Stimulatory effects on Na+ transport in renal epithelia induced by extracts of Nigella arvensis are caused by adenosine
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
* Author for correspondence (e-mail: willy.vandriessche{at}med.kuleuven.ac.be)
Accepted 21 August 2002
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Summary |
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Key words: Nigella arvensis extract, sodium transport, adenosine, renal epithelia, short-circuit current, transepithelial capacitance, transepithelial conductance
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Introduction |
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For a long time, plant remedies, including nigella, have been used to treat
diabetes. It has been proposed that the anti-diabetic action of the nigella
extracts may, at least partly, be mediated through decreased hepatic
gluconeogenesis (al-Awadi et al.,
1991). Traditionally, these seeds are well known for their action
on stone dissolution in the kidney and bladder. Therefore, we wished to
investigate the effects of N. arvensis at the molecular level in
renal A6 cells.
We have demonstrated the effects of extracts of N. arvensis (NA) seeds on transepithelial Na+ transport in a distal tubule cell line, A6, isolated from the kidney of the toad Xenopus laevis, by recording short-circuit current (Isc), transepithelial conductance (GT) and transepithelial capacitance (CT) and by analyzing the fluctuations induced by a reversible blocker of the apical Na+ channel [6-chloro-3,5-diaminopyrazine-2-carboxamide (CDPC)]. Analysis of the purified active fraction by mass spectrometry demonstrated the presence of adenosine as the single organic compound in the NA extract that had a stimulatory effect on Na+ transport.
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Materials and methods |
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Capacitance measurements
In a previous report from our laboratory
(Van Driessche et al., 1999),
we described in detail the equipment used and extensively discussed the
theoretical background of the measurement of transepithelial capacitance
(CT). Briefly, in our study, we used sine-wave analysis at
2 kHz, 2.7 kHz, 4.1 kHz, 5.4 kHz and 8.2 kHz. The data in the present paper
illustrate records at 4.1 kHz. Phase shift and amplitude ratio between the
voltage and current signal was calculated using regression analysis. With
these data, we calculated the parameters of the equivalent circuit of the
epithelium represented by a simple RC network that consists of a series
resistance, transepithelial capacitance and its equivalent parallel
resistance. The graphical interface (Labview, National Instruments, Austin,
TX, USA) enabled real-time display of transepithelial conductance
(GT), short-circuit current (Isc) and
CT.
Model calculations based on a lumped two-membrane model demonstrated that,
in the high-frequency range, CT equals the equivalent
capacitance of the series arrangement of the apical (Cap)
and basolateral (Cb1) capacitance
(Van Driessche et al., 1999):
1/CT=1/Cap+1/Cb1.
As Cb1 is approximately 12 times larger than
Cap (Erlij et al.,
1994
), changes in CT will mainly reflect
alterations at the apical membrane, i.e. the result of endo- and exocytotic
processes at that border.
Noise analysis
As we wanted to investigate the effect of the NA extract on the
kinetics of the Na+ channel in A6 epithelia, fluctuation analysis
of Isc was applied using increasing concentrations of
CDPC. The macroscopic current, which equals Isc,
fluctuates around its mean value. These oscillations are, in fact, the sum of
small currents through many channels that switch randomly between an open and
a closed state. Their transition states depend on voltage, temperature and
blocker concentration. Interaction of the blockers (amiloride or CDPC) with
the Na+ channels induces interruptions of the current through the
individual channels and consequently causes a third, blocked state. Analysis
of such a process gives power-density spectra (PDS) with a single Lorentzian
noise, which can be described by the following equation:
![]() | (1) |
We used a pulse protocol similar to that described by Blazer-Yost et al.
(1998). The apical surface was
alternately exposed to 10 µmol l-1 and 40 µmol l-1
CDPC for 5 min. Current noise at both blocker concentrations was amplified,
digitized and Fourier transformed to yield PDS during each 5 min period. The
amiloride-insensitive current (Iami) was measured by
blocking the channels at the apical side with 50 µmol l-1
amiloride. The blocker-sensitive macroscopic current
was calculated as:
.
The single-channel currents in the presence of 10 µmol l-1
CDPC were regarded as
single-channel currents in the absence of blocker, as they do not differ
significantly (Blazer-Yost et al.,
1998
) according to:
![]() | (2) |
Channel density at 10 µmol l-1 CDPC
is given by the following
equation:
.
In the absence of CDPC, channel density (No) is calculated
as:
![]() | (3) |
![]() | (4) |
Methods of extraction and identification of the active principal
molecule of NA
Preparation of the plant extract
The decoction was prepared by boiling 5 g of dried and pulverized
Nigella arvensis seeds in 100 ml distilled water for 10 min. Using
filter paper, the plant extract was then filtered (filtrate I). Subsequently,
a sample (2 ml) of NA extract was filtered through a polyvinylidene
difluoride (PVDF) syringe filter with a pore size of 0.45 µm (Alltech
Europe, Laarne, Belgium) (filtrate II). In addition, the PVDF filter was
washed with 1 ml 5% isopropanol in high-pressure liquid chromatography (HPLC)
water (filtrate III). Filtrates II and III were evaporated in a Savant
Speed-Vac concentrator (Savant Instruments, Hicksville, NY, USA) and then
dissolved in 2.5 ml and 100 µl of distilled water, respectively. These
fractions were used at a concentration of 250 µll-1 to analyze
activity on the Na+ channel. Filtrate II showed the maximum
activity on Na+ transport.
Fast protein liquid chromatography (FPLC)
A sample (2.5 ml) of filtrate II was lyophilized in a Savant Speed-Vac
concentrator. The sample was dissolved in 300 µl of FPLC column buffer (100
mmoll-1 ammonium bicarbonate in HPLC water, pH 7.5), and 250 µl
was loaded onto a Superdex 200 gel-filtration column (Marsha Pharmacia Biotech
AB, SE751-84, Upscale, Sweden). Flow rate was 0.5 ml min-1, and
fractions of 250 µl were collected. Elution of the various compounds was
established by monitoring the absorbance at 280 nm. Each fraction was
lyophilized and analyzed for its activity on the Na+ channel. The
highest stimulating activity on Na+ transport was detected in
fraction 108. It should be noted that the volatile column buffer allowed for a
complete lyophilization without subsequent generation of high salt
concentrations.
Reverse-phase HPLC (RP-HPLC)
Fraction 108 was further purified by performing RP-HPLC using a C2/C18
column (µRPC C2/C18 SC2.1/10 column, Amersham Pharmacia Biotech,
Buckinghamshire, UK) connected to a SMART system (Amersham Pharmacia Biotech).
Operating conditions were as follows: solvent A, 0.1% trifluoracetic acid
(TFA) in HPLC water, solvent B, 95% acetonitrile in 0.1% TFA. Column
conditions were as follows: 0% solvent B for 7 min followed by a linear
gradient to 70% solvent B in 83 min at a flow rate of 80 µl
min-1.
The sample was injected onto the reverse-phase column, which had previously been equilibrated in solvent A. The column was washed with 420 µl of solvent A. Subsequently, a linear gradient from 0% to 70% acetonitrile in solvent A was performed. Elution was monitored at three wavelengths (215 nm, 254 nm and 280 nm), and peak fractions of fraction 108 were collected manually in 500 µl Eppendorf tubes based on the absorbance at 215 nm. Each fraction was lyophilized, dissolved in analysis buffer (Ringer solution; see composition below) and analyzed for its activity on Na+ transport in the renal epithelial cells. Activity was demonstrated in a 215 nm peak eluting at 20% acetonitrile, which corresponded to fraction 12. To identify the molecular identity of the active principle, this fraction was further analyzed by mass spectrometry.
Mass spectrometry
Mass spectrometry analysis was performed on a Perkin Elmer API 3000
LC/MS/MS system (PE Biosystems, Foster City, CA, USA) equipped with a
nanospray (Protana Engineering, Odense M, Denmark) at a flow rate of 1 µl
h-1. Calibration was performed externally with a polypropylene
glycol test solution (Perkin Elmer test-kit) and horse heart myoglobin (16.95
kDa). 2 µl of the prepared fraction was introduced into the nanospray.
Scans were made between m/z 5 and m/z 2000. Data from 50 shots to 100 shots
were averaged to obtain the final spectrum.
Solutions
In all experiments, the apical and basolateral Ringer solutions contained
102 mmoll-1 Na+, 2.5 mmoll-1 K+,
2.5 mmoll-1 HCO3-, 1 mmoll-1
Ca2+ and 104 mmoll-1 Cl- (pH 8; osmolality,
200 mosmol kg-1 H2O). For the experiments in which we
investigated Cl- secretion, the apical solution was NaCl-free and,
instead, contained 69 mmoll-1 N-methyl-D-glucamine
sulphate [(NMDG)2SO4]; osmolality was 180 mosmol
kg-1 H2O. At the end of this type of experiment, we
removed Cl- from the basolateral solution, which contained 102
mmoll-1 Na+, 2.5 mmoll-1 K+, 2.5
mmoll-1 HCO3-, 1 mmoll-1
Ca2+ and 52 mmoll-1 SO42- (the
osmolality was adjusted to 200 mosmol kg-1 H2O with
sucrose).
Amiloride (50 µmoll-1; Sigma, St Louis, MO, USA) was used to determine the amiloride-sensitive component of the Isc. CDPC (Aldrich Chemical, Milwaukee, WI, USA; stock solution in dimethyl sulfoxide) was used in concentrations of up to 100 µmoll-1. Nigella arvensis was brought from Fès, Morocco. The lyophilized NA extract was used in most experiments at a concentration of 250 µll-1. We chose to use this concentration of NA extract because an absolute maximal stimulation was recorded with 500 µll-1 (see Results) and because of the limited access to the extract. Adenosine (1 µmoll-1, 9-ß-D-ribofuranosyladenine) was also purchased from Sigma.
Statistics
For pooled data, means ± S.E.M. were calculated. Statistical
significance was evaluated using a Student's t-test.
P<0.05 was accepted as significant.
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Results |
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To further verify a possible effect of NA extract on Cl- secretion, we performed experiments with a Cl- gradient directed from the basolateral to the apical side (Fig. 2B). Apical NaCl-Ringer was replaced by (NMDG)2SO4-Ringer. No effect on Isc and GT was observed after addition of NA extract to the basolateral side. However, the increase in CT was comparable with the effect observed in Fig. 1, demonstrating that the NA extract exerted its effect under these conditions. At the end of the experiment, basolateral NaCl-Ringer was replaced by Na2SO4-Ringer to check the Cl- current, but practically no effect was observed. These data show that, with both protocols (Fig. 2A,B), the NA extract does not activate a Cl- pathway.
Noise analysis parameters: blocker rate coefficients and
determination of iNa, NT and Po values in
control conditions and in the presence of NA extract
Fig. 3 depicts the different
steps that lead to the determination of the ON and OFF rates
(kob and kbo kinetics) of the
interaction of CDPC with the Na+ channel.
Fig. 3A shows the inhibition of
Isc caused by apical application of increasing
concentrations of CDPC, ranging from 10 µmol l-1 to 100 µmol
l-1, before and after basolateral stimulation with NA
extract. The observed relative instability of Isc after
the application of different CDPC concentrations has been reported and
discussed in the literature (Baxendale-Cox
et al., 1997). It was attributed to the feedback regulation of
Na+ transport and therefore becomes more pronounced at higher
transport rates, as observed after stimulation of Isc by
the NA extract. Similarly, after washout of CDPC from the apical
solution, just before NA extract application, relatively high
transient overshoots in Isc were observed. Typically,
amiloride inhibited Na+ transport rapidly and completely when
applied at the end of the experiment following the highest dose of CDPC.
|
Fig. 3B illustrates that
2fc data correlate linearly with the CDPC
concentrations. Therefore, kob and kbo
can be determined by linear regression analysis using the following equation:
2
fc=kob[CDPC]+kbo
(see Materials and methods). The ON and OFF rates for CDPC
during the control period were consistent with the rates previously reported
(Jans et al., 2000
). Treatment
with NA extract did not change kob and slightly
increased kbo. Mean values of the kinetic parameters
(kob, kbo and
KB) are presented in
Table 2.
|
As Na+ channels in the apical membrane of A6 cells are rate
limiting for transepithelial Na+ transport
(Granitzer et al., 1991), the
increase in Isc observed during application of NA
extract could result from a rise in iNa and/or
NT and/or Po. To resolve this
question, we performed noise analysis experiments.
Fig. 4A illustrates typical
Isc responses to basolateral NA extract in such
an experiment where apical [CDPC] was switched alternately between 10 µmol
l-1 and 40 µmol l-1 every 5 min. It should be noted
that basolateral NA was added in the presence of 10 µmol
l-1 apical CDPC and that the first 40 µmol l-1 CDPC
pulse was executed approximately 10 min after addition of the NA
extract. Isc increased from 8.62 µA cm-2 to
22.12 µA cm-2, and GT increased from 0.20 mS
cm-2 to 0.36 mS cm-2. In addition, indicated on these
tracings is the consistent finding that the Isc in these
studies is amiloride sensitive, as shown by the depression of
Isc and GT by 50 µmol
l-1 amiloride at the end of the experiment.
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We used noise analysis to determine the contributions of iNa, Po and NT to the Isc. Current noise PDS were alternately measured during exposure to 10 µmol l-1 and 40 µmol l-1 CDPC, thus providing the So and fc values of the blocker-induced Lorentzians. Fig. 4B summarizes the influence of NA extract on Isc, iNa, NT and Po. NA extract increased Isc from 8.3±0.44 µA cm-2 to 21.36±0.71 µA cm-2. The most important factor involved in the activation of Isc after application of NA extract was NT, which increased from 0.75±0.06 µm-2 to 3.54±0.14 µm-2. The relatively small decrease in iNa from 0.42±0.01 pA to 0.36±0.01 pA during exposure to NA extract probably represents the immediate response to depolarization of the apical membrane by activation of the Na+ permeability. Po in the presence of basolateral NA decreased from 0.29±0.02 to 0.18±0.01. Results are presented as means ± S.E.M. (N=6).
Identification of the principal active component of the NA
extract
Fig. 5A shows the elution
position (fraction 108) of the activity during the separation of the
NA extract by FPLC on a Superdex 200 column. This corresponds to
compounds with a molecular mass of <2000 Da. Fraction 108, in turn, was
separated using RP-HPLC (Fig.
5B). Fraction 12 of the separation of fraction 108, corresponding
to a 215 nm peak eluting at 20% acetonitrile, showed maximum stimulatory
effect on Isc (data not shown). Therefore, it was taken
for further identification. Functional tests with other fractions of FPLC and
RP-HPLC did not demonstrate a stimulatory activity.
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Mass spectrometry analysis (Fig. 6) allowed the identification of a 268-mass compound as adenosine (Mr 267.24), from which adenine (Mr 135.15) is further derived. The compounds with other masses could be identified as contaminants generated during the purification procedure.
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The possible role of adenosine was functionally tested by verifying its effect on Na+ transport and by subsequently investigating the effect of NA extract on Na+ transport (Fig. 7). The addition of NA extract 30 min after an adenosine response (Fig. 7) did not show an additive effect on apical membrane Na+ influx. We obtained similar effects on Na+ transport with adenosine. Moreover, adenosine blunted the effect of NA extract. The NA extract concentrations were chosen to elicit maximal responses. These data suggest that the stimulation by both agents occurs through a common signaling pathway and confirm that adenosine is the single organic compound in the NA extract that increased Na+ transport. Fig. 7 represents the mean of six experiments.
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Discussion |
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It is remarkable that the activation of Na+ transport by
NA extract is exclusively due to an increase of apical Na+
channels, whereas the iNa and Po
decreased. The decrease in iNa probably results from cell
depolarization, as previously reported
(Van Driessche and Zeiske,
1985). In the present study, the depolarization was caused by an
increase in apical membrane Na+ conductance
(Fig. 4A). Possible mechanisms
that could explain the decrease of Po by 38% are, as yet,
unknown. Despite appreciable NA-extract-related decreases of
Po, the net stimulation of transport occurs as a result of
a considerable increase in No. Studies with forskolin,
which is known to increase cytosolic cyclic adenosine monophosphate (cAMP),
have shown similar effects (Els et al.,
1991
), suggesting a possible role for cAMP in the regulation of
Po. It is also possible that cytosolic Ca2+
causes this decrease in Po. In recent experiments with
ionomycin, elevation of cytosolic Ca2+ induced by this ionophore
led to a decrease of Po in A6 epithelia
(Helman et al., 1998
).
To elucidate the mechanisms of action of the NA extract on transepithelial Na+ transport in A6 cells, we attempted to identify the principle active compound(s) by using biochemical approaches: FPLC, RP-HPLC and mass spectrometry. The molecule was identified as adenosine (Fig. 6). The function tests with adenosine confirmed that it stimulates active Na+ transport in A6 epithelia and that its action blunted the effect of the NA extract (Fig. 7).
Adenosine regulates both Na+ uptake and Cl- secretion
in A6 cells, as in other epithelia. It has been reported that the regulation
of Cl- secretion is mediated by A1 adenosine receptors
located at the apical cell surface and transduced by Ca2+ release
from intracellular stores (Banderali et
al., 1999; Schwiebert et al.,
1992
). Similar results were shown by a metabolically stable
analogue of adenosine N6- cyclopentyladenosine (CPA),
which binds to both the A1 and A2 adenosine receptors in
A6 cells (Casavola et al.,
1996
) but has a higher affinity for the A1 receptors.
When CPA was added to the apical side, it induced an increase in
Cl- conductance by acting on the A1 receptors. Recently,
Di Sole et al. (1999
) reported
results indicating that stimulation of A3 receptors induced an
elevation of cytosolic Ca2+ in A6/C1 cells. These
findings were confirmed by Reshkin et al.
(2000
), who demonstrated the
presence of A3 receptors in the apical membrane. When added to the
basolateral side, adenosine increases Na+ transport by interacting
with basolaterally located A2 receptors. The activation of
Na+ conductance occurs through an adenylate cyclase-dependent
mechanism (Lang et al., 1985
).
Moreover, Dobbins et al.
(1984
) have demonstrated that
adenosine and some of its analogues increase cAMP levels, which results in
secondary Cl- secretion, indicating the presence of an
A2 adenosine receptor on rabbit ileum mucosal cells that activates
adenylate cyclase. On the other hand, adenosine stimulation of electrogenic
Na+ transport in renal cells has been demonstrated to occur, at
least in part, through Ca2+-dependent signal transduction events
and not through regulation of adenylate cyclase
(Hayslett et al., 1995
). The
observation that increased levels of intracellular Ca2+ correlated
with a two- to threefold increase in inositol (1,4,5)-trisphosphate suggests
that Ca2+ was released from intracellular stores. Furthermore,
Kurtz (1988) has demonstrated that, in isolated juxtaglomerular cells,
activation of A1 receptors is associated with an elevation of
cyclic guanosine-3',5'-monophosphate (cGMP) but not with changes
in either cytosolic Ca2+ or cAMP, suggesting the involvement of yet
another second messenger system for adenosine. An effect of adenosine on
intracellular Ca2+ was also found in experiments where
A3 receptors were stimulated by
2-chloro-N6-(3-iodobenzyl)-adenosine-5'-methyluronamide,
which activates Cl- secretion by Ca2+ and cAMP-regulated
channels.
In the present study, we did not find Cl- secretion induced by
adenosine or NA extract in A6 cells
(Fig. 2). On the other hand, we
have evidence that cAMP and/or Ca2+ activate Cl-
channels in the same clone of A6 cells
(Atia et al., 1999;
Zeiske et al., 1998
). It is
puzzling that, in this study, we found a marked activation of Na+
absorption without affecting Cl- secretion. The magnitude of the
effect on Na+ transport suggests that, if the stimulation occurs
through cAMP, its rise in cytosolic concentration should be significant and
probably sufficient to activate Cl- secretion. However, in
experiments where the additive effect of forskolin and adenosine was tested,
we found that forskolin could still markedly activate Na+ transport
in tissues pretreated with adenosine, whereas the effect of adenosine after
forskolin addition was rather small (F. Atia, unpublished observation). So, it
remains possible that the cAMP levels reached by adenosine or NA
extract treatment are still too small to activate Cl- conductance
but are sufficiently large enough to activate Na+ transport. This
issue requires further investigation and leaves the possibility open of the
involvement of a cAMP-independent pathway.
The present study is of importance because of the widespread use of spices and other plant products by humans. Plants have always been used in traditional medicine. Their effects may provide a source of inspiration for the development of new drugs based on careful scientific studies that are required to avoid adverse effects that may occur. In fact, there are numerous plants, including nigella, awaiting further investigation of their therapeutic potential.
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Acknowledgments |
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