The vasorelaxation of cerebral arteries by carbon monoxide.

Carbon monoxide (CO) is known to increase cerebral blood flow, but the effect of CO on the vascular tone of large cerebral arteries is uncertain. We tested whether CO affects cerebral artery tone by measuring tension generated by ex vivo segments of dog basilar artery upon exposure to CO. In cerebral artery segments contracted with either KCl or prostaglandin F(2alpha), CO caused a concentration-related relaxation beginning with a concentration of 57 microM. Relaxation did not occur if CO was administered in the presence of bubbling carboxygen (95% O(2):5% CO(2)), which reduces greater than 99% of CO from the solution. Furthermore, the CO-induced relaxation of cerebral artery segments was reduced in the presence of the guanylyl cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ, 10 microM)or the potassium channel blocker tetraethylammonium (TEA, 1 mM). Neither ODQ nor TEA completely eliminated the relaxation caused by CO and there was no additive effect if ODQ and TEA were administered together. These results suggest that cerebral arteries are directly relaxed by CO and that this relaxation depends upon the activation of guanylyl cyclase and the opening of potassium channels.

Vasospasm in monkeys resolves because of loss of and encasement of subarachnoid blood clot.

BACKGROUND AND PURPOSE: We studied in monkeys why vasospasm resolves after subarachnoid hemorrhage (SAH). METHODS: Monkeys underwent angiography and right (n=17) or bilateral (n=8) SAH. Animals with bilateral SAH underwent angiography 1, 3, 5, and 7 days later. Animals with right SAH underwent angiography 7 days later. The clot was then not removed (n=5), removed and replaced with fresh clot (n=7), or removed and not replaced (n=5). At the same time on day 7, the removed clot (n=12) or fresh clot (n=5) was placed on the left side. Angiography was repeated every 2 days until day 14. RESULTS: SAH caused significant vasospasm on day 7 that resolved by day 14. Removal of clot on day 7 resulted in more rapid resolution of vasospasm. Placement of fresh clot onto arteries that had already been exposed to clot for 7 days produced vasospasm that persisted without resolving for an additional 7 days. Placement of 7-day-old clot from the right onto previously unexposed left arteries or of clot from blood removed from an animal 7 days after SAH caused significantly more rapid onset of vasospasm compared with de novo vasospasm. Microscopic examination of the clots showed they were surrounded by macrophages 7 days after SAH. Arterial compliance and contractility were reduced in relation to duration of the exposure of arteries to clot. CONCLUSIONS: Vasospasm resolves because of loss of subarachnoid blood clot. We hypothesize that reduced spasmogen release from the clot contributes to resolution of vasospasm. There was no response in the cerebral arteries that rendered them less responsive to the subarachnoid clot.

Role of adenosine 5'-triphosphate in vasospasm after subarachnoid hemorrhage: human investigations.

OBJECTIVE: Adenosine 5'-triphosphate (ATP) is a vasoactive compound found in high concentrations inside erythrocytes. This compound may contribute to vasospasm after subarachnoid hemorrhage (SAH). We assessed the hypothesis that ATP contributes to vasospasm in humans. METHODS: ATP and hemoglobin concentrations were measured in cerebrospinal fluid (CSF) from humans with SAH and in blood incubated in vitro. The vasoactivity of the human CSF samples and of fractionated (fractions with molecular weight greater than or less than 10 kDa) and unfractionated blood incubated in vitro was assessed by application of samples to canine basilar artery segments under isometric tension. RESULTS: ATP in human CSF declined within 72 hours of SAH to concentrations too low to contract cerebral arteries. Vasoactivity of human CSF correlated with the concentration of hemoglobin. The vasoactivity of incubated erythrocyte hemolysates remained high despite a decline in ATP concentrations. Fractionation of incubated erythrocyte hemolysates showed that for incubation periods up to 7 days, all vasoactivity was in a fraction of molecular weight greater than 10 kDa. CONCLUSION: ATP is unlikely to contribute to vasospasm because the concentrations in CSF after SAH in humans are not high enough to cause vasospasm after 72 hours. The vasoactivity of erythrocyte hemolysate is not related to the ATP or ferrous hemoglobin content but may be related to the total hemoglobin content. Therefore, ATP is unlikely to be a major cause of clinically significant delayed vasospasm.

Hemoglobin increases endothelin-1 in endothelial cells by decreasing nitric oxide.

We determined whether ferrous hemoglobin increases endothelin-1 (ET-1) secretion from bovine cerebral artery endothelial cells and the mechanisms involved. Exposure of endothelial cells to hemoglobin caused dose-dependent increases in pre-proET-1 mRNA and peptide. The increase in ET-1 peptide was inhibited by cycloheximide or actinomycin D whereas only cycloheximide decreased basal ET-1 release. N(G)-nitro-l-arginine significantly increased ET-1 concentration and reduced hemoglobin stimulation of ET-1 release. 8-Bromo-cGMP did not alter basal ET-1 concentration but suppressed hemoglobin-induced ET-1 production. Methemoglobin and S-nitrosylated methemoglobin were less potent inducers of ET-1 release. In summary, hemoglobin increases ET-1 in cerebral endothelial cells by mechanisms that involve transcription and translation. Nitric oxide production inhibits ET-1 production. Ferrous hemoglobin increases ET-1 by binding nitric oxide and abolishing this inhibitory pathway although other mechanisms are involved since N(G)-nitro-l-arginine reduces hemoglobin-induced ET-1 release.

Effects of hemoglobin on heme oxygenase gene expression and viability of cultured smooth muscle cells.

Ferrous Hb contributes to cerebral vasospasm after subarachnoid hemorrhage, although the mechanisms involved are uncertain. The hypothesis that cytotoxic effects of ferrous Hb on smooth muscle cells contribute to vasospasm was assessed. Cultured rat basilar artery smooth muscle cells were exposed to pure Hb, dog erythrocyte hemolysate, or Hb breakdown products; and heme oxygenase (HO-1 and HO-2) and ferritin mRNA and protein were measured. Cytotoxicity was assessed by lactate dehydrogenase release and fluorescence assays. Pure Hb or hemolysate caused dose- and time-dependent increases in HO-1 mRNA and protein. Hemin was the component of Hb that increased HO-1 mRNA. Cycloheximide inhibited the increase in HO-1 mRNA in response to hemin. Ferritin protein heavy chain but not mRNA increased upon exposure of cells to Hb. Hemin and ferric but not ferrous Hb were toxic to smooth muscle cells. Toxicity was increased by exposure to Hb plus tin protoporphyrin IX. In conclusion, exposure of smooth muscle cells to Hb induces HO-1 mRNA and protein through pathways that involve new protein synthesis. Hemin is the component of Hb that induces HO-1. Hemin and ferric but not ferrous Hb are toxic.

Thrombin reduces cerebral arterial contractions caused by cerebrospinal fluid from patients with subarachnoid hemorrhage.

BACKGROUND AND PURPOSE: We observed that the second application of cerebrospinal fluid (CSF) from subarachnoid hemorrhage (SAH) patients onto cerebral arterial segments in vitro produces a greater contraction than does the initial application. It was hypothesized that the difference between the first and second applications of SAH CSF was due to the activity of thrombin. METHODS: Canine vertebrobasilar artery was removed under general anesthesia, cut into rings, and suspended in tissue culture baths so as to measure isometric tension. CSF was taken from patients 1 to 3 days after SAH via ventricular drains. CSF was administered in 10(-5) to 10(-1) dilutions. The thrombin antagonist hirudin (5 U) was administered before CSF in some experiments. The arterial tension response to pure oxyhemoglobin (10(-4) to 3.2 g/dL) and thrombin (10(-4) to 3.2 U/mL), administered alone or in combination, was also examined. RESULTS: Hirudin increased arterial tension generated on the initial application of SAH CSF but had no effect on the tension generated by the second application of the SAH CSF, suggesting that thrombin limits the tension generated by vasoconstrictive agents in the CSF. Thrombin and pure oxyhemoglobin administered together produced less tension than that generated in response to oxyhemoglobin administered alone; no additive response was observed by coadministering the 2 vasoconstrictive agents. CONCLUSIONS: In the presence of oxyhemoglobin, thrombin acts to reduce cerebral arterial tension. This interaction between thrombin and hemoglobin may account for the observation that the second application of CSF from SAH patients onto cerebral arterial segments in vitro produces a greater contraction than does the initial application.

Subarachnoid hemorrhage as a cause of an adaptive response in cerebral arteries.

OBJECT: It is not known whether the factors responsible for vasospasm after subarachnoid hemorrhage (SAH) cause the cerebral arteries to be narrowed independent of the subarachnoid blood clot or whether the continued presence of clot is required for the entire time of vasospasm. The authors undertook the present study to investigate this issue. METHODS: To distinguish between these possibilities, bilateral SAH was induced in monkeys. The diameters of the monkeys' cerebral arteries were measured on angiograms obtained on Days 0 (the day of SAH), 1, 3, 5, 7, and 9. The subarachnoid blood clot was removed surgically on Day 1, 3, or 5 or, in control animals, was not removed until the animals were killed on Day 7 or 9. The concentrations of hemoglobins and adenosine triphosphate (ATP), substances believed to cause vasospasm, were measured in the removed clots and the contractile activity of the clots was measured in monkey basilar arteries in vitro. If the clot was removed 1 or 3 days after placement, vasospasm was significantly diminished 4 days after clot removal. Clot removal on Day 5 had no marked effect on vasospasm. There was a significant decrease over time in hemoglobin and ATP concentrations and in the contractile activity of the clots, although substantial hemoglobin and contractile activity was still present on Day 7. CONCLUSIONS: The authors infer from these results that vasospasm requires the presence of subarachnoid blood for at least 3 days, whereas by Day 5 vasospasm is less dependent on subarachnoid blood clot. Because the clot still contains substantial amounts of hemoglobin and contractile activity after 5 days, there may be an adaptive response in the cerebral arteries that allows them to relax in the presence of the stimulus that earlier caused contraction.

Heme oxygenase-1 and ferritin are increased in cerebral arteries after subarachnoid hemorrhage in monkeys.

Hemoglobin is a key factor in the production of cerebral vasospasm. Metabolism of hemoglobin involves breakdown of heme by heme oxygenase (HO) and sequestration of the released iron in ferritin. We determined whether subarachnoid hemorrhage induces these proteins in cerebral arteries and, if so, in which cells they are produced. Whether the changes correlated with vasospasm also was investigated. Subarachnoid hemorrhage was created in monkeys, and vasospasm was assessed by angiography in cohorts of animals killed 3, 7, or 14 days after the hemorrhage. Ferritin and HO-1 messenger ribonucleic acid (mRNA) and protein were measured by competitive reverse transcription-polymerase chain reaction and Western blotting in hemorrhage-side and control-side cerebral arteries and brain tissue. The location of these proteins was determined by immunohistochemistry. There was significant vasospasm 3 and 7 days but not 14 days after subarachnoid hemorrhage. There were no significant changes in mRNA for HO-1 or ferritin in cerebral arteries or brain tissue at any time. There was a significant increase in HO-1 and ferritin protein in hemorrhage-side compared with control-side cerebral arteries at 3, 7, and 14 days. The increase in HO-1 protein was maximal at 3 days, whereas the increase in ferritin protein was maximal at 7 days. There was no detectable increase in HO-1 or ferritin protein in brain tissue at any time. Immunohistochemistry localized HO-1 protein and ferritin to cells in the adventitia of the arterial wall. We show that subarachnoid hemorrhage is associated with a significant increase in HO-1 and ferritin proteins in cerebral arteries that begins at least as early as 3 days after the hemorrhage and that persists for up to 14 days.

Immediate early gene expression in vascular smooth-muscle cells synergistically induced by hemolysate components.

OBJECT: Vasospasm after subarachnoid hemorrhage is associated with changes in modulators of vascular tone in the arterial wall and is related to the presence of erythrocyte hemolysate in the subarachnoid space. The purpose of this study was to determine the compounds in erythrocyte hemolysate that are responsible for changing smooth-muscle cell gene expression. METHODS: Rat aorta smooth-muscle cells were exposed to erythrocyte hemolysate in vitro and the effects on immediate early gene messenger (m)RNA levels were determined by competitive reverse transcriptase-polymerase chain reaction. Message levels for c-fos, jun B, and c-jun were increased in the presence of hemolysate, reaching maximum expression between 30 and 60 minutes, whereas the level of jun D mRNA was unaffected. Increasing doses of hemolysate caused greater expression of c-fos and jun B, but not c-jun. Adenosine triphosphate and hemoglobin, possible spasmogens present in hemolysate, caused much smaller and more rapid increases in c-fos expression than whole hemolysate. Size fractionation showed that all of the c-fos mRNA-inducing activity of hemolysate was recovered with molecules greater than 6 kD. Following separation of hemolysate proteins by hydrophobic interaction chromatography, only one of the three fractions had partial activity. Recombining the three fractions, however, yielded greater c-fos activation than any combination of two. CONCLUSIONS: Multiple high-molecular-weight components present in erythrocytes have synergistic effects on gene expression in smooth-muscle cells. The differences in patterns of gene induction suggest that multiple signaling pathways are activated.

Role of protein tyrosine phosphorylation in erythrocyte lysate-induced intracellular free calcium concentration elevation in cerebral smooth-muscle cells.

OBJECT: Tyrosine kinases play an important role in the regulation of systemic vascular smooth-muscle tone. The authors studied the involvement of protein tyrosine kinase activity in erythrocyte lysate-mediated signal transduction in cerebral smooth-muscle cells. METHODS: Tyrosine kinase phosphorylation and intracellular free Ca++ ([Ca++]i) were measured in rat aortic and basilar artery smooth-muscle cells by using Western blot and fura 2-acetoxymethyl ester microfluorimetry. Erythrocyte lysate enhanced tyrosine phosphorylation in cultured rat aortic and basilar smooth-muscle cells and induced a rapid transient and a prolonged plateau phase of [Ca++]i response in rat basilar smooth-muscle cells. The tyrosine kinase inhibitors genistein and tyrphostin A51 (administered at concentrations of 30 or 100 microM) attenuated both phases of erythrocyte lysate-induced [Ca++]i elevation. Erythrocyte lysate was separated into low- (<10 kD, which contains adenine nucleotides) and high- (>10 kD, which contains hemoglobin) molecular-weight fractions; these fractions were tested separately in these cells. The low-molecular-weight fraction produced a similar [Ca++]i response to that of erythrocyte lysate and the high-molecular-weight fraction produced a small response. The [Ca++]i responses from both fractions were inhibited by tyrosine kinase inhibitors. CONCLUSIONS: To the authors' knowledge, this is the first report to show that tyrosine phosphorylation may be involved in erythrocyte lysate-induced signal transduction and [Ca++]i responses in cerebral smooth-muscle cells.

Effects of cell-permeant calcium chelators on contractility in monkey basilar artery.

Vasospasm after traumatic or aneurysmal subarachnoid hemorrhage is associated with smooth muscle contraction, a process that results in part from increased intracellular calcium in smooth muscle cells. These experiments tested the hypothesis that chelation of intracellular calcium with the cell-permeant calcium chelator, 1,2-bis-(2-aminophenoxy)ethane-N,N,N',N'-tetracetic acid acetoxymethyl ester (BAPTA-AM), decreases smooth muscle contraction in response to agents that cause contraction by increasing intracellular calcium. Effects of BAPTA-AM on vasoconstriction induced by KCl, prostaglandin F2alpha (PGF2alpha), caffeine, and erythrocyte hemolysate were tested on monkey basilar artery under isometric tension. BAPTA-AM, 30 and 100 micromol/L, caused a significant decrease in resting tension in rings with and without endothelium (30 micromol/L; 8+/-6% [n.s.] and 14+/-5%, 100 micromol/L; 19+/-3% and 32+/-6%,p < 0.05, paired t test). Contractions to caffeine were significantly decreased by 30 micromol/L BAPTA-AM and were abolished at 100 micromol/L in rings with and without endothelium (p < 0.05). BAPTA-AM, 100 micromol/L, competitively inhibited contractions to PGF2alpha. BAPTA-AM, 100 micromol/L, significantly decreased the maximum contractions to KCI in rings with and without endothelium (p < 0.05). There were no significant effects of BAPTA-AM on contractions induced by hemolysate in rings with endothelium but in rings without endothelium, BAPTA-AM, 100 micromol/L, significantly inhibited contractions. In rings with endothelium contractions to hemolysate could be significantly reduced by BAPTA-AM plus indomethacin or indomethacin alone, suggesting that hemolysate releases an eicosanoid from the endothelium by a pathway that is not inhibited by BAPTA. These results suggest that the ability of BAPTA-AM to inhibit smooth muscle contractions will depend on the agonists mediating the contraction. In response to erythrocyte hemolysate, loading of endothelial cells with BAPTA-AM increases the release of a vasoconstricting eicosanoid from these cells that counteracts the decreased contraction caused by loading of smooth muscle cells with BAPTA-AM.

Effects of erythrocyte lysate of different incubation times on intracellular free calcium in rat basilar artery smooth-muscle cells.

OBJECT: The purpose of this study was to characterize substance(s) in the erythrocytes that increase intracellular free Ca++ concentration ([Ca++]i) in smooth-muscle cells and that therefore may be involved in the pathogenesis of vasospasm. METHODS: Because vasospasm occurs days after subarachnoid hemorrhage (SAH), the authors studied the effects of aged human erythrocyte hemolysate and its low-molecular-weight (LMW) and high-molecular-weight (HMW) fractions on [Ca++]i in freshly isolated rat basilar artery smooth-muscle cells. Fresh hemolysate (Day 0) produced a biphasic response consisting of a transient peak and a sustained plateau increase in [Ca++]i, whereas hemolysate prepared from cells incubated for 3, 7, or 14 days induced only a transient response without a sustained phase. The effect of hemolysate declined with increasing incubation time. The HMW fraction and purified human oxyhemoglobin (OxyHb) did not evoke a response. The LMW fraction from Days 3, 7, or 14 produced no response at low concentrations (< 10%) and a transient response at high concentrations (> 20%), and the effect diminished with increasing incubation time. Unfractionated hemolysate or the LMW fraction of hemolysate incubated for 21 days produced no response. The combination of the 10% LMW fraction from Day 3 plus the 10% HMW fraction (Days 3. 7, 14, or 21) transiently increased [Ca++]i,. However, [Ca++]i was not changed by the 10% LMW fraction from Day 14 plus the 10% HMW fraction from Day 3 or 14. In the presence of OxyHb, [Ca++]i was increased by the 10% LMW fraction on Days 3 and 7, but not by the LMW fraction from Days 14 or 21. CONCLUSIONS: The decline over time in the effect of hemolysate on [Ca++]i indicates either that the time that substances are released from erythrocytes is important in the generation of vasospasm or that this experimental system as used is not representative of conditions present after SAH. The data indicate that the ability to elevate [Ca++]i in smooth-muscle cells with hemolysate is provided by multiple substances, including OxyHb. These substances may interact during specific times after incubation of erythrocytes in vitro.

U74389G prevents vasospasm after subarachnoid hemorrhage in dogs.

OBJECTIVE: Oxygen-derived free radicals may contribute to vasospasm after the rupture of an intracranial aneurysm through direct vasoconstricting effects occurring within the arterial wall or, secondarily, by causing lipid peroxidation in the subarachnoid erythrocytes with secondary induction of vasoconstriction. U74389G is a potent inhibitor of lipid peroxidation and a scavenger of oxygen-derived free radicals. This study determined the relative contributions of oxygen-derived free radicals and lipid peroxidation to vasospasm in the double-hemorrhage dog model. METHODS: Sixteen dogs underwent baseline (Day 0) cerebral angiography and induction of subarachnoid hemorrhage by two injections of blood into the cisterna magna 2 days apart. They were randomized to receive drug vehicle (n=8) or U74389G (n=8, 3 mg/kg of body weight/d) intravenously. Drug administration and end point analysis were blinded. The end points were angiographic vasospasm, as assessed by comparison of angiograms obtained before and 7 days after subarachnoid hemorrhage, and the levels of malondialdehyde and salicylate hydroxylation products (dihydroxybenzoic acids) in cerebrospinal fluid and of malondialdehyde in subarachnoid blood clots and basilar arteries 7 days after hemorrhage. RESULTS: Comparisons within groups of Day 0 and Day 7 angiograms and between groups of angiograms obtained at Day 7, showed significant vasospasm in animals in the vehicle group (mean+/-standard error, 51%+/-4) but not in the U74389G group (25%+/-11, P < 0.05, unpaired t test). High-pressure liquid chromatographic assays of malondialdehyde and dihydroxybenzoic acids in cerebrospinal fluid, subarachnoid blood clots, and basilar arteries showed no significant differences between groups. CONCLUSION: The significant prevention of vasospasm by U74389G without change in levels of indicators of free radical reactions suggests that the effect of the drug is related to other processes occurring in the arterial wall and that cerebrospinal fluid levels of oxygen radicals and lipid peroxides are not useful markers of vasospasm.

Adenosine triphosphate causes vasospasm of the rat femoral artery.

OBJECTIVE: Adenosine 5'-triphosphate (ATP) causes vasoconstriction by activation of P2-purinoceptors on vascular smooth muscle cells. Erythrocytes contain ATP at a concentration (1.6 mmol/L) that contracts smooth muscle. Previous studies of hemoglobin solutions did not assess whether the vasoactivity was caused by ATP rather than or in addition to hemoglobin. It was hypothesized that the hemolysis of erythrocytes that occurs after subarachnoid hemorrhage releases ATP in concentrations that cause vasospasm. METHODS: Thirty-eight rats were randomly assigned to undergo placement of one of the following compounds in a silastic elastomer cuff around each femoral artery: 1) agarose gel (n = 8); 2) dog erythrocyte hemolysate (n = 8); 3) purified human hemoglobin (Hemolink; Hemosol, Inc., Toronto, Canada; n = 8); 4) ATP (n = 8); or 5) clotted autologous blood (n = 6). The amounts of hemoglobins and adenine nucleotides in the compounds were measured by spectrophotometry and high pressure liquid chromatography. Hemolysate, purified hemoglobin, and ATP were mixed with agarose gel to create an artificial clot. Rats were killed and fixed by perfusion at physiological blood pressure 7 days after perivascular cuff and spasmogen placement. Vasospasm was assessed by image analysis of cross sections of fixed femoral arteries. Arteries were assessed for histopathological changes on 3-point scales. RESULTS: There was significant variance in arterial diameters among groups (mean diameter +/- standard deviation: agarose gel, 0.29 +/- 0.06; purified hemoglobin, 0.28 +/- 0.04; hemolysate, 0.24 +/- 0.05; ATP, 0.25 +/- 0.05; clotted blood, 0.24 +/- 0.01; P < 0.05, analysis of variance, n = 11-20). Animals exposed to clotted blood, hemolysate that contained ATP, or ATP, developed vasospasm, whereas purified hemoglobin and agarose did not cause vasospasm. Endothelial proliferation and perivascular inflammation were more severe (P < 0.05) in arteries exposed to clotted blood, purified hemoglobin, and hemolysate. CONCLUSION: These results suggest that ATP may be a vasospastic substance released by erythrocyte hemolysis. The concentration of ATP in impure solutions of hemoglobin is too low to account for the vasoactivity of these solutions. The discrepancy between arterial narrowing and histopathological changes suggests that either histopathological changes may not be an important correlate of arterial vasospasm or that other substances are important in vasospasm.

Expression of the oligodendrocyte-myelin glycoprotein by neurons in the mouse central nervous system.

The oligodendrocyte-myelin glycoprotein (OMgp) is a 110-kDa glycosylphosphatidylinositol-linked protein that was initially identified as a myelin-specific protein but whose precise function remains unknown. In this study, immunohistochemistry, western blots, in situ hybridization, and northern blots were used to determine the distribution of OMgp in the mouse brain. OMgp is present in a concentration detectable on western blots in the brains of newborn mice, and its concentration gradually increases until day 24 of life. OMgp mRNA is also present in amounts detectable on northern blots in the brains of newborn mice, and its concentration gradually increases until day 21 of life, after which the concentration diminishes a little. Most of the OMgp in the mouse brain appears to be expressed in diverse groups of neurons, but it is particularly prominent in large projection neurons such as the pyramidal cells of the hippocampus, the Purkinje cells of the cerebellum, motoneurons in the brainstem, and anterior horn cells of the spinal cord. However, OMgp is not confined to these cells and is expressed in cells in the white matter as well. The OMgp gene is placed within an intron of the neurofibromatosis type I gene and on the opposite strand. This organization raises the possibility that there may be a relationship between the functions of the products of the two genes. In support of this possibility, we show that within the mouse CNS OMgp and neurofibromin are expressed in the same cell types.

Prevention of vasospasm after subarachnoid hemorrhage in dogs by continuous intravenous infusion of PD156707.

This randomized, blinded study tested the prophylactic effect of PD156707, a nonpeptide competitive antagonist of endothelin A receptors, against vasospasm after subarachnoid hemorrhage in dogs. Twenty-two dogs were allocated on day 0 to undergo cerebral angiography followed by injection of arterial blood (0.5 ml/kg) into the cisterna magna. Dogs had central venous catheters implanted for continuous infusion of drug vehicle (n = 10) or PD156707 (n = 12). Cisternal blood injection was repeated on day 2. Drug levels were measured in plasma on days 2, 4, 6, and 7 and in cerebrospinal fluid (CSF) on days 2 and 7. Angiography was repeated on day 7 to assess vasospasm. After angiography on day 7, acute effects of infusion of PD156707, 100 mg, or drug vehicle on established vasospasm were assessed. Analysis of physiological variables within (analysis of variance) groups across time and between (unpaired t-test) groups at each time showed that drug-treated animals had significantly increased heart rate on day 7 compared to day 0 (p < 0.005). Comparison of basilar artery diameters at day 7 showed that PD156707 significantly decreased the degree of basilar artery vasospasm (placebo: -47 +/- 5% reduction [mean +/- SE] versus PD156707: -28 +/- 7%, p < 0.05, unpaired t-test). There was, however, significant vasospasm when comparing within groups (paired t-test, placebo: p < 0.0001, PD156707: p < 0.005). Mean plasma PD156707 levels (322 +/- 123 ng/ml) were adequate to block responses of endothelin-1 on endothelin A receptors in vitro although CSF levels (11 +/- 7 ng/ml) were substantially lower. Infusion of PD156707 into the basilar artery on day 7 caused a small but significant 10 +/- 3% (paired t-test, p < 0.01) increase in diameter compared to placebo (3 +/- 3% increase, p = 0.32). This infusion also was associated with a substantial increase in CSF drug levels to 19 +/- 9 mg/ml. These results suggest that endothelin A receptors mediate some of the vasospasm that occurs after SAH in dogs and that blockade of these receptors may be a beneficial treatment for vasospasm.