INVOLVEMENT OF ETA AND ETB RECEPTORS IN THE ACTIVATION OF PHOSPHOLIPASE-D BY ENDOTHELINS IN CULTURED RAT CORTICAL ASTROCYTES

1 This study was performed to characterize the receptor subtypes invol ved in the endothelin stimulation of phospholipase D (PLD) in rat cort ical astrocytes in primary culture. PLD activity was determined by mea suring the formation of [P-32]phosphatidylbutanol in [32P]orthophospha te prelabelled cells stimulated in the presence of 25 mM butanol. 2 Th e agonists endothelin-1 (ET-1), endothelin-3 (ET-3), sarafotoxin 6e (S 6c) and IRL 1620 elicited PLD activation in a concentration-dependent manner. The potencies of ET-1, ET-3 and S6c were similar. The maximal effects evoked by the ETB-preferring agonists, ET-3, S6c and IRL 1620, were significantly lower than the maximal response to the non-selecti ve agonist ET-1. 3 The response to 1 nM ET-1 was inhibited by increasi ng concentrations of the ET, receptor antagonist BQ-123 in a biphasic manner. A high potency component of the inhibition curve (24.2 +/- 3.5 % of the ET-1 response) was defined at low (up to 1 mu M) concentratio ns of BQ-123, yielding an estimated K-i value for BQ-123 of 21.3 +/- 2 .5 nM. In addition, the presence of 1 mu M BQ-123 significantly reduce d the maximal response to ET-1 but did not change the pD(2) value. 4 I ncreasing concentrations of the ETB selective antagonist BQ-788 inhibi ted the S6c response with a K-i of 17.8 +/- 0.8 nM. BQ-788 also inhibi ted the effect of ET-1, although, in this case, two components were de fined, accounting for approximately 50% of the response, and showing K -i values of 20.9 +/- 5.1 nM and 439 +/- 110 nM, respectively. The ET- 1 concentration-response curve was shifted to the right by 1 mu M BQ-7 88, also revealing two components. Only one of them, corresponding to 69.8 +/- 4.4% of the response, was sensitive to BQ-788 which showed a K-i value of 28.8 +/- 8.9 nM. 5 Rapid desensitization was achieved by preincubation with ET-1 or S6c. In cells pretreated with S6c neither E T-3 nor S6c activated PLD, but ET-1 still induced approximately 40% of the response shown by non-desensitised cells. This remaining response was insensitive to BQ-788, but fully inhibited by BQ-123. 6 In conclu sion, endothelins activate PLD in rat cortical astrocytes acting throu gh both ETA and ETB receptors, and this response desensitizes rapidly in an apparently homologous fashion. The percentage contribution of ET A and ETB receptors to the ET-1 response was found to be approximately 20% and 80%, respectively, when ETB receptors were not blocked, and 3 0-50% and 50-70%, respectively, when ETB receptors were inhibited or d esensitized. These results may be relevant to the study of a possible role of PLD in the proliferative effects shown by endothelins on cultu red and reactive astrocytes.