CHARACTERISTICS OF NUCLEOTIDE RECEPTORS THAT CAUSE ELEVATION OF CYTOPLASMIC CALCIUM IN IMMORTALIZED RAT-BRAIN ENDOTHELIAL-CELLS (RBE4) AND IN PRIMARY CULTURES

Citation
M. Nobles et al., CHARACTERISTICS OF NUCLEOTIDE RECEPTORS THAT CAUSE ELEVATION OF CYTOPLASMIC CALCIUM IN IMMORTALIZED RAT-BRAIN ENDOTHELIAL-CELLS (RBE4) AND IN PRIMARY CULTURES, British Journal of Pharmacology, 115(7), 1995, pp. 1245-1252
Citations number
45
Categorie Soggetti
Pharmacology & Pharmacy
ISSN journal
00071188
Volume
115
Issue
7
Year of publication
1995
Pages
1245 - 1252
Database
ISI
SICI code
0007-1188(1995)115:7<1245:CONRTC>2.0.ZU;2-7
Abstract
1 A dual-wavelength microfluorimetric method using Fura-2 as calcium i ndicator was applied to cells from an immortalized cell line of rat br ain microvascular endothelial cells (RBE4), and to primary cultured ra t brain endothelial cells. 2 In RBE4 cells, a brief (20 s) pulse of ex tracellular ATP (100 mu M) induced a transient increase in the cytopla smic calcium level ([Ca2+](i)). Control responses to 100 mu M ATP cons isted of a ratio increase of 0.64 +/- 0.03 (mean +/- s.e., n = 51). Re sponses were seen at a concentration of 2.5 mu M and were maximal at 1 00-1000 mu M. When extracellular calcium was chelated with EGTA, the t ransient increase in [Ca2+](i) was not affected. The results are consi stent with Ca2+ mobilization from intracellular stores. 3 The purinoce ptor involved belongs to the P-2 subtype, since the agonist potency or der among the adenine nucleotides was ATP>ADP>AMP. Moreover, the incre ase in [Ca2+](i) evoked by ATP was partially inhibited by the P-2Y ant agonist, suramin but was not affected by 8-phenyltheophylline, a P-1-p urinoceptor antagonist. The strong desensitization observed with repea ted applications of ATP is also typical of a P-2 receptor. 4 2-Methylt hio-ATP (2meS-ATP 100 mu M), a P-2Y agonist, elevated [Ca2+](i) in onl y 17% of the cells tested; however, 2meS-ATP was found to antagonize t he effect of ATP in all cells tested. The increase in [Ca2+](i) evoked by ATP was inhibited by 500 s application of the P-2Y purinoceptor an tagonist, Reactive Blue 2 at 10 mu M, while 60 s application of 100 mu M was ineffective. 5 The uracil nucleotide, UTP (100 mu M) was as eff ective as ATP in increasing [Ca2+](i). The effects of ATP and UTP were not additive. Cells desensitized to the action of ATP (or UTP) were u nable to respond to UTP (or ATP). 6 alpha,beta Methylene-ATP (alpha,be ta meATP 100 mu M), a Pix agonist, elevated [Ca2+](i) in only 40% of t he cells tested. In these cells it was less effective than ATP in incr easing [Ca2+](i). 7 Cells desensitized to the action of ADP responded, to a smaller extent, to ATP. In contrast, cells desensitized to the a ction of ATP were unable to respond to ADP. 8 On primary cultures of b rain endothelial cells the increase in [Ca2+](i) in response to extrac ellular ATP (100 mu M) and UTP (100 mu M) was of an equivalent amplitu de, and similar to the response in RBE4 cells. The pattern of desensit ization was also similar to that in RBE4 cells. 9 This comparative stu dy indicates that in well-characterized brain microvascular endothelia l cells that retain brain endothelial characteristics, the major class of nucleotide receptor is of the P-2U type. The implications for phys iology are discussed.