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 involved in the endothelin stimulation of phospholipase D (PLD) in rat cortical astrocytes in primary culture. PLD activity was determined by measuring the formation of [³²P]phosphatidylbutanol in [³²P]orthophosphate prelabelled cells stimulated in the presence of 25 mM butanol.
2. The agonists endothelin-1 (ET-1), endothelin-3 (ET-3), sarafotoxin 6c (S6c) 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 ET_B-preferring agonists, ET-3, S6c and IRL 1620, were significantly lower than the maximal response to the non-selective agonist ET-1.
3. The response to 1 nM ET-1 was inhibited by increasing concentrations of the ET_A 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 μM) concentrations of BQ-123, yielding an estimated K_i value for BQ-123 of 21.3±2.5 nM. In addition, the presence of 1 μM BQ-123 significantly reduced the maximal response to ET-1 but did not change the pD₂ value.
4. Increasing concentrations of the ET_B selective antagonist BQ-788 inhibited the S6c response with a K_i of 17.8±0.8 nM. BQ-788 also inhibited the effect of ET-1, although, in this case, two components were defined, 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 μM BQ-788, 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 ET-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 conclusion, endothelins activate PLD in rat cortical astrocytes acting through both ET_A and ET_B receptors, and this response desensitizes rapidly in an apparently homologous fashion. The percentage contribution of ET_A and ET_B receptors to the ET-1 response was found to be approximately 20% and 80%, respectively, when ET_B receptors were not blocked, and 30–50% and 50–70%, respectively, when ET_B receptors were inhibited or desensitized. These results may be relevant to the study of a possible role of PLD in the proliferative effects shown by endothelins on cultured and reactive astrocytes.