Rabbit esophageal cell cytoplasmic pH regulation: role of Na+-H+ antiport and Na+-dependent HCO, transport systems THOMAS J. LAYDEN, LARRY SCHMIDT, LOUIS AGNONE, PHILLIP LISITZA, JANET BREWER, AND JAY L. GOLDSTEIN Department of Medicine, University of Illinois at Chicago, and West Side Veterans Affairs Medical Center, Chicago, Illinois 60612 Layden, Thomas J., Larry Schmidt, Louis Agnone, Phillip Lisitza, Janet Brewer, and Jay L. Goldstein. Rabbit esophageal cell cytoplasmic pH regulation: role of Na+-H+ antiport and Na+-dependent HCQ; transport systems. Am. J. Physiol. 263 (Gastrointest. Liver Physiol. 26): G407G413,1992.-Regulation of cytoplasmic pH (pHi) of esophageal cells assumes importance as these cells can be exposed to mucosally absorbed acid during gastroesophageal reflux episodes. In this study, we examined whether esophageal cells possess pHi transport systems. Esophageal cells were harvested utilizing a gentle trypsin technique that yielded 2-5 x IO6 cells per esophagus. Cells were attached to a glass cover slip that had been pretreated with rat-tail collagen, and pHi was measured continuously in a spectrofluorometer utilizing 2’,7’-bis(Z-carboxyethyl)-5( -6) -carboxyfluoroscein acetoxymethyl ester as a pH-sensitive fluorescent probe. The basal pHi of cells exposed to a Na+-containing solution averaged 7.52 t 0.20 (n = 6). The pHi declined slightly but not significantly to 7.46 t 0.12 with the addition of 5% CO, and 28 mM NaHCO,. When H2 4,4’-diisothiocyanatostilbene-2,2’-disulfonic acid (DIDS; 0.5 mM) was added, pHi was unchanged. However, addition of low4 M amiloride caused pHi to decrease to 7.29 t 0.18 (P < 0.01). When cells were acidified (pHi 6.3-7.0) using a NH,Cl (20 mM) pulse technique, pHi was rapidly restored toward neutrality in the presence of a HCO,-free external Na+ concentration ([Na+],)-containing solution (pH units/min = 0.26 t 0.12; n = 8). Alkalinization was completely blocked with 10m4 M amiloride. In the presence of 10e4 M amiloride, 28 mM NaHC03, and 5% C02, acidified cells also alkalinized, although at a slower rate (0.11 t 0.04 pH units/min; n = 16). Alkalinization was blocked by 60-70% in the presence of 0.5 mM DIDS (n = 8). Alkalinization did not occur when [Na’], was omitted from the external HCO; concentration ([HCO&)-containing media. When cells were depleted of cytoplasmic Cl ([Cl-Ii) before acidification, there was a significant reduction in [Na+],dependent [HCO& alkalinization (pH units/min = 0.02 t 0.01; n = 8). These studies indicate that esophageal cells from rabbits possess both a Na+-H+ antiport as well as an [Na+],-dependent, [HCO&-[Cl-], transport system. esophagus; gastroesophageal reflux; chloride ion-bicarbonate transport GASTROESOPHAGEAL REFLUX DISEASE the esophageal mucosa is intermittently bathed with the acid contents of the stomach. Clearance of intracavitary acid is dependent on motility and neutralization of H+ by salivary bicarbonate ([HCO;]; see Ref. 27). In most individuals, these clearance mechanisms are sufficient to prevent diffusion of H+ into the mucosa with resultant tissue injury. Compared with the numerous studies that deal with these intraluminal defense mechanism, relatively little attention has been given to cellular defense mechanism(s) that protect cells against cytoplasmic acidification. In fact, the membrane transport systems
IN
that regulate esophageal cytoplasmic pH (pHi) are poorly defined. In most cell types, pHi is regulated by a membrane transporter that exchanges extracellular Na+ for intracellular H+, i.e., Na+-H+ antiport (9, 10, 13). Studies from our laboratory indicate that isolated esophageal cells from New Zealand White rabbits possess such a transport system, which appears to have a role in regulating basal pHi (20). In other cell types, pHi has been shown to be regulated by a transporter that exchanges HCO, for Cl- and/or by a transport system that moves HCO; into the cell in conjunction with external Na+ concentration ([Na+],) with or without exchange for cytoplasmic Cl ([Cl-Ii; 6, 14, 15, 19, 21, 22, 26, 28). Neither of the HCO; transport systems is inhibited by amiloride, but in many tissues they are inhibited by the anion exchange stilbene inhibitors 4,4’-diisothiocyanatostilbene-2,2’-disulfonic acid (DIDS) and/or 4-acetamido-4’-isothiocyanatostilbene2,2’-disulfonic acid. In some cell types, these transporters regulate basal pHi (2,14), are important mechanisms in restoring pHi when the cell becomes acidified (11, l4), and function to transport H+ and HCO; equivalents (11, 26). In the present study, we determined whether isolated esophageal cells from rabbits possess HCO, transport system(s) and whether they function to regulate basal cell pHi and/or restore pHi in acidified cells. The overall importance of these transporters in regulating pHi is compared with the role of the Na+-H+ antiport. In contrast to our earlier study that examined pHi regulation in a mixed population of both basal and squamous cells that were suspended in solution (20), in these studies we examined pHi regulation in a uniform population of basal cells that had been attached to collagen-coated cover slips. These studies confirm and extend on our observation of the importance of the Na+-H+ antiport in regulation of pHi and indicate that basal cells also possessa [Na+],-dependent HCO, transport system. METHODS Isolation of Esophageal
Cells
Nonfasted male New Zealand White rabbits (2-3 kg; Johnson Rabbit Ranch, Moorsville, IN) were used in all studies. Use of these animals and the experimental design were approved by the Animal Care Committee at the West Side Veterans Affairs Medical Center. The esophagus was removed after a lethal intravenous injection of 3-5 ml of pentobarbital sodium (50 mg/ ml). The esophagus was placed in an ice-cold (4°C) Ringer solution, and the muscle layer was removed. As previously described (ZO), the stripped esophagus was then placed in a Ca2+and Mg2+-free isosmotic saline solution [extracellular pH (pH,) G407
Downloaded from www.physiology.org/journal/ajpgi by ${individualUser.givenNames} ${individualUser.surname} (129.186.138.035) on January 19, 2019.
G408
CYTOPLASMIC
PH REGULATORY
7.4; 37”C] consisting of crystalline bovine pancreas trypsin (1 mg/ml; Calbiochem, San Diego, CA) and DNAse (0.2 mg/ml; Sigma, St. Louis, MO). The solution was agitated slowly with a magnetic mixer for 30 min. The submucosa was then gently stripped from the underlying mucosa. The mucosa with the basal cell layer exposed was cut into small pieces (3 mm2) and transferred to a test tube containing the above solution without trypsin or DNAse. The solution was vortexed 3 times at 5 s each to release cells. Tissue fragments were removed and the solution centrifuged at 1,000 g for 10 min to pellet the cells. A cell count was obtained and assessment of cell viability was performed using 0.2% trypan blue. Measurement
of
Intracellular
pH
pHi was monitored using the pH-sensitive fluorescent dye 2’,7’-bis(:!-carboxyethyl)-5(-6)-carboxyfluoroscein acetoxymethyl ester (BCECF) (Molecular Probes, Eugene, OR; see Refs. 11, 13, 20). Cells were labeled with BCECF by suspending them at 23°C for 45 min in a BCECF solution (0.02 mg/ esophagus) consisting of (in mM) 114 NaCl, 3.5 KCl, 1.8 CaC12, 0.5 MgC12, 2 Na,HPO,, and 5 dextrose buffered to pH, 7.4 with 20 mM tris(hydroxymethyl)aminomethane (Tris)-2-(N-morpholino)ethanesulfonic acid (MES; Ringer solution). Cells were then centrifuged (1,000 g) twice in the BCECF-free 130-mM choline chloride solution, pH, 7.4. Cells were viewed under a microscope with fluorescent capability (Leitz Laborlux 12 Microscope, FRG) to assess the extent of cell labeling and to assure labeling of >95% of the cells. For each experimental result, we used cells harvested from two to four rabbits. BCECF-labeled cells were then suspended in 1.5 ml of Ringer solution buffered to pH, 7.4. The solution containing the cells was placed in plastic Leighton tubes (Cambridge, MA) that contained plastic cover slips that were previously treated with rat-tail collagen. Briefly, 20 ~1 of rat-tail collagen prepared by the technique of Cereijido et al. (5) was spread over two-thirds of a plastic cover slip. Slides were allowed to dry for 30-60 min while being exposed to an infrared heat lamp. BCECF-labeled cells were incubated with the slide for 30 min at 23°C in the dark. The cover slips were then washed gently with Ringer solution and carefully cut. The extent of surface attachment was assessed by examining the preparation under the fluorescent microscope. The cover slip was placed in a slide holder that was positioned in a four-sided clear 2.0 ml disposable plastic cuvette (Kartell). The cover slip was positioned in the spectrofluorometer in such
SYSTEMS
a manner to be at a 45” angle to the excitation beam. The cuvette was perfused at 12 ml/min with varying solutions using a Harvard peristaltic pump (see Table 1). The inlet was at the base of the cuvette and the exit port at the top was connected to suction. A pH curve correlating pHi and BCECF fluorescence activity ratios was established at the end of each experiment using the nigericin technique as previously described (20). Fluorescent activity was measured in a PTI-Alpha Scan Spectrofluorometer (Princeton, NJ), which was controlled by a Tatung TC S-5000 computer using an alpha scan control program, 1.050. Excitation wavelengths were 500 and 440 nm, and the emission wavelength was 530 nm. At 500 nm, the BCECF signal is pH sensitive but it is not at 440 nm (9, 10, 20). The slit width was adjusted to give 50% maximum excitation wavelengths at 500 nm. Data were expressed as a ratio of signals at 500/440 nm (20). Cellular
Acidification
Cells were initially perfused for 10 min with a 119 mM choline chloride solution (Table 1; solution B). Cells were acidified utilizing the NH&l pulse technique (4, 13). In this technique, the initial perfusing solution was rapidly changed to a solution containing 20 mM NH&l; NaCl was substituted in equimolar amounts for choline chloride, and mannitol was eliminated. This solution also contained 10V4 M amiloride (Sigma) and was buffered to pH, 7.4 with 20 mM Tris-MES. After 5 min, the NH,Cl was removed leading to a rapid cytoplasmic acidification with pHi values in the range 6.3-7.0. Cells were then exposed to various solutions as seen in Table 1 to assess cellular alkalinization rates in the presence or absence of [Na+10 and/or external HCO; concentration ([HCO&). Amiloride (10m4 M) was added to all HCO,-containing experiments to negate the activity of the Na+-H+ antiport. As such, changes in pHi would more directly reflect activity of a HCO; transport system. The anion exchange inhibitor, DIDS (Sigma), was used at concentrations of either 0.1 or 0.5 mM. The effect of [Cl-Ii depletion on Na+/HCO; transport was assessed in attached BCECF-loaded cells that had been nominally depleted of [Cl-Ii by incubation for 40 min in an extracellular [Cl-] ([Cl-1,)-f ree solution (solution F). These studies were performed in an attempt to assess whether [Na+],-dependent HCO, transport was dependent on Cl- (i.e, [Cl-Ii[HCO,], transporter). Cells were then acidified utilizing 10 mM
Table 1. Composition of buffered salt solutions Solutions A (+Na+)
Na+ K’ Mg2+ Ca2+ Clsoy Choline Gluconate HPO, Dextrose HCO,
122 5 1 1
128 1 1 5
co2
Mannitol Amiloride
(low4
M)
36 t
C (+Na+/+HCO;)
B (-Na+)
-
-
150 5 1
8 1 1 128 1 119
8 1 1 128 1 147
1
128 1 -
-
1 5
1 5 28 5% +
36 -
Values are given in millimolar. Various solutions Perfusing solution pH was constantly measured morpholino)ethanesulfonic acid.
D (-Na+/+HCO,)
were using
perfused into a pH meter
1 5 28 5% a 2.0-ml cuvette and maintained
E (-Cl-/+HCO,)
150 5
F (-Cl-/-HCO,)
128 5
-
-
126 1 5 28 5% -
126 1 5 36
at a rate of 12 ml/min using a Harvard peristaltic at 7.4 using tris(hydroxymethyl)aminomethane/%(N-
pump.
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CYTOPLASMIC
PH REGULATORY
SYSTEMS
G409
(NH&SO, in solution F (Table 1); mannitol was removed. After (NH&SO, wasremoved, cellswere exposedto solution E to assesswhether [Na+],-dependent HCO, alkalinization occurred in the nominal absenceof Cl-. To directly compare results and to assurethat preincubation did not affect pHi recovery, experiments were performed using cells obtained from the sameharvest except that they were preincubated for 40 min in a Cl--replete solution (solution A plus low4 M amiloride). Cellswere then acidified using NHICl asindicated above. After acidification, they were exposedto solution C. Basal pH, Role of [Na+l,
and [HCO,-/,
In this set of experiments, attached cells were initially exposed to solution A for 5 min (n = 6). The solution was then changed to solution C containing 5% CO, and 28 mM NaHCO, (without amiloride), and pHi wasmonitored. HzDIDS (0.5 mM) wasthen addedwhile pHi wasmonitored. After 5 min, 10e4 M amiloride was added. In another set of experiments (n = 3), we reversed the addition of amiloride (low4 M) and HsDIDS
(0.5 mM).
The effect of [Cl-], removal (solution E) on basal pHi was alsoassessed, and results were compareddirectly with pHi measurementsin the presenceof [Cl-Ii (solution C). Both solutions contained 5% COz and 28 mM NaHCO,.
Fig. 1. Round cells (basal cells) labeled with the fluorescent dye 2’,7’bis(2-carboxyethyl)-5(-6)-carboxyfluoroscein acetoxymethyl ester (BCECF) are seen to occupy >50% of the surface area of a plastic cover slip, which was precovered with rat-tail collagen (A). Where there is no rat-tail collagen attached (B), fluorescent cells are rarely seen. (Original magnification X100).
Statistics
Means f SD are provided. The paired or unpaired Student’s t test wasusedas appropriate to test for significance within and
between groups, respectively. RESULTS
Technique of Cell Attachment
and pHi Measurement
With the technique of esophageal cell isolation, we were able to obtain 2-5 X lo6 cells/esophagus of which >90% were round or basal cells. The remainder were squamous cells. More than 95% of all cells excluded trypan blue and >95% took up and retained the vital stain BCECF. As noted in Fig. 1, which depicts a plastic cover slip that is partially coated with rat-tail collagen, -50% of the collagen-coated portion is covered with cells. In general, 2550% of the collagen-coated area was covered with cells in each experiment. If there was a lesser degree of attachment ( [Cl-], gradient. This gradient favors HCO; movement into the cell. A similar Cl- dependence was observed in monkey kidney epithelia cells (BS-C-l) by Jentsch et al. (14). As this Cl- gradient dependence was not seen in the absence of extracellular
SYSTEMS
HCO; (Fig. 7) and as DIDS inhibited this process, it appears that esophageal cells can express either a Na+dependent or Na+-independent [HCO,],-[Cl-]; transporter at or near physiological pH values if the Na+-H+ antiport is blocked and there is a favorable [Cl-Ii-[ Cl-], gradient. These data indicate that in both the acidified state and the basal state the Na+-H+ antiport appears to have more of a dominant effect in restoring and maintaining pHi. However, these studies also indicate that esophageal pHi-regulating systems also include a DIDS-sensitive HCO; transport system that is dependent on [Na+], and requires Cl-. This system may have a physiological as well as a protective role because the luminal surface (mucosa) of the esophagus is bathed with salivary HCO; and because the rate of salivary HCO; flow increases in acidreflux disease (12). It is well established from earlier in vitro studies in Ussing chambers that 30-40% of Na+ absorption is not inhibited by mucosal-placed amiloride (25). Although not previously tested for in Ussing chamber transport studies, it is conceivable that the remaining amiloride-insensitive Na+ absorption is in part dependent on a [Na+],-dependent Cl-- [HCO,], transporter. The confirmation of this hypothesis will require examination and localization of these transporters to squamous cells in the stratum spinosum, which contain tight junctions and may be involved in vectoral Na+ transport. As basal cells are nonpolar (i.e., lack tight junction), transporters in basal cells are clearly not important in Na+ absorption but probably function more in pHi and cell volume regulation. In summary, the present results indicate basal esophageal cells from rabbits possess both an amiloride-sensitive Nat--H+ antiport and a DIDS-sensitive [Na+],-dependent HCO,-Clexchanger. These transporters may have important physiological roles in protecting cells from acidification resulting from gastroesophageal acid reflux disease. We thank Carol Lane, Marilyn Smith, and Patricia Randle for their dedicated help in completing this manuscript. This work was supported by a Veterans Affairs Medical Research Merit Review Grant to T. J. Layden. Address for reprint requests: T. J. Layden, Section of Digestive and Liver Diseases, Univ. of Illinois at Chicago, 840 S. Wood St., M/C 787, Chicago, IL 60612. Received
20 June
1991; accepted
in final
form
23 April
1992.
REFERENCES 1. Aronson, P. S., M. A. Suhm, and J. Nee. Interaction of external H+ with the Na+-H+ exchanger in renal microvillus membrane vesicles. J. Biol. Chem. 258: 6767-6771, 1983. Bierman, A. J., E. J. Cragoe, S. W. De-Laat, and W. H. Moolenaar. Bicarbonate determines cytoplasmic pH and suppresses mitogen-induced alkalinization in fibroblastic cells. J. Biol. Chem. 263: 15253-15256, 1988. Binder, H. J., G. Strange, H. Murer, B. Steiger, and H. P. Hauri. Sodium-protein exchange in colon brush border membranes. Am. J. Physiol. 251 (Gastrointest. Liver Physiol. 14): G382-G388, 1986. Boron, W. F. Intracellular pH regulation in epithelial cells (Abstract). Annu. Rev. Physiol. 48: 377, 1986. Cereijido, M., E. S. Robbins, W. J. Dolan, C. A. Rotunno, and D. D. Sabatini. Polarized monolayers formed by epithelial cells on a permeable and translucent support. J. Cell Biol. 77: 852-883, 1978.
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CYTOPLASMIC
PH REGULATORY
6. Fineman, I., D. Hart, and E. P. Nord. Intracellular pH regulates Na+-independent Cl--base exchange in JTC-12 (proximal tubule) cells. Am. J. Physiol. 258 (Renal Fluid Electrolyte Physiol. 27): F883-F892, 1990. 7. Foster, E. S., P. F. Dudeja, and T. A. Brasitus. Na-H exchange in rat colonic brush-border membrane vesicles. Am. J. Physiol. 250 (Gastrointest. Liver Physiol. 13): G781-G790, 1986. 8. Gleeson, D., N. D. Smith, and J. L. Boyer. Bicarbonatedependent and -independent intracellular pH regulatory mechanisms in rat hepatocytes: evidence for Na+-HCO; cotransport. J. Clin. Invest. 84: 312-321, 1989. 9. Grinstein, S., S. Cohen, and A. Rothstein. Cytoplasmic pH regulation in thymic lymphocytes by an amiloride-sensitive Na+/H+ antiport. J. Gen. Physiol. 83: 341-348, 1984. 10. Grinstein, S., and A. Rothstein. Mechanism of regulation of the Na+/H+ exchanger. J. Membrane BioZ. 90: l-10, 1986. 11. Hart, D., and E. P. Nord. Polarized distribution of Na+/H+ antiport and Na+/HCO; cotransport in primary cultures of renal inner medullary collecting dual cells. J. BioZ. Chem. 266: 23742382, 1991. J. F., W. J. Dodd, and W. J. Hogan. Salivary response 12. Helm, to a esophageal acid in normal subjects and patients with reflux esophagitis. Gastroenterology 93: 1393-1398, 1987. R. M., J. Graf, and J. L. Boyer. Na+-H+ ex13. Henderson, change regulates intracellular pH in isolated rat hepatocyte couplets. Am. J. Physiol. 252 (Gastrointest. Liver Physiol. 15): G109G113, 1987. T. J., I. Janicke, D. Sorgenfrei, S. K. Keller, and 14. Jentsch, M. Wiederholt. The regulation of intracellular pH in monkey kidney epithelial cells (BSC-1): role of Na+/H+ antiport, Na+HCO,-(NaCO,) symport and Cl-/HCO; exchange. J. BioZ. Chem. 281: 12120-12127, 1986. 15. Jentsch, T. J., P. Schwartz, B. S. Schill, B. Langner, A. P. Lepple, S. K. Keller, and M. Wiederholt. Kinetic properties of the sodium bicarbonate (carbonate) symport in monkey kidney epithelial cells (BSC-1): interactions between Na+, HCO, and pH. J. BioZ. Chem. 261: 10673-10679, 1986. 16. Kikuchi, K., N. N. Abumrad, and F. K. Grisham. Na+/H+ exchange by brush-border membrane vesicles of human ileal small intestine. Gastroenterology 95: 388-393, 1988. 17. Knickelbein, R. P., S. Aronson, C. M. Schron, J. Seifter, and J. W. Dobbins. Sodium and chloride transport across rabbit
18.
19.
20.
21.
22.
23.
24.
25.
26.
27.
28.
29.
SYSTEMS
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ileal brush border. II. Evidence for Cl-HC03 exchange and mechanism of coupling. Am, J. Physiol. 249 (Gastrointest. Liver Physiol. 12): G236-G245, 1985. Kurtz, I., and K. Golchini. Na+-dependent Cl--HCO~ exchange in Madin-Darby Canine kidney cells: role in intracellular pH regulation. J. BioZ. Chem. 262: 4516-4520, 1987. L’Allemain, G., S. Paris, and J. Pouyssegur. Role of a Na+dependent Cl-/HCO, exchange in regulation of intracellular pH in fibroblasts. J. Biol. Chem. 260: 4877-4883, 1985. Layden, T. J., L. A. Agnone, L. N. Schmidt, B. Hakim, and J. L. Goldstein. Rabbit esophageal cells possess an Na+,H+ antiport. Gastroenterology 99: 909-917, 1990. Madshus, I. H., and S. Olsnes. Selective inhibition of sodiumlinked and sodium-independent bicarbonate/chloride antiport in vero cells. J. Biol. Chem. 262: 7486-7491, 1987. Nord, E. P., S. E. S. Brown, and E. D. Crandall. Cl-/ HCO; exchange modulates intracellular pH in rat type II alveolar epithelial cells. J. BioZ. Chem. 263: 5599-5606, 1988. Orlando, R. C., D. W. Powell, and C. N. Carney. Pathophysiology of acute acid injury in rabbit esophageal epithelium. J. CZin. Invest. 68: 286-293, 1981. Orlando, R. C., J. C. Bryson, and D. W. Powell. Mechanisms of H+ injury in rabbit esophageal epithelium. Am. J. Physiol. 246 (Gastrointest. Liver Physiol. 9): G718-G724, 1984. Powell, D. W., S. M. Morris, and D. D. Boyd. Water and electrolyte transport by rabbit esophagus. Am. J. Physiol. 229: 438-443, 1975. Reid, I. R., R. Civitelli, S. L. Westbrook, L. V. Avioli, and K. A. Hrauska. Cytoplasmic pH regulation in canine renal proximal tubule cells. Kidney Int. 31: 1113-1120, 1987. Richter, J. E., and D. 0. Castell. Gastroesophageal reflux: pathogenesis, diagnosis and therapy. Ann. Intern. Med. 97: 93103, 1982. Soleimani, M., S. M. Grassi, and P. S. Aronson. Stoichiometry of Na+-HCO; cotransport in basolateral membrane vesicles isolated from rabbit renal cortex. J. CZin. Invest. 79: 1276-1780, 1987. Sundaram, U., R. G. Knickelbein, and J. W. Dobbins. pH regulation in ileum: Na+ -H+ and Cl--HCO; exchange in isolated crypt and villus cells. Am. J. Physiol. 260 (Gastrointest Liver PhysioZ. 23): G440-G449, 1991.
Downloaded from www.physiology.org/journal/ajpgi by ${individualUser.givenNames} ${individualUser.surname} (129.186.138.035) on January 19, 2019.
IN
that regulate esophageal cytoplasmic pH (pHi) are poorly defined. In most cell types, pHi is regulated by a membrane transporter that exchanges extracellular Na+ for intracellular H+, i.e., Na+-H+ antiport (9, 10, 13). Studies from our laboratory indicate that isolated esophageal cells from New Zealand White rabbits possess such a transport system, which appears to have a role in regulating basal pHi (20). In other cell types, pHi has been shown to be regulated by a transporter that exchanges HCO, for Cl- and/or by a transport system that moves HCO; into the cell in conjunction with external Na+ concentration ([Na+],) with or without exchange for cytoplasmic Cl ([Cl-Ii; 6, 14, 15, 19, 21, 22, 26, 28). Neither of the HCO; transport systems is inhibited by amiloride, but in many tissues they are inhibited by the anion exchange stilbene inhibitors 4,4’-diisothiocyanatostilbene-2,2’-disulfonic acid (DIDS) and/or 4-acetamido-4’-isothiocyanatostilbene2,2’-disulfonic acid. In some cell types, these transporters regulate basal pHi (2,14), are important mechanisms in restoring pHi when the cell becomes acidified (11, l4), and function to transport H+ and HCO; equivalents (11, 26). In the present study, we determined whether isolated esophageal cells from rabbits possess HCO, transport system(s) and whether they function to regulate basal cell pHi and/or restore pHi in acidified cells. The overall importance of these transporters in regulating pHi is compared with the role of the Na+-H+ antiport. In contrast to our earlier study that examined pHi regulation in a mixed population of both basal and squamous cells that were suspended in solution (20), in these studies we examined pHi regulation in a uniform population of basal cells that had been attached to collagen-coated cover slips. These studies confirm and extend on our observation of the importance of the Na+-H+ antiport in regulation of pHi and indicate that basal cells also possessa [Na+],-dependent HCO, transport system. METHODS Isolation of Esophageal
Cells
Nonfasted male New Zealand White rabbits (2-3 kg; Johnson Rabbit Ranch, Moorsville, IN) were used in all studies. Use of these animals and the experimental design were approved by the Animal Care Committee at the West Side Veterans Affairs Medical Center. The esophagus was removed after a lethal intravenous injection of 3-5 ml of pentobarbital sodium (50 mg/ ml). The esophagus was placed in an ice-cold (4°C) Ringer solution, and the muscle layer was removed. As previously described (ZO), the stripped esophagus was then placed in a Ca2+and Mg2+-free isosmotic saline solution [extracellular pH (pH,) G407
Downloaded from www.physiology.org/journal/ajpgi by ${individualUser.givenNames} ${individualUser.surname} (129.186.138.035) on January 19, 2019.
G408
CYTOPLASMIC
PH REGULATORY
7.4; 37”C] consisting of crystalline bovine pancreas trypsin (1 mg/ml; Calbiochem, San Diego, CA) and DNAse (0.2 mg/ml; Sigma, St. Louis, MO). The solution was agitated slowly with a magnetic mixer for 30 min. The submucosa was then gently stripped from the underlying mucosa. The mucosa with the basal cell layer exposed was cut into small pieces (3 mm2) and transferred to a test tube containing the above solution without trypsin or DNAse. The solution was vortexed 3 times at 5 s each to release cells. Tissue fragments were removed and the solution centrifuged at 1,000 g for 10 min to pellet the cells. A cell count was obtained and assessment of cell viability was performed using 0.2% trypan blue. Measurement
of
Intracellular
pH
pHi was monitored using the pH-sensitive fluorescent dye 2’,7’-bis(:!-carboxyethyl)-5(-6)-carboxyfluoroscein acetoxymethyl ester (BCECF) (Molecular Probes, Eugene, OR; see Refs. 11, 13, 20). Cells were labeled with BCECF by suspending them at 23°C for 45 min in a BCECF solution (0.02 mg/ esophagus) consisting of (in mM) 114 NaCl, 3.5 KCl, 1.8 CaC12, 0.5 MgC12, 2 Na,HPO,, and 5 dextrose buffered to pH, 7.4 with 20 mM tris(hydroxymethyl)aminomethane (Tris)-2-(N-morpholino)ethanesulfonic acid (MES; Ringer solution). Cells were then centrifuged (1,000 g) twice in the BCECF-free 130-mM choline chloride solution, pH, 7.4. Cells were viewed under a microscope with fluorescent capability (Leitz Laborlux 12 Microscope, FRG) to assess the extent of cell labeling and to assure labeling of >95% of the cells. For each experimental result, we used cells harvested from two to four rabbits. BCECF-labeled cells were then suspended in 1.5 ml of Ringer solution buffered to pH, 7.4. The solution containing the cells was placed in plastic Leighton tubes (Cambridge, MA) that contained plastic cover slips that were previously treated with rat-tail collagen. Briefly, 20 ~1 of rat-tail collagen prepared by the technique of Cereijido et al. (5) was spread over two-thirds of a plastic cover slip. Slides were allowed to dry for 30-60 min while being exposed to an infrared heat lamp. BCECF-labeled cells were incubated with the slide for 30 min at 23°C in the dark. The cover slips were then washed gently with Ringer solution and carefully cut. The extent of surface attachment was assessed by examining the preparation under the fluorescent microscope. The cover slip was placed in a slide holder that was positioned in a four-sided clear 2.0 ml disposable plastic cuvette (Kartell). The cover slip was positioned in the spectrofluorometer in such
SYSTEMS
a manner to be at a 45” angle to the excitation beam. The cuvette was perfused at 12 ml/min with varying solutions using a Harvard peristaltic pump (see Table 1). The inlet was at the base of the cuvette and the exit port at the top was connected to suction. A pH curve correlating pHi and BCECF fluorescence activity ratios was established at the end of each experiment using the nigericin technique as previously described (20). Fluorescent activity was measured in a PTI-Alpha Scan Spectrofluorometer (Princeton, NJ), which was controlled by a Tatung TC S-5000 computer using an alpha scan control program, 1.050. Excitation wavelengths were 500 and 440 nm, and the emission wavelength was 530 nm. At 500 nm, the BCECF signal is pH sensitive but it is not at 440 nm (9, 10, 20). The slit width was adjusted to give 50% maximum excitation wavelengths at 500 nm. Data were expressed as a ratio of signals at 500/440 nm (20). Cellular
Acidification
Cells were initially perfused for 10 min with a 119 mM choline chloride solution (Table 1; solution B). Cells were acidified utilizing the NH&l pulse technique (4, 13). In this technique, the initial perfusing solution was rapidly changed to a solution containing 20 mM NH&l; NaCl was substituted in equimolar amounts for choline chloride, and mannitol was eliminated. This solution also contained 10V4 M amiloride (Sigma) and was buffered to pH, 7.4 with 20 mM Tris-MES. After 5 min, the NH,Cl was removed leading to a rapid cytoplasmic acidification with pHi values in the range 6.3-7.0. Cells were then exposed to various solutions as seen in Table 1 to assess cellular alkalinization rates in the presence or absence of [Na+10 and/or external HCO; concentration ([HCO&). Amiloride (10m4 M) was added to all HCO,-containing experiments to negate the activity of the Na+-H+ antiport. As such, changes in pHi would more directly reflect activity of a HCO; transport system. The anion exchange inhibitor, DIDS (Sigma), was used at concentrations of either 0.1 or 0.5 mM. The effect of [Cl-Ii depletion on Na+/HCO; transport was assessed in attached BCECF-loaded cells that had been nominally depleted of [Cl-Ii by incubation for 40 min in an extracellular [Cl-] ([Cl-1,)-f ree solution (solution F). These studies were performed in an attempt to assess whether [Na+],-dependent HCO, transport was dependent on Cl- (i.e, [Cl-Ii[HCO,], transporter). Cells were then acidified utilizing 10 mM
Table 1. Composition of buffered salt solutions Solutions A (+Na+)
Na+ K’ Mg2+ Ca2+ Clsoy Choline Gluconate HPO, Dextrose HCO,
122 5 1 1
128 1 1 5
co2
Mannitol Amiloride
(low4
M)
36 t
C (+Na+/+HCO;)
B (-Na+)
-
-
150 5 1
8 1 1 128 1 119
8 1 1 128 1 147
1
128 1 -
-
1 5
1 5 28 5% +
36 -
Values are given in millimolar. Various solutions Perfusing solution pH was constantly measured morpholino)ethanesulfonic acid.
D (-Na+/+HCO,)
were using
perfused into a pH meter
1 5 28 5% a 2.0-ml cuvette and maintained
E (-Cl-/+HCO,)
150 5
F (-Cl-/-HCO,)
128 5
-
-
126 1 5 28 5% -
126 1 5 36
at a rate of 12 ml/min using a Harvard peristaltic at 7.4 using tris(hydroxymethyl)aminomethane/%(N-
pump.
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CYTOPLASMIC
PH REGULATORY
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G409
(NH&SO, in solution F (Table 1); mannitol was removed. After (NH&SO, wasremoved, cellswere exposedto solution E to assesswhether [Na+],-dependent HCO, alkalinization occurred in the nominal absenceof Cl-. To directly compare results and to assurethat preincubation did not affect pHi recovery, experiments were performed using cells obtained from the sameharvest except that they were preincubated for 40 min in a Cl--replete solution (solution A plus low4 M amiloride). Cellswere then acidified using NHICl asindicated above. After acidification, they were exposedto solution C. Basal pH, Role of [Na+l,
and [HCO,-/,
In this set of experiments, attached cells were initially exposed to solution A for 5 min (n = 6). The solution was then changed to solution C containing 5% CO, and 28 mM NaHCO, (without amiloride), and pHi wasmonitored. HzDIDS (0.5 mM) wasthen addedwhile pHi wasmonitored. After 5 min, 10e4 M amiloride was added. In another set of experiments (n = 3), we reversed the addition of amiloride (low4 M) and HsDIDS
(0.5 mM).
The effect of [Cl-], removal (solution E) on basal pHi was alsoassessed, and results were compareddirectly with pHi measurementsin the presenceof [Cl-Ii (solution C). Both solutions contained 5% COz and 28 mM NaHCO,.
Fig. 1. Round cells (basal cells) labeled with the fluorescent dye 2’,7’bis(2-carboxyethyl)-5(-6)-carboxyfluoroscein acetoxymethyl ester (BCECF) are seen to occupy >50% of the surface area of a plastic cover slip, which was precovered with rat-tail collagen (A). Where there is no rat-tail collagen attached (B), fluorescent cells are rarely seen. (Original magnification X100).
Statistics
Means f SD are provided. The paired or unpaired Student’s t test wasusedas appropriate to test for significance within and
between groups, respectively. RESULTS
Technique of Cell Attachment
and pHi Measurement
With the technique of esophageal cell isolation, we were able to obtain 2-5 X lo6 cells/esophagus of which >90% were round or basal cells. The remainder were squamous cells. More than 95% of all cells excluded trypan blue and >95% took up and retained the vital stain BCECF. As noted in Fig. 1, which depicts a plastic cover slip that is partially coated with rat-tail collagen, -50% of the collagen-coated portion is covered with cells. In general, 2550% of the collagen-coated area was covered with cells in each experiment. If there was a lesser degree of attachment ( [Cl-], gradient. This gradient favors HCO; movement into the cell. A similar Cl- dependence was observed in monkey kidney epithelia cells (BS-C-l) by Jentsch et al. (14). As this Cl- gradient dependence was not seen in the absence of extracellular
SYSTEMS
HCO; (Fig. 7) and as DIDS inhibited this process, it appears that esophageal cells can express either a Na+dependent or Na+-independent [HCO,],-[Cl-]; transporter at or near physiological pH values if the Na+-H+ antiport is blocked and there is a favorable [Cl-Ii-[ Cl-], gradient. These data indicate that in both the acidified state and the basal state the Na+-H+ antiport appears to have more of a dominant effect in restoring and maintaining pHi. However, these studies also indicate that esophageal pHi-regulating systems also include a DIDS-sensitive HCO; transport system that is dependent on [Na+], and requires Cl-. This system may have a physiological as well as a protective role because the luminal surface (mucosa) of the esophagus is bathed with salivary HCO; and because the rate of salivary HCO; flow increases in acidreflux disease (12). It is well established from earlier in vitro studies in Ussing chambers that 30-40% of Na+ absorption is not inhibited by mucosal-placed amiloride (25). Although not previously tested for in Ussing chamber transport studies, it is conceivable that the remaining amiloride-insensitive Na+ absorption is in part dependent on a [Na+],-dependent Cl-- [HCO,], transporter. The confirmation of this hypothesis will require examination and localization of these transporters to squamous cells in the stratum spinosum, which contain tight junctions and may be involved in vectoral Na+ transport. As basal cells are nonpolar (i.e., lack tight junction), transporters in basal cells are clearly not important in Na+ absorption but probably function more in pHi and cell volume regulation. In summary, the present results indicate basal esophageal cells from rabbits possess both an amiloride-sensitive Nat--H+ antiport and a DIDS-sensitive [Na+],-dependent HCO,-Clexchanger. These transporters may have important physiological roles in protecting cells from acidification resulting from gastroesophageal acid reflux disease. We thank Carol Lane, Marilyn Smith, and Patricia Randle for their dedicated help in completing this manuscript. This work was supported by a Veterans Affairs Medical Research Merit Review Grant to T. J. Layden. Address for reprint requests: T. J. Layden, Section of Digestive and Liver Diseases, Univ. of Illinois at Chicago, 840 S. Wood St., M/C 787, Chicago, IL 60612. Received
20 June
1991; accepted
in final
form
23 April
1992.
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