteries on extracellular Ca 2+ in developing endothelin-l-induced contractions. 23 Therefore, reduced dependence on extracellular Ca 2+ after subarachnoid hemorrhage and reduced involvement of L-type Ca 2+ channels in Ca 2+ entry after nonhemorrhagic cerebral ischemia lead to impaired cerebrovascular effects of Ca 2+ entry blockers like nicardipine. This could have clinical relevance if these drugs are used in the management of cerebrovascular diseases. In conclusion, transient global cerebral ischemia in the goat induces changes in the constrictor effects of endothelin-1 on the MCA that depend both on the duration of the ischemic insult and the subsequent reperfusion. The major change in reactivity was an increase in the potency of the peptide observed 1 week after 20 minutes of ischemia. At this time, the involvement of dihydropyridine-sensitive L-type Ca 2+ channels in Ca 2+ entry induced by endothelin-1 to develop contraction is reduced. This is indicated by a lower inhibitory effect of nicar-
21
CEREBROVASCULAR REACTIVITY AFTER ISCHEMIA
dipine in reperfused arteries, despite the higher dependency of contractions on extracellular Ca 2+ when compared with normal arteries. We suggest that the increased reactivity to endothelin-1 could have pathophysiological relevance and the reduced effect of nicardipine could have therapeutic relevance in cerebral ischemia.
Acknowledgment: The authors are grateful to Maria del Carmen Tirados and Maria del Carmen M~fiez for their technical assistance.
15.
16.
17.
18.
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31.
with acute ischemic stroke. Am J Hypertens 1994;7:10851089. Wei GZ, Zhang J, Sheng SL, et al. Increased plasma endothelin-1 concentration in patients with acute cerebral infarction and actions of endothelin-1 on pial arterioles of the rat. Chin Med J 1993;106:917-921. Ziv I, Fleminger G, Djaldetti R, et al. Increased plasma endothelin-1 in acute ischemic stroke. Stroke 1992;23:10141016. Buunk G, Van der Hoeven JG, Fr61ich M, et al. Cerebral vasoconstriction in comatose patients resuscitated from a cardiac arrest? Intensive Care Med 1996;22:1191-1196. Patel TR, Galbraith S, McAuley MA, et al. Endothelinmediated vascular tone following focal cerebral ischemia in the cat. J Cereb Blood Flow Metab 1996;16:679-687. Barone FC, White RF, Elliott JD, et al. The endothelin receptor antagonist SB217242 reduces cerebral focal ischemic brain injury. J Cardiovasc Pharmacol 1995;26:$404$407, (suppl 3). Spatz M, Yasuma Y, Strasser A, et al. Cerebral postischemic hypoperfusion is mediated by ETA receptors. Brain Res 1996;726:242-246. Feuerstein G, Gu JL, Ohlstein EH, et al. Peptidic endothelin-1 receptor antagonist, BQ-123, and neuroprotection. Peptides 1994;15:467-469. Salom JB, Torregrosa G, Miranda FJ, et al. Effects of endothelin-1 on the cerebrovascular bed of the goat. Eur J Pharmaco11991;192:39-45. Alabadi JA, Salom JB, Torregrosa G, et al. Changes in the cerebrovascular effects of endothelin-1 and nicardipine after experimental subarachnoid hemorrhage. Neurosurgery 1993;33:707-715. Torregrosa G, Barber~ MD, Centeno JM, et al. Characterization of the cortical laser-Doppler flow and hippocampal degenerative patterns after global cerebral ischaemia in the goat. Pflfigers Arch 1998;435:662-669. Alabadi JA, Torregrosa G, Miranda FJ, et al. Impairment of the modulatory role of nitric oxide on the endothelin-1elicited contraction of cerebral arteries: A pathogenetic factor in cerebral vasospasm after subarachnoid hemorrhage? Neurosurgery 1997;41:245-253. Salom JB, Barber~ MD, Centeno JM, et al. Relaxant effects of sodium nitroprusside and NONOates in goat middle cerebral artery: Delayed impairment by global ischemiareperfusion. Nitric Oxide 1999;3:85-93. Kohzuki M, Onodera H, Yasujima M, et al. Endothelin receptors in ischemic rat brain and Alzheimer brain. J Cardiovasc Pharmacol 1995;26:$329-$331, (suppl 3). Touzani O, Galbraith S, Siegl P, et al. Endothelin-B receptors in cerebral resistance arterioles and their functional significance after focal cerebral ischemia in cats. J Cereb Blood Flow Metab 1997;17:1157-1165. Saito T, Fushimi E, Abe T, et al. Augmented contractile response to endothelin and blunted endothelium-dependent relaxation in post-ischemic reperfused coronary arteries. Jpn Circ J 1992;56:657-670. Zaugg CE, Zhu P, Simper D, et al. Differential effects of endothelin-1 on normal and postischemic reperfused myocardium. J Cardiovasc Pharmacol 1993;22:$367-$370, (suppl 8). Komuro T, Miwa S, Zhang XF, et al. Physiological role of Ca2~-permeable nonselective cation channel in endothelin-l-induced contraction of rabbit aorta. J Cardiovasc Pharmacol 1997;30:504-509.
We have examined the effects of global ischemia and subsequent reperfusion on the reactivity of goat middle cerebral artery to endothelin-1. The participation of extracellular Ca 2+ in endothelin-l-induced contractions and the ability of the Ca 2+ entry blocker, nicardipine, to counteract them after ischemia were also assessed. Concentration-response curves to endothelin-1 (10-12 - 3 • 10 s tool/L) obtained in arteries from goats subjected to either 5 or 10 minutes of ischemia and 7-day reperfusion did not significantly differ from curves obtained in arteries from sham-operated goats, Concentration-response curves to endothelin-1 in arteries from goats subjected to 20 minutes of ischemia and 7-day reperfusion, however, showed significantly higher pECs0 (negative logarithm to base 10 of the concentration of endothelin-1 producing 50% of the maximal effects) value (P < .01) than in arteries from sham-operated goats. The effect of 20 minutes of ischemia was assessed at various reperfusion times. The maximal effects (Ema• values of the concentration-response curves to endothelin-1 were significantly reduced (P < .01) 1 and 3 days after ischemia when compared with sham-operated animals. The concentration-response curve to endothelin-1 was displaced to the left 7 days after the ischemia. Two weeks after the ischemia, the Ernaxvalue was again significantly reduced (P < .01). When arteries obtained from goats 7 days after 20 minutes of ischemia were incubated in Ca2+-free medium or nicardipine (10-l~ and 10 s mol/L), the concentration-response curve to endothelin-1 was significantly inhibited (P < .01) both in terms of pECs0 and of Ema• In conclusion, there was an increase in the endothelin-1 potency 1 week after 20 minutes of ischemia. At this time, the involvement of dihydropyridine-sensitive L-type Ca2+ channels in Ca2+ entry induced by endothelin-1 to develop contraction is reduced when compared with normal arteries. We suggest that the increased reactivity to endothelin-1 could have pathophysiological relevance, and the reduced effect of nicardipine could have therapeutical relevance in cerebral ischemia. Key Words: Endothelin-l~Ca2+ chann e l - M i d d l e cerebral artery--Cerebral ischemia--Goat.
From the Centro de Investigaci6n, Hospital Universitario La Fe, Departamento de BiologiaAnimal, and Departamento de Fisiologia, Universidad de Valencia,Valencia,Spain. Received March 8,1999; accepted July 1, 1999. Partially supported by a grant from Fondo de Investigaci6n Sanitaria (FIS 95/1668). J.M.C. and M.O. hold research fellowships from Ministeriode Educaci6n y Cultura and Conselleriade Cultura, Educaci6 i Ci6ncia,respectively. Address reprint requests to Juan B. Salom, PhD, Centro de Investigaci6n, Hospital Universitario La Fe, Ave Campanar 21, E46009, Valencia,Spain. Copyright 9 2000by National Stroke Association 1052-3057/00/0901-000353.00/0
16
Endothelins are potent constrictors of cerebral arteries that are isolated from various species, including humans. 1 Abluminal local application of endothelin-1 to the middle cerebral artery (MCA) in rats results in profound and long-lasting reductions in local cerebral blood flow (CBF), focal cerebral ischemia, and development of cerebral infarction.2,3 Intrastriatal injection of endothelin-1 in rats produces ischemic lesions caused by vasoconstriction of cerebral microvessels.4 Endothelins are believed to be involved in the pathophysiology of most c o m m o n cerebrovascular diseases, such as subarachnoid hemorrhage, cerebral ischemia, and
Journal of Stroke and Cerebrovascular Diseases, Vol, 9, No. 1 (January-February), 2000: pp 16-21
17
CEREBROVASCULAR REACTIVITY AFTER ISCHEMIA
migraine. -~In regard to the involvement of endothelin-1 in the neuropathological consequences of cerebral ischemia, immunoreactive endothelin-1 is increased in rat brain, 6,7 and in rabbit brain and plasma s after focal ischemia. After global ischemia, concentration of endothelin-1 increases in gerbil forebrain, 9,1~ rat striatum, 6 and rat hippocampus, u,12 in which selective neuronal death takes place. 13In the clinical setting, plasma endothelin-1 is increased in patients with acute ischemic stroke 14-16 and in patients resuscitated from cardiac arrest. 17The use of endothelin-1 receptor antagonists has added to the evidence that endothelin-I has a role in the pathophysiology of cerebral ischemia. Intravenous administration of PD156707 (a nonselective receptor antagonist) increases cerebral perfusion and reduces ischemic damage in feline focal ischemia. ~8 Oral administration of SB217242 (a nonselective receptor antagonist) also reduces cerebral focal ischemic injury in the rat. 19 Finally, BQ-123 (selective endothelin A receptor antagonist) reverses late postischemic hypoperfusion 2~ and increases hippocampal CA1 neuron survival 21 in gerbils subjected to transient global ischemia. We have previously shown that endothelin-1 reduces CBF in goats by causing contraction of the cerebral arteries, which depends partially on the activation of Ca 2+ influx through dihydropyridine-sensitive channels. 22 After experimental subarachnoid hemorrhage, the cerebral vasoconstrictor effects of endothelin-I are increased, and dependence on extracellular Ca 2+ is reduced. 23 To gain insight into the role of endothelin-1 in the pathophysiology of cerebral ischemia, we have examined, in this study, the effects of global ischemia and subsequent reperfusion on the reactivity of goat MCA to endothelin-1. Participation of extracellular Ca 2+ in endothelin-l-induced contractions and the ability of the Ca 2+ entry blocker, nicardipine, to counteract them after ischemia were also assessed.
Materials a n d M e t h o d s Experiments were performed in accordance with the guidelines from the Council of the European Economic Community (86 / 609 / EEC, Article 5, Appendix II), promulgated by the Spanish legislature on March 14, 1988 (R.D. 223/1988).
and surgical procedures have been reported previously. 24 Briefly, the external carotid arteries were exposed bilaterally by means of an incision made along each mandible. Snare-type occluders were placed around the external carotid arteries, cephalad to the mandibular and occipital branches, and tunneled subcutaneously to be exteriorized on the back of the horns. The following parameters were continuously monitored: cortical perfusion (laser-Doppler probe on the left dorsolateral cortex); intracranial pressure (16-gauge 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 (the blood pressure pulse by means of a tachymeter). After a period of stabilization after surgery, transient global cerebral ischemia (5, 10, or 20 minutes) was achieved under anesthesia by occlusion of the 2 external carotid arteries and simultaneous external compression of the jugular veins by means of a neck tourniquet. Successful ischemia was assessed by a reduction of cortical perfusion by more than 80% and flattening of brain waves seen on the electrocorticogram. The reperfusion period began when the occlusions were released and was monitored for 2 hours. The animals were then allowed to recover from anesthesia. The animals were allowed to reperfuse for 1, 3, 7, or 14 days. Sham-operated goats, in which the surgical procedures were followed but ischemia was not induced and the different reperfusion times were allowed, served as the control. The number of animals used in each experimental group was, as follows: 7 sham-operated goats (included in the 4 different reperfusion groups), 4 goats subjected to 5 minutes of ischemia, 4 goats subjected to 10 minutes of ischemia, and 18 goats subjected to 20 minutes of ischemia (which were included in the 4 different reperfusion groups). The 7 sham-operated goats were distributed, as follows: 1 goat in the 1-day reperfusion group, 1 goat in 3-day reperfusion, 4 goats in 7-day reperfusion, and 1 goat in the 14-day reperfusion group. Finally, the 18 goats subjected to 20 minutes of ischemia were distributed, as follows: 4 goats in the 1-day reperfusion group, 4 goats in 3-day reperfusion, 7 goats in 7-day reperfusion, and 3 goats in the 14-day reperfusion group.
Induction of Transient Global Cerebral Ischemia Thirty-three female goats (30 to 35 kg) were sedated with 10 m g / k g ketamine (Ketolar; Parke-Davis, Barcelona, Spain) administered intramuscularly (i.m.) and anesthetized with 2% sodium thiopental (Tiobarbital; Palex, Ja6n, Spain) administered intravenously (i.v.). After gastric and tracheal intubation, anesthesia was maintained by means of a ventilator (Themel VT/3, Valencia, Spain), supplying 1.5% halothane (Fluothane; Zeneca Farma, Pontevedra, Spain) in 80% N20 and 20% 02. Details on the dynamic anatomy of the blood supply to the goat's brain
Isometric Tension Recording in Isolated Arteries The goats, sedated and anesthetized as described previously, were killed by i.v. injection of 30 mEq KC1 (UCB Pharma, Barcelona, Spain). The whole brain was rapidly removed. The 2 MCAs were dissected free and cut into a total of 8 to 10 cylindrical segments, measuring 4 m m in length and about 1 m m in outer diameter. Each segment was prepared for isometric tension recording in an organ bath. Two stainless-steel L-shaped pins (125 ~m in diameter) were introduced through the arterial lumen. One pin
18
J.B. S A L O M E T AL.
was fixed to the organ bath wall; 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% o2 and 5% co2 to give a pH of 7.3 to 7.4. The temperature was kept at 37~ A resting tension of 1 g was applied to the arterial segments, and they were allowed to equilibrate for 60 to 90 minutes. Tension was readjusted when necessary, and the bath fluid was changed every 15 minutes before beginning the experiments. The contractile capacity of the arterial segments was examined by exposure to a high-K + (50 mmol/L) RingerLocke solution. The effects of endothelin-1 (10 12 3 • 10 8 mol/L) were studied by cumulative addition to the organ bath. The influx of extracellular Ca 2+, on stimulation with endothelin-1, was studied by obtaining concentration-response curves to the peptide after incubation in Ca2+-free medium for 20 minutes. The involvement of dihydropyridine-sensitive Ca 2+ channels in endothelin-1induced responses was assessed by obtaining concentration-response curves to the peptide after incubation with the Ca 2+ entry blocker, nicardipine (10 10or 10 8 mol/L), for 20 minutes. Only 1 concentration-response curve was performed in each arterial segment. _
Statistical Analysis The contractile responses to endothelin-1 were expressed as a percentage of the previous contraction induced by 50 mmol/L of KC1. Maximal effects (Ema• and the concentration of endothelin-1 producing 50% of the Er~ax (EC50) were calculated for each concentrationresponse curve. The pEC50 was calculated as the negative logarithm to base 10 of the EC50 for statistical analysis. The Dunnett test was used for comparisons with the control. Probability values of less than 5% were considered statistically significant.
Drugs and Solutions Endothelin-1 (Peptide Institute Inc, Osaka, Japan) was dissolved in 0.1% aqueous acetic acid and diluted in a phosphate-buffered saline solution with 0.05% bovine serum albumin, fraction V (Sigma Chemicals Co, St Louis, MO). Nicardipine (Ferrer Int, Barcelona, Spain) was dissolved in twice-distilled water and diluted in saline solution. The Ringer-Locke solution had the following composition: 120 mmol/L of NaC1, 5.4 mmol/L of KC1, 2.2 mmol/L of CaC12, 10 mmol/L of MgC12, 25 mmol/L of NaHCO3, and 5.6 mmol/L of glucose. In KCl-depolarizing solution, NaC1 was replaced by an equimolar amount of KC1. The Ca2+-free solution was prepared by omitting CaC12. The phosphate-buffered saline solution had the
following composition: 150 mmol/L of NaC1, and 10 mmol/L of NaH2PO4.
Results Endothelin-1 (10-12 - 3 x 10-s tool/L) induced concentration-dependent contractions of goat MCAs were obtained from sham-operated goats, as well as from goats subjected to different times of global cerebral ischemia and reperfusion. Reactivity to endothelin-1 was not significantly different in MCAs from sham-operated goats killed at 1, 3, 7, and 14 days after surgery. The effects of ischemia duration were assessed after 1 week of reperfusion. Concentration-response curves to endothelin-1 obtained in arteries from goats subjected to either 5 or 10 minutes of ischemia did not significantly differ, either in terms of pECs0 or in terms of E.... from curves obtained in arteries from sham-operated goats (Fig 1 and Table 1). Concentration-dependent contractions induced by endothelin-1 in arteries from goats subjected to 20 minutes of ischemia showed significantly higher pECs0 value (P < .01) than in arteries from sham-operated goats. The Emax value, however, remained unchanged (Table 1). Therefore, the concentration-response curve to endothelin-1 was slightly displaced to the left in arteries from goats subjected to 20 minutes of ischemia and 1 week of reperfusion with respect to arteries from shamoperated animals (Fig 1). The effect of 20 minutes of ischemia was assessed at various reperfusion times. The Emaxvalues of the concen--0---I---rn---II--
180 160 O ~" o z E O E F- o On o z
Sham-0perated 5 min 10 min 20 min
140 120
ZI
100 80
Se(~
60
~
40
~
20
q
I
i
i
f
I
i
I
12
11
10
9
8
7
ENDOTHELIN-1 (-log mol/L)
Figure1. Concentration-response curves to endothelin-1 in MCA isolated from sham-operated goats (n = 37) and from goats subjected to transient global cerebral ischemia of 5 (n = 32), 10 (n = 36), or 20 (n = 23) minutes duration. All goats were allowed 7 days of reperfusion. The symbols represent mean values of contraction expressed as a percentage of previous response to 50 mmol/L KCI. The vertical bars show SD.
19
CEREBROVASCULAR REACTIVITY AFTER ISCHEMIA
Table 1. Effects o f ischemia duration on
Table 2. Effects o f repeffusion duration on
concentration-response curve parameters f o r endothelin- 1
concentration-response curve parameters f o r endothelin- 1
in M C A f r o m goats qfter 1 week o f reperfusion
in M C A from goats subjected to 20 minutes o f ischemia
pECso Sham-operated 5-min ischemia 10-rain ischemia 20-rain ischemia
9.6 9.6 9.8 10.2
-+ 0.3 + 0.5 -+ 0.6 _+ 0.5*
Emax (%)
n
125 107 135 116
37 32 36 23
_+ 38 + 18 _+ 36 _+ 38
pECso Sham-operated 1-day reperfusion 3-day reperfusion 7-day reperfusion 14-day reperfusion
NOTE: Values are expressed as mean _+ SD. *Significantly different from sham-operated goats (P < .01).
-6 E E o ~o
- - O - - Control
- - O - - Sham-operated - - t - - 1 day - - r n - - 3 days
160
O
140
z
40-
~
"6
E
O E t- o O Lo o
80-
O
--13-- Nicardipine 10 -1~ mol/L --I--
Nicardipine 10 -8 mol/L
T
T
140 120 -
O
100-
60-
Ca2+-free medium
v
-
gr O o Z
60 29 29 23 30
_+ 34 + 24* _+ 16" -4- 38 -+ 15"
Our results show that transient global cerebral ischemia in the goat induces changes in the reactivity of the MCA to endothelin-1, which d e p e n d both on the duration of the ischemic insult and the subsequent reperfusion. Up to 10 minutes of ischemia did not induce changes in the reactivity of MCA to endothelin-1. A 20-minute ischemic insult was necessary to produce an increase in the reactivity to endothelin-1, consisting of a higher contractile potency of the peptide on the MCA. In regard to the reperfusion time, the increase in reactivity to endothelin-1 appeared 7 days after ischemia, whereas, after shorter (1 and 3 days) and longer (14 days) reperfusion periods,
120 z O Io
121 103 97 116 93
Discussion
--I--
O
_+ 0.3 + 0.3 _+ 0.3 -4- 0.5* -+ 0.3
n
NOTE: Values are expressed as mean _+ SD. *Significantly different from sham-operated goats (P < .01).
tration-response curves to endothelin-1 were significantly reduced (P < .01) 1 and 3 days after ischemia when compared with sham-operated animals (Fig 2 and Table 2). Then, as mentioned previously, the concentrationresponse curve to endothelin-1 was displaced to the left 1 week after the ischemic episode. Two weeks after the ischemia, the Emax value was again significantly reduced (P < .01) with respect to sham-operated goats (Fig 2 and Table 2). When arteries obtained from goats 7 days after 20 minutes of ischemia were incubated in Ca2+-free medium, the concentration-response curve to endothelin-1 was significantly inhibited (P < .01; Fig 3), both in terms of pECs0 and of Emax (Table 3). Incubation with the Ca 2+ entry blocker, nicardipine (10 ]0, 10 8 mol/L), also induced significant inhibition (P < .01) of the concentration-response curves to endothelin-1 (Fig 3 and Table 3).
160
9.6 9.7 9.7 10.2 9.7
E ..... (%)
Iz
O O
t-
100 8060-
O
40-
"6
2o-
20 O0 i
12
1
i
i
i
i
10
9
8
7
t
t
t
i
t
12
11
10
9
8
ENDOTHELIN-1 (-log mol/L) ENDOTHELIN-1 (-log mol/L) Figure 2. Concentration-response curves to endothelin-1 in MCA isolated from sham-operated goats (n - 60) and from goats subjected to 20 minutes of transient global cerebral ischemia, followed by 1 (n = 29), 3 (n = 29), 7 (n = 23), or 14 (n = 30) days of reperfusion. The symbols represent mean values of contraction expressed as a percentage of previous response to 50 mmol/L KC1. The vertical bars show SD.
Figure 3. Concentration-response curves to endothelin-1 in MCA isolated from goats subjected to 20 minutes of transient global cerebral ischemia, followed by 7 days of reperfusion. Curves were obtained in control conditions (n - 23), in Ca2+-free medium (n - 7), or during incubation with the Ca2~ entry blocker, nicardipine 10 7o mol/L (n = 8), or 10 8 mol/L (n - 8). The symbols represent mean values of contraction expressed as a percentage of previous response to 50 mmol/L KCI. The vertical bars show SD.
20
J.B. SALOM ET AL.
Table 3. Effects of Ca2+ removal and nicardipine on concentration-response curve parameters for endothelin-1 in MCA from goats subjected to 20 minutes of ischemia and 1-week of reperfusion
Control Ca2+-free medium Nicardipine 10-1~mol/L Nicardipine 10 8 mol/L
pECs0
Emax(%)
n
10.2 • 0.5 9.3 -+ 0.3* 9.1 _+ 0.4* 9.2 --_ 0.3*
116 --+ 38 48 • 17" 7.4 • 20* 65 • 21"
23 7 8 8
NOTE: Values are expressed as mean _+SD. *Significantly different from control (P < .01). the reactivity of MCA to endothelin-1 was reduced in terms of efficacy (maximum effect) of the peptide. Therefore, the increase in reactivity induced by ischemia showed up acutely I week after the ischemic insult. We have previously shown that there is a functional interaction between endothelin-1 and nitric oxide (NO) in goat MCA, consisting of a partial counteraction of the contractile response to endothelin-1 by NO. 25 Moreover, we have recently found that the relaxant effects of NO are impaired in MCA from goats subjected to 20 minutes of global cerebral ischemia and 7-day reperfusion. 26 Therefore, impairment of the counteracting effect of NO is in line with the potentiation of endothelin-l-induced contractions observed in this study. Other investigators have observed that cerebral ischemia induces changes in the presence and function of brain endothelin receptors. In the gerbil, transient forebrain ischemia results in a 15% reduction in endothelin receptor density 60 minutes after reperfusion. 1~In the rat brain, the endothelin-binding sites are remarkably increased in the hippocampal CA1 field and dentate gyrus, ventral thalamic nucleus, and cortical vessels 4 and 7 days after transient forebrain ischemia. 27 Finally, endothelin B receptor-mediated dilatation, elicited by the direct adventitial application of BQ-3020 onto cat pial arterioles, is lost within 30 minutes of focal ischemia. 28 Therefore, our results and those from other investigators indicate that early and delayed changes in endothelin receptors of brain tissue and blood vessels, together with increases in the synthesis and availability of endothelin-1, 6,8,9,16could contribute to the pathophysiology of cerebral ischemia. In a previous study, 24 we have shown that delayed neuronal death in the hippocampal CA1 subfield of the goat appears 7 days after 20 minutes of ischemia but not after shorter ischemia insults (5 or 10 minutes). Therefore, there is a temporal correlation (7 days) between the appearance of hyperreactivity to endothelin-1 and delayed neuronal death induced by transient global ischemia in the goat. Moreover, there is a coincidence in the severity of ischemia (20 minutes) causing hyperreactivity and delayed neuronal death. A direct causal relationship, however, between maintained cerebral hypoperfusion
eventually induced by hyperreactivity to endothelin-1 and delayed neuronal death can not be inferred. Additional studies using selective blockers of endothelin-1 receptors in vivo to counteract hyperreactivity to endothelin-1 could help to clarify this point. A postischemic increase in the contractile response to endothelin-1 seems not to be restricted to the cerebrovascular bed. Augmented reactivity to endothelin-1 has been reported in dog isolated left anterior descending coronary artery after myocardial ischemia and reperfusion. 29Moreover, decreases in coronary flow induced by endothelin-1 are enhanced in isolated perfused rat hearts after ischemia and reperfusion. 3~ Therefore, endothelin-1 seems to be involved in the pathophysiology of ischemic insults both in heart and brain. Our results show that enhanced endothelin-l-induced contractions in the MCAs of goats subjected to ischemia and reperfusion depend on extracellular Ca2+ to a higher extent than in normal arteries. Whereas incubation in Ca2+-free medium reduces contractions to 58% of control in normal arteries, 22the response was reduced to a 41% in this study. The inhibitory effects, however, of nicardipine on endothelin-l-induced contractions were lower in this study in reperfused arteries than in normal arteries. 22 Therefore, involvement of dihydropyridine-sensitive Ltype Ca R+channels in Ca 2+ entry induced by endothelin-1 is reduced by ischemia and reperfusion. It has been shown that Ca2+-permeable nonselective cation channels can contribute to endothelin-l-induced contraction of rabbit aorta. 31 Such a compensatory mechanism of Ca 2+ entry could be increased after ischemia and reperfusion in goat MCAs. We have previously reported that the ability of nicardipine to inhibit the cerebrovascular effects of endothelin-1 is impaired after experimental subarachnoid hemorrhage in the goat, caused by reduced dependence of the cerebral arteries on extracellular Ca 2+ in developing endothelin-l-induced contractions. 23 Therefore, reduced dependence on extracellular Ca 2+ after subarachnoid hemorrhage and reduced involvement of L-type Ca 2+ channels in Ca 2+ entry after nonhemorrhagic cerebral ischemia lead to impaired cerebrovascular effects of Ca 2+ entry blockers like nicardipine. This could have clinical relevance if these drugs are used in the management of cerebrovascular diseases. In conclusion, transient global cerebral ischemia in the goat induces changes in the constrictor effects of endothelin-1 on the MCA that depend both on the duration of the ischemic insult and the subsequent reperfusion. The major change in reactivity was an increase in the potency of the peptide observed 1 week after 20 minutes of ischemia. At this time, the involvement of dihydropyridine-sensitive L-type Ca 2+ channels in Ca 2+ entry induced by endothelin-1 to develop contraction is reduced. This is indicated by a lower inhibitory effect of nicar-
21
CEREBROVASCULAR REACTIVITY AFTER ISCHEMIA
dipine in reperfused arteries, despite the higher dependency of contractions on extracellular Ca 2+ when compared with normal arteries. We suggest that the increased reactivity to endothelin-1 could have pathophysiological relevance and the reduced effect of nicardipine could have therapeutic relevance in cerebral ischemia.
Acknowledgment: The authors are grateful to Maria del Carmen Tirados and Maria del Carmen M~fiez for their technical assistance.
15.
16.
17.
18.
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