Previous reports have demonstrated that endothelin promotes proliferation of brain astrocytes,
47,48 vascular smooth muscle cells,
49 Swiss 3T3 fibroblasts,
50 and type I astrocytes
51 through Ca
2+ signaling and that this mitogenic effect occurs through ETA.
47,48 In cultured human ONHAs, ETA antagonism completely blocked ET-1–induced increases of [Ca
2+]
i; however, pretreatment of the cells with PD142893, a mixed ETA/ETB antagonist blocked the increase in ET-1–induced intracellular calcium level by only 60% to 75%.
10 We observed that ETA, but not ETB receptor blockade prevented increased [Ca
2+]
i in WT ONHAs. Interestingly, in TSL ONHAs we observed enhanced ET-1–induced [Ca
2+]
i increases compared with wild type ONHAs that possessed both ETB and ETA receptors. The mechanism for this finding is unclear because ETA and ETB receptors are both G-protein coupled receptors linked to phosphoinositol signaling that leads to increased IP
3 and, therefore, release of Ca
2+ from IP
3-sensitive intracellular stores.
17,47 ETA receptor upregulation in the TSL ONHAs or desensitization are possible explanations. Furthermore, these results may be due to a novel ET receptor or heterodimerization of the ETB and ETA receptors that results in a unique ligand signaling cascade, as described by Prasanna and colleagues.
10,20,30 We previously suggested that there may be overlap in the mitogenic function of ETB and ETA receptors.
32 However, our proliferation and calcium-imaging results indicate that ETB and ETA may have separate signaling pathways which independently induce ONHA proliferation. Interestingly, we observed no change in [Ca
2+]
i with 10
−9 M ET-1; however, significant ONHA proliferation occurred at this concentration. Although it is possible that [Ca
2+]
i is not coupled with ET-1 induced proliferation in ONHAs, given the extensive evidence from other models identifying an association between [Ca
2+]
i and proliferation, our findings are likely due to differences in our experimental paradigms. [Ca
2+]
i changes were observed after only brief (1 minute) exposure to ET-1 whereas proliferation was assessed after continuous ET-1 exposure for 48 or 72 hours. Therefore [Ca
2+]
i changes may occur with 10
−9 M ET-1 after prolonged exposure. Future studies should investigate whether impairment of ET-1–induced [Ca
2+]
i elevation prevents proliferation and attempt to elucidate the signaling mechanism by which ETB promotes ONHA proliferation.