Receptor changes in cerebral arteries after subarachnoid haemorrhage

Subarachnoid haemorrhage (SAH), occurring with a delay of 4-10 days is linked to cerebral vasospasm (CVS), a pathological constriction of the cerebral arteries. Several agents have been suggested as being responsible - amongst these perhaps 5-hydroxytryptamine (5-HT) and endothelin-1 (ET-1) are the most prominent, given their ability to elicit powerful constriction of arteries. Investigating both 5-HT and ET receptors we observed distinct changes in the receptor phenotype after experimental SAH - namely upregulation of the ETB and 5-HT1B receptors - linked to a higher sensitivity to the endogenous agonists. This multiple receptor upregulation may explain the failure in treating CVS using single receptor antagonists, and may also significantly change our understanding of the effector mechanism behind CVS. So far only the ET and 5-HT receptors have been studied in this regard, but other receptor systems may also undergo changes.     

内皮素-1诱导培养大鼠大脑皮质神经元凋亡的初步研究

目的观察内皮素(endothelin,ET)-1有无直接诱导原代培养神经元凋亡的作用,以及其作用通过的ET受体亚型。方法神经元培养取自新生SD大鼠大脑皮质。培养5 d后分别加入0.2 nmol/L,20 nmol/L ET-1处理24 h,用Annexin V、Hoechst 33258染色半定量测定细胞凋亡。再用流式细胞仪分别定量检测ET受体A拮抗剂(BQ123)或ET受体B拮抗剂(BQ788)对20 nmol/L ET-1诱导神经元凋亡的效果。结果0.2 nmol/L ET-1未显示诱导培养神经元凋亡的作用;20 nmol/L ET-1处理后24 h,培养神经元凋亡率明显高于对照组(P<0.01);BQ123和BQ788分别部分阻断了20 nmol/L ET-1诱导神经元凋亡的作用(P<0.01),但阻断效果不完全。结论20 nmol/L ET-1可直接诱导培养大鼠大脑皮质神经元凋亡,其作用可能是通过其A受体和B受体亚型共同实现的。     

Selective up-regulation of 5-HT(1B/1D) receptors during organ culture of cerebral arteries.

5-Hydroxytryptamine (5-HT) is thought to be involved in migraine headache and the pathophysiology of cerebrovascular diseases. Previous data show that organ culture induces a phenotypic change in cerebral vessels. Therefore we investigated if these changes also applied for the vasoconstrictive 5-HT receptors. Rat cerebral arteries express 5-HT2 receptors. Using organ culture we observed a phenotypic change with a selective up-regulation of 5-HT(1B/1D) receptors. This was revealed by an increased sensitivity to the selective 5-HT(1B/1D) agonist 5-CT after organ culture (pEC50(fresh) 5.6+/-0.2 and pEC50(cultured) 6.8+/-0.4). The response was inhibited by the 5-HT(1B/1D) selective antagonist GR55562 (pEC50(fresh) 5.1+/-0.2 and pEC50(cultured) 6.0+/-0.3). The organ model might mimic the phenotypic changes during cerebrovascular diseases.     

Expression of vascular endothelial growth factor (VEGF) in rat brain after subarachnoid haemorrhage and endothelin receptor blockage with BQ-123

Cerebral vasospasm is one of the most severe complications of subarachnoid haemorrhage (SAH), leading to pathological changes in the vessel wall itself and in the nervous tissue, due to ischaemia of endothelial cells and neurones. Amongst the known substances inducing vasospasm, the most potent spasmogenic effect is exerted by endothelin-1 (ET1). The constriction of cerebral arteries and obliteration of capillaries highly stimulates the secretion of growth factors by endothelial cells and induces compensatory formation of collateral circulation in response to brain ischaemia. Expression of vascular endothelial growth factor (VEGF), the main factor responsible for angiogenesis and vascular permeability, was found to be increased in hypoxic cells (irrespective of the cause of hypoxia) as well as in neoplastic cells in the brain. The aim of the study was to determine whether chronic vasospasm and hypoxia of endothelial cells stimulate expression of VEGF, and whether blockage of the endothelin receptor ET(A) reduces this expression. The SAH was induced experimentally in male Wistar rats and the ET(A) receptor antagonist--BQ-123 was administered into the cisterna magna. After 48 hours the brain was removed and expression of VEGF studied immunohistochemically on paraffin sections. We found that hypoxia of endothelial cells, induced by chronic vasospasm after SAH, caused increased expression of VEGF in brain vessels and neurones of the cerebral hemispheres, brain stem and cerebellum. After administration of the endothelin receptor antagonist BQ-123, no changes in VEGF expression in the brain were found.     

Characterization of the potent combined endothelin(A/B)-antagonist PD 142893 on cerebral vessels

A disturbed balance between endothelin (ET)-1 and nitric oxide (NO) seems to play a key role in the development of delayed cerebral vasospasm following subarachnoidal hemorrhage. Therefore, the effect of PD 142893 one of the first potent ET(A)- and ET(B)-receptor antagonists was characterized on the contraction and relaxation induced by ET-1 and bigET-1 on rat basilar artery (BA). Concentration-effect curves (CECs) were constructed by cumulative application of ET-1 or big ET-1 on BA ring segments with (E+) and without (E-) functionally intact endothelium. The effect of PD 142893 was determined by the modified pK(b) value and the shift between the CECs. PD 142893 inhibited the contraction by ET-1 and bigET-1. The pK(b)-values were for ET-1: 5.17 (E+) and 5.15 (E-) and for big ET-1: 5.34 (E+) and 5.57 (E-), respectively. A significant relaxation of pre-contracted segments by ET-1 or big ET-1 was neither observed in the presence nor in the absence of the receptor antagonist. The present data suggest a competitive inhibition of the ET(A)-receptor mediated contraction of cerebral arteries by PD 142893. The ET(B)-dependent relaxation of the cerebrovasculature is inhibited by PD 142893 at least in a comparable amount of contraction.     

Characterization of the potent combined endothelin(A/B)-antagonist PD 142893 on cerebral vessels

Abstract

A disturbed balance between endothelin (ET)-1 and nitric oxide (NO) seems to play a key role in the development of delayed cerebral vasospasm following subarachnoidal hemorrhage. Therefore, the effect of PD 142893 one of the first potent ET(A)- and ET(B)-receptor antagonists was characterized on the contraction and relaxation induced by ET-1 and bigET-1 on rat basilar artery (BA). Concentration-effect curves (CECs) were constructed by cumulative application of ET-1 or big ET-1 on BA ring segments with (E+) and without (E–) functionally intact endothelium. The effect of PD 142893 was determined by the modified pK_b value and the shift between the CECs. PD 142893 inhibited the contraction by ET-1 and bigET-1. The pK_b-values were for ET-1: 5.17 (E+) and 5.15 (E–) and for big ET-1: 5.34 (E+) and 5.57 (E–), respectively. A significant relaxation of pre-contracted segments by ET-1 or big ET-1 was neither observed in the presence nor in the absence of the receptor antagonist. The present data suggest a competitive inhibition of the ET(A)-receptor mediated contraction of cerebral arteries by PD 142893. The ET(B)-dependent relaxation of the cerebrovasculature is inhibited by PD 142893 at least in a comparable amount of contraction.     

Experimental subarachnoid hemorrhage induces changes in the levels of hippocampal NMDA receptor subunit mRNA

NMDA receptors may play a crucial role in nerve cell death following subarachnoid hemorrhage (SAH). Changes in NMDA receptor-mediated transmission appear before neuronal death in rodent models of transient ischemia, and NMDA receptor function is known to be dependent on subunit composition. Here, we have investigated whether mRNA expression of the NMDA receptor subunits is altered in the hippocampal formation 3-5 h following experimental SAH, and correlated these early alterations to subsequent delayed cell death. SAH was induced by intraluminal perforation of the internal carotid artery intracranially, and cerebral blood flow (CBF) was bilaterally monitored by laser-Doppler flowmetry. Early changes in NMDA receptor subunit mRNA and early nerve cell death were analyzed at 3-5 h after SAH, and delayed nerve cell death was analyzed at 2-7 days after SAH. Duration of ipsilateral CBF reduction below 30% of baseline (CBF30) was predictive of ipsilateral delayed nerve cell death in the CA1 2-7 days after SAH. At CBF30 > 9 min, we found downregulation of mRNA for NR2A, NR2B, and NR3B at 3-5 h after SAH, whereas the levels of NR1 mRNA were unaffected. The downregulation of NR2A and NR2B mRNA may result in a reduced NMDA receptor function. Such reduction may be sufficient to provide neuroprotection in the dentate gyrus, where no cell death appears, but insufficient to rescue neurons in the hippocampus proper following SAH.     

Effects of S-nitrosoglutathione on acute vasoconstriction and glutamate release after subarachnoid hemorrhage.

BACKGROUND AND PURPOSE: Subarachnoid hemorrhage (SAH) causes acute vasoconstriction that contributes to ischemic brain injury shortly after the initial bleed. It has been theorized that decreased availability of nitric oxide (NO) may contribute to acute vasoconstriction. Therefore we examined the effect of the NO donor N-nitroso glutathione (GSNO) on acute vasoconstriction and early ischemic glutamate release after experimental SAH. METHODS: SAH was induced by the endovascular suture method in anesthetized rats. GSNO (1 micromol/L/kg, n=31) or saline (n=21) was injected 5 minutes after SAH. Sham-operated rats received GSNO (1 micromol/L/kg, n=5) 5 minutes after sham surgery. Arterial and intracranial pressures, cerebral blood flow (CBF), and extracellular glutamate release were measured serially for 60 minutes after SAH. SAH size was determined, and vascular measurements were made histologically. RESULTS: GSNO had no effect on resting blood pressure, intracranial pressure, cerebral perfusion pressure, or CBF in sham-operated animals. However, administration of GSNO after SAH was associated with significantly increased CBF (161.6+/-26.6% versus saline 37.1+/-5.5%, 60 minutes after SAH, P<0.05), increased blood vessel diameter (internal carotid artery [ICA] 285.0+/-16.5 microm versus saline 149.2+/-14.1 microm, P<0.01), decreased vessel wall thickness (ICA12.9+/-0.7 microm versus saline 25.1+/-1.6 microm, P<0.01), and decreased extracellular glutamate levels (3315.6+/-1048.3% versus saline469. 7+/-134.3%, P<0.05). Blood pressure decreased transiently, whereas intracranial pressure, cerebral perfusion pressure, and SAH size were not affected. CONCLUSIONS: These results suggest that GSNO can reverse acute vasoconstriction and prevent ischemic brain injury after SAH. This further implies that acute vasoconstriction contributes significantly to ischemic brain injury after SAH and is mediated in part by decreased availability of NO.     

Evidence that 20-HETE contributes to the development of acute and delayed cerebral vasospasm

Recent studies have indicated that arachidonic acid (AA) is metabolized by the cytochrome P450 4A (CYP4A) enzymes in cerebral arteries to produce 20-hydroxyeicosatetraenoic acid (20-HETE) and that this compound has effects on cerebral vascular tone that mimic those seen following subarachnoid hemorrhage (SAH). In this regard, 20-HETE is a potent constrictor of cerebral arteries that decreases the open state probability of Ca(2+)-activated K(+) channels through activation of protein kinase C (PKC). It increases the sensitivity of the contractile apparatus to Ca(2+) by activating PKC and rho kinase. The formation of 20-HETE is stimulated by angiotensin II (AII), endothelin, adenosine triphosphate (ATP) and serotonin, and inhibited by NO, CO and superoxide radicals. Inhibitors of the formation of 20-HETE block the myogenic response of cerebral arterioles to elevations in transmural pressure in vitro and autoregulation of cerebral blood flow (CBF) in vivo. 20-HETE also plays an important role in modulating the cerebral vascular responses to vasodilators (NO and CO) and vasoconstrictors (AII, endothelin, serotonin). Recent studies have indicated that the levels of 20-HETE in cerebrospinal fluid (CSF) increase in rats, dogs and human patients following SAH and that inhibitors of the synthesis of 20-HETE prevent the acute fall in CBF in rats and reverse delayed vasospasm in both dogs and rats. This review examines the evidence that an elevation in the production of 20-HETE contributes to the initial fall in CBF following SAH and the later development of delayed vasospasm.     

Cardiovascular effects of centrally applied endothelin-1 1-31 and its relationship to endothelin-1 1-21 in rats

Endothelin-1(1-31) (ET-1(1-31)) is a novel member of the endothelin family, which comprises 31 amino acids and derived from the selective hydrolysis of big ET-1 by chymase. Although ET-1(1-31) has been reported to be involved in biological effects via direct or indirect (converting to ET-1(1-21)) mechanisms, the cardiovascular effects of central ET-1(1-31) are not fully identified. The present study was designed to comparatively investigate the cardiovascular effects of intracerebroventricular (icv) application of ET-1(1-31) or ET-1(1-21) in anesthetized rats. Injection (icv) of ET-1(1-31) (500 pmol) produced a biphasic blood pressure response: an initial increase (from 118+/-8 to 138+/-14 mmHg, P<0.05) followed by a sustained decrease in BP (from 118+/-8 to 58+/-9 mmHg, P<0.05), which was very similar to BP response to icv injection of big ET-1 (500 pmol) or ET-1(1-21) (25 pmol)(.) The cardiovascular effects of icv injection of ET-1(1-31) or ET-1(1-21) were completely antagonized by ET(A) receptor antagonist BQ123 but not ET(B) receptor antagonist BQ788. Furthermore, pretreatment with ET converting enzyme inhibitor phosphoramidon (10 nmol) abolished the cardiovascular effects evoked by icv injection of ET-1(1-31) or big ET-1. In conclusion, the current data showed that central ET-1(1-31) produced the similar cardiovascular effects as those of central ET-1(1-21), and suggesting that the central cardiovascular effects of ET-1(1-31) resulted from it converting to ET-1(1-21) and then activating ET(A) receptors.     

Effects of a new magnesium-rich artificial cerebrospinal fluid on contractile 5-hydroxytryptamine and endothelin receptors in rat cerebral arteries

ABSTRACT

Objectives Cerebral vasospasm after subarachnoid haemorrhage (SAH) is associated with cerebrovascular contractile receptor upregulation resulted from haemolysis in the subarachnoid space. This study developed a new magnesium-rich artificial cerebrospinal fluid (MACSF) formula and investigated its effects on receptor-mediated contraction in rat basilar arteries.

Methods Clear and haemorrhagic cerebrospinal fluid (CSF) were collected from patients with hydrocephalus or SAH. MACSF was freshly prepared using clinical intravenous injections. Rat basilar arteries were segmented and incubated with clear CSF, haemorrhagic CSF or MACSF. The contractile responses were studied by myograph. The messenger ribonucleic acid (mRNA) and protein expression of 5-hydroxytryptamine 1B (5-HT_1B), endothelin subtype B (ET_B) and endothelin subtype A (ET_A) receptors were evaluated by real-time polymerase chain reaction (PCR) and Western blot analyses.

Results Haemorrhagic CSF exposure shifted the contractile curves induced by 5-hydroxytryptamine (5-HT), sarafotoxins 6c (S6c) and endothelin-1 (ET-1) leftward with increased maximal contraction values. Furthermore, mRNA and protein expression were markedly elevated for 5-HT_1B, ET_B and ET_A receptors on arteries exposed to haemorrhagic CSF. However, the contractile responses to 5-HT, S6c or ET-1 and expression of 5-HT_1B, ET_B and ET_A receptors in rat cerebral arteries exposed to MACSF remained unaffected compared to those exposed to clear CSF. Besides, unlike normal saline which can inactive in-vitro vessels, MACSF can maintain their physiological activity.

Conclusion Haemorrhagic CSF induces upregulation of 5-HT_1B, ET_B and ET_A receptors in rat cerebral arteries. However, MACSF can maintain in-vitro rat basilar arteries in good physiological activity and normal expression of contractile 5-HT and ET receptors.     

Bradykinin and phorbol ester but not 5-HT2B receptor activation stimulate phospholipase D activity in the rat stomach fundus.

1. Serotonin has been implicated as a mediator involved in migraine headache, an effect that may involve central 5-HT2B receptor activation. 5-HT2B receptor signal transduction in controversial. 2. Rat stomach fundus contraction to serotonin has been used as a model for 5-HT2B receptor activation. Serotonin-induced contractility involves intracellular calcium release and activation of protein kinase C without stimulation of phosphoinositide (PI) hydrolysis. 3. Since phospholipase D (PLD) activation results in phosphatidic acid production, which can release intracellular calcium and provide diacylglycerol for PKC activation, the purpose of this study was to determine whether the 5-HT2B receptor coupled to PLD activation using the rat stomach fundus as a model system. 4. Using phosphatidylethanol production to measure PLD activity, both bradykinin (0.01-1 microM) and phorbol dibutyrate (PDBu, 1 microM) stimulated PLD activity in rat stomach fundal strips, indicating that this tissue possesses an active PLD system. 5. Under identical conditions, 5-hydroxytryptamine (5-HT) failed to stimulate PLD activity over a concentration range (1 nM-1 microM) documented to induce 5-HT2B receptor-mediated contraction in rat stomach fundus. Thus, the 5-HT2B contractile receptor in rat stomach fundus is not coupled to PLD activation, whereas both bradykinin and phorbol ester do couple to PLD.     

Changes in contractile response and effect of a calcium antagonist, nimodipine, in isolated intracranial arteries of baboon following experimental subarachnoid hemorrhage

Isolated pial arteries from a previously well-characterized model of experimental subarachnoid hemorrhage (SAH) in baboon were tested for their contractile response to 5-hydroxytryptamine (5-HT), norepinephrine (NE), and prostaglandin F2 alpha (PGF2 alpha) and the effect of the calcium antagonist, nimodipine. Autologous blood was injected cisternally at three times with one-day intervals to a total amount of 11.5-29.5 ml (mean: 18.5 ml), and the animals were killed 7 days after the first injection. Untreated animals served as controls. The degree of maximum contraction (EAm) with 5-HT and NE in the control situation was for the three arteries tested in the order middle cerebral greater than anterior cerebral greater than basilar artery. Experimental SAH markedly increased EAm, by 190-370 percent above control values (depending on type of vessel) for 5-HT and 170-185 percent for NE. In addition, the sensitivity to 5-HT was significantly increased, as evidenced by a left-shift of the concentration-response curve. Previous exposure of the artery to 10(-6) M nimodipine reduced the contractile response of both amines to approximately half, the inhibition being slightly less pronounced post-SAH. When vessels were contracted beforehand with the amines or with PGF2 alpha, followed by administration of increasing amount of nimodipine (10(-9) M to 10(-6) M), a concentration-dependent relaxation was obtained by up to 60 percent of the original level. This relaxing effect was significantly less following SAH in the experiments with NE and PGF2 alpha compared to 5-HT; the contraction in the presence of 5-HT did not differ before and after experimental SAH. The experiments show that SAH markedly enhances the intrinsic activity for both 5-HT and NE. Nimodipine inhibits the contractile response less efficiently following experimental SAH. The difference in the responsiveness to 5-HT on the one hand, and to NE and PGF2 alpha on the other, could be due to differences in the blood-induced alterations of those calcium channels that are influenced by the calcium antagonist, nimodipine.     

Mechanisms underlying potentiation of endothelin-1-induced myofilament Ca(2+) sensitization after subarachnoid hemorrhage

Increased vascular smooth muscle contractility has an important role in the development of cerebral vasospasm after subarachnoid hemorrhage (SAH). Myofilament Ca²⁺ sensitivity is a major determinant of smooth muscle contractility. We investigated changes in the Ca²⁺-sensitizing effect of endothelin-1 (ET-1) and the mechanisms underlying ET-1-induced Ca²⁺ sensitization after SAH using a rabbit SAH model. After SAH, the contractile response to ET-1 was enhanced, and the ET_A receptor expression was upregulated in the basilar artery. In α-toxin-permeabilized preparations, ET-1 induced enhanced and prolonged contraction after SAH, suggesting that ET-1-induced Ca²⁺ sensitization is potentiated after SAH. Endothelin-1-induced Ca²⁺ sensitization became less sensitive to inhibitors of Rho-associated coiled-coil protein kinase (ROCK) and protein kinase C (PKC) after SAH. The expression of PKCα, ROCK2, PKC-potentiated phosphatase inhibitor of 17 kDa (CPI-17) and myosin phosphatase target subunit 1 (MYPT1) was upregulated, and the level of phosphorylation of CPI-17 and MYPT1 was elevated after SAH. This study demonstrated for the first time that the Ca²⁺-sensitizing effect of ET-1 on myofilaments is potentiated after SAH. The increased expression and activity of PKCα, ROCK2, CPI-17, and MYPT1, as well as the upregulation of ET_A receptor expression are suggested to underlie the enhanced and prolonged Ca²⁺ sensitization induced by ET-1.     

The effects of hyponatraemia and subarachnoid haemorrhage on the cerebral vasomotor responses of the rabbit

Impairment of cerebral autoregulation and development of hyponatraemia are both implicated in the pathogenesis of delayed cerebral ischaemia and infarction following subarachnoid haemorrhage (SAH) but the pathophysiology and interactions involved are not fully understood. We have studied the effects of hyponatraemia and SAH on the cerebral vasomotor responses of the rabbit. Cerebrovascular reactivity to hypercapnia and cerebral autoregulation to trimetaphan-induced hypotension were determined in normal and hyponatraemic rabbits before and 6 days after experimental SAH produced by two intracisternal injections of autologous blood. Hyponatraemia (mean plasma sodium of 119 mM) was induced gradually over 48 h by administration of Desmopressin and intraperitoneal 5% dextrose. Sham animals received normal saline. The cerebrovascular reactivity (% change +/- SD in cortical CBF/mm Hg PaCO2, measured by hydrogen clearance) of hyponatraemic (4.8 +/- 3.0%) and SAH (1.3 +/- 2.0%) animals was significantly less (p less than 0.05) than control (11.6 +/- 4.0%) and sham (8 +/- 2.0%) animals, whereas the reactivity of hyponatraemic-SAH animals was preserved (9.8 +/- 6.0%). Hyponatraemia and SAH alone each significantly impaired CBF autoregulation but their combined effects were not additive. Systemic hyponatraemia impairs normal cerebral vasomotor responses but does not augment the effects of experimental SAH in the rabbit.     

Effects of morphine and naloxone on cerebral blood flow and metabolism in experimental subarachnoid hemorrhage

Objective - Naloxone is reported to improve the clinical condition of patients with subarachnoid hemorrhage (SAH). If this effect is vascular determined is unknown, wherefore the influence of morphine and naloxone on cerebral blood flow (CBF) and metabolic rate of oxygen (CMRO_z) after SAH was evaluated. Material and methods - Two groups of 8 rats each with SAH and 2 corresponding groups of controls were investigated. CBF was calculated by the intracarotid ¹³³Xenon method and CMRO₂ as the product of CBF and the difference between systemic arterial and cerebral venous oxygen content. Results - In controls morphine, 1 mg/kg administered intravenously, decreased CBF by 25% ( P < 0.001) without changing the CBF/CMRO₂ ratio. In animals with SAH CBF was decreased by 32% ( P < 0.001) and CBF/CMRO₂ ratio by 38% ( P < 0.01). Naloxone, 40ug/kg administered intravenously neither influenced CBF nor the CBF/CMRO₂ ratio in the 2 groups. Conclusion - The reported clinical effect of naloxone after SAH can, according to our results, not be explained by changing the relationship between CBF and metabolism.     

Cerebrovascular characterization of the novel nonpeptide endothelin-A receptor antagonist LU 208075

Enhanced cerebrovascular resistance under pathologic conditions, like cerebral vasospasm after subarachnoid hemorrhage, seems to be caused by the vasocontractile effect of endothelin-1 (ET-1). Therefore, the effect of the novel and ET(A) receptor selective antagonist LU 208075 was characterized by the contraction and relaxation induced by ET-1 and bigET-1 on rat basilar artery. Basilar artery ring segments with (E+) and without (E-) functionally intact endothelium were prepared to measure the isometric force. Concentration-effect curves were constructed by cumulative application of ET-1 or bigET-1 in the presence of LU 208075 (10(-7)M, 10(-6)M, and 10(-5)M). The effect of LU 208075 was determined by the pA(2) value. The contraction by ET-1 and bigET-1 was inhibited by LU 208075 in a dose-dependent manner. The pA(2) values for ET-1 and for bigET-1 were 6.51 +/- 0.39 (E+) and 6.67 +/- 0.43 (E-), and 7.03 +/- 0.32 (E+) and 7.24 +/- 0.31 (E-) respectively. The E(max) values for bigET-1 but not for ET-1 were reduced significantly in the presence of LU 208075. A significant relaxation by ET-1 or bigET-1 was observed only in the presence of LU 208075. This relaxation was inhibited by LU 208075 in higher concentrations, with pA(2) values of 5.68 +/- 0.05 (ET-1) and 5.50 +/- 0.39 (bigET-1). The current data correlate with a competitive inhibition of ET(A) receptor-mediated contraction and relaxation, caused by ET(B) receptor activation on cerebral vessels by LU 208075. The selectivity for the ET(A) receptor was approximately sevenfold. Furthermore, the results may suggest an inhibition of the functional ET-converting enzyme activity by LU 208075.