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MS 9156 Received 18 January 1999; accepted 4 February 1999.
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ABSTRACT |
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 TopAbstract IntroductionMethodsResultsDiscussionReferences |
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INTRODUCTION |
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In general, cell volume is actively maintained despite variations in extracellular osmolarity or transmembrane ion fluxes. Two types of volume-regulatory mechanisms have been identified: regulatory volume increase (RVI), which occurs in response to cell shrinkage, and regulatory volume decrease (RVD), seen after cell swelling (Hoffmann, 1991). Both RVI and RVD occur as a consequence of the movement of osmotically active solutes, which are followed by water. Therefore, by accumulating solutes, cells can increase their volume and, by voiding solutes, cells can decrease their volume. In RVI, cells typically accumulate Na+ and Cl- ions, while in RVD they typically void K+, Cl- and HCO3-. Studies investigating the mechanisms underlying volume regulation in renal cells have demonstrated that RVD involves the activation of both conductive and cotransport mechanisms for K+, Cl- and HCO3- (Kirk et al. 1987; Volkl & Lang, 1988; Hoffmann, 1991).
Several previous investigations into the mechanisms underlying RVD have utilized hypotonic shock to initiate cell swelling. Such studies in renal epithelia have demonstrated that hypotonic shock-induced RVD involves the activation of both conductive and cotransport mechanisms for K+, Cl- and HCO3- (Kirk et al. 1987; Volkl & Lang, 1988; Hoffmann, 1991). Single proximal tubule cells isolated from frog kidney are also capable of hypotonic shock-induced RVD (Robson & Hunter, 1994b). In these cells, hypotonic shock-induced cell swelling activates non-selective stretch-activated channels (SACs). These allow Ca2+ to move into the cell leading to a rise in intracellular Ca2+. Ca2+ then acts either directly, on Ca2+-sensitive channels, or indirectly, through activation of protein kinase C (PKC) and phosphorylation, to activate K+ and Cl- channels. K+ and Cl- exit the cell, followed by water, and cell volume goes down.
Although hypotonic shock is a popular tool for the examination of the mechanisms underlying RVD, in the physiological context, most cells will never be exposed to such an extreme alteration in osmolarity. This begs the question as to what is the physiological role for volume regulatory mechanisms? One suggestion is that, in both secretory and reabsorptive epithelia, volume-regulatory mechanisms may play a role in the maintenance of intracellular composition and cell volume in the face of changes in transepithelial transport (Beck et al. 1994; Schultz, 1994). In keeping with this, a recent study has demonstrated that RVD can be elicited in single proximal tubules isolated from frog kidney in response to Na+-alanine cotransport-induced cell swelling (Mounfield & Robson, 1998). However, while the volume responses of the cells are similar to those observed on exposure to a hypotonic shock, i.e. an initial swelling phase followed by recovery, the cellular mechanisms involved in alanine-induced RVD seem to be different. Whilst hypotonic shock-induced RVD is dependent on the presence of extracellular Ca2+ (Robson & Hunter, 1994b), alanine-induced RVD involves the release of Ca2+ from intracellular stores (Mounfield & Robson, 1998). However, despite the fact that it occurs in the absence of extracellular Ca2+, alanine-induced RVD is inhibited by Gd3+, suggesting that activation of a Gd3+-sensitive conductance is required for volume regulation to occur.
At the present time three volume-sensitive conductances have been identified which may play a role in hypotonic shock-induced RVD. One conductance is a DIDS-sensitive Cl- conductance and may provide a Cl- efflux pathway for hypotonic shock-induced RVD (Robson & Hunter, 1994a, 1997). The other two conductances are gadolinium (Gd3+) sensitive and Ca2+ permeable and may provide Ca2+ influx pathways during hypotonic shock-induced RVD (Robson & Hunter, 1994c). The aim of the following study was to examine whether stimulation of Na+-alanine cotransport modulated the activity of these two Gd3+-sensitive conductances.
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METHODS |
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Cell isolation
Single proximal tubule cells were isolated by enzyme digestion from kidneys of Rana temporaria as described previously (Hunter, 1989). Frogs were killed by decapitation and the brain and spinal cord destroyed. Kidneys were removed and perfused with a divalent cation-free Ringer isolation solution which contained 101 mM NaCl, 3 mM KCl and 10 mM Hepes (titrated to pH 7·4 with NaOH) to promote dissociation of the tight junctions. Kidneys were then injected with a mixture of collagenase and pronase, minced and then shaken in a water bath for 10 min. After this period the tissue suspension was removed and single cells sheared from the kidney fragments by trituration. Enzyme digestion was halted by a series of centrifugation and re-suspension steps and the final cell suspension stored on ice. This technique resulted in the generation of a mixed population of single cells derived from the whole kidney. Proximal cells were identified from their 'Snowman' like appearance (Hunter, 1990).
Patch clamp
A suspension of single cells was placed in a Perspex bath on the stage of an inverted microscope (Olympus IX70 or Nikon Diaphot) and standard patch clamp techniques employed to investigate whole cell currents (Hamill et al. 1981). Voltage protocols were driven from an IBM-compatible computer equipped with a Digidata or TL-1 interface, using the pCLAMP software, Clampex (Axon Instruments). Recordings were made using a List EPC-7 amplifier. To reduce stray capacitance and associated noise, patch pipettes were coated with Sylgard (Dow Corning Corp.). Whole-cell patches were obtained via the basolateral aspect of the cells and currents saved directly onto the hard disk of the computer following low-pass filtering at 5 kHz. Cell potential was held at -40 mV and then initially stepped to potentials between +100 and -100 mV in -20 mV steps. This was followed by a second fixed step to +100 mV (Fig. 1). We have previously reported that the voltage- and time-dependent conductance GVD takes several seconds to completely deactivate. Therefore, to avoid problems of incomplete deactivation, an interpulse interval of 15 s was utilized. Mean steady-state currents were derived at the end of each potential step using the pCLAMP software, Clampfit (Axon Instruments) and Excel version 7.0 (Microsoft). Reversal potentials (Vrev) of ohmic and rectifying currents were calculated by linear or polynomial regression, respectively. The time constant of activation of GVD (
act) was derived from a single exponential fit to the rising phase of the current. As the activation of GVD is voltage dependent, the effect of L-alanine on this conductance was determined by looking at the changes in whole cell currents at +100 and +80 mV (potentials at which the conductance was activated). These currents were also normalized with respect to the surface area of the cell. In contrast, over the range +20 to -100 mV, potentials at which GVI is typically observed, currents were ohmic. Therefore these data are given as whole cell conductance (G20-100, see Whole-cell current characteristics in Results) and were normalized with respect to the area of the cell. Cell area was calculated from the capacity transients seen in response to a +20 mV potential step, and membrane capacitance assumed to be 1 µF cm-2.
Solutions
The pipette solution was a low Ca2+, high K+ amphibian Ringer solution that contained (mM): 100 KCl, 1 MgCl2 and 10 Hepes (titrated to pH 7·4 with KOH). Pipette Ca2+ was not buffered so as to allow changes in intracellular Ca2+. The control bath solution was a high Ca2+, high Na+ amphibian Ringer solution that contained (mM): 97 NaCl, 3 KCl, 2 CaCl2, 1 MgCl2, 10 Hepes (titrated to pH 7·4 with NaOH) and 10 mannitol. The osmolality of all solutions was measured (Roebling osmometer) and adjusted to within 1 mosmol (kg H2O)-1 of 215 mosmol (kg H2O)-1 with water or mannitol as appropriate. All chemicals were obtained from Sigma, and were of analytical grade.
Experimental protocol
Whole-cell patches were obtained whilst cells were superfused with the control solution and then 5 mM L-alanine added to the bathing solution (substitution of mannitol). Once currents reached a steady state L-alanine was washed from the bath using control Ringer solution. L-Alanine activated a voltage-dependent current, which looked similar in characteristics to GVD (see Results); therefore a second series of experiments examined the effect of 10 µM Gd3+ on the alanine-activated current in paired cells. To determine whether the activation of GVD by L-alanine was dependent on a change in cell volume, paired cells were exposed to L-alanine initially in the absence and then in the presence of a hyperosmotic solution (control Ringer solution plus 40 mM mannitol).
To test whether the stimulatory effect of L-alanine was due to a direct action of L-alanine itself on the conductance, whole cell patches were obtained as described previously. However, either 5 mM mannitol or 5 mM L-alanine was added to the pipette solution. Currents were recorded in the control Ringer solution and then with 10 µM Gd3+ added to the bathing solution.
Statistics
All values are given as the mean ± 1 S.E.M. Significance was tested using Student's t test and significance assumed at the 5 % level.
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RESULTS |
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Whole cell current characteristics
As previously reported (Robson & Hunter, 1994c), two distinct current types are present in the whole cell recordings, resulting in outward rectification. At potentials more positive than +60 mV, currents demonstrated a voltage- and time-dependent activation, characteristic of GVD (Fig. 1). On the other hand, the currents between +20 and -100 mV, voltages at which GVI is evident, were time independent.
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Upper panel: left, whole cell currents recorded in the absence of L-alanine; right, whole cell currents from the same cell recorded in the presence of 5 mM L-alanine. The lower panel shows (left) the I-V curves recorded from these traces ( | ||
Effect of L-alanine on whole cell currents
L-Alanine increased the magnitude and altered the Vrev of whole cell currents in a depolarizing direction, consistent with activation of the Na+-alanine cotransporter (Fig. 1). The Vrev of currents recorded in the absence of alanine was -36·0 ± 4·91 mV (n = 15). On addition of L-alanine to the bath the Vrev shifted to +12·9 ± 4·22 mV. On washout of alanine, Vrev did not recover completely, although it returned to within 83 % of the pre-alanine level (-27·6 ± 4·5 mV). L-Alanine also reversibly increased whole cell currents at +100 and +80 mV (Fig. 2), potentials at which GVD was activated. Similarly, at those potentials where GVI is typically observed, +20 to -100 mV, whole cell currents were also increased in a reversible manner by L-alanine (Fig. 2). G20-100 increased from 17·8 ± 2·6 µS cm-2 in control solution to 33·8 ± 3·1 µS cm-2 in the presence of L-alanine.
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Current (in nA cm-2) is shown against bath solution. The upper panel shows data obtained at those potentials where GVD was activated, while the lower panel shows potentials at which GVI was activated. The asterisks indicate a significant difference to control for all potentials tested. | ||
L-Alanine increased the rate of activation of GVD (Fig. 3). In the absence of alanine the act at +100 mV was 899·4 ± 151·6 ms (n = 18). On switching to 5 mM L-alanine it was significantly reduced to 365·0 ± 26·6 ms. In addition, L-alanine decreased the potential at which GVD was observed (Fig. 4).
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The figure shows whole cell current (pA) against time (ms) for current traces obtained from a representative cell in the absence and presence of L-alanine and the exponential fit for each trace. | ||
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The graphs show the mean ± S.E.M. whole cell current recorded at each potential over the range +100 to +20 mV either in the absence (A) or presence (B) of L-alanine (n = 15). In both graphs | ||
Effect of Gd3+ on the L-alanine-stimulated currents
The whole cell currents have several components, including currents from the Na+-alanine cotransporter, GVD, GVI and leak currents. To determine GVD and GVI, Gd3+ was added to the bath solution. The presence of GVD was indicated by Gd3+-induced decreases in the whole cell currents recorded at +100 and +80 mV (Table 1 and Fig. 5). The presence of GVI was indicated by Gd3+-induced decreases in G20-100: 16·3 ± 2·9 and 9·23 ± 1·33 µS cm-2 (n = 14) in the absence and presence of Gd3+, respectively. Over the range +20 to -100 mV, the Gd3+-sensitive current demonstrated characteristics similar to those reported previously (Robson & Hunter, 1994c). The Vrev, -29·4 ± 7·6 mV (n = 14), was not significantly different to the Vrev of GVI (Robson & Hunter, 1994c). In addition, the slopes of the I-V curves of the two Gd3+-sensitive currents were not significantly different: 0·29 ± 0·08 nS (n = 14) in the present experiments versus 0·26 ± 0·03 nS (n = 5) in the previously reported experiments.
Table 1. Effect of Gd3+ and hypertonic shock (Hyper) on whole cell currents
| Vc (mV) | +100 mV | +80 mV |
| Control (nA cm-2) | 3404 ± 449 | 1890 ± 301 |
| Gd3+ (nA cm-2) | 1164 ± 210 * | 823 ± 176 * |
| Control (nA cm-2) | 3011 ± 401 | 1177 ± 210 |
| Hyper (nA cm-2) | 2668 ± 509 | 1431 ± 298 |
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A and B, I-V curves recorded in the same cell in the absence (A) or presence (B) of 10 µM Gd3+. In both graphs | ||
In paired cells, the increase in whole cell current magnitude typically observed with L-alanine at +100 and +80 mV was significantly inhibited in the presence of Gd3+ (Fig. 5). In the absence of Gd3+, L-alanine increased whole cell current by 2155 ± 407 nA cm-2 at +100 mV and 1130 ± 264 nA cm-2 at +80 mV (n = 14). These increases were abolished by Gd3+: 6·35 ± 184 and -167 ± 90 nA cm-2 at +100 and +80 mV, respectively. In contrast, the alanine-induced increase in G20-100 was unaffected by the addition of Gd3+ to the bathing solution: 15·8 ± 2·1 versus 13·2 ± 1·0 µS cm-2 (n = 14), in the absence and presence of Gd3+, respectively.
Reversal potential of alanine-activated current. The Vrev of the alanine-activated current was +45·5 ± 5·4 mV. With Gd3+ this shifted in a positive direction to +123·2 ± 16 mV (n = 14) (Fig. 5).
Effect of hypertonic shock
Addition of a hypertonic solution to the bath had no effect on the currents recorded at +100 and +80 mV (n = 9) (Table 1). G20-100 was similarly unaffected by the hypertonic solution: 11·1 ± 1·1 and 13·9 ± 2·5 µS cm-2 (n = 9) in control and hypertonic solutions, respectively.
In paired cells, the increase in whole cell current observed at +100 and +80 mV with L-alanine was significantly reduced in the presence of a hypertonic solution (Fig. 6). Under control conditions the increase in current was 2865 ± 460 and 1430 ± 183 nA cm-2 (n = 9), at +100 and +80 mV, respectively. In the presence of a hypertonic bath solution, current increases were only 1611 ± 511 and 511 ± 249 nA cm-2 (n = 9), at +100 and +80 mV, respectively. The alanine-induced increase in G20-100 was unaffected by the presence of a hypertonic solution: 16·5 ± 1·98 versus 16·5 ± 1·96 µS cm-2 (n = 9).
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I-V curves recorded in the same cell in the absence (A) or presence (B) of a hypertonic solution. In both graphs | ||
Effect of pipette L-alanine on GVD
With 5 mM mannitol in the pipette solution the Gd3+-sensitive currents recorded at +100 and +80 mV were 2318 ± 529 and 1012 ± 272 nA cm-2 (n = 12), respectively. These were not significantly different from the Gd3+-sensitive currents recorded with 5 mM L-alanine in the pipette solution: 2269 ± 1156 and 716 ± 307 nA cm-2 (n = 5).
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DISCUSSION |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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The addition of L-alanine to single proximal tubule cells isolated from frog kidney increased the magnitude of whole cell currents and also depolarized the reversal potential of these currents. This is consistent with activation of the Na+-alanine cotransporter, which would be expected to drive the Vrev of whole cell currents towards the equilibrium potential for Na+, a positive value in this study. However, while a large part of the L-alanine-activated current was composed of the co-transporter current, alanine also activated a Gd3+-sensitive conductance. In the absence of Gd3+ the Vrev of the alanine-activated current was +45·5 mV, while in the presence of Gd3+ it shifted closer to the equilibrium potential for Na+ (+123 mV). Therefore, the alanine-activated current is composed of at least the cotransporter current and the current flowing through a Gd3+-sensitive current.
The study provides evidence that, on stimulation of Na+-alanine cotransport, it is the voltage-dependent, Gd3+-sensitive conductance GVD that is activated. There are several pieces of evidence that support this statement. In the first instance, Gd3+, an inhibitor of GVD (Robson & Hunter, 1994c), prevented the alanine-induced increase in whole cell current at +100 and +80 mV. Second, the time constant of activation of GVD was reduced on exposure to alanine, thus the conductance activated faster. Third, the threshold potential at which GVD was observed was shifted from +80 mV in control cells to +60 mV in the presence of alanine. Finally, the shape of the Gd3+-sensitive component of the alanine-activated current, Fig. 5D, is characteristic of GVD, i.e. outwardly rectifying.
Stimulation of substrate transport in a variety of cell types leads to the activation of several different types of ion channels. In hepatocytes activation of Na+-alanine cotransport causes a loss of Cl- from the cells, which appears to be a consequence of the activation of outwardly rectifying Cl- channels (Wang & Wondergem, 1993; Lidofsky & Roman, 1997). Similarly, addition of glucose to rat pancreatic 
-cells or glycine to Ehrlich ascites tumour cells activates a Cl--selective conductance (Hudson & Schultz, 1988; Best, 1997). In contrast, in proximal tubule cells isolated from Rana pipiens Na+-coupled transport activates a mechanosensitive K+ conductance, while in intestinal cells evidence exists for the activation of a H+ conductance (Cemerikic & Sackin, 1993; MacLeod & Hamilton, 1997). GVD seems to be distinct from the previously described substrate-activated H+ and K+ conductances. It is not H+ permeable, nor does it discriminate between Na+ and K+ (Robson & Hunter, 1994c). However, it is 3 times more selective for anions over cations (Robson & Hunter, 1994c). This is similar to the Cl- channel activated by Na+-coupled glycine transport in Ehrlich ascites tumour cells, which is 11 times more selective for Cl- over K+ (Hudson & Schultz, 1988). The greater anion-to-cation selectivity of GVD suggests that its role in Ca2+ entry may be limited, and it may function as a Cl- efflux pathway.
The characteristics of GVD, slow time-dependent activation and activation by depolarization, are similar to a stretch-activated channel observed previously on the basolateral membrane of frog proximal cells (Hunter, 1990). This single channel is activated over a physiological potential range. If GVD represents this channel, it is possible that its voltage dependence has been shifted, either by use of the whole cell configuration or by the loss of some intracellular factor that regulates the conductance. Certainly, the voltage dependence of GVD is shifted to more negative potentials on Na+-alanine cotransport stimulation. Taken with the fact that the volume response to Na+-alanine cotransport stimulation does not require extracellular Ca2+, it seems likely that GVD could play a role as a Cl- efflux pathway in alanine-induced RVD.
The mechanism by which Na+-alanine cotransport stimulation activates GVD may be related to cell volume. The response to L-alanine was blunted in the presence of a hypertonic bath solution. This manoeuvre would be expected to minimize the change in cell volume induced by L-alanine uptake (Lau et al. 1985). In our previous study, we had shown that GVD may be regulated by changes in cell volume induced by osmotic shock, with an increase in GVD on hypotonic shock (Robson & Hunter, 1994c). Therefore it is possible that the activation of GVD by L-alanine also occurred as a consequence of cell swelling (Mounfield & Robson, 1998). Certainly, in the presence of a hypertonic solution, which would be expected to reduce alanine-induced cell swelling, the activation of GVD was reduced.
Swelling-induced activation could be due to either a direct or indirect effect of changes in the volume of the cell. Cation non-selective, Gd3+-sensitive channels (or stretch-activated channels) are mechanosensitive, i.e. they are sensitive to the tension of the cell membrane, and are therefore directly sensitive to changes in cell volume (Guharay & Sachs, 1984; Christensen, 1987; Gustin et al. 1988; Marchenko & Sage, 1997). Although GVD is an anion-selective conductance and would appear not to fall into the stretch-activated channel category, a recent study has identified a stretch-activated Cl- conductance (Sato & Koumi, 1998), suggesting that stretch sensitivity is not just the domain of cation-selective channels. Indirect activation mechanisms for Gd3+-sensitive conductances include voltage dependence and modulation by ATP and/or intracellular Ca2+ (Silberberg & Magleby, 1997; Lidofsky et al. 1997). It is unlikely that any of these modulators were important in the L-alanine-induced activation of GVD in the current study. In the first instance, experiments were carried out on voltage-clamped cells, although a role for Na+-alanine cotransport-induced depolarization in the intact cell cannot be ruled out as GVD is a depolarization-activated conductance. Second, there was no ATP in the pipette solution. Finally, even though pipette Ca2+ was not buffered, a previous study has demonstrated that GVD is not sensitive to changes in intracellular Ca2+ (Robson & Hunter, 1994c).
In contrast to the activation of GVD, the activity of the voltage-independent, Gd3+-senstive conductance, GVI, was not affected by stimulation of Na+-alanine cotransport. Although alanine approximately doubled G20-100, this increase in conductance was unaffected by Gd3+, suggesting that it simply reflected the increase in inward current expected on stimulation of the electrogenic Na+-alanine cotransporter. This lack of effect of Gd3+ was, however, not simply due to a lack of GVI in the cells, as addition of Gd3+ to the control solution inhibited a conductance with similar characteristics to GVI. This lack of an effect of Na+-alanine cotransport on GVI is unsurprising, given the selectivity characteristics of the conductance. GVI is 4 times more selective for cations over anions and in addition is Ca2+ permeable (Robson & Hunter, 1994c). Taking into account electrochemical driving forces, on activation it would be expected to allow K+ efflux and Na+ and Ca2+ influx. Indeed, it is thought to play an important role as a Ca2+ entry pathway during hypotonic shock-induced RVD, as such RVD is inhibited in the presence of Gd3+ or absence of extracellular Ca2+ (Robson & Hunter, 1994b). In contrast however, alanine-induced RVD appears to be independent of the extracellular Ca2+ concentration as it is unaffected by the removal of extracellular Ca2+ (Mounfield & Robson, 1998). Instead it depends on the release of Ca2+ from internal stores (Mounfield & Robson, 1998). If alanine-induced RVD does not require the movement of extracellular Ca2+ into the cell it follows that GVI, a conductance associated with the influx of extracellular Ca2+, need not be activated by stimulation of Na+-alanine cotransport.
In conclusion, stimulation of Na+-alanine cotransport in single proximal tubule cells activates the voltage-activated and anion-selective conductance GVD but is without effect on the voltage-independent and cation-selective conductance GVI. This lack of responsiveness of GVI to L-alanine, coupled with the absence of a role for extracellular Ca2+ in alanine-induced RVD, is consistent with the hypothesis that GVI may act as a Ca2+ entry pathway during volume regulation induced by hypotonic shock. In contrast, even though GVD is Ca2+ permeable, its activation by L-alanine suggests that it may have a different role to GVI and instead may function as a Cl- efflux pathway during RVD induced by alanine or hypotonic shock.
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REFERENCES |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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| Beck, J. S., Laprade, R. & Lapointe, J.-Y. (1994). Coupling between transepithelial Na+ transport and basolateral K+ conductance in renal proximal tubule. American Journal of Physiology 266, F517-527 | [Medline] |
| Best, L. (1997). Glucose and alpha-ketoisocaproate induce transient inward currents in rat pancreatic beta cells. Diabetologica 40, 1-6. | |
| Cemerikic, D. & Sackin, H. (1993). Substrate acitvation of mechanosensitive, whole cell currents in renal proximal tubule. American Journal of Physiology 264, F697-714 | [Medline] |
| Christensen, O. (1987). Mediation of cell volume regulation by calcium influx through stretch-activated channels. Nature 330, 66-68 | [Medline] |
| Guharay, F. & Sachs, F. (1984). Stretch-activated single ion channel currents in tissue-cultured embryonic chick skeletal muscle. The Journal of Physiology 352, 685-701 | [Abstract] |
| Gustin, M. C., Zhou, X. L., Martinac, B. & Kung, C. (1988). A mechanosensitive ion channel in the yeast plasma membrane. Science 242, 762-765 | [Medline] |
| Hamill, O. P., Marty, A., Neher, E., Sakmann, B. & Sigworth, F. J. (1981). Improved patch-clamp techniques for high-resolution current recording from cells and cell-free membrane patches. Pflügers Archiv 391, 85-100 | [Medline] |
| Hoffmann, E. K. (1991). Cell swelling and volume regulation. Canadian Journal of Physiology and Pharmacology 70, S310-313. | |
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| Hunter, M. (1989). Isolation of single proximal cells from frog kidneys. The Journal of Physiology 416, 13-13P. | |
| Hunter, M. (1990). Stretch-activated channels in the basolateral membrane of single proximal cells of frog kidney. Pflügers Archiv 416, 448-453 | [Medline] |
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| Lau, K. R., Hudson, R. L. & Schultz, S. G. (1985). Effect of hypertonicity on the increase in basolateral conductance of Necturus small intestine in response to Na+-sugar cotransport. Biochimica et Biophysica Acta 855, 193-196. | |
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| Lidofsky, S. D., Sostman, A. & Fitz, J. G. (1997). Regulation of cation-selective channels in liver cells. Journal of Membrane Biology 157, 231-236 | [Medline] |
| MacLeod, R. J. & Hamilton, J. R. (1997). Activation of H+ conductance is required for volume regulation after L-alanine absorption in villus cells. Gastroenterology 112, A381 (abstract). | |
| Marchenko, S. M. & Sage, S. O. (1997). A novel mechanosensitive cationic channel from the endothelium of rat aorta. The Journal of Physiology 498, 419-425 | [Abstract] |
| Mounfield, P. R. & Robson, L. (1998). The role of Ca2+ in volume regulation induced by Na+-coupled alanine uptake in single proximal tubule cells isolated from frog kidney. The Journal of Physiology 510, 145-153 | [Abstract/Full Text] |
| Robson, L. & Hunter, M. (1994a). Role of cell volume and protein kinase C in regulation of a Cl- conductance in single proximal tubule cells of Rana temporaria. The Journal of Physiology 480, 1-7 | [Abstract] |
| Robson, L. & Hunter, M. (1994b). Volume regulatory responses in frog isolated proximal cells. Pflügers Archiv 428, 60-68 | [Medline] |
| Robson, L. & Hunter, M. (1994c). Volume-activated, gadolinium-sensitive whole cell currents in single proximal cells of frog kidney. Pflügers Archiv 429, 98-106 | [Medline] |
| Robson, L. & Hunter, M. (1997). Regulation of an outwardly rectifying Cl- conductance in single proximal tubule cells isolated from frog kidney. The Journal of Physiology 498, 409-417 | [Abstract] |
| Sato, R. & Koumi, S. I. (1998). Characterizaton of the stretch-activated chloride channel in isolated human atrial myocytes. Journal of Membrane Biology 163, 67-76 | [Medline] |
| Schultz, S. G. (1994). The 'pump-leak' parallelism in Necturus enterocytes: some cellular and molecular insights. Renal Physiology and Biochemistry 17, 134-137. | [Medline] |
| Silberberg, S. D. & Magleby, K. L. (1997). Voltage-induced slow activation and deactivation of mechanosensitive channels in Xenopus oocytes. The Journal of Physiology 505, 551-569 | [Abstract] |
| Volkl, H. & Lang, F. (1988). Ionic requirements for regulatory cell volume decrease in renal straight tubules (proximal). Pflügers Archiv 412, 1-6 | [Medline] |
| Wang, K. & Wondergem, R. (1993). Redistribution of hepatocyte chloride during L-alanine uptake. Journal of Membrane Biology 135, 237-244 | [Medline] |
This work was supported by the Wellcome Trust and the National Kidney Research Fund.
Corresponding author
L. Robson: Department of Biomedical Science, University of Sheffield, Western Bank, Sheffield S10 2TN, UK.
Email: l.robson{at}sheffield.ac.uk
Author's email address
M. Hunter: m.hunter@leeds.ac.uk
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, control currents; 
, plus L-alanine) and (right) the alanine-activated current.