Mechanism of alkalosis-induced constriction of rat cerebral penetrating arterioles

Neuroscience Research 70 (2011) 98–103

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Mechanism of alkalosis-induced constriction of rat cerebral penetrating arterioles Yuhui Li, Tetsuyoshi Horiuchi ∗ , Takahiro Murata, Kazuhiro Hongo Department of Neurosurgery, Shinshu University School of Medicine, Asahi 3-1-1, Matsumoto 390-8621, Japan

a r t i c l e

i n f o

Article history: Received 28 August 2010 Received in revised form 22 December 2010 Accepted 11 January 2011 Available online 21 January 2011 Keywords: Cerebral circulation Na+ /H+ exchanger Na+ /K+ -adenosine triphosphatase Na+ /Ca2+ exchanger Alkalosis Constriction Mechanism

a b s t r a c t Cerebral arterioles are in close contact with the supplied tissue and are strong regulators of cerebrovascular tone. Transient ischemia can cause brain intracellular alkalosis producing vasoconstriction. However, the mechanisms of alkalosis-induced cerebral arteriolar constriction are poorly understood. Here, we determined the vascular responses to alkalosis under different conditions by monitoring the internal diameter of pressurized penetrating arterioles isolated from the rat cerebrum with an operating microscope. The roles of Na+ /H+ exchanger (NHE), Na+ /Ca2+ exchanger (NCX), Na+ /K+ -adenosine triphosphatase (NKA), and potassium (K+ ) channels during alkalosis were examined using specific inhibitors. Our results indicated that the extent of constriction of the penetrating arterioles was dependent on alkaline pH. Moreover, the alkalosis-induced vasoconstriction was significantly attenuated by inhibitors of NHE, NCX, and NKA, but not K+ channel inhibitors. Therefore, we concluded that NHE, NKA, and NCX are important regulators involved in alkalosis-induced vasoconstriction of rat cerebral penetrating arterioles. © 2011 Elsevier Ireland Ltd and the Japan Neuroscience Society. All rights reserved.

1. Introduction Brain intracellular alkalosis has been observed in experimental models of adult stroke (Mabe et al., 1983) in which longer periods of ischemia were associated with earlier and more prolonged periods of alkalosis (Chopp et al., 1990). The cerebral arterioles, which are in close contact with the supplied tissue, are known to act as strong regulators of cerebrovascular tone (Rosenblum and Kontos, 1974). These cerebral arterioles present vasomotor responses of dilation or contraction under conditions of acidosis or alkalosis, respectively (Apkon and Boron, 1995; Austin and Wray, 1993, 1994, 1995; Dacey and Duling, 1982; Wray, 1988). Recently, it was demonstrated that acidosis-induced dilation of rat cerebral arterioles and middle cerebral artery (MCA) was mediated by nitric oxide and ATP-sensitive potassium channels (KATP channels) and Ca2+ -activated potassium channels (KCa channels) (Horiuchi et al., 2002; Lindauer et al., 2003). However, the precise mechanisms of alkalosis-induced arteriole vasoconstriction remain unclarified.

Abbreviations: NHE, Na+ /H+ exchanger; NKA, Na+ /K+ -adenosine triphosphatase; NCX, Na+ /Ca2+ exchanger; VSMCs, vascular smooth muscle cells; MCA, middle cerebral artery; KCa channel, calcium-activated potassium channel; KATP channel, ATP-sensitive potassium channel; [Ca2+ ]i , intracellular calcium; [Na+ ]i , intracellular sodium; [K+ ]o , extracellular K+ ; pHi , intracellular pH; pHo , extracellular pH; HMA, 5-N,N hexamethylene-amiloride; DCB, 3 ,4 -dichlorobenzamil; TEA, tetraethylammonium ion; SE, standard error. ∗ Corresponding author. Tel.: +81 263 37 2690; fax: +81 263 37 0480. E-mail address: [email protected] (T. Horiuchi).

The Na+ /H+ exchanger (NHE) is a family of membrane proteins in mammalian cells that regulates intracellular pH (pHi ) and intracellular Na concentration ([Na+ ]i ) by extruding H+ from, and taking up Na+ into cells (Noel and Pouyssegur, 1995; Wakabayashi et al., 1997). NHE acts as the major contributor to the Na+ influx pathway in vascular smooth muscle cells (VSMCs) (Little et al., 1986). It was hypothesized that an increase in [Na+ ]i mediated by NHE may induce vasoconstriction, which may be mainly attributable to the interaction of Na+ /Ca2+ exchanger (NCX) (Bova et al., 1990). Other mechanisms, such as membrane potential, Ca2+ channels, plasma membrane and sarcoplasmic reticulum Ca2+ -ATPases, would also be affected (Batlle et al., 1991; Bova et al., 1990; Nabel et al., 1988; Tepel et al., 1998; Zhu et al., 1994). Furthermore, Na+ /K+ -adenosine triphosphatase (NKA) mediates the 2:3 exchange of [Na+ ]i for extracellular potassium ([K+ ]o ) and results in hyperpolarization. Then, the transmembrane Na+ gradient will activate NHE and result in Na+ influx (Kuro-o, 1996; Wakabayashi et al., 1997). However, it was unclear whether these factors were involved in alkalosisinduced constriction of cerebral arterioles. Therefore, the present study was designed to clarify the factors involved in alkalosis-induced vasoconstriction with isolated rat cerebral penetrating arterioles. 2. Materials and methods 2.1. Animal preparation The Animal Ethics Committee at the Shinshu University School of Medicine approved the experimental protocols implemented in this study.

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Y. Li et al. / Neuroscience Research 70 (2011) 98–103

Male Sprague–Dawley rats obtained from Japan SLC, with a mean body weight of 385.3 ± 13.6 g, were used. 2.2. Dissection and isolated vessel preparation Dissection, isolation, and cannulation techniques of rat intracerebral penetrating arterioles were previously reported in detail (Dacey and Duling, 1982; Horiuchi et al., 2002; Saesue et al., 2004). The rats were anesthetized with pentobarbital sodium (65 mg/kg IP) and decapitated. The head was rapidly dissected. The brain was carefully removed and placed in a dissection chamber filled with 3-(N-morpholino) propanesulfonic acid (MOPS)-buffered saline (see below for composition). A long unbranched penetrating arteriole was isolated from the MCA using ultrafine microdissection scissors. The isolated arteriole was carefully transferred from the dissection chamber to a 10 mL vessel cannulation chamber containing two glass micropipettes. One end of the unbranched penetrating arteriole was cannulated with a perfusion pipette and secured with a nylon suture (diameter ∼10 ␮m), and the other end of the vessel was occluded by a glass pipette with a nylon suture. The vessel was then filled with MOPS solution through the perfusion pipette and the luminal pressure was maintained at 60 mmHg (Baumbach et al., 1989; Faraci and Heistad, 1990; Hudetz et al., 1987); no luminal perfusion was given in all experiments (Dacey and Duling, 1982). The vessel chamber was mounted on the stage of a microscope (Olympus BX51; Olympus, Tokyo, Japan), and the maximal passive internal diameter of the arteriole was monitored continuously. The vessel chamber was heated from room temperature to the optimal temperature between 37.5 ◦ C and 38.5 ◦ C, which was maintained throughout the experiment. The organ bath was continuously perfused with MOPS solution using a peristaltic pump at a rate of 6 mL/min. After an equilibration period, spontaneous arteriolar tone developed at pH 7.3, and control diameter was measured. Vessels that did not constrict to within 80% of the passive diameter were excluded from further analyses (Dacey and Duling, 1982).

of any inhibitor. Dose-dependent vascular responses of alkalosis were observed. To determine the role of NHE in alkalosis-induced constriction, in the second series of experiments, the internal diameter was measured at pH 7.3 and 7.6 before administration of any inhibitor (as a control). Then, vascular responses to alkalosis with 0.3 and 3 ␮mol/L 5-N,N hexamethylene-amiloride (HMA) were examined at pH 7.3 and 7.6. In the third series of experiments, the internal diameter was measured at pH 7.3 and 7.6 before administration of any inhibitor (as a control). Then, the contribution of NKA to the alkalosisinduced vascular response was studied. The effects of 0.1 mmol/L ouabain were evaluated at pH 7.3 and 7.6. In the fourth series of experiments, 3 ␮mol/L DCB was used to determine the role of NCX in alkalosis-induced constriction with same means. To determine the involvement of K+ channels in alkalosisinduced constriction, we used two K+ channel inhibitors in the last series of experiments: 3 ␮mol/L glibenclamide specific for KATP channels and 1 mmol/L tetraethylammonium (TEA) ions specific for KCa channels. Samples were incubated with each antagonist for at least 20 min. 2.6. Statistical analysis Only one arteriole was used from each rat brain. All data were presented as the means ± standard error (SE) of the mean, and “n” indicated the number of vessels used in the experiments. To compare the inhibitory effect on alkalosis-induced vasoconstriction, the magnitude of vasomotor responses was expressed as relative percent change in diameter. Single comparisons were made with Student’s paired t test as appropriate. For comparison of the various treatments, the results were compared by ANOVA, followed by the Student–Newman–Keuls test. In all analyses, P < 0.05 was taken to indicate statistical significance.

2.3. Drugs and solutions

3. Results

MOPS-buffered saline with the following composition (in mmol/L) was used: NaCl 144, KCl 3.0, CaCl2 2.5, MgSO4 1.4, pyruvate 2.0, glucose 5.0, EDTA 0.02, NaH2 PO4 1.21, and MOPS 2.0. Ouabain, glibenclamide, DCB, TEA, and HMA were purchased from Sigma (St. Louis, MO).

3.1. Reactiveness of dissected penetrating arterioles

2.4. Vessel diameter measurements The internal diameter of vessels was determined using an objective lens (10×), a photo-eyepiece lens (10×), and a monochrome charge-coupled device camera (KCB-270A; KOCOM Co., Ltd., Seoul, South Korea) and was displayed on a TV monitor (TM-150S; Nihon Victor, Iwai, Japan). Changes in this internal diameter in response to vasoactive agents were measured manually with a custommade diameter detection device calibrated with a stage micrometer (Nikon Instech Co., Ltd., Kanagawa, Japan), and recorded on a video recorder (HR-S300; Nihon Victor) and a strip chart recorder (VP6712A; Nihon National Instruments, Tokyo, Japan) (Saesue et al., 2004). 2.5. Experimental protocol Alkalosis-induced constriction was studied by adding NaOH to MOPS-buffered saline at the time of perfusion to raise the extracellular pH. In the first series of experiments, the internal diameter was measured at pH 7.3, 7.4, 7.5, and pH 7.6 without administration

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Given that alkalosis occurs at arterial pH values over 7.45, pH 7.3 was used as a physiological control. As shown in Fig. 1, the passive maximum diameter of isolated arterioles was 69.5 ± 2.6 ␮m under conditions of room temperature and pH 7.3. As the temperature was raised to 38 ◦ C, vessels developed spontaneous tone and constricted to an average diameter of 54.8 ± 2.3 ␮m. These observations indicated that the arteriolar competence was unaffected by dissection. All subsequent experiments were conducted at 38 ◦ C. All vessels contracted markedly at pH 7.6 and presented an average diameter of 43.4 ± 2.1 ␮m (constricted 21.1 ± 1.4%, n = 35, Fig. 1). Thus, the alkaline-dependent constriction of arterioles was confirmed. 3.2. Alkaline pH-dependent constriction response of penetrating arterioles To investigate the influence of alkalosis on vasoconstriction response in detail, arteriolar diameters were determined under different pH conditions. As indicated in Fig. 2, the vessel internal diameters contracted markedly at all extracellular pH values ranging from 7.4 to 7.6 compared with those at pH 7.3. The vessels contracted to 48.4 ± 2.6 ␮m (pH 7.3), 44.4 ± 2.8 ␮m (pH 7.4), 40.9 ± 3.2 ␮m (pH 7.5), and 36.9 ± 3.5 ␮m (pH 7.6). Notably, the extent of contraction and extracellular pH showed a positive relationship (pH 7.4 group vs. pH 7.3 group, P < 0.05; pH 7.5 group vs.

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Fig. 1. Microphotographs of penetrating arterioles isolated from the rat cerebrum. The spontaneous tone and vasoconstriction are presented according to changes in temperature and pH.

pH 7.4 group, P < 0.05; pH 7.6 group vs. pH 7.5 group, P < 0.05) (n = 7; Fig. 2). Therefore, we chose pH 7.6 as a representative condition of alkalosis and omitted the pH 7.4 and pH 7.5 groups from the subsequent experiments.

(both P < 0.05). Both lower and higher doses of HMA significantly attenuated but did not abolish the vasoconstriction in response to alkalosis (n = 7; Fig. 3).

3.3. Effects of NHE inhibitor on alkalosis-induced vasoconstriction

Next, the vasoconstriction response was examined with extraluminal application of 0.1 mmol/L ouabain, which efficiently inhibits NKA. Transient vasoconstriction induced by ouabain itself was monitored at pH 7.3 and then the diameter returned to the control level within 15 min. This was consistent with the results of our previous study (Saesue et al., 2004). As shown in Fig. 4, the relative rate of change in arteriole diameter treated with ouabain was decreased by approximately 70% compared with that under control conditions (P < 0.01, n = 6).

We first determined the function of NHE in alkalosis-induced vasoconstriction by extraluminal application of HMA, an effective inhibitor of NHE. It is notable that HMA itself caused a certain amount of vasoconstriction at pH 7.3 (11.7% and 39.4% at concentrations of 0.3 ␮mol/L and 3 ␮mol/L HMA, respectively) (Saesue et al., 2004). As indicated in Fig. 3, during alkalosis, the relative rate of change in diameter of arterioles treated with 0.3 ␮mol/L HMA was decreased by about 30% compared with that under control conditions (P < 0.05). On the other hand, the relative rate of change in diameter of arterioles treated with 3 ␮mol/L HMA was decreased by about 70% compared with that under control conditions and by 50% compared with that in the presence of 0.3 ␮mol/L HMA

3.4. Effects of NKA inhibitor on alkalosis-induced vasoconstriction

3.5. Effects of NCX inhibitor on alkalosis-induced vasoconstriction DCB is an effective inhibitor of NCX. Extraluminal treatment with 3 ␮mol/L DCB itself did not affect the vessel diameter at

55 50 Diameter (µm)

*

45

*◆

*◆◇

40 35 30 7.3

control

7.4

7.5

7.6

pH

Fig. 2. Determination of arteriole diameters under conditions of different extracellular pH from 7.3 to 7.6. Asterisks indicates significant differences from control diameter (*P < 0.05). , Significantly different from the diameter at pH 7.4 ( P < 0.05). ♦, Significantly different from the diameter at pH 7.5 (♦ P < 0.05).

Fig. 3. Effects of HMA on alkalosis-induced vasoconstriction. The relative changes in diameter were quantified as described in Section 2. Results are expressed as means ± standard error (SE) (n = 7). Asterisks indicate significant differences between control and HMA treatment groups (*P < 0.05).  P < 0.05, Significant difference between the effects of 0.3 ␮mol/L and 3 ␮mol/L HMA.

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Relative Change in Diameter (%)

30

101

3.6. Effects of KATP channel inhibitor and KCa channel inhibitor on alkalosis-induced vasoconstriction

20

10

*

0 Control

Ouabain (0.1 mmol/L)

Fig. 4. Effects of ouabain on alkalosis-induced vasoconstriction. The relative changes in diameter were quantified as described in Section 2. The results are expressed as means ± SE (n = 6). The asterisk indicates significant difference between control and ouabain treatment groups (*P < 0.01).

A previous study indicated that KATP channels were involved in acidosis-induced dilation of rat cerebral arterioles (Horiuchi et al., 2002). Here, we examined whether these channels also contribute to alkalosis-induced vasoconstriction using the same dose of glibenclamide, a specific KATP channel inhibitor, as used in the acidosis experiment. The results indicated that extraluminal application of glibenclamide did not affect the arteriolar diameter (n = 5; Table 1). In addition, the effects of other potassium channels in the alkalosis experiment were examined. The KCa channels are a large family of potassium channels that are abundant in vascular smooth muscle. These channels are activated by elevated concentrations of intracellular calcium in response to calcium influx (Faraci and Heistad, 1998). In the present study, extraluminal application of 1 mmol/L TEA, a KCa channel blocker, had no effect under either basal conditions or alkalosis (n = 6; Table 1). These observations were consistent with the suggestion that KCa channels are present but silent in cerebral arterioles (Horiuchi et al., 2001; Lang et al., 1997; Paterno et al., 1996). Other potassium channels, such as voltage-dependent K+ channels and inward rectifier K+ channels, were also not involved in alkalosis-induced constriction (data not shown).

20

Relative Change in Diameter (%)

4. Discussion The results of the present study revealed the mechanism of alkalosis-induced constriction in rat cerebral penetrating arterioles. We confirmed the previous observation that penetrating arterioles were sensitive to pH changes in extracellular alkalosis (Takayasu and Dacey, 1989). Notably, alkalosis caused a dosedependent decrease in diameter of isolated rat cerebral arterioles. The alkalosis-induced vasoconstriction was significantly attenuated by inhibitors of NHE, NCX, and NKA, but not by K+ channel inhibitors. Therefore, we concluded that constriction of cerebral arterioles in response to alkalosis was mediated by NHE, as well as NKA and NCX (Fig. 6).

10 *

4.1. pHo and alkalosis

0

DCB (3 μ mol/L)

Control

Fig. 5. Effects of DCB on alkalosis-induced vasoconstriction. The relative changes in diameter were quantified as described in Section 2. Results are expressed as means ± SE (n = 4). The asterisk indicates a significant difference from control constriction (*P < 0.01).

pH 7.3. Fig. 5 shows that the relative rate of change in arteriole diameter on treatment with DCB was decreased by approximately 50% compared with that under control conditions (P < 0.01, n = 4).

Vascular tone is known to be affected by changes in pHo or pHi . Previous studies indicated that in isolated rat intracerebral arterioles, a bathing medium pH of 7.6 induces constriction by 23–24% (Takayasu and Dacey, 1989). Similarly, an alkaline pH-dependent constriction response of penetrating arterioles was observed in the present study (Figs. 1 and 2). These results indicated that pHo was an important regulator of vascular tone in the cerebral microcirculation, and the extent contraction and pHo showed a positive relationship.

Table 1 The effects of two kinds of K+ channel inhibitors on alkalosis induced vasoconstriction. Administrated of K+ channel inhibitor

Control (without any inhibitor) Arteriolar diameter (␮m) n

pH 7.3

Glibenclamide (3 ␮mol/L) 5 45.0 ± 5.0 TEA (1 mmol/L) 6 52.2 ± 3.2

Constriction (%)

pH 7.6

Arteriolar diameter (␮m) pH 7.3

pH 7.6

Constriction (%)

34.4 ± 4.7

23.7 ± 4.4

43.8 ± 3.8

34.2 ± 4.8

23.6 ± 5.1

43.5 ± 2.6

16.6 ± 0.5

47.3 ± 3.4

39.8 ± 3.6

16.3 ± 1.9

Values are expressed as mean ± SE; n is number of observations.

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Fig. 6. Overview of the factors involved in alkalosis-induced vasoconstriction. The activated NKA or changed extracellular pH will excite NHE and result in increased [Na+ ]i , which will excite NCX and therefore change the [Ca2+ ]i and vascular smooth muscle tension.

4.2. Roles of NHE and NCX in vasoconstriction in response to alkalosis Note that NHE works according to the Na+ and H+ gradients by exchanging extracellular Na+ for intracellular H+ with a tightly coupled 1:1 stoichiometry. Under the basal state in rat cerebral penetrating arterioles, NHE is activated and regulates the arteriolar tone through its vasodilator effect (Saesue et al., 2004). In the present study, vasoconstriction induced by extracellular alkalosis was significantly attenuated by extraluminal application of NHE inhibitor (Fig. 3). Under conditions of extracellular alkalosis, NHE may be activated by decreased extracellular H+ , which would result in increased [Na+ ]i . Moreover, it was reported that both K+ and Ca2+ fluxes can also be altered by Na+ influx (Wakabayashi et al., 1997). In addition, increases in pHo are directly and/or indirectly related to alkalosis-induced constriction via Ca2+ channels in VSMCs (Klockner and Isenberg, 1994). It has been suggested that excessive activation of NHE after hypoxia or ischemia was responsible for brain intracellular alkalosis (Wakabayashi et al., 1992). If NHE activity remains high for a sufficient period of time, increased NHE can lead to intracellular Ca2+ overload by reversing the NCX (Trudeau et al., 1999), and then intracellular Ca2+ elevation can exacerbate the process of cell death (Choi, 1987; Stout et al., 1998). It was reported that NHE inhibitor reduced brain injury in a model of neonatal ischemia (Kendall et al., 2006), and exerted a neuroprotective effect in cortical neurons in vitro and ischemia-induced cerebral infarct in vivo (Lee et al., 2009). We hypothesized that NHE blockade may be beneficial to cell survival after hypoxia or ischemia. Therefore, NHE is involved in alkalosis-induced constriction, probably followed by increased [Ca2+ ]i mediated by NCX and increased vascular smooth muscle tension. Our results also suggested that NHE inhibitors may play a role in improving microcirculation under conditions of extracellular alkalosis. Meanwhile, our results also suggested that there must be some other factors involved in this reaction, as the vasoconstriction response was not abolished despite NHE inhibition by high-dose HMA.

et al., 1988). The direct vasoconstrictor effect of cardiac glycosides is mediated by NCX through the regulation of cytosolic-free [Ca2+ ]i under conditions of [Na+ ]i loading, followed by a sequence of reactions of inhibition of NKA activity, increased [Na+ ]i , and increased Ca2+ entry (or decreased Ca2+ extrusion) (Nabel et al., 1988). Our previous study suggested that NCX may be inactive under basal conditions and works in Ca2+ influx mode in stimulated rat cerebral arterioles (Saesue et al., 2004). In the present study, the transient vasoconstriction caused by ouabain was probably due to the increased Ca2+ influx through NCX (Saesue et al., 2004). In addition, our study showed that under conditions of extracellular alkalosis, extraluminal application of ouabain or DCB significantly attenuated alkalosis-induced constriction. The transmembrane sodium gradient by NKA stimulates NHE (Kuro-o, 1996; Wakabayashi et al., 1997). We supposed that inhibition of NKA would lead to elevated [Na+ ]i , tending to reduce NHE-mediated Na+ influx. This declined in Na+ influx can attenuate Ca2+ entry-induced vasoconstriction. These results suggested that NKA and NCX are involved in alkalosis-induced constriction, which may be coordinated by NHE in isolated rat cerebral arterioles. Our results also indicated that inhibitors of these channels probably play a role in improvement of microcirculation under conditions of extracellular alkalosis. 4.4. Roles of potassium channels in constriction in response to alkalosis Previous studies confirmed that K+ channels play a major role in acidosis-induced vasodilation (Horiuchi et al., 2002; Lindauer et al., 2003). Therefore, we examined whether K channels were also involved in alkalosis-induced vasoconstriction. KATP channels are distributed in both large cerebral arteries and pial arterioles (Faraci and Heistad, 1998; Faraci and Sobey, 1998; Lang et al., 1997; Nagao et al., 1996). These channels are activated by several stimuli, such as reductions in intracellular ATP, PO2 , and pH (Faraci and Heistad, 1998; Faraci and Sobey, 1998). In rat cerebral penetrating arterioles, KATP channels are present but inactive under resting conditions (Horiuchi et al., 2001; Janigro et al., 1997; Lindauer et al., 2003; Nguyen et al., 2000). It was reported that 3 ␮mol/L glibenclamide was sufficient and specific for KATP channels because this concentration abolished KATP channel opener-induced dilation (Horiuchi et al., 2001; Nguyen et al., 2000). KCa channels are abundant in vascular smooth muscle and are activated by both elevated concentrations of intracellular calcium and membrane depolarization (Faraci and Heistad, 1998; Faraci and Sobey, 1998). These channels act as modulators of vasoconstrictor responses as well as mediators of vasodilation. It is generally accepted that KCa channels are present but silent in cerebral arterioles (Horiuchi et al., 2001; Lang et al., 1997; Paterno et al., 1996; Wei et al., 1996). In the present study, extraluminal application of 3 ␮mol/L glibenclamide (KATP channel inhibitor) and 1 mmol/L TEA (KCa channel inhibitor) had no effect under basal conditions or conditions of extracellular alkalosis. Other potassium channels, such as KV and KIR , were also not involved in alkalosis-induced constriction (data not shown). These results indicated that potassium channels are silent under resting conditions and are not involved in alkalosis-induced vasoconstriction of isolated rat cerebral penetrating arterioles.

4.3. Roles of NKA and NCX in constriction in response to alkalosis 4.5. Physiological and pathophysiological consequences It is known that the inhibition of NKA by ouabain, a cardiac glycoside, increases [Na+ ]i and has a direct vasoconstrictor effect on the vascular smooth muscle (Lee et al., 1980; McRitchie et al., 1976; Saesue et al., 2004). It was reported that pretreatment with ouabain promotes Na+ -dependent Ca2+ influx in cultured VSMCs (Nabel

Brain intracellular alkalosis has been observed in experimental models of adult stroke (Mabe et al., 1983). Moreover, extracellular alkalosis of VSMCs reduces the diameter of cerebral arterioles, thereby decreasing the circulation and exacerbating

Y. Li et al. / Neuroscience Research 70 (2011) 98–103

brain ischemia. In the present study, inhibition of NHE, NKA, and NCX significantly attenuated alkalosis-induced constriction. Therefore, our results suggested that inhibitors of these ion channels are probably beneficial to improve cerebral blood flow after hypoxia or ischemia. In conclusion, the inhibition of NHE, NKA, and NCX, but not KATP or KCa , significantly attenuated alkalosis-induced constriction of isolated rat cerebral penetrating arterioles. NHE in arterioles appears to be activated under both basal conditions and conditions of extracellular alkalosis. A previous study showed that NHE activation is essential to restore physiological pH following intracellular acidosis by extruding H+ . The high [Na+ ]i level contributes to activation of NCX, which leads to an increase in [Ca2+ ]i (Masereel et al., 2003). Therefore, we hypothesized that NHE may be activated by decreased extracellular pH. Thus, NHE plays an important role in alkalosis-induced vasoconstriction coordinated by NKA and NCX (Fig. 6). This result suggests that the NHE inhibitor, HMA, may have therapeutic potency against ischemic brain injury, possibly through the suppression of intracellular Ca2+ influx. Further studies are required to elucidate the signaling mechanism involved in the neuroprotective effect of HMA. Acknowledgements This research was supported by a Grant-in-Aid for Scientific Research (22591582) and Japan Brain Foundation (2009). References Apkon, M., Boron, W.F., 1995. Extracellular and intracellular alkalinization and the constriction of rat cerebral arterioles. J. Physiol. 484, 743–753. Austin, C., Wray, S., 1993. Extracellular pH signals affect rat vascular tone by rapid transduction into intracellular pH changes. J. Physiol. 466, 1–8. Austin, C., Wray, S., 1994. A quantitative study of the relation between intracellular pH and force in rat mesenteric vascular smooth muscle. Pflugers Arch. 427, 270–276. Austin, C., Wray, S., 1995. The effects of extracellular pH and calcium change on force and intracellular calcium in rat vascular smooth muscle. J. Physiol. 488, 281–291. Batlle, D.C., Godinich, M., LaPointe, M.S., Munoz, E., Carone, F., Mehring, N., 1991. Extracellular Na+ dependency of free cytosolic Ca2+ regulation in aortic vascular smooth muscle cells. Am. J. Physiol. 261, C845–856. Baumbach, G.L., Siems, J.E., Faraci, F.M., Heistad, D.D., 1989. Mechanics and composition of arterioles in brain stem and cerebrum. Am. J. Physiol. 256, H493–501. Bova, S., Goldman, W.F., Yauan, X.J., Blaustein, M.P., 1990. Influence of Na+ gradient on Ca2+ transients and contraction in vascular smooth muscle. Am. J. Physiol. 259, H409–423. Choi, D.W., 1987. Ionic dependence of glutamate neurotoxicity. J. Neurosci. 7, 369–379. Chopp, M., Chen, H., Vande Linde, A.M., Brown, E., Welch, K.M., 1990. Time course of postischemic intracellular alkalosis reflects the duration of ischemia. J. Cereb. Blood Flow Metab. 10, 860–865. Dacey Jr., R.G., Duling, B.R., 1982. A study of rat intracerebral arterioles: methods, morphology, and reactivity. Am. J. Physiol. 243, H598–606. Faraci, F.M., Heistad, D.D., 1990. Regulation of large cerebral arteries and cerebral microvascular pressure. Circ. Res. 66, 8–17. Faraci, F.M., Heistad, D.D., 1998. Regulation of the cerebral circulation: role of endothelium and potassium channels. Physiol. Rev. 78, 53–97. Faraci, F.M., Sobey, C.G., 1998. Role of potassium channels in regulation of cerebral vascular tone. J. Cereb. Blood Flow Metab. 18, 1047–1063. Horiuchi, T., Dietrich, H.H., Hongo, K., Goto, T., Dacey Jr., R.G., 2002. Role of endothelial nitric oxide and smooth muscle potassium channels in cerebral arteriolar dilation in response to acidosis. Stroke 33, 844–849. Horiuchi, T., Dietrich, H.H., Tsugane, S., Dacey Jr., R.G., 2001. Role of potassium channels in regulation of brain arteriolar tone: comparison of cerebrum versus brain stem. Stroke 32, 218–224. Hudetz, A.G., Conger, K.A., Halsey Jr., J.H., Pal, M., Dohan, O., Kovach, A.G., 1987. Pressure distribution in the pial arterial system of rats based on morphometric data and mathematical models. J. Cereb. Blood Flow Metab. 7, 342–355.

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