ET-1 induced Elevation of intracellular calcium in clonal neuronal and embryonic kidney cells involves endogenous endothelin-A receptors linked to phospholipase C through Gαq/11

Abstract

Endothelin-1 (ET-1) is a pain mediator, elevated in skin after injury, which potentiates noxious thermal and mechanical stimuli (hyperalgesia) through the activation of ETA (and, perhaps, ETB) receptors on pain fibers. Part of the mechanism underlying this effect has recently been shown to involve potentiation of neuronal TRPV1 by PKCɛ. However, the early steps of this pathway, which are recapitulated in HEK 293 cells co-expressing TRPV1 and ETA receptors, remain unexplored. To clarify these steps, we investigated the pharmacological profile and signaling properties of native endothelin receptors in immortalized cell lines including HEK 293 and ND7 model sensory neurons. Previously we showed that in ND7/104, a dorsal root ganglia-derived cell line, ET-1 elicits a rise in intracellular calcium ([Ca2+]in) which is blocked by BQ-123, an ETA receptor antagonist, but not by BQ-788, an ETB receptor antagonist, suggesting that ETA receptors mediate this effect. Here we extend these findings to HEK 293T cells. Examination of the expression of ETA and ETB receptors by RT-PCR and [125I]-ET-1 binding experiments confirms the slight predominance of ETA receptor binding sites and messenger RNA in both ND7/104 and HEK 293T cells. In addition, selective agonists of the ETB receptor (sarafotoxin 6c, BQ-3020 or IRL-1620) do not induce a transient increase in [Ca2+]in. Furthermore, reduction of ETB mRNA levels by siRNA does not abrogate calcium mobilization by ET-1 in HEK 293T cells, corroborating the lack of an ETB receptor role in this response. However, in HEK 293 cells with low endogenous ETA mRNA levels, ET-1 does not induce a transient increase in [Ca2+]in. Observation of the [Ca2+]in elevation in ND7/104 and HEK 293T cells in the absence of extracellular calcium suggests that ET-1 elicits a release of calcium from intracellular stores, and pretreatment of the cells with pertussis toxin or a selective inhibitor of phospholipase C (PLC) point to a mechanism involving Gαq/11 coupling.These results are consistent with the hypothesis that a certain threshold of ETA receptor expression is necessary to drive a transient [Ca2+]in increase in these cells and that this process involves release of calcium from intracellular stores following Gαq/11 activation.