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RESEARCH |
1 UMIB, Instituto de Ciências Biomédicas de Abel Salazar (ICBAS), Universidade do Porto, L. Prof. Abel Salazar, 2, 4099-003 Porto, Portugal2 Laboratório de Biologia Celular, ICBAS-UP, 4099-003 Porto, Portugal3 Departamento de Genética, FM-UP, 4200-319 Porto, Portugal4 REQUIMTE, Departamento de Química, FCT-UNL, 2829-516 Caparica, Portugal5 Laboratório de Fisiologia Geral, ICBAS-UP, 4099-003 Porto, Portugal
Correspondence should be addressed to P F Oliveira who is now at Laboratório de Fisiologia dos Gâmetas e Transporte Iónico, ICBAS, LNIV (ala H), Rua dos Lagidos, Lugar da Madalena, 4485-655 Vairão, Vila do Conde, Portugal; Email: transp.bio{at}mail.icav.up.pt
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Abstract |
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Introduction |
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This cellular parameter is kept mainly through the net balance between production and elimination of protons and by intracellular buffers (Roos & Boron 1981). Cells possess in their plasmatic membrane a wide range of ion transporters that participate in pHi regulation, among which are the basic and acidic particles membrane transporters (Boron 2004). These transporters directly involved on the movement basic and acidic particles across the membrane are classified as acid extruders (Na+–H+ exchangers, Na+-driven HCO3–/Cl– transporters, Na+/HCO3– co-transporters, and V-ATPases) or acid loaders (Na+-independent HCO3–/Cl– transporters and Na+/HCO3– co-transporters), depending on the direction of movement of those particles (Boron 2004).
The goal of this work was to provide a first assessment on the participation of the different membrane transporters (Na+–H+ exchangers, Na+-driven and Na+-independent HCO3–/Cl– transporters, Na+/HCO3– co-transporter, Na+/K+/Cl– co-transporters, and V-ATPases) in the regulation of the pHi of human Sertoli cells. The approach used in this study was to follow pHi of cultured human Sertoli cells in Na+, K+, or Cl– replete- and free mediums and on pHi recovery in the presence of specific inhibitors (e.g., amiloride, DIDS (4, 4'-diisothiocyanostilbene disulfonic acid), PSA (picrylsulfonic acid), bumetanide, and concanamycin A) after an intracellular acidosis. The acidosis was achieved by adding sodium propionate to cells. To follow the pHi transients, the pH-sensitive fluorescent probe 2', 7'-bis-(2-carboxyethyl) -5-(and-6)-carboxyfluorescein (BCECF) was used.
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Acidification and recovery
Cells equilibrated in Sertoli Ringer were suddenly exposed to sodium propionate Ringer causing a rapid decrease in pHi, corresponding to an average fall of 0.63±0.01 (n=5) units (Fig. 1; Table 1). The half-time of the acidification process was 
295 s. Following the acidification, pHi slowly recovered to 100% of its initial value with an initial recovery rate of 0.0069±0.0002 pH units/s (Table 1).
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Addition of amiloride (1 mM), an inhibitor of the Na+/H+ exchange, used at doses from 0.1 to 1 mM (LD50 (rat/oral)=36–85 mg/Kg; LD50 (mouse/oral)=56 mg/Kg; IC50=5–25 µM; Delvaux et al. 1990, Otani et al. 1990, Ahearn et al. 1994, Good & George 1995, Ahearn et al. 1999), caused a similar decrease on the initial pHi recovery rates (Table 1). On the other hand, the recovery extension in the presence of amiloride was significantly lower (56% of the initial value) than in the presence of DIDS as can be seen in Fig. 1 and Table 1.
PSA (0.5 mM), a specific inhibitor of the Na+-driven HCO3–/Cl– exchanger, used at the concentration 0.5 mM (LD50=not available; IC50=not available; Knauf & Rothstein 1971, Madshus & Olsnes 1987, Frelin et al. 1988, Marvao et al. 1994), also affected the pHi recovery. Although to a smaller extent than DIDS, which also inhibits this membrane transporter, PSA caused a 21% decrease in pHi recovery and an 81% decrease in the initial recovery rate (Fig. 1; Table 1).
Concanamycin A (10 µM), a specific inhibitor of the V-type ATPases, used at concentrations from 1 to 5 µM (LD50 (mouse/oral)=21 mg/Kg; IC50=0.2–0.7 µM; Muroi et al. 1993, Huss et al. 2002, Oliveira et al. 2004), caused a very small, although significant, decrease in the pHi recovery extension, as can be seen in Fig. 1 and Table 1. On the other hand, in the presence of this inhibitor the initial pHi recovery rate was significantly lower than in the control situation (77% of the initial value; Table 1).
Bumetanide (10 µM) was the only inhibitor of a non-acid/base membrane transporter used. Used at the concentration 0.1 mM, it specifically inhibits the Na+–K+–Cl– co-transporters (LD50 (rat/oral)>6000 mg/Kg; LD50 (mouse/oral)>4000 mg/Kg; IC50=0.4–5 µM; Paris & Pouyssegur 1986, Vigne et al. 1994, Gamba 2005). The inhibition of this transporter did not affect the pHi recovery extension, although the initial recovery rate was significantly decrease to 52% of the control value (Fig. 2; Table 1).
Effects of external Na+, K+, and Cl– on pHi
Cytoplasmic pH was also monitored in the presence and absence of external Na+, K+, and Cl–. Isosmotic removal of external Na+ (by replacement with K+) or K+ (by replacement with Na+) did not cause any effects on pHi (Fig. 3). On the other hand, isosmotic removal of Cl– (by replacement with gluconate) caused a rapid increase in pHi of 0.13±0.02 units (Fig. 3). Reperfusing of external Cl– caused a recovering of pHi to values identical of the control situation.
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Discussion |
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The nature of the ion membrane transporters involved in pHi regulation in Sertoli cell is not very clear in the literature. As the ability of the Sertoli cell to regulate pHi is an important aspect of its physiology, especially because this cellular parameter may play a major role on the response to hormonal stimulation as well as on the determination of the pH of the seminiferous fluid, the enlightenment of the participation of diverse membrane transporters on the mechanisms of pHi regulation in these cells is of great relevance and was the main purpose of this study. For that, cells were loaded with a pH-sensitive fluorescent probe (BCECF) and subjected to an acid load in the presence of specific inhibitors.
Under the experimental conditions used, human Sertoli cells presented a mean value of pHi of 7.05±0.01. Amiloride, a high-affinity inhibitor of the Na+/H+ exchanger (Ahearn et al. 1994, 1999) significantly decreased the pHi initial recovery rate and extension (Table 1). These results are consistent with the participation of the Na+/H+ exchanger on pHi regulation mechanisms.
DIDS is an inhibitor of several bicarbonate transport systems (Boron et al. 1997, Boron 2001), namely the ones that involve base loading at the expense of the Na+ gradient, such as the Na+/HCO3– co-transporters and the Na+-driven HCO3–/Cl– exchanger. In the presence of this inhibitor, the initial pHi recovery rate and recovery extension were drastically reduced (to 17% of the control value and 39% of the initial value respectively), therefore we could presuppose that one or both these transporters could be involved in the pHi regulation after an acid load.
In order to further elucidate the participation of these two bicarbonate transporters on the pHi recovery we used PSA, a specific inhibitor of the Na+-driven HCO3–/Cl– exchanger (Knauf & Rothstein 1971, Madshus & Olsnes 1987). This compound significantly reduced the pHi initial recovery rate and recovery extension after the acid load. Therefore, we could assume the presence of the Na+-driven HCO3–/Cl– exchangers on the membrane of the human Sertoli cells. Nevertheless, the magnitude of the effect of PSA on the recovery extension was much smaller than that of DIDS, suggesting to us the presence of the Na+/HCO3– co-transporters on the plasma membrane of these cells and their participation in pHi regulation after an acid load.
Although proton pumps have been described in a wide variety of plasma membranes of acid-secreting epithelial cells (Harvey 1992, Martinez-Zaguilan et al. 1993, Ehrenfeld & Klein 1997, Rebelo da Costa et al. 1999, Oliveira et al. 2004), its presence has not yet been confirmed in Sertoli cells (Herak-Kramberger et al. 2001). The present study indicates that the pHi recovery mechanisms in Sertoli cells involve the action of the H+ pump, although to a small extent, as a statistically significant difference was observed on initial pHi recovery rate and extension after the acid load, in the presence of concanamycin A, a specific inhibitor of this kind of pump (Huss et al. 2002).
Bumetanide is a specific inhibitor of the Na+–K+–Cl– co-transporter, expressed in a broad spectrum of tissues and implicated in cell volume regulation and in ion transport by secretory epithelial tissue (O'Grady et al. 1987), namely in the seminiferous epithelium (Pace et al. 2000). The presence of this inhibitor did not affect the recovery extension of the pHi. As one can see, this membrane transporter is not directly implicated on the extrusion or loading of acid particles thus, unless it had a major role on the maintenance of the intracellular ionic gradients in human Sertoli cells, its effect on pHi recovery should not be very significant, as is the case. Nevertheless, the presence of bumetanide on the external bathing solution causes a decrease on initial pHi recovery rate, probably due to the referred disturbance of the intracellular ionic gradients.
Finally, ionic replacement experiments were performed in order to further access on the presence of the several membrane transporters discussed. Only the removal of chloride ion caused a shift to basic values of the basal pHi. Neither the removal of external Na+, nor of K+, caused any effect. The reasoning of these results could be the following: the removal of external Cl– affected the functioning of either the Na+-independent HCO3–/Cl– exchanger or the Na+-driven HCO3–/Cl– exchanger that will lead to the intracellular accumulation of bicarbonate and consequent rise of pHi.
In conclusion, human Sertoli cells, in the presence of external Na+ and bicarbonate, seem to regulate pHi after an acid load mainly by the action of an Na+-driven HCO3–/Cl– exchanger and a Na+/HCO3– co-transporter and also by the action of the Na+/H+ exchanger (Fig. 4). Also, our work, although it suggests the presence in Sertoli cells of V-type ATPases, points to a minor participation on these mechanisms of the regulation of pHi after an acid load. In addition, human Sertoli cells seem to also have on their plasma membranes the Na+–K+–Cl– co-transporter, which, as one could expect, does not have a major role on pHi regulation mechanisms but its absence (inhibition) affects those mechanisms, probably due to ionic gradient alterations.
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Materials and Methods |
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Ethical issues
Human Sertoli cells were obtained from testicular biopsies of infertile patients under treatment for recovery of male gametes at the IVF Unit of the Department of Genetics, Faculty of Medicine and University of Porto.
Patients with normal spermatogenesis were selected. In accordance with the Guidelines of the Local, National and European Ethical Committees, in the present study only cells left in the tissue culture plates after treatment of the patients by intracytoplasmic sperm–spermatid injection were used. In all cases, Sertoli cells were used only after informed patient consent.
Sertoli cell culture
Each biopsy (0.1 to 0.2 g), once obtained, was transferred to sperm preparation medium (SPM; Medicult, Copenhagen, Denmark) containing penicillin and streptomycin until cell isolation.
Sertoli cells were isolated using an adaptation of the enzymatic procedure adapted from the one described by Welsh & Wiebe (1975), modified by Majumdar et al. (1995) and evaluated by Valdés-González et al. (2005).
Tissue samples were washed twice in cold HBSSf (calcium- and magnesium-free HBSS, containing 50 U/ml of penicillin and 50 µg/ml streptomycin sulfate (pH 7.4)) and minced in HBSSf (2.5 ml per 0.1 g tissue) in a glass-stoppered Erlenmeyer, shaken vigorously during 1 min to disperse tubules. The tissue was left to settle for 5 min on ice, and the supernatant was discarded. This procedure was repeated twice to mechanically remove red blood cells and free Leydig cells. The resulting pellet was digested in 5 ml of HBSS with collagenase type I (200 U; C0130, Sigma) and DNAse (100 U; D4263, Sigma) continuously shaken (100 r.p.m.) at 32 °C during 25–35 min. The formed aggregate was removed, washed in HBSSf and discarded. The washing HBSSf was added to the cellular suspension resulting from the digestion.
The resulting suspension was washed twice and left to settle completely at 4 °C. The resulting pellet was suspended in 5 ml HBSSf with 1 mg pancreatin (P3292, Sigma) and DNAse (100 U; D4263) and digested at 32 °C with continuous shaking (100 r.p.m.) during 15–25 min. The new aggregate formed was discarded and 0.1 ml fetal bovine serum (FBS) was added to the cellular suspension, which was left to rest at 4 °C for 5 min. The suspension was then centrifuged at 100 g during 5 min. The pellet was gently suspended in 5 ml HBSSf. This procedure was repeated twice and the resulting pellet was suspended in 5 ml HBSSf. This suspension was passed through a glass Pasteur pipette in order to loosen germ cells from the clusters, and then pelleted at 200 g for 5 min. This procedure was repeated twice. The resulting pellet was suspended Sertoli culture medium (DMEM +Ham's F-12 [HF12]; 1:1, containing 50 U/ml penicillin and 50 µg/ml streptomycin sulfate, 0.5 µg/ml fungizone, and 5% heat inactivated FBS) and forced through a 19 G needle, in order to disaggregate large Sertoli clusters.
For culture of Sertoli cells, the concentration of clusters on the cellular suspension obtained from the procedure described above was adjusted to 1000 clusters/ml plated on 25 cm2 culture flasks (Cell+, Sarsted), and incubated at 37 °C in an atmosphere of 5% CO2: 95% O2. The day of plating was considered day 0 of culture. The cultures were left undisturbed until day 2. Sertoli cell isolation procedures had an average yield of 5x104 cell per gram of tissue.
Intracellular pH measurements
Cells were loaded with the fluorescent probe during 15 min at 37 °C with 1 ml Sertoli Ringer (NaCl 130 mM; KCl 5 mM; MgCl2 1 mM; CaCl2 2 mM; NaHCO3 10 mM; Glucose 1 mM; HEPES 10 mM (pH 7.4)) containing 1 µM BCECF–AM (Molecular Probes). The cells were transferred to the imaging chamber and washed with Sertoli Ringer using a gravity perfusion system (1 ml/min).
The fluorescence intensities, excited at 490 and 440 nm (emission 515 nm), were continuously measured with an epifluorescence system (DeltaRam, PTI) while the cells were perfused with different solutions.
Background fluorescence was determined at the end of each experiment by removing the cells and perfusing the empty chamber with Sertoli Ringer. The background fluorescence intensity was measured for each wavelength.
The ratios (F490/F440) were calculated after subtracting the background fluorescence intensities for each measurement at each wavelength.
The calibration procedures were done using the method described by Thomas (1986). In order to convert the fluorescence signals into pHi values, at the end of each protocol a in vivo calibration procedure was performed, using solutions of known pH (5.5 and 9.0), to which nigericin (10 µM) was added.
To convert the measured fluorescence ratio (F490/F440) in intracellular pH values, the following equation was used:
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To determine the BCECF Kd value, we performed in vivo measurements of the fluorescence intensity signals emitted at 515 nm, for the excitation wavelengths of 490 and 440 nm, in cells loaded with BCECF and perfused with a series of five calibration solutions of increasing pH (5.5, 6.5, 7.4, 8.0, and 9.0) where nigericin (10 µM) was added previously. The pH data were plotted as a function of
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Intracellular buffering capacity
In order to characterize changes in pHi, cells were acidified with sodium propionate Ringer (NaCl 90 mM; KCl 5 mM; MgCl2 1 mM; CaCl2 2 mM; NaHCO3 10 mM; glucose 1 mM; HEPES 10 mM; 40 mM sodium propionate (pH 7.4)) or potassium propionate Ringer (KCl 95 mM; MgCl2 1 mM; CaCl2 2 mM; KHCO3 10 mM; glucose 1 mM; HEPES 10 mM; 40 mM potassium propionate (pH 7.4)). At physiological pH, propionate is in equilibrium with its non-ionized form propionic acid that rapidly diffuses into the cell and promptly ionizes causing an intracellular acidosis. The response to this intracellular acid load was followed in time.
The cells were initially perfused with Sertoli Ringer and, after a steady-state period of 180 s, the propionate was added. The subsequent alkalinization (pHi recovery) of the cells was followed in the presence or absence of specific inhibitors (amiloride 1 mM, bumetanide 100 µM, DIDS 0.5 mM, PSA 0.5 mM, and concanamycin A 10 µM).
Effect of external ion removal on pHi
The cells were initially perfused with Sertoli Ringer and, after a steady-state period of 180 s, Na+, K+, or Cl– were removed from the external medium and replaced isosmotically by K+, Na+, or gluconate respectively. Sodium-free medium was composed by KCl 135 mM, MgCl2 1 mM, CaCl2 2 mM, KHCO3 10 mM, glucose 1 mM, HEPES 10 mM (pH 7.4). Potassium free-medium was composed by NaCl 135 mM, MgCl2 1 mM, CaCl2 2 mM, NaHCO3 10 mM, glucose 1 mM, HEPES 10 mM (pH 7.4). Chloride free-medium was composed by sodium gluconate 130 mM, potassium gluconate 135 mM, magnesium gluconate 1 mM, calcium gluconate 2 mM, NaHCO3 10 mM, glucose 1 mM, HEPES 10 mM (pH 7.4). The pHi of the cells was followed in the presence or absence of the specific ions.
Analysis of results
All data are presented as arithmetic means±S.E.M. of five replicates of five different biopsies. The initial pHi recovery rate was determined by linear regression of the data.
For statistical analysis a one-way ANOVA was performed followed by a Dunn's multiple comparison test. Results were considered significantly different from control value if P<0.05.
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Declaration of interest |
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Funding |
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Received August 24, 2008
First decision October 8, 2008
Revised manuscript received November 18, 2008
Accepted November 21, 2008
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References |
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