Stimulatory effects on Na+ transport in renal epithelia induced by extracts of Nigella arvensis are caused by adenosine.
2002
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 ( 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 μl l -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 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 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.
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