Department of Biomedical Science, University of Sheffield, Sheffield S10 2TN, United Kingdom
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ABSTRACT |
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Hyposmotically induced swelling of frog renal proximal tubule
cells activates a DIDS-sensitive, outwardly rectifying Cl
conductance via a conventional protein kinase C (PKC). This study examines whether Na+-alanine cotransport similarly
activates a DIDS-sensitive Cl
conductance in frog renal
proximal tubule cells. On stimulation of Na+-alanine
cotransport, the DIDS-sensitive current
(IDIDS-Ala) increased markedly over time.
IDIDS-Ala exhibited outward rectification, a
Na+/Cl
selectivity ratio of 0.19 ± 0.03, and an anion selectivity sequence Br
= Cl
> I
> gluconate
. Activation of IDIDS-Ala
was dependent on ATP hydrolysis and PKC-mediated phosphorylation and
was inhibited by hyperosmotic conditions. Activation could be not
ascribed to a conventional PKC isoform, as
IDIDS-Ala was not affected by removing
Ca2+ or by phorbol ester treatment, suggesting a role for a
nonconventional PKC isoform, either novel or atypical. We conclude that
Na+-alanine cotransport activates a DIDS-sensitive
Cl
conductance via a nonconventional PKC isoform. This
contrasts with the hyposmotically activated Cl
conductance, which requires conventional PKC activation.
cell volume; chloride channels; cotransport; 4,4' diisothiocyanostilbene-2,2' disulfonic acid; protein kinase C
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INTRODUCTION |
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AS EPITHELIAL CELLS
ABSORB and secrete large quantities of solutes and water, the
composition of their intracellular environment and volume is constantly
under challenge. Accordingly, epithelial cells possess mechanisms to
regulate excessive changes in their volume (14). In most
cells, swelling activates solute exit pathways that drive the loss of
excess cell water, allowing cell volume to return to preswollen levels.
This is termed a regulatory volume decrease (RVD). Indeed, cell
swelling induced by hyposmotic media in renal proximal tubule cells of
different species activates both conductive and cotransport pathways,
allowing the efflux of K+, Cl and/or
HCO
Exposure of single frog renal proximal tubule cells to a hypotonic
shock leads to cell swelling followed by RVD (26). The RVD
is inhibited by barium (an inhibitor of K+ channels), DIDS
(an anion transport inhibitor), and gadolinium (Gd3+, an
inhibitor of stretch-activated channels) (26). On the
basis of these findings, it was suggested that hyposmotically
stimulated RVD was mediated by K+, Cl, and
nonselective stretch-activated channels (SACs). Indeed, two whole cell
conductances that are activated by hyposmolality and inhibited by
Gd3+ have been attributed to SACs in these cells
(27). An investigation into the identity of the
swelling-activated K+ conductance is ongoing (30,
32). In addition, a Cl
conductance that is
sensitive to DIDS and whose activity increases under hyposmotic
conditions (GDIDS-Hypo) has also been
characterized (28, 29). GDIDS-Hypo
exhibits outward rectification with an anion selectivity sequence
I
> Br
> Cl
(29). Furthermore, activation is dependent on ATP
hydrolysis and phosphorylation, mediated by a protein kinase C (PKC)-
and Ca2+-dependent mechanism (28, 29).
However, cells in vivo are rarely subjected to large changes in medium osmolality, except in the cases of specialized epithelia (e.g., renal medullary cells) or systemically during certain pathological conditions (6). In the case of renal proximal tubule cells, the more pertinent challenges to cell volume are changes in the rate of transepithelial flux of substrate (36). In general, transepithelial substrate flux is typified by concentrative, secondary active uptake from the tubule lumen followed by facilitated efflux at the basolateral face (34). The majority of concentrative substrate uptake from the lumen is energized by the Na+ gradient (3, 13, 15, 25, 33, 35). Any disparity in uptake at the apical membrane and efflux at the basolateral membrane will lead to changes in intracellular composition, and, consequently, cell volume. Therefore, cells must possess mechanisms to counteract excessive changes in their volume due to altered substrate uptake, a situation analogous to hyposmotically induced cell swelling and subsequent volume regulation (14).
In several cell types, Na+-amino acid cotransport has been found to induce cell swelling and subsequent volume regulation (8, 16, 18, 24). However, some apparent differences exist between the signaling of volume regulation stimulated by Na+-amino acid uptake and hyposmolality. In guinea pig jejunum and frog renal proximal tubule cells, RVD induced by hyposmotic shock is dependent on extracellular Ca2+, whereas RVD induced by Na+-alanine uptake is dependent on intracellular Ca2+ (19, 24, 26). Also, RVD in guinea pig jejunal enterocytes induced by Na+-alanine uptake is PKC dependent, whereas hyposmotically activated RVD is PKC insensitive (20).
The RVD that is observed after Na+-alanine cotransport stimulation in single proximal tubule cells isolated from frog kidney has previously been shown to be inhibited by DIDS, suggesting that cotransport-induced cell swelling activates an anion efflux pathway (24). A recent study has identified an anion-selective whole cell conductance (GVD) in single proximal tubule cells that is activated by stimulation of Na+-alanine cotransport, although its DIDS sensitivity was not investigated (31). The activity of GVD is both voltage and time dependent. It activates at potentials more positive than +60 mV and shows a characteristic slow time course of activation on stepping cell potential. As GVD is three times more selective for anions than cations, it could provide the anion efflux pathway during Na+-alanine-induced volume regulation. However, GVD is only observed at very positive, nonphysiological potentials. Therefore, another, as yet unidentified, anion conductance could be activated at more physiological potentials by stimulation of Na+-alanine cotransport. Certainly, hypotonic shock activates a DIDS-sensitive anion conductance at physiological potentials that has very different properties from those of GVD (28, 29). Therefore, the purposes of the following study were to investigate the effects of Na+-alanine cotransport on DIDS-sensitive currents in frog renal proximal tubule cells and the DIDS sensitivity of GVD.
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METHODS |
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Single proximal tubule cells were isolated from frog kidney by enzyme digestion, modified from the method of Hunter (9). Briefly, Rana temporaria were killed by decapitation, the brain and spinal column were destroyed, and the kidneys were removed. The excised kidneys were then perfused for 30 min with a divalent cation-free amphibian Ringer solution, containing (in mM) 101 NaCl, 3 KCl, and 10 NaHEPES (pH 7.4), to disrupt cell-to-cell tight junction integrity. The kidneys were then perfused retrogradely with 10 ml in total of the above solution plus collagenase (130 U/ml) and pronase (0.8 U/ml) to digest connective tissue. The kidneys were minced and further incubated in the enzyme mixture for 10 min at 20°C and gassed with 100% O2. A mixed cell population was liberated by trituration, resuspended in experimental bath solution, and kept on ice until required. Single proximal tubule cells were identified by their polarized morphology (26).
Suspensions of single cells were placed in a Perspex superfusion bath
at room temperature on the stage of an inverted microscope (Olympus
IX70). Whole cell currents were investigated by using standard
patch-clamp techniques (5). Patch pipettes were pulled from soda lime glass and had resistances of ~5 M when filled with
pipette solution. Whole cell patches were obtained via the basolateral
membrane, with access resistance being <25 M
. Success of achieving
the whole cell configuration was dependent on the effectiveness of
daily cell isolation and bath and pipette solutions, but when
conditions were optimum success rates of up to 90% were possible. In
all cases, patched cells were free floating. Voltage protocols were
driven from an IBM-compatible computer with a Digidata 1200 interface
using pCLAMP software Clampex6 (Axon Instruments). Cell recordings were
made via a Heka EPC-7 patch-clamp amplifier, subjected to low-pass
filtering at 5 kHz and saved directly to the computer hard drive. Data
were expressed as either current normalized to the cell capacitance or
conductance per unit cell area. Cell area was calculated from
capacitance, using the assumption that membrane capacitance
corresponded to 1 µF/cm2. Cell capacitance was
estimated from cancellation of slow capacitance transients, with mean
capacitance being 55.8 ± 1.6 pF (n = 142). To aid
capacitance transient cancellation, all patch pipettes were coated with
Sylgard (Dow Corning).
Solutions
Unless stated in the text, bath and pipette solutions were asymmetrical with regard to Na+ and ClExperimental Protocol: Effect of Alanine and Mechanism of Activation
When the whole cell configuration was obtained, residual currents were allowed to run down to steady state over ~2 min. Cells were clamped at a holding potential ofNa+ dependence and stereospecificity of IDIDS-Ala activation. When the sodium dependence of the activation of IDIDS-Ala was examined, NaCl in bath and pipette was replaced with equimolar amounts of N-methyl-D-glucamine (NMDG) Cl, and all solutions were titrated to pH 7.4 with free NMDG. The stereospecificity of activation by alanine was examined by the addition of 5 mM D-alanine to the bath (substitution of mannitol).
Osmotic sensitivity of IDIDS-Ala activation. The osmotic sensitivity of IDIDS-Ala activation was tested by exposing cells to 5 mM L-alanine under hyperosmotic conditions throughout (addition of 40 mM mannitol to bathing solutions).
ATP dependence of IDIDS-Ala activation. The dependence of IDIDS-Ala activation on ATP was investigated. The pipette contained either 2 mM MgATP (control), no ATP (addition of 2 mM Cs gluconate), 2 mM 5'-adenylylimidodiphosphate (AMP-PNP, nonhydrolysible ATP analog), or 2 mM MgATP plus 25 U/ml AP (to catalyze dephosphorylation).
PKC dependence of
IDIDS-Ala activation.
The dependence of IDIDS-Ala on PKC was examined
by inclusion of 10 µM PKC-ps (a broad-range PKC inhibitor), 0.6 µM
Gö 6983 (an inhibitor of PKC isoforms ,
,
,
, and
, but not µ) (4), or 2 µM Gö 6850 (an
inhibitor of PKC isoforms
,
,
,
,
, and
)
(7) in the pipette. In a second series of experiments,
activation of PKC was examined by including 0.1 µM phorbol
12-myristate 13-acetate (PMA, activator of conventional and novel
isoforms) in the bath (7). To allow full activation of
PKC, cells were preincubated in experimental solutions for ~10 min.
Ca2+ dependence of IDIDS-Ala activation. To test the dependence of IDIDS-Ala on extracellular Ca2+, CaCl2 was omitted from the bathing solution. To test the dependence of IDIDS-Ala activation on intracellular Ca2+, 5 mM EGTA was included in the pipette solution to buffer intracellular Ca2+.
Experimental Protocol: Properties of IDIDS-Ala
Na+/Cl selectivity
ratio.
The Na+/Cl
selectivity ratio of the
DIDS-sensitive current was examined in the asymmetrical solutions
described earlier, with extracellular and intracellular concentrations
of Na+ being 98 and 29 mM, respectively, and those of
Cl
being 98 and 20 mM, respectively. Clamp potential was
stepped between +40 mV and
100 mV, in
10-mV steps in the absence
and presence of L-alanine. The current sensitive to 100 µM DIDS was measured under both circumstances. The reversal potential
(Vrev) of IDIDS-Ala was
determined by using polynomial regression with either 80 mM
Cs-gluconate or 80 mM NMDG-gluconate in the pipette. The
Na+/Cl
selectivity ratio was calculated by
using the Goldman equation. This calculation used the measured
selectivity ratio for gluconate (see RESULTS) and, as the
Vrev of the conductance did not alter between
Cs+ and NMDG+ pipette solutions (see
RESULTS), permeation of Cs+ through the
conductance was assumed to be minimal and was omitted from the
selectivity calculation.
Current-voltage relationship.
The current-voltage relationship of IDIDS-Ala
was investigated by using symmetrical Cl solutions. The
bath solution was a high-Na+, high-Ca2+
amphibian Ringer containing (in mM) 95 NaCl, 2 CaCl2, 1 MgCl2, 5 mannitol, and 10 HEPES, titrated to pH 7.4 with
NaOH. The pipette contained a low-Na+, nominally
Ca2+-free amphibian Ringer containing (in mM) 99 CsCl, 0.5 EGTA, 2 MgATP, and 10 HEPES, titrated to pH 7.4 with NaOH. On
achievement of the whole cell configuration, residual currents were
allowed to run down to steady state. Whole cell potential was held at
70 mV and stepped between +15 and
60 mV in
5-mV steps. DIDS sensitivity was tested before 5 mM L-alanine superfusion
(control) and when currents had activated to a steady state in the
presence of L-alanine. Outward slope conductance was
determined between +15 and +5 mV, whereas inward slope conductance was
determined between
5 and
60 mV. Conductance was expressed as a
function of cellular area (µS/cm2).
Anion selectivity.
Anion selectivity was determined, in the presence of 5 mM
L-alanine, from changes in the Vrev
of IDIDS-Ala when 95 mM NaCl in the bathing
solution was substituted with 95 mM NaBr, NaI, or Na-gluconate. Clamp
potential was varied between +15 and 60 mV in
5-mV steps in the
presence of Cl
, Br
, or I
, or
varied between +70 and
20 mV in
10-mV steps in the presence of
gluconate
, all from a holding potential of
70 mV. When
the +70 to
20-mV voltage step protocol was employed, each voltage
step was separated by 10 s, as opposed to 1 s, to allow
inactivation of the voltage-activated conductance
GVD, which is activated at by potentials over
+60 mV (27). To rule out any contamination by
GVD in the gluconate selectivity calculation,
the effect of DIDS on GVD was examined at +70 mV
in the presence of L-alanine. Current was recorded
initially on stepping the potential to +70 mV and then within 20 ms of
the end of the potential step, in the absence and presence of 100 µM
DIDS. The difference between the current recorded initially on stepping
the potential and the current recorded at the end of the potential step
(after full activation of GVD) was
taken as an index of GVD (see Fig. 4,
inset). Selectivity was determined from shifts in
Vrev by using the Goldman equation.
Vrev was calculated from polynomial regression
of IDIDS-Ala. For all selectivity calculations, the Vrev for the experimental anion was compared
with the Vrev measured in the presence of
Cl
immediately before bath solutions were changed.
Vrev was corrected for junction potential
changes associated with bath Cl
substitution. Junction
potential changes were determined by using a flowing 3 M KCl reference
electrode and were found to be 1.8, 10.1, and 10.7 mV for substitution
of Cl
with Br
, I
, and
gluconate
, respectively.
Dose response to DIDS.
To test the nature of the inhibition of
IDIDS-Ala induced by DIDS, the effect of a range
of concentrations of DIDS, from 2 to 500 µM, was tested for their
potency of inhibition. The bath and pipette solutions were the
asymmetrical solutions described earlier. Whole cell currents were
allowed to activate to steady state in response to 5 mM
L-alanine. The voltage protocol utilized a holding
potential of 70 mV and was stepped to +40, 0, and
40 mV. The data
were fitted to the Hill equation (Eq. 1)
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(1) |
Gd3+ sensitivity of
IDIDS-Ala.
A previous study has demonstrated that the hyposmotically activated
Cl conductance is inhibited by Gd3+
(28). The sensitivity of IDIDS-Ala
to Gd3+ was examined at a clamp potential of +39 mV after
the current had activated to a steady state in the presence of 5 mM
L-alanine. The magnitude of the DIDS-sensitive current was
measured before and during exposure of the cells to 100 µM
GdCl3.
Role of PKC in Na+-Alanine-Induced RVD
The length of intact cells was monitored by using a photodiode array as described by Mounfield and Robson (24). Briefly, the length of cells adhered to a coverslip was determined by measuring the distance between the changes in light intensity that occur at the start and end of the cell. The bathing solution was an amphibian Ringer containing (mM) 90 NaCl, 3 KCl, 2 CaCl2, 1 MgCl2, 15 mannitol, and 10 HEPES, titrated to pH 7.4 with NaOH. RVD induced by 5 mM L-alanine (equimolar substitution of mannitol) was examined under the control circumstance, in the presence of 0.1 µM PMA or in the presence of 2 µM Gö 6850 (a cell-permeable inhibitor of PKC isoformsSimultaneous Cell Length and Patching
To determine whether stimulation of Na+-alanine cotransport altered cell length in the whole cell configuration, simultaneous measurement of cell length and whole cell current recordings were carried out in response to addition of 5 mM L-alanine to the extracellular solution. Whole cell recordings were obtained by patching the side of free-floating cells and current recorded atStatistical Significance
Statistical significance was determined by using Student's t-test or ANOVA, as appropriate, and was assumed at the 5% level. All values are expressed as means ± SE. ![]() |
RESULTS |
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Na+-Alanine Cotransport Current
Application of 5 mM L-alanine to frog renal proximal tubule cells induced large instantaneous increases in the whole cell currents when recorded at
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Mechanism of Activation of IDIDS-Ala
Effect of alanine on IDIDS-Ala. In control conditions, the DIDS-sensitive current recorded at +39 mV was small (Fig. 1). On application of 5 mM L-alanine, there was a progressive increase in the DIDS-sensitive current to a plateau, which subsequently decreased when L-alanine was removed from the bath solution (Fig. 1). In paired cells, the DIDS-sensitive current was 0.04 ± 0.02 pA/pF in the control bath, increasing to 2.64 ± 0.49 pA/pF in the presence of 5 M L-alanine, 36 ± 3 min after L-alanine superfusion was started (n = 10). Subsequent removal of L-alanine from the bathing solution caused the DIDS-sensitive current to decrease slowly again to a steady state of 0.86 ± 0.22 pA/pF, which was significantly lower than the peak DIDS-sensitive current in the presence of L-alanine, but remained significantly higher than prestimulation values. The DIDS-sensitive current activated by alanine, IDIDS-Ala, was taken as the difference between the DIDS-sensitive current recorded under the control conditions and at steady state in the presence of alanine (Table 1).
Na+ dependence and stereospecificity of
IDIDS-Ala activation.
Application of 5 mM L-alanine to proximal tubule cells
activated IDIDS-Ala. This activation was
dependent on the presence of extracellular Na+ and was
stereospecific. When NMDG+ was substituted for
Na+, IDIDS-Ala was still capable of
activation. However, this was significantly reduced compared with
Na+-containing solutions (Table 1). Five millimolar
D-alanine significantly activated
IDIDS-Ala, although at a level reduced from that
induced by L-alanine (Table 1). Furthermore, the magnitude
of IDIDS-Ala activation varied proportionately
with either L- or D-alanine cotransport
currents (Fig. 2). When
IDIDS-Ala was calculated as a function of the
Na+-alanine cotransport current
(IDIDS-Ala divided by the
Na+-alanine cotransport current), there was no significant
difference between the DIDS-sensitive current activated by either
L- or D-alanine. (IDIDS-Ala = 1.23 ± 0.17 and
1.10 ± 0.24 pA/pF per pA/pF Na+-alanine cotransport
current for L- and D-alanine, respectively.)
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Osmotic sensitivity of IDIDS-Ala activation. IDIDS-Ala activation was completely blocked under hyperosmotic conditions compared with isosmotic conditions (Table 1).
ATP dependence of IDIDS-Ala activation. Activation of IDIDS-Ala was dependent on the presence of ATP (Table 1). Omission of MgATP from the pipette solution abolished activation of IDIDS-Ala. Activation of IDIDS-Ala was similarly inhibited by replacement of pipette MgATP with 2 mM AMP-PNP. The addition of 25 U/ml AP to the pipette solution, to stimulate dephosphorylation, in the presence of 2 mM MgATP gave rise to significant activation of IDIDS-Ala in response to 5 mM L-alanine, although activation was reduced compared with 2 mM MgATP alone.
PKC dependence of IDIDS-Ala activation. Activation of IDIDS-Ala was dependent on the stimulation of PKC (Table 1). The broad range PKC inhibitor PKC-ps (10 µM) significantly blunted IDIDS-Ala activation. The PKC isoform-specific inhibitors Gö 6983 (0.6 µM) and Gö 6850 (2 µM) also inhibited activation. However, inclusion of 0.1 µM PMA in bath and pipette solutions had no effect on activation.
Ca2+ dependence of IDIDS-Ala activation. Removal of extracellular Ca2+ had no effect on IDIDS-Ala (Table 1). This was also true for increased buffering of intracellular Ca2+ by increasing pipette EGTA from 0.5 to 5 mM (Table 1).
Properties of IDIDS-Ala
Na+/Cl
selectivity ratio.
In asymmetrical solutions with Cs-gluconate in the pipette, the
Vrev of IDIDS-Ala was
18.8 ± 1.4 mV (n = 6). This was not significantly different from the Vrev recorded
when Cs-gluconate was replaced with NMDG-gluconate (
20.9 ± 2.6 mV, n = 4). Combining these data gave a mean
Vrev of
19.6 ± 1.2 mV and a
Na+/Cl
selectivity ratio of 0.19 ± 0.03 (n = 10).
Current-voltage relationship.
In symmetrical solutions, IDIDS-Ala demonstrated
outward rectification (Fig. 3).
GDIDS-Ala: out and
GDIDS-Ala: in were 95.99 ± 10.64 and 30.33 ± 3.63 µS/cm2,
respectively (n = 24). The reversal potential of
IDIDS-Ala in symmetrical Cl was
found to be 4.31 ± 0.42 mV. This was not significantly different from 4.17 mV, which corresponds to the calculated reversal potential, taking the partial Na+ permeability into account.
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Anion selectivity.
The anion selectivity sequence of IDIDS-Ala was
Br = Cl
> I
>>
gluconate (Table 2).
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Contribution of GVD.
The voltage- and time-dependent conductance GVD
was not sensitive to DIDS (Fig. 4). There
was no significant difference between the activation of
GVD in the absence and presence of DIDS. The magnitude of current flow through GVD at +70 mV
was 11.1 ± 1.9 pA/pF in the absence of DIDS and 11.9 ± 1.5 pA/pF in its presence (n = 5).
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Dose-response to DIDS.
Inhibition of IDIDS-Ala by DIDS was dose
dependent (Fig. 5). There were no
significant differences between the Hill coefficients determined at
+40, 0, and 40 mV: 1.6 ± 0.4, 1.0 ± 0.2, and 1.8 ± 0.7, respectively (n = 7). These values were also not
significantly different from one. However, inhibition was voltage
dependent. The Kd values determined at +40, 0, and
40 mV were 4.8 ± 1.2, 13.0 ± 3.1, and 26.9 ± 3.1 µM, respectively (n = 7). These values were
significantly different from each other
(F2,18 = 21.06).
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Sensitivity of IDIDS-Ala to Gd3+. IDIDS-Ala was inhibited by Gd3+. Maximally activated DIDS-sensitive current before addition of Gd3+ to the bath was 3.76 ± 1.28 pA/pF (n = 5). This was reduced to 1.54 ± 0.64 pA/pF in the presence of Gd3+ (n = 5).
Role of PKC in Na+-Alanine-Induced RVD
Addition of L-alanine to the cells initially increased cell length to a peak (Fig. 6). This was followed by decrease in length back toward prealanine levels (Fig. 6). Neither the degree of cell swelling (a), nor the magnitude of RVD during L-alanine superfusion (b), nor its rate (T1/2) was affected by PMA (Table 3). However, RVD was inhibited by the presence of the PKC inhibitor Gö 6850 (Table 3).
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Simultaneous Cell Length and Patching
Addition of L-alanine to the extracellular solution increased both the whole cell current recorded at
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DISCUSSION |
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Application of alanine to frog renal proximal tubule cells induced instantaneous currents. The induced current was found to be Na+ dependent and stereoselective for L-alanine over D-alanine. These findings suggest that the instantaneous current generated by alanine is due to Na+-alanine cotransport, in common with mammalian proximal tubule (3, 13). Indeed, the instantaneous current generated by application of alanine (Fig. 1) cannot be attributed to a K+ conductance as K+ was absent from all solutions. Similarly, the current cannot be attributed to a nonselective conductance, GVI, identified in these cells as it has been shown to be unaffected by alanine application (31). Furthermore, DIDS or Gd3+ did not inhibit the alanine current.
Stimulation of the Na+-alanine cotransporter subsequently
caused reversible activation of a predominantly
Cl-selective conductance,
IDIDS-Ala. There are several pieces of evidence
that support this conclusion: 1)
IDIDS-Ala was more selective for anions, with a
Na+/Cl
selectivity ratio of 0.19, suggesting
that the conductance was five times more selective for Cl
than Na+; 2) the conductance was sensitive to
the anion transport blocker DIDS; 3) removal of
extracellular Na+ or addition of the poorly transported
isomer of alanine (D-alanine) reduced the activation of
IDIDS-Ala; and 4) the magnitude of
IDIDS-Ala activation was proportional to the
Na+-alanine cotransport current, which was found to be the
case irrespective of whether cells were exposed to L- or
D-alanine. Given that D-alanine is poorly
metabolized by cells, these findings would suggest that activation of
IDIDS-Ala by alanine was directly due to
Na+-alanine cotransport, rather than secondary metabolism
of alanine leading to activation. This appears to contrast with
the finding that D-alanine caused no significant swelling
of frog renal proximal tubule cells (24). However, given
that D-alanine induces only 10% of the increase in
IDIDS-Ala compared with L-alanine, a
comparable increase in cell length induced by D-alanine
would be ~0.05 µm. This value is on the limit of detection of the
optical technique used in the previous study to assess cell volume
changes (26). Taken together, these data show that
stimulation of Na+-alanine cotransport activated a
predominantly Cl
-selective conductance in the cells.
Properties of the DIDS-sensitive conductance.
The DIDS-sensitive current activated during application of 5 mM
L-alanine exhibited outward rectification in symmetrical
Cl solutions and was sensitive to DIDS in a
concentration-dependent fashion. These characteristics are also shared
by several volume-sensitive Cl
conductances in different
cell types that are activated in hyposmotic conditions
(37), including frog renal proximal tubule cells (29).
Mechanism of activation of the DIDS-sensitive conductance. Activation of IDIDS-Ala was inhibited by a hypertonic bath solution, suggesting that activation was dependent on cell swelling. Indeed, simultaneous cell length and patching experiments demonstrated that stimulation of Na+-alanine cotransport during whole cell patching was still capable of increasing cell length. However, it should be noted that the magnitude of swelling observed in patched cells (Fig. 7) was approximately four times that observed in intact cells. This can be explained by the fact that K+ was replaced in the pipette solution by Cs+, thereby meaning that the patched cells would be incapable of undergoing RVD. Indeed, the magnitude of cell swelling observed in patched cells is comparable to that in intact cells that fail to exhibit RVD (24).
Activation of IDIDS-Ala was dependent on ATP hydrolysis. This is in common with several other volume-sensitive ClComparison with other anion-selective conductances in the cells.
Previous studies have identified two other anion conductances in these
cells. One of the conductances, GVD, is also
activated by stimulation of Na+-alanine cotransport and is
predominantly Cl selective (3 times more selective for
anions over cations) (27, 31). However, it is unlikely
that IDIDS-Ala is attributable to
GVD. There are several pieces of evidence to
support this. In the first instance, GVD is only
observed at potentials more positive than +60 mV and shows a
characteristic slow, time-dependent activation on stepping cell
potential (Fig. 4). IDIDS-Ala was active at all
potentials tested and did not show any time-dependent activation on
stepping cell potential. In addition, the activation of
GVD by alanine does not require intracellular
ATP (31). Activation of IDIDS-Ala
has an absolute requirement for ATP. Finally,
IDIDS-Ala is inhibited by DIDS, whereas
GVD is DIDS insensitive.
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ACKNOWLEDGEMENTS |
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We are grateful to the Wellcome Trust and the National Kidney Research Fund for financial support. We also thank Judith Hartley for excellent technical assistance.
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FOOTNOTES |
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Address for reprint requests and other correspondence: L. Robson, Dept. of Biomedical Science, Univ. of Sheffield, Western Bank, Sheffield S10 2TN, UK (Email: l.robson{at}sheffield.ac.uk).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 5 September 2000; accepted in final form 7 December 2000.
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