SHEEP ALVEOLAR AND PERITONEAL MACROPHAGES: EVALUATION OF THE

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1 The Journal of Experimental Biology 198, (1995) Printed in Great Britain The Company of Biologists Limited EFFECTS OF BAFILOMYCIN A 1 ON CYTOSOLIC ph OF SHEEP ALVEOLAR AND PERITONEAL MACROPHAGES: EVALUATION OF THE ph-regulatory ROLE OF PLASMA MEMBRANE V-ATPases THOMAS A. HEMING 1,4, DANIEL L. TRABER 2,3, FRANK HINDER 3 AND AKHIL BIDANI 1,2 Departments of 1 Internal Medicine, 2 Physiology and Biophysics and 3 Anesthesiology and 4 Marine Biomedical Institute, University of Texas Medical Branch, Galveston, TX , USA Accepted 3 May 1995 The role of plasma membrane V-ATPase activity in the regulation of cytosolic ph () was determined for resident alveolar and peritoneal macrophages (m ) from sheep. Cytosolic ph was measured using 2,7 biscarboxyethyl-5,6-carboxyfluorescein (BCECF). The baseline of both cell types was sensitive to the specific V-ATPase inhibitor bafilomycin A 1. Bafilomycin A 1 caused a significant (approximately 0.2 ph units) and rapid (within seconds) decline in baseline. Further, bafilomycin A 1 slowed the initial rate of recovery (d/dt) from intracellular acid loads. oride had no effects on baseline, but reduced d/dt (acid-loaded nadir Summary <) by approximately 35 %. Recovery of was abolished by co-treatment of m with bafilomycin A 1 and amiloride. These data indicate that plasma membrane V- ATPase activity is a major determinant of regulation in resident alveolar and peritoneal m from sheep. Sheep m 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 values. Key words: vacuolar-type H + -ATPase, intracellular ph, ovine, leukocyte, bafilomycin A 1. 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 (m ), plasma membrane V-ATPases are important for the regulation of intracellular ph () (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 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 m from mice (Swallow et al. 1988, 1990a; Tapper and Sundler, 1992b). The activity of plasma membrane V-ATPases in m of other animal species is uncertain. Further, no study to date has examined the plasma membrane V-ATPase activities of more than one m 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 regulation. For example, V-ATPase activity is the primary determinant of baseline and recovery from acute intracellular acid loads in resident alveolar m 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 m 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 m. These latter authors found that V-ATPase plays a secondary role in recovery from intracellular acid loads and that inhibition of V-ATPase activity at a phe of 7.35 does not alter baseline. Thus, it is possible that the relative importance of plasmalemmal V-ATPase for m regulation is specific to the test animal species or to the source tissue of the m. To address these issues, the present

2 1712 T. A. HEMING AND OTHERS study examined the significance of V-ATPase activity for regulation in resident alveolar and peritoneal m from sheep. Materials and methods Sheep (body mass, kg) were anesthetized with ketamine (5 mg kg 1 ) and then killed by intravenous injection of saturated KCl. Immediately thereafter, resident alveolar and peritoneal m were obtained by bronchoalveolar and peritoneal lavages, respectively, with l of sterile physiological saline (composition in mmol l 1 : 127 NaCl, 5 KCl, 9 Na 2 HPO 4, 2 NaH 2 PO 4 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 Hepesbuffered saline (composition in mmol l 1 : 135 NaCl, 5 KCl, 1 CaCl 2, 1 MgSO 4, 2 KH 2 PO 4, 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 NaHCO 3 and 5 % fetal bovine serum, and were incubated for 2 h in plastic culture dishes at 37 C in a humidified atmosphere of 5 % CO 2. Supernatants and nonadherent cells were then discarded. The adherent cells were removed by gentle scraping, pelleted by centrifugation, and finally suspended in nominally CO 2 -free RPMI-1640 (25 mmol l 1 Hepes, ph 7.4) without serum. These cells were called adherence-enriched m. The remaining bronchoalveolar cells were directly transferred to nominally CO 2 -free RPMI (25 mmol l 1 Hepes, ph 7.4, serum-free) and studied without adherence-enrichment. These latter cells were termed crude alveolar m. The final cell suspensions (i.e. adherenceenriched and crude m ) contained approximately 10 6 m ml 1, of which 86±3 % were viable by Trypan Blue exclusion. The of cells in suspension was measured at 37 C in the nominal absence of CO 2 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 of crude alveolar m was 7.15±0.03 (mean ± S.E.M., N=7) at a phe of 7.4. The baseline decreased significantly when phe was reduced to 6.5. Baseline was 1±0.06 (N=5) at the lower phe (Fig. 1A). These values agree with previous measurements for the alveolar m of rats and rabbits (Bidani et al. 1989; Murphy and Forman, 1993). The baseline of crude alveolar m was unaffected by 1 mmol l 1 amiloride, an inhibitor of Na + transporters including the Na + /H + exchanger (Benos, 1982) (Fig. 1A). Baseline was sensitive to 10 mol l 1 bafilomycin A 1, a specific V-ATPase inhibitor (Bowman et al. 1988) (Fig. 1A). Bafilomycin A 1 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 m (Lukacs et al. 1990). Overall, the data show that plasma membrane V-ATPase had a predominant role in setting the baseline of sheep alveolar m. The effects of amiloride and bafilomycin A 1 on sheep m are consistent with previous findings for unstimulated and stimulated (phorbol-ester-treated or endotoxin-treated) resident alveolar m from rats and rabbits (Murphy and Forman, 1993; Bidani et al. 1994; Bidani and Heming, 1995a; Heming and Bidani, 1995). The baseline of adherence-enriched peritoneal m was 8±0.05 (N=4) at a phe of 7.4. The baseline of adherence-enriched peritoneal m (phe 7.4) was similar to that of adherence-enriched alveolar m (Fig. 1C) which, in turn, was similar to that of crude alveolar m (Fig. 1A). Thus, the adherence-enrichment procedure itself had no detectable effects on m acid base status. Treatment of adherenceenriched peritoneal m with 10 mol l 1 bafilomycin A 1 caused baseline 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 m. The effects of amiloride on peritoneal m were not examined. These data show that V-ATPase activity had a significant role in setting the baseline of resident sheep peritoneal m at a phe of 7.4. Conversely, Swallow et al. (1990b) found that bafilomycin A 1 had no effects on the baseline of murine thioglycolate-elicited peritoneal m under similar experimental conditions. It is plausible that Na + /H + exchange in murine thioglycolateelicited peritoneal m (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 m 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 m with that of thioglycolate-elicited murine m is complicated by thioglycolate-elicitation. Whatever the reason(s) for published differences in the bafilomycin-sensitivity of m, it is clear that one must look beyond differences in the source tissue of the m (alveolar versus peritoneal) or experimental animal species for a full explanation of those disparities. To evaluate the role of V-ATPase in mediating recovery from intracellular acid challenges, alveolar m were acidloaded using sodium propionate (phe 7.4) as described previously (Bidani et al. 1989, 1994). Similar studies with peritoneal m were not possible because of the limited numbers of m recovered from peritoneal lavage fluid. Crude alveolar m were titrated with sodium propionate to minimum acid-loaded values ( acid ) between 6.66 and 3. Cytosolic ph then recovered back towards the baseline value.

3 Bafilomycin effects on sheep macrophage ph 1713 The initial rate of recovery (d/dt) was computed by linear regression of a 30 s segment of the digitized data record that immediately followed achievement of acid. The d/dt of control m (i.e. in the absence of bafilomycin A 1 and amiloride) was 0.110±0.022 ph units min 1 at acid of 6.71±0.08 (N=3). Preliminary studies detected a significant phe 7.4 phe 6.5 B C Cont Baf Cont Baf phe 7.4 phe Time (s) Alveolar Peritoneal A Baf Baf amiloride-sensitive component (presumably Na + /H + exchange) to this recovery process, which was equivalent to 35±8 % of the control d/dt (N=3; acid 6.66). These results are consistent with published information that the setpoint of the Na + /H + antiporter (i.e. the value below which allosteric activation of Na + /H + exchange by cytosolic H + is detectable) in resident m is at a phe of 7.4 (Tapper and Sundler, 1992b; Bidani et al. 1994). During studies of V-ATPasemediated recovery (see below), m were treated with 1 mmol l 1 amiloride to eliminate the contribution of this antiporter to the recovery process. Fig. 2A illustrates the responses of amiloride-treated alveolar m to an intracellular acid challenge at a phe of 7.4. The amiloride-resistant rate of recovery (i.e. V-ATPasemediated recovery) was 0.080±0.013 ph units min 1 at a acid of 6.66±0.03 (N=7). V-ATPase-mediated d/dt varied significantly with acid (Fig. 2B). V-ATPasemediated d/dt was significantly faster at a acid of 6.66 than at more alkaline acid values. A similar trend of increases in V-ATPase-mediated d/dt with cytosolic acidification has been reported in rabbit alveolar m over a comparable range of acid (Bidani et al. 1994). In murine resident peritoneal m, Tapper and Sundler (1992b) reported that V-ATPase-mediated recovery from ammoniaprepulse acid loads was unaffected by acid over a more acidic range ( acid = ) and was significantly impaired at acid values below 6.1. However, V-ATPase-mediated d/dt cannot be directly related to V-ATPase activity (i.e. net rate of V-ATPase-mediated H + extrusion or J H ) without knowledge of intracellular buffering power ( ) and the m volume-to-surface area ratio (V/S), that is, J H =d/dt V/S. The latter parameters were not measured in sheep m. Previous studies have shown that increases with progressive cytosolic acidification of murine peritoneal m (Tapper and Sundler, 1992a) and rabbit alveolar m (Bidani et al. 1994). Under such circumstances, J H is more sensitive to cytosolic acidification than is apparent from changes in d/dt with decrements in acid. Thus, the observed relationship between acid and d/dt probably underestimates the effects of acid on V-ATPase activity in sheep m. Furthermore, the use of d/dt to monitor V-ATPase activity assumes that recovery reflects H + flux without a Cont Baf Cont Baf Fig. 1. Cytosolic ph () of sheep alveolar and peritoneal macrophages (m ). (A) of crude alveolar m showing the effects of extracellular ph (phe), amiloride (; 1 mmol l 1, 10 min treatment) and bafilomycin A 1 (Baf; 10 mol l 1, 10 min treatment). Cont, control. Data are means + S.E.M. (N=5 7). Significantly different from control at the same phe (paired Student s t-test, P<0.05). Significantly different from comparable treatment at a phe of 7.4 (paired Student s t-test, P<0.05). (B) Representative time courses for of crude alveolar m, showing the effects of 1 mmol l 1 amiloride and 10 mol l 1 bafilomycin A 1. The inhibitors were added at time zero. (C) Effects of bafilomycin A 1 (10 mol l 1, 10 min treatment) on of adherence-enriched peritoneal and alveolar m (phe 7.4). Data are means + S.E.M. (N=4).

4 1714 T. A. HEMING AND OTHERS A B + Baf 100 oride-resistant 80 recovery rate (%) oride- and bafilomycin-resistant min acid Fig. 2. V-ATPase-mediated recovery from acute intracellular acid loads in crude alveolar m (phe 7.4). (A) Representative time courses for of amiloride-treated m challenged with sodium propionate (arrows), showing the effects of bafilomycin A 1 (Baf; 10 mol l 1, added with weak acid)., 1 mmol l 1 amiloride. (B) Initial rate of recovery (d/dt) as a function of the acid- loaded nadir ( acid ). Data are means ± S.E.M. (N=5 7) and are expressed relative to paired values for amiloride-resistant d/dt at a acid of Significantly different from amiloride-resistant recovery at a acid of 6.66 (paired Student s t-test, P<0.05). Rates of combined amiloride- and bafilomycinresistant recovery were not statistically different from zero. 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 following exposure to weak acid is hindered by a persistent influx of weak acid driven by transporter-mediated H + extrusion. During 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 d/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 recovery was effectively abolished by m co-treatment with 1 mmol l 1 amiloride and 10 mol l 1 bafilomycin A 1 (Fig. 2B). These results provide strong evidence (a) that the amiloride-resistant d/dt reflected V-ATPase-mediated H + extrusion and (b) that the observed recovery reflected an increment in net H + extrusion rather than a change in propionate flux. 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). References BENOS, D. J. (1982). oride: a molecular probe of sodium transport in tissues and cells. Am. J. Physiol. 242, C131 C145. BIDANI, A., BROWN, S. E. S. AND HEMING, T. A. (1994). ph i regulation in alveolar macrophages: relative roles of Na + /H + antiport and H + - ATPase. Am. J. Physiol. 266, L681 L688. BIDANI, A., BROWN, S. E. S., HEMING, T. A., GURICH, R. AND DUBOSE, T. D., JR (1989). Cytoplasmic ph in pulmonary macrophages: recovery from acid load is Na + independent and NEM sensitive. Am. J. Physiol. 257, C65 C76. BIDANI, A. AND HEMING, T. A. (1995a). Effects of bafilomycin A 1 on functional capabilities of LPS-activated alveolar macrophages. J. Leukoc. Biol. 57, BIDANI, A. AND HEMING, T. A. (1995b). Kinetic analysis of cytosolic ph regulation in alveolar macrophages: V-ATPase mediated responses to a weak acid. Am. J. Physiol. (in press). BOWMAN, E. J., SIEBERS, A. AND ALTENDORF, K. (1988). Bafilomycins: a class of inhibitors of membrane ATPases from microorganisms, animal cells and plant cells. Proc. natn. Acad. Sci. U.S.A. 85, CHATTERJEE, D., CHAKRABORTY, M., LEIT, M., NEFF, L., JAMSA- KELLOKUMPU, S., FUCHS, R., BARTKIEWICZ, M., HERNANDO, N. AND BARON, R. (1992). The osteoclast proton pump differs in its pharmacology and catalytic subunits from other vacuolar H + - ATPases. J. exp. Biol. 172, CHEN, L. C., QU, Q. S., GORDON, T., AMDUR, M. O. AND FINE, J. M. (1992). Characterization of intracellular ph (ph i) regulation in human, guinea pig and rabbit alveolar macrophages. Am. Rev. Resp. Dis. 145, A651 (Abstract). GLUCK, S. AND NELSON, R. (1992). The role of V-ATPase in renal epithelial H + transport. J. exp. Biol. 172, GRINSTEIN, S., NANDA, A., LUKACS, G. AND ROTSTEIN, O. (1992). V- ATPases in phagocytic cells. J. exp. Biol. 172, HEMING, T. A. AND BIDANI, A. (1995). Effects of myristate phorbol

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