Role of α-adrenoceptors and prostacyclin in the enhanced adrenergic reactivity of goat cerebral arteries after ischemia-reperfusion

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Research Report

Role of α-adrenoceptors and prostacyclin in the enhanced adrenergic reactivity of goat cerebral arteries after ischemia-reperfusion Luis Monge, Nuria Fernández⁎, Adely Salcedo, Ángel Luis García-Villalón, Godofredo Diéguez Departamento de Fisiología, Facultad de Medicina, Universidad Autónoma, 28029 Madrid, Spain

A R T I C LE I N FO

AB S T R A C T

Article history:

To analyze ischemia-reperfusion effects on the cerebrovascular adrenergic response, the

Accepted 28 May 2010

left middle cerebral artery (MCA) of anesthetized goats was occluded for 120 min and

Available online 4 June 2010

reperfused for 60 min. Isolated segments from the left (ischemic) and right (control) MCA exhibited isometric constriction in response to noradrenaline (10− 8–10− 4 M, in the presence

Keywords:

of β-adrenoceptors blockade), phenylephrine (α1-adrenoceptors agonist, 10− 8–10− 4 M), B-HT-

Brain ischemia

920 (α2-adrenoceptors agonist, 10− 7–3 × 10− 3 M) or tyramine (indirect sympatheticomimetic

Cerebrovascular reactivity

amine, 10− 8–10− 4 M), but this constriction was greater in ischemic arteries. The cyclooxygenase

α-adrenoceptors

(COX) inhibitor meclofenamate (10− 5 M) augmented the response to noradrenaline only in

COX-2

control arteries. The prostacyclin (PGI2) synthesis inhibitor tranylcypromine (TCP, 10− 5 M)

Endothelium

increased the response to noradrenaline in control arteries and reduced it in ischemic arteries.

Prostaglandins

The thromboxane A2 (TXA2) synthase inhibitor furegrelate (10− 6 M) did not modify the noradrenaline effect in both types of arteries, whereas the TXA2 receptor antagonist SQ 29 548 (10− 5 M) and the COX-2 inhibitor NS-398 (10− 6 M) decreased the response to noradrenaline only in ischemic arteries. PGI2 caused a small relaxation in control arteries and a small contraction in ischemic arteries. α-Adrenoceptors and COX-2 protein expression and the metabolite of PGI2 were augmented in ischemic arteries. Therefore, ischemia-reperfusion may increase the cerebrovascular responsiveness to noradrenaline, through upregulation of α-adrenoceptors and increased COX-2-derived PGI2 exerting a vasoconstrictor action. After ischemiareperfusion, noradrenaline might increase PGI2 production thus contributing to adrenergic vasoconstriction and/or PGI2 would potentiate the noradrenaline effects. © 2010 Elsevier B.V. All rights reserved.

⁎ Corresponding author. Departamento de Fisiología, Facultad de Medicina, Universidad Autónoma, Arzobispo Morcillo, 2, 28029 Madrid, Spain. Fax: +34 91 497 5478. E-mail address: [email protected] (N. Fernández). Abbreviations: MCA, middle cerebral artery; COX, cyclooxygenase; PGI2, prostacyclin; TCP, tranylcypromine; TXA2, thromboxane A2; NPY, neuropeptide Y 0006-8993/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2010.05.091

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1.

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Introduction

Brain ischemia-reperfusion can produce damage and dysfunction of cerebral blood vessels, in addition to that of nervous tissue. There are studies showing that even short periods (≤30 min) of cerebral ischemia followed by reperfusion can induce endothelial dysfunction (Mayhan et al., 1988; Rosenblum, 1997) and longer periods (60 min) might also increase the endothelial production of vasoconstrictor prostanoids (Sánchez et al., 2006). These adverse effects of ischemia-reperfusion could alter the adrenergic response of cerebral vasculature. This might have significant implications for regulation of blood supply to ischemic-reperfused brain area as experimental and clinical cerebral ischemia can be accompanied by increased catecholamine levels in plasma (Hachinski et al., 1986) and cerebrospinal fluid (Meyer et al., 1974). This catecholamine release by acting on cerebral vasculature may be detrimental to the extent of brain ischemia (Nellgard et al., 1999). Both α1-adrenoceptors antagonists, by inhibiting vascular smooth muscle contraction (He et al., 2008), and α2-adrenoceptors agonists (Zhang, 2004) and antagonists (Gustafson et al., 1990) have been shown to lessen ischemia-induced neuronal damage. Cerebral vasculature appears to receive a dense adrenergic innervation, and although the functional significance of this innervation remains to be a controversial topic, there are in vivo and in vitro studies suggesting that cerebral vasculature exhibits a basal adrenergic constrictor tone and constricts to exogenous and endogenous noradrenaline by direct α-adrenoceptors activation (Edvinsson and Krause, 2002; Goadsby and Edvinsson, 1997; Jordan et al., 2000). Recent evidence suggests that in humans sympathetic innervation produces a basal tone and vasoconstriction in cerebral vasculature (Van Lieshout and Secher, 2008) and that this innervation plays a role in cerebral autoregulation (Hamner et al., 2010). On the other hand, the contraction of cerebral arteries in response to noradrenaline may be modulated by the endothelium (Edvinsson and Krause, 2002). Studies to examine the effects of ischemia-reperfusion on cerebrovascular response to adrenergic stimulation are very few, and the available results are inconclusive. One study performed in anesthetized newborn pigs shows that 20 min of global cerebral ischemia induced by intracranial hypertension followed by 2–3 h or 24 h of reperfusion did not affect the reactivity of pial arteries in situ to topical application of noradrenaline (Leffler et al., 1989b). Another study in rats shows that the long-term inhibition of NO synthesis with L -NAME limits infarct expansion by a reduction in the vasoconstrictor response to noradrenaline and serotonin after prolonged occlusion of the left middle cerebral artery (MCA), without reperfusion (Serercombe et al., 2001). The present study was performed to analyze the effects of ischemia-reperfusion on the cerebrovascular adrenergic reactivity, analyzing the role of α-adrenoceptors and prostanoids in this reactivity. Ischemia-reperfusion was induced in anesthetized goats by inducing 120-min occlusion of the MCA, followed by 60-min reperfusion, and vascular adrenergic response was examined in isolated arteries. Previous studies suggest that in the goat, adrenergic mechanisms are involved

in the regulation of the cerebral circulation (Alborch et al., 1977; Diéguez et al., 1998), and that ischemia-reperfusion induces endothelial dysfunction (Sánchez et al., 2006; Salcedo et al., 2009).

2.

Results

2.1.

Hemodynamic changes during ischemia and reperfusion

In 29 animals, during MCA occlusion mean systemic arterial pressure was decreased by 12 ± 2% (p < 0.01), and it was further decreased by 16 ± 2% during reperfusion (p < 0.01). Heart rate during MCA occlusion and reperfusion was not significantly distinct from the control. In 10 of these animals, blood flow in the left MCA was abolished during arterial occlusion as expected. Immediately after the release of this occlusion, left MCA flow increased markedly, then it was progressively recovering and at 60 min after the start of reperfusion it remained increased by 44 ± 11% (p < 0.05). At this time, left MCA resistance was decreased by 27 ± 6% (p < 0.01). The hemodynamic values during control, left MCA occlusion and reperfusion are summarized in Table 1. Systemic blood gases and pH did not change significantly during ischemia and reperfusion when compared to control conditions (these data are not shown).

2.2.

In vitro arterial response

KCl (100 mM) contracted resting arteries, and this contraction was similar at the beginning and at the end of the experiments. Also, this contraction was not statistically different between control and ischemic arteries. Mean contraction was 2020 ± 97 mg in control arteries (82 segments, 29 animals) and 1817 ± 92 mg (79 segments, 29 animals) in ischemic arteries (p > 0.05). Noradrenaline (10− 8–10− 4 M) produced a concentrationdependent contraction in control and ischemic arteries pretreated with propranolol (10− 7 M). The sensitivity to noradrenaline was similar in both types of arteries, but the

Table 1 – Values for mean systemic arterial pressure, middle cerebral artery (MCA) flow, MCA resistance and heart rate obtained in 29 anesthetized goats under control conditions at the end of the MCA arterial occlusion (ischemia) and at 60 min of reperfusion. Control Ischemia Reperfusion Mean arterial pressure (mm Hg) MCA flow a (mL min− 1) MCA resistance a (mm Hg mL–1 min) Heart rate (beats min− 1)

99 ± 2 ⁎⁎

95 ± 1 ⁎⁎

2.62 ± 0.15 44 ± 2.36

0 ∞

3.78 ± 0.5 ⁎ 32 ± 4.31 ⁎⁎

77 ± 2

77 ± 3

113 ± 2

Values are mean ± SEM. a The values correspond to 10 of the 29 animals. ⁎⁎ p < 0.01 compared with its control. ⁎ p < 0.05 compared with its control.

79 ± 3

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NS-398 (10− 6 M) did not affect significantly the response to noradrenaline in control arteries, while it decreased it in ischemic arteries (Table 2, Fig. 3). Table 2 shows the pD2 values and the maximal contraction for noradrenaline, phenylephrine, B-HT-920, tyramine and NPY in control and ischemic arteries, as well as the pD2 values and the maximal contraction for noradrenaline in the presence of meclofenamate, TCP, SQ 29 548, furegrelate and NS-398. In the arteries tested, the thromboxane A2 analogue U46619 contracted control and ischemic arteries, and the tone achieved was 743 ± 69 mg (36 control segments, 5 animals) and 471 ± 65 mg (28 ischemic segments, 5 animals) (p < 0.01). In the presence of this tone, ADP (10− 8–10− 4 M) caused a concentration-dependent relaxation that was significantly lower in ischemic than in control arteries, without altering significantly the sensitivity. The maximal relaxation (% of the induced tone with U46619) for ADP was 90 ± 4 (control arteries) and 76 ± 5 (ischemic arteries) (p < 0.05), and the pD2 values were 6.48 ± 0.07 (control arteries) and 6.42 ± 0.10 (ischemic arteries) (p > 0.05). Under basal conditions, PGI2 (10− 6–10− 5 M) produced a small relaxation in control arteries and a small contraction in ischemic arteries, and these effects were statistically different for the dose of 10− 5 M (p < 0.05). In the presence of the inhibitor of PGI2 IP-receptors RO 1138452 both types of arteries exhibited a much higher contraction to PGI2 (Fig. 4).

maximal contraction was significantly higher (p < 0.01) in ischemic than in control vessels (Table 2, Fig. 1). Propranolol by itself did not modify resting tone in control and ischemic arteries. Phenylephrine (10− 8–10− 4 M) induced a concentration-dependent contraction, and both the sensitivity and maximal contraction were higher in ischemic than in control arteries (p < 0.05) (Table 2, Fig. 1). B-HT-920 (10− 7–3 × 10− 3 M) produced a concentration-dependent contraction and the maximal contraction, but not the sensitivity was higher (p < 0.05) in ischemic than in control arteries (Table 2, Fig. 1). Tyramine (10− 8–10− 4 M) induced a concentration-dependent contraction in control and ischemic arteries, and both the sensitivity and the maximal contraction to tyramine were significantly higher (p < 0.05) in ischemic than in control vessels (Table 2, Fig. 1). Neuropeptide Y (NPY, 10− 10–10− 7 M) induced a concentration-dependent contraction that was similar in control and ischemic arteries (Table 2). Meclofenamate (10− 5 M) augmented the contraction without changing the sensitivity to noradrenaline in control arteries, whereas it did not modify both the sensitivity and contraction in ischemic vessels (Table 2, Fig. 2). TCP (10− 5 M) augmented the response to noradrenaline in control arteries, whereas it inhibited this response in ischemic vessels. This inhibitor of PGI2 synthesis did not change significantly the sensitivity to noradrenaline in both types of vessels (Table 2, Fig. 3). SQ 29 548 (10− 5 M) decreased the maximal response, without changing the sensitivity to noradrenaline in ischemic arteries, and it did not change the effects of this neurotransmitter in control arteries (Table 2, Fig. 3). Furegrelate (10− 6 M) did not modify significantly the response of both control and ischemic arteries to noradrenaline (Table 2, Fig. 3).

2.3. Measurements of prostanoid production by enzyme immunoassay Basal production of 6-keto-PGF1α (metabolite of PGI2) was significantly higher in ischemic arteries than in control arteries (p < 0.05). Basal TXB2 production (metabolite of TXA2) and 13,14dihydro-15-keto PGF2α production (metabolite of PGF2α) did not change significantly in arteries after ischemia-reperfusion in comparison to control conditions.

Table 2 – Values for maximal contraction (Emax) and pD2 obtained with noradrenaline, phenylephrine, B-HT-920, tyramine and NPY in control and ischemic arteries, in the absence and in the presence of the different treatments tested. Control arteries Emax (%KCl)

Ischemic arteries pD2

Emax (%KCl)

pD2

Noradrenaline Phenylephrine B-HT-920 Tyramine NPY

21 ± 2 25 ± 3 4 ± 0.4 13 ± 3 31 ± 6

5.80 ± 0.10 4.73 ± 0.14 3.85 ± 0.18 4.32 ± 0.19 8.07 ± 0.05

(22,14) (7,5) (4,2) (8,5) (17,9)

38 ± 2 ⁎⁎ 37 ± 1 ⁎ 19 ± 2 ⁎ 29 ± 7 ⁎ 30 ± 6

5.83 ± 0.14 5.40 ± 0.06 3.92 ± 0.19 5.31 ± 0.19 8.19 ± 0.06

(19,12) (9,5) ⁎⁎ (4,2) (5,4) ⁎ (16,9)

Noradrenaline + treatment with Meclofenamate TCP SQ 29 548 Furegrelate NS-398

59 ± 4 †† 34 ± 3 † 23 ± 3 21 ± 5 15 ± 3

5.73 ± 0.13 5.68 ± 0.18 5.85 ± 0.09 6.29 ± 0.12 5.90 ± 0.12

(9,4) (8,6) (9,6) (11,6) (7,4)

48 ± 4 17 ± 3⁎⁎,†† 21 ± 3 † 24 ± 4 11 ± 3 ††

5.54 ± 0.14 5.56 ± 0.23 6.00 ± 0.15 6.07 ± 0.10 6.26 ± 0.16

(7,4) (10,6) (10,6) (7,6) (8,4)

Values are mean ± SEM. In parentheses, number of arterial segments and animals, respectively. ⁎ p < 0.05 compared with control arteries in the corresponding conditions. ⁎⁎ p < 0.01 compared with control arteries in the corresponding conditions. † p < 0.05 compared with its corresponding untreated arteries. †† p < 0.01 compared with its corresponding untreated arteries.

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Fig. 1 – Summary of the contractile responses to noradrenaline, tyramine, phenylephrine and B-HT-920 in goat middle cerebral arteries under control conditions (○) and after ischemia-reperfusion (●). *p < 0.05; **p < 0.01 between control and ischemic arteries. The prostanoids production in control and ischemic arteries are summarized in Table 3.

2.4. Expression of α1A-adrenoceptor, α2A-adrenoceptor and COX-2 proteins α1A-adrenoceptor and α2A-adrenoceptor proteins expression were significantly higher in ischemic-reperfused arteries than in control arteries. The images and results found showed single bands and the degree of augmentation found in ischemic arteries was comparable for the two subtypes of αadrenoceptors (Fig. 5). COX-2 protein expression was detected matching the commercial standard. Its expression was also significantly higher in ischemic than in control arteries (Fig. 6).

3.

Discussion

In the present study, we report for the first time that brain ischemia-reperfusion resulted in upregulation of α-adrenoceptors and increased COX-2-derived PGI2 exerting a vasoconstrictor action. The results in hemodynamic changes obtained after 2 h of ischemia followed by 1 h of reperfusion in anesthetized goats compare to those obtained previously in our laboratory after 1 h of ischemia and 1 h of reperfusion (Sánchez et al., 2006) both of them showing a postischemic cerebral vasodilatation. We found that cerebral arteries previously exposed in vivo to ischemia-reperfusion exhibited an enhanced in vitro cerebral vasoconstrictor response to exogenous

Fig. 2 – Summary of the contractile responses to noradrenaline in goat middle cerebral arteries under control conditions (left) and after ischemia-reperfusion (right), in arteries untreated (○, ●) and arteries treated with meclofenamate (10− 5 M) (▲,4). *p < 0.05; **p < 0.01 between treated and untreated arteries.

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Fig. 3 – Summary of the contractile responses to noradrenaline in goat middle cerebral arteries under control conditions (left) and after ischemia-reperfusion (right), in arteries untreated (○,●), and arteries treated with (a) TCP (10− 5 M) (□,■), (b) SQ 29 548 (10− 5 M) (4,▲), (c) furegrelate (10− 6 M) (◊,♦) or (d) NS 398 (10− 6 M) (∇,▼). *p < 0.05; **p < 0.01 between treated and untreated arteries.

noradrenaline, phenylephrine (agonist for α1-adrenoceptors) and B-HT-920 (agonist for α2-adrenoceptors), as well as to endogenous noradrenaline released from perivascular nerve terminals with tyramine (indirect sympatheticomimetic amine). Also, we found that in both control and ischemicreperfused arteries the response to phenylephrine was higher than to B-HT-920 and that in ischemic arteries increased the response to both phenylephrine and B-HT-920. Since the contraction in response to potassium was similar in control and ischemic arteries, the higher response to adrenergic

stimuli appears to be specific and not due to increased contractility of the vascular smooth muscle. Also, as the contractile response to NPY was not changed after ischemiareperfusion, we consider that the augmented response to adrenergic stimulation after this condition may be specific for this type of stimulus. However, this change in reactivity of cerebral vasculature after ischemia-reperfusion does not seem to occur only with adrenergic stimulation but also with endothelin-1, as previously found in our laboratory (Salcedo et al., 2009). The augmented arterial response to

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Fig. 4 – Summary of the response to PGI2 in untreated, control (○) and ischemic-reperfused (●) arteries and treated with the inhibitor of PGI2 IP-receptors RO1138452 (10− 5 M), control (□) and ischemic-reperfused (■) arteries. *p < 0.05; between untreated control and ischemic arteries; **p < 0.01 between treated and untreated corresponding arteries.

adrenergic stimulation correlates with the enhanced expression of α1- and α2-adrenoceptors found in cerebral arteries after ischemia-reperfusion, as measured with western blot analysis. Therefore, our results suggest that the augmented cerebrovascular responsiveness to the adrenergic stimulation after ischemia-reperfusion may be related to upregulation of α-adrenoceptors. As our results about expression of both α1- and α2-adrenoceptors proteins are increased in ischemic arteries, and the effects of phenylephrine and BHT-920 are also increased in this type of arteries we consider that upregulation may occur in both α1- and α2-adrenoceptors. Observations made by others indicate that hypoxia is a stimulus that increases gene expression of α1-adrenoceptors in arterial blood vessels (Eckhart et al., 1996). Our results, however, contrast with those performed in anesthetized newborn pigs where 20 min of global cerebral ischemia induced by intracranial hypertension, followed by 2–3 h or 24 h of reperfusion did not affect the reactivity of pial arteries to noradrenaline (Leffler et al., 1989b), and also with those performed in rats where long-term inhibition of NO synthesis with L-NAME reduced the in vitro cerebral vasoconstrictor response to noradrenaline and the vasodilator response to

Table 3 – Basal production of prostanoids (pg mL− 1 mg tissue) in control and ischemic arteries measured by enzyme immunoassay.

Metabolite of PGI2 (6-keto-PGF1α) Metabolite of TXA2 (TXB2) Metabolite of PGF2α (13,14-dihydro15-keto PGF2α)

Control arteries

Ischemic arteries

403 ± 55 24 ± 6 7.3 ± 1.2

565 ± 56 ⁎ 21 ± 3 8.4 ± 0.5

Values are mean ± SEM. ⁎ p < 0.05 between control and ischemic arteries.

acetylcholine, after occlusion of the left middle cerebral artery for 8–9 weeks, without reperfusion (Sercombe et al., 2001). The discrepancies between these two studies (Leffler et al., 1989b; Sercombe et al., 2001) and ours may be related to differences in the duration of ischemia, the presence or not of reperfusion, the procedure to examine vascular response, and/or perhaps in species used. As the role of sympathetic innervation in the regulation of cerebral blood flow may vary between animal species, and this role in humans remains a controversial topic, extrapolation of our present data to the participation of sympathetic regulation in the effects of ischemia-reperfusion on human cerebral vasculature should be made with caution. However, recent studies in human cerebral circulation reporting the presence of a significant sympathetic basal tone and sympathetic mediated vasoconstriction (van Lieshout and Secher, 2008), as well as a consistent sympathetic contribution in cerebral autoregulation (Hamner et al., 2010), support the idea that our present findings might be of significance. As our experiments were performed in endothelium-intact arteries, we cannot exclude that other factors such as endothelial dysfunction may also contribute to the observed augmented adrenergic cerebrovascular constrictor response after ischemia-reperfusion. In this sense, it has been reported that ischemia-reperfusion can induce endothelial dysfunction in cerebral blood vessels (Mayhan et al., 1988; Rosenblum, 1997; Sánchez et al., 2006), a phenomenon that was also observed in our laboratory (present study, Salcedo et al., 2009; Sánchez et al., 2006) as indicated by the reduced cerebrovascular relaxation in response to ADP after ischemia-reperfusion, and that the endothelium or endothelial factors may counteract the adrenergic cerebral vasoconstriction (Edvinsson and Krause, 2002). There are studies in vivo (Bauknight et al., 1992; Diéguez et al., 1998, Fernández et al., 2001) and in vitro (Wagerle et al., 1995), suggesting that NO acts at postjunctional level in the vessel wall and inhibits the cerebral vasoconstriction in response to noradrenaline. Furthermore, nitric oxide can also act pre-junctionally to enhance neurotransmitter release (Mbaku et al., 2000). With regard to prostanoids, the present data suggest that their role in the vasoconstrictor response to noradrenaline is altered after ischemia-reperfusion. In control arteries the nonspecific COX inhibitor meclofenamate and the specific PGI2 synthesis inhibitor TCP, but not the specific inhibitor of COX-2 NS-398, augmented the response to noradrenaline, suggesting that under normal conditions a vasodilator prostanoid (probably PGI2), released through COX-1, may inhibit the cerebral adrenergic vasoconstriciton. In ischemic-reperfused arteries meclofenamate did not affect the response to noradrenaline, but both TCP and NS-398 reduced this response. As SQ 29 548 but not furegrelate also inhibited the effects of noradrenaline in ischemic arteries, we suggest that after ischemia-reperfusion a prostanoid activating TP-receptors may be involved in the observed increased noradrenaline effects and that this prostanoid may be PGI2. In the present study, we have also found that PGI2 induced a small relaxation in normal arteries but a small contraction in ischemic arteries, and a much higher contraction was observed in both types of arteries after inhibiting PGI2 IP-receptors. The absence of effects of furegrelate on the response to noradrenaline

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Fig. 5 – Representative Western blots showing expression of α1A-adrenoceptor (ADRA-1A) (left) and α2A-adrenoceptor (ADRA-2A) (right) and corresponding β-actin, in segments from control and ischemic-reperfused middle cerebral arteries. The lower trace of each panel shows the bar graph summarizing the immunoblot data. Densitometric results (mean ± SEM) are expressed as the ratio between the signal for ADRA-1A or ADRA-2A protein and the signal for β-actin. *p < 0.05. Number of animals is indicated in parentheses.

probably is not due to the dose used for this inhibitor was not sufficient because same dosage of furegrelate has been shown to inhibit the enhanced phenylephrine contraction in rat aorta (Pecanha et al., 2010) and the electrical field stimulationinduced contraction in rat mesenteric arteries (del Campo et al., 2008). The observed effects with SQ 29 548 may be surprising, but it has been reported that cerebral ischemia may eliminate the prostanoid vasodilator system, as noradrenaline, hypotension and hypercapnia increased the concentrations of cortical periarachnoid vasodilator prostanoids in control anesthetized newborn pigs, whereas after ischemia this type of stimuli did not affect the production of these prostanoids (Leffler et al., 1989a,b). With these studies in mind (Leffler et al., 1989a,b) and considering that PGI2 may cause contraction in ischemic cerebral arteries (present data), we can suggest that a contracting role of PGI2 may be involved in the cerebral adrenergic vasoconstriciton after ischemiareperfusion. Prostanoids can be produced by isoforms COX-1 and COX-2, and we found increased production of metabolites of PGI2, without changes in production of metabolites of TXA2 and PGF2α as well as overexpression of COX-2 in ischemic arteries with regard to control arteries. These data suggest that the observed increased production of PGI2 may occur via COX-2 in cerebral arteries after ischemia-reperfusion. We propose that the augmented α-adrenergic response after

ischemia-reperfusion might be related to noradrenaline increase the production of PGI2 the vasoconstrictor action of which would contribute to the action of noradreanline on α-adrenoceptors and/or PGI2 might potentiate the effects of noradrenaline by acting at any of the steps that follow α-adrenergic activation (see Fig. 7). In fact, we also found that in normal arteries the response to noradrenaline was increased by meclofenamate and TCP (inhibitor of PGI2 synthesis), suggesting that PGI2 is also involved in the α-adrenergic response of cerebral blood vessels, but in normal conditions this prostanoid would act as vasodilator and would inhibit the adrenergic vasoconstriction. There are studies suggesting that noradrenaline by stimulating α-adrenoceptors, promotes the release of PGI2 in rabbit aorta (Boeynaems et al., 1987; Nebigil and Malik, 1990), which may take place in smooth muscle cells (Nebigil and Malik, 1990). To our knowledge, this is the first study showing this effect of ischemia-reperfusion on the role of prostanoids in the adrenergic cerebral vasoconstriction. It has been reported that during hypertension and aging PGI2 becomes a contracting prostaglandin via activation of TP-receptors (Gluais et al., 2005; Blanco-Rivero et al., 2005; Félétou et al., 2009). Also, brain ischemia may induce a strong increase in COX2 expression in neurons, blood vessels and inflammatory cells infiltrating the injured brain (Iadecola et al., 1999) which occurs as early as 30 min of reperfusion (Planas et al., 1995).

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Fig. 6 – Representative Western blots showing expression of COX-2 and corresponding β-actin, in segments from control and ischemic-reperfused middle cerebral arteries. The lower trace shows the bar graph summarizing the immunoblot data. Densitometric results (mean ± SEM) are expressed as the ratio between the signal for COX-2 protein and the signal for β-actin. *p < 0.05. Number of animals is indicated in parentheses.

Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996), and the use of animals was approved by the local Animal Research Committee. Anesthesia was induced with an intramuscular injection of 10 mg kg− 1 ketamine hydrochloride and i.v. administration of 2% thiopental sodium. After orotracheal intubation, ventilation with a mixture of oxygen and isoflurane was adjusted to maintain normocapnia and a stable level of anesthesia by use of a respirator (New England Medical Instruments Inc. Medway, MA, USA). After removal of the left horn, a 4 × 4-cm window was made in the skull, the dura was opened, and a main branch of the left MCA at the cortical surface was exposed. Then, a snare-type occluder was placed around this artery to induce 120-min occlusion followed by release of this occlusion to allow 60 min of reperfusion. Systemic arterial pressure was measured through a polyethylene catheter placed in one femoral artery and connected to a Statham transducer (Statham Instruments Inc., Oxnard, CA, USA). In all the animals, systemic arterial pressure and heart rate were simultaneously recorded on a Grass model 7 polygraph, and blood samples from the femoral artery were taken periodically to measure pH, pCO2 and pO2 by standard electrometric methods (Radiometer, ABL TM5, Copenhagen, Denmark). In 10 of these animals, an electromagnetic flow transducer (Biotronex Laboratory Inc., Silver Spring, MD, USA) was also placed on the left MCA, proximal to the occluder, to measure blood flow throughout the ischemia-reperfusion period. At 60 min of reperfusion, the animals were sacrificed with an overdose of i.v. thiopental sodium and potassium chloride. Then, the brain was removed and pial branches of the left MCA, 5 mm distal to the occluder (ischemic arteries), and pial branches of the right MCA (control arteries) were dissected free for in vitro and biochemical analysis.

4.2. In conclusion, our results suggest that ischemia-reperfusion induces an enhancement in the cerebral vasoconstrictor response to endogenous and exogenous noradrenaline, which may be related to (1) upregulation of α-adrenoceptors and (2) increased COX-2-derived PGI2 exerting a vasoconstrictor effect. We propose the hypothesis that ischemia-reperfusion leads to an altered role of PGI2, which underlies the increased adrenergic response of cerebral vasculature after ischemia-reperfusion. In this condition, noradrenaline might increase the production of PGI2 which would contribute to the α-adrenergic vasoconstriction, and/or PGI2 might amplify the effects of noradrenaline by acting at any of the steps that follow α-adrenergic stimulation. These cerebrovascular alterations may be lead to dysregulation of blood perfusion of the brain ischemic-reperfused area, thus contributing to detrimental effects of ischemia-reperfusion.

4.

Experimental procedures

4.1.

Induction of ischemia-reperfusion

In this study, 29 adult, female goats (38–57 kg) were used. The investigation conformed the Guide for the Care and Use of

In vitro functional study

Control and ischemic arteries were cut into cylindrical segments 3 mm in length. Both types of artery segments had similar external diameter (0.4–0.6 mm). Each arterial segment was prepared for isometric tension recording in a 4-mL organ bath at 37 °C, containing modified Krebs–Henseleit solution with the following composition (in millimolar): NaCl, 115; KCl, 4.6; KH2PO4, 1.2; MgSO4, 1.2; CaCl2, 2.5; NaHCO3, 25; glucose, 11. The solution was equilibrated with 95% oxygen and 5% carbon dioxide to give a pH 7.3–7.4. Briefly, the method consists of passing through the lumen of the vascular segment of two fine stainless steel pins, 90 μm in diameter. One pin is fixed to the organ bath wall, while the other is connected to a strain gauge for isometric tension recording, thus permitting the application of passive tension in a plane perpendicular to the long axis of the vascular cylinder. The recording system included a Universal Transducing Cell UC3 (Statham Instruments Inc.) and a Statham Microscale Accessory UL5 (Statham Instruments Inc.). Changes in isometric force were recorded on a Macintosh computer by use of Chart v3.6/s software and a MacLab/8e data acquisition system (ADInstruments Ltd., UK). A previously determined optimal passive tension of 1 g was applied to the vascular segments, and they were allowed to equilibrate for 60– 90 min.

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Fig. 7 – Schematic representation of a portion of arterial smooth muscle cell, showing the steps involved in the arterial contraction to noradrenaline (NA), and the possible effects of ischemia-reperfusion on α-adrenergic vasoconstriction together with action of the inhibitors used on formation/effects of products derived from COX (+, activation; −, inhibition; =, no change). a) Normal conditions (left): NA causes arterial contraction mainly by (1) activating α-adrenoceptors, which is followed by (2) increasing cytosolic Ca2+ concentration by influx through Ca2+ channels and release from sarcoplasmic reticulum (SR), and (3) binding of Ca2+ to, and sliding of contractile myofilaments. This vasoconstriction might be inhibited by PGI2 exerting as vasodilator, and its release might be increased after α-adrenergic stimulation. b) Ischemia-reperfusion (right): The contraction to NA and the formation of PGI2, exerting as vasoconstrictor, are increased. Hypothesis: this increased response to NA might be due to (1) upregulation of α-adrenoceptors, and (2) NA increases further the formation of PGI2, the contractile effect of which would add up to the α-adrenergic vasoconstriction, and/or PGI2 would act at the level of any of the steps involved in the α-adrenergic activation, thus potentiating the NA cerebrovascular effects. PLC = phospholipase C; IP3 = inositol triphosphate; meclo = meclofenamate; IP = PGI2 receptor; TP = TXA2 receptor.

The ability of each vascular preparation to contract was tested twice under resting conditions with KCl (100 mM): first time before applying any treatment, and second time at the end of the experiment after 3–4 washouts. After the first test with KCl the solution was renewed by repeated washouts, and then, the response to exogenous noradrenaline (10− 8–10− 4 M), and endogenous noradrenaline released from perivascular nerve terminals with tyramine (indirect sympatheticomimetic amine, 10 − 8 –10 − 4 M) were obtained under resting conditions in ischemic and control arteries. To examine to role of α1- and α2-adrenoceptors in the adrenergic response, we also tested the effects of phenylephrine (selective α1-adrenoceptor agonist, 10− 8–10− 4 M) and B-HT-920 (selective α2-adrenoceptor agonist, 10− 7–3 × 10− 3 M) in both types of arteries. To analyze the role of prostanoids in the response to noradrenaline, the contraction to this neurotransmitter was recorded in the presence of the cyclooxygenase inhibitor meclofenamate (10− 5 M), the PGI2 synthesis inhibitor TCP (10− 5 M), the TP-receptor antagonist SQ 29 548 (10− 5 M), the TXA2 synthase inhibitor furegrelate (10− 6 M) or the COX-2 inhibitor NS-398 (10− 6 M). In each artery, only one concentration–response curve was determined for noradrenaline, phenylephrine, B-HT-920 or tyramine. In every condition, the arteries tested with noradrenaline were treated with propranolol (10− 7 M) to block a possible β-adrenoceptor effect of noradrenaline. Meclofenamate, TCP, SQ 29 548, furegrelate or NS-398 were applied to the

organ bath for 30–35 min before the responses to noradrenaline were tested. At the end of the experiments, control and ischemic arteries from 5 goats were contracted with the thromboxane A2 analogue U46619 (3 × 10− 7–10− 6 M) and then exposed to ADP (10− 8–10− 4 M) to test the endothelium-dependent relaxation. In 4 animals, the response to PGI2 (10− 6–10− 5 M) was examined in control and ischemic arteries under basal tone, untreated or treated with the inhibitor of PGI2 IP-receptors RO 1138452 (10− 5 M). To test whether the changes observed with noradrenaline, phenylephrine, B-HT-920 and tyramine were or not specific for this type of stimuli, we also examined the effects of NPY (10− 10–10− 7 M) in both control and ischemic arteries. NPY is a transmitter of cerebrovascular sympathetic nerve terminals acting on a family of G-protein-coupled receptors named Y. In non-treated segments from 9 animals, the response to NPY was also tested after noradrenaline concentration–response curve. To this, the artery was washed for 40–50 min before adding the cumulative concentrations of NPY. The contraction to noradrenaline, phenylephrine, B-HT-920, tyramine, NPY and PGI2 is expressed as percentage of the mean contraction achieved after the two tests with 100 mM of KCl. The EC50 values for the concentration–response curves to noradrenaline, phenylephrine, B-HT-920, tyramine and NPY were calculated as the concentration producing 50% of the maximum

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effect by geometric interpolation. The relaxation to ADP is expressed as percentage of the tone achieved with U46619. Drugs used were L(−)-norepinephrine bitartrate salt monohydrate, L(−)-phenylephrine hydrochloride, B-HT-920 dihydrochloride, tyramine hydrochloride, NPY (sheep), meclofenamate (2[1,6-dicloro-3-methylphenyl-amino]benzoic acid sodium salt), PGI2 (prostaglandin I2, sodium salt), tranylcypromine, furegrelate, U46619 (9, 11-dideoxy α, 9α-epoxymethanoprostaglandin F2α) and ADP (adenosine 5′diphosphate, sodium salt), all were obtained from SigmaAldrich Inc. (Saint Louis, MO, USA); SQ 29 548 was obtained from ICN (Holland) and NS 398 (N[-2-(cyclohexyloxy)-4-nitrophenyl]-methanesulfonamide) and RO1138452 (CAY10441) were purchased from Cayman Chemical Co. (Ann Arbor, MI, USA). All drugs were dissolved in distilled water and further diluted in isotonic saline.

4.3.

Production of prostanoids

The production of TXA2, PGI2 and PGF2α in arterial tissue was assayed by measuring the stable metabolites TXB2, 6-ketoPGF1α and 13,14-dihydro-15-keto PGF2α, respectively, using the respective enzyme immunoassay kit (Cayman Chemical). Control and ischemic arteries from 4 animals were obtained and pre-incubated for 30 min in 5 mL of Krebs–Henseleit solution at 37 °C, continuously gassed with a 95% O2–5% CO2 mixture (stabilization period). This was followed by two washout periods of 10 min in a bath of 0.2 mL of Krebs– Henseleit solution and then incubated for 10 min more after which the medium samples were collected. The assays were carried out according to the manufacturer's instructions. Results were expressed as pg prostanoid mL− 1 mg tissue.

quantified with Scion Image software (NIH, Bethesda, MD, USA). Protein expression data were normalized to the β-actin expression, which was determined on the same membranes.

4.5.

Statistical analysis

The effects of left MCA occlusion and reperfusion on blood flow, mean systemic arterial pressure, heart rate and blood gases and pH were evaluated as changes in absolute values and percentages by applying one-way, repeated-measures analysis of variance (ANOVA) followed by Student's t-test for paired data. To evaluate the sensitivity of control and ischemic arteries to noradrenaline, phenylephrine, B-HT-920, tyramine and NPY, as well as ADP, the pD2 of each concentration–response curve for these substances was calculated as the negative logarithm of the EC50. Statistical comparisons of Emax and pD2 values between ischemic and control arteries, and between untreated and treated arteries were made using unpaired Student's t-test. Comparisons of the effects of noradrenaline obtained in control arteries as well as in ischemic arteries under the different conditions tested were made using analysis of variance (ANOVA) followed by Dunnett test. Data for Western Blot analysis were compared in ischemic and control arteries using the non parametric Mann–Whitney test. Data for ELISA analysis were compared using repeatedmeasures ANOVA followed by Newman–Keuls multiple comparison test. All results are reported as mean ± SEM. A p value < 0.05 was considered significant.

Acknowledgments 4.4. Western blot analysis for α-adrenoceptors and COX-2 proteins Commercially available antibodies were used for detection by Western blotting of the α1A-adrenoceptors (α1A-AR [C-19], Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA ), α2A-adrenoceptors (α2A-AR [C-19], Santa Cruz) and COX-2 (Cayman Chemical) and β-actin (Sigma-Aldrich). Frozen control and ischemic MCA segments were homogenized in ice-cold Tris buffer (pH 7.4) containing a protease inhibitor cocktail (Sigma-Aldrich). After centrifugation (14,000×g for 15 min at 4 °C), the protein concentration in the supernatant was determined at 750 nm in a Shimadzu UV-160A spectrophotometer (Shimadzu Corporation, Japan). Equal amounts of protein were resolved and separated by SDS–polyacrylamide gel electrophoresis (SDS–PAGE) and the proteins were then transferred to PVDF membranes by wet electroblotting. The membranes were then blocked for 2 hours at room temperature in blocking buffer (Sigma-Aldrich) and incubated overnight at 4 °C with primary antibodies. The membranes were washed three times for 10 min each at room temperature in blocking buffer and incubated for 90 min with horseradish peroxidase conjugate anti-mouse IgG or horseradish peroxidase conjugate anti-donkey IgG as the secondary antibody. Immunolabelling was visualized by enhanced chemoluminiscence (ECL) using the ECL reagent (Amersham, Arlington Heights, IL, USA) and the membranes were exposed to Hyperfilm (Amersham), which was scanned and

The authors are grateful to Ms. H. Fernández-Lomana and E. Martínez for their technical assistance. This work was supported, in part, by the Ministerio de Ciencia e Innovación (SAF2008-01410) and the Fundación Médica Mútua Madrileña Automovilista (2008).

REFERENCES

Alborch, E., Gómez, B., Diéguez, G., Marín, J., Lluch, S., 1977. Cerebral blood flow and vascular reactivity after removal of the superior cervical sympathetic ganglion in the goat. Circ. Res. 41, 278–282. Bauknight Jr., G.C., Faraci, F.M., Heistad, D.D., 1992. Endothelium-derived relaxing factor modulates noradrenergic constriction of cerebral arterioles in rabbits. Stroke 23, 1522–1525. Boeynaems, J.M., Demolle, D., Galand, N., 1987. Adrenergic stimulation of vascular prostacyclin: role of alpha 1-receptors in smooth muscle cells. Eur. J. Pharmacol. 144, 193–200. Blanco-Rivero, J., Cachofeiro, V., Lahera, V., Aras-Lopez, R., Márquez-Rodas, I., Salaices, M., Xavier, F.E., Ferrer, M., Balfagón, G., 2005. Participation of prostacyclin in endothelial dysfunction induced by aldosterone in normotensive and hypertensive rats. Hypertension 46, 107–112. del Campo, L., Sagredo, A., Aras-López, R., Balfagón, G., Ferrer, M., 2008. Orchidectomy increases the formation of non-endothelial

BR A I N R ES E A RC H 1 3 4 6 ( 2 01 0 ) 1 2 1 –1 31

thromboxane A2 and modulates its role in the electrical field stimulation-induced response in rat mesenteric artery. J. Endocrinol. 197, 371–379. Diéguez, G., Fernández, N., Sánchez, M.A., García-Villalón, A.L., Monge, L., Gómez, B., 1998. Adrenergic reactivity after inhibition of nitric oxide synthesis in the cerebral circulation of awake goats. Brain Res. 813, 381–389. Eckhart, A.D., Zhu, Z., Arendshorst, W.J., Faber, J.E., 1996. Oxygen modulates alpha 1B-adrenergic receptor gene expression by arterial but not venous vascular smooth muscle. Am. J. Physiol. 271, H1599–H1608. Edvinsson, L., Krause, D.N., 2002. Catecholamines, In: Edvinsson, L., Krause, D.N. (Eds.), Cerebral blood flow and metabolism, 2nd ed. Lippincott Williams and Wilkins, Philadelphia, pp. 191–211. Félétou, M., Verbeuren, T.J., Vanhoutte, P.M., 2009. Endothelium-dependent contractions in SHR: a tale of prostanoid TP and IP receptors. Br. J. Pharmacol. 156, 563–574. Fernández, N., Martínez, M.A., Monge, L., García-Villalón, A.L., Diéguez, G., 2001. Adrenergic vasoconstrictor activity in the cerebral circulation after inhibition of nitric oxide synthesis in conscious goats. Auton. Neurosci. 89, 16–23. Gluais, P., Lonchampt, M., Morrow, J.D., Vanhoutte, P.M., Feletou, M., 2005. Acetylcholine-induced endothelium-dependent contractions in the SHR aorta: the Janus face of prostacyclin Br. J. Pharmacol. 146, 834–845. Goadsby, P.J., Edvinsson, L., 1997. Extrinsic innervation: transmitters, receptors, and functions. The sympathetic nervous system. In: Welch, K.M.A., Caplan, L.R., Reis, D.J., Siesjö, B.K., Weir, B. (Eds.), Primer on Cerebrovascular Diseases. London, Academic Press, pp. 60–63. Gustafson, I., Westerberg, E., Wieloch, T., 1990. Protection against ischemia-induced neuronal damage by the alpha 2-adrenoceptor antagonist idazoxan: influence of time of administration and possible mechanisms of action. J. Cereb. Blood Flow Metab. 10, 885–894. Hachinski, V.C., Smith, K.E., Silver, M.D., Gibson, C.J., Ciriello, J., 1986. Acute myocardial and plasma catecholamine changes in experimental stroke. Stroke 17, 387–390. Hamner, J.W., Tan, C.O., Lee, K., Cohen, M.A., Taylor, J.A., 2010. Sympathetic control of the cerebral vasculature in humans. Stroke 41, 102–109. He, Z., Huang, L., Wu, Y., Wang, J., Wang, H., Guo, L., 2008. DDPH: improving cognitive deficits beyond its alpha 1-adrenoceptor antagonism in chronic cerebral hypoperfused rats. Eur. J. Pharmacol. 588, 178–188. Iadecola, C., Forster, C., Nogawa, S., Clark, H.B., Ross, M.E., 1999. Cyclooxygenase-2 immunoreactivity in the human brain following cerebral ischemia. Acta Neuropathol. 98, 9–14. Jordan, J., Shannon, J.R., Diedrich, A., Black, B., Costa, F., Robertson, D., Biaggioni, I., 2000. Interaction of carbon dioxide and sympathetic nervous system activity in the regulation of cerebral perfusion in humans. Hypertension 36, 383–388. Leffler, C.W., Beasley, D.G., Busija, D.W., 1989a. Cerebral ischemia alters cerebral microvascular reactivity in newborn pigs. Am. J. Physiol. 257, H266–H271. Leffler, C.W., Busija, D.W., Beasley, D.G., Armstead, W.M., Mirro, R., 1989b. Postischemic cerebral microvascular responses to

131

norepinephrine and hypotension in newborn pigs. Stroke 20, 541–546. Mayhan, W.G., Amundsen, S.M., Faraci, F.M., Heistad, D.D., 1988. Responses of cerebral arteries after ischemia and reperfusion in cats. Am. J. Physiol. 255, H879–H884. Mbaku, E.N., Zhang, L., Duckles, S.P., Buchholz, J., 2000. Nitric-oxide synthase-containing nerves facilitate adrenergic transmitter release in sheep middle cerebral arteries. J. Pharmacol. Exp. Ther. 293, 397–402. Meyer, J.S., Welch, K.M., Okamoto, S., Shimazu, K., 1974. Disordered neurotransmitter function. Demonstration by measurement of norepinephrine and 5-hydroxytryptamine in CSF of patients with recent cerebral infarction. Brain 97, 654–664. Nebigil, C., Malik, K.U., 1990. Prostaglandin synthesis elicited by adrenergic stimuli in rabbit aorta is mediated via alpha-1 and alpha-2 adrenergic receptors. J. Pharmacol. Exp. Ther. 254, 633–640. Nellgard, B., Mackensen, G.B., Sarraf-Yazdi, S., Miura, Y., Pearlstein, R., Warner, D.S., 1999. Pre-ischemic depletion of brain norepinephrine decreases infarct size in normothermic rats exposed to transient focal cerebral ischemia. Neurosci. Lett. 275, 167–170. Pecanha, F.M., Wiggers, G.A., Briones, A.M., Pérez-Girón, J.V., Miguel, M., García-Redondo, A.B., Vasallo, D.V., Alonso, M.J., Salaices, M.J., 2010. The role of cyclooxygenase (COX)-2 derived prostanoids on vasoconstrictor responses to phenylephrine is increased by exposure to low mercury concentration. Physiol. Pharmacol. 61, 29–36. Planas, A.M., Soriano, M.A., Rodríguez-Farré, E., Ferrer, I., 1995. Induction of cyclooxygenase-2 mRNA and protein following transient focal ischemia in the rat brain. Neurosci. Lett. 200, 187–190. Rosenblum, W.I., 1997. Selective impairment of response to acetylcholine after ischemia/reperfusion in mice. Stroke 28, 448–451. Salcedo, A., Fernández, N., García Villalón, A.L., Monge, L., Narváez Sánchez, R., Diéguez, G., 2009. Role of angiotensin II in the response to endothelin-1 of goat cerebral arteries after ischemia-reperfusion. Vascul. Pharmacol. 50, 160–165. Sánchez, A., Fernández, N., Monge, L., Salcedo, A., Climent, B., García-Villalón, A.L., Diéguez, G., 2006. Goat cerebrovascular reactivity to ADP after ischemia-reperfusion. Role of nitric oxide, prostanoids and reactive oxygen species. Brain Res. 1120, 114–123. Sercombe, R., Issertial, O., Seylaz, J., Pinard, E., 2001. Effects of chronic L-NAME treatment on rat focal cerebral ischemia and cerebral vasoreactivity. Life Sci. 69, 2203–2216. van Lieshout, J.J., Secher, N.H., 2008. Point:counterpoint: sympathetic activity does/does not influence cerebral blood flow. Point: sympathetic activity does influence cerebral blood flow. J. Appl. Physiol. 105, 1364–1366. Wagerle, L.C., Moliken, W., Russo, P., 1995. Nitric oxide and beta-adrenergic mechanisms modify contractile responses to norepinephrine in ovine fetal and newborn cerebral arteries. Pediatr. Res. 38, 237–242. Zhang, Y., 2004. Clonidine preconditioning decreases infarct size and improves neurological outcome from transient forebrain ischemia in the rat. Neuroscience 125, 625–631.