The role of Ca2+ in volume regulation induced by Na+-coupled alanine uptake in single proximal tubule cells isolated from frog kidney

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The role of Ca²⁺ in volume regulation induced by Na⁺-coupled alanine uptake in single proximal tubule cells isolated from frog kidney

P. R. Mounfield and L. Robson

Department of Biomedical Science, University of Sheffield, Western Bank, Sheffield S10 2TN, UK

Received 20 February 1998; accepted 16 March 1998.

ABSTRACT

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Abstract
Introduction
Methods
Results
Discussion
References

It has been suggested that epithelial cells maintain cell volume and function, in the face of changes in the rate of transepithelial transport, by activation of volume-regulatory pathways.

The aim of the following study was to examine directly the effect of an alteration in Na⁺-coupled alanine transport on cell length in single proximal tubule cells isolated from frog kidney.

An optical technique was used to examine the change in cell length induced by 5 mM L-alanine.

On addition of L-alanine to the bath there was an initial increase in cell length to a peak value. This was followed by two types of response. In eighteen out of thirty-one cells a typical volume-regulatory response was observed. The remaining cells showed no volume regulation.

Volume regulation was not affected by the removal of extracellular Ca²⁺. The mean degrees of recovery were 159 � 21 % (n = 18) and 144 � 18 % (n = 8) in the presence and absence of Ca²⁺, respectively.

Volume regulation was inhibited by depletion of intracellular Ca²⁺ stores, or in the presence of either Gd³⁺ or DIDS. The mean degrees of regulation were 55�4 � 9�2 % (n = 7), 68�2 � 18�8 % (n = 7) and 69�1 � 14�3 % (n = 7), respectively.

The alanine-induced increases in cell length were both stereospecific and Na⁺ dependent.

The evidence suggests that volume regulation induced by Na⁺-coupled alanine uptake may be dependent on the release of Ca²⁺ from intracellular stores. This is in contrast to volume regulation induced by hypotonic shock, which appears to require extracellular Ca²⁺. Results obtained using a hypotonic shock should, therefore, be viewed with caution.

INTRODUCTION

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Abstract
Introduction
Methods
Results
Discussion
References

Cell volume regulation is the means by which cells prevent large changes in their volume. There are two types of volume-regulatory mechanisms: regulatory volume increase (RVI) which acts to increase cell volume after cell shrinkage, and regulatory volume decrease (RVD) which acts to reduce cell volume after cell swelling. These recovery mechanisms are a consequence of the activation of solute transport pathways, entry pathways for Na⁺ and Cl^- during RVI, and exit pathways for K⁺, Cl^- or HCO₃^- during RVD. Many renal cells demonstrate RVD in response to hypotonic shock (HS-RVD). Such RVD is dependent on the activation of both conductive and cotransport mechanisms for K⁺, Cl^- and HCO₃^- (Kirk, DiBona & Schafer, 1987a; Volkl & Lang, 1988; Spring & Hoffman, 1992). A previous study in single proximal tubule cells isolated from frog kidney has shown that these cells also demonstrate HS-RVD (Robson & Hunter, 1994a). A model for RVD in these cells has been proposed (Robson & Hunter, 1994a). On cell swelling there is activation of 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 and phosphorylation, to activate K⁺ and Cl^- channels. K⁺ and Cl^- exit the cell, followed by water, and cell volume goes down. Three volume-sensitive conductances have been identified in the cells which may play a role in HS-RVD. Two conductances seem to be attributable to SACs (Robson & Hunter, 1994b), and the third conductance is a volume-sensitive Cl^- conductance (Robson & Hunter, 1994c, 1997b). The identity of the volume-sensitive K⁺ conductance is currently under investigation (Robson, Hunter & Wragg, 1996; Robson & Hunter, 1997a).

Physiologically, volume regulation is thought to play a role in maintaining epithelial cell volume and function in the face of changes in the rate of transepithelial transport. For example, the renal proximal tubule reabsorbs Na⁺, Cl^- and solutes which have been filtered at the glomerulus. Transport by this cell is ultimately driven by a Na⁺-K⁺ pump located on the basolateral membrane. This pumps three Na⁺ ions out of the cell in exchange for two K⁺ ions; the K⁺ which enters then leaves via a basolateral K⁺ conductance. The function of the pump is to maintain a low intracellular Na⁺ concentration, which provides the driving force for the uptake of Na⁺ across the apical membrane, coupled to Cl^- and solutes, such as amino acids (Ullrich, Rumrich & Kloss, 1974; Lang, Messner & Rehwald, 1986; Kimmich, Randles & Wilson, 1994; Aronson, 1996). Na⁺ exits the cell via the Na⁺-K⁺ pump, while Cl^- and solutes leave via a basolateral conductance pathway and facilitated transport systems, respectively (Cramer, Pardridge, Hirayama & Wright, 1992; Seki, Taniguchi, Uwatoko, Suzuki & Kurokawa, 1995). The net effect is the transepithelial transport of Na⁺, Cl^- and solutes. The rate of Na⁺ transport across the proximal tubule is extremely high (Schultz, 1992), with an amount of Na⁺ equivalent to the cell content transported every 20 s. Such a high rate of transport has implications for cell function, as any mismatch between rates of transport at the apical and basolateral membranes will lead to changes in cell content and, ultimately, cell volume. However, despite variable apical Na⁺ transport rates, proximal tubule cells maintain their composition, cell volume and, ultimately, cell function (Schultz & Hudson, 1986; Beck, Laprade & Lapointe, 1994). They do this by carefully matching transport rates at the apical and basolateral membranes. This is known as pump-leak coupling (Schultz & Hudson, 1986). The identity of the mechanism which couples transport at the two membranes is unknown. However, several possibilities have been suggested, e.g. ATP, pH, Ca²⁺ and cell volume regulation, or a combination of these (Beck et al. 1994; Schultz, 1994).

How could volume regulation provide the link in pump- leak coupling? The idea is that an increase in the apical uptake of Na⁺ and amino acids causes an increase in cell volume. This increase in cell volume subsequently activates volume-sensitive solute exit pathways located on the basolateral membrane, e.g. K⁺ and Cl^- channels. Opening of these channels would allow net solute efflux from the cell and a subsequent decrease in cell volume. The net effect would be maintenance of cell volume with upregulation of transport at the apical and basolateral membranes.

Only a few studies exist which have investigated directly the effect of changes in Na⁺ cotransport on cell volume in epithelia. In Ehrlich ascites tumour cells and guinea-pig jejunal enterocytes, activation of Na⁺-amino acid cotransport has been shown to produce cell swelling, and in the enterocytes this is subsequently followed by volume recovery (Hudson & Schultz, 1988; MacLeod, Lembessis & Hamilton, 1992a). Work has shown that this volume recovery is dependent on both an increase in intracellular Ca²⁺ and Cl^- loss from the cell (MacLeod et al. 1992a; MacLeod, Lembessis & Hamilton, 1992b). In contrast, there are many studies which examine the mechanisms underlying RVD by exposing cells to a hypotonic shock (Lopes & Guggino, 1987; Lambert, Hoffmann & Christensen, 1987; Ubl, Murer & Kolb, 1989; Banderali & Roy, 1992; Robson & Hunter, 1994a). The aims of the following study were threefold: (1) to examine the mechanism(s) underlying alanine-induced swelling in single proximal tubule cells isolated from frog kidney, (2) to investigate whether these cells demonstrate RVD in response to alanine-induced cell swelling (AS-RVD), and (3) to examine the role of Ca²⁺ in AS-RVD (Robson & Hunter, 1994a).

METHODS

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Abstract
Introduction
Methods
Results
Discussion
References

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 to promote dissociation of the tight junctions (Table 1, Isolation). The kidneys were then subjected to partial digestion with collagenase and pronase, which 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).

Measurement of cell length

Cell length was measured continuously using a modified version of an optical technique which has been reported previously (Boyett, Moore, Jewell, Montgomery, Kirby & Orchard, 1988). A 10 µl sample of the proximal cell suspension was placed in a perspex experimental chamber which contained approximately 200 µl of control Ringer solution. This chamber was situated on the stage of an inverted microscope (Olympus IX70). Cells were continuously superfused with Ringer solutions throughout the duration of the experiment. The optical technique for measurement of cell length used a linear photodiode array (Reticon, UK), which was attached to the camera port of the microscope. The photodiode array measured changes in light intensity across the bath. Therefore, by aligning the array along the longitudinal axis of the cell, the change in light intensity which occurred at the cell membrane-bath interfaces could be detected, i.e. the start and end of the cell. The output from the array gave a raw cell image which was then sent to an Interface (R. Montgomery, London) where it was processed using analogue circuitry to give cell length. To obtain cell length the raw image was observed on an oscilloscope (Gould 400 Series) and this image aligned so that the first and last peaks crossed a single fixed threshold. The point where the rising phase of the first peak crossed the threshold was taken as the start of the cell, and the point where the falling phase of the last peak crossed the threshold was taken as the end of the cell. The distance between these points was converted to a DC voltage (0�66 ms = 1 V). This output voltage (1 V = 20 µm) was low-pass filtered (5 Hz) and displayed on a chart recorder (Gould TA240S). The system was calibrated by projecting the image of a stage-piece graticule. In some experiments the photodiode array was aligned across the basolateral sphere of the cells to examine changes in cell width.

Experimental protocols

Validation. In a previous study investigating the effect of hypotonic shock on single proximal tubule cells isolated from frog kidney, two thresholds were used to determine cell length (Robson & Hunter, 1994a). To allow comparison between the data obtained in that study and the current study, which used only one threshold, validation experiments were carried out to confirm that the sensitivity of the one-threshold technique was not different from that of the two-threshold technique. Cells were initially superfused with control Ringer solution (Table 1, Control 2), and then exposed to hypotonic shock by the removal of 40 mM mannitol.

Regulatory volume decrease. Experiments examined the response of the cells to alanine. Cells were initially superfused with a Hepes-buffered control amphibian Ringer solution (Table 1, Control 1), and then exposed to 5 mM L-alanine. In all experiments described, when alanine was added to the bathing solution 5 mM mannitol was removed to maintain osmolality. In a second series of experiments, changes in cell width were examined in response to 5 mM L-alanine.

Response to L-alanine - stereospecificity. Cells were superfused with control Ringer solution (Table 1, Control 1), and then exposed to either 5 mM L-alanine or 5 mM D-alanine (5 mM mannitol was removed to maintain osmolality).

Response to L-alanine - Na⁺ dependence. Cells were superfused with control Ringer solution and then exposed to a Na⁺-free solution, where 90 mM NaCl was replaced with 90 mM N-methyl-D-glucamine chloride (NMDG-Cl^-; Table 1, 0 Na⁺). Once a steady length was attained, cells were exposed to 5 mM L-alanine in the absence of extracellular Na⁺.

Role of Ca²⁺ in RVD and effects of blockers. To examine the role of extracellular Ca²⁺, cells were superfused with a 0 Ca²⁺ solution (Table 1, 0 Ca²⁺), and then exposed to 5 mM L-alanine. Removing extracellular Ca²⁺ has been shown previously to inhibit HS-RVD in these cells (Robson & Hunter, 1994a). In addition, the role of intracellular Ca²⁺ stores was also examined. Cells were initially superfused with 0 Ca²⁺ Ringer solution and then exposed to a solution containing 10 mM caffeine (removal of 10 mM mannitol) and 1 µM thapsigargin. Caffeine stimulates Ca²⁺ loss from intracellular stores by activating Ca²⁺ channels present on the store membrane, while thapsigargin prevents the uptake of Ca²⁺ back into the stores by inhibiting a Ca²⁺-ATPase (Tinel, Wehner & Sauer, 1994; Brodin, Rytved & Nielson, 1994). Once cell length attained a new steady state, 5 mM L-alanine was added to the bathing solution.

Table 1. Composition of basic experimental solutions

Solution	Isolation *	Control 1 *	0 Na⁺	0 Ca²⁺ *	Control 2 *	HCO₃^- ²
NaCl	101	90	-	90	50	66�2
KCl	3	3	3	3	3	3
CaCl₂	-	2	2	-	2	2
MgCl₂	-	1	1	1	1	1
Hepes	10	10	10	10	10	-
Mannitol	-	15	15	15	84	23�6
EGTA	-	-	-	2	-	-
NMDG	-	-	90	-	-	-
NaHCO₃	-	-	-	-	-	23�8

* Titrated to pH 7�4 with NaOH. dagger

Titrated to pH 7�4 with HCl. ² Continuously bubbled with 5 % CO₂ in O₂. NMDG, N-methyl-D-glucamine. All concentrations are millimolar.

To examine possible transport mechanisms involved in RVD, cells were superfused with Ringer solution in the presence of 10 µM gadolinium (Gd³⁺), an inhibitor of SACs (Yang & Sachs, 1989) or 100 µM DIDS, an anion channel blocker (Robson & Hunter, 1994c). Gd³⁺ and DIDS have been shown previously to inhibit HS-RVD (Robson & Hunter, 1994a).

Exposure of cells to 0 Ca²⁺, DIDS or Gd³⁺ alone has previously been shown to have no effect on cell length (Robson & Hunter, 1994a). Therefore, in these experiments cells were placed directly into control Ringer solution in the absence of extracellular Ca²⁺, or in the presence of DIDS or Gd³⁺, and then exposed to alanine. In contrast, the effects of caffeine and thapsigargin have not been previously investigated. Therefore, in these experiments cells were initially superfused with control Ringer solution and then exposed to caffeine and thapsigargin before subsequent exposure to alanine when a steady cell length was attained.

Effect of HCO₃^- on alanine-induced RVD. Previous experiments using a hypotonic shock have provided evidence that frog proximal tubule is composed of two distinct cell populations. In Hepes-buffered Ringer solution only half of the cells demonstrated HS-RVD (Robson & Hunter, 1994a), while in HCO₃^--buffered Ringer solution all of the cells were capable of HS-RVD (Robson & Hunter, 1994b). In the current study, AS-RVD was also only observed in 50 % of the cells tested in Hepes-buffered Ringer solution (see Results). Experiments therefore also examined the effect of alanine in the presence of a HCO₃^--buffered solution (Table 1, HCO₃^-).

Solutions

All chemicals were obtained from Sigma. The osmolality of all solutions was checked (Roebling osmometer, Camlab, UK) and adjusted to within 1 mosmol kg^-1 of 215 mosmol kg^-1 using water or mannitol.

Statistics

Results are given as means � S.E.M. with the number of observations (n). Significance was tested by use of Student's t test, analysis of variance or the $chi$ ² test, as appropriate, and assumed at the 5 % level.

RESULTS

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Abstract
Introduction
Methods
Results
Discussion
References

Validation - effect of hypotonic shock

As observed previously, two types of response were observed. In 48 % of cells (10 out of 21) cell length increased to a peak, followed by recovery back towards the pre-shock level. In control Ringer solution, cell length was 21�1 � 0�5 µm (n = 10). This increased by 2�14 � 0�3 % on hypotonic shock, followed by a fall of 2�05 � 0�4 %, corresponding to a mean degree of recovery of 100�9 � 18�3 %. These changes were not significantly different from the changes reported previously using the two-threshold system: 1�77 � 0�37, 1�47 � 0�32 and 88�1 � 14�7 % (n = 9) (Robson & Hunter, 1994a).

Regulatory volume decrease - cell length

On addition of 5 mM L-alanine to the bath solution there was an initial increase in cell length to a peak value, followed by two types of response: (1) a volume-regulatory response where cell length subsequently fell back towards the control level; (2) maintenance of peak length until cells were placed in control Ringer solution. Figure 1 shows typical traces from cells demonstrating these responses. Peak cell length was defined as the maximum length attained by the cells on exposure to alanine, while steady-state length was defined as the length at which no further changes in cell length could be observed. Of the cells tested, 58�1 % (18 out of 31) demonstrated a typical RVD response (Table 2). In the remaining 41�9 % of cells (13 out of 31), no volume regulation was observed. Cell length was initially 19�7 � 0�6 µm (n = 13). On the addition of alanine it rose by 0�65 � 0�13 µm (P = 0�0003). Cell length subsequently recovered to 19�9 � 0�6 µm when cells were placed in control Ringer solution. The increase in length to a peak in the non-regulating cells was significantly greater than that seen in the regulating cells (0�65 � 0�13 vs. 0�28 � 0�07 µm, respectively; P = 0�01).

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Figure 1. Effect of addition of L-alanine
The figure shows two cell length traces obtained from a cell which demonstrated AS-RVD (top) and a cell which did not demonstrate AS-RVD (bottom).

Table 2. The effect of inhibitors on RVD

	Length (µm)
Conditions	Control	Peak	Steady	No. RVD (%)
Control	20�2 � 0�6	0�28 � 0�07	-0�11 � 0�04	58�1
(n = 18)
0 Ca²⁺	19�5 � 0�7	0�30 � 0�06	-0�11 � 0�05	66�7
(n = 8)
0 Ca²⁺ stores	20�3 � 1�1	0�29 � 0�07	0�14 � 0�06 *	58�3
(n = 7)
10 µM Gd³⁺	20�5 � 0�5	0�55 � 0�17	0�28 � 0�18 *	63�6
(n = 7)
100 µM DIDS	19�7 � 0�8	0�26 � 0�05	0�11 � 0�06	53�9
(n = 7)

Values are given as means � S.E.M. for regulating cells. Peak and steady lengths are expressed relative to control length, where control cell length is represented by 0. The n numbers in the left-hand column refer to the number of cells demonstrating AS-RVD. In the right-hand column, the number of regulating cells is expressed as a percentage of the total. * P < 0�025 compared with control length. dagger

P < 0�01 compared with control length.

Regulatory volume decrease - cell width

Two types of response were also observed when changes in cell width were examined on addition of 5 mM L-alanine to the bath solution. Of the cells tested, 41 % (7 out of 17) demonstrated RVD. Cell width was initially 16�2 � 0�77 µm (n = 7). This increased by 0�38 � 0�11 µm to a peak (P = 0�01) and was followed by width recovery, a fall of 0�44 � 0�15 µm (P = 0�02). In the remaining cells (10 out of 17), cell width was initially 15�9 � 0�5 µm. On the addition of alanine this increased to a peak which was maintained throughout the duration of the exposure, a mean increase of 0�64 � 0�12 µm (P = 0�0003). Width only recovered on placing cells back in alanine-free Ringer solution (16�0 � 0�43 µm).

Response to L-alanine - stereospecificity

The initial mean length of cells in control Ringer solution was 22�7 � 0�9 µm (n = 9). Addition of L-alanine to the bath produced a significant increase in cell length to a peak value of 23�2 � 0�9 µm (n = 9, P = 0�001). A typical trace is shown in Fig. 2. The initial length of cells exposed to D-alanine was 23�5 � 0�9 µm. Replacing mannitol with D-alanine did not result in a significant change: 23�5 � 0�9 µm in the presence of D-alanine (n = 6), Fig. 2.

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Figure 2. Stereospecificity of response to alanine
Typical traces demonstrating the effect of L-alanine (top) and D-alanine (bottom) on cell length.

Response to L-alanine - Na⁺ dependence

As no distinction could be made between cell types, i.e. RVD or non-RVD, in the Na⁺-free experiments, the change in length in response to alanine in Na⁺-free Ringer solution was compared with the mean increase in length to a peak of both RVD and non-RVD cells obtained in Na⁺-containing control experiments.

Superfusing cells with 0 Na⁺ Ringer solution resulted in a significant fall in cell length of 0�40 � 0�11 µm (P = 0�005), from 18�5 � 0�4 to 18�1 � 0�5 µm (n = 9). On the subsequent addition of alanine, the increase in cell length normally observed in the presence of Na⁺ was completely abolished: -0�18 � 0�04 µm (n = 9) vs. 0�44 � 0�07 µm (n = 31) (P < 0�0001).

Role of Ca²⁺ in RVD and effects of blockers

Caffeine and thapsigargin alone produced a biphasic response in cell length. In control Ringer solution, cell length was 20�16 � 0�69 µm (n = 12). On the addition of caffeine and thapsigargin, length initially rose to a peak (P = 0�002) followed by recovery to a new steady-state level (P = 0�0001), 20�48 � 0�67 and 20�24 � 0�69 µm, respectively.

Exposure to L-alanine in the absence of extracellular Ca²⁺, after Ca²⁺ store depletion or in the presence of Gd³⁺ or DIDS produced the two types of response observed under control conditions, with approximately half of the cells demonstrating RVD (Table 2). There was no significant difference between the proportion of cells demonstrating RVD between the groups ( $chi$ ²₄ = 0�62).

In the absence of extracellular Ca²⁺, exposure to L-alanine produced a similar RVD response to the control circumstance (Table 2). The mean degree of recovery was not significantly different from the control circumstance, 144 � 18 % (n = 8) vs. 159 � 21 % (n = 18). However, after intracellular Ca²⁺ store depletion or in the presence of Gd³⁺ or DIDS, RVD was inhibited (F_4,42 = 5�84, Table 2). The mean degrees of recovery were 55�4 � 9�2 % in the absence of Ca²⁺ stores, 68�2 � 18�8 % in the presence of Gd³⁺, and 69�1 � 14�3 % in the presence of DIDS.

Table 2. The effect of inhibitors on RVD

	Length (µm)
Conditions	Control	Peak	Steady	No. RVD (%)
Control	20�2 � 0�6	0�28 � 0�07	-0�11 � 0�04	58�1
(n = 18)
0 Ca²⁺	19�5 � 0�7	0�30 � 0�06	-0�11 � 0�05	66�7
(n = 8)
0 Ca²⁺ stores	20�3 � 1�1	0�29 � 0�07	0�14 � 0�06 *	58�3
(n = 7)
10 µM Gd³⁺	20�5 � 0�5	0�55 � 0�17	0�28 � 0�18 *	63�6
(n = 7)
100 µM DIDS	19�7 � 0�8	0�26 � 0�05	0�11 � 0�06	53�9
(n = 7)

P < 0�01 compared with control length.

Effect of HCO₃^- on alanine-induced RVD

In HCO₃^--buffered Ringer solution, all cells tested demonstrated AS-RVD (13 out of 13; Fig. 3).

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Figure 3. Efect of HCO₃^- on alanine-induced RVD
Cell length changes in response to 5 mM alanine in the presence of either Hepes- (top) or HCO₃^--buffered (bottom) Ringer solution. Control, peak and steady-state cell lengths are expressed as a percentage of control. The dotted lines show results obtained from all cells tested, while the continuous lines show the mean data.

DISCUSSION

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Abstract
Introduction
Methods
Results
Discussion
References

Validation

The changes in cell length and degree of volume regulation observed during hypotonic shock in this study were not significantly different from those observed in a previous study. This suggests that the use of only one threshold to determine cell length instead of two did not alter the sensitivity of the optical system, and a comparison can therefore be made between data obtained in this study and the previous study (Robson & Hunter, 1994a).

Response to L-alanine

Addition of L-alanine to single proximal tubule cells isolated from frog kidney produced cell swelling, consistent with uptake of alanine into the cell. This swelling was both stereospecific, with only the L-isomer of alanine causing swelling, and Na⁺ dependent, suggesting that alanine uptake occurred via the apical Na⁺-alanine cotransporter. As experiments were carried out on single cells, the basolateral membrane was also exposed to alanine. Therefore, a contribution to the length change by the basolateral facilitated alanine transporter cannot be completely ruled out. However, the lack of a measurable effect of alanine in the absence of extracellular Na⁺ would suggest that any role is relatively minor.

Regulatory volume decrease

In the presence of Hepes-buffered Ringer solution, approximately half of the cells tested demonstrated alanine-induced RVD. There are several pieces of evidence which suggest that the presence of non-regulating cells did not simply reflect a problem with cell viability. In the first instance, regulating and non-regulating cells have been observed previously in frog proximal cells following hypotonic shock-induced cell swelling of similar magnitude: an increase in length to a peak by 1�4 and 1�8 % with alanine and hypotonic shock, respectively (Robson & Hunter, 1994a). Secondly, RVD was observed in half of the cells when either cell length or width was recorded, suggesting that non-RVD cells were not simply a consequence of changes in the width of the cell in the absence of length changes. Thirdly, all cells were capable of AS-RVD in the presence of a HCO₃^--buffered Ringer solution. This is consistent with previous work in which all cells demonstrated HS-RVD in the presence of HCO₃^- (Robson & Hunter, 1994a). As for HS-RVD, the data suggest that there are two populations of proximal tubule cells, one which voids Cl^- during AS-RVD and one which voids HCO₃^-.

The alanine-induced RVD observed in the cells is in contrast to a study carried out on rabbit proximal tubule, where the simultaneous addition of 5�5 mM glucose and 6 mM alanine caused cell swelling which was not followed by volume regulation (Beck, Breton, Lapreade & Giebisch, 1991). The reason for this difference is unclear at the present time, although it is unlikely to be due simply to a difference between mammalian and amphibian tissue, as recent work in the laboratory has shown that mouse proximal tubule cells also demonstrate glucose- and alanine-induced RVD (K. Balloch, unpublished observations).

The two phases of length change in the regulating cells (increase then recovery) are typical of RVD responses observed in both amphibian and mammalian proximal tubule in response to hypotonic shock (Lopes & Guggino, 1987; Kirk, Schafer & DiBona, 1987b; Volkl & Lang, 1988; Robson & Hunter, 1994a). Additionally, the peak length attained by the regulating cells was significantly smaller than that attained by the non-regulating cells. This suggests that in the regulating cells, activation of RVD pathways acted both to restore the volume of the cell and also to minimize initial volume changes.

The degree of recovery, 159 %, is larger than that observed in response to a hypotonic shock in these cells (88 %; Robson & Hunter, 1994a). Such a difference may reflect the involvement of different signalling pathways in RVD (see later). Indeed, a previous study has also demonstrated larger AS-RVD compared with HS-RVD in jejunal enterocytes (MacLeod et al. 1992a).

Role of Ca²⁺ in RVD and effects of blockers

As under control conditions, addition of alanine in the absence of extracellular Ca²⁺, after depletion of Ca²⁺ stores or in the presence of Gd³⁺ or DIDS produced typical RVD responses in approximately half of the cells tested.

In the absence of extracellular Ca²⁺ there was no effect on RVD; the same degree of regulation was observed in the presence and absence of extracellular Ca²⁺. Therefore, AS-RVD was not dependent on the influx of Ca²⁺ into the cell. This is in contrast to HS-RVD, where omission of extracellular Ca²⁺ reduced the degree of regulation fourfold (Robson & Hunter, 1994a). Such dependence of HS-RVD on extracellular Ca²⁺ is characteristic of many cell types, e.g. rabbit proximal tubule and guinea-pig enterocytes (McCarty & O'Neil, 1991; MacLeod et al. 1992a). Of interest is the inhibition of AS-RVD by Gd³⁺. Gd³⁺ has been shown to inhibit HS-RVD in these cells, probably by inhibiting Ca²⁺ influx into the cell via SACs (Robson & Hunter, 1994a). As AS-RVD is independent of extracellular Ca²⁺, how is Gd³⁺ inhibiting AS-RVD? Whole-cell patch-clamp studies have revealed that in addition to inhibiting two Ca²⁺-permeable volume-sensitive conductances (Robson & Hunter, 1994b), Gd³⁺ also inhibits a volume- and DIDS-sensitive Cl^- conductance (Robson & Hunter, 1994c). It is feasible, therefore, that Gd³⁺ inhibits AS-RVD by blocking Cl^- efflux. Indeed, in the presence of DIDS, AS-RVD was inhibited.

In the presence of caffeine and thapsigargin, AS-RVD was reduced threefold, suggesting an important role for the release of Ca²⁺ from intracellular stores. This is in contrast to HS-RVD, where extracellular Ca²⁺ is required for volume regulation to occur (Robson & Hunter, 1994a). The depletion of the Ca²⁺ stores did not completely inhibit RVD. This could be due to several reasons, e.g. incomplete activation of caffeine-sensitive Ca²⁺ channels, the presence of caffeine-insensitive Ca²⁺ release pathways or the presence of Ca²⁺-insensitive solute efflux pathways. The actual mechanism which couples the solute-induced increase in cell volume to the release of Ca²⁺ is unknown. However, at the present time three distinct types of Ca²⁺ release mechanisms from intracellular stores have been identified which may play a role in AS-RVD. The first is the ryanodine-gated Ca²⁺ release channel (Hazama & Okada, 1990). This channel is both caffeine and Ca²⁺ activated and is thought to release Ca²⁺ from the stores in response to a rise in intracellular Ca²⁺, which enters the cell across the plasma membrane. However, this channel would be unlikely to play a role in frog proximal tubule AS-RVD, as removal of extracellular Ca²⁺ in these cells has no effect. The second is the inositol 1,4,5-trisphosphate (IP₃)-gated channel (Suzuki et al. 1990). This channel is not activated by caffeine and would, therefore, be unlikely to represent the Ca²⁺ efflux pathway activated in this study. However, a potential role cannot be completely dismissed as many cells contain both ryanodine- and IP₃-sensitive Ca²⁺ channels (Tinel, Wehner & Kinne, 1997). The final mechanism is a novel G-protein-coupled, arachidonic acid-activated Ca²⁺ channel (Tinel et al. 1997). Further work involving the measurement of intracellular Ca²⁺ will be required to identify if any of these mechanisms are important in AS-RVD.

In conclusion, single renal proximal tubule cells isolated from frog kidney are capable of RVD in response to Na⁺-coupled alanine uptake. Unlike HS-RVD, this response is not dependent on the presence of extracellular Ca²⁺, but instead seems to be dependent on the release of Ca²⁺ from intracellular stores. That such a large difference may exist in the signalling pathways for AS-RVD and HS-RVD demonstrates that, in the renal proximal tubule at least, hypotonic shock may not be an appropriate tool for investigating how changes in Na⁺-coupled solute transport modulate the activity of volume-sensitive transport pathways. Results obtained with such a protocol should, therefore, be viewed with caution.

				H. J. Han, S. H. Park, and Y. J. Lee Signaling cascade of ANG II-induced inhibition of {alpha}-MG uptake in renal proximal tubule cells Am J Physiol Renal Physiol, April 1, 2004; 286(4): F634 - F642. [Abstract] [Full Text] [PDF]

				I. D. Millar, J. A. Hartley, C. Haigh, A. A. Grace, S. J. White, J. D. Kibble, and L. Robson Volume regulation is defective in renal proximal tubule cells isolated from KCNE1 knockout mice Exp Physiol, March 1, 2004; 89(2): 173 - 180. [Abstract] [Full Text] [PDF]

				I. D. Millar and L. Robson Na+-alanine uptake activates a Cl{-} conductance in frog renal proximal tubule cells via nonconventional PKC Am J Physiol Renal Physiol, May 1, 2001; 280(5): F758 - F767. [Abstract] [Full Text] [PDF]