Stimulatory effects on Na+ transport in renal epithelia induced by extracts of Nigella arvensis are caused by adenosine

Abstract

SUMMARY Effects of the extract of Nigella arvensis (NA) seeds on transepithelial Na+ transport were studied in cultured A6 toad kidney cells by recording short-circuit current (Isc),transepithelial conductance (GT), transepithelial capacitance (CT) and fluctuation in Isc. Apical application of NA extract had merely a small stimulatory effect on Na+ transport, whereas basolateral administration markedly increased Isc, GT and CT. A maximal effect was obtained at 500 μl l-1 of lyophilized NA extract. The increase in CT suggests that the activation of Isc occurs through the insertion of transport sites in the apical membrane. In experiments performed in the absence of Na+transport [apical Na+ was replaced by N-methyl-D-glucamine(NMDG+)], basolateral NA extract did not affect Isc and GT, indicating that Cl- conductance was not influenced. Noise analysis of Isc using 6-chloro-3,5-diaminopyrazine-2-carboxamide(CDPC) showed that NA extract reduced single-channel current(iNa) and decreased channel open probability(Po) but evoked a threefold increase in channel density(NT), which confirms the insertion of Na+channels. The separation of the compounds in the crude extract of NAwas performed by fast protein liquid chromatography (FPLC) on a Superdex 200 gel-filtration column and by reverse-phase high-pressure liquid chromatography(RPHPLC) on an μRPC C2/C18 SC2.1/10 column connected to a SMART system. Analysis of the purified active fraction by mass spectrometry demonstrated the presence of adenosine as the single organic compound in the extract that had a stimulatory effect on Na+ transport. In a separate series of experiments, we confirmed that 1 μmol l-1 adenosine had similar effects on the parameters of Na+ transport as did the NAextract. The action of adenosine was further identified by experiments in which NA extract was added after adenosine. In these experiments, NA extract did not affect Isc, GT or CT. These results clearly demonstrate an essential role of adenosine in the stimulatory action of NA extract.