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(Stroke. 1998;29:175-180.)
© 1998 American Heart Association, Inc.

Original Contributions

Control of Vascular Tone by Endogenous Endothelin-1 in Human Pial Arteries

Eric Thorin, PhD; Thanh-Dung Nguyen, BSc; Alain Bouthillier, MD

From the Institut de Cardiologie de Montréal, Centre de Recherche (E.T., T.-D.N.), Montreal, and Département de Neurochirurgie, Hôpital Notre-Dame, Montréal, Canada.

Correspondence to Eric Thorin, Institute de Cardiologie de Montréal, Centre de Recherche, 5000 rue Belanger Est, Montréal (PQ) H1T 1C8, Canada. E-mail thorin{at}icm.umontreal.ca

	Abstract

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Background and Purpose—Endothelin-1 (ET) has been shown to be involved in human pathological conditions, but its physiological function remains to be elucidated. The aim of this work was to assess whether endothelium-derived ET was involved in the overall responsiveness of freshly isolated human pial arteries.

Methods—Samples of cerebral cortex, otherwise discarded, were obtained during tumor or epileptic lesion resections (n=10 donors). Arterial segments were isolated and mounted on a microvessel myograph.

Results—Inhibition of nitric oxide (NO) formation with N-nitro-L-arginine (L-NA, 100 µmol/L) increased basal tone by 7±1%E_max (n=5). This increase in tone was fully abolished in the presence of BQ123 (1 µmol/L; ET_A receptor antagonist, P<.05) but potentiated by a subthreshold concentration of exogenous ET (1 nmol/L; 33±8%E_max; P<.05). In the presence of L-NA, serotonin (10 µmol/L)–induced tone was doubled compared with the control response (P<.05) but reduced by 90% in the presence of BQ123 (P<.05). In the absence of L-NA, BQ123 prevented serotonin-induced tone (n=3). Oxymetazoline, a selective ₂-adrenergic receptor agonist, induced an endothelium-dependent relaxation of preconstricted human pial arteries. The relaxation was partially sensitive to NO synthase inhibition and fully prevented by the addition of ET, whereas substance P–induced relaxation was preserved. Glibenclamide (1 µmol/L), an inhibitor of ATP-sensitive K⁺ channels and tetraethylammonium (1 mmol/L), an inhibitor of Ca²⁺-activated K⁺ channels had no effect on oxymetazoline-induced relaxation.

Conclusions—The results of this study suggest first that ET is involved in the tonic response induced by NO synthase inhibition; second, part of the contractile response induced by serotonin is endothelium-dependent and sensitive to BQ123; and third, the data suggest that activation of ₂-adrenergic receptors generated an endothelium-dependent relaxation that was selectively inhibited by exogenous ET.

Key Words: endothelins • endothelium • nitric oxide synthase • pial arteries

	Introduction

Top Abstract Introduction Methods Results Discussion References Introduction  References

 
Endothelin-1 is a potent endothelium-derived constricting factor first identified in the medium of cultured endothelial cells.¹ It is a small peptide (21 amino acids) in which secretion is regulated by numerous factors. Most stimuli (such as -thrombin,² insulin,³ oxidized low-density lipoprotein,⁴ and hemodynamic shear stress⁵ ) regulate ET release at the level of gene transcription. The expression of preproET mRNA is regulated by mechanisms that involve receptor-mediated mobilization of Ca²⁺ and activation of protein kinase C in endothelial cells.⁶ Secretion of ET is also calcium-dependent. Serotonin⁷ and angiotensin II^{8 9} have an immediate effect on ET release in various resistance arteries. PreproET and ET are stored in intracellular vesicles of cultured bovine aortic endothelial cells,¹⁰ suggesting that this could be a target for some stimuli.^{11 12}

The involvement of ET in the control of the cerebrovascular tone is uncertain. The reactivity of small arteries is regulated by numerous factors, including membrane potential,¹³ pressure, and shear stress^{14 15} as well as factors released by the endothelium.^{16 17 18 19} In cerebral arteries, all these factors are associated with the regulation of cerebral blood flow.²⁰ A recent report showed that the diameter of canine cerebral arteries was enhanced 24 hours after in vivo injection of BQ123, an ET_A receptor antagonist.²¹ However, it is accepted that ET is critical in pathological states only.²² ET-1 levels increase in patients with subarachnoid hemorrhage²³ and coronary heart disease.^{24 25} Antagonism of ET receptors successfully prevented the appearance of cerebral vasospasms^{26 27} and the development of early atherosclerotic lesions²⁸ in animal models. However, there is no direct demonstration of the involvement of ET in the regulation of human cerebrovascular tone in physiological conditions.

The purpose of the present study was to assess whether endothelium-derived ET was involved in the overall responsiveness of freshly isolated human pial arteries by comparing responses to agonists after selective inhibition of various endothelium-derived factors with responses obtained after endothelial denudation of human pial arteries. The results reported in this manuscript demonstrate that ET may regulate human cerebrovascular tone during agonist stimulation and in conditions in which NO production is blocked.

	Methods

Top Abstract Introduction Methods Results Discussion References Introduction  References

 
Human pial arteries (382±21 µm, 69 rings), otherwise discarded, were obtained from 10 patients (who were 14, 16, 29, 29, 32, 35, 35, 37, 44, and 46 years of age; 8 males, 2 females) during neurosurgical resection of brain tumor (2 male patients) or epileptic lesion. None of the patients were diabetic or hypertensive, and none had coronary heart disease. Only normal arteries (those not feeding the tumor or included in the epileptic lesion) were used. They were transported in the laboratory in ice-cold PSS containing indomethacin (10 µmol/L, inhibitor of cyclooxygenase) and of the following composition (mmol/L): NaCl 130, KCl 4.7, KH₂PO₄ 1.18, MgSO₄ 1.17, NaHCO₃ 14.9, EDTA 0.026, glucose 10, and aerated with 12% O₂/5% CO₂/83% N₂ (pH 7.4). Segments of 2 mm long were mounted on 30 µm tungsten wires in resistance artery myograph (IMF) and either studied 1 hour, 8 hours, or 16 hours after surgery. No significant changes in reactivity were observed after 8 or 16 hours of surgery as reported by others.^{13 27} At least 6 segments from a patient were used. Two segments were used per protocol, one being a control. After a 1-hour stabilization period, arterial segments were challenged with a 40 mmol/L KCl PSS with 15-minute wash-out periods until a stable contractile response, representing the optimal tension, was reached (usually between three and four challenges). Arterial segments were contracted with angiotensin II (0.1 µmol/L) or 40 mmol/L KCl PSS and acetylcholine (1 µmol/L)– or substance P (0.1 µmol/L)–induced dilation tested.

The endothelium was removed mechanically by a human hair. The effectiveness of endothelium removal was confirmed by the absence of dilation induced by acetylcholine (1 µmol/L) and substance P (0.1 µmol/L) in preconstricted arteries.

To prepare K⁺-rich solutions, equimolar amounts of NaCl were replaced with KCl. The drugs used were acetylcholine hydrochloride, glibenclamide, indomethacin, L-NA, oxymetazoline hydrochloride, serotonin hydrochloride, tetraethylammonium (Sigma), ET1, angiotensin II, substance P (Peninsula), and pinacidil (Research Biochemicals International). Stock solutions of drugs were dissolved in physiological saline except for indomethacin and pinacidil, which were dissolved in ethanol, and glibenclamide, which was dissolved in dimethyl sulfoxide. Solutions were prepared daily and kept on ice except for glibenclamide, which was kept at room temperature.

Results are expressed as mean±SEM. In all experiments, n equals the number of patients. Vasoconstrictions are expressed as % of the maximal response (E_max) obtained in the presence of 127 mmol/L KCl PSS at the end of each individual experiment; vasorelaxations are expressed as the % inhibition of the preconstricting tone. Statistical differences between means were determined by ANOVA followed by Scheffé's F test. In appropriate conditions, an unpaired Student's t test was applied. A value of P<.05 was accepted as significant for differences between groups of data.

	Results

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In all patients, acetylcholine (1 µmol/L) and substance P (0.1 µmol/L) induced relaxation of 32±9% and 82±5%, respectively, of segments preconstricted with 40 mmol/L KCl PSS (64±8% E_max). When arterial rings were preconstricted with angiotensin II (1 µmol/L; 19±7% E_max, n=3), acetylcholine induced a significantly greater relaxation (64±6%, P<.05) compared with the level of relaxation obtained in depolarized conditions, whereas substance P induced a maximal relaxation.

After removal of the endothelium (n=6), relaxations to acetylcholine (-9±4%) and substance P (-15±22%) were abolished (P<.05).

Effect of NO Synthase Inhibition, BQ123, Exogenous ET, and Endothelial Denudation on Basal and Serotonin-Induced Tone
Preincubation (45 minutes) of the arterial rings in the presence of L-NA (inhibitor of NO synthase) significantly increased basal tone (7.4±1.0%E_max, P<.05; n=5). This constricting effect was abolished by endothelial denudation.

In intact arteries, the response induced by L-NA was abolished by BQ123 (ET_A receptor antagonist; 0±0%E_max, P<.05 versus L-NA alone), whereas BQ123 alone had no effect on basal tone. In contrast, L-NA–induced tone was potentiated (33±8%E_max, P<.05 versus previous experimental conditions) by prior addition of exogenous ET at a concentration (1 nmol/L) that had no significant direct constricting effect.

Serotonin induced contraction of isolated pial arteries; all results are summarized in Table 1. In the presence of L-NA, serotonin-induced tone was 1.7-fold greater than in control conditions. The combined addition of L-NA (100 µmol/L) and BQ123 (1 µmol/L) reduced serotonin-induced tone by 90%. Furthermore, this contraction was not sustained (Fig 1, upper trace). In the presence of BQ123 alone, serotonin failed to induce a contractile response (n=3, data not shown). In the presence of L-NA and exogenous ET (1 nmol/L), the contraction induced by serotonin was 1.6 times higher than the tone produced in the presence of L-NA alone. Finally, removal of the endothelium did not significantly potentiate the contraction induced by serotonin compared with the response obtained in control conditions. In denuded arteries, responses were sustained. However, the contraction was significantly lower than the response obtained in intact arteries in the presence of L-NA. L-NA or BQ123 did not affect the contraction induced by serotonin in the absence of endothelium (data not shown).

View this table: [in this window] [in a new window]   Table 1. Constrictions Induced by Serotonin (10 µmol/L; %Emax) Before (Control) or After Inhibition of NO Formation with L-NA (100 µmol/L) Alone or Combined With Either BQ123 (1 µmol/L) or Exogenous ET (1 nmol/L)

View larger version (16K): [in this window] [in a new window]   Figure 1. Partial recording of two experiments performed on isolated segments of human pial artery. The top recording shows the inhibitory effect of BQ123 on the contractile response induced by a single concentration of serotonin (5-HT, 10 µmol/L). The lower trace shows the concentration-dependent relaxant effect of oxymetazoline (OXY, 0.01 to 30 µmol/L) of arterial segments preconstricted with serotonin.

Role of Endothelium-Derived Factors on the Relaxation Mediated by Oxymetazoline of Human Pial Arteries
Oxymetazoline (₂-adrenoceptor agonist) had no direct contractile effect on arterial rings with or without endothelium. However, it induced a concentration-dependent relaxation of human pial arterial rings preconstricted with serotonin (Fig 1, lower trace); this response was endothelium dependent (Fig 2).

View larger version (24K): [in this window] [in a new window]   Figure 2. Relaxation mediated by oxymetazoline (0.01 to 30 µmol/L) of human pial arterial segments preconstricted with serotonin (10 µmol/L) in control conditions (n=3), after endothelial removal (-Endo; n=3), or inhibition of NO production with L-NA (100 µmol/L; n=5). Results are expressed as mean±SEM. n represents the number of donors. *P<.05 versus control. ¶P<.05 versus L-NA.

In the presence of L-NA, the relaxation induced by oxymetazoline was significantly decreased but not abolished (Fig 2).

Since BQ123 antagonized the preconstricting tone induced by serotonin (see above), it was not possible to obtain a concentration-dependent relaxation of arterial rings pretreated with L-NA and BQ123. But the combined addition of L-NA and exogenous ET potentiated responses to serotonin; the addition of cumulative concentrations of oxymetazoline caused no relaxation (Fig 3), whereas substance P (0.1 µmol/L) still produced a significant relaxation (88±1%), although it slightly decreased compared with the response obtained in the absence of exogenous ET (103±2%; n=3, P<.05).

View larger version (18K): [in this window] [in a new window]   Figure 3. Relaxation mediated by oxymetazoline (0.01 to 30 µmol/L) of human pial artery segments preconstricted with serotonin (10 µmol/L) after inhibition of NO production with L-NA (100 µmol/L; n=5) alone or combined with the addition of exogenous ET-1 (ET, 1 nmol/L; n=3). Results are expressed as mean±SEM. n represents the number of donors. *P<.05 versus L-NA.

To investigate the possible involvement of EDHF, which would mediate ₂-adrenergic receptor–dependent relaxation by activating smooth muscle K⁺ conductance, we studied the effect of TEA (1 mmol/L), an inhibitor of Ca²⁺-activated K⁺ (K_ca) channels, and glibenclamide (1 µmol/L), an inhibitor of ATP-sensitive K⁺ (K_ATP) channels, on vascular reactivity after inhibition of NO using L-NA. TEA and glibenclamide were added 20 minutes before testing the relaxant effect of oxymetazoline. In the presence of TEA or glibenclamide, the relaxation mediated by oxymetazoline was not modified (Fig 4).

View larger version (22K): [in this window] [in a new window]   Figure 4. Effect of TEA (1 mmol/L) and glibenclamide (GLI, 1 µmol/L) on the relaxation induced by oxymetazoline of human pial arteries preconstricted with serotonin (10 µmol/L; n=4 to 6 per group) in the presence of L-NA (l-NA, 100 µmol/L). n represents the number of donors.

Relaxations induced by pinacidil, an ATP-sensitive K⁺ channel agonist, represented 19±4%, 68±5%, and 92±6% of relaxation at 1, 3, and 10 µmol/L, respectively. In the presence of glibenclamide, relaxations were decreased to 0±0%, 0±0%, and 54±4% of relaxation at 1, 3, and 10 µmol/L, respectively (n=3 to 4, P<.05 versus in the absence of glibenclamide).

	Discussion

Top Abstract Introduction Methods Results Discussion References Introduction  References

 
The results of this study suggest first that ET is involved in the tonic response induced by NO synthase inhibition; Second, part of the contractile response induced by serotonin is endothelium-dependent and sensitive to BQ123; and third, the data suggest that activation of ₂-adrenergic receptors generated an endothelium-dependent relaxation that was selectively inhibited by exogenous ET and insensitive to K_ca and K_ATP channel inhibition.

Acetylcholine mediated endothelium-dependent relaxation of a similar amplitude as reported before and was sensitive to NO synthase inhibition.³³ It is important to note that inhibition of NO production increased basal tone. In experimental conditions in which isometric myographs are used, arterial segments do not develop myogenic tone by opposition to what is observed in isobaric conditions.³⁴ This can be demonstrated by the addition of sodium nitroprusside or papaverine, which do not relax isometrically mounted vessels. Consequently, it is likely that any increase in tone observed in the presence of L-NA is induced by endothelium-derived constricting factors, since L-NA had no effect in denuded arteries. This hypothesis is further supported by the inhibitory effect of BQ123, suggesting that ET is actively involved in the constriction induced by L-NA. It would also confirm and give a functional correlate to previous studies showing that NO actively inhibits the release of stimulated endothelium-derived ET.^{35 36 37 38} However, it is possible that NO masks the constricting influence of ET in our experimental conditions without directly affecting ET production.³⁵ In canine cerebral arteries, cyclooxygenase derivatives have been shown to be involved in both basal and agonist-stimulated tone in similar experimental conditions.^{18 39 40 41} Since all experiments were performed in the presence of indomethacin, by blocking the production of vasoactive cyclooxygenase products, we could not confirm that these previous findings are applicable to humans.

Thus, this first set of data suggests that basal tone of human pial arteries is actively regulated by the endothelium. Although we previously reported that both constricting and dilating endothelial factors were involved in the regulation of the overall vascular tone of the rat tail artery,⁴² this is the first time that ET is shown to be physiologically involved in the regulation of the human cerebrovascular tone.

In the presence of an intact endothelium, the contraction mediated by serotonin was reduced by BQ123, suggesting an endothelium-dependent component of the serotoninergic response that may involve ET. The concentration of BQ123 used in this study has been shown to antagonize angiotensin II–induced endothelium-dependent contraction of the isolated rat tail artery.⁸ As mentioned in the introduction, most stimuli regulate ET release at the level of gene transcription. However, our finding that serotonin has an immediate effect on ET release is not without precedent.^{7 8 9}

The importance of endothelium-derived ET in the net contractile response to serotonin is actively counteracted by NO. Inhibition of NO synthase significantly potentiated the contractile response, and this contraction was highly sensitive to ET_A-receptor antagonism. Although exogenous ET potentiated serotonin-induced contraction, a mechanism previously described in isolated human arteries,⁴³ this specific experiment by itself does not imply that ET is involved in the regulation of the vascular tone. However, taken together, our data strongly support the idea that the endothelium influences the human cerebrovascular tone by releasing both dilating and constricting factors. It is therefore likely that serotonin has a dual effect: it induces endothelium-derived ET secretion that potentiates the contraction induced by stimulation of smooth muscle serotoninergic receptors.

Intuitively, however, we would have expected that BQ123 would have decreased the contractile response induced by serotonin to a level of tone similar to that in the contraction obtained in denuded arteries; but this was clearly not the case, since BQ123 almost abolished the contraction induced by serotonin in the presence of an intact endothelium. Therefore, if we assume that the endothelium-dependent component of the contractile response, that is to say ET, was successfully antagonized by BQ123, we can propose at least one possible explanation to justify this apparent discrepancy: an endothelium-derived relaxing factor, other than NO or a cyclooxygenase product, efficiently antagonizes the contraction induced by stimulating smooth muscle cell serotoninergic receptors. The release of EDHF¹⁹ has been shown to be involved in the regulation of the human cerebrovascular tone. Alkayed and coworkers¹⁶ showed that miconazole, a cytochrome P450 inhibitor, decreased rat cerebral blood flow, reinforcing the idea that NO is not the sole regulator of the vascular tone. It is therefore likely that in our experimental conditions, an endothelium-derived relaxing factor, possibly EDHF, counteracts smooth muscle cell contraction. We will not be able to confirm this hypothesis until selective inhibitors of EDHF become available.⁴⁴

It has been reported that oxymetazoline induced endothelium-dependent relaxation of rabbit cerebral arteries through a selective activation of ₂-adrenergic receptors and via inhibition of ET release.⁷ Contrary to findings in rabbits, inhibition of NO production significantly attenuated the relaxant properties of oxymetazoline (Fig 1). Activation of endothelial ₂-adrenergic receptors induced NO release, triggering relaxation of large conductance arteries.^{45 46} In rat cerebral arteries, NO has been shown to have a permissive role on the relaxation induced by ₂-adrenergic receptor agonists.⁴⁷ However, as in our experimental conditions, NO appears not to be the only factor contributing to the ₂-adrenergic receptor–mediated relaxation as previously reported by others.⁴⁶

Since the preconstricting tone is highly dependent on ET release, we hypothesized that oxymetazoline may cause relaxation of serotonin-preconstricted human pial arteries by decreasing ET production, counterbalancing the stimulatory effect of serotonin. It has been reported that oxymetazoline decreased ET production from cultured human pial artery ECs and isolated segments of rabbit middle cerebral artery.⁷ Since BQ123 abolished the contractile response to serotonin (Fig 1, Table 1), we were unable to construct relaxant concentration-response curves to oxymetazoline in these conditions. Thus, we postulated that if our hypothesis was valid, the addition of exogenous ET would not only potentiate the preconstricting tone but would also selectively antagonize the relaxation mediated by activation of ₂-adrenergic receptors. Indeed, by adding ET we believed we would artificially inhibit the relaxant pathway, ie, the decrease in endothelial ET release by ₂-adrenergic receptor occupation. As shown by Fig 3, the relaxation mediated by oxymetazoline was fully antagonized by ET, whereas substance P still induced a potent relaxation. Substance P has been shown to cause relaxation of human cerebral arteries by stimulating the release of both NO and EDHF¹⁹ ; this would suggest that EDHF is not the mediator of the ₂-adrenergic receptor–dependent pathway. Rather, a functional inhibition of ET release is likely, as previously reported in rabbit cerebral arteries and cultured human pial artery ECs.⁷

This hypothesis is reinforced by the absence of effect of two well characterized inhibitors of potassium channels (Fig 4). TEA is an inhibitor of K_Ca channels^{32 48 49} ; a TEA-sensitive pathway has been shown to be a key regulator of the mesenteric and brain circulations.^{19 30 50} In some vascular preparations, receptor-mediated endothelium-dependent smooth muscle cell relaxation could be blocked by inhibitors of K_Ca channels but not K_ATP channels in the absence of NO production.^{32 51 52 53} The absence of effect of GLI is therefore not surprising.

In conclusion, our results reveal for the first time that ET may have a relevant physiological function in the human cerebral circulation in vitro. They show that NO actively counteracts ET production and/or action. They also suggest that receptor-activated ET regulation may play an important role in the overall control of the local cerebrovascular tone. It is noteworthy that in humans, serotonin has been proposed to be involved in the pathogenesis of migraine, and it has been suggested that treatment with ET receptor antagonists may be an efficient therapy for some forms of migraine.^{54 55 56 57 58} Although we acknowledge the possible limitations of this study due to the limited numbers of experiments performed in some protocols, altogether these studies suggest that there may be a link between ET, serotonin, and certain cerebrovascular disorders in humans.

	Selected Abbreviations and Acronyms

 

EDHF = endothelium-derived hyperpolarizing factor ET = endothelin L-NA = N-nitro-L-arginine NO = nitric oxide PSS = physiological salt solution TEA = tetraethylammonium

Received May 1, 1997; revision received September 8, 1997; accepted October 7, 1997.

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Editorial Comment

Frank M. Faraci, PhD, Guest Editor

Department of Internal Medicine, Cardiovascular Division, University of Iowa College of Medicine, Iowa City, Iowa

	Introduction

Top Abstract Introduction Methods Results Discussion References Introduction  References

 
Endothelium exerts a major influence on tone of underlying vascular muscle by production and release of potent relaxing and contracting factors.^{1 2} The endothelium-derived contracting factor that has received the most attention is endothelin, a peptide originally isolated from porcine aortic endothelium.³

Vascular effects of endothelin are mediated through activation of two receptors, endothelin-A and endothelin-B receptors. In general, endothelin-A receptors are expressed in vascular muscle and mediate contraction to endothelin. In contrast, endothelin-B receptors are expressed on endothelium and can mediate endothelium-dependent relaxation. Although these concepts were based initially on findings made with use of vessels from animal models, the same mechanisms have been described in cerebral arteries from humans.⁴

The results of the present study are interesting because they suggest an additional effect of endothelin. Based on data obtained with use of human pial arteries, the findings suggest that endothelin may selectively modulate responses to other vasoactive stimuli. For example, relatively low concentrations of endothelin impaired endothelium-dependent relaxation to oxymetazoline (an ₂-adrenergic receptor agonist), but not substance P (a peptide that produces endothelium-dependent relaxation independent of adrenergic receptors). The mechanism that produces this effect is not obvious, since both oxymetazoline and substance P produce an endothelium-dependent response that is mediated largely by nitric oxide. The finding that endothelin may alter responses to other vasoactive stimuli is consistent with previous work in this area.⁵ The effect may also not be unique to endothelin, because low concentrations of thromboxane also alter responses of human cerebral arteries to other vasoconstrictors.⁶

An additional interesting aspect of the present study relates to potential interaction of endothelin with nitric oxide. Previous studies suggest that in addition to being a powerful vasodilator, nitric oxide also inhibits gene expression and/or synthesis of endothelin.⁵ In the present study, pharmacological inhibition of nitric oxide synthase appeared to unmask a constrictor effect of endogenous endothelin. An implication of this finding is that endothelin may exert a greater influence on vascular tone under pathophysiological conditions which are associated with impairment of the nitric oxide signaling pathway.

	Selected Abbreviations and Acronyms

 

EDHF = endothelium-derived hyperpolarizing factor ET = endothelin L-NA = N-nitro-L-arginine NO = nitric oxide PSS = physiological salt solution TEA = tetraethylammonium

Received May 1, 1997; revision received September 8, 1997; accepted October 7, 1997.

	References

Top Abstract Introduction Methods Results Discussion References Introduction  References

 
1. Faraci FM, Heistad DD. Regulation of the cerebral circulation: role of endothelium and potassium channels. Physiol Rev. In press.

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