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