INTRODUCTION

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EARLY CLEARANCE STUDIES on snakes suggest that the proximal tubules may have a role in maintenance of systemic acid-base homeostasis similar to the proximal tubules of mammals (8), although this role has yet to be determined definitively. In whatever way reptilian proximal tubules may help to maintain systemic acid-base homeostasis, they must at least deal with systemic acid or base loads while maintaining their own cellular acid-base homeostasis. Because nothing was known about the maintenance of intracellular pH (pH_i) or the possible acid or base fluxes at the luminal and basolateral membranes in reptilian tubules, we performed an initial study of pH_i and its possible regulation by acid or base fluxes at the basolateral membrane in snake proximal tubules (17). To examine the factors involved in regulation of pH_i at the basolateral membrane independent of those at the luminal membrane, we worked with isolated tubules with oil-filled lumens (17). To compare the data with studies on mammalian proximal tubules (12, 19), we used HEPES-buffered bathing medium nominally free of HCO₃ maintained at pH 7.4. Under these circumstances, the resting pH_i in both the proximal portion and the distal portion of the snake proximal tubules was ~7.1 (17), essentially the same as in mammalian proximal tubules under the same conditions (12, 19). The response to an NH₄Cl pulse was also qualitatively the same as that observed in mammalian proximal tubules (17). However, the mechanisms involved in the recovery of pH_i from the acid value produced by the NH₄Cl pulse were far from clear. Recovery was not influenced by amiloride or DIDS (17). However, the rate of recovery and the resting pH_i were depressed by the removal of Na⁺ in the distal portion but not the proximal portion of the proximal tubule (17). These data provided no clear evidence of roles for a basolateral Na⁺/H⁺ exchanger or basolateral Na⁺-dependent or Na⁺-independent HCO₃ transporters in the regulation of pH_i in these tubules (17).

Because it is difficult to assess the role of possible HCO₃ transporters in the absence of a known quantity of HCO₃, we undertook to reexamine the regulation of pH_i in the presence of HCO₃ in the present study. Also, because the results of the Na⁺ replacement experiments suggested that its apparent regulatory effect could be the result of changes in membrane potential, we also examined the effects of manipulations of membrane potential on regulation of pH_i. Finally, we examined the effects of inhibitors of several additional possible pathways for basolateral regulation of pH_i after an NH₄Cl pulse. However, the data provided no clear evidence for basolateral regulation of pH_i by one of the commonly recognized acid or base transport pathways.

	METHODS

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Animals and dissection of tubules. Garter snakes (Thamnophis spp.) were obtained from Kons Scientific, Germantown, WI, and maintained as described previously (9, 10). The snakes were decapitated, and their kidneys were quickly removed and placed in chilled (4°C), oxygenated Ringer solution (see below and Table 1 for composition). Entire snake proximal tubules were dissected from renal tissue without the aid of enzymatic agents (9). Snake proximal tubules can be divided into two segments, a proximal-proximal segment and a distal-proximal segment, on the basis of differences in their ability to transport para-aminohippurate (10), although cells throughout the proximal tubule appear to be structurally identical (W. H. Dantzler and R. B. Nagle, unpublished observations). In this study, we examined pH_i in each segment independently. However, in most experiments, we studied a proximal-proximal segment and a distal-proximal segment from the same kidney.

To determine pH_i regulation at the basolateral membrane alone during NH₄Cl pulse experiments, we filled the lumens of individual tubules with mineral oil. In our previous study, we found that oil-filled lumens do not interfere with measurements of resting pH_i and do permit evaluation of pH_i regulation at the basolateral membrane without complications arising from transport at the luminal membrane (17). All dissections were performed at 4°C, but all experiments were performed at 25°C.

Ringer composition. The components of the snake Ringer solutions used in these studies are shown in Table 1. Solutions were buffered with HEPES (25 mM) or with HEPES (20 mM) and HCO₃ (5 mM). Solution 1 was the basic solution used for dissection, for initial incubation with pH-sensitive fluorescent dye (see Measurement of pH_i in single renal tubules), and for all control pH_i measurements in the nominal absence of HCO₃ ("HEPES-buffered solution"). It was also used for measurements of pH_i in the nominal absence of HCO₃ but with the addition of ethylisopropylamiloride (EIPA), Schering 28080, tetrapentylammonium (TPeA), and tetraethylammonium (TEA). Solution 2 was the standard solution used for all control pH_i measurements in the presence of HCO₃ ("HEPES-HCO₃-buffered solution"). It was also used for measurements of pH_i in the presence of HCO₃ and with the addition of EIPA, DIDS, bafilomycin, and Schering 28080. Solutions 3-11 involved modifications in solutions 1 and 2 designed to examine the effects of Na⁺, Cl, K⁺, and Ba²⁺ on pH_i in the presence and absence of HCO₃ (see RESULTS). The pH of each solution was adjusted to 7.4 with 1 N NaOH, 1 N KOH, or Tris base, as appropriate. When 20 mM NH₄Cl was present in the medium the concentration of NaCl was reduced by an equimolar amount to maintain the osmolality and ionic strength approximately constant. The osmolality of all solutions was ~290 mosmol/kgH₂O (Table 1) and was checked regularly with a vapor pressure osmometer. Solutions buffered with HEPES alone were continuously bubbled with 100% O₂ and were assumed to be nominally HCO₃-free in the absence of tissue. All the solutions containing both HEPES and HCO₃ were bubbled with 95% O₂-5% CO₂.

Measurement of pH_i in single renal tubules. We used the pH-sensitive fluorescent dye 2',7'-bis(carboxyethyl)-5,6-carboxyfluorescein (BCECF) to measure pH_i in a manner similar to that described by others (19). For these measurements, we used a dual-wavelength spectrofluorimeter built around an Olympus IMT-2 inverted epifluorescence microscope. A 100-W mercury arc lamp was used as an excitation source, and specific excitation wavelengths for measurement of pH using BCECF were selected by a filter wheel mounted to the shaft of a high-speed motor. This filter wheel was composed of hemispheric filters centered at 445 nm (isobestic wavelength) and 495 nm. As the wheel spun, sequential excitation wavelengths were transmitted. The selected excitation light was directed to the sample by a matched dichroic mirror. To prevent photodamage to the dye-loaded cells from the excitation light, a neutral density filter (ND 2, Oriel, Stratford, CT) was placed in front of the illumination site. The emitted fluorescent light passed through the dichroic mirror and a wavelength-specific emission filter (530 nm for BCECF). The fluorescent light emitted from a selected region of the sample was collected by a Hamamatsu HC120-03 photomultiplier tube operated in photon-counting mode. The dark current of the photomultiplier tube was zeroed out. Collection of fluorescent light was synchronized to the wheel rotation and excitation filter position. Synchronization, speed selection, and data collection were controlled by a microcomputer running custom software. The integrated average of 30 measurements per second was collected at 1-s intervals.

Individual tubules were held in an appropriate bathing chamber on the stage of the microscope and incubated with the acetoxymethyl ester (AM) form of BCECF (3-6 µM) for 45 min at 25°C. The AM form of BCECF readily enters the cells where the ester is cleaved by nonspecific esterases yielding the impermeant, fluorescent form of the dye. After the loading period, the bath was replaced with identical dye-free solution for at least 5 min before beginning an experiment. For collection of fluorescent light, the microscope was equipped with a Zeiss 63× Neofluor oil-immersion objective (1.25 numerical aperture). Wavelength-specific fluorescence was collected as described above over a 15-32 µm diameter area of nonperfused (oil-filled lumen) or perfused tubule. The ratio of fluorescence at 495/445 nm was then used as a measurement of pH_i to eliminate influence of changes in dye content or cell shape. Calibration of the pH sensitivity of intracellular BCECF was performed for each tubule at the end of each experiment. This involved monitoring the 495/445 nm ratio at various values of pH_i by incubating the tubule in a solution with a high K⁺ concentration containing 10-12 µM of the ionophore nigericin (which exchanges K⁺ for H⁺ and sets pH_i to approximate extracellular pH) (24). The calibration curve was linear between pH 6.5 and 8.0. Autofluorescence was insignificant compared with the fluorescence from BCECF and was taken into account by the calibration procedure.

Exposure to NH₄Cl pulse. To alter pH_i, we exposed single tubules for 30-60 s to 20 mM NH₄Cl in the bathing medium (7, 15, 21, 22). NH₃ diffuses across cell membranes much more readily than NH⁺₄ (11). Therefore, NH₃ rapidly enters the tubule cells and combines with free intracellular H⁺ to form NH⁺₄ and alkalinize the cell interior. In principle, pH_i should increase until the intracellular NH₃ concentration ([NH₃]_i) is equal to the extracellular NH₃ concentration. NH⁺₄ enters the cells more slowly than NH₃, leading to a gradual decrease in pH_i over the exposure period. When NH₄Cl is then removed from the bathing medium, free NH₃ diffuses rapidly from the cells, leaving behind free H⁺ and producing rapid acidification of the cell interior.

Determination of rate of pH_i change, total buffering capacity, NH₃ flux across the basolateral membrane, and permeability of basolateral membrane to NH₃ during NH₄Cl pulse experiments. As in our previous study in HEPES buffer alone (17), we examined more quantitatively the factors involved in the changes in pH_i during the NH₄Cl pulse experiments. For this purpose, we measured the rate of change of pH_i (dpH_i/dt) and calculated the total buffering capacity (_t), NH₃ flux across the basolateral membrane (J_NH3), and the permeability of the basolateral membrane to NH₃ (P_NH3) during the initial alkalinization (addition of NH₄Cl to the bath) and acidification (removal of NH₄Cl from the bath). These calculations were performed as in our previous studies (7, 17, 15, 21) and are described briefly below.

dpH_i/dt was measured directly. The intrinsic buffering capacity (_i, mM H⁺/pH U) was then calculated from the following equation

(1)

where [H⁺]_i is the change in the amount of H⁺ in the cells by virtue of the NH₃ loading or removal, and pH_i is the change in pH_i. _t was then determined from _i by including the buffering capacity of the intracellular HCO₃ (14, 22). In HCO₃-free solutions _t = _i.

J_NH3 (nmol · cm² · s¹) was calculated from the following equation

(2)

where the surface area (S) and tubular volume (V) are calculated from the measured tubule length, and diameter and dpH_i/dt and pH_i are as defined above. [NH₃]_i, the change in [NH₃]_i, equals the total amount of NH₃ that has moved across the basolateral membrane. On the assumption, as noted above, that intracellular NH⁺₄ is formed from NH₃ that has entered the cell and is reduced through the movement of NH₃ from the cell, we can calculate [NH₃]_i from the change in the intracellular NH₃ and NH⁺₄ concentrations at maximum pH_i after NH₄Cl addition and at resting pH_i before NH₄Cl removal. In this calculation, the relative intracellular concentrations of NH₃ and NH⁺₄ are determined from the pH_i with the Henderson-Hasselbalch equation on the assumption that the pK_a for the reaction NH₃ + H⁺NH⁺₄ equals 9.4 at 25°C (11). Finally, P_NH3 (cm/s) was calculated from the following relationship

(3)

in which the terms have been defined above.

Protocol for experiments. A single tubule from a single snake was used for each experiment. The total experimental period after loading with dye was 30 min, a time found to be appropriate to maintain sufficient dye in the tubule so that the counts remained high enough for accurate measurements. The protocol usually involved three experimental manipulations accompanied by two control manipulations. Experimental measurements were bracketed by control measurements. If, at any point, pH_i failed to return to control or if the tubule shifted so that focus was not maintained at the same position (a very uncommon occurrence), the experiment was terminated. Also, if the calibration curve at the end of each experiment was not linear over the appropriate range, the experiment was discarded. The control values before and after experimental manipulations were averaged together by computer for comparison with the experimental values for that tubule only.

Chemicals. Nigericin, bafilomycin A₁, and DIDS were purchased from Sigma. EIPA and BCECF were purchased from Molecular Probes. All other chemicals were purchased from standard sources and were of the highest purity available. Schering 28080 was a generous gift from the Schering Corporation both to us directly and via Dr. Raul Martinez-Zaguilan, Texas Tech University Health Science Center, Lubbock, TX.

Statistics. Values are summarized as means ± SE (n = no. of tubules with each tubule from a different animal). Significant differences between values were determined with Student's t-test for paired or unpaired data, as appropriate. Linear regression analyses were performed for calibrations as required. In all analyses, differences were considered statistically significant when P < 0.05.

	RESULTS

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Control measurements of resting pH_i. We measured control resting pH_i in both proximal-proximal and distal-proximal segments in HEPES-buffered and HEPES-HCO₃-buffered media (solutions 1 and 2; Table 1) before studying alterations in pH_i. The results are shown in Table 2. The only apparent difference in resting pH_i produced by changes in the buffer system occurred in the distal-proximal tubules where pH_i was somewhat (although not significantly) higher in the presence than in the absence of HCO₃. Because resting pH_i in distal-proximal tubules tended to be higher in the presence than in the absence of HCO₃, we studied a series of distal-proximal tubules in which we measured resting pH_i repeatedly in each individual tubule in the presence or absence of HCO₃. In these tubules there was no significant difference in resting pH_i in the presence or absence of HCO₃. Resting pH_i was 7.13 ± 0.06 in the presence of HCO₃ and 7.13 ± 0.07 in the absence of HCO₃ (mean ± SE; 8 tubules). It is noteworthy that the resting pH_i under both sets of circumstances in these tubules was virtually identical to that in the other set of distal-proximal segments in the absence of HCO₃ and to that in proximal-proximal tubules in either the presence or absence of HCO₃ (Table 2).

Response of pH_i to exposure to NH₄Cl pulse. The response to a NH₄Cl pulse was qualitatively the same as that observed previously (Table 2; Figs. 1 and 2) (17). Moreover, the response was quantitatively the same in each segment whether HCO₃ was present or not (Table 2).

View larger version (16K): [in this window] [in a new window]   Fig. 1.   Intracellular pH (pHi) and its response to an NH4Cl pulse in a single snake proximal-proximal tubule in HEPES-HCO3 buffer. Boxes above tracing show periods when control buffer is present and when NH4Cl has been added.

View larger version (24K): [in this window] [in a new window]   Fig. 2.   pHi in a single snake distal-proximal tubule in HEPES-HCO3 buffer. Boxes above tracing indicate presence of control buffer, NH4Cl pulse, and recovery from NH4Cl pulse in control buffer, DIDS, and ethylisopropylamiloride (EIPA).

dpH_i/dt, _t, J_NH3, P_NH3 during NH₄Cl pulse experiments. The values for these determinations made during changes in pH_i produced by adding and then removing NH₄Cl are shown for both tubule segments in the presence and absence of HCO₃ in Table 3. For each tubule segment, the major significant change produced by a change in the buffering system occurred in the _t (Table 3). As expected, _t was significantly greater in the presence of HCO₃ than in the absence of HCO₃. However, the rate of change in pH_i in proximal-proximal segments during the addition of NH₄Cl was significantly greater in the absence of HCO₃ than in the presence of HCO₃.

View this table: [in this window] [in a new window]   Table 3.   dpHi/dt, t, JNH3, and PNH3

Effects of various treatments on rate of recovery from minimum pH_i to control resting pH_i. We examined dpH_i/dt from the minimum value after removal of NH₄Cl to the control resting value. As indicated previously (17), in these tubules with oil-filled lumens, recovery of pH_i can only take place through ion fluxes across the basolateral membrane. A number of basolateral Na⁺-coupled acid or base transporters have been identified in various segments of amphibian or mammalian proximal renal tubules (12). These include 1) Na⁺/H⁺ exchange that can be inhibited by amiloride or amiloride derivatives (5, 12); 2) Na⁺-coupled Cl/HCO₃ exchange moving HCO₃ into the cells (3, 12, 13) that can be inhibited by DIDS and other disulfonic stilbene compounds (11); and 3) electrogenic Na⁺-HCO₃-CO²₃ cotransport moving HCO₃ out of the cells (1, 6, 12, 20, 25) that can also be inhibited by DIDS (6, 12). Basolateral Na⁺-independent Cl/HCO₃ exchange moving HCO₃ out of the cells that can be inhibited by DIDS has also been described (2, 3, 23).

Because nothing was known about the basolateral regulation of pH_i in snake proximal tubules, we began in our previous study by examining the effect of removing Na⁺ from the HEPES-buffered bathing medium on the rate of recovery of pH_i (17). As reported in that study, the removal of Na⁺ significantly reduced the rate of recovery in the distal-proximal segment, but not the proximal-proximal segment (Table 4). In the present study, we repeated these experiments in HEPES-HCO₃-buffered medium. However, when all the Na⁺ was removed from the bathing medium (solution 3, Table 1), there was no effect on dpH_i/d_t in either tubule segment (Table 5).

View this table: [in this window] [in a new window]   Table 5.   Effect of treatments on dpHi/dt from acid pHi to control resting pHi in HEPES-HCO3 buffer

To pursue further a possible role for inorganic ions in regulation of pH_i, we examined the effects of removing Cl (solutions 4 and 5, Table 1) or both Na⁺ and Cl (solutions 6 and 7, Table 1) on the rate of recovery of pH_i. However, neither the removal of Cl alone nor the removal of Na⁺ and Cl together had any effect on dpH_i/dt in either tubule segment in the presence of either HEPES or HEPES-HCO₃ buffer (Tables 4 and 5).

In our previous study, because the effect of Na⁺ removal suggested that basolateral Na⁺/H⁺ exchange might be important in pH_i regulation, we examined the effect of amiloride, the well-known inhibitor of this exchange on recovery of pH_i (17). However, even 1 mM amiloride failed to alter dpH_i/dt in that study (17). Nevertheless, in the present study, despite the apparent lack of effect of Na⁺ removal, we examined the effect of EIPA, an amiloride analog that is a more potent inhibitor of Na⁺/H⁺ exchange than amiloride itself, on the rate of recovery of pH_i in the presence of Na⁺ (standard control solutions 1 and 2, Table 1). The presence of EIPA (100 µM) significantly inhibited dpH_i/dt in the distal-proximal segment in HEPES-HCO₃ buffer but not in HEPES buffer (Fig. 2; Tables 4 and 5). EIPA had no effect in proximal-proximal segments in either buffer (Tables 4 and 5).

As noted above, a number of the basolateral acid-base transporters identified in other species are sensitive to DIDS. However, in our previous study of snake tubules in HEPES-buffered medium without added HCO₃, DIDS had no effect on the recovery of pH_i after acidification (17). In the current study, we examined the effect of DIDS again in the presence of HCO₃ (standard control solution 2, Table 1). The results show that in HEPES-HCO₃-buffered medium, DIDS (100 µM) significantly depressed dpH_i/dt in the distal-proximal but not the proximal-proximal tubule (Table 5; Fig. 2).

As pointed out above, at least one basolateral mechanism that might be involved in regulation of pH_i is electrogenic. This is the Na⁺-HCO₃-CO²₃ cotransporter that moves HCO₃ out of the cells. Its continued function would tend to slow the rate of pH_i recovery. Because it is electrogenic, its function and thus its ability to delay the rate of recovery would be reduced if the basolateral membrane potential were reduced. Our previous studies on snake renal tubules showed that increasing the K⁺ concentration in the bathing medium to 75 mM reduces the basolateral membrane potential by ~75% (from about 60 to about 15 mV), and the addition of 5 mM Ba²⁺ to the bathing medium reduces it by ~50% (17, 18). Therefore, we examined the effect on pH_i recovery of raising the K⁺ concentration of the bathing medium to 75 mM (solutions 8 and 9, Table 1) and of adding 5 mM Ba²⁺ to the bathing medium (solutions 10 and 11, Table 1). Neither of these maneuvers had any effect on dpH_i/dt in either tubule segment in the presence of HEPES buffer alone or in the presence of HEPES-HCO₃ buffer (Tables 4 and 5).

Because so few maneuvers had any effect on dpH_i/dt, we considered other possible basolateral regulatory mechanisms that might play a role in the rate of recovery and could be inhibited by various treatments. One of these might be a basolateral H⁺-ATPase that transports H⁺ out of the cells. Because this process, if present, should be inhibited by bafilomycin, we examined the effect of bafilomycin (1 µM) in the presence of HCO₃ (standard control solution 2, Table 1) on the rate of recovery. However, bafilomycin had no effect on dpH_i/dt in either tubule segment (Table 5).

Recently, an electroneutral K⁺/NH⁺₄ exchanger has been described in cultured inner medullary collecting duct cells (4). Although this transporter normally moves NH⁺₄ into the cells in exchange for K⁺, it can function in reverse mode (4). It appeared possible that such an exchanger might function at the basolateral membrane of these snake tubule cells and could play a role in the observed recovery of pH_i after an NH₄Cl pulse. It also appeared possible that a basolateral H⁺-K⁺-ATPase might function in these cells to move H⁺ from the cells during the observed recovery, although such a basolateral transporter has not been described. Both the newly described K⁺/NH⁺₄ exchanger and the known H⁺-K⁺-ATPase are inhibited by Schering 28080. Therefore, we examined the effect of this compound on the rate of recovery (standard control solutions 1 and 2, Table 1). We first used 10-20 µM (sufficient to inhibit H⁺-K⁺-ATPase) and then 100 µM (sufficient to inhibit the K⁺/NH⁺₄ exchanger) (4). However, Schering 28080 had no effect on dpH_i/dt in either tubule segment at either concentration (Tables 4 and 5).

Previous studies indicated that efflux of organic cations (e.g., TEA) across the basolateral membrane of these snake renal tubules occurs by a carrier-mediated process that is not influenced by membrane potential (16). It also appeared possible that recovery of pH_i after the NH₄Cl pulse might involve NH₄ exit from the cells via this pathway. This exit step, at least for model organic cations such as TEA, can be completely inhibited by the addition of very low concentrations (25 µM) of TPeA to the bathing medium (16). To determine if basolateral efflux of NH⁺₄ via this carrier might play a role in the recovery of pH_i after the NH₄Cl pulse, we examined the effect of TPeA on the rate of recovery (standard control solution 1, Table 1). In initial experiments, 25 µM TPeA had no effect on dpH_i/dt (data not shown). Therefore, we increased the concentration to 250 µM, 2.5 mM, and finally to 20 mM. However, as shown in Table 4, even a concentration of 20 mM did not reduce dpH_i/dt.

It also appeared possible that, during the recovery phase, NH⁺₄ moved out of the cells through K⁺ channels. This did not seem likely in view of the lack of effect of Ba²⁺ and TPeA (which, like TEA, might be expected to block K⁺ channels). However, we decided to explore the possibility further by determining the effect of 20 mM TEA on the rate of recovery (standard control solution 1, Table 1). As shown in Table 4, 20 mM TEA had no effect on dpH_i/dt.

Again, because of the lack of evidence for a specific mechanism involved in the recovery of pH_i from acidification after the NH₄Cl pulse, we decided to determine if recovery could occur under extreme conditions. First, we checked to see if it still occurred when we replaced everything in the bathing medium except HEPES with sucrose. Although the recovery rates were low in both control and experimental setups in these experiments, recovery still occurred when all constituents except HEPES were replaced with sucrose and there was no significant difference from control (Table 4). Second, we determined if the response to an NH₄Cl pulse was altered by reducing the temperature from 25 to 4°C. Because we could not change the temperature rapidly enough to examine the effect of cooling on the rate of recovery alone, we simply measured pH_i and determined the effect of an NH₄Cl pulse on it at 4°C. Under these circumstances, resting pH_i was reduced to ~6.6 (data not shown). The addition of NH₄Cl produced alkalinization, presumably from simple diffusion of NH₃ into the cells, but no acidification occurred during the NH₄Cl addition or after its removal (data not shown). The pH_i simply returned to control after NH₄Cl removal (data not shown). Thus acidification and recovery from acidification are temperature dependent.

Effect of EIPA and DIDS on resting pH_i. Because both EIPA and DIDS reduced the rate of recovery of pH_i from the acid to the resting value in distal-proximal segments in HEPES-HCO₃-buffered medium, we also checked to see whether these agents affected resting pH_i in distal-proximal segments in this medium. However, neither EIPA (100 µM) nor DIDS (100 µM) had any statistically significant effect on resting pH_i in these segments in the HEPES-HCO₃-buffered medium (control resting pH_i, 7.37 ± 0.05; resting pH_i with EIPA, 7.32 ± 0.08; resting pH_i with DIDS, 7.26 ± 0.04; n = 9).

	DISCUSSION

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In the present study, we continued our examination of pH_i regulation in snake proximal renal tubules in the presence as well as the absence of HCO₃ in the extracellular bathing medium. As in our previous study (17), we limited our examination of possible acid/base transporters involved in such regulation to those located at the basolateral membrane by working with tubules with oil-filled lumens.

Resting pH_i in some distal-proximal segments in the presence of HCO₃ was higher than the resting pH_i in distal-proximal segments in the absence of HCO₃. However, when examined carefully, resting pH_i was found to be essentially the same in both tubule segments and was unchanged in a given segment whether HCO₃ was present or absent. Nevertheless, the total buffering capacity was higher in the presence than in the absence of HCO₃ in both proximal-proximal and distal-proximal tubules. Thus, in both tubule segments, resting pH_i appeared to be regulated to a similar extent whether or not HCO₃ was present in the bathing medium.

As in our previous study on snake renal tubules (17), we examined the factors affecting the rate of recovery of pH_i from the minimum after an NH₄Cl pulse to gain insight into basolateral acid/base transporters that might help regulate pH_i. The control rate of recovery was similar in both tubule segments in both buffer systems and was similar to that observed in our earlier study (17). Thus it appears that pH_i is regulated in some fashion by ion fluxes across the basolateral membrane. In our earlier study with HEPES-buffered solution only (17), removal of Na⁺ from the bathing medium depressed the rate of recovery in the distal-proximal but not the proximal-proximal segments. This observation suggested that an Na⁺-dependent basolateral transporter (e.g., Na⁺/H⁺ exchanger or Cl/HCO₃ exchanger) might be involved in regulation of pH_i in the distal-proximal segment. However, neither amiloride (even in a concentration of 1 mM) nor DIDS (100 µM) had any effect on the rate of recovery in the presence of Na⁺ (17). Therefore, these data did not support the presence of any known Na⁺-dependent acid/base transporters at the basolateral membrane of these tubules. Instead, because Na⁺ removal from the bathing medium also depressed resting pH_i in the distal-proximal segment in HEPES-buffered medium, it appeared possible that the effects of Na⁺ removal reflected changes in the membrane potential (17).

In the current study, we examined the effects of Na⁺ and other ions, possible acid/base transport inhibitors, and changes in membrane potential on recovery of pH_i in both HEPES-buffered and HEPES-HCO₃-buffered media. In contrast to the previous study with HEPES-buffered medium alone, the removal of Na⁺ from the HEPES-HCO₃-buffered medium in the current study had no effect on the rate of recovery in either segment. Similarly, in the present study, the removal of Cl or of Na⁺ and Cl together had no effect on the rate of recovery in either segment in the presence or absence of HCO₃. Although these data did not support the presence of any Na⁺- or Cl-dependent acid or base transporter at the basolateral membrane of these tubule segments, we nevertheless examined the effects of EIPA and DIDS on the rate of recovery. Both of these agents significantly reduced the rate of recovery in the distal-proximal segment only and only in the HEPES-HCO₃-buffered medium.

The interpretation of these data is extremely difficult. Although it might be expected that a basolateral Na⁺/H⁺ exchanger would be present in these tubule segments, the lack of effect of Na⁺ replacement (at least in the current study) would seem to rule out the role of such a transporter in regulating pH_i. However, Na⁺ removal did reduce the rate of recovery of pH_i in the distal-proximal segment alone in HEPES-buffered medium in the previous study (17). This effect apparently cannot simply be a result of a change in the membrane potential because factors known to alter the membrane potential, albeit in the opposite direction, had absolutely no effect on the rate of recovery in either HEPES-buffered or HEPES-HCO₃-buffered medium. Moreover, EIPA reduced the rate of recovery in the distal-proximal segment in the presence of Na⁺ in HEPES-HCO₃-buffered medium in the present study, although neither EIPA in the present study nor amiloride in the previous study (17) had any effect on the rate of recovery in this segment in the presence of Na⁺ in HEPES-buffered medium alone. It is possible that EIPA has some effect other than one on an Na⁺/H⁺ exchanger that could produce this depression in the rate of recovery of pH_i. In any case, the data do not provide consistent support for the role of a basolateral Na⁺/H⁺ exchanger in the regulation of pH_i in the distal-proximal segment of these tubules.

Another possible mechanism for basolateral regulation of pH_i could be an H⁺-ATPase located in the basolateral membrane that transports H⁺ out of the cells. However, such a transporter has not generally been reported for vertebrate proximal renal tubules. Moreover, the lack of effect of bafilomycin on the rate of recovery of pH_i strongly militates against the involvement of a basolateral H⁺-ATPase in the regulation of pH_i. Similarly, a basolateral H⁺-K⁺-ATPase could move H⁺ out of the cells and K⁺ into the cells. Again, however, there is no report of this enzyme being located in the basolateral membrane of vertebrate proximal tubules, and the lack of effect of Schering 28080 appears to eliminate this possibility.

Much of the recovery of pH_i from an HN₄Cl pulse could, of course, involve the exit of NH⁺₄ per se from the cells across the basolateral membrane, especially because recovery can occur at control rates in a simple HEPES-buffered sucrose solution. We explored several pathways by which basolateral NH⁺₄ exit might occur, but the evidence supported none of them. First, the lack of effect of even 100 µM Schering 28080 on the rate of recovery of pH_i apparently eliminated the recently described K⁺/NH⁺₄ exchanger (4). Second, the failure of blockade of our previously described basolateral organic cation efflux pathway (16) to alter the rate of recovery apparently eliminated this route for NH⁺₄ efflux. Third, the lack of effect of even 20 mM TEA (as well as 20 mM TPeA and 5 mM Ba²⁺) on the rate of recovery appeared to rule out NH⁺₄ exit via larger K⁺ channels. Therefore, at present we have no definitive information about the basolateral mechanisms that might be involved in regulation of pH_i or even in simple recovery from an NH₄Cl pulse in either segment of snake renal proximal tubules.

Perspectives