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(Stroke. 1997;28:2053-2059.)
© 1997 American Heart Association, Inc.

Articles

Extracellular Nucleotide–Induced [Ca²⁺]_i Elevation in Rat Basilar Smooth Muscle Cells

Bogdan Sima, BA; Bryce K. A. Weir, MD; R. Loch Macdonald, MD, PhD; He Zhang, MD, PhD

From the Section of Neurosurgery, Department of Surgery, University of Chicago (Ill).

Correspondence to H. Zhang, MD, PhD, Deborah Research Institute, 20 Pine Mill Rd, Brown Mills, NJ 08015-1799. E-mail jzhang{at}cybernet.net

	Abstract

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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 mobilization in freshly isolated cells was monitored using fura 2 microfluorimetry.

Result Extracellular nucleotides produced a concentration-dependent biphasic [Ca²⁺]_i response, a large transient peak followed by a slowly decaying plateau. The potency of nucleotides to raise [Ca²⁺]_i was ATPSUDPATPUDPTTP, indicating that P_2u 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 Ca²⁺-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 P_2u receptors to mobilize [Ca²⁺]_i in rat basilar smooth muscle cells.

Key Words: rats • nucleotides • muscle, smooth

	Introduction

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Cerebral vasospasm is a major cause of mortality and morbidity after SAH.^{1 2} Cerebral vasospasm is characterized by a persistent narrowing of the major cerebral arteries that is resistant to most vasodilators.^{3 4} Even though significant advances have been made, especially in the clinical management of vasospasm, the pathogenesis or etiology of vasospasm remains unclarified. Oxyhemoglobin and some smaller molecules both have been proposed as the causes for vasospasm.^{5 6} We have suggested that adenine nucleotides released from blood clots after SAH may be involved in the pathogenesis of cerebral vasospasm.^{7 8}

There is a widespread appreciation for the potent and diverse interactions of extracellular nucleotides with various body systems. Extracellular nucleotides influence neurotransmission, inflammatory and immune responses, platelet aggregation and secretion, and pulmonary and cardiac functions.⁹ Extracellular nucleotides play an important role in the regulation of vascular tone, including cerebral arteries,^{7 8} by activation of P₂ receptors. Subtypes of P₂ receptors have been classified by numerous studies in peripheral arteries⁹ ; however, little information is available for P₂ receptor subtypes and their Ca²⁺ mobilization pathways in major cerebral smooth muscle cells. Using [Ca²⁺]_i microfluorimetry, we found that P_2u receptors mediated the effect of nucleotides on [Ca²⁺]_i in rat basilar smooth muscle cells.

	Materials and Methods

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Cell Isolation
Rat basilar smooth muscle cells were isolated as previously described and the protocols approved by the Animal Care and Use Committee of the University of Chicago.⁷ Briefly, Sprague-Dawley female rats were anesthetized with Metofane and decapitated. The basilar arteries were immediately removed to a medium consisting of (in mmol/L except as noted): 130 NaCl, 5 KCl, 0.8 CaCl₂, 1.3 MgCl₂, 5 glucose, 10 HEPES, penicillin (100 units/mL), and streptomycin (0.1 g/L). Subsequently, the arteries were cleaned of connective tissue and small side branches, then cut into 0.2-mm rings and incubated at room temperature in a medium containing 0.2 mmol/L CaCl₂, collagenase (type II, 0.5 g/L), elastase (0.5 g/L), hyaluronidase (type IV-S 0.5 g/L), and deoxyribonuclease I (0.1 g/L). After 1 hour the rings were triturated gently, plated on glass coverslips, and stored at 4°C for 2 hours for recovery in the buffer described above containing 0.8 mmol/L CaCl₂, essentially fatty acid–free bovine serum albumin (2 g/L), and trypsin inhibitor (0.5 g/L).

[Ca²⁺]_i Microfluorimetry
The buffer solution for [Ca²⁺]_i measurement was (in mmol/L): 145 NaCl, 2 CaCl₂, 3 KCl, 1 MgCl₂, 10 HEPES, and 10 glucose, and the pH was adjusted to 7.4 with NaOH. Cells were loaded with the fluorescence indicator fura 2-AM (3 µmol/L) for 30 minutes at room temperature in the extracellular buffer solution.^{7 10} After loading, cells were rinsed and the coverslips were then placed in the bottom of a Plexiglas perfusion chamber (volume approximately 600 µL) with two openings at each end for perfusion and aspiration. Cells were perfused for 20 minutes before the experiment to allow deesterification of the dye.

Digital [Ca²⁺]_i imaging was performed by video microfluorimetry using an intensified charge-coupled device (CCD) camera (Hamamatsu) coupled to a Nikon Diaphot microscope (40x Fluor objective, Nikon Inc) and software (Universal Imaging Corp) on a 486 personal computer. Sample illumination was supplied by a 150 W xenon arc lamp, and excitation wavelengths were selected by computer control of a filter wheel. Fluorescence imaging was obtained with alternating excitation wavelengths of 340 and 380 nm, and an emission wavelength of 510 nm through the CCD camera. Typically 4 to 8 frames at each wavelength were averaged to produce ratio images. Data from regions of interest were displayed in real time and logged to hard disk. Background fluorescence obtained from a cell-free position of the same slip was subtracted from all recordings before calculation of the 340/380 ratio. EGTA (0.5 mmol/L) was included in all experiments using a Ca²⁺-free extracellular buffer.

Subsequent analysis converted the ratio to the [Ca²⁺]_i according to the following calibration method. Standardized chelated Ca²⁺-free and high-Ca²⁺ buffers (Molecular Probes) were mixed in 10 varying proportions, with 2 µmol/Lfura 2 salt added, covering the range of Ca²⁺ from 7.9 to 2860 nmol/L. Ratio values for 340 nm to 380 nm fluorescence were determined from droplets of these standard mixtures of known Ca²⁺, with the objective focused approximately 20 µm above the glass coverslip and with background from a fura 2–free solution. The relationship of the Ca²⁺ to the 340 nm–to–380 nm ratio was fitted by an iterative regression to the predicted relationship for fura 2¹¹ to determine R_min, R_max, and the apparent K_d. These values were then used to convert ratio data to approximate [Ca²⁺]_i values.^{7 10}

Reagents
Fura 2-acetoxymethyl ester (fura 2-AM) was purchased from Molecular Probes; SK&F96365, suramin sodium salt, and thapsigargin from Calbiochem; PPADS from Research Biochemicals; and ATP, ADP, AMP, adenosine, UTP, UDP, UMP, uridine, GTP, TTP, ITP, CTP, ATPS, NCDC, pertussis toxin, cell isolation enzymes, and all other chemicals from Sigma Chemical Co.

Data Analysis
Data are expressed as mean±SD. Statistical differences between the control groups and other groups were compared using one-way ANOVA and then Tukey-Kramer multiple comparisons procedure (95% lower and upper confidence interval) if significant variance was found. A value of P<.05 was considered statistically significant. In all figures drug exposures and solution changes are indicated by the horizontal lines.

	Results

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We noticed in our preliminary experiments that the [Ca²⁺]_i response began to "rundown" after about 30 minutes, so experiments reported here were conducted within 30 minutes. To avoid possible interactions among the agonists tested, only one dose of one agonist was tested in each experiment.

General Effects of Nucleotides
Application of ATP, UTP, TTP (100 nmol/L to 1 mmol/L), and at higher concentrations CTP, GTP, or ITP (0.1 to 1 mmol/L), produced a transient [Ca²⁺]_i peak followed by a steady-state [Ca²⁺]_i plateau that decreased slowly to a level slightly above the resting level after 20-minute application in rat basilar smooth muscle cells (examples are shown in Fig 2A and B of 5-minute application of UTP). In Ca²⁺-free buffer solution, ATP, UTP, and TTP produced a transient [Ca²⁺]_i peak that returned to the baseline rapidly without a plateau phase, which suggests that the [Ca²⁺]_i peak was Ca²⁺ released from intracellular stores and that the [Ca²⁺]_i plateau was Ca²⁺ that entered from the extracellular space (see Fig 3A). The effect of these nucleotides on [Ca²⁺]_i was dose-dependent, completely reversible by washout, and repeatable.

View larger version (26K): [in this window] [in a new window]   Figure 2. Effect of pertussis toxin (PTX) and NCDC on [Ca2+]i responses to UTP. A, left, UTP (100 µmol/L) produced a biphasic [Ca2+]i elevation in the presence of extracellular Ca2+ in a rat basilar smooth muscle cell. Right, UTP (100 µmol/L) produced a smaller [Ca2+]i response in a cell pretreated with pertussis toxin (1200 ng/mL) for 10 hours (right). B, left, UTP (100 µmol/L) produced a biphasic [Ca2+]i elevation in the presence of extracellular Ca2+ in a rat basilar smooth muscle cell. Right, NCDC (10 µmol/L), preincubated for 10 minutes, abolished the [Ca2+]i response to UTP (100 µmol/L). C, Summary of the inhibitory effect of pertussis toxin and NCDC. Increased [Ca2+]I indicates the net value of increased [Ca2+]i (elevated [Ca2+]i–resting level). *, **, and *** indicate P<.05, P<.01, and P<.001, respectively (ANOVA).

View larger version (24K): [in this window] [in a new window]   Figure 3. Effects of thapsigargin (TSG) on [Ca2+]i responses to UTP. A, left, UTP (100 µmol/L) produced a transient [Ca2+]i response in the absence of extracellular Ca2+. Right, Thapsigargin (100 nmol/L) produced a transient [Ca2+]i response in the absence of extracellular Ca2+. UTP (100 µmol/L) failed to produce a further [Ca2+]i response in the presence of thapsigargin. B, Summary of the effects of thapsigargin on [Ca2+]i responses to UTP. UTP produced a smaller, but not more significant, [Ca2+]i peak in the absence of extracellular Ca2+ than that seen in the presence of extracellular Ca2+. Thapsigargin abolished the [Ca2+]i peak response to UTP. ** and *** indicate P<.01 and P<.001, respectively (compared with control responses to UTP; ANOVA).

Effects of Different Nucleotides
We tested the effect of different nucleotides to raise [Ca²⁺]_i in cells. Each dose of each nucleotides was tested separately as described above, and the peak and plateau [Ca²⁺]_i responses to nucleotides both were used for the calculation. The maximum responses to ATPS, UTP, UDP, ATP, TTP, and ITP were obtained, and the ED₅₀ values for these nucleotides were calculated from nonlinear regression. The –log₁₀ ED₅₀ values were ATPS 6.5, UTP 6.1, UDP 5.6, ATP 5.8, TTP 5.4, and ITP 4.6, respectively. Since the responses to CTP and GTP failed to reach the maximum and since UMP and uridine failed to produce marked effects, their ED₅₀ values were not calculated. Fig 1A and B shows the dose-dependent elevation of [Ca²⁺]_i to different nucleotides. The potency of nucleotides to raise [Ca²⁺]_i was UTP (selective agonist for P_2u receptors) ATP (selective agonist for P₂ receptors) TTP (Fig 1A, peak responses; 1B, plateau responses). GTP and CTP failed to produce marked response at concentrations below 1 mmol/L. Fig 1C (peak responses) and 1D (plateau responses) shows the potency of ATPS and uracil nucleotides to raise [Ca²⁺]_i. ATPS (selective agonist for P_2u receptors) UTPUDP. UMP and uridine failed to induce any marked response. Since UTP, UDP, and ATPS are selective agonists for P_2u but not for P_2x or P_2y receptors and since UTP, UDP, and ATPS were equally (or more) potent to ATP, these studies indicate the predominance of P_2u receptors in rat basilar smooth muscle cells.

View larger version (34K): [in this window] [in a new window]   Figure 1. Effect of nucleotides and uridine on [Ca2+]i in rat basilar smooth muscle cells. A and B, Peak and plateau [Ca2+]i responses to CTP, GTP, ITP, TTP, ATP, and UTP (1 nmol/L - 1 mmol/L). C and D, Peak and plateau [Ca2+]i responses to ATPS, UTP, UDP, UMP and uridine (1 nmol/L - 1 mmol/L). Each agent and each dose were tested separately. n indicates the number of cells tested for each concentration. Values were expressed as mean and SD. Increased Peak indicates the net maximum [Ca2+]i response (=Peak–Resting level) and Increased Plateau indicates the net [Ca2+]i values (=Plateau - Resting level) 3 minutes after application of nucleotides. The potency of nucleotides to raise [Ca2+]i was UTPATPTTP>ITPGTPCTP. [Ca2+]i elevation induced by UTP, ATP, and TTP was significantly higher at 10 to 100 µmol/L levels than those of CTP and GTP (P<.05, ANOVA) but not that of ITP (P>.05, ANOVA). Nosignificant difference was found among the effects of UTP, ATP, and TTP (P>.05, ANOVA).The potency of pyrimidines to raise [Ca2+]i was ATPS UTP UDPUMPuridine. [Ca2+]i elevation induced by ATPS, UTP, and UDP was significantly higher at 1 µmol/L to -1 mmol/L levels than those of UMP and uridine (P<.05, ANOVA). No significant difference was found among the effects of ATPS, UTP, and UDP (P>.05, ANOVA).

Signal Transduction Pathways of P_2u Receptors
Since UTP was one of the most potent agonists among these nucleotides, we used UTP as the agonist to study signal transduction pathways in rat basilar smooth muscle cells. The [Ca²⁺]_i responses to UTP (100 µmol/L) in cells pretreated with pertussis toxin (1200 ng/mL) for 10 hours¹² were compared with those of control cells. As shown in Fig 2A and C, pretreatment with pertussis toxin (Fig 2A, right) markedly reduced the [Ca²⁺]_i responses to UTP. The PLC inhibitor NCDC (10 µmol/L)¹³ was tested and found, after 15 minutes' incubation with cells, to abolish the [Ca²⁺]_i responses to UTP (100 µmol/L, Fig 2B and C).

Since activation of PLC produces inositol 1,4,5-triphosphate (IP₃), which releases Ca²⁺ from internal stores, we tested the effect of UTP on thapsigargin-sensitive Ca²⁺ stores. UTP (100 µmol/L) produced a transient [Ca²⁺]_i peak response without plateau phase in Ca²⁺-free external solution (Fig 3A, left) . In the absence of external Ca²⁺, thapsigargin (100 nmol/L), a well-known Ca²⁺-pump inhibitor, produced a transient [Ca²⁺]_i rise (leak) that lasted about 5 to 7 minutes before falling to a level similar to the resting level (Fig 3A, right). The following application of UTP (100 µmol/L), in the presence of thapsigargin, failed to induce any marked [Ca²⁺]_i elevation (Fig 3A, right). Thapsigargin produced a prolonged [Ca²⁺]_i elevation in the presence of external Ca²⁺ in our previous studies.¹⁴ [Ca²⁺]_i peak and plateau responses induced by UTP in the presence and absence of external Ca²⁺ and the inhibitory effect of thapsigargin on UTP-induced [Ca²⁺]_i elevation in the absence of external Ca²⁺ are compared in Fig 3B.

The pathways for Ca²⁺ entry activated by UTP were studied by using the receptor-operated Ca²⁺ influx blocker SK&F96365 and the voltage-dependent Ca²⁺ channel blocker verapamil. SK&F96365 (5 µmol/L; Fig 4A, left) markedly and verapamil (1 µmol/L, Fig 4A, right) partially reduced the [Ca²⁺]_i plateau phase induced by UTP without significant effect on the [Ca²⁺]_i peak. Lanthanum (100 µmol/L, Fig 4B), an inorganic Ca²⁺ channel blocker, abolished both peak and plateau responses induced by UTP, consistent with our previous report that lanthanum completely abolishes the [Ca²⁺]_i response to ATP-containing erythrocyte lysate.⁷ The effects of Ca²⁺ entry blockers are summarized in Fig 4B.

View larger version (25K): [in this window] [in a new window]   Figure 4. Effect of Ca2+ influx blockers on [Ca2+]i response to UTP. A, left, SK&F96365 (5 µmol/L) applied 10 seconds before and then together with UTP (100 µmol/L) markedly reduced the [Ca2+]i plateau but not peak response induced by UTP. Right, Verapamil (1 µmol/L), under the same protocol as SK&F96365, partially reduced [Ca2+]i plateau but not peak response induced by UTP. B, Summary of effects of Ca2+ influx blockers on UTP-induced [Ca2+]i response under the same protocol as described in A above. Lanthanum (100 µmol/L) was applied and found to abolish both peak and plateau [Ca2+]i responses to UTP. ** and *** indicate P<.01 and P<.001, respectively, compared with control-UTP response; ANOVA.

P₂-Purinoceptor Antagonists
[Ca²⁺]_i response to UTP was further examined using P₂ receptor antagonists. Suramin, a selective P₂ receptor antagonist, and PPADS, a selective P_2x receptor antagonist,¹⁵ were incubated with cells for 5 minutes before UTP (100 µmol/L) was applied (Fig 5A). PPADS at 10 µmol/L failed to reduce the effect of UTP (Fig 5B), the concentration that inhibited the effect of ATP on P_2x receptors.¹⁵ PPADS at 30 µmol/L partially reduced the plateau but not the peak [Ca²⁺]_i response induced by UTP. At higher concentration, PPADS (100 µmol/L) markedly reduced both the peak and plateau responses. Pre-incubation with suramin (1 µmol/L) significantly reduced both the peak and plateau responses induced by UTP (Fig 5C). However, increasing the suramin concentration to 100 µmol/L failed to further reduce the [Ca²⁺]_i response to UTP (Fig 5C).

View larger version (22K): [in this window] [in a new window]   Figure 5. Effect of P2 receptor antagonists suramin and PPADS. A, left, Preincubation of PPADS (30 µmol/L) for 3 minutes partially reduced the peak and plateau [Ca2+]i responses to UTP (100 µmol/L). Right, Preincubation with suramin (1 µmol/L) for 3 minutes markedly reduced both peak and plateau [Ca2+]i responses to UTP. B, Summary of the dose-dependent inhibitory effect of PPADS on the [Ca2+]i response to UTP (100 µmol/L). ** indicates P<.01 (compared with control [Ca2+]i response; ANOVA). C, Summary of the dose-dependent inhibitory effect of suramin on the [Ca2+]i response to UTP (100 µmol/L). ** indicates P<.01 (compared with control [Ca2+]i response; ANOVA).

	Discussion

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In the present study, we have demonstrated that UTP, ATP, TTP, and, at higher concentrations, ITP, GTP, and CTP increase [Ca²⁺]_i in rat basilar smooth muscle cells. Selective P_2u receptor agonists ATPS, UTP and UDP produced [Ca²⁺]_i responses similar to those of ATP, consistent with our previous report that P_2u receptors mediate effects of nucleotides in rat basilar artery smooth muscle cells, since a selective agonist of P_2x receptor ß-Methylene-ATP and selective agonist of P_2y receptor 2-Methylthio-ATP failed to markedly raise [Ca²⁺]_i.⁷ Using pertussis toxin, NCDC, and thapsigargin, we have shown that the P_2u receptor in these cells was coupled to G protein and, by activation of PLC, released Ca²⁺ from intracellular thapsigargin-sensitive stores. A similar mechanism of Ca²⁺ mobilization by activation of P_2u receptors has been reported previously in other tissues.⁹ The partial retention of the [Ca²⁺]_i response to UTP after 10 hours of treatment with a high concentration of pertussis toxin (1200 ng/mL) indicates that other G proteins that are not sensitive to pertussis toxin may also be involved in the UTP-induced [Ca²⁺]_i response. It has been established that PLC can be activated either by an -subunit of the pertussis toxin-insensitive G_q/G_II family or the ß-dimer of the pertussis toxin–sensitive G_i2.¹⁶ NCDC at 10 µmol/L completely abolished the [Ca²⁺]_i response to UTP, indicating UTP-induced intracellular Ca²⁺ mobilization was mediated by activation of PLC in these cells. Furthermore, we have demonstrated that 1 µmol/L suramin inhibited the [Ca²⁺]_i response to UTP, consistent with our previous finding that suramin reduced the effect of ATP-containing erythrocyte lysate on [Ca²⁺]_i in rat basilar smooth muscle cells⁷ and contraction in dog basilar artery.⁸ PPADS, a selective antagonist for P_2x receptors, failed to significantly reduce the effect of UTP at concentrations that blocked the effect of P_2x receptors in other tissues.¹⁵ The inhibitory effect of PPADS at 100 µmol/Lcould be due to nonspecific effects. We have observed previously that PPADS possessed limited effects on either [Ca²⁺]_i⁷ or contraction produced by ATP-containing erythrocyte lysate.⁸ Whether the effect of PPADS at 30 µmol/L that significantly reduced the plateau but not the peak [Ca²⁺]_i response is due to its blockade of P_2x receptors, which may contribute partially to the plateau phase, needs further investigation.

It has been established that P_2u (or P_2y2¹⁷ ) receptors are G protein–coupled receptors and that signal transduction is mediated by the receptor–G protein–PLC-IP₃ system, which mobilizes internal Ca²⁺ stores.⁹ Store depletion triggers Ca²⁺ entry via voltage-independent Ca²⁺ pathways, possibly by diffusible chemical messengers.¹⁸ Ca²⁺ entry through voltage-dependent Ca²⁺ channels may also partially contribute to UTP-induced [Ca²⁺]_i elevation.⁹ We have demonstrated that both receptor-operated Ca²⁺ influx inhibitor SK&F96365 and voltage-dependent Ca²⁺ channel blocker verapamil markedly reduced the Ca²⁺ entry induced by UTP, consistent with other reports using peripheral vasculature.⁹ SK&F96365 but not verapamil significantly reduced the Ca²⁺ entry induced by erythrocyte lysate in cultured endothelial cells in our previous study.¹⁰ Lanthanum completely abolished both peak and plateau [Ca²⁺]_i responses to UTP in this study, consistent with our previous results that lanthanum abolished [Ca²⁺]_i response to erythrocyte lysate.⁷ The mechanism by which lanthanum blocks both peak and plateau [Ca²⁺]_i responses is not clear but may be explained by its multiple actions against Ca²⁺ entry via membrane Ca²⁺ channels, Na⁺/Ca²⁺ exchangers, and Ca²⁺-ATPase pumps.⁷ Cytosolic Ca²⁺ binds to calmodulin, and the Ca²⁺-calmodulin complex subsequently activates myosin light chain kinase that phosphorylates the light chain of myosin, thus activating the Mg²⁺-ATPase activity, which promotes the formation of myosin-actin crossbridges.¹⁹ Several recent investigations indicated that ATP and UTP elevate [Ca²⁺]_i and contract cerebral arteries and that these effects of ATP and UTP were prevented by P₂ receptor antagonists.^{7 8 20 21 22 23 24} The P_2u receptor is believed to be activated by both ATP and UTP,⁹ although some studies have suggested the existence of two different receptors for ATP and UTP.²³ Cross-desensitization would be a better way to distinguish these two types of receptors.²⁵ In our preliminary studies, ATP (0.1 to 1 mmol/L, 5 minutes) failed to produce additional [Ca²⁺]_i response in the presence of UTP (0.1 to 1 mmol/L, pretreated with cells for 1 or 5 minutes), and UTP failed to produce additional [Ca²⁺]_i response in the presence of ATP, indicating that possibly only one type of receptor mediated the effect of nucleotides in rat basilar smooth muscle cells.

Cytoplasmic ATP may be released after cell lysis or selective permeabilization of the plasma membrane. This permeabilization can occur in various types of cells, including the smooth muscle cells,⁹ in the absence of irreversible damage such as hypoxia. Exocytosis of secretory granules such as platelet-dense bodies (>600 mmol/L ATP) also contributes to the presence of extracellular ATP, as well as other nucleotides such as ADP (±400 mmol/L), GTP, and UTP.²⁶ After aneurysmal SAH, ATP, which could be released from blood clots, would contact major cerebral arteries from the adventitia side, activate P_2x and P_2u receptors in smooth muscle cells, and may produce vasospasm.^{7 8 27} Since a high level of ATP, ADP, and UTP exists in blood cells,²⁶ these blood cells may play an important role in the pathogenesis of vasospasm.^{7 28} ATP and ADP produce contraction by activation of P₂ receptors or by releasing vasocontractile agents such as eicosanoids from cerebral endothelial cells.^{21 22 29} Presumably, these contractile actions of blood cells during vasospasm may be further amplified by SAH-induced endothelial dysfunction. Endothelial degeneration and disruption attenuated the production of prostacyclin and nitric oxide and attenuated the ADP-induced relaxation.²⁸ However, damaging endothelial cells with oxyhemoglobin enhanced the production of endothelins.¹⁴ Oxyhemoglobin in erythrocyte lysate, which chelates nitric oxide, may also potentiate the contractile effect of ATP-containing erythrocyte lysate.

The ability of UTP, UDP, ATP, and TTP to elevate [Ca²⁺]_i indicates that multiple nucleotides may have vasoactive effects in cerebral vasculature. The vasoconstrictive properties of UTP and its possible role in chronic cerebral vasospasm have been investigated. UTP induced long-lasting contractions of isolated human brain arteries up to 20 to 24 hours.³⁰ Both UTP and UDP produced vasoconstriction of canine cerebral arteries and intracisternal application of UTP produced cerebral vasospasm in dogs.²⁰ The vasoconstrictive effect of different nucleotides was tested in rabbit basilar artery. The rank order of potency of the pyrimidine nucleotides was UTP=UDPUMP=CTP; that of the purine nucleotides was ATPS>AMP-PNP>ATP> ADP>2-methylthio-ATP=,ß-methylene-ATP=ß,-methylthio-ATP.²³ UTP produced much stronger contractions than ATP in canine basilar arteries.⁸ We have obtained a similar effect for nucleotides to raise [Ca²⁺]_i in rat basilar smooth muscle cells in this study. Since UTP, UDP, and ATPS are all selective agonists for P_2u receptors, these studies indicate a predominance of P_2u receptors in cerebral arteries. ATP, however, may be a primary candidate for eliciting vascular responses such as vasospasm after SAH due to its abundance relative to the other nucleotides inside all cells and especially in red blood cells and platelets.⁹ ATP produced endothelium-dependent contraction by releasing endothelium-derived contracting factors, possibly thromboxane A₂, and endothelium-independent relaxation by activation of P₁ receptors in canine basilar artery.²¹ The endothelium-independent and endothelium-dependent contractions induced by ATP were mediated by P_2x and P_2y receptors in canine basilar arteries, respectively.²² In cannulated rabbit basilar artery, application of ATP or UTP from adventitia side caused contraction but not relaxation.²³ Application of ATP directly into canine basilar arteries in the canine model of vasospasm, in an attempt to relieve vasospasm, further aggravated vasospasm and caused disruption of endothelium.³¹ Adenine nucleotides were detected in erythrocyte lysate and were suggested to mediate the effect of erythrocyte lysate on [Ca²⁺]_i and contraction in cerebral arteries.^{7 8}

There are challenges to the hypothesis that UTP and ATP mediate the smooth muscle contraction observed in vasospasm. Extracellular ectonucleotidases rapidly metabolize ATP and other nucleotides.⁹ To elicit vasoconstriction, the release of UTP or ATP must overcome degradation by extracellular nucleotides hydrolyzing enzymes. However, subsequent metabolism of ATP to di- and monophosphates and adenosine by ectonucleotidases may enhance the effects of ATP.³² Future studies to determine the time course of the concentrations of nucleotides in cerebrospinal fluid and blood clots during vasospasm may provide answers to these questions.

	Selected Abbreviations and Acronyms

 

[Ca2+]i = intracellular Ca2+ concentration NCDC = 2-nitro-4-carboxyphenyl N,N-diphenylcarbamate PLC = phospholipase C PPADS = pyridoxal-phosphate-6-azophenyl-2',4'-disulphonic acid SAH = subarachnoid hemorrhage

	Acknowledgments

 
This work was partially supported by a Grant-in-Aid to Dr Zhang from the American Heart Association and by National Institutes of Health grant 3 RO1 NS25946-07 to Dr Weir.

Received March 7, 1997; revision received June 10, 1997; accepted June 11, 1997.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 
1. Kassell NF, Sasaki T, Colohan AR, Nazar G. Cerebral vasospasm following aneurysmal subarachnoid hemorrhage. Stroke. 1985;16:562-572.[Abstract/Free Full Text]

2. Kiwak KJ, Heros RC. Cerebral vasospasm after subarachnoid hemorrhage. Trends Neurol Sci. 1987;10:89-92.

3. Bevan JA, Bevan RD. Arterial wall changes in chronic cerebrovasospasm: in vitro and in vivo pharmacological evidence. Ann Rev Pharmacol Toxicol. 1988;28:311-329.[Medline] [Order article via Infotrieve]

4. Mayberg MR, Okada T, Bark DH. The significance of morphological changes in cerebral arteries after subarachnoid hemorrhage. J Neurosurg. 1990;72:624-633.

5. Aoki T, Takenaka K, Suzuki S, Kassell N, Sagher O, Lee K. The role of hemolysate in the facilitation of oxyhemoglobin-induced contraction in rabbit basilar arteries. J Neurosurg. 1994;81:261-266.[Medline] [Order article via Infotrieve]

6. Cosentino F, Katusic ZS. Does endothelin-1 play a role in the pathogenesis of cerebral vasospasm? Stroke. 1994;25:904-908.[Abstract]

7. Zhang H, Weir B, Marton L, Macdonald RL, Bindokas V, Miller R, Brorson J. Mechanisms of hemolysate-induced calcium elevation in cerebral smooth muscle cells. Am J Physiol. 1995;269:H1874-H1890.[Abstract/Free Full Text]

8. Sima B, Macdonald RL, Marton LS, Weir B, Zhang J. Effect of P₂-purinoceptor antagonists on hemolysate- and ATP-induced contractions of dog basilar artery in vitro. Neurosurgery. 1996;39:815-822.[Medline] [Order article via Infotrieve]

9. Dubyak GR, El-Moatassim C. Signal transduction via P₂-purinergic receptors for extracellular ATP and other nucleotides. Am J Physiol. 1993;265:C577-C606.[Abstract/Free Full Text]

10. Zhang H, Weir B, Macdonald RL, Marton L, Solenski N, Kwan J, Lee K. Mechanisms of [Ca²⁺]_i elevation induced by erythrocyte components in endothelial cells. J Pharmacol Exp Ther. 1996;277:1501-1509.[Abstract/Free Full Text]

11. Grynkiewicz G, Poenie M, Tsien R. A new generation of Ca²⁺ indicators with greatly improved fluorescence properties. J Biol Chem. 1985;260:3440-3450.[Abstract/Free Full Text]

12. Sjölander A, Grönroos E, Hammarström S, Andersson T. Leukotriene D₄ and E₄ induce transmembrane signaling in human epithelial cells. J Biol Chem. 1990;265:20976-20981.[Abstract/Free Full Text]

13. Yuan Y, Granger HJ, Zawieja DC, DeFily DV, Chilian WM. Histamine increases venular permeability via a phospholipase C-NO synthase-guanylate cyclase cascade. Am J Physiol. 1993;264:H1734-H1739.[Abstract/Free Full Text]

14. Macdonald RL, Wang X, Zhang J, Marton L. Molecular changes with subarachnoid hemorrhage and vasospasm. In: Raffel C, Harsh G, eds. The Molecular Biology of Neurosurgical Disease. Baltimore, Md: Williams & Wilkins; 1997:278-293.

15. Ziganshin AU, Hoyle CH, Bo X, Lambercht G, Mutschler E, Baumert HG, Burnstock G. PPADS selectively antagonizes P_2x-purinoceptor-mediated responses in the rabbit urinary bladder. Br J Pharmacol. 1993;110:1491-1495.[Medline] [Order article via Infotrieve]

16. Clapham DE, Neer EJ. New roles for G-protein ß dimers in transmembrane signaling. Nature. 1993;365:403-406.[Medline] [Order article via Infotrieve]

17. Barnard EA, Burnstock G, Webb TE. G protein-coupled receptors for ATP and other nucleotides: a new receptor family. Trends Pharmacol Sci. 1994;15:67-70.[Medline] [Order article via Infotrieve]

18. Fasolato C, Innocenti B, Pozzan T. Receptor-activated Ca²⁺ influx: how many mechanisms for how many channels? Trends Pharmacol Sci. 1994;15:77-83.[Medline] [Order article via Infotrieve]

19. Missiaen L, De Smedt H, Droogmans G, Himpens B, Casteels R. Calcium ion homeostasis in smooth muscle. Pharmacol Ther. 1992;56:191-231.[Medline] [Order article via Infotrieve]

20. Shirasawa Y, White RP, Robertson JT. Mechanisms of the contractile effect induced by uridine 5-triphosphate in canine cerebral arteries. Stroke. 1983;14:347-354.[Abstract/Free Full Text]

21. Shirahase H, Usui H, Manabe K, Kurahashi K, Fukiwara M. Endothelium-dependent contraction and -independent relaxation induced by adenine nucleotides and nucleoside in the canine bisilar artery. J Pharmacol Exp Ther. 1988;247:1152-1157.[Abstract/Free Full Text]

22. Shirahase H, Usui H, Shimaji H, Kurahashi K, Fukiwara M. Endothelium-independent and endothelium-dependent contractions mediated by P_2x- and P_2y-purinoceptors in canine basilar arteries. J Pharmacol Exp Ther. 1991;258:683-688.

23. Von Khgelgen I, Starke K. Evidence for two separate vaso-constriction-mediating nucleotide receptors, both distinct from the P2x-receptors, in rabbit basilar artery: a receptor for pyrimidine nucleotides and a receptor for purine nucleotides. Naunyn Schmiedeberg's Arch Pharmacol. 1990;341:538-546.[Medline] [Order article via Infotrieve]

24. Ikeuchi Y, Nishizaki T. ATP activates the potassium channel and enhances cytosolic Ca²⁺ release via a P_2y purinoceptor linked to pertussis toxin-insensitive G-protein in brain artery endothelial cells. Biochem Biophys Res Commun. 1995;215:1022-1028.[Medline] [Order article via Infotrieve]

25. Yang S, Buxton ILO, Probert CB, Talbot JN, Bradley ME. Evidence for a discrete UTP receptor in cardiac endothelial cells. Br J Pharmacol. 1996;117:1572-1578.[Medline] [Order article via Infotrieve]

26. Motte S, Communi D, Pirotton S, Boeynaems J-M. Involvement of multiple receptors in the actions of extracellular ATP: the example of vascular endothelial cells. Int J Biochem Cell Biol. 1995;27:1-7.[Medline] [Order article via Infotrieve]

27. Ralevic V, Burnstock G. Roles of P₂-purinoceptors in the cardiovascular system. Circulation. 1991;84:1-14.[Abstract/Free Full Text]

28. Akopov S, Sercombe R, Seylaz J. Cerebrovascular reactivity: role of endothelium/platelet/leukocyte interactions. Cerebrovasc Brain Metab Rev. 1996;8:11-94.[Medline] [Order article via Infotrieve]

29. Dominiczak AF, Quilley J, Bohr EF. Contraction and relaxation of rat aorta in response to ATP. Am J Physiol. 1991;261:H243-H251.[Abstract/Free Full Text]

30. Urquilla PR. Prolonged contraction of isolated human and canine cerebral arteries induced by uridine 5'-triphosphate. Stroke. 1978;9:133-136.[Abstract/Free Full Text]

31. Haciyakupoglu S, Kaya M, Cetinalp E, Yucesoy A. Effect of prostacyclin and adenosine triphosphate on vasospasm of canine basilar artery. Surg Neurol. 1985;24:126-140.[Medline] [Order article via Infotrieve]

32. Gerwins P, Fredholm BB. ATP and its metabolite adenosine act synergistically to mobilize intracellular calcium via the formation of inositol 1,4,5-trisphostate in a smooth muscle cell line. J Biol Chem. 1992;267:16081-16087.[Abstract/Free Full Text]

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