V-type H+-ATPase in the human eccrine sweat duct: immunolocalization and functional demonstration

D. Granger1, M. Marsolais1, J. Burry2, and R. Laprade1

1 Groupe de Recherche en Transport Membranaire, Université de Montréal, Montreal, Quebec, Canada H3C 3J7; and 2 Unilever Research, Port Sunlight, UK CH63 3JW


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

We investigated for the presence of a vacuolar-type H+-ATPase (V-ATPase) in the human eccrine sweat duct (SD). With the use of immunocytochemistry, an anti-V- ATPase antibody showed a strong staining at the apical membrane and a weaker one in the cytoplasm. Cold preservation followed by rewarming did not alter this staining pattern. With the use of the pH-sensitive dye 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein on isolated and perfused straight SD under HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>-free conditions and in the absence of Na+, proton extrusion was determined from the recovery rate of intracellular pH (dpHi/dt) following an acid load. Oligomycin (25 µM), an inhibitor of F-type ATPases, decreased dpHi/dt by 88 ± 6%, suggesting a role for an ATP-dependent process involved in pHi recovery. Moreover, dpHi/dt was inhibited at 95 ± 3% by 100 nM luminal concanamycin A, a specific inhibitor of V-ATPases, whereas 10 µM bafilomycin A1, another specific inhibitor of V-ATPases, was required to decrease dpHi/dt by 73%. These results strongly suggest that a V-ATPase is involved in proton secretion in the human eccrine SD.

proton pump; intracellular pH measurement; concanamycin A; bafilomycin A1; oligomycin


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

THE SECRETORY PORTION of the eccrine sweat gland generates a fluid called the primary secretion. The composition of this isotonic fluid is similar to an ultrafiltrate of the plasma and has a pH of 7.4. However, as it flows through the ductal portion of the gland, this fluid is modified by the reabsorption of solutes such as Na+, Cl-, lactate, and HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>, leaving a hypotonic sweat (4, 32). Current models of ionic transport in the human eccrine sweat duct propose the presence of luminally located Na+ and Cl- channels and basolaterally located Na+-K+-ATPase, as well as K+ and Cl- channels (4, 2, 35, 32, 33, 36). The pH of final sweat is a function of sweat rate, being as acidic as pH 5 when the sweat rate is low and increasing as sweat rate increases (22). The acidity of sweat implies that sweat duct cells secrete protons across the apical membrane, and the presence of an electrogenic proton-secreting pump at this membrane has been proposed (3, 27, 34). Indeed, vacuolar-type H+-ATPases (V-ATPases) have been shown to be responsible for the acidification in many organelles of eukaryotic cells, including clathrin-coated vesicles, lysosomes, endosomes, and vacuoles of plants and fungi (31). Moreover, V-ATPases are highly expressed in the apical membrane of specialized epithelial cells in the kidney (13, 24), epididymis, and vas deferens (14), where they play a major role in the acidification of urine and luminal fluid of the reproductive tract (11, 16, 12, 23).

Recently, in the sweat duct, we have demonstrated by both immunolocalization and intracellular pH (pHi) measurements that both Na+/H+ exchanger isoforms 1 (NHE1) and 3 (NHE3) were absent from the luminal membrane, whereas NHE1 was present at the basolateral membrane (25). In addition, an immunolocalization study showed the presence of an apical V-ATPase (7).

Therefore, the aim of the present work was to further the recent immunolocalization studies and to demonstrate the functional activity of the apical V-ATPase in the sweat duct. For this purpose, we microperfused straight sweat ducts in vitro and measured the effect of oligomycin, an inhibitor of mitochondrial ATP synthase (F0F1) (39) and specific inhibitors of V-ATPases, bafilomycin A1 (8) and concanamycin A (19-21), on basal pHi and on the pHi recovery rate from an intracellular acid load. Both HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> and Na+ were absent from the solutions to eliminate the possible contributions of these ions to proton transport.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Immunocytochemistry

Tissue fixation. Specimens of human healthy skin (female) were taken, after esthetic surgery, with consent from patients and ethics committee approval. Skin was conserved at 4°C in Dulbecco's phosphate-buffered saline containing CaCl2 and MgCl2 from Sigma Chemical (St. Louis, MO) until use. Sections of human skin were fixed for 1 h in 2% paraformaldehyde, 75 mM lysine, and 10 mM sodium periodate (PLP) at room temperature. Slices (3 mm) were cut with scissors and fixed overnight at 4°C in fresh PLP. The tissue was moved in phosphate buffer containing 1.9 mM NaH2PO4, 8.1 mM Na2HPO4, 0.9% NaCl, and 0.02% NaN3, pH 7.4 (PBS), until the cryostat step.

Effect of temperature. In another series of experiments, we fixed skin samples after a rewarming period of 0, 15, 30, and 60 min at 37°C in Dulbecco's phosphate buffer. The fixation step for this experiment was done at 37°C in a PLP solution.

Cryostat sectioning. Sections infiltrated with 30% sucrose and frozen in liquid nitrogen were covered with a drop of Tissue-Tek embedding medium (Sakura, Torrance, CA) and cut at a thickness of 5 µm on a cryostat -25°C. Sections were picked up onto Fisher Superfrost Plus-charged glass slides and stored at -20°C.

Antibodies. The proton pump antibody is a rabbit antiserum raised against the COOH-terminal 14 amino acids of the 31-kDa subunit (E subunit) of the V-ATPase (13) (provided by Dr. Dennis Brown, Massachusetts General Hospital, Charlestown, MA), used at a 1:100 dilution. The secondary antibody, a goat anti-rabbit IgG-FITC (ImmunoResearch, West Grove, PA) was used at 7.5 µg/ml. Negative controls, including omission of primary antibodies, and preimmune serum were performed. Sections of rat kidney were used as positive control.

Incubation procedure. The incubation procedures were based on the protocol described by Brown et al. (15). The standard procedure and also the antigen unmasking procedure, in which an incubation with 1% SDS was performed, were completed (15). The incubation of sections with antibodies was carried out overnight at 4°C for the anti-proton pump antibody and for 2 h at room temperature for the secondary antibody.

Photography. Tissues were viewed at ×400 magnification with a Nikon Fluor 40/1.3 oil objective in a Nikon epifluorescence microscope with fluorescein filters (excitation 450-490 nm, barrier 510 nm). Sections were photographed in black and white with a Nikon FX-35DX camera on Kodak TMax 400 film at 1600 ASA.

Materials and reagents. Sodium nitrate was purchased from Anachemica Chemicals (Montreal, Canada). BDH Laboratories (Poole, England) supplied L-lysine monohydrochloride. All other chemical products were purchased from Sigma Chemical.

Measurements of pHi

Isolation and microperfusion of sweat duct. Eccrine sweat glands were isolated by using a previously described shearing technique (28). Portions of straight reabsorptive duct were dissected with sharpened forceps and microperfused in vitro, as previously described for kidney tubule (1). Visual criteria for luminal perfusion and duct viability were validated by using basolateral membrane potential measurements that were stable for >1 h and that responded to luminal Na+ and Cl- substitutions. Bath solution temperature was kept at 37°C, and the flow of the bath solution (flow rate >1 ml/min) was aimed directly at the sweat duct as described by Macri et al. (29). The luminal perfusion rate was high enough to eliminate axial changes in the luminal fluid composition.

pHi. pHi was measured with the fluorescent probe 2', 7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF). The acetoxymethyl ester form of the dye (BCECF-AM) was added to the bath solution at a final concentration of 5 µM, and loading was allowed in the nonperfused bath for 5-10 min at 25°C. After loading, the bath was perfused with control solution at 37°C for at least 5 min before an experiment was begun. The duct was alternately excited at 450 and 500 nm, and the emitted fluorescence was monitored at 530 nm with the photomultiplier-based spectrofluorimeter PTI D104 (Photon Technology International, London, Ontario) linked to a computer. The fluorescence ratio (F500/F450) corrected for autofluorescence was calculated and converted to pHi at the end of each experiment by calibration with the high-K+ nigericin method (38).

Acid loading. Sweat ducts were H+ loaded with the ammonia prepulse technique. NH4Cl (20 mM) was added to the bath solution for 30-60 s and then removed, resulting in abrupt intracellular acidification.

Solutions and chemicals products. The HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>-free control solution used to perfuse and bathe the sweat ducts contained (in mM) 114 NaCl, 25 sodium gluconate, 2.5 K2HPO4, 1 MgCl2, 1 CaCl2, 5 glucose, and 4 sodium lactate. Osmolality was adjusted to 300 mosmol/kgH2O with mannitol and pH adjusted to 7.4 with Tris-HEPES. For experiments done in the absence of Na+, sodium lactate and NaCl were replaced by N-methyl-D-glucamine (NMDG)-lactate and -chloride, respectively.

BCECF-AM was obtained from Molecular Probes (Eugene, OR) and stored in a freezer as a 1 mM stock in 95% ethanol. NH4Cl was purchased from Fisher Scientific (Fair Lawn, NJ). Bafilomycin A1, diluted in dimethyl sulfoxide (DMSO) and stored at 4°C, was the gift of Dr. S. Pathak (SmithKline Beecham). Concanamycin A (also known as Folimycin) was from Sigma Chemical and was diluted in DMSO and stored at -20°C. The oligomycin used was a mixture of oligomycins A, B, and C (~65% oligomycin A) diluted in ethanol (Sigma Chemical). The final concentration of DMSO or ethanol in any solution did not exceed 0.1% and had no effect on pHi. All other chemical products were purchased from Sigma Chemical.

Statistics. Results are presented as means ± SE. Statistical significance was analyzed by using the paired or unpaired Student t-test. Significance was accepted at P <=  0.05.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
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Distribution of V-ATPase in the Sweat Duct

Immunostaining. To determine the cellular localization of proton pump in the human eccrine sweat duct, we used a rabbit anti-V-ATPase antibody on cryosections (5 µm) of PLP-fixed human skin. Comparison between phase-contrast and fluorescent images (Fig. 1, A and B) shows that the luminal cells of the sweat duct are heavily labeled at their apical pole. Also, cytoplasmic staining is shown, suggesting a redistribution of V-ATPase into cytoplasmic vesicles. In the secretory coil, we obtained a discontinued and weaker apical staining compared with that shown in the reabsorptive sweat duct (Fig. 1D). Moreover, no difference appeared in V-ATPase distribution when we used an SDS unmasking technique for antigen retrieval epitope (15), although the intensity of staining was slightly increased (not shown). Control incubation with the use of preimmune rabbit serum gave no detectable staining (not shown). Sections of rat kidney were used as positive control and gave results identical to those previously published by Brown and coworkers (12, 13).


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Fig. 1.   Immunofluorescence staining with a polyclonal anti-proton pump antibody directed against the 31-kDa subunit (E subunit) of the vacuolar-type H+-ATPase (V-ATPase) was applied on 5-µm cryosections of sodium periodate (PLP)-fixed human skin. A and C: correspondent phase-contrast microscopy showing the same regions of V-ATPase immunolocalization in sweat ducts (B) and in the secretory portion (D), respectively. Bars, 15 µm.

Effect of temperature. In kidney-intercalated cells, it has been shown that cold preservation followed by rewarming to 37°C induces a marked redistribution of proton pumps into endocytotic vesicles (10). However, no significant difference in the distribution of proton pumps was identified on secretory coil and sweat duct cells after a period of rewarming preceding tissue fixation, compared with control (without rewarming). In all samples (rewarming during 0, 15, 30, and 60 min), a strong V-ATPase staining, identical to that shown in Fig. 1B, was always present at the apical membrane of sweat ducts.

Functional Activity of the Proton Pump in the Sweat Duct

Acid load and pHi recovery. To investigate the functional activity of protons pumps, we monitored the rate of pHi recovery, following an acute acid load induced by a 20 mM NH<UP><SUB>4</SUB><SUP>+</SUP></UP> pulse, on perfused sweat ducts. We measured the recovery rate of pHi (dpHi/dt) in the absence of HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> to eliminate the contribution of potential bicarbonate transporters. Under control conditions, we measured a baseline pHi of 7.33 ± 0.03 (n = 41). Addition of 20 mM bath NH4Cl followed by its withdrawal produced a sharp fall of pHi with subsequent recovery within 3 min (Fig. 2, A and B, first pulse). Na+ was then replaced by NMDG, in both bath and lumen, to inhibit all potential Na+/H+ exchangers, such as the basolateral NHE1 that we recently identified in this tissue (25). Under these conditions, after a strong decrease of pHi due to the reversal of the basolateral Na+/H+ exchanger, a second NH4Cl pulse was applied. Two types of response were obtained following the acid load: 1) a slower pHi recovery, decreased by 65 ± 6% (n = 14) with respect to control, which occurred in most of the sweat ducts (14 of 21 tested) (see Figs. 2A and 6A); and 2) no significant pHi recovery following the acid load, which occurred in about one-third of the ducts tested (Fig. 2B).


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Fig. 2.   Two different intracellular pH (pHi) recovery responses in the absence of Na+, following an acid load, on perfused sweat ducts. A: a decreased recovery rate (second pulse, 0 Na+) compared with that in control solution (first pulse, Ctl) was observed on 14 of 21 tested sweat ducts. B: in contrast, in the other ducts no recovery was observed in the absence of Na+. Acid load, indicated by the shaded portion of the protocol, was by NH4Cl prepulse. All solutions were HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> free.

ATPase inhibitors. To investigate whether the pHi recovery in response to the acid load in the absence of Na+ and HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> is ATP dependent, we tested the effect of oligomycin, an inhibitor of mitochondrial ATP-synthase (F-type ATPases) (39). Addition of oligomycin (25 µM) to bath and luminal solutions had no effect on basal pHi but strongly inhibited pHi recovery after the acid load (Figs. 3 and 6B). A rapid recovery of pHi was observed when control solutions (containing Na+) were reperfused in lumen and bath, likely due to the activity of the basolateral Na+/H+ exchanger.


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Fig. 3.   Effect of bilateral oligomycin (Oligo; 25 µM) on pHi recovery, following an acid load, in Na+- and HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>-free solutions. Acid load was by NH4Cl prepulse. Control solution contained 139 mM Na+.

Because the previous result suggests that pHi recovery is ATP dependent, we tested different inhibitors of V-ATPases. Figure 4 shows a typical tracing where addition of luminal bafilomycin A1, a specific inhibitor of V-ATPases (8), inhibits dpHi/dt. On average, 1 µM bafilomycin A1 produced an inhibition of 28% (n = 3), whereas with 10 µM bafilomycin A1, a 73% inhibition (n = 4) was observed (see Fig. 6B). Additional experiments at 0.1 µM led to inhibition of 28 and 40%. The effect of 10 µM bafilomycin A1 observed in Fig. 4 is the same when it is applied immediately after the control NH<UP><SUB>4</SUB><SUP>+</SUP></UP> pulse (not shown) and eliminates the possibility of a run-down effect. No change of steady-state pHi occurred when bafilomycin A1 was added to the luminal solution.


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Fig. 4.   Effect of luminal bafilomycin A1 (1 and 10 µM) on pHi recovery, following an acid load, in Na+- and HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>-free solutions. Acid load was by NH4Cl prepulse.

A third inhibitor tested was concanamycin A, another specific inhibitor of V-ATPases (19-21). As shown in Fig. 5, addition of 100 nM concanamycin A in the luminal solution led to a slow acidification of 0.16 pH units within 5 min. Moreover, no pHi recovery was observed (n = 4) following the acid load, suggesting total inhibition of the V-ATPase by concanamycin A (Figs. 5 and 6B). As in Fig. 3, a recovery of pHi was observed with the reintroduction of Na+ in bath and luminal solutions.


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Fig. 5.   Effect of luminal concanamycin A (100 nM) on pHi recovery, following an acid load, in Na+- and HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>-free solutions. Acid load was by NH4Cl prepulse. Control solution contained 139 mM Na+.



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Fig. 6.   Summary of effects of inhibitors on pHi recovery, following an acid load, in HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>-free solutions. A: recovery rate in presence (control) and absence of Na+ (0 Na+), observed on 14 of 21 tested sweat ducts. B: relative recovery rate with respect to 0 Na+ condition in the presence of luminal bafilomycin A1 (1 and 10 µM), oligomycin (25 µM), and concanamycin A (100 nM). Data are presented as means ± SE; n = no. of experiments. Results are statistically very significant (**P < 0.01) or significant (*P < 0.05) with respect to 0 Na+ condition.


    DISCUSSION
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Distribution of V-ATPase

Using an antibody against the 31-kDa subunit of the V-ATPase, we confirmed the presence of a V-ATPase in human eccrine sweat gland. In the duct, our results show that V-ATPases are more concentrated at the apical membrane but are also present within distinct structures into the cytoplasm, possibly acidic organelles (Fig. 1B). In addition, secretory coil cells present a weak, discontinued apical staining (Fig. 1D). It is likely that only one type of coil cells (either clear or dark cells) possesses V-ATPases, but in our preparation it was not possible to distinguish which one. Our results confirm those of a previous study (7), which demonstrated the presence of a V-ATPase at the apical membrane of the sweat duct. However, in contrast with our finding of discrete and localized staining of the cytoplasm, the previous study showed a strong and uniform staining of cytoplasm of the luminal cells layer of the duct. This difference, which still persisted with the SDS unmasking technique (15), may be due to the different methods of sample preparation, fixation, and immunohistochemistry.

Effect of Temperature on V-ATPase Distribution

In contrast to kidney intercalated cells, cold preservation followed by rewarming to 37°C does not lead to any difference in the distribution of V-ATPases in sweat duct compared with ducts fixed without rewarming. Indeed, a complete disappearance of apical proton pump staining in intercalated cells was shown following this treatment, which induced a marked redistribution of the V-ATPases into endocytotic vesicles (10). In the kidney, it was demonstrated that the cytoskeleton is disrupted by cold while the proton pumps remain at the apical membrane. However, when kidney collecting ducts are rewarmed, the pumps are temporally internalized into vesicles until the cytoskeleton is regenerated, within ~1 h. It therefore appears that V-ATPases of the sweat ducts are not internalized following rewarming at 37°C and remain at the apical membrane.

Functional Characterization of V-ATPase

Effect of Na+ removal. In the absence of Na+, dpHi/dt decreased by 65 ± 5% in most sweat ducts (14 of 21 tested), in agreement with the presence of a basolateral Na+/H+ exchanger (Fig. 2A) (25). However, for the other 7 ducts, we measured little or no pHi recovery (Fig. 2B). It is unlikely that these ducts were damaged, because a pHi recovery was observed as soon as Na+ was added to the bath solution. The most probable hypothesis for the different responses upon an acid load is that an axial heterogeneity may exist through the ductal portion of the sweat gland, as in the kidney tubule (9), although no clear differences in staining patterns for V-ATPases appear in sections of sweat ducts tested with immunochemistry.

Effect of oligomycin. The nearly complete inhibition of pHi recovery by oligomycin demonstrates that the transporter involved in pHi recovery after the acid load in the absence of Na+ and HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> requires ATP (Fig. 3). Indeed, oligomycin very rapidly decreases the level of cellular ATP. The inhibitory effect of oligomycin on pHi recovery does not exclude the possibility that an H+-K+-ATPase, also requiring ATP and playing a role in luminal acidification such as in the gastrointestinal tract (5) and kidney (40), could be involved in this response. However, a previous study of Reddy and Quinton (36) based on electrophysiology and intracellular K+ measurements concluded that neither a K+ conductance nor a K+-dependent carrier transport system exists at the apical membrane of the sweat duct.

Effect of bafilomycin A1. It has been shown that inhibition of V-ATPases occurs at nanomolar concentrations of bafilomycin A1. However, it also has been reported that 10 µM bafilomycin A1 is necessary to obtain half-maximal inhibition of the V-ATPase in the Malpighian tubules of an ant (18). It has been proposed that accessibility to the target sites of the H+-ATPase may be responsible for the different affinities observed in different preparations. Also, one cannot exclude the possibility of slightly different binding sites to explain these differences. Our results already show an inhibition at 100 nM that reaches 73% at 10 µM. Therefore, together with the evidence reported above (36), the fact that 50 µM bafilomycin is necessary to inhibit 50% of the gastric H+-K+-ATPase (30) makes it unlikely that the effect of bafilomycin is on an H+-K+-ATPase.

Effect of concanamycin A. Compared with bafilomycin A1, concanamycin A showed a much stronger inhibition of 95% of pHi recovery at 100 nM (Figs. 5 and 6B). In addition, concanamycin A is the only tested inhibitor that produced an effect on steady-state pHi, with an acidification of 0.16 pH units in 5 min. This result demonstrates that the apical V-ATPase in sweat duct is active at resting cell pH.

Dröse et al. (20, 21) were the first to demonstrate that concanamycins are even more potent inhibitors than the bafilomycins A1 of the activity of the Kdp-ATPase from Escherichia coli and the V-ATPase from Neurospora crassa, with an IC50 between two and five times lower and with a slightly different active site. Similarly, concanamycin A requires one-tenth the dose of bafilomycin A1 for inhibiting lysosome acidification and V-ATPase activity (37). It has been proposed that the increased sensitivity of V-ATPases for concanamycin A could be based on the larger flexibility of the 18-member macrolide ring (20, 41). Alternatively, others have demonstrated that concanamycin, with its additional sugar moiety, is more stable than bafilomycin (6, 20). Similarly, the lower potency of bafilomycin A1 has been proposed to be linked to limited accessibility to its target site (26). Because the inhibitory processes of bafilomycin and concanamycin are not yet elucidated and roles of all subunits of V-ATPases are not all understood, the explanations at the present time must remain hypothetical.

Because a V-ATPase has been shown to be one of the most important transporters involved in acid secretion in the distal nephron (17, 23) and epididymis (11, 16, 12), we suggest that it probably plays a major role in the acidification of sweat in eccrine sweat glands.

Summary

The current study confirms that luminal sweat duct cells express high levels of the vacuolar H+-ATPase (31-kDa subunit) at their apical membrane, whereas secretory cells show a discontinued apical staining. Cold preservation followed by rewarming does not alter the staining pattern. In addition, in the absence of HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>- and Na+-dependent proton transport, we have demonstrated an apical proton secretion activity that is much more sensitive to concanamycin A than to bafilomycin A1. Our results with all tested inhibitors strongly suggest that the inhibition of pHi recovery is due to the inhibition of a V-ATPase. Because no other proton transporter activity has yet been identified at the apical membrane of the human eccrine sweat duct, and particularly because V-ATPase expression is very low in the secretory portion of the gland, we suggest that the apical V-ATPase is probably involved in active luminal acidification of sweat in the duct.


    ACKNOWLEDGEMENTS

We thank the surgeons and patients of Institut de Polychirurgie de Montréal and those of the plastic surgery department of Hôpital Notre-Dame (Montreal, Canada) for providing the human skin. We thank Dr. Philippe Mayers for precious advice with respect to immunolocalization and Dr. Sylvie Breton (Massachusetts General Hospital) for helpful discussions and comments on the manuscript.


    FOOTNOTES

This work was supported by Unilever Research (Port Sunlight, UK).

This study was presented at Experimental Biology, Orlando, FL, March 31-April 4, 2001, and was published as an abstract.

Address for reprint requests and other correspondence: R. Laprade, Université de Montréal, GRTM, C.P. 6128, Succursale Centre-ville, Montreal, Quebec, Canada H3C 3J7 (E-mail: Raynald.Laprade{at}umontreal.ca).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

10.1152/ajpcell.00319.2001

Received 17 July 2001; accepted in final form 29 January 2002.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Beck, J, and Laprade R. Evidence against a proton pump in rabbit proximal convoluted tubule. Am J Physiol Renal Fluid Electrolyte Physiol 264: F175-F180, 1993[Abstract/Free Full Text].

2.   Bijman, J. Transport processes in the eccrine sweat gland. Kidney Int 32 Suppl21: S109-S112, 1987[ISI].

3.   Bijman, J, and Quinton PM. Influence of abnormal Cl permeability on sweating in cystic fibrosis. Am J Physiol Cell Physiol 247: C3-C9, 1984[Abstract/Free Full Text].

4.   Bijman, J, and Quinton PM. Lactate and bicarbonate uptake in the sweat duct of cystic fibrosis and normal subjects. Pediatr Res 21: 79-82, 1987[Abstract].

5.   Binder, HJ, Sangan P, and Rajendran VM. Physiological and molecular studies of colonic H+,K+-ATPase. Semin Nephrol 19: 405-414, 1999[ISI][Medline].

6.   Bindseil, KU, and Zeeck A. Metabolic products of microorganisms. Part 265. Prelactones C and B, oligoketides from Streptomyces producing concanamycins and bafilomycins. Helv Chim Acta 76: 150-157, 1993[ISI].

7.   Bovell, DL, Clunes MT, Roussa E, Burry J, and Elder HY. Vacuolar-type H+-ATPase distribution in unstimulated and acetylcholine-activated isolated human eccrine sweat. Histochem J 32: 409-413, 2000[ISI][Medline].

8.   Bowman, EJ, Siebers A, and Altendorf K. Bafilomycins: a class of inhibitors of membrane ATPases from microorganisms, animal cells and plant cells. Proc Natl Acad Sci USA 85: 7972-7976, 1988[Abstract].

9.   Bradley, SE, and Coelho JB. Nephron heterogeneity and the evaluation of single nephron function. Prog Biochem Pharmacol 9: 2-12, 1974[Medline].

10.   Breton, S, and Brown D. Cold-induced microtubule disruption and relocalization of membrane proteins in kidney epithelial cells. J Am Soc Nephrol 9: 155-166, 1998[Abstract].

11.   Breton, S, Smith PJS, Lui B, and Brown D. Acidification of male reproductive tract by a bafilomycin-sensitive H+ATPase. Nat Med 2: 470-473, 1996[ISI][Medline].

12.   Brown, D, and Breton S. H+V-ATPase-dependent luminal acidification in the kidney collecting duct and the epididymis/vas deferens: vesicle recycling and transcytotic pathways. J Exp Biol 203: 137-145, 2000[Abstract].

13.   Brown, D, Hirsch S, and Gluck S. Localization of a proton-pumping ATPase in rat kidney. J Clin Invest 82: 2114-2126, 1988[ISI][Medline].

14.   Brown, D, Lui B, Gluck S, and Sabolic I. A plasma membrane proton ATPase in specialized cells of rat epididymis. Am J Physiol Cell Physiol 263: C913-C916, 1992[Abstract/Free Full Text].

15.   Brown, D, Lydon J, McLaughlin M, Stuart-Tilley A, Tyszkowski R, and Alper S. Antigen retrieval in cryostat tissue sections and cultured cells by treatment with sodium dodecyl sulfate (SDS). Histochem Cell Biol 105: 261-267, 1996[ISI][Medline].

16.   Brown, D, Smith PJ, and Breton S. Role of V-ATPase-rich cells in acidification of the male reproductive tract. J Exp Biol 200: 257-262, 1997[Abstract/Free Full Text].

17.   Capasso, G, Malnic G, Wang T, and Giebisch G. Acidification in mammalian cortical distal tubule. Kidney Int 45: 1543-1554, 1994[ISI][Medline].

18.   Dijkstra, S, Lohrmann E, Van Kerkhove E, and Greger R. Characteristics of the luminal proton pump in Malpighian tubules of the ant. Renal Physiol Biochem 17: 27-39, 1994[Medline].

19.   Dröse, S, and Altendorf K. Bafilomycins and concanamycins as inhibitors of V-ATPases and P-ATPases. J Exp Biol 200: 1-8, 1997[Abstract/Free Full Text].

20.   Dröse, S, Bindseil KU, Bowman EJ, Siebers A, Zeeck A, and Altendorf K. Inhibitory effect of modified bafilomycins and concanamycins on P- and V-type adenosinetriphosphatases. Biochemistry 32: 3902-3906, 1993[ISI][Medline].

21.   Dröse, S, Boddien C, Gassel M, Ingenhorst G, Zeeck A, and Altendorf K. Semisynthetic derivatives of concanamycin A and C, as inhibitors of V- and P-type ATPases: structure-activity investigations and developments of photoaffinity probes. Biochemistry 40: 2816-2825, 2001[ISI][Medline].

22.   Emrich, HM, and Oelert H. pH value and total ammonia in human sweat. Pflügers Arch 290: 311-314, 1966.

23.   Gluck, S, Kelly S, and Al-Awqati Q. The proton translocating ATPase responsible for urinary acidification. J Biol Chem 257: 9230-9233, 1982[Abstract/Free Full Text].

24.   Gluck, S, Underhill DM, Iyori M, Holliday LS, Kostrominova TY, and Lee BS. Physiology and biochemistry of the kidney vacuolar H+-ATPase. Annu Rev Physiol 58: 427-445, 1996[ISI][Medline].

25.   Granger, D, Marsolais M, Burry J, and Laprade R. Regulation of intracellular pH by the human eccrine sweat duct: involvement of a Na+/H+ exchanger (Abstract). Suppl Mol Biol Cell 11: 229A, 2000.

26.   Hanada, H, Moriyama Y, Maeda M, and Futai M. Kinetic studies of chromaffin granule H+-ATPase and effects of bafilomycin A1. Kinetic studies of chromaffin granule H+-ATPase and effects of bafilomycin A1. Biochem Biophys Res Commun 170: 873-878, 1990[ISI][Medline].

27.   Kaiser, D, Songo-Williams R, and Drack E. Hydrogen ion and electrolyte excretion of the single human sweat gland. Pflügers Arch 349: 63-72, 1974[ISI][Medline].

28.   Lee, CM, Jones CJ, and Kealy T. Biochemical and ultrastructural studies of human eccrine sweat glands isolated by shearing and maintained for seven days. J Cell Sci 72: 259-274, 1984[Abstract].

29.   Macri, P, Breton S, Beck JS, Cardinal J, and Laprade R. Basolateral K+, Cl-, and HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> conductances and cell volume regulation in rabbit PCT. Am J Physiol Renal Fluid Electrolyte Physiol 264: F365-F376, 1993[Abstract/Free Full Text].

30.   Mattsson, JP, Väänänen K, Wallmark B, and Lorentzon P. Omeprazole and bafilomycin, two proton pump inhibitors: differentiation of their effects on gastric, kidney and bone H+-translocating ATPases. Biochim Biophys Acta 1065: 261-268, 1991[ISI][Medline].

31.   Mellman, I, Fuchs R, and Helenius A. Acidification of the endocytic and exocytic pathways. Annu Rev Biochem 55: 663-700, 1986[ISI][Medline].

32.   Quinton, PM. Effects of some ion transport inhibitors on secretion and reabsorption in intact and perfused single human sweat glands. Pflügers Arch 391: 309-313, 1981[ISI][Medline].

33.   Quinton, PM. Missing Cl- conductance in cystic fibrosis. Am J Physiol Cell Physiol 251: C649-C652, 1986[Abstract/Free Full Text].

34.   Quinton, PM. Physiology of sweat secretion. Kidney Int 32 Suppl 21: S102-S108, 1987[ISI].

35.   Quinton, PM, and Tormey JM. Localization of Na/K-ATPase sites in the secretory and reabsorptive epithelia of perfused eccrine sweat glands: a question to the role of the enzyme in secretion. J Membr Biol 29: 383-399, 1976[ISI][Medline].

36.   Reddy, MM, and Quinton PM. Intracellular potassium activity and the role of potassium in transepithelial salt transport in the human reabsorptive sweat duct. J Membr Biol 119: 199-210, 1991[ISI][Medline].

37.   Tapper, H, and Sundler R. Bafilomycin A1 inhibits lysosomal, phagosomal, and plasma membrane H+-ATPase and induces lysosomal enzyme secretion in macrophages. J Cell Physiol 163: 137-144, 1995[ISI][Medline].

38.   Thomas, JA, Buchsbaum RN, Zimniak A, and Racker E. Intracellular pH measurements in Ehrlich ascites tumor cells utilizing spectroscopic probes generated in situ. Biochemistry 18: 2210-2218, 1979[ISI][Medline].

39.   Whittam, R, Wheeler KP, and Blake A. Oligomycin and active transport reactions in cell membranes. Nature 203: 720-724, 1964[ISI].

40.   Wingo, CS, and Smolka AJ. Function and structure of H-K-ATPase in the kidney. Am J Physiol Renal Fluid Electrolyte Physiol 269: F1-F16, 1995[Abstract/Free Full Text].

41.   Woo, JT, Shinohara C, Sakai K, Hasumi K, and Endo A. Isolation, characterization and biological activities of concanamycins as inhibitors of lysosomal acidification. J Antibiot (Tokyo) 45: 1108-1116, 1992[ISI][Medline].


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