Journal of Experimental Biology vol:205 issue:Pt 23 pages:3729-37
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 (I(sc)), transepithelial conductance (G(T)), transepithelial capacitance (C(T)) and fluctuation in I(sc). Apical application of NA extract had merely a small stimulatory effect on Na(+) transport, whereas basolateral administration markedly increased I(sc), G(T) and C(T). A maximal effect was obtained at 500 microll(-1) of lyophilized NA extract. The increase in C(T) suggests that the activation of I(sc) 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 I(sc) and G(T), indicating that Cl(-) conductance was not influenced. Noise analysis of I(sc) using 6-chloro-3,5-diaminopyrazine-2-carboxamide (CDPC) showed that NA extract reduced single-channel current (i(Na)) and decreased channel open probability (P(o)) but evoked a threefold increase in channel density (N(T)), which confirms the insertion of Na(+) channels. The separation of the compounds in the crude extract of NA was 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 micro RPC 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 micromol l(-1) adenosine had similar effects on the parameters of Na(+) transport as did the NA extract. 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 I(sc), G(T) or C(T). These results clearly demonstrate an essential role of adenosine in the stimulatory action of NA extract.