ABSTRACT
The role of plasma membrane V-ATPase activity in the regulation of cytosolic pH (pHi) was determined for resident alveolar and peritoneal macrophages (mϕ) from sheep. Cytosolic pH was measured using 2′7′-biscarboxyethyl-5,6-carboxyfluorescein (BCECF). The baseline pHi of both cell types was sensitive to the specific V-ATPase inhibitor bafilomycin A1. Bafilomycin A1 caused a significant (approximately 0.2 pH units) and rapid (within seconds) decline in baseline pHi. Further, bafilomycin A1 slowed the initial rate of pHi recovery (dpHi/dt) from intracellular acid loads. Amiloride had no effects on baseline pHi, but reduced dpHi/dt (acid-loaded pHi nadir <6.8) by approximately 35 %. Recovery of pHi was abolished by co-treatment of mϕ with bafilomycin A1 and amiloride. These data indicate that plasma membrane V-ATPase activity is a major determinant of pHi regulation in resident alveolar and peritoneal mϕ from sheep. Sheep mϕ also appear to possess a Na+/H+ exchanger. However, Na+/H+ exchange either is inactive or can be effectively masked by V-ATPase-mediated H+ extrusion at physiological pHi values.
Introduction
Vacuolar-type H+-ATPases (V-ATPases) are a constituent element of the plasma membrane of many animal cells, including osteoclasts, renal tubular cells, macrophages and metastatic tumor cells (Chatterjee et al. 1992; Gluck and Nelson, 1992; Grinstein et al. 1992; Martinez-Zaguilan et al. 1993). Plasma membrane V-ATPases extrude intracellular H+ and, in cells such as osteoclasts and renal tubular cells, serve to acidify specialized extracellular compartments (Chatterjee et al. 1992; Gluck and Nelson, 1992). In macrophages (mϕ), plasma membrane V-ATPases are important for the regulation of intracellular pH (pHi) (Swallow et al. 1988; Bidani et al. 1989). In these latter cells, plasma membrane V-ATPase protects pH-sensitive cell functions from the adverse effects of acidic microenvironments (e.g. interstitial fluids of tumors and abscesses) (Swallow et al. 1990b; Murphy and Forman, 1993; Bidani and Heming, 1995a) and from agonist-induced increases in metabolic acid production (Heming and Bidani, 1995).
Plasma membrane V-ATPase activity has been detected in resident alveolar m ϕ from rabbits, rats, guinea pigs and humans (Bidani et al. 1989; Chen et al. 1992; Murphy and Forman, 1993), and in resident and thioglycolate-elicited peritoneal mϕ from mice (Swallow et al. 1988, 1990a; Tapper and Sundler, 1992b). The activity of plasma membrane V-ATPases in m ϕ of other animal species is uncertain. Further, no study to date has examined the plasma membrane V-ATPase activities of more than one mϕ type from any single animal species. These are meaningful concerns because of a lack of consensus about the relative importance of plasma membrane V-ATPases for m ϕ pHi regulation. For example, V-ATPase activity is the primary determinant of baseline pHi and pHi recovery from acute intracellular acid loads in resident alveolar mϕ from rabbits (Bidani et al. 1989, 1994). In addition, inhibition of V-ATPase activity causes a significant and rapid cytosolic acidification of rat and rabbit resident alveolar mϕ at an extracellular pH (pHe) of 7.4 (Murphy and Forman, 1993; Bidani et al. 1994; Bidani and Heming, 1995a). Conversely, Swallow et al. (1988, 1990a,b) have shown that Na+/H+ exchange is the primary determinant of cellular acid–base status in murine resident and thioglycolate-elicited peritoneal mϕ These latter authors found that V-ATPase plays a secondary role in pHi recovery from intracellular acid loads and that inhibition of V-ATPase activity at a pHe of 7.35 does not alter baseline pHi. Thus, it is possible that the relative importance of plasmalemmal V-ATPase for mϕ pHi regulation is specific to the test animal species or to the source tissue of the mϕ To address these issues, the present study examined the significance of V-ATPase activity for pHi regulation in resident alveolar and peritoneal mϕfrom sheep.
Materials and methods
Sheep (body mass, 34–56 kg) were anesthetized with ketamine (5 mg kg-1) and then killed by intravenous injection of saturated KCl. Immediately thereafter, resident alveolar and peritoneal mϕ were obtained by bronchoalveolar and peritoneal lavages, respectively, with 1.0–1.5 l of sterile physiological saline (composition in mmol l-1: 127 NaCl, 5 KCl, 9 Na2HPO4, 2 NaH2PO4 and 5 D-glucose; pH 7.4). Cells in the lavage solutions were recovered by centrifugation (250 g, 10 min, 4 ° C) and washed twice in a sterile Hepes-buffered saline (composition in mmol l-1: 135 NaCl, 5 KCl, 1 CaCl2, 1 MgSO4, 2 KH2PO4, 5 D-glucose and 6 Hepes; pH 7.4). The peritoneal cells and a sample of the bronchoalveolar cells were transferred to a complete medium (RPMI-1640, Gibco, Grand Island, NY; pH 7.4), containing 25 mmol l-1 NaHCO3 and 5 % fetal bovine serum, and were incubated for 2 h in plastic culture dishes at 37 ° C in a humidified atmosphere of 5 % CO2. Supernatants and non-adherent cells were then discarded. The adherent cells were removed by gentle scraping, pelleted by centrifugation, and finally suspended in nominally CO2-free RPMI-1640 (25 mmol l-1 Hepes, pH 7.4) without serum. These cells were called adherence-enriched mϕ. The remaining bronchoalveolar cells were directly transferred to nominally CO2-free RPMI-1640 (25 mmol l-1 Hepes, pH 7.4, serum-free) and studied without adherence-enrichment. These latter cells were termed crude alveolar mϕ. The final cell suspensions (i.e. adherence-enriched and crude mϕ) contained approximately 106 mϕ ml-1, of which 86±3 % were viable by Trypan Blue exclusion. The pHi of cells in suspension was measured at 37 ° C in the nominal absence of CO2 using 2′,7′-biscarboxyethyl-5,6-carboxyfluorescein (BCECF; Molecular Probes, Eugene, OR), as described previously (Bidani et al. 1989, 1994).
Results and discussion
The baseline pHi of crude alveolar mϕ was 7.15±0.03 (mean ± S.E.M., N=7) at a pHe of 7.4. The baseline pHi decreased significantly when pHe was reduced to 6.5. Baseline pHi was 6.81±0.06 (N=5) at the lower pHe (Fig. 1A). These values agree with previous pHi measurements for the alveolar mϕ of rats and rabbits (Bidani et al. 1989; Murphy and Forman, 1993). The baseline pHi of crude alveolar mϕ was unaffected by 1 mmol l-1 amiloride, an inhibitor of Na+ transporters including the Na+/H+ exchanger (Benos, 1982) (Fig. 1A). Baseline pHi was sensitive to 10 μmol l-1 bafilomycin A1, a specific V-ATPase inhibitor (Bowman et al. 1988) (Fig. 1A). Bafilomycin A1 caused a significant cytosolic acidification of 0.16±0.01 and 0.23±0.02 pH units at pHe values of 7.4 and 6.5, respectively (N=5). The half-time of the bafilomycin-induced acidification was 37±7 s at a pHe of 7.4 and 41±9 s at a pHe of 6.5 (Fig. 1B). The rapidity of the bafilomycin-induced acidification suggests that the affected transporters were located on the plasma membrane, rather than in intracellular vesicular compartments. Bafilomycin-induced collapse of vesicular pH gradients (e.g. phagosomal pH gradients) requires more than 10 min in intact mϕ (Lukacs et al. 1990). Overall, the data show that plasma membrane V-ATPase had a predominant role in setting the baseline pHi of sheep alveolar mϕ. The effects of amiloride and bafilomycin A1 on sheep mϕ are consistent with previous findings for unstimulated and stimulated (phorbol-ester-treated or endotoxin-treated) resident alveolar mϕ from rats and rabbits (Murphy and Forman, 1993; Bidani et al. 1994; Bidani and Heming, 1995a; Heming and Bidani, 1995).
The baseline pHi of adherence-enriched peritoneal mϕ was 7.08±0.05 (N=4) at a pHe of 7.4. The baseline pHi of adherence-enriched peritoneal mϕ (pHe 7.4) was similar to that of adherence-enriched alveolar mϕ (Fig. 1C) which, in turn, was similar to that of crude alveolar mϕ (Fig. 1A). Thus, the adherence-enrichment procedure itself had no detectable effects on mϕ acid–base status. Treatment of adherence-enriched peritoneal mϕ with 10 μmol l-1 bafilomycin A1 caused baseline pHi to decrease by 0.18±0.01 units (Fig. 1C) with a half-time of 33±7 s (N=4). These results agree with those for sheep alveolar mϕ. The effects of amiloride on peritoneal mϕ were not examined. These data show that V-ATPase activity had a significant role in setting the baseline pHi of resident sheep peritoneal mϕ at a pHe of 7.4. Conversely, Swallow et al. (1990b) found that bafilomycin A1 had no effects on the baseline pHi of murine thioglycolate-elicited peritoneal mϕ under similar experimental conditions. It is plausible that Na+/H+ exchange in murine thioglycolate-elicited peritoneal mϕ (Swallow et al. 1988, 1990a) compensated for inhibition of plasma membrane V-ATPase in those cells. However, it also is noteworthy that the plasma membrane V-ATPase of resident and thioglycolate-elicited peritoneal ϕ respond differently to stimulating agents (e.g. endotoxin) (Swallow et al. 1991). In other words, thioglycolate-elicitation may alter the characteristics of plasma membrane V-ATPase. Thus, it is conceivable that comparison of the bafilomycin-sensitivity of resident sheep mϕ with that of thioglycolate-elicited murine mϕ is complicated by thioglycolate-elicitation. Whatever the reason(s) for published differences in the bafilomycin-sensitivity of mϕ pHi, it is clear that one must look beyond differences in the source tissue of the mϕ (alveolar versus peritoneal) or experimental animal species for a full explanation of those disparities.
To evaluate the role of V-ATPase in mediating pHi recovery from intracellular acid challenges, alveolar mϕ were acid-loaded using sodium propionate (pHe 7.4) as described previously (Bidani et al. 1989, 1994). Similar studies with peritoneal mϕ were not possible because of the limited numbers of m ϕrecovered from peritoneal lavage fluid. Crude alveolar mϕ were titrated with sodium propionate to minimum acid-loaded pHi values (pHiacid) between 6.66 and 6.83. Cytosolic pH then recovered back towards the baseline value.
The initial rate of pHi recovery (dpHi/dt) was computed by linear regression of a 30 s segment of the digitized data record that immediately followed achievement of pHiacid. The dpHi/dt of control m ϕ (i.e. in the absence of bafilomycin A1 and amiloride) was 0.110±0.022 pH units min-1 at pHiacid of 6.71±0.08 (N=3). Preliminary studies detected a significant amiloride-sensitive component (presumably Na+/H+ exchange) to this recovery process, which was equivalent to 35±8 % of the control dpHi/dt (N=3; pHiacid≈6.66). These results are consistent with published information that the pHi setpoint of the Na+/H+ antiporter (i.e. the pHi value below which allosteric activation of Na+/H+ exchange by cytosolic H+ is detectable) in resident mϕ is ⩽6.8 at a pHe of 7.4 (Tapper and Sundler, 1992b; Bidani et al. 1994). During studies of V-ATPase-mediated pHi recovery (see below), mϕ were treated with 1 mmol l-1 amiloride to eliminate the contribution of this antiporter to the pHi recovery process.
Fig. 2A illustrates the responses of amiloride-treated alveolar mϕ to an intracellular acid challenge at a pHe of 7.4. The amiloride-resistant rate of pHi recovery (i.e. V-ATPase-mediated recovery) was 0.080±0.013 pH units min-1 at a pHiacid of 6.66±0.03 (N=7). V-ATPase-mediated dpHi/dt varied significantly with pHiacid (Fig. 2B). V-ATPase-mediated dpHi/dt was significantly faster at a pHiacid of 6.66 than at more alkaline pHiacid values. A similar trend of increases in V-ATPase-mediated dpHi/dt with cytosolic acidification has been reported in rabbit alveolar mϕ over a comparable range of pHiacid (Bidani et al. 1994). In murine resident peritoneal mϕ Tapper and Sundler (1992b) reported that V-ATPase-mediated pHi recovery from ammonia-prepulse acid loads was unaffected by pHiacid over a more acidic range (pHiacid=6.1–6.6) and was significantly impaired at pHiacid values below 6.1. However, V-ATPase-mediated dpHi/dt cannot be directly related to V-ATPase activity (i.e. net rate of V-ATPase-mediated H+ extrusion or JH) without knowledge of intracellular buffering power (β) and the mϕ volume-to-surface area ratio (V/S), that is, JH=dpHi/dt X β V/S. The latter parameters were not measured in sheep m ϕ. Previous studies have shown β that increases with progressive cytosolic acidification of murine peritoneal mϕ (Tapper and Sundler, 1992a) and rabbit alveolar mϕ (Bidani et al. 1994). Under such circumstances, JH is more sensitive to cytosolic acidification than is apparent from changes in dpHi/dt with decrements in pHiacid. Thus, the observed relationship between pHiacid and dpHi/dt probably underestimates the effects of pHiacid on V-ATPase activity in sheep mϕ.
Furthermore, the use of dpHi/dt to monitor V-ATPase activity assumes that pHi recovery reflects H+ flux without a contribution from the fluxes of other acid–base equivalents. This is not true when cells are acid-loaded using weak acids (Bidani and Heming, 1995b). Recovery of pHi following exposure to weak acid is hindered by a persistent influx of weak acid driven by transporter-mediated H+ extrusion. During pHi recovery in the present study, it is expected that H+ and propionate left the cell and propionic acid was recycled back into the cell. Thus, the observed dpHi/dt must be regarded as a conservative indicator of the rate of V-ATPase-mediated H+ extrusion. With this in mind, it is important to note that pHi recovery was effectively abolished by m ϕ co-treatment with 1 mmol l-1 amiloride and 10 μ,mol l-1 bafilomycin A1 (Fig. 2B). These results provide strong evidence (a) that the amiloride-resistant dpHi/dt reflected V-ATPase-mediated H+ extrusion and (b) that the observed pHi recovery reflected an increment in net H+ extrusion rather than a change in propionate flux.
ACKNOWLEDGEMENTS
Serina Flores and Tammy Wheeler provided technical assistance. The studies were supported by grants from the Shriner’s Hospital for Crippled Children, the Moody Foundation (Galveston) and the American Lung Association (Texas Affiliate).