Stimulatory effects on Na+ transport in renal epithelia induced by extracts of Nigella arvensis are caused by adenosine
Fatima Atia1,
Irina Mountian2,
Jeannine Simaels2,
Etienne Waelkens3 and
Willy Van Driessche2,*
1 Université Sidi Mohamed Ben Abdellah, Faculté des Sciences,
UFR: Physiologie — Pharmacologie, Fès, Morocco
2 Laboratory of Physiology, KU Leuven, Campus Gasthuisberg, B-3000 Leuven,
Belgium
3 Laboratory of Biochemistry, KU Leuven, Campus Gasthuisberg, B-3000 Leuven,
Belgium
View larger version (17K):
[in a new window]
|
Fig. 1. Effects of the Nigella arvensis (NA) extract on short-circuit
current (Isc), transepithelial conductance
(GT) and transepithelial capacitance
(CT) in cultured monolayers of A6 epithelia under
Na+-transporting conditions. The A6 cells were incubated in 102
mmoll-1 NaCl-Ringer solutions on both surfaces. 250
µll-1 NA extract was applied either to the apical side
(dashed line) or basolateral side (solid line) of the monolayer. At the end of
the experiment, Na+ current was inhibited by apical 50
µmoll-1 amiloride (Ami). The traces are mean values from six
tissues; dotted lines represent means ± S.E.M.
|
|
View larger version (17K):
[in a new window]
|
Fig. 2. Effects of the Nigella arvensis (NA) extract on Cl-
secretion. (A) Epithelia were incubated in 102 mmoll-1 NaCl-Ringer
solutions on both apical (Ap) and basolateral (B1) sides;
N-methyl-D-glucamine chloride (NMDGCl)-Ringer was then added apically
30 min before the NA extract (250 µll-1) was
administrated basolaterally. 50 µmoll-1 amiloride (Ami) was
added at the end of the experiment. (B) Same experimental conditions as in A,
but NMDG2SO4-Ringer was applied apically 30 min before
the NA extract was added. At the end of the experiment, basolateral
Cl- was replaced by SO42-.
|
|
View larger version (22K):
[in a new window]
|
Fig. 4. Effects of the Nigella arvensis (NA) extract on
short-circuit current (Isc), single-channel current
(iNa), transepithelial conductance
(GT), total number of channels (NT)
and open-channel probability (Po). (A) Pulse protocol of
6-chloro-3,5-diaminopyrazine-2-carboxamide (CDPC)-induced noise before and
after addition of the NA extract basolaterally. CDPC-induced noise
was recorded by switching the apical CDPC concentration ([CDPC]Ap)
alternately from 10 µmol l-1 to 40 µmol l-1 every
5 min. 50 µmol l-1 amiloride (Ami) was administered apically at
the end of the experiment. Solution conditions were as in
Fig. 1. (B)
Isc, iNa, NT and
Po during control (open bars) and after exposure to
NA extract (filled bars). The values were calculated using a
three-state model. * The values of the control and the NA
extract are significantly different (P<0.05) for all
parameters.
|
|
View larger version (17K):
[in a new window]
|
Fig. 5. Identification of the active compound in the Nigella arvensis
(NA) extract. (A) Fast protein liquid chromatography (FPLC) profile
of the NA extract. The elution position of fraction 108, showing
activity, is indicated by an arrow. (B) Further separation of fraction 108 by
reverse-phase high-pressure liquid chromatography (RP-HPLC). The activity was
demonstrated in a 215 nm peak eluting at 20% acetonitrile (fraction 12,
indicated by an asterisk). The linear elution gradient is also shown.
|
|
View larger version (10K):
[in a new window]
|
Fig. 6. Mass spectrum of fraction 12. The presence of three distinct molecules with
masses of 316, 288 and 268 are visible. The compounds with masses of 316 and
288 could be identified as contaminants generated during the purification
procedure. The 268-mass compound was identified as adenosine
(Mr 267.24).
|
|
View larger version (18K):
[in a new window]
|
Fig. 7. Time courses of short-circuit current (Isc),
transepithelial conductance (GT) and transepithelial
capacitance (CT) during basolateral (Bl) serial
stimulation with adenosine and the Nigella arvensis (NA)
extract. First, 1 µmol l-1 adenosine was added at the
basolateral side, followed by addition of 250 µl l-1 NA
extract under transporting conditions. The same solution conditions were used
as in Fig. 1. The traces are
mean values from six tissues; dotted lines represent means ± S.E.M.
|
|
CiteULike 
Complore 
Connotea 
Del.icio.us 
Digg 
Reddit 
Technorati 
Twitter What's this?
© The Company of Biologists Ltd 2002
fc data at different doses of apical CDPC
([CDPC]Ap). The slope and intercept of the linear regressions for
the control (2