Relationship between sodium transport and intracellular in isolated perfused rabbit proximal convoluted tubule
ATP
J. S. BECK, S. BRETON, H. MAIRBAURL, R. LAPRADE, AND G. GIEBISCH Department of Cellular and Molecular Physiology, Yale University School of Medicine, New Haven, Connecticut 06510; and Membrane Transport Research Group, University of Montreal, Montreal, Quebec H3C 37J, Canada
BECK, J. S., S. BRETON, H. MAIRBAURL, R. LAPRADE, AND G. GIEBISCH. Relationship between sodium transport and intracellular ATP in isolated perfused rabbit proximal convoluted tubule. Am. J. Physiol. 261 (Renal Fluid Electrolyte Physiol. 30): F634-F639, 1991.-The effect of alterations in sodium transport on cell ATP content and pH in the isolated perfused proximal convoluted tubule (PCT) of the rabbit was examined. Stimulating sodium transport by the addition of luminal glucose and alanine decreased cell ATP from 4.44 t 0.93 to 2.69 pH by 0.13 t 0.02 t 0.62 mM (n = 4), increased intracellular (n = 7), and increased cell volume by 0.10 t 0.02 nl/mm (n = 4). Blocking the sodium pump with 10B4 M strophanthidin in tubules in which sodium transport had been stimulated increased cell ATP from 2.04 t 0.24 to 2.42 t 0.32 mM (n = 6). In parallel experiments the same dose of strophanthidin depolarized the basolateral membrane from -52.6 t 1.9 to -6.4 t 1.6 mV, depolarized the transepithelial potential from -3.2 t 0.3 to -0.1 t 0.1 mV, and reduced the basolateral membrane potassium transference number from 0.47 to 0.26 indicating a reduction in basolateral potassium conductance. Since strophanthidin caused a cell alkalinization of 0.15 k 0.03, this latter effect cannot be due to changes of intracellular pH. Strophanthidin caused no change in cell volume over the period studied, suggesting that stretch-activated potassium channels are not involved either. Instead, potassium conductance inhibition may be the result of the closure of ATP-sensitive potassium channels. These same channels might thus be partly responsible for the increase in potassium conductance commonly observed during stimulation of sodium transport.
potassium conductance; potential; strophanthidin
sodium
pump;
pH; cell volume;
cell
of the regulation of potassium channels of the basolateral membrane of the renal proximal tubule remains incompletely understood. Patch-clamp studies of the basolateral membrane have found calcium-insensitive potassium-selective channels (9, 12, 25, 31), stretch-activated potassium-selective channels (13, 29, 30), and pH-sensitive potassium-selective channels (24). These observations are in accordance with the results of microelectrode studies of the basolateral membrane of intact proximal tubules, which demonstrated an increase in relative potassium conductance after cell swelling (7, 44), no effect of elevation of cell calcium (6), and an increase during intracellular alkalinization (15, 33). Microelectrode studies have also shown that a marked correlation exists between sodium pump (sodium-potasTHE NATURE
F634
0363-6127/91
$1.50 Copyright
sium-ATPase) activity and potassium selectivity: increases in sodium transport that stimulate sodium pump activity enhance the basolateral potassium conductance (7, 17, 18, 21), and a reduction in sodium pump activity, either by sodium pump blockers (19,22,38) or by cooling (38), reduces the basolateral potassium conductance. Sodium pump activity and potassium conductance may be functionally linked by ATP, since changes in sodium pump activity may modulate cytoplasmic levels of ATP through altered hydrolysis rate. Furthermore, ATP-sensitive potassium channels are known to exist in a variety of tissues including cardiac muscle cells, pancreatic ,8cells, and skeletal muscle cells (see Ref. 2 for review). In addition there is evidence that such ATP-sensitive potassium channels may also exist in renal epithelia including cells of the rat collecting duct (40), rabbit thick ascending limb (42), Amphiuma diluting segment (II), and cultured proximal tubule (23). In the present study we combine electrophysiological approaches with measurements of ATP concentrations in single proximal tubules to assessthe possibility that the transport-related fluctuations of the basolateral potassium conductance occur in parallel with changes in intracellular ATP. METHODS
General Female New Zealand White rabbits (1.5-2.0 kg) were anesthetized with pentobarbitone sodium (35 mg/kg) given through an ear vein. After exposure of the left kidney, the renal artery was cannulated, and 20 ml of cold (4-10°C) preservation fluid consisting of (in mM) 56 Na2HP04, 13 NaH2P04, and 140 sucrose were flushed through the kidney at 100 cmHzO. The kidney was removed and slices -0.5 mm thick were cut coronally and stored in the preservation fluid at 4°C. This procedure provides excellent preservation of proximal tubular function for periods of at least 24 h (27). Sl or S2 segments of PCT from the midcortex were dissected from the slices using ~80 magnification and were set up for microperfusion as previously described (5). Briefly, the tubules were mounted on conventional microperfusion apparatus (J. White; Bradbury Park, MD) and perfused by gravity at a rate >20 nl/min. Bath solution was passed through the 200-~1 chamber in which the tubule was mounted at a rate of 10 ml/min. The bath and perfusion solutions contained (in mM) 114 NaCl, 25 NaHC03, 2.5
0 1991 the American
Physiological
Society
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SODIUM
TRANSPORT
AND
K2HP04, 1.2 MgC12, 2.0 CaC12, 5.5 glucose, 6 alanine, 4.0 sodium lactate, and 1.0 sodium citrate. ATP and Cell Volume Measurements
For ATP measurements tubules of 1.3-2.6 (1.73 t 0.08; n = 20) mm long were used. Due to variability of cell ATP concentrations between animals paired experiments were performed such that the ATP content of two tubules from each kidney was measured and compared. Care was taken to minimize the amount of tubule held within each holding pipette. Before ATP analysis, the volume of the tubule was estimated by recording images at ~2 to measure tubule length and x40 to measure cell volume. Images were recorded with an MT1 NC-70 camera in conjunction with a Panasonic TQ-2026F optical disk recorder. Images were played back onto a Sony PVM 1271Q monitor and analyzed using Imagepro software (Cybernetics) (32). ATP metabolism was halted by manually, and rapidly, flushing the perfusion chamber with ice-cold bath solution. The holding pipette of the perfusion system, with the tubule still attached, was lifted from the chamber and dipped for 4 min into 50 ~1 of 2% perchloric acid contained in a glass microscope slide mounted on a cold metal block (perchloric acid was used rather than trichloroacetic acid, since the latter was found to cause some luminescence quenching). After lifting the tubule from the slide, the perchloric acid was brought to a pH of 7.7 with 1 M KOH and diluted with 10 mM tris( hydroxymethyl)aminomethane (Tris) buffer, pH 7.7, to a final volume of 360 ~1. The ATP content of this solution was measured with a Turner luminometer 7D-20e (Turner Designs) using a firefly luciferase activity assay kit (Sigma), which was sensitive to subpicomolar concentrations of ATP. ATP concentration is expressed as ATP content per liter of cell volume. Intracellular
pH
Intracellular pH was measured using the pH-sensitive fluorescent dye 2’,7’-bis(2-carboxyethyl)5(6)-carboxyfluorescein (BCECF) (Molecular Probes). A 5 mM stock solution of BCECF in ethanol was used to make a final concentration of 5 PM in control bath solution. Microperfused tubules were loaded at 30°C for 5-8 min. Fluorescence emission at 530 nm resulting from dual-wavelength excitation light (450 and 500 nm) from a Spex CNIII spectrofluorometer was detected with a photomultiplier tube. From these measurements a fluorescence excitation ratio for the 450- and 500-nm light was obtained. Background fluorescence (measured prior to BCECF loading) was subtracted, and the intracellular pH was calculated from calibration of the dye by equilibrating the tubule with solutions containing 120 mM potassium and 10 PM nigericin at different levels of pH. Electrophysiology
The basolateral membrane potential (&,I) was measured using glass microelectrodes fabricated from borosilicate tubing (1 mm OD and 0.5 mm ID; Frederick Haer) using a horizontal electrode puller (PD-5; Narashige, Tokyo, Japan). These were filled with 1 M KC1 and had
INTRACELLULAR
F635
ATP
resistances of 90-150 MR The intraluminal perfusion pipette was used as an electrode to measure the transepithelial potential ( Vte). Both intraluminal and intracellular electrodes were connected to WPI M701 amplifiers (WPI; New Haven, CT) via Ag-AgCl pellets. Transepithelial and basolateral membrane potentials were displayed on a Gould brush recorder. To obtain a cell impalement, the microelectrode was positioned on a cell close to the perfusion pipettes with a hydraulic micromanipulator (MO 103 Narashige) and gently tapped. The following criteria were used to accept impalements: 1) the impalement was associated with a rapid voltage deflection, 2) it remained stable (+2 mV) for at least 1 min, and 3) the electrode potential returned to a value within 4 mV of the zero baseline after withdrawal. The apparent basolateral potassium transference number (tK) was estimated from the voltage deflection caused by an elevation of bath potassium from 5 to 20 mM (potassium replacing sodium). The apparent transference number was calculated as tK = V/61.5dog(
[e]/[i])
where V is initial change in potential, [e] is new concentration of potassium (i.e., 20 mM), and [i] is control concentration of potassium (i.e., 5 mM). Protocols
Sodium transport was manipulated in two ways; in one protocol sodium transport was increased by adding 5.5 mM glucose and 6 mM alanine to the luminal perfusate from a situation where they had been isosmotically replaced with mannitol. In the other protocol 0.1 mM strophanthidin (from a 0.1 M stock solution in dimethyl sulfoxide) was added to the bathing medium to inhibit the sodium pump. In the latter experiments both glucose and alanine were always present in the luminal perfusate. Data are expressed as means t SE. A paired Student’s t test was used where appropriate. RESULTS
Effects of Sodium Transport on Intracellular pH
In the absence of luminal glucose and alanine, cell pH was 7.07 t 0.02 (n = 7). Stimulation of sodium transport by the luminal addition of glucose and alanine led to a sustained alkalinization of 0.13 t 0.02 (P < 0.002). This response was almost fully reversible, with pH returning to 7.11 after removal of glucose and alanine. A representative trace is shown in Fig. 1. Strophanthidin (0.1 mM) led to a qualitatively similar effect, with pH increasing by 0.15 t 0.03 (P < 0.02) after 3 min from a control value of 7.08. On removal of strophanthidin, pH fell to 7.03 t 0.09 within 5 min. A representative trace is shown in Fig. 2. Effects of Sodium Transport on Intracellular ATP
Stimulation of sodium transport for a period of 8 min [at which time basolateral potassium conductance has
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F636
SODIUM
TRANSPORT
AND
The volume of tubules exposed to luminal glucose and alanine or bath strophanthidin was measured both prior to and at the end of the experimental maneuver. The effect of increasing sodium transport for 8 min was an increase in cell volume from 1.09 t 0.07 to 1.19 t 0.09 nl/mm (P c 0.02) (Fig. 4). No significant change in cell volume was observed when tubules had been exposed to 0.1 mM strophanthidin for 3.5 min (Fig. 4).
7.200
.-
7.100
a 7.000
LL
t 6.900
IW. transport alanine
ATP
Effects of Sodium Transport on Cell Volume
Glu/Alo
I
INTRACELLULAR
Zmin.
1
1. Representative
trace showing on intracellular pH. During were added to luminal perfusate.
effect of stimulation “Glu/Ala” period,
of sodium glucose and
-W-
Stroph.
Effects of Strophanthidin
on Tubule Electrophysiology
The observation that tK increases during stimulation of sodium transport with glucose and alanine has been published previously (7). Strophanthidin (0.1 mM) abolished the transepithelial potential and depolarized the basolateral membrane. tK was significantly reduced. All of these changes were reversible (Fig. 5; Table 1). DISCUSSION
Effects of Sodium Transport on Cell ATP
6.5 ’ 2 min. PIG. 2. Representative trace showing effect transport on intracellular pH. During “Stroph” was added to bathing medium at concentration
of inhibition of sodium period, strophanthidin of 0.1 mM.
(n=4)
(n=6)
P(O.05
P(O.05
This is the first study in which the cell ATP of the perfused tubule has been estimated. The values of ATP we report are in good agreement with the value of -3 mM previously reported for nonperfused mouse proximal tubules (36) and dog cortical tubules in suspension (1). Although nuclear magnetic resonance measurements have failed to find an effect of altered sodium transport on kidney ATP (45), a study in suspensions of cortical tubules (which are mainly comprised of proximal tubules) found that cell ATP rose when the sodium pump was inhibited by ouabain and fell when the sodium pump was reactivated with potassium after its removal (3). The findings of the present study are in agreement with these observations, since we have also observed that inhibition of pump activity increases cell ATP, whereas enhancement of pump activity reduces cell ATP. The observed changes of cell ATP concentration may 1.6
P ( 0.05
I--
N.S.
7 \
‘ill G/A
-
G/A
+
G/A
E
1.4
c -
+
G/A
+
Stroph.
IW. 3. Effects of alterations of sodium transport on cell ATP. In the G/Acondition glucose and alanine were not present in luminal perfusate. In the G/A+ condition glucose and alanine had been added to luminal perfusate. Left, effect of addition of glucose and alanine to luminal perfusate; right, effect of pump inhibition by strophanthidin (Stroph) in the presence of luminal glucose and alanine. Comparisons made with paired t tests.
-6 ' =
1.2
0"
1.0
51.-
0.8
G/A
been found to increase (7)] led to a reduction in cell ATP from 4.44 t 0.93 to 2.69 t 0.62 mM (P < 0.05) (Fig. 3). This effect was found to be reversible (data not shown). Inhibition of sodium transport with 0.1 mM strophanthidin for a period of 3.5 min (which was the- time required for maximal cell depolarization; see Fig. 5) led to an increase in cell ATP from 2.04 t 0.24 to 2.42 t 0.32 mM (P c 0.05) (Fig. 3).
-
G/A
-t
G/A
-I0.1
G/A -t mM Stroph.
FIG. 4. Effects of alterations of sodium transport on cell volume. Nomenclature as for Fig. 3. Cell volume is expressed in nanoliter per millimeter length of tubule. Left, effect of addition of glucose and alanine to luminal perfusate; right, effect of sodium pump blockade with strophanthidin in presence of luminal glucose and alanine. Each line represents volume measurements from same tubule. Dotted lines join mean data. Significance of any change is indicated above each protocol.
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SODIUM TRANSPORT AND INTRACELLULAR
0 Vbl (mv)
-20 -40
- 60
Stroph. FIG. 5. Representative trace showing effects of sodium pump inhibition on tubule electrophysiology. During Stroph period 0.1 mM of strophanthidin was added to bathing medium. Top, basolateral membrane potential ( Vi,,); bottom, transepithelial potential ( Vte). Pairs of step increases in bath potassium from 5 to 20 mM were performed before, during, and after application of strophanthidin as indicated by vertical lines.
TABLE 1. Electrophysiological effects of 0.1 mM strophanthidin applied to bath medium for 3.5 min Initial Control -52.6+1.9t 0.47t0.06t -3.2+0.3t
During Strophanthidin -6.4t1.6 0.26kO.06 -0.lkO.l
Final Control -47.0+2.o”r 0.40*0.07* -2.5+0.3t
not only reflect direct effects on ATP hydrolysis due to pump activity. In the case of increased sodium transport by cotransported solutes the intracellular concentrations of glucose and alanine probably increase (35). Phosphorylation of these substances may occur, although for glucose this will be extremely limited due to the low levels of hexokinase activity in this nephron segment (37). Indeed Gullans et al. (10) have convincingly demonstrated that the increased oxygen consumption mediated by glucose transport in the proximal tubule is related to enhanced sodium transport rather than glucose metabolism. In addition, the availability of substrates for ATP production should be considered. Both acute inhibition of the sodium pump and sudden stimulation of sodium transport lead to an increase in intracellular sodium (20, 39) and a cell depolarization (5, 7, 16-18, 20, 21). This tends to reduce not only the chemical driving force for sodium-coupled entry of luminal solutes but, in the case of electrogenic transport, also reduces the electrical driving force. Such cotransported solutes would include lactate, citrate, and phosphate (14), the first of which is known to be avidly used for ATP synthesis in the proximal tubule (36). Thus ATP production may be limited by substrate transport into the cell. This may account for some of the reduction in ATP observed during stimulation of sodium transport and may also explain the relatively small increase of ATP after inhibition of sodium transport by strophanthidin (although it should also be noted that pump inhibition by strophanthidin
ATP
F637
was maintained for only 3 min, whereas pump stimulation with glucose and alanine proceeded for 8 min). Could changes of cell volume be responsible for changes in ATP concentrations? Although, in the case of blockade of the sodium pump with strophanthidin, there was no change in cell volume associated with the rise of ATP, an increase in cell volume of 9% was observed during the stimulation of sodium transport elicited by glucose and alanine. Although this volume change accounts, at best, for a 9% reduction by dilution in ATP concentration, it does not explain the 39% reduction observed. In conclusion, our observations concerning a linkage between sodium transport and intracellular ATP suggest an involvement of alterations in ATP breakdown by the sodium pump. Effects of Sodium Transport on Potassium Conductance PH. In the present study we observed that 0.; mM strophanthidin causes a cell depolarization, abolition of Vte (and therefore abolition of equivalent short-circuit current), and reduction in tk. These findings are in good agreement with previous observations in which another strophanthus glycoside, ouabain, had similar effects in frog (22, 39), Necturus (19), and mouse (38) proximal tubules. The reduction in tk almost certainly reflects a reduction in absolute potassium conductance (rather than increase in other conductive pathways), since a previous study found that 10 PM strophanthidin caused an increase in the ratio of basolateral membrane resistance to apical membrane resistance (4). However, it should be noted that approximately one-third of this change is caused by a reduction in intracellular potassium activity (22). The reduction of tk cannot be caused by cell pH changes, because an increase in pH was observed during sodium pump inhibition (Fig. 2). Such a pH shift would be expected to increase tk. In the present investigation, a sustained alkalinization was observed during the increase in sodium transport. This pH change can certainly account for part of the increase in potassium conductance observed under the same experimental condition (7). An electrophysiological study in fused proximal cells has shown that intracellular pH is voltage dependent and that this dependence is due to the 4,4’-diisothiocyanostilbene-2,2’-disulfonic acid-sensitive electrogenic, basolateral sodium bicarbonate cotransporter (41). This would suggest that the alkalinizations observed in the present study in response to enhanced or blocked sodium transport arise passively from the effects of the associated cell depolarizations on the basolateral sodium bicarbonate cotransporter. pH changes cannot always account for increases in tk during enhanced sodium transport. Whereas in one study an increase in sodium transport in frog proximal tubules using phenylalanine led to a transient alkalinization (20)) experiments using rabbit proximal tubules demonstrated a transient alkalinization followed by acidification ( 17). Cell uolume. Cell volume cannot be a factor in the reduction in tk observed during pump inhibition, since no change in cell volume was observed at the time at
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F638
SODIUM
TRANSPORT
AND
which tk was measured (Figs. 4 and 5). In any case, prolonged exposure to sodium pump inhibitors causes cell swelling (8, 43) that should enhance tk (7, 44). However, changes in cell volume could be a contributing factor to the increased potassium conductance observed during stimulation of sodium transport as discussed (7, 18) dell ATP. In the present study we found that cell ATP increases when sodium pump rate is reduced and decreases when sodium pump rate is increased. It is not currently possible to measure cytoplasmic levels of ATP, and we cannot be certain that measured changes in total cell ATP are directly correlated with changes in ATP at the site of potassium channels. Indeed some degree of compartmentation is likely to exist in mitochondria (26) and in pools located at the plasma membrane (28). Nevertheless, if the directional changes in ATP to some extent reflect alterations in the rate of hydrolysis of ATP by the sodium pump, then modifications in ATP concentration at the membrane would be expected to occur and influence ATP-sensitive potassium channels. Evidence for the existence of such channels in the proximal tubule is the finding that ATP prevents the rundown of excised potassium channels from opossum kidney cells (a model for the proximal tubule) (23), which is a feature of ATPsensitive potassium channels (2). In addition, studies indicate that sulfonylureas, agents that selectively block ATP-sensitive potassium channels, inhibit the basolatera1 potassium conductance of the rabbit proximal tubule (34), as does exogenously applied ATP (K. Tsuchiya and P. Welling, personal communication). Summary We have found a qualitative correlation between cell ATP and basolateral potassium conductance in the isolated perfused rabbit proximal convoluted tubule; increased sodium transport (known to increase the potassium conductance) reduced cell ATP, and decreased sodium transport (which reduced the potassium conductance) increased ATP. In addition, we have ruled out an influence of cell volume and intracellular pH as factors modulating the potassium conductance when the sodium pump is inhibited. Both of these factors may, however, play a role in enhancing the basolateral potassium conductance during stimulation of sodium transport. These findings are consistent with a role for ATP as a second messenger linking sodium pump activity to basolateral potassium conductance. We acknowledge the superb technical assistance provided by Bernadette Wallendorf and dedicated secretarial work provided by Louise Lefort. We are also indebted to Dr. J. Hoffman for his kind loan of the luminometer and to Drs. J. Hoffman and P. Vinay for helpful discussion during this work. Address for reprint requests: J. Beck, University of Montreal, Membrane Transport Research Group, Montreal H3C 357, Quebec, Canada. Received
20 November
1990; accepted
in final
form
1 April
1991.
REFERENCES 1. AMMANN, H., J. NOEL, Y. BOULANGER, AND P. VINAY. Relationship between intracellular ATP and the sodium pump activity in dog renal tubules. Can. J. Physiol. Pharmacol. 68: 57-67, 1989.
INTRACELLULAR
ATP
2. ASHCROFT, F. M. Adenosine 5’-triphosphate-sensitive potassium channels. Annu. Rev. Neurosci. 11: 97-118, 1988. 3. BALABAN, R. S., L. J. MANDEL, S. P. SOLTOFF, AND J. M. STOREY. Coupling of active ion transport and aerobic respiratory rate in isolated renal tubules. Proc. Natl. Acad. Sci. USA 77: 447-451, 1980. BECK, J. S. Transient changes in the basolateral cell membrane potential difference of the rabbit proximal tubule (PhD thesis). Leeds, UK: University of Leeds, 1987. BECK, J. S., AND D. J. POTTS. Acetazolamide and transient responses of basolateral membrane potential of rabbit kidney proximal tubules perfused in vitro. J. Physiol. Land. 416: 337-348, 1989. BECK, J. S., S. BRETON, R. LAPRADE, AND G. GIEBISCH. Volume regulation and intracellular calcium in rabbit proximal convoluted tubule. Am. J. Physiol. 260 (Renal Fluid Electrolyte Physiol. 29): F861-F867,1991. 7. BECK, J. S., AND D. J. POTTS. Cell swelling, co-transport activation and potassium conductance in isolated perfused rabbit kidney proximal tubules. J. Physiol. Land. 425: 369-378, 1990. 8. GAGNON, J., D. OUIMET, H. NGUYEN, R. LAPRADE, C. LEGRIMELLEC, S. CARRIERE, AND J. CARDINAL. Cell volume regulation in the proximal convoluted tubule. Am. J. Physiol. 243 (Renal Fluid Electrolyte Physiol. 12): F408-F415, 1982. 9. G~GELEIN, H., AND R. GREGER. Properties of single K’ channels in the basolateral membrane of rabbit proximal straight tubules. Pfluegers Arch. 410: 288-295, 1987. 10. GULLANS, S. R., S. I. HARRIS, AND L. J. MANDEL. Glucosedependent repiration in suspensions of rabbit cortical tubules. J. Membr. Biol. 78: 257-262, 1984. 11. HUNTER, M., AND G. GIEBISCH. Calcium-activated K-channels of Amphiuma early distal tubule: inhibition by ATP. Pfluegers Arch. 412: 331-333. 12. HUNTER, M., K. KAWAHARA, AND G. GIEBISCH. Potassium channels along the nephron. Federation Proc. 45: 2723-2726, 1986. 13. KAWAHARA, K. A stretch activated K’ channel in the basolateral membrane of Xenopus kidney proximal tubule cells. Pfluegers Arch. 415: 624-629, 1990. 14. KINNE, R. Transport function of renal cell membranes: sodium cotransport systems. In: Renal Biochemistry, edited by R. K. H. Kinne. New York: Elsevier, 1985. 15. KUWAHARA, M., K. ISHIBASHI, R. KRAPF, F. C. RECTOR, AND C. A. BERRY. Effect of lumen pH on cell pH and cell potential in rabbit proximal tubules. Am. J. Physiol. 256 (Renal Fluid Electrolyte Physiol. 25): F1075-F1083, 1989. 16. LANG, F., G. MESSNER, AND W. REHWALD. Electrophysiology of sodium-coupled transport in proximal renal tubules. Am. J. Physiol. 250 (Renal Fluid Electrolyte Physiol. 19): F953-F962, 1986. 17. LAPOINTE, J.-Y., AND M. DUPLAIN. Regulation of basolateral membrane potential after stimulation of Na’ transport in proximal tubules. J. Membr. Biol. 120: 165-192, 1991. 18. LAPOINTE, J.-Y., L. GARNEAU, P. D. BELL, AND J. CARDINAL. Membrane crosstalk in the mammalian proximal tubule during alterations in transepithelial sodium transport. Am. J. Physiol. 258 (Renal Fluid Electrolyte Physiol. 27): F339-F345, 1990. 19. MATSUMURA, Y., B. COHEN, W. B. GUGGINO, AND G. GIEBISCH. Regulation of the basolateral potassium conductance of the Necturus proximal tubule. J. Membr. Biol. 79: 153-161, 1984. 20. MESSNER, G., A. KOLLER, AND F. LANG. The effect of phenylalanine on intracellular pH and sodium activity in proximal convoluted tubule cells of the frog kidney. Pfluegers Arch. 404: 145-149, 1985. 21. MESSNER, G., H. OBERLEITHNER, AND F. LANG. The effect of phenylalanine on the electrical properties of proximal tubule cells in the frog kidney. Pfluegers Arch. 404: 138-144, 1985. 22. MESSNER, G., W. WANG, M. PAULMICHL, H. OBERLEITHNER, AND F. LANG. Ouabain decreases apparent K+-conductance in proximal tubules of the amphibian kidney. Pfluegers Arch. 404: 131-137, 1985. 23. OHNU-SHOSAKU, T., T. KUBOTA, J. YAMAGUCHI, M. FUKASE, T. FUJITA, AND M. FUJIMOTO. Reciprocal effects of Ca” and MgATP on the ‘run-down’ of the K’ channels in opossum kidney cells. Pfluegers Arch. 413: 562-564, 1989. 24. OHNU-SHOSAKU, T., T. KUBOTA, J. YAMAGUCHI, AND M. FUJIMOTO. Regulation of inwardly rectifying K’ channels by intracel-
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TRANSPORT
AND
lular pH in opossum kidney cells. Pfluegers Arch. 416: 138-143, 1990. PARENT, L., J. CARDINAL, AND R. SAUVI?. Single-channel analysis of a K channel at the basolateral membrane of rabbit proximal convoluted tubule. Am. J. Physiol. 254 (Renal Fluid Electrolyte Physiol. 23): F105-F113, 1988. PFALLER, W., W. G. GUDER, G. GSTRAUNTHALER, P. KOTANKO, I. JEHART, AND S. PURSCHEL. Compartmentation of ATP within renal proximal tubular cells. Biochim. Biophys. Acta 805: 152-157, 1984. PIRIE, S. C., AND D. J. POTTS. Application of cold flush preservation to in vitro microperfusion studies of kidney tubules. Kidney Int. 28: 982-984,1985. PROVERBIO, F., D. G. SHOEMAKER, AND J. F. HOFFMAN. Functional consequences of the membrane pool of ATP associated with the human red blood cell Na/K Pump. In: The Nu+, K+-Pump, Part A: Molecular Aspects, edited by J. C. Skou, J. G. Norby, A. B. Maunsbach and M. Esmann. New York: Liss, 1988. SACKIN, H. Stretch-activated potassium channels in renal proximal tubule. Am. J. Physiol. 253 (Renal Fluid Electrolyte Physiol. 22): F1253-F1262,1987. SACKIN, H. A stretch-activated K+ channel sensitive to cell volume. Proc. Natl. Acad. Sci. USA 86: 1731-1735, 1989. SACKIN, H., AND L. G. PALMER. Basolateral potassium channels in renal proximal tubule. Am. J. Physiol. 253 (Renal Fluid Electrolyte Physiol. 22): F476-F487, 1987. SCHILD, L., P. S. ARONSON, AND G. GIEBISCH. Basolateral transport pathways for K and Cl in the rabbit proximal tubule: effects on cell volume. Am. J. Physiol. 260 (Renal Fluid Electrolyte Physiol. 29): FlOl-Fl09,1990. STEELS, P. S., AND E. L. BOULPAEP. pH-dependent electrical properties and buffer permeability of the Necturus renal proximal tubule cell. J. Membr. Biol. 100: 165-182, 1987. TSUCHIYA, K., P. A. WELLING, AND G. GIEBISCH. Pharmacological evidence for an ATP-sensitive K conductance in the rabbit proximal tubule basolateral membrane (Abstract). J. Am. Sot. Nephrol. 1: 693, 1990. TUNE, B. M., AND M. B. BURG. Glucose transport by proximal renal tubules. Am. J. Physiol. 221: 580-585, 1971.
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ATP
F639
36. UCHIDA, S., AND H. ENDOU. Substrate specificity to maintain cellular ATP along the mouse nephron. Am. J. Physiol. 255 (Renal Fluid Electrolyte Physiol. 24): F977-F983, 1988. 37. VANDEWALLE, A., G. WIRTHENSOHN, H.-G. HEIDRICH, AND W. G. GUDER. Distribution of hexokinase and phosphoenolpyruvate carboxykinase along the rabbit nephron. Am. J. Physiol. 240 (Renal Fluid Electrolyte Physiol. 9): F492-F500, 1981. 38. VOLKL, H., J. GEIBEL, R. GREGER, AND F. LANG. Effects of ouabain and temperature on cell membrane potentials in isolated perfused straight proximal tubules of the mouse kidney. Pfluegers Arch. 407: 252-257,1986. 39. WANG, W., G. MESSNER, H. OBERLEITHNER, F. LANG, AND P. DEETJEN. The effect of ouabain on intracellular activities of K’, Na+, Cl-, H’, and Ca2+ in proximal tubules of frog kidneys. Pfluegers Arch. 401: 6-13, 1984. 40. WANG, W., A. SCHWAB, AND G. GIEBISCH. Regulation of smallconductance K+ channel in apical membrane of rat cortical collecting tubule. Am. J. Physiol. 259 (Renal Fluid Electrolyte Physiol. 28): F494-F502,1990. 41. WANG, W., Y. WANG, S. SILBERNAGL, AND H. OBERLEITHNER. Fused cells of frog proximal tubule: II. voltage-dependent intracellular pH. J. Membr. Biol. 101: 259-265, 1989. 42. WANG, W., S. WHITE, J. GEIBEL, AND G. GIEBISCH. A potassium channel in the apical membrane of rabbit thick ascending limb of Henle’s loop. Am. J. Physiol. 258 (Renal Fluid Electrolyte Physiol. 27): F244-F253,1990. 43. WELLING, P. A., M. A. LINSHAW, AND L. P. SULLIVAN. Effect of barium on cell volume regulation in rabbit proximal straight tubules. Am. J. Physiol. 249 (Renal Fluid Electrolyte Physiol. 18): F20-F27,1985. 44. WELLING, P. A., AND R. G. O’NEIL. Cell swelling activates basolateral membrane Cl and K conductances in rabbit proximal tubule. Am. J. Physiol. 258 (Renal Fluid Electrolyte Physiol. 27): F951F962,1990. studies of renal 45. WOLFF, S. D., C. ENG, AND R. S. BALABAN. NMR phosphate metabolites in vivo: effects of hydration and dehydration. Am. J. Physiol. 255 (Renal Fluid Electrolyte Physiol. 24): F581-F589,1988.
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ATP
J. S. BECK, S. BRETON, H. MAIRBAURL, R. LAPRADE, AND G. GIEBISCH Department of Cellular and Molecular Physiology, Yale University School of Medicine, New Haven, Connecticut 06510; and Membrane Transport Research Group, University of Montreal, Montreal, Quebec H3C 37J, Canada
BECK, J. S., S. BRETON, H. MAIRBAURL, R. LAPRADE, AND G. GIEBISCH. Relationship between sodium transport and intracellular ATP in isolated perfused rabbit proximal convoluted tubule. Am. J. Physiol. 261 (Renal Fluid Electrolyte Physiol. 30): F634-F639, 1991.-The effect of alterations in sodium transport on cell ATP content and pH in the isolated perfused proximal convoluted tubule (PCT) of the rabbit was examined. Stimulating sodium transport by the addition of luminal glucose and alanine decreased cell ATP from 4.44 t 0.93 to 2.69 pH by 0.13 t 0.02 t 0.62 mM (n = 4), increased intracellular (n = 7), and increased cell volume by 0.10 t 0.02 nl/mm (n = 4). Blocking the sodium pump with 10B4 M strophanthidin in tubules in which sodium transport had been stimulated increased cell ATP from 2.04 t 0.24 to 2.42 t 0.32 mM (n = 6). In parallel experiments the same dose of strophanthidin depolarized the basolateral membrane from -52.6 t 1.9 to -6.4 t 1.6 mV, depolarized the transepithelial potential from -3.2 t 0.3 to -0.1 t 0.1 mV, and reduced the basolateral membrane potassium transference number from 0.47 to 0.26 indicating a reduction in basolateral potassium conductance. Since strophanthidin caused a cell alkalinization of 0.15 k 0.03, this latter effect cannot be due to changes of intracellular pH. Strophanthidin caused no change in cell volume over the period studied, suggesting that stretch-activated potassium channels are not involved either. Instead, potassium conductance inhibition may be the result of the closure of ATP-sensitive potassium channels. These same channels might thus be partly responsible for the increase in potassium conductance commonly observed during stimulation of sodium transport.
potassium conductance; potential; strophanthidin
sodium
pump;
pH; cell volume;
cell
of the regulation of potassium channels of the basolateral membrane of the renal proximal tubule remains incompletely understood. Patch-clamp studies of the basolateral membrane have found calcium-insensitive potassium-selective channels (9, 12, 25, 31), stretch-activated potassium-selective channels (13, 29, 30), and pH-sensitive potassium-selective channels (24). These observations are in accordance with the results of microelectrode studies of the basolateral membrane of intact proximal tubules, which demonstrated an increase in relative potassium conductance after cell swelling (7, 44), no effect of elevation of cell calcium (6), and an increase during intracellular alkalinization (15, 33). Microelectrode studies have also shown that a marked correlation exists between sodium pump (sodium-potasTHE NATURE
F634
0363-6127/91
$1.50 Copyright
sium-ATPase) activity and potassium selectivity: increases in sodium transport that stimulate sodium pump activity enhance the basolateral potassium conductance (7, 17, 18, 21), and a reduction in sodium pump activity, either by sodium pump blockers (19,22,38) or by cooling (38), reduces the basolateral potassium conductance. Sodium pump activity and potassium conductance may be functionally linked by ATP, since changes in sodium pump activity may modulate cytoplasmic levels of ATP through altered hydrolysis rate. Furthermore, ATP-sensitive potassium channels are known to exist in a variety of tissues including cardiac muscle cells, pancreatic ,8cells, and skeletal muscle cells (see Ref. 2 for review). In addition there is evidence that such ATP-sensitive potassium channels may also exist in renal epithelia including cells of the rat collecting duct (40), rabbit thick ascending limb (42), Amphiuma diluting segment (II), and cultured proximal tubule (23). In the present study we combine electrophysiological approaches with measurements of ATP concentrations in single proximal tubules to assessthe possibility that the transport-related fluctuations of the basolateral potassium conductance occur in parallel with changes in intracellular ATP. METHODS
General Female New Zealand White rabbits (1.5-2.0 kg) were anesthetized with pentobarbitone sodium (35 mg/kg) given through an ear vein. After exposure of the left kidney, the renal artery was cannulated, and 20 ml of cold (4-10°C) preservation fluid consisting of (in mM) 56 Na2HP04, 13 NaH2P04, and 140 sucrose were flushed through the kidney at 100 cmHzO. The kidney was removed and slices -0.5 mm thick were cut coronally and stored in the preservation fluid at 4°C. This procedure provides excellent preservation of proximal tubular function for periods of at least 24 h (27). Sl or S2 segments of PCT from the midcortex were dissected from the slices using ~80 magnification and were set up for microperfusion as previously described (5). Briefly, the tubules were mounted on conventional microperfusion apparatus (J. White; Bradbury Park, MD) and perfused by gravity at a rate >20 nl/min. Bath solution was passed through the 200-~1 chamber in which the tubule was mounted at a rate of 10 ml/min. The bath and perfusion solutions contained (in mM) 114 NaCl, 25 NaHC03, 2.5
0 1991 the American
Physiological
Society
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SODIUM
TRANSPORT
AND
K2HP04, 1.2 MgC12, 2.0 CaC12, 5.5 glucose, 6 alanine, 4.0 sodium lactate, and 1.0 sodium citrate. ATP and Cell Volume Measurements
For ATP measurements tubules of 1.3-2.6 (1.73 t 0.08; n = 20) mm long were used. Due to variability of cell ATP concentrations between animals paired experiments were performed such that the ATP content of two tubules from each kidney was measured and compared. Care was taken to minimize the amount of tubule held within each holding pipette. Before ATP analysis, the volume of the tubule was estimated by recording images at ~2 to measure tubule length and x40 to measure cell volume. Images were recorded with an MT1 NC-70 camera in conjunction with a Panasonic TQ-2026F optical disk recorder. Images were played back onto a Sony PVM 1271Q monitor and analyzed using Imagepro software (Cybernetics) (32). ATP metabolism was halted by manually, and rapidly, flushing the perfusion chamber with ice-cold bath solution. The holding pipette of the perfusion system, with the tubule still attached, was lifted from the chamber and dipped for 4 min into 50 ~1 of 2% perchloric acid contained in a glass microscope slide mounted on a cold metal block (perchloric acid was used rather than trichloroacetic acid, since the latter was found to cause some luminescence quenching). After lifting the tubule from the slide, the perchloric acid was brought to a pH of 7.7 with 1 M KOH and diluted with 10 mM tris( hydroxymethyl)aminomethane (Tris) buffer, pH 7.7, to a final volume of 360 ~1. The ATP content of this solution was measured with a Turner luminometer 7D-20e (Turner Designs) using a firefly luciferase activity assay kit (Sigma), which was sensitive to subpicomolar concentrations of ATP. ATP concentration is expressed as ATP content per liter of cell volume. Intracellular
pH
Intracellular pH was measured using the pH-sensitive fluorescent dye 2’,7’-bis(2-carboxyethyl)5(6)-carboxyfluorescein (BCECF) (Molecular Probes). A 5 mM stock solution of BCECF in ethanol was used to make a final concentration of 5 PM in control bath solution. Microperfused tubules were loaded at 30°C for 5-8 min. Fluorescence emission at 530 nm resulting from dual-wavelength excitation light (450 and 500 nm) from a Spex CNIII spectrofluorometer was detected with a photomultiplier tube. From these measurements a fluorescence excitation ratio for the 450- and 500-nm light was obtained. Background fluorescence (measured prior to BCECF loading) was subtracted, and the intracellular pH was calculated from calibration of the dye by equilibrating the tubule with solutions containing 120 mM potassium and 10 PM nigericin at different levels of pH. Electrophysiology
The basolateral membrane potential (&,I) was measured using glass microelectrodes fabricated from borosilicate tubing (1 mm OD and 0.5 mm ID; Frederick Haer) using a horizontal electrode puller (PD-5; Narashige, Tokyo, Japan). These were filled with 1 M KC1 and had
INTRACELLULAR
F635
ATP
resistances of 90-150 MR The intraluminal perfusion pipette was used as an electrode to measure the transepithelial potential ( Vte). Both intraluminal and intracellular electrodes were connected to WPI M701 amplifiers (WPI; New Haven, CT) via Ag-AgCl pellets. Transepithelial and basolateral membrane potentials were displayed on a Gould brush recorder. To obtain a cell impalement, the microelectrode was positioned on a cell close to the perfusion pipettes with a hydraulic micromanipulator (MO 103 Narashige) and gently tapped. The following criteria were used to accept impalements: 1) the impalement was associated with a rapid voltage deflection, 2) it remained stable (+2 mV) for at least 1 min, and 3) the electrode potential returned to a value within 4 mV of the zero baseline after withdrawal. The apparent basolateral potassium transference number (tK) was estimated from the voltage deflection caused by an elevation of bath potassium from 5 to 20 mM (potassium replacing sodium). The apparent transference number was calculated as tK = V/61.5dog(
[e]/[i])
where V is initial change in potential, [e] is new concentration of potassium (i.e., 20 mM), and [i] is control concentration of potassium (i.e., 5 mM). Protocols
Sodium transport was manipulated in two ways; in one protocol sodium transport was increased by adding 5.5 mM glucose and 6 mM alanine to the luminal perfusate from a situation where they had been isosmotically replaced with mannitol. In the other protocol 0.1 mM strophanthidin (from a 0.1 M stock solution in dimethyl sulfoxide) was added to the bathing medium to inhibit the sodium pump. In the latter experiments both glucose and alanine were always present in the luminal perfusate. Data are expressed as means t SE. A paired Student’s t test was used where appropriate. RESULTS
Effects of Sodium Transport on Intracellular pH
In the absence of luminal glucose and alanine, cell pH was 7.07 t 0.02 (n = 7). Stimulation of sodium transport by the luminal addition of glucose and alanine led to a sustained alkalinization of 0.13 t 0.02 (P < 0.002). This response was almost fully reversible, with pH returning to 7.11 after removal of glucose and alanine. A representative trace is shown in Fig. 1. Strophanthidin (0.1 mM) led to a qualitatively similar effect, with pH increasing by 0.15 t 0.03 (P < 0.02) after 3 min from a control value of 7.08. On removal of strophanthidin, pH fell to 7.03 t 0.09 within 5 min. A representative trace is shown in Fig. 2. Effects of Sodium Transport on Intracellular ATP
Stimulation of sodium transport for a period of 8 min [at which time basolateral potassium conductance has
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F636
SODIUM
TRANSPORT
AND
The volume of tubules exposed to luminal glucose and alanine or bath strophanthidin was measured both prior to and at the end of the experimental maneuver. The effect of increasing sodium transport for 8 min was an increase in cell volume from 1.09 t 0.07 to 1.19 t 0.09 nl/mm (P c 0.02) (Fig. 4). No significant change in cell volume was observed when tubules had been exposed to 0.1 mM strophanthidin for 3.5 min (Fig. 4).
7.200
.-
7.100
a 7.000
LL
t 6.900
IW. transport alanine
ATP
Effects of Sodium Transport on Cell Volume
Glu/Alo
I
INTRACELLULAR
Zmin.
1
1. Representative
trace showing on intracellular pH. During were added to luminal perfusate.
effect of stimulation “Glu/Ala” period,
of sodium glucose and
-W-
Stroph.
Effects of Strophanthidin
on Tubule Electrophysiology
The observation that tK increases during stimulation of sodium transport with glucose and alanine has been published previously (7). Strophanthidin (0.1 mM) abolished the transepithelial potential and depolarized the basolateral membrane. tK was significantly reduced. All of these changes were reversible (Fig. 5; Table 1). DISCUSSION
Effects of Sodium Transport on Cell ATP
6.5 ’ 2 min. PIG. 2. Representative trace showing effect transport on intracellular pH. During “Stroph” was added to bathing medium at concentration
of inhibition of sodium period, strophanthidin of 0.1 mM.
(n=4)
(n=6)
P(O.05
P(O.05
This is the first study in which the cell ATP of the perfused tubule has been estimated. The values of ATP we report are in good agreement with the value of -3 mM previously reported for nonperfused mouse proximal tubules (36) and dog cortical tubules in suspension (1). Although nuclear magnetic resonance measurements have failed to find an effect of altered sodium transport on kidney ATP (45), a study in suspensions of cortical tubules (which are mainly comprised of proximal tubules) found that cell ATP rose when the sodium pump was inhibited by ouabain and fell when the sodium pump was reactivated with potassium after its removal (3). The findings of the present study are in agreement with these observations, since we have also observed that inhibition of pump activity increases cell ATP, whereas enhancement of pump activity reduces cell ATP. The observed changes of cell ATP concentration may 1.6
P ( 0.05
I--
N.S.
7 \
‘ill G/A
-
G/A
+
G/A
E
1.4
c -
+
G/A
+
Stroph.
IW. 3. Effects of alterations of sodium transport on cell ATP. In the G/Acondition glucose and alanine were not present in luminal perfusate. In the G/A+ condition glucose and alanine had been added to luminal perfusate. Left, effect of addition of glucose and alanine to luminal perfusate; right, effect of pump inhibition by strophanthidin (Stroph) in the presence of luminal glucose and alanine. Comparisons made with paired t tests.
-6 ' =
1.2
0"
1.0
51.-
0.8
G/A
been found to increase (7)] led to a reduction in cell ATP from 4.44 t 0.93 to 2.69 t 0.62 mM (P < 0.05) (Fig. 3). This effect was found to be reversible (data not shown). Inhibition of sodium transport with 0.1 mM strophanthidin for a period of 3.5 min (which was the- time required for maximal cell depolarization; see Fig. 5) led to an increase in cell ATP from 2.04 t 0.24 to 2.42 t 0.32 mM (P c 0.05) (Fig. 3).
-
G/A
-t
G/A
-I0.1
G/A -t mM Stroph.
FIG. 4. Effects of alterations of sodium transport on cell volume. Nomenclature as for Fig. 3. Cell volume is expressed in nanoliter per millimeter length of tubule. Left, effect of addition of glucose and alanine to luminal perfusate; right, effect of sodium pump blockade with strophanthidin in presence of luminal glucose and alanine. Each line represents volume measurements from same tubule. Dotted lines join mean data. Significance of any change is indicated above each protocol.
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SODIUM TRANSPORT AND INTRACELLULAR
0 Vbl (mv)
-20 -40
- 60
Stroph. FIG. 5. Representative trace showing effects of sodium pump inhibition on tubule electrophysiology. During Stroph period 0.1 mM of strophanthidin was added to bathing medium. Top, basolateral membrane potential ( Vi,,); bottom, transepithelial potential ( Vte). Pairs of step increases in bath potassium from 5 to 20 mM were performed before, during, and after application of strophanthidin as indicated by vertical lines.
TABLE 1. Electrophysiological effects of 0.1 mM strophanthidin applied to bath medium for 3.5 min Initial Control -52.6+1.9t 0.47t0.06t -3.2+0.3t
During Strophanthidin -6.4t1.6 0.26kO.06 -0.lkO.l
Final Control -47.0+2.o”r 0.40*0.07* -2.5+0.3t
not only reflect direct effects on ATP hydrolysis due to pump activity. In the case of increased sodium transport by cotransported solutes the intracellular concentrations of glucose and alanine probably increase (35). Phosphorylation of these substances may occur, although for glucose this will be extremely limited due to the low levels of hexokinase activity in this nephron segment (37). Indeed Gullans et al. (10) have convincingly demonstrated that the increased oxygen consumption mediated by glucose transport in the proximal tubule is related to enhanced sodium transport rather than glucose metabolism. In addition, the availability of substrates for ATP production should be considered. Both acute inhibition of the sodium pump and sudden stimulation of sodium transport lead to an increase in intracellular sodium (20, 39) and a cell depolarization (5, 7, 16-18, 20, 21). This tends to reduce not only the chemical driving force for sodium-coupled entry of luminal solutes but, in the case of electrogenic transport, also reduces the electrical driving force. Such cotransported solutes would include lactate, citrate, and phosphate (14), the first of which is known to be avidly used for ATP synthesis in the proximal tubule (36). Thus ATP production may be limited by substrate transport into the cell. This may account for some of the reduction in ATP observed during stimulation of sodium transport and may also explain the relatively small increase of ATP after inhibition of sodium transport by strophanthidin (although it should also be noted that pump inhibition by strophanthidin
ATP
F637
was maintained for only 3 min, whereas pump stimulation with glucose and alanine proceeded for 8 min). Could changes of cell volume be responsible for changes in ATP concentrations? Although, in the case of blockade of the sodium pump with strophanthidin, there was no change in cell volume associated with the rise of ATP, an increase in cell volume of 9% was observed during the stimulation of sodium transport elicited by glucose and alanine. Although this volume change accounts, at best, for a 9% reduction by dilution in ATP concentration, it does not explain the 39% reduction observed. In conclusion, our observations concerning a linkage between sodium transport and intracellular ATP suggest an involvement of alterations in ATP breakdown by the sodium pump. Effects of Sodium Transport on Potassium Conductance PH. In the present study we observed that 0.; mM strophanthidin causes a cell depolarization, abolition of Vte (and therefore abolition of equivalent short-circuit current), and reduction in tk. These findings are in good agreement with previous observations in which another strophanthus glycoside, ouabain, had similar effects in frog (22, 39), Necturus (19), and mouse (38) proximal tubules. The reduction in tk almost certainly reflects a reduction in absolute potassium conductance (rather than increase in other conductive pathways), since a previous study found that 10 PM strophanthidin caused an increase in the ratio of basolateral membrane resistance to apical membrane resistance (4). However, it should be noted that approximately one-third of this change is caused by a reduction in intracellular potassium activity (22). The reduction of tk cannot be caused by cell pH changes, because an increase in pH was observed during sodium pump inhibition (Fig. 2). Such a pH shift would be expected to increase tk. In the present investigation, a sustained alkalinization was observed during the increase in sodium transport. This pH change can certainly account for part of the increase in potassium conductance observed under the same experimental condition (7). An electrophysiological study in fused proximal cells has shown that intracellular pH is voltage dependent and that this dependence is due to the 4,4’-diisothiocyanostilbene-2,2’-disulfonic acid-sensitive electrogenic, basolateral sodium bicarbonate cotransporter (41). This would suggest that the alkalinizations observed in the present study in response to enhanced or blocked sodium transport arise passively from the effects of the associated cell depolarizations on the basolateral sodium bicarbonate cotransporter. pH changes cannot always account for increases in tk during enhanced sodium transport. Whereas in one study an increase in sodium transport in frog proximal tubules using phenylalanine led to a transient alkalinization (20)) experiments using rabbit proximal tubules demonstrated a transient alkalinization followed by acidification ( 17). Cell uolume. Cell volume cannot be a factor in the reduction in tk observed during pump inhibition, since no change in cell volume was observed at the time at
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F638
SODIUM
TRANSPORT
AND
which tk was measured (Figs. 4 and 5). In any case, prolonged exposure to sodium pump inhibitors causes cell swelling (8, 43) that should enhance tk (7, 44). However, changes in cell volume could be a contributing factor to the increased potassium conductance observed during stimulation of sodium transport as discussed (7, 18) dell ATP. In the present study we found that cell ATP increases when sodium pump rate is reduced and decreases when sodium pump rate is increased. It is not currently possible to measure cytoplasmic levels of ATP, and we cannot be certain that measured changes in total cell ATP are directly correlated with changes in ATP at the site of potassium channels. Indeed some degree of compartmentation is likely to exist in mitochondria (26) and in pools located at the plasma membrane (28). Nevertheless, if the directional changes in ATP to some extent reflect alterations in the rate of hydrolysis of ATP by the sodium pump, then modifications in ATP concentration at the membrane would be expected to occur and influence ATP-sensitive potassium channels. Evidence for the existence of such channels in the proximal tubule is the finding that ATP prevents the rundown of excised potassium channels from opossum kidney cells (a model for the proximal tubule) (23), which is a feature of ATPsensitive potassium channels (2). In addition, studies indicate that sulfonylureas, agents that selectively block ATP-sensitive potassium channels, inhibit the basolatera1 potassium conductance of the rabbit proximal tubule (34), as does exogenously applied ATP (K. Tsuchiya and P. Welling, personal communication). Summary We have found a qualitative correlation between cell ATP and basolateral potassium conductance in the isolated perfused rabbit proximal convoluted tubule; increased sodium transport (known to increase the potassium conductance) reduced cell ATP, and decreased sodium transport (which reduced the potassium conductance) increased ATP. In addition, we have ruled out an influence of cell volume and intracellular pH as factors modulating the potassium conductance when the sodium pump is inhibited. Both of these factors may, however, play a role in enhancing the basolateral potassium conductance during stimulation of sodium transport. These findings are consistent with a role for ATP as a second messenger linking sodium pump activity to basolateral potassium conductance. We acknowledge the superb technical assistance provided by Bernadette Wallendorf and dedicated secretarial work provided by Louise Lefort. We are also indebted to Dr. J. Hoffman for his kind loan of the luminometer and to Drs. J. Hoffman and P. Vinay for helpful discussion during this work. Address for reprint requests: J. Beck, University of Montreal, Membrane Transport Research Group, Montreal H3C 357, Quebec, Canada. Received
20 November
1990; accepted
in final
form
1 April
1991.
REFERENCES 1. AMMANN, H., J. NOEL, Y. BOULANGER, AND P. VINAY. Relationship between intracellular ATP and the sodium pump activity in dog renal tubules. Can. J. Physiol. Pharmacol. 68: 57-67, 1989.
INTRACELLULAR
ATP
2. ASHCROFT, F. M. Adenosine 5’-triphosphate-sensitive potassium channels. Annu. Rev. Neurosci. 11: 97-118, 1988. 3. BALABAN, R. S., L. J. MANDEL, S. P. SOLTOFF, AND J. M. STOREY. Coupling of active ion transport and aerobic respiratory rate in isolated renal tubules. Proc. Natl. Acad. Sci. USA 77: 447-451, 1980. BECK, J. S. Transient changes in the basolateral cell membrane potential difference of the rabbit proximal tubule (PhD thesis). Leeds, UK: University of Leeds, 1987. BECK, J. S., AND D. J. POTTS. Acetazolamide and transient responses of basolateral membrane potential of rabbit kidney proximal tubules perfused in vitro. J. Physiol. Land. 416: 337-348, 1989. BECK, J. S., S. BRETON, R. LAPRADE, AND G. GIEBISCH. Volume regulation and intracellular calcium in rabbit proximal convoluted tubule. Am. J. Physiol. 260 (Renal Fluid Electrolyte Physiol. 29): F861-F867,1991. 7. BECK, J. S., AND D. J. POTTS. Cell swelling, co-transport activation and potassium conductance in isolated perfused rabbit kidney proximal tubules. J. Physiol. Land. 425: 369-378, 1990. 8. GAGNON, J., D. OUIMET, H. NGUYEN, R. LAPRADE, C. LEGRIMELLEC, S. CARRIERE, AND J. CARDINAL. Cell volume regulation in the proximal convoluted tubule. Am. J. Physiol. 243 (Renal Fluid Electrolyte Physiol. 12): F408-F415, 1982. 9. G~GELEIN, H., AND R. GREGER. Properties of single K’ channels in the basolateral membrane of rabbit proximal straight tubules. Pfluegers Arch. 410: 288-295, 1987. 10. GULLANS, S. R., S. I. HARRIS, AND L. J. MANDEL. Glucosedependent repiration in suspensions of rabbit cortical tubules. J. Membr. Biol. 78: 257-262, 1984. 11. HUNTER, M., AND G. GIEBISCH. Calcium-activated K-channels of Amphiuma early distal tubule: inhibition by ATP. Pfluegers Arch. 412: 331-333. 12. HUNTER, M., K. KAWAHARA, AND G. GIEBISCH. Potassium channels along the nephron. Federation Proc. 45: 2723-2726, 1986. 13. KAWAHARA, K. A stretch activated K’ channel in the basolateral membrane of Xenopus kidney proximal tubule cells. Pfluegers Arch. 415: 624-629, 1990. 14. KINNE, R. Transport function of renal cell membranes: sodium cotransport systems. In: Renal Biochemistry, edited by R. K. H. Kinne. New York: Elsevier, 1985. 15. KUWAHARA, M., K. ISHIBASHI, R. KRAPF, F. C. RECTOR, AND C. A. BERRY. Effect of lumen pH on cell pH and cell potential in rabbit proximal tubules. Am. J. Physiol. 256 (Renal Fluid Electrolyte Physiol. 25): F1075-F1083, 1989. 16. LANG, F., G. MESSNER, AND W. REHWALD. Electrophysiology of sodium-coupled transport in proximal renal tubules. Am. J. Physiol. 250 (Renal Fluid Electrolyte Physiol. 19): F953-F962, 1986. 17. LAPOINTE, J.-Y., AND M. DUPLAIN. Regulation of basolateral membrane potential after stimulation of Na’ transport in proximal tubules. J. Membr. Biol. 120: 165-192, 1991. 18. LAPOINTE, J.-Y., L. GARNEAU, P. D. BELL, AND J. CARDINAL. Membrane crosstalk in the mammalian proximal tubule during alterations in transepithelial sodium transport. Am. J. Physiol. 258 (Renal Fluid Electrolyte Physiol. 27): F339-F345, 1990. 19. MATSUMURA, Y., B. COHEN, W. B. GUGGINO, AND G. GIEBISCH. Regulation of the basolateral potassium conductance of the Necturus proximal tubule. J. Membr. Biol. 79: 153-161, 1984. 20. MESSNER, G., A. KOLLER, AND F. LANG. The effect of phenylalanine on intracellular pH and sodium activity in proximal convoluted tubule cells of the frog kidney. Pfluegers Arch. 404: 145-149, 1985. 21. MESSNER, G., H. OBERLEITHNER, AND F. LANG. The effect of phenylalanine on the electrical properties of proximal tubule cells in the frog kidney. Pfluegers Arch. 404: 138-144, 1985. 22. MESSNER, G., W. WANG, M. PAULMICHL, H. OBERLEITHNER, AND F. LANG. Ouabain decreases apparent K+-conductance in proximal tubules of the amphibian kidney. Pfluegers Arch. 404: 131-137, 1985. 23. OHNU-SHOSAKU, T., T. KUBOTA, J. YAMAGUCHI, M. FUKASE, T. FUJITA, AND M. FUJIMOTO. Reciprocal effects of Ca” and MgATP on the ‘run-down’ of the K’ channels in opossum kidney cells. Pfluegers Arch. 413: 562-564, 1989. 24. OHNU-SHOSAKU, T., T. KUBOTA, J. YAMAGUCHI, AND M. FUJIMOTO. Regulation of inwardly rectifying K’ channels by intracel-
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SODIUM
TRANSPORT
AND
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