Evidence for a role of Trp proteins in the oxidative stress-induced membrane conductances of porcine aortic endothelial cells

Objective: Expression of homologues of the Drosophila transient receptor po tential (Trp) protein has recently been demonstrated for vascular endotheli um. Some Trp isoforms such as Trp3, are known to constitute cation conducta nces with biophysical properties similar to those of the endothelial oxidan t-activated cation conductance. Therefore we tested whether Trp proteins pr ovide the molecular basis of the oxidant-induced membrane currents in porci ne aortic endothelial cells (ECAP). Methods: Expression of the Trp3 isoform in ECAP was tested by RT-PCR and subsequent southern blot analysis. In ord er to knock-out the function of endogenous Trp channels, ECAP were transien tly transfected to express NTRP3, a dominant negative :fragment of Trp3. Ox idative-stress was introduced by exposure of cells to tert-butylhydroperoxi de (tBHP; 400 mu M), and membrane currents as well as membrane potential we re recorded using the conventional whole cell patch-clamp technique. Result s: RT-PCR experiments demonstrated the expression of a Trp3 isoform in ECAP . The oxidant tert.-butylhydroperoxide (tBHP) completely depolarized endoth elial cells by activation of a cation conductance which allowed significant Na+ inward currents at negative potentials (mean inward current 462 pA at -80 mV). The tBHP-induced currents resembled Trp-related currents in terms of cation selectivity, La3+ sensitivity and lack of voltage dependence. Exp ression of the N-terminal fragment of hTrp3 (NTRP3), but not of a C-termina l fragment of hTrp3 (CTRP3), abolished the oxidant-induced cation current a nd reduced membrane depolarization. Conclusion: Our results strongly sugges t Trp proteins as the molecular basis of endothelial oxidant-activated cati on channels. It is concluded that Trp proteins play an important role in th e redox sensitivity of the vascular endothelium. (C) 1999 Elsevier Science BN. All rights reserved.