title: ET-1 induced Elevation of intracellular calcium in clonal neuronal and embryonic kidney cells involves endogenous endothelin-A receptors linked to phospholipase C through Gα(q/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 ET(A) (and, perhaps, ET(B)) 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 ET(A) 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 ([Ca(2+)](in)) which is blocked by BQ-123, an ET(A) receptor antagonist, but not by BQ-788, an ET(B) receptor antagonist, suggesting that ET(A) receptors mediate this effect. Here we extend these findings to HEK 293T cells. Examination of the expression of ET(A) and ET(B) receptors by RT-PCR and [(125)I]-ET-1 binding experiments confirms the slight predominance of ET(A) receptor binding sites and messenger RNA in both ND7/104 and HEK 293T cells. In addition, selective agonists of the ET(B) receptor (sarafotoxin 6c, BQ-3020 or IRL-1620) do not induce a transient increase in [Ca(2+)](in). Furthermore, reduction of ET(B) mRNA levels by siRNA does not abrogate calcium mobilization by ET-1 in HEK 293T cells, corroborating the lack of an ET(B) receptor role in this response. However, in HEK 293 cells with low endogenous ET(A) mRNA levels, ET-1 does not induce a transient increase in [Ca(2+)](in). Observation of the [Ca(2+)](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 ET(A) receptor expression is necessary to drive a transient [Ca(2+)](in) increase in these cells and that this process involves release of calcium from intracellular stores following Gαq/11 activation.
authors.affiliation: Pain Research Center, Department of Anesthesiology, Perioperative and Pain Medicine, Brigham & Women's Hospital, Boston, MA 02115, USA; Centre des Sciences du Goût et de l'Alimentation, UMR6265 CNRS, F-21000 Dijon, France.
authors.forename: Jean-Pierre
authors.initials: JP
authors.lastname: Montmayeur
authors.forename: Travis P
authors.initials: TP
authors.lastname: Barr
authors.forename: Sarah A
authors.initials: SA
authors.lastname: Kam
authors.forename: Samuel J
authors.initials: SJ
authors.lastname: Packer
authors.forename: Gary R
authors.initials: GR
authors.lastname: Strichartz
mesh_terms: D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002460:Cell Line; D003593:Cytoplasm / Q000378:metabolism*; D019332:Endothelin-1 / Q000378:metabolism*; D043802:GTP-Binding Protein alpha Subunits, Gq-G11 / Q000378:metabolism*; D005786:Gene Expression Regulation; D057809:HEK293 Cells; D006801:Humans; D007668:Kidney / Q000166:cytology; D051379:Mice; D051381:Rats; D044022:Receptor, Endothelin A / Q000235:genetics / Q000378:metabolism*; D044023:Receptor, Endothelin B / Q000235:genetics / Q000378:metabolism; D011984:Sensory Receptor Cells / Q000378:metabolism; D010738:Type C Phospholipases / Q000378:metabolism*
chemical_list: D019332:Endothelin-1; D044022:Receptor, Endothelin A; D044023:Receptor, Endothelin B; D010738:Type C Phospholipases; D043802:GTP-Binding Protein alpha Subunits, Gq-G11; D002118:Calcium