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.

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.

Multimedia computer database for neurosurgery.

OBJECTIVE: There is a need for an efficient mechanism of storing and analyzing neurosurgical clinical, imaging, and operative data to facilitate clinical audit, research, education, and preparation of scientific presentations. METHODS: A computer database was developed to meet this need. The recorded data include diagnoses, digitized neuroimaging studies, operative details (with intraoperative video clips), transcranial Doppler studies, outcomes, complications, admissions, and clinic visits. The anatomy, pathology, and clinical presentation are recorded for each diagnosis. RESULTS: The database provides an audit of neurosurgery cases, which includes admission Glasgow Coma Scale score, length of intensive care and hospital stays, Glasgow Outcome Scale score, and complications. Clinical research is facilitated by flexible search strategies based on the anatomy, pathology, or clinical presentation of diseases, or any of the recorded intraoperative or outcome factors. The system can be used to assess the influence on outcome of factors, such as transcranial Doppler velocity, intraoperative blood pressure, and the use of ventricular drainage, intraoperative angiography, or temporary clipping. The database can be used to track patients with untreated or partially treated conditions, such as incidental or incompletely coiled aneurysms. The recorded images and video clips are used for teaching and producing multimedia presentations and reports. The database is designed to enable secure Internet connections among institutions so that outcomes and complications can be compared among surgeons and institutions. CONCLUSION: This multimedia computer database facilitates clinical audit, research, teaching, and presentation activities.

Effects of posterior fossa decompression with and without duraplasty on Chiari malformation-associated hydromyelia.

OBJECTIVE: The optimal surgical treatment of Chiari malformation is unclear, especially in patients with hydromyelia. Various surgical approaches have included suboccipital craniectomy, syringostomy, obex plugging, syringosubarachnoid shunting, and fourth ventriculosubarachnoid shunting. The purpose of this study is to differentiate extradural and intradural approaches in the treatment of Chiari I malformation. METHODS: We reviewed the medical records and magnetic resonance imaging (MRI) scans of 34 surgical corrections' of Chiari malformation performed at our institution from 1988 to 1998. The age and sex of the patient, the presence of hydromyelia, the type of surgery (duraplasty or nonduraplasty), and the clinical outcome were determined. RESULTS: Eleven patients underwent posterior fossa decompression (PFD) and C1 laminectomy without duraplasty. Eight (73%) of these patients had an improvement in symptoms. Seven of the 11 patients had hydromyelia. Of the six patients who underwent follow-up MRI, three (50%) had a decrease in the size of the hydromyelia, and all three had clinical improvement. We also noted a morphometric increase in posterior fossa volume on postoperative MRI scans in these three patients, which was not observed in those without improvement. Two of the three patients whose hydromyelia did not decrease on follow-up MRI scans worsened clinically, and one underwent a reoperation with duraplasty. Twenty-three patients underwent combined PFD, C1 laminectomy, and duraplasty. Twenty (87%) of these patients had improvement. Twelve of the patients who underwent duraplasty had hydromyelia; nine underwent follow-up MRI. All nine of these patients (100%) had a decrease in the cavity size, including eight with clinical improvement. There were 10 minor complications (seroma, 4; superficial infection, 3; cerebrospinal fluid leak, 2; aseptic meningitis and occipital nerve pain, 1) when the dura was opened, compared with one superficial wound infection that resolved in patients who underwent PFD only. CONCLUSION: PFD, C1 laminectomy, and duraplasty for the treatment of Chiari I malformation may lead to a more reliable reduction in the volume of concomitant hydromyelia, compared with PFD and C1 laminectomy alone. However, there seems to be a subset of patients whose symptoms will resolve and whose hydromyelic cavity will decrease with the removal of bone only. These patients seem to undergo a volumetric increase in the posterior fossa. Further studies are needed to better characterize these patients, to determine which patients with Chiari I malformation are better served with bony decompression only, and which will require duraplasty to resolve their hydromyelia.

Intraoperative angiography of brain arteriovenous malformations.

OBJECTIVE: The gold standard for documentation of surgical cure of a brain arteriovenous malformation (AVM) is a postoperative angiogram. Intraoperative angiography also has been used for assessing surgical obliteration of AVMs. The objective of this work is to determine the incidence of unexpected residual AVM in patients undergoing intraoperative angiography after brain AVM surgery, the incidence of false-negative intraoperative angiography, and whether there are any identifiable factors that would predict such an occurrence. METHODS: Patient age and sex, AVM location and size, clinical presentation of the AVM, day of surgery after hemorrhage, whether embolization was performed preoperatively, presence of intraoperative brain swelling or substantial bleeding, and postoperative course were recorded prospectively on 34 consecutive patients who underwent surgery for brain AVMs. Intraoperative angiography was performed after the surgeon thought that the AVM was completely obliterated. The incidence of unexpected residual AVM and false-negative intraoperative angiography was determined. Factors predicting these findings were identified by multivariate analysis. RESULTS: Twenty-five of 34 patients underwent intraoperative angiography to assess the extent of resection, and two patients underwent the examination to localize the AVM. Postoperative angiograms were obtained for 26 patients. Intraoperative angiography showed unexpected residual AVM in 2 (8%) of 25 patients. In two patients, intraoperative angiography was useful to locate a small AVM in the wall of a hematoma cavity. Three patients (18%) whose intraoperative angiograms had not shown AVM had postoperative angiograms that showed residual or recurrent AVM. One (11%) of nine patients who had only postoperative angiography had an unexpected residual nidus; the patient underwent a reoperation and successful resection. There were no significant clinical or radiological features that predicted the intraoperative angiographic finding of residual AVM or of false-negative intraoperative angiogram. CONCLUSION: Intraoperative angiography is useful to demonstrate residual AVM in about 8% of patients undergoing AVM resection. It can be used to localize small AVMs, but other methods for localization may be as useful and may avoid the risks and cost of additional angiography. Intraoperative angiography does not replace postoperative angiography to confirm AVM removal because of false-negative findings, which occurred in 18% of patients in this series.

Safety of perioperative subcutaneous heparin for prophylaxis of venous thromboembolism in patients undergoing craniotomy.

OBJECTIVE: To determine whether perioperative subcutaneous heparin is safe to use for patients undergoing craniotomy and to determine the incidence of venous thromboembolism in patients undergoing craniotomy. METHODS: Perioperative prophylaxis with subcutaneous heparin, 5000 U every 12 hours, was begun at induction of anesthesia for craniotomy and continued for 7 days postoperatively or until the patient was ambulating. Entry criteria to the study included patient age over 18 years and no evidence of deep vein thrombosis (DVT) preoperatively as judged by lower limb duplex ultrasound. Patients were excluded if they had duplex evidence of DVT or clinical evidence of pulmonary embolus (PE) preoperatively, had hypersensitivity to heparin or related products, had sustained a penetrating head injury, or refused informed consent. Any patient undergoing craniotomy was eligible, including patients with a ruptured aneurysm or arteriovenous malformation and those with spontaneous intracranial hemorrhage. Patients underwent duplex study 1 week after surgery and 1 month of clinical follow-up. Records were also kept on 68 nonstudy patients who refused consent. All patients were treated with lower limb pneumatic compression devices. RESULTS: One hundred six patients were treated. No differences were noted between study and nonstudy patients in some individual risk factors for DVT or PE, such as obesity, smoking, paralysis, infection, pregnancy or postpartum state, varicose veins, heart failure, or previous DVT or PE. Significantly more (43 of 106) patients in the study group had a history of risk factors for DVT or PE, particularly malignancy, however, compared with nonstudy patients (20 of 68 patients; chi2, P < 0.01). There were no differences between groups in intraoperative blood loss, transfusion requirements, or postoperative platelet counts. Four clinically significant hemorrhages occurred during surgery in patients receiving heparin. Three resulted from intraoperative aneurysm rupture and one from intraventricular bleeding during resection of an arteriovenous malformation. These events were believed to be related to known complications of these operations, not to heparin. Of the study patients, two developed symptomatic DVT and one developed a nonfatal PE during the 1-month postoperative period. One additional study patient developed DVT below the popliteal veins, which was not treated. Four study patients developed DVT 1 to 2 months after surgery. In nonstudy patients, three developed DVT and two developed PE (one fatal, one nonfatal). CONCLUSION: Perioperative heparin may be safe to administer to patients undergoing craniotomy, but a larger study is needed to demonstrate efficacy.

Cerebrovascular contractility after clot removal in monkey subarachnoid haemorrhage.

1. The effects of mechanical clot removal during early surgery on pharmacological cerebrovascular reactivity after subarachnoid haemorrhage (SAH) were investigated in the monkey. 2. Contractions to potassium chloride, 5-hydroxytryptamine and noradrenaline in rings of proximal parts of middle cerebral arteries (MCP), surrounded with clot, and basilar arteries (BAP), far from the clot, were examined 7 days after SAH, in which an autologous blood clot was bilaterally placed around major cerebral arteries. 3. Compared with the sham-operated group, contractions in the clot removal groups at 48 and 72 h after SAH were reduced in MCP and enhanced in BAP. 4. These results suggest that divergent vascular contractility may occur according to the distance between artery and clot if the clot is removed later than 48 h after SAH.

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.

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.

Cigarette smoking as a cause of aneurysmal subarachnoid hemorrhage and risk for vasospasm: a report of the Cooperative Aneurysm Study.

OBJECT: Cigarette smoking is associated with aneurysmal subarachnoid hemorrhage (SAH) and subsequent vasospasm. The purpose of this study was to quantify this association. METHODS: Nearly 3500 patients with SAH from North America and Europe have been enrolled in five different multicenter, controlled studies coordinated at the Neuroclinical Trials Center of the Virginia Neurological Institute at the University of Virginia. Among the prospective data gathered were whether the patient smoked at the time of their most recent SAH and the evolution of angiographic vasospasm. The rate of smoking in the patients enrolled in the studies was compared with the expected rate by using a chi-square statistic adjusted for age and gender, in the general population in the United States (U.S.) and Europe. In virtually all age and gender subgroups, and for the combined populations in the five clinical trials, patients with SAH reported current smoking rates 2.5 times higher than expected based on U.S. and European national surveys (p < 0.0001). Cigarette smoking was also associated with younger age at onset of SAH (5-10 years, p < 0.0001) and increased incidence of clinically confirmed vasospasm (p < 0.005). CONCLUSIONS: The findings of a significantly increased representation of current cigarette smokers in the study populations and significant association with younger age at the time of SAH and increased incidence of vasospasm concur with recent reports of smoking as a significant risk factor for ruptured aneurysms and subsequent vasospasm.

Surgical treatment of middle cerebral artery aneurysms.

Middle cerebral artery (MCA) aneurysms account for approximately 20% of aneurysmal subarachnoid hemorrhage (SAH). An understanding of MCA anatomy and variations is the key to successful treatment of MCA aneurysms, which may occur on the M1 segment, at the division of the main trunk or on distal branches. Patients may present with SAH, mass effect, epilepsy, or ischemia. MCA aneurysms associated with large temporal hematomas may be approached through the superior temporal gyrus. Other aneurysms are best approached with a medial or lateral transSylvian approach.

Pregnancy and intracranial aneurysms.

Aneurysmal subarachnoid hemorrhage during pregnancy is rare but is an important cause of maternal mortality. Physiologic changes in pregnancy may predispose to aneurysm formation and rupture. In general, neurosurgical considerations take precedence over obstetric considerations. Ruptured aneurysms should be treated as they would be in patients who are not pregnant. Unruptured aneurysms should be treated if they are symptomatic or enlarging. Other aneurysms should be treated on an individual basis.

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.

Erythrocyte lysate releases Ca2+ from IP3-sensitive stores and activates Ca(2+)-dependent K+ channels in rat basilar smooth muscle cells.

Erythrocyte lysate increases intracellular Ca2+ ([Ca2+]i), contracts cerebral arteries and has been suggested to be the causative agent for cerebral vasospasm. However, the mechanism of erythrocyte lysate-induced [Ca2+]i mobilization is not clear. This study was undertaken to investigate the action of erythrocyte lysate on [Ca2+]i mobilization by monitoring [Ca2+]i and the Ca(2+)-dependent K+ channels (KCa) in freshly isolated rat basilar artery smooth muscle cells. In a [Ca2+]i imaging study, erythrocyte lysate produced a biphasic response, a transient peak and a prolonged plateau [Ca2+]i elevation. In the absence of external Ca2+, erythrocyte lysate induced only a transient peak [Ca2+]i response without a marked plateau phase, indicating the [Ca2+]i was Ca2+ released from internal stores. Erythrocyte lysate-induced plateau [Ca2+]i response was resistant to nicardipine, a voltage-dependent Ca2+ channel blocker, but was abolished by EGTA. Elevation of [Ca2+]i induced by erythrocyte lysate contracted smooth muscle cells. In the electrophysiological study, elevation of [Ca2+]i by erythrocyte lysate increased KCa currents in whole-cell patch-clamp configuration. This effect of erythrocyte lysate on KCa was blocked by heparin, an antagonist of IP3 receptors. We conclude that erythrocyte lysate releases Ca2+ from IP3-sensitive intracellular stores and produces Ca2+ entry from voltage-independent Ca2+ pathways.

Quantitative imaging study of extent of surgical resection and prognosis of malignant astrocytomas.

OBJECTIVE: This study used quantitative radiological imaging to determine the effect of surgical resection on postoperative survival of patients with malignant astrocytomas. Previous studies relied on the surgeons' impressions of the amount of tumor removed, which is a less reliable measure of the extent of resection. METHODS: Information concerning possible prognostic factors was collected for 75 patients undergoing magnetic resonance imaging or computed tomography preoperatively and within 10 days postoperatively. Image analysis of the neuroradiological studies was conducted to quantify pre- and postoperative total tumor volumes and enhancing volumes. Univariate and multivariate proportional hazards models were used to analyze the regression of survival regarding 22 covariates that might affect survival. The covariates that were entered included age, gender, tumor grade, cumulative radiation dose, chemotherapy, seizures as a first symptom, Karnofsky performance status at presentation, pre- and postoperative total and enhancing tumor volumes, ratio of pre- to postoperative total and enhancing tumor volumes, tumor location, surgeon's impression of the degree of resection, and subsequent surgery. RESULTS: There were 23 patients with anaplastic astrocytomas and 52 with glioblastomas multiforme. The estimated mean survival time was 27 months for patients undergoing gross total resection, 33 months for subtotal resection, and 13 months for open or stereotactic biopsy. Five factors that were significant predictors of survival in multivariate analysis were tumor grade, age, Karnofsky performance status, radiation dose, and postoperative complications (P < 0.05). In univariate analysis, tumor grade, radiation dose, age, Karnofsky status, complications, presence of enhancing tumor in postoperative imaging, and postoperative volume of enhancing tumor were significantly associated with survival (P < 0.05). CONCLUSION: We conclude that the most important prognostic factors affecting survival of patients with anaplastic astrocytomas and glioblastomas multiforme are tumor grade, age, preoperative performance status, and radiation therapy. Postoperative complications adversely affect survival. Aggressive surgical resection did not impart a significant increase in survival time. Surgical resection may improve survival, but its importance is less than that of other factors and may be demonstrable only by larger studies.

Extracellular nucleotide-induced [Ca2+]i elevation in rat basilar smooth muscle cells.

BACKGROUND AND PURPOSE: Extracellular nucleotides play an important role in the regulation of vascular tone and may be involved in cerebral vasospasm after subarachnoid hemorrhage. The objective of this study was to investigate the receptor subtypes for nucleotides and their mechanisms of [Ca2+]i mobilization in cerebral vasculature. METHODS: Rat basilar smooth muscle cells were isolated by an enzymatic method. [Ca2+]i response, a large transient peak followed by a slowly decaying plateau. The potency of nucleotides to raise [Ca2+]i was ATP gamma S > or = UDP > or = ATP approximately UDP approximately TTP, indicating that P2u receptors were expressed in the rat basilar smooth muscle cells. The effect of UTP to release Ca2+ from internal stores was reduced by pertussis toxin, by the phospholipase C inhibitor 2-nitro-4-carboxyphenyl N,N-diphenylcarbamate, and by the Ca(2+)-pump inhibitor thapsigargin. The Ca2+ entry induced by UTP was partially attenuated by the receptor-operated Ca2+ channel blocker SK&F96365 and by the voltage-dependent Ca2+ channel blocker verapamil. P2 receptor antagonists suramin and, at higher concentrations, pyridoxal-phosphate-6-azophenyl-2',4'-disulphonic acid reduced the effect of UTP. CONCLUSIONS: The results are the first demonstration that nucleotides activate G protein-coupled P2u receptors to mobilize [Ca2+]i in rat basilar smooth muscle cells.

P2 purinoceptors in cultured bovine middle cerebral artery endothelial cells.

Extracellular adenosine triphosphate (ATP) plays an important role in the regulation of endothelial function. However, its receptors and their signal-transduction pathways in major cerebral arterial endothelial cells are largely unknown. This study was undertaken functionally to classify the P2 purinoceptors in cultured bovine middle cerebral artery endothelial cells by using [Ca2+]i microfluorimetry. The rank order of potency to increase [Ca2+]i was 2-methylthio-ATP approximately ATP approximately uridine triphosphate (UTP) > adenosine diphosphate (ADP) >> adenosine monophosphate (AMP) > alpha,beta-methylene-ATP > adenosine, suggesting that the effect was mediated by both P2y and P2u receptors. ATP, 2-methylthio-ATP, and UTP mobilized Ca2+ from intracellular stores and triggered Ca2+ entry. The effects of ATP, 2-methylthio-ATP, and UTP were reduced by phospholipase C inhibitor 2-nitro-4-carboxyphenyl-N,N-diphenylcarbamate (NCDC), but only the effects of ATP and UTP were attenuated by pertussis toxin, indicating that P2y and P2u receptors may activate the same effector mechanisms by coupling to different G proteins. The [Ca2+]i entry caused by UTP was significantly reduced by the receptor-regulated Ca2+ channel blocker SK&F 96365, by P-450 inhibitor econazole and by inorganic Ca2+ entry blocker lanthanum. P2-receptor antagonists suramin, pyridoxal-phosphate-6-azophenyl-2',4'-disulfonic acid (PPADS), and reactive blue 2 reduced the effects of ATP and 2-methylthio-ATP, but not those of UTP, in a concentration-dependent manner. These studies suggest a coexistence of P2y and P2u receptors in cultured bovine middle cerebral artery endothelial cells.