Invited Symposium: Cerebral Artery Pharmacology and Physiology
Extracellular nucleotides influence neurotransmission, inflammatory and immune responses, platelet aggregation and secretion, and pulmonary and cardiac function. Extracellular nucleotides play an important role in the regulation of vascular tone including cerebral arteries, by activation of P2 receptors. Subtypes of P2 receptors have been classified by numerous studies in peripheral arteries, 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 P2u receptors mediated the effect of nucleotides on [Ca2+]i in rat basilar smooth muscle cells.
Materials and Methods
Digital [Ca2+]i imaging was performed by video microfluorimetry using an intensified CCD camera (Hamamatsu, Bridgewater, NJ) coupled to a Nikon Diaphot microscope (40x Fluor objective, Nikon Inc. New York) and software (Universal Imaging Corp. West Chester, PA) 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.
General Effects of Nucleotides
Effect of Different Nucleotides
Signal Transduction Pathways of P2u Receptors
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&F 96365 (5 :M) markedly and verapamil (1 :M) partially reduced the [Ca2+]i plateau phase induced by UTP without significant effect on the [Ca2+]i peak. Lanthanum (100 mM), an inorganic Ca2+ channel blocker, abolished both peak and plateau responses induced by UTP.
P2-purinoceptor Antagonists [Ca2+]i response to UTP was further examined by using P2 receptor antagonists. Suramin, a selective P2 receptor antagonist, and PPADS, a selective P2x receptor antagonist15 were incubated with cells for 5 min before UTP (100 :M) was applied. PPADS at 10 :M failed to reduce the effect of UTP, the concentration which inhibited the effect of ATP on P2x receptors15. Pre-incubation with suramin (1 :M) significantly reduced both the peak and plateau responses induced by UTP.
Discussion and Conclusion
Furthermore, we have demonstrated that 1 :M suramin inhibited the [Ca2+]i response to UTP, consistent with our previous founding that suramin reduced the effect of ATP-containing erythrocyte lysate on [Ca2+]i in rat basilar smooth muscle cells and contraction in dog basilar artery. PPADS, a selective antagonist for P2x receptors, failed to significantly reduce the effect of UTP at concentrations that blocked the effect of P2x receptors in other tissues.
It has been established that P2u (or P2y217) 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. Store depletion triggers Ca2+ entry via voltage-independent Ca2+ pathways, possibly by diffusible chemical messengers. Ca2+ entry through voltage-dependent Ca2+ channels may also partially contribute to UTP-induced [Ca2+]i elevation. We have demonstrated that both receptor-operated Ca2+ influx inhibitor SK&F 96365 and voltage-dependent Ca2+ channel blocker verapamil markedly reduced the Ca2+ entry induced by UTP, consistent with other reports using peripheral vasculatures. SK&F 96365 but not verapamil significantly reduced the Ca2+ entry induced by erythrocyte lysate in cultured endothelial cells in our previous study. 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. 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. Cytosolic Ca2+ binds to calmodulin, and the Ca2+-calmodulin complex subsequently activates myosin light chain kinase which phosphorylates the light chain of myosin thus activating the Mg2+-adenosine triphosphatase activity, which promotes the formation of myosin-actin cross-bridges. 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.
Cytoplasmic ATP may be released following cell lysis or selective permeabilization of the plasma membrane. This permeabilization can occur in various types of cells, including the smooth muscle cells, in the absence of irreversible damage, such as hypoxia. Exocytosis of secretory granules such as platelet dense bodies (> 600 mM ATP) also contributes to the presence of extracellular ATP, as well as other nucleotides such as ADP (" 400 mM), GTP and UTP. After aneurysmal SAH, ATP, which could be released from blood clot, would contact major cerebral arteries from the adventitia side, activate P2x and P2u receptors in smooth muscle cells and may produce vasospasm. Since a high level of ATP, ADP and UTP exists in blood cells, these blood cells may play important roles in the pathogenesis of vasospasm. ATP and ADP produce contraction by activation of P2 receptors or by releasing vasocontractile agents such as eicosanoids from cerebral endothelial cells.
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 20-24 hrs. Both UTP and UDP produced vasoconstriction of canine cerebral arteries and intracisternal application of UTP produced cerebral vasospasm in dogs. The vasoconstrictive effect of different nucleotides was tested in rabbit basilar artery. The rank order of potency of the pyrimidine nucleotides was UTP = UDP >> UMP =CTP; that of the purine nucleotides was ATP(S > AMP-PNP > ATP > ADP > 2-methylthio-ATP = ",$-methylene-ATP = $,(-methylthio-ATP23. UTP produced much stronger contractions than ATP in canine basilar arteries. 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 ATP(S are all selective agonists for P2u receptors, these studies indicate a predominance of P2u receptors in cerebral artery. ATP, however, may be a primary candidate for eliciting vascular responses such as vasospasm following SAH due to its abundance relative to the other nucleotides inside all cells and especially in red blood cells and platelets. 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. The endothelium-independent and endothelium-dependent contractions induced by ATP were mediated by P2x and P2y receptors in canine basilar artery respectively. In cannulated rabbit basilar artery, application of ATP or UTP from adventitia side caused contraction but not relaxation. Application of ATP directly into canine basilar artery in the canine model of vasospasm, in an attempt to relieve vasospasm, further aggravated vasospasm accompanied by disruption of endothelium. 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.
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. In order 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. Future studies determining the time course of the concentrations of nucleotides in cerebrospinal fluid and blood clot during vasospasm may provide answers to these questions.
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|Zhang, J; Bogdan Sima, B; MacDonald, RL; Weir, B; (1998). P2 Receptors In Cerebral Arteries. Presented at INABIS '98 - 5th Internet World Congress on Biomedical Sciences at McMaster University, Canada, Dec 7-16th. Invited Symposium. Available at URL http://www.mcmaster.ca/inabis98/laher/zhang0702/index.html|
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