Luminal carbonic

pH in the amphibian distal tubule: effects of anhydrase and carbonic anhydrase inhibitors

GABRIELLE Institut Necker




Planelles, Gabrielle, Francoise Discala, and Takis Anagnostopoulos. Luminal pH in the amphibian distal tubule: effects of carbonic anhydrase and carbonic anhydrase inhibitors. Am. J. Physiol. 263 (Regulatory Integrative Comp. Physiol. 32): R1254-R1259, 1992.-To better delineate acidbase transport properties in the distal tubule (DT) of Necturus in vivo, we 1) studied the effects of peritubular (pt) isohydric increase of PCO~ and [HCO,],, on luminal pH (pH& and 2) measured the steady-state pHIU under various experimental conditions. The experiments were carried out on initial (DTi) or distal (DT,) loops of the DT in control state and then during intravenous infusion of carbonic anhydrase (CA) or CA inhibitors (CAI). In control state, isohydric increase of PCO~ and [HCO,],, results in transient acidification of the DTi lumen, whereas in DTd lumen the same maneuver yields sustained (plateau) acidification. Under systemic infusion of CAI, isohydric increase of PCO~ and [HCO& lowers pHI, (sustained fall of pHI,) in DTi and DTd, whereas under CA infusion both segments exhibit only transient acidification. During intravenous infusion with benzolamide DTi steady-state pH,, falls, suggesting that this maneuver inhibits a functional luminal CA, in contrast to the DTd, whose pH,, remains unaltered. Intravenous infusion of CA significantly increases steady-state DT, pHlu; by contrast, steady-state pH,, in DTi does not change. These data are consistent with the presence of functional luminal CA in the DTi, whereas the DTd segment lacks the luminal enzyme. acid-base




AMPHIBIAN DISTAL NEPHRON is a major site of urinary acidification (13, 25). The mechanisms underlying acid-base transport in the amphibian distal tubule have been explored on an in vitro preparation in Amphiuma (21) and in the Necturus kidney in vivo (15, 16). In the latter species, we have established that the transfer of H+ equivalents into the lumen is mediated, at least in part, by a K-H pump (16), while absorption of base-equivalents is carried out via a basolateral Na-(HCO,),,, cotransport (15). It is also well known that in proton-secreting segments the presence of luminal carbonic anhydrase is instrumental for rapid transcellular HC03 absorption, whereas lack of luminal carbonic anhydrase is attended by luminal acid disequilibrium pH, favoring ammonium secretion (for reviews, see Ref. 12 and Ref. 6). Because it has been established that the amphibian distal tubule (DT) absorbs bicarbonate (15) and that ammonium secretion in the amphibian kidney occurs in the distal tubule (24), it was important to find out whether carbonic anhydrase (CA) activity is present in the luminal cell membrane in the DT of Necturus in vivo, to better delineate the role of this segment in acid-base homeostasis. Our data indicate that the initial portion of the accessible DT in Necturus is endowed with functional luminal CA, whereas the distal portion of this segment (still, the accessible portion) lacks this enzyme. THE


National de la Sante et de la Recherche Mkdicale, Enfants-Malades, 75730 Paris Cedex 15, France


Unit6 323, Centre Hospitalier



Adult Necturi of either sex, purchased from Nasco, were kept in tap water and fed three times a week. Initial anesthesia was achieved by - 15 min immersion in a solution of tricaine methane-sulfonate (0.78 g/l); thereafter this solution was diluted fivefold throughout the experiment. Dissection of the animal, exposure of the kidney surface, and continuous delivery of a physiological Ringer solution on the surface of the kidney have been described elsewhere (17). Determination and Luminal






Transepithelial potential difference ( VTE) and luminal pH (pH1,) were measured simultaneously with double-barreled microelectrodes. The technique for construction of such electrodes, including introduction of a droplet of H-sensitive resin (I) in the tip of the silanized barrel and calibration procedures for these electrodes, has been described in detail elsewhere (17). Briefly, one of the channels of a double microelectrode is exposed to N,N-dimethyltrimethylsilylamine (Fluka) for 7 min. After baking (2 h at IZOOC), a droplet of the H+ ion exchanger is introduced into the silanized channel and then allowed to reach the tip of the electrode after a few hours. Then the selective barrel is backfilled with the following solution (in mM): 67 NaCl, 40 KH,PO*, and 23 NaOH, pH 7.0. The nonselective barrel is backfilled with 1 M KCl. Before the experiment, double-selective microelectrodes are tested in a physiological Ringer solution (in mM, 82 NaCl, 3 KCl, 1 MgC12, and 1.8 CaCl,) buffered with tris(hydroxymethyl)aminomethane (trizma base; 50 mM) from pH 6.0 to 8.0. When necessary, the tip was gently beveled using a microgrinder (De Marco Eng) to improve the response time. We used microelectrodes displaying a slope comprised between 50 and 58 mV/unit pH. To detect possible changes in electrode properties with time, the slope was systematically checked before and after each impalement, on the surface of the kidney, by changing for 30 s the superfusion solution [N-tris(hydroxymethyl)methyl-Z-aminoethanesulfonic acid (TES) IO mM buffered Ringer solution] from pH 7.5 to 7.0, then back to pH 7.5. Paired conventional and selective potentials were fed into the input of an ultrahigh impedance electrometer (World Precision Instruments, model 223, New Haven, CT); the differential signal (i.e., the chemical H potential) is provided automatically. The outputs (V and pH) were recorded to a multipen chart recorder (Sefram, Servofram Enertec). Impalement of the DT lumen with double-barreled pH microelectrodes was achieved on the basis of the following criteria: 1) VTE is lumen negative (14), and 2) pH,, is more acid than that of the proximal tubule and the early distal tubule (EDT) (16, 17). We impaled DT loops lying lateral but close to the glomerulus and also DT loops located more externally from the craniocaudal axis. The former follow the EDT and will be designated by DTi (initial DT); the latter correspond to more distal sites of DT (DT,). Preliminary experiments in which Silastic (Canton Biomed, Boulder, CO) was injected through the glomerulus confirmed this distribution.

R1254 0363-6119/92 $2.00by ${individualUser.givenNames} Copyright 0 1992 The American Physiological Society( on January 20, 2019. Downloaded from ${individualUser.surname}





To ascertain whether the DT is endowed with luminal carbonic anhydrase (CA],), we challenged this segment by suddenly increasing peritubular PCO~ while recording pH,,. Because CO, is a highly diffusible species, Pcoz equilibrium between perfusing solution and luminal fluid is warranted. Entry of CO, into the lumen brings about a new equilibrium, according to the relationships Hz0 -+ H+ + OH- and OH- + CO, --+ HCO;; the second reaction is relatively slow (IO) but is considerably accelerated (--6,000-fold) in presence of CA (9). The rise of PCO~ was carried out through a peritubular (pt) perfusion solution, whose composition (in mM) was 63.8 NaCl, 3 KCl, 1.8 CaCl,, 1 MgCIB, and 26.4 NaHCO,, equilibrated in vitro with a gas mixture (3 kPa C02-20 kPa 02-77 kPa N2) at pH 7.6. This solution was delivered via a glass micropipette (OD 1.1 mm, ID 0.45 mm) into a peritubular capillary vessel, adjacent to a DT loop, to be impaled; the micropipette was connected upstream to a polyethylene (PE) tube, both of which were filled with the above artificial solution. A gravimetric system allowed delivery of this solution, when necessary. To avoid CO, loss, the PE catheter and the pipette (except for the first cm from its tip) were protected by a larger, gastight tube in a concentric fashion: a 3-kPa CO, gas mixture (see above) was continuously delivered between the concentric tubes to avoid pH changes in the perfusion solution. This was checked on occasion during each experiment by shortly delivering this solution into a peritubular capillary impaled with a pH microelectrode. The imposed PCO~ is higher than the physiological PCO~ of Necturus: blood PCO~ content in Necturus is Cl kPa CO, (23), whereas the artificial solution has been equilibrated with 3 kPa CO, at pH 7.6, the physiological peritubular blood pH for amphibia (2). Thus this disturbance amounts to isohydric increase of PCO~ and [HCO,],,. To rule out contamination from fluid originating in earlier parts of the nephron, in some experiments we blocked the lumen upstream with castor oil while recording DT pH,, and VTE during isohydric increase of PCO~ and wQ31,t in peritubular capillaries. Experimental


To substantiate whether the DT is endowed with luminal CA, we challenged this preparation with systemic infusion of this enzyme or with CA inhibitors (CAI). Studied parameters were 1) steady-state pHI, and 2) time-dependent changes of pHI, during a short exposure (- 1-2 min) to isohydric increase of PCO~ and [HCO,],,, carried out by peritubular capillary perfusion. Single loops were studied in paired fashion, before and after systemic infusion with CA or CAI; three experimental groups were defined. Group 1 (5 animals, n = 11 tubules) was treated with systemic infusion containing acetazolamide (ACZ): prime, IO mg/kg body wt; sustaining, 1 mg kg-l h-l. Group 2 (7 animals, n = 16 tubules) received systemic infusion supplemented with benzolamide (BZL): prime, 32 mg/kg body wt; sustaining, 3.2 mg kg-l 9h-l. Group 3 (6 animals, n = 10 tubules) was defined by systemic infusion containing CA, 40 mg/kg; sustaining, 4 rngm kg-l *h-l. Systemic delivery was carried out through the hepatic or iliac vein. One to three superficial DT loops were studied in a single animal, before (control state), then during intravenous infusion of ACZ, BZL, or CA (experimental state). When possible, one double-selective microelectrode was used throughout the experiment. The experimental state starts 30 min after prime injection and ends up to 2 h later. To rule out that volume expansion during the experimental period (resulting from intravenous infusion) neither altered the steady-state pHI, nor the time-dependent changes of pH,, during isohydric increase of PCO~ and ww31,tt we checked in two animals (n = 6 tubules) that these l




parameters infusion.


were not altered before and after intravenous

R1255 vehicle

Chemicals Acetazolamide and benzolamide were generous gifts from Theraplix and Lederle Laboratories, respectively. CA (from bovine erythrocytes) was purchased from Sigma. Proton cocktail was manufactured by Dr. A. Kurkdjian, according to the procedure described by Ammann et al. (1). Statis tics The data are expressed as means t SE. Student’s used to define the level of significance.

t test was


Control Period Steady-state pHl, and VTE. Simultaneous measurements of pHIU and VTE show that steady-state pH,, is 6.72 t 0.04 in DTi and VTE is -20.6 t 2.7 mV (n = 20); by contrast, in DTd, pH,, is 6.40 t 0.07 and VTE is -36.9 t 3.1 mV (n = 17). Time-dependent pHIU profile during isohydric increase of Pco2 and [HCO,],,. DTi. peritubular exposure to an


solution buffered to physiological pH, at high and [HCO&, results in an initial acidification (peak), followed by recovery to a value close to the steadystate pH1,. Switching off the peritubular perfusion (capillary blood recovery) yields a mirror image, i.e., an alkaline overshoot, followed by relaxation toward steady-state PHI,. A representative recording from this series is illustrated in Fig. 1A. DTd. the above maneuver brings about a different pH1, profile in the DTd; pH,, falls to reach a lower pH1, plateau. Stopping the peritubular perfusion fails to generate alkaline overshoot. An experiment from this series is represented in Fig. 1B. Injection of an oil block in the lumen upstream did not alter the above-described time-dependent changes in pH,, during isohydric increase of PCO~ and [HCOJPt in peritubular capillaries (n = 3 animals, 5 DTi and 3 DTd, data not shown), indicating that changes in pH,, on increasing PCO~ and [HCOJpt are genuine in situ effects. PCO~

Effects of ACZ

In this series, we measured during control period 1) the steady-state pH1, and 2) the time-dependent changes of pH1, during isohydric increase of PCO~ and [HCOJPt; then the same parameters were assessed during systemic exposure to ACZ. In the presence of ACZ, the steady-state pH1, of DTi loops increased from 6.63 t 0.05 to 7.07 t 0.07 (n = 5, P < 0.02) as did DTd loops, from 6.35 t 0.14 to 6.95 t 0.06 (n = 5, P < 0.01); see also Table 1. Furthermore, in presence of ACZ, isohydric increase of PCO~ and [HCO& results in plateau acidification in both DTi and DTd loops. Effects of BZL

In a second series of comparable experiments, we assessed the same parameters during control state, then, during systemic infusion of BZL. Like the ACZ experiment, isohydric increase of PCO~ and [HCO& in the

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pH 6.1

pH 6.3 65. Fig. 1. Effect of isohydric increase of PCO~ (3 kPa) and peritubular HCO, concentration ([HCO,],,; 26.4 mM) on luminal pH (pH1,) (top) and transepithelial potential difference ( VTE) (bottom). Thick horizontal bars, peritubular exposure to isohydric increase of PCO~ and [HCO&; thin horizontal bar, time scale. Due to position of pens on the recorder, pH1, trace slightly precedes VTE trace in real time. A: effects of isohydric increase of PCO~ and [HCO,],, on an initial distal tubule (DTi) loop; B: effects of isohydric increase of PCO~ and [HCO& on a more distal distal tubule (DTJ loop.

67. 71. 75. mV 0 -20 -lli lmin


Table 1. Summary of the steady-state luminal pH and transepithelial potential difference measurements Steady-State Control

Group DTi


pHIU; State

Steady-State Experimental

pHIU; State



Steady-State VTE; Control State, mV



Group 2 (BZL) DTi DTd

Steady-State Experimental

VTE; State,




6.63t0.05 6.35t0.14

7.07t0.07 6.95t0.06

5 6

* *

-23.4t6.1 -31.0k3.6

-27.Okl.9 -49.8t6.2

5 6

NS *

6.74kO.06 6.48kO.10

6.65t0.06 6.57t0.10

10 6

* NS

-18.9t3.4 -34.3t2.3

-21.7t2.6 -31.3zkl.9

10 6


Group 3 (CA) -19.4t7.1 -12.8k5.8 DTi 6.78kO.06 6.74t0.04 5 NS 5 NS -28.0t6.2 6.35kO.09 6.69t0.05 5 * -47.0t8.3 5 NS DT-I Values are means t SE; n, no. of animals; pHI,, luminal pH; V TE, transepithelial potential difference; ACZ, acetazolamide; BZL, benzolamide; CA, carbonic anhydrase. Statistics were performed with paired t test. NS, nonsignificant; * P < 0.05.

presence of BZL brings about plateau luminal acidification in both DTi and DTd loops. Figure 2 illustrates the pattern of a DTi segment during isohydric increase of PCO~ and [HCO&, before (control) and under intravenous BZL infusion, in paired fashion. The steady-state pH1, during intravenous BZL administration decreases in the DTi (from 6.74 t 0.06 to 6.65 t 0.06, n = 10, P c 0.03) whereas in DTd, pHI, fails to change (6.48 t 0.1 vs. 6.57 t 0.1, n = 6, NS; see also Table 1).

venous infusion of CA. There was no change in steady-state pH,, (CA infusion VS. control state) in DTi loops (6.78 t 0.06 VS. 6.74 t 0.04, respectively, n = 5, not significant), whereas DTd loops display an increase of pHI,, from 6.35 t 0.09 to 6.69 t 0.05 (n = 5, P < 0.04; see also Table 1). DISCUSSION

This study is focused on the distribution and functional properties of the CA along the DT of Necturus. In the third experimental series, the effects of CA in- Two parameters are considered: 1) the pHI, profile during under various fusion on pH,, were studied. Isohydric increase of PCO~ isohydric increase of Pco,, and [HCO& and [HCOJPt in presence of CA elicits a transient fall of experimental conditions (paired comparison between no pHlu, followed by recovery of pH,, to its steady-state addition vs. iv infusion of ACZ, BZL, or CA), and 2) the value, in both DTi and DTd segments. Figure 3 illustrates steady-state pHIU in control state, then during systemic these changes in a DTd segment before and during intradelivery of ACZ, BZL, or CA. Effects of CA

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96.5 pH .67. m 69.

pH6.7 69. 71. 73 . 75 mI mV 0

Fig. 2. A: effect of isohydric increase of PCO~ and on a DTi segment. B: same disturbances on wm1,t same DTi loop during continuous intravenous infusion supplemented with benzolamide (BZL). Note difference in steady-state pHI, between the 2 conditions (control vs. BZL). Symbols and notation as in Fig. 1.


L -20



imin Localization of Luminal in Distal Tubule

lmin Carbonic


Time-dependent pH1, profiles, recorded during isohydric increase of PCO~ and [HCOJpt, were assessed in control state and then during infusion of CA or CAIs. Because of the high diffusibility of COz, this gas freely permeates renal cell membranes (19); thus an increase of peritubular Pco2 is attended by equal rise of luminal Pco~. The gas COz, entering the lumen, is hydrated to yield carbonic acid; this acid load generates immediate acidification followed by a plateau phase. Plateau pH1, results from a balance between the acid loading rate (i.e., CO2 hydration and, possibly, cellular secretion of H+ equivalents) and HC03 influx into the lumen (i.e., H&JO3 dissociation, paracellular HC03 diffusion, and possibly cellular base-equivalent secretion). In the absence of systemic infusion (control state), isohydric increase of PCO~ and [HCO& results in DTi transient acidification followed by pH,, recovery from this disturbance (Fig. lA), whereas in DTd the same maneuver yields a fall of pH,, (“plateau acidification”) without significant recovery during this state (Fig. 1B). The simultaneous presence of both pHI, profiles in single animals, under control conditions, rules out influences associated with different metabolic and/or hormonal states. As stated above, the plateau pH,, expresses a dual mechanism, acid loading rate vs. base loading rate; in the presence of CA],, HC03 influx into the lumen and subsequent dissociation in OH- and CO2 leads to pHI, recovery toward the control value. These time-dependent pH,, profiles in DTi and DTd were reexamined after systemic infusion of CA1 or CA.

Two distinct CAIs were used in this study. ACZ inhibits both cytoplasmic and membrane-bound CA (11, 18), whereas the low dissociation constant of BZL prevents it from entering the cell; thus BZL is considered as an inhibitor of luminal CA (8,18), even if supplied by systemic infusion (3), because it is secreted into the proximal tubular lumen (7). To rule out a loss of selectivity in luminal vs. cytosolic CA inhibition by using 32 mg/kg body wt (prime dose) BZL, we checked that similar results were obtained with a lower concentration, 3.2 mg/kg body wt bolus (n = 3 DTi, data not shown). That under BZL, as well as in presence of ACZ, isohydric increase of PCO~ and [HCOslpt yields plateau acidification in both DTi and DTd lumen (thereby converting the DTi profile to that of DTd) strongly suggeststhat the time-dependent profile of pHI, depends on presence (or absence) of luminal CA. To further substantiate this claim, we initiated a systemic infusion of CA to provide functional CA in contact with the luminal surface of cell membrane, as shown by others (3, 4). Under these conditions (presence of luminal CA), isohydric increase of PCO~ and [HCOJpt shifts the pHIU pattern of DTd to that of DTi. These data strongly suggest that in control state (no systemic infusion) the luminal CA is located only in the DTi. The steady-state pH1, under control conditions and during intravenous infusion of CA or CA1 supports the above conclusion. Steady-state pHI, is by definition at equilibrium in presence of CA and, reciprocally, lack (inhibition) of luminal CA should yield disequilibrium pH. Our approach does not allow determination of (dis)equilibrium pH, which would require simultaneous measurements of pH and total COz in situ (3, 22). We simply

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61. 63. 65.

l6.6 PH ------i/ -_-; _.-._._ ..-_--L--_ ---

_t-1 Fig. 3. A: effect of isohydric increase of PCO~ and [HCO,],, in a DTd segment. B: same experimental maneuver was repeated on the same DTd loop under continuous intravenous carbonic anhydrase (CA) delivery. Note the difference in steady-state pH,, between these 2 conditions (control vs. CA). Symbols and notation as in Fig. 1.

oh@--+- -f-y---- -;j -..-- -- -i -.,-----:---





mV O-






-. 4 L/ .---, ! i

-0 mV



.- 25

t :



recorded steady-state pH1, in control and then experimental (ACZ, BZL, CA) states. Table 1 shows that systemic infusion of CA raises the steady-state pHI, in DTd, suggesting dissipation of acid disequilibrium pH; by contrast, BZL significantly decreases steady-state pHI, in DTi, suggesting induction of acid disequilibrium pH. Such observations support our conclusions regarding the distribution of luminal CA along the DT. Proton



The data confirm that H+ secretion proceeds along the whole DT. In the absence (or during inhibition) of luminal CA, H+ secretion should result in acid disequilibrium pH; indeed, we observed a fall of steady-state pHI,. Nevertheless, it should be stressed that our observations on steady-state pH1, cannot be considered as reliable estimates of equilibrium or disequilibrium pH, since they rely on the assumption of extremely high CO, diffusibility under all experimental conditions. That ACZ brought about a significant rise of steadystate pHIU in both DTi and DTd suggests that both segments are endowed with intracellular CA: decrease of endogenous production of H+ during inhibition of the intracellular enzyme must lower the rate of acid secretion if H+ secretion is totally or even partly CA dependent. If a fraction of the acid secretion is CA independent, the steady-state pHI,, in presence of ACZ, may also include a component of acid disequilibrium pH. Elimination

of Upstream


It could be argued that pHIU profiles observed during isohydric increase of PCO~ and [HCO& and/or differ-

ences in steady-state pH,, between tubules under control conditions vs. systemic infusion with CA or CA1 may not reflect in situ events. However, neither the time-dependent changes of pHI, following isohydric increase of PCO~ and [HC031pt nor steady-state pHI, are different when comparing free-flow and oil-block single tubules. This direct approach could be applied only in control state, since drugs (CA or CAI) delivered by systemic infusion would not have access to the tubular lumen of DT in the presence of an oil droplet upstream. However, two arguments indicate that the observed changes in steady-state pH,, in the presence of BZL or CA reflect in situ events. First, the decrease in steadystate DTi pH,, under BZL is not ascribed to upstream events, because the inhibition of CA], in proximal and diluting tubules would rather increase the HC03-filtered load (7). Thus the BZL-associated fall of pHIU in DTi may have been underestimated, but it is qualitatively correct. Second, the CA-associated increase of DTd steadystate pH1, does not reflect an upstream effect because CA fails to alter the PHI, of DTi, which immediately precedes the DTd. These arguments converge to validate our interpretation that we are dealing with events occurring in DTi and DTd in situ. Perspectives

This study, carried out on the distribution of CAI, along the DT of Necturus, provides new information on the properties of the DT. It reveals axial heterogeneity between DTi and DTd with regard to CAI, distribution. This finding is consonant with previous observations on

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distal nephron, which displays axial morphological heterogeneity (20). Our study suggests that functional heterogeneity occurs along the Necturus distal tubule. A comparison of physiological role and transport mechanisms between the amphibian DT and the mammalian distal convoluted and/or connecting tubule may be premature yet this correspondence could be considered in the future. In the DT of Necturus, DTi pHIU falls on exposure to a luminal CA inhibitor, whereas in the DTd PHI, increases on exogenous supply of CA, denoting active acid secretion. The presence of CAI, in DTi favors HC03 absorption (6), thereby increasing the rate of H+ equivalent secretion. Because this segment is endowed with an apical K-H-ATPase (16) and a basolateral Na-(HC03)n,l cotransport (ES), it provides an interesting model epithelium for further in vivo acid-base transport studies. The physiological role of DTd on acid-base homeostasis is still speculative. By analogy with the mammalian nephron (5, 6), the absence of CA1, may favor ammonium secretion, which is restricted to the amphibian distal nephron (24). Further studies, including measurements of (dis)equilibrium pH are required to implement this hypothesis.


The authors are grateful to Michele Poitou for expert secretarial assistance. Address for reprint requests: G. Planelles, INSERM U.323, CHU Necker Enfants-Malades, 156 rue de Vangirard, 75730 Paris Cedex 15, France. Received

21 October

1991; accepted

in final


19 June






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Luminal pH in the amphibian distal tubule: effects of carbonic anhydrase and carbonic anhydrase inhibitors.

To better delineate acid-base transport properties in the distal tubule (DT) of Necturus in vivo, we 1) studied the effects of peritubular (pt) isohyd...
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