Proton efflux from rat intestinal basal-lateral membrane vesicles is stimlated by ATP and NA+

Camp. Biochmm. Phuiol. Printed in Great Biitain

Vol. 93A.

No. 4. pp. 845-850,

1989 Maxwell

PROTON EFFLUX BASAL-LATERAL STIMULATED AARON

The George

0300.9629189 $3.00 + 0.00 Pergamon Macmillan plc

FROM RAT INTESTINAL MEMBRANE VESICLES IS BY ATP AND NA’

J. MOE,* JENNIFERA. HOLLYWOODand MICHAELJ. JACKSON? Washington

University Medical Center, Washington, Telephone: (202) 994-20037

DC 20037, USA

(Received 23 January 1989) efflux were studied in inside-out rat ATP-stimulated ” Na uptake and “‘C-methylamine intestinal basal-lateral membrane vesicles (BLMV). 2. Uptake of 22Na by basal-lateral membrane vesicles was stimulated by addition of ATP and by an acidic vesicle interior. 3. Efflux of “‘C-methylamine was stimulated by ATP and Na+. 4. “C-methylamine efflux was not influenced by vanadate or amiloride by themselves but was inhibited by the presence of both agents. 5. These data are consistent with a basal-lateral proton translocation mechanism which may be responsible for alkalinization of the lateral intercellular space and implicates the Na+-pump in this mechanism Abstract-l.

INTRODUCTION

When rat small intestine is incubated in vitro, a characteristic pattern of weak electrolyte transport has been observed in the absence of transmural gradients of electrochemical potential. Weak acids are transported in the mucosal to serosal direction and weak bases are transported in the serosal to mucosal direction (Jackson et al., 1974). It has been proposed that the asymmetries in weak electrolyte movements are dependent on alkalinization of the epithelial lateral intercellular space (Jackson et al., 1974; Jackson, 1987). It has been shown (Jackson, 1984) that the alkalinizing process cannot be ascribed to movements of buffer species, such as bicarbonate, or to gradients of electrical or chemical potential, and it has been suggested (Jackson, 1984) that the process may be associated with a proton or hydroxyl translocating function of the sodium pump. The present studies were undertaken to examine transport properties of intestinal basal-lateral membranes that may be associated with the development of pH gradients. The results demonstrate a proton transport process that is dependent on the presence of sodium and ATP. MATERIALS

cervical dislocation for each membrane batch prepared. The small intestine was severed approximately 10 cm distal to the pylorus and flushed with 50 ml of ice-cold 150 mM NaCl solution. The intestine was severed approximately 3 cm proximal to the ileo-cecal junction and everted on a stainless steel rod. Intestines were incubated in (125 ml/intestine) 150mM NaCl solution, containing I mM dithiothreitol (solution changed three times) for 30 min in a shaking ice bath. One end of each intestine was ligated and transferred to 100 ml of 37’C isolation solution containing (in mM) 110 NaCI, 9.9 KH,PO,, 8 Na,citrate, 6.4 Na,HPO,, 1.8 KCI, 5 D-glucose, 0.5 glutamine, 0.5 fi-OH-butyrate, 0.3 acetoacetate, 0.25 g% bovine serum albumin and 0.15 g% hyaluronidase, pH 7.2. The intestines were filled to distension with 37°C isolation medium containing (in mM) 112.5 NaCI, 9.4 KH,PO,, 8.2 Na,citrate, 6.6 Na,HPO,, 1.8 KCI, 5 D-glucose, 0.5 glutamine, 0.5 /?-OH-butyrate, and 0.3 acetoacetate, pH 7.2, and the end ligated to close the intestine. lntestines were incubated 15 min in a 37 C shaking water bath. Following the incubation, the epithelial cells were collected in a glass dish by gently rubbing the intestines between thumb and forefinger. The cells from all four intestines were resuspended in IOOml of ice-cold isolation buffer containing 250 mM sorbitol, 12 mM NaCI, 0.5 mM ethylenediaminetetraacetic acid (EDTA), 0.1 mM phenylsuifonyl fluoride, 2.5 mg/lOO ml trypsin inhibitor, and 5 mM histidine/imidazole, pH 7.2. Isolation of basal-lateral membrane vesicles (BLMV)

AND METHODS

BLMV were isolated by a modification of the method of Mircheff et al. (1985). All steps in the isolation procedure were carried out at 4°C or on ice. Cells were disrupted by homogenizing with a Tekmar Tissuemizer (Tekmar Company, Cincinnati, OH, USA) setting No. 45, 15 min. The homogenate was centrifuged (3000g for 10min) and the supernatant aspirated and saved. The resulting pellet was resuspended in 25 ml isolation buffer and homogenized as above and the homogenate was centrifuged at 750g for 10 min. The supernatant was aspirated and combined with the first supernatant and centrifuged at 70,OOOg for 60 min. The resulting pellet was resuspended in 10 ml of isolation buffer and homogenized 10 strokes with a glass-Teflon homogenizer at I200 rpm. The homogenate was placed on

Animal and epithelial cell isolafion Intestinal epithelial cells were isolated from male SpragueDawley rats (200-250 g) by a modified procedure combining an enzymatic treatment (Kimmich, 1970) and incubation buffers (Towler et al., 1978). Four rats were killed by *Present address: Department of Pediatrics, Washington University School of Medicine, Children’s Hospital, 400 S. Kingshighway, St. Louis, MO 63110, USA. tAll correspondence should be addressed to: Dr Michael J. Jackson, Office of Research and Sponsored Programs, The George Washington University Medical Center, 2300 Eye St. NW, Washington, DC 20037, USA. 845

846

AARONJ. MOE et (11.

top of a discontinuous sucrose gradient (30%/~% w/w sucrose) and centrifuged at 100,OOOgfor 90min. Marker enzyme analysis indicated the highest enrichment of basal-lateral membranes in the top band of the gradient. The BLMV from the density gradient were diluted in several volumes of isolation buffer and centrifuged at 70,OOOgfor 40 min. The final pellet was suspended in 3 ml of isolation buffer and frozen in liquid nitrogen for transport assays.

ization of the alkalinization process to a specific cellular component. Ideally. such a probe would be simple to use, give reliable quantitative results, and result in continuous monitoring over time. The pH-sensitive fluorescent dye, 9-aminoacridine (Moe and Jackson, 1987). would meet the above criteria but had to be rejected because of nonspecific quenching from ATP. It was known that subsequent experiments would have to include ATP because the alkalization process described by Jackson (1984) was ATP-dependent. Another consideration is that BLMV isolation procedures Marker enzyme.7 result in a mixed population of rightside-out (orientation the Basal-lateral membrane purification was assayed by same as in rlico) and inside-out (orientation opposite of vanadate-sensitive K+-stimulated phosphatase by moni- in tizlo) and open membranes. lntravesicular pH could be toring the hydrolysis of p-nitrophenyl phosphate in monitored in the two former cases but ATP-dependent buffer containing lOmM MgSO,, IOOmM KCl. lOmM changes in intravesicular pH would be observable only in cysteine, 50 mM Trishydroxymethylaminomethane Tristhe inside-out population of vesicles because these would be HCI, pH 7.8. Vanadate insensitive phosphatase was deterthe only group of intact vesicles with ATP binding sites mined in the presence of 1mM vandate, a known inhibitor exposed. In the intact epithelium the ATP-dependent of the Na+/K’-ATPase. The brush border membrane alkalinization process results in alkalinization of the lateral marker was alkaline phosphatase, as measured by hydrolyinter~llular space (Jackson, 1984) which corresponds to sis of p-nitrophenyl phosphate (Sigma enzyme assay no. alkalinization of the intravesicular space for inside-out 104). The mitochondrial membrane marker was succinate BLMV. Given these considerations, the logical choice for dehydrogenase (EC 1.3.99.1), as measured by reduction of a pH probe would be a weak acid such as 5,5-dimethyl-2,4p-iodonitrotetrazolium violet (Pennington, 1961). The endooxazolidine-dione (DMO) (Boron and Roos, 1976) since plasmic reticulum marker was NADPH-cytochrome-c alkalinization of the intravesicular space would be reflected reductase (EC 1.6.2.4) (Masters PI al., 1966). Protein was in increased uptake of DMO. It would then be possible to assayed by the method of Lowry er ni. (1951), as modified observe the generation of a H’ gradient, where there was by Markwell ef al. (I 978). none present, by addition of ATP and sodium to BLMV and measuring DMO uptake over time. Unfortunately, reliable measurement of DMO and several other weak acids was not Trunsport assal‘s possible in the present study with the membrane filtration Transport was assayed by the membrane filtration technique because of low uptake relative to filter binding. technique (Kimmich, 1975; Murer et al., 1974). Standard Therefore, intravesicular pH changes were monitored with preload and transport buffers were selected for maximum the weak base, methylamine; or more precisely, the dissiNa+-pump activity (Boumendil-Podevin and Podevin, pation of imposed H+ gradients was monitored under 1983). Preioad buffer containing 150 mM mannitol, 100 mM various conditions by measuring methylamine disappearKCI, 10 mM N-2-hydroxvethvl-ni~razine-N-2-ethanesulance. Because methylamine is a weak base (pK, = 10.6) it fonic acid Hepes-Tris, pfi S.O-td PH 7.5, were preloaded accumulates in the BLMV to levels higher than its external as described previously (Moe and Jackson, 1987). The concentration when intravesicular H’ concentration is standard transport buffer contained 300 mM mannitol, greater than the external H’ concentration. This was not 4mM NaCl, and lOmM Hepes-Tris, pH 7.5. Buffer advantageous for the design of experiments in the present osmolarities were monitored with an Advanced Instruments study since the putative alkalization process should decrease (model 3W) osmometer. Transport reactions were stopped the intravesicular H+ concentration. Thus, an experimental by addition of 1201.~1assay buffer containing SO-2OO~g protocol was developed which was somewhat cum~rsome BLMV protein and 0.25 PCi of 22NaCl (10~1000 mCi/mg and indirect but did provide useful information. A H+ Na, Amersham, Arlington Heights, IL) or “C-methylamine gradient was imposed on the BLMV by preioading with low (50 mCi/mmol, ICN, Irvine, CA) to 2 ml ice-cold 200 mM DH buffer. When &aced in buffer of higher nH. ‘Y-methvlKC1 (stop solution) and filtered immediately. Filters (milli- amine was concentrated inside the BLMV in a pH-dep&pore, 0.45 @rn pore size) were washed with two additional dent manner (Fig. I). The Ii+ gradient would begin to 2 ml aliquots of stop solution. Washed filters containing the dissipate at a constant rate dependent on the permeability trapped BLMV, or without BLMV for filter binding deterof the BLMV to H’. Dissipation of the H’ gradient was minations, were placed in scintillation vials with 1Oml reflected in decreased methylamine uptake (i.e. increased of cocktail (Opti-Fluor”, United Technologies Packard, efflux) over time. If the theory is correct, and the putative USA) and counted many times and the average of three alkalization process is a component of the basal-lateral replications determined. Specific uptakes were calculated as membrane, addition of ATP, Na+ and conversely inhibitors follows: such as ouabain and vanadate, should effect loss of “‘Cmethylamine in a manner consistent with increases and pmol:mg protein = sample cpm-filter binding! decreases in the rate of dissipation of the imposed HA (cpm:pmol) (mg protein). gradient. The cpm/pmol was obtained by counting 20 ~1 of isotope solution containing a known concentration of substrate. RESULTS L

_

.

Methylamine eJYux e.uperiments

Enzyme

characterization

It may be useful to clarify some points about the experimenta approach taken in this study. The major goal of the study was to examine transport properties of the basal-lateral membrane which might be associated with development of H+ gradients. This goal derives directly from previous studies in this laboratory which examined weak electrolyte fluxes in intact intestinal epithelium. It was essential to find a probe to monitor intravesicular pH in order to design experiments to extend this work to local-

Results of marker enzyme analyses indicate enrichment of BLMV relative to protein (Table 1). The basal-lateral membrane marker, vanadate-sensitive K”-stimulated phosphatase was enriched 12.4 fold relative to protein. Activity of measured marker enzymes for contaminating membranes in the BLMV fraction did not change or were decreased relative to protein.

Basal~lateral Table

1.Marker

proton

transport

847

enzymes for various membranes in the BLMV fraction Marker enzymes

~Specific activity* % Recovered activity+ Enrichment factor2 n membrane batches

K’-stimulated phosphatase

Alkaline phosphatase

Succinate dehydrogenase

13.9& I.1 17.2 k 2.2 12.4* I.1 (14)

10.6 * I.4 I.3 kO.4

27.9 + IO I.0 * 0.5 0.6 i 0.2 (5)

I .o+ 0.2 (7)

NADPH-cyt-r reductase 0.6 f 0.1 2.5 i 0.8 I.1 f0.3 (8)

*Values are means + SE in nmol/mg proteinjmin. +Percentage of total recovered enzyme in the BLMV fraction, :Percentage of enzyme activity in BLMV fraction relative to the percentage protein recovered in BLMV fraction.

A first step to validate the BLMV model determine whether ATP-dependent Na+

was to uptake

could be observed, especially under the H+ gradient conditions which would be necessary for subsequent methylamine experiments. BLMV were preloaded with buffer containing 1OOmM KC1 and fluxes measured in external buffer containing 4mM NaCl and 2 mM MgATP. Therefore, in rightside-out BLMV the binding sites for Na+, K+, and ATP would not be appropriately exposed. Although efl?ux of “C-methylamine would occur from both populations of membranes, effects from addition of ATP would be limited to the inside-out ~pulation. The ATP-stimulation of ‘ZNa uptake (Table 2) indicated functional inside-out BLMV. ATP-dependent *‘Na uptake decreased from 2.5 nmol/mg protein/min in the absence of a H” gradient to 1.6 nmol/mg protein/min in the presence of a H+ gradient. This result is consistent with the known effect of pH on the Na+/K+-ATPase. In the absence of a pH gradient (pH, = pH, = 7.5) there was ATP-stimulated uptake of “Na by BLMV (Table 2). Ouabain (1 mM) was partially effective as an inhibitor of the ATP-dependent **Na uptake, whereas the presence of vanadate (1 mM) reduced 22Na uptake to levels observed in the absence of ATP. The lower effectiveness of ouabain was probably not the result of vesicle sidedness since BLMV were preloaded with ouabain in addition to externai ouabain. The presence of an outwardly directed pH gradient, stimulated **Na uptake in the presence and absence of ATP and vanadate and perhaps reduced ATP-dependent “Na uptake. The presence of 1 mM amiloride reduced **Na+ uptake in the presence

and absence

amine uptake was related to the magnitude of the pH gradient across the membrane and thus to the intravesicular pH. The effects of inhibitors and ion species on methylamine efflux from BLMV in the presence of a pH gradient are presented in Table 2. Data in Table 2 are for methylamine remaining in BLMV after time and therefore greater efflux is indicated by smaller numbers. Addition of ATP (2 mM) enhanced methylamine efflux with 84% of pH-gradient-dependent methylamine lost by 0.1 min of incubation. By 0.5 min after addition of ATP, methylamine efflux was 123% of the pH-gradient dependent methylamine uptake, which indicates transient alkalinization of the intravesicular space relative to the external PH. The control plus ATP was the only condition where observed methylamine loss exceeded the pHgradient-dependent methylamine uptake. The ATP effect on methylamine efflux was decreased in the absence of Na+ and in the presence of amiloride plus

z

400-

E B c: ‘;

200-

s p. \ I

of ATP.

I

4

5

6

PRELOAD

Uptake and ejj7u.u of “C-metlylamine BLMV were preloaded with various pH buffers and the uptake of 14C-methylamine determined in transport buffer, pH 7.5 (Fig. 2). As Fig. 2 illustrates, methylamine uptake was inversely related to pH of the preload buffer. This indicates that methyl-

f

6

pli

Fig. I. Uptake (2min of ‘QZ-methylamine (42pM)) by BLMV preloaded at different pHs. Preload and transport buffers (pH 7.5) constituents are given in the text. The figure shows the preload buffer pH and not the calculated intravesicular pH. Points represent means and SEs for three membrane batches and, where absent, SEs were smaller than the symbol.

Table 2. Uptake of **Na (4 mM) by BLMV +ATP

PH, 7.5 7.5 6.0 6.0

(4) (I) (3) (I)

5.6 + 0.7 5.0 7.7 * 0.8 6.7

+ATP + ouabain

+ATP + vo,

+ATP + amiloride

4.4 it 0.6

- ATP 3.1 rt:0.3 2.5 6.1 t 0.2

2.1 5.8 + 0.9 1.8

- ATP + ouabain

-ATP + vo,

-ATP + amiloride

3.1 & 0.3 2.3 6.2 & 0.9 0

Data are means i SE in nmoljmg proteinjmin for the number of membrane batches in parentheses. Uptake (30 see) was determined in pH 7.5 transport buffer and test agents where added, were at the following concentrations: 2 mM ATP, I mM VO, (vanadate), I mM ouabain and I mM amiloride.

AARON J. MOE et al

X48

Table 3. Transport of “Cmethylamine

from acid preloaded BLMV

Time after addition of ATP Treatment _____.-. - ATP (control) +ATP + ATP. -sodium + ATP. + vanadate + ATP. +amilorlde + ATP, + vanadate iamiloride

0.1 min

~~~~ 232’ 167’ 229. 176t l65t

+ + + i +

35 (18%) 5 (84%) 5 (22%) 5 i68%; 4 (64%)

l92*t k I6 (49%)

0.5 min 232* f 12X** 190’ + 159t + 134t &

33 (18%I3 (123%) I7 (55%) 5 (is%, 3 (100%)

193’ * 13 (48%)

Values are means k SE in pmol/mg protein, n = 4. BLMV were preloaded in pH 6.0 buffer and mcubated with ‘“C-methylamme I min prior to addition of ATP or inhibitors. A zero sample was taken at I min and an equilibrium sample at 60 min. The pH-dependent uptake was determined by uptake zero sample-uptake 60 mm In parentheses are the percentage elllux which was calculated % Efflux = (zero sample-uptake)~pH-dependent UPtake; x 100. ‘I” Times in the table are times after the I min preincubation and after addltioo of test buffers or water in the cahe of control. *SMeans withln a column not bearing the same superscript differ (P < 0.1).

vanadate. Addition of amiloride or vanadate separately did not significantly affect the ATP stimulated methylamine efflux. DISCUSSION

The success of the BLMV purification scheme was assessed by enrichments and yields of appropriate marker enzymes. The basal-lateral marker was vanadate-sensitive K+-stimulated phosphatase (a partial reaction of the Na+/K+ ATPase) which is an accepted marker enzyme (Murer et al., 1976; Hopfer, 1987). K+-independent phosphatase is commonly measured in the presence of 100 mM NaCl but, alternatively, I mM ouabain can be used as an inhibitor (Garrahan, 1969). Vanadate is known to be a potent inhibitor of Nat/K’-ATPase (Nechay and Saunders, 1969; Grantham and Glynn. 1979). Vanadate replaced ouabain in the present study because rat intestinal Na+/K+-ATPase is relatively insensitive to ouabain (Allen and Schwartz. 1969). The relative enrichment of 12.4-fold (Table 1) is comparable with published enrichments of 12-fold for K+-stimulated phosphatase (Murer ef al., 1976) 1Zfold for Na+/K+-ATPase (Mircheff and Wright, 1976) IO-fold (Mircheff, 1980) 13-fold (Del Castillo and Robinson, 1982) for other isolation schemes. The 17% yield of K+-stimulated phosphatase activity (Table 1) is similar to results from other laboratories. Contamination of the BLMV fraction from brush borders and endoplasmic reticulum was minor as indicated by no enrichment of alkaline phosphatase or NADPH-cytochrome-c reductase. Enrichment of succinate dehydrogenase was less than one (0.6) indicating nonsignificant contamination from mitochondira (Table 1). Therefore, the marker enzyme data demonstrate successful isolation of BLMV. The finding that inhibition of ATP-stimulated Z2Na uptake was incomplete with 1 mM ouabain (Table 2) is in agreement with the observation that rat tissues are relatively insensitive to this agent (Allen and Schwartz, 1969). It is also consistent with the finding that net fluxes of weak electrolytes are incompletely inhibited by ouabain (Jackson, 1984). ATP-stimulated “Na uptake will occur only in inside-out BLMV because external ATP does not have access to

the ATP-binding site in rightside-out vesicles and Na+ is pumped out of such vesicles. Although we have not assessed the proportion of inside-out vesicles in our preparation, the magnitude of ATPstimulated uptake of 22Na would indicate a proportion large enough to obtain reliable measurement of isotope fluxes from the inside-out population. BLMV isolated from guinea pig intestine were 60% insideout (Del Castillo and Robinson, 1982) and 70% inside-out from rat kidney (Marin et al., 1986). ATP-stimulated 22Na uptake was measured in the presence of a pH gradient because a pH gradient was necessary to the protocol of the subsequent r4Cmethylamine efflux experiment. The presence of a H+ gradient did not abolish ATP-stimulated 22Na uptake but there was a dramatic increase in ATP-independent 22Na uptake when the intravesicular H+ concentration was increased (Table 2). This phenomenon suggests the possibility of a Na+/H+ exchanger in the basal-lateral membrane. Further evidence for a Na+/H+ exchanger is the result that ATP-independent “Na uptake is completely inhibited by 1 mM amiloride (Table 2). A basal-lateral Na+/H+ exchange mechanism in response to acid loading, has recently been demonstrated in a cell line (Monroe et al., 1987). It seems unlikely that this phenomenon could result from brush border contamination given the relatively low alkaline phosphatase activity in this preparation. The objective of the present study was to examine the possibility of a basal-lateral mechanism for H+ (or OH-) translocation consistent with the development of a high-pH compartment, as described previously (Jackson, 1987). Although we use the term proton translocation throughout this paper, it should be recognized that similar observations would be obtained if the underlying alkahnization was the result of hydroxyl transport and we use the former term only for convenience. It should also be pointed out that the 14C-methylamine method, as used in the present study, does not give quantitative results. The data presented in Fig. 1 simply illustrate the fact that “C-methylamine uptake is higher than BLMV are preloaded with acid and is consistent with the mechanism for distribution of weak acids and bases across biological membranes.

Basal~lateral proton transport Measurements of “‘C-methylamine uptake would have had to have been done under steady-state conditions in order to obtain estimates of actual intravesicular pH, and this was not practical under the conditions of these experiments. Results of “C-methylamine efflux (Table 3) indicate that the rate of efflux from acid preloaded BLMV is influenced by Na+ and ATP. In the absence of ATP (i.e. control) there was an 18% loss of “C-methylamine in the first 0.5 min of incubation. Addition of ATP increased methylamine efflux so that 84% and 123% of pH-dependent methylamine uptake had escaped by 0.1 and 0.5 min, respectively. “C-methylamine efflux greater than 100% would indicate a transient alkalinization of the intravesicular space. The influence of Nat is clearly demonstrated by ‘JC-methylamine efflux which is not different from control when Na+ is absent from the medium but loss is significantly greater in the presence of Na+. These data are consistent with a role for the Na+/K+ATPase in H+ translocation since such a process would require both ATP and Na+. These data are consistent with findings on intestinal weak electrolyte 1984) and implicate this transport (Jackson, phenomenon with the mechanism for alkalinization of the intravesicular space since, in the present study, alkalinization of the intravesicular space would be consistent with alkalinization of the lateral intercellular space. These data are also consistent with the finding of an ATP-dependent hyperpolarization of renal BLMV (Boumendil-Podevin and Podevin, 1983). These authors found that under conditions similar to the present study, the ATP-dependent was abolished by the prohyperpolarization tonophore, dinitrophenol, and was insensitive to vanadate. They concluded that the BLMV possesses an electrogenic proton pump, independent of the Na+/K+-ATPase. Our results, estimating changes in intravesicular pH more directly, would agree with a proton translocation function and would not rule out Na+-pump involvement. The inability of vanadate to inhibit methylamine efflux (Table 3) may be due to the presence of a Na+/H+ exchanger. Increasing the intravesicular H+ concentration resulted in increased ‘2Na uptake which was amiloride inhibitable (Table 2). It is reasonable to suppose that the opposite effect may also occur. When ATP-dependent Na+ uptake is inhibited by vanadate, there is a compensating increase in Na+/H+ exchange. Conversely, ATP-dependent proton translocation may compensate for decreased Na+/H+ exchange in the presence of amiloride (Table 3). This interpretation is supported by the observed decrease in “‘C-methylamine efflux when both amiloride and vanadate are present as inhibitors. The significance of this Na +/H + exchanger in ciao is unknown since, in an intact epithelium, the Na+ and H’ gradients would presumably drive the exchange in the opposite direction (i.e. Na+ uptake and H+ extrusion) from the present study. This mechanism (Na+/H+ exchange) could not be responsible for alkalinization of the lateral intercellular space, but may have a regulatory role in opposing the alkalinization process and preventing intracellular acidification. Recently, it has been demonstrated that basal-lateral Na+/H+ exchange is

849

responsible for recovery of intracellular pH in acid loaded epithelial cells (Montrose et al., 1987). The present study demonstrates that a proton translocation mechanism exists in the intestinal basallateral membrane which is consistent with the alkalinization of the lateral-intercellular space proposed in studies with weak electrolyte transport (Jackson, 1984). The findings of proton translocation which is dependent on Na+ and ATP, and which is inhibited by vanadate in the presence of amiloride, implicates the Na+/K+-ATPase, and further studies are required to delineate the biochemical mechanism(s) of these relationships. Acknowledgemenl-This work was supported by National Institute for General Medical Sciences (GM22369). REFERENCES

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ization of brush border membrane vesicles from pig small intestine. Comp. Biochem. Physiol. 88A, 51 l-517. Montrose M. H., Friedrich T. and Murer H. (1987) Measurement of intracellular pH in single LLC-PK, cells: recovery from acid load via basolateral Na+/H+ exchange. J. Membr. Biol. 97, 63-78. Murer H., Hopfer U., Kinne-Saffran E. and Kinne R. (1974) Glucose transport in isolated brush-border and lateral basal plasma membrane vesicles from intestinal epithelial cells. Biochem. biophys. Acta 345, 170-179. Murer H., Amman E., Biber J. and Hopfer U. (1976) The surface membrane of the small intestinal epithelial cell. 1. Localization of adenyl cyclase. Biochim. biophys. Acra 433, 509-5 19. Nechay B. R. and Saunders J. P. (1978) Inhibition of vanadium of sodium and potassium dependent adenosine triphosphatase derived from animal and human tissues. J. Enr. Pathol. Toxicol. 2, 247-262. Pennington R. J. (1961) Biochemistry of dystrophic muscle: mitochondrial tetrazolium reductase and adenosine triphosphatase. Biochemical J. 80, 649454. Towler C. M., Pugh-Humphreys G. P. and Porteus J. W. (1978) Characterization of columnar absorptive epithelial cells isolated from rat jejunum. J. Cell Sci. 29, 53--75.