Intracellular Endothelin Type B Receptor-driven Ca2+ Signal Elicits Nitric Oxide Production in Endothelial Cells

Abstract

Background:The intracrine nature of endothelin-1 is largely correlated with nuclear signaling events. Results: In endothelial cells, endothelin-1 acting on endolysosomal ET B receptors increases cytosolic Ca 2ϩ and nitric oxide. Conclusion: Endolysosomal ET B receptors are functional. Significance: We identify a new pathway for ET-1-induced intracrine signaling and provide the first evidence that intracellular G protein-coupled receptors are involved in redox signaling.

Differential Activation of Cultured Neonatal Cardiomyocytes by Plasmalemmal Versus Intracellular G Protein-coupled Receptor 55

“…However, the Ca 2ϩ response pattern was strikingly different in the two paradigms. Extracellular administration of LPI resulted in a relatively slow response that reached a peak within 1-2 min and continued with a plateau phase, whereas LPI microinjection induced a fast and transitory effect similar to that reported upon activation of other intracellular GPCRs, such as those for angiotensin II (29,33,62) or endothelin 1 (28). We have noticed likewise discrepancies in the plasmalemmal-initiated versus intracellularly initiated Ca 2ϩ responses by the G protein-coupled estrogen receptor GPER/GPR30 (34,63), which is reportedly expressed at both sites (64,65).…”

“…The accumulating evidence pointing to endolysosomes as intracellular locations where GPCRs initiate signaling (19,24,28,69) prompted us to probe for colocalization of GFP-tagged GPR55 and RFP-tagged Rab7, an endolysosomal-associated small GTPase (46). We noticed extensive colocalization of GPR55 and Rab7.…”

“…Calcium Imaging-Measurements of [Ca 2ϩ ] i were performed as described previously (27)(28)(29). Cells were incubated with 5 M Fura-2 AM (Invitrogen) in Hanks' balanced salt solution at room temperature for 45 min, in the dark, washed three times with dye-free Hanks' balanced salt solution, and then incubated for another 45 min to allow for complete de-esterification of the dye.…”

“…Intracellular Microinjection-Injections were performed using the Femtotips II, InjectMan NI2, and FemtoJet systems (Eppendorf) as reported previously (24,28,29). Pipettes were backfilled with an intracellular solution containing 110 mM KCl, 10 mM NaCl, 20 mM HEPES (pH 7.2), and dimethyl sulfoxide 0.004% (31) or the compounds to be tested.…”

Visualization of nitric oxide production by individual platelets during adhesion in flowing blood

“…41 We also showed that platelet NO production by individual platelets temporally follows the increase in cytosolic Ca 11 concentration, because in the majority of platelets (.90%), fluorescence for NO was preceded by an intracellular Ca 11 increase, similarly to what was previously shown in smooth muscle cells 42 and endothelial cells. [43][44][45][46] The lag time between the 2 events in platelets (33 6 9.5 s) seems to be longer than that reported in vascular endothelial cells stimulated with bradykinin, in which a delay of ;5 seconds was observed, 45 or in human umbilical cord endothelial cells stimulated with histamine, 46 or in porcine aortic endothelial cells stimulated with thrombin, where almost no lag phase between the 2 events was observed. 47 These apparent discrepancies could be explained considering the different roles played by endothelial-released and platelet-released NO.…”