0300-9629/91 $3.00f0.00 0 1991Pergamon Press plc

Camp. Eiochem.Phpiol.Vol.IOOA,No. 2,pp.335-341,1991 Printed in Great Britain

UNCOUPLED BASAL SODIUM ABSORPTION AND CHLORIDE SECRETION IN PRAIRIE DOG (CYNOMYS LUD0VKWVUS) GALLBLADDER JOEL

J. ROSLYN,*MOHAMMAD Z. ABEDIN,KIMBERLYD. SAUNDERS, JOEA. CATES, SETHD. STRICHARTZ,MICHAELALPERIN,MICHAELFROM@ and CARLOSE. PALANTS

Surgical Services, Sepulveda Veterans Administration Medical Center, Sepulveda, CA and the Department of Surgery, UCLA School of Medicine, Los Angeles, CA 90024, U.S.A. Telephone: (213) 8256156; TInstitut fur Klinische Physiologie, Freie Universitat Berlin, 1000 Berlin 45, Federal Republic of Germany; and SDepartment of Medicine, Brooklyn VA and SUNY Health Science Center, New York, U.S.A. (Received 3 December 1990)

Abstract-l.

Prairie dog gallbladders mounted in a Ussing-type chamber and bathed with symmetrical Ringer’s solutions exhibited a transepithelial resistance (R,) of 51 f 5 R cm*, a lumen negative potential

difference (V,,) of 11.5 f 0.7 mV and a short-circuit current (I,) of 6.9 + 0.3 pEq/hr/cm2. 2. Radioisotopic ion flux experiments revealed that the basal I, of 6.9 f 0.3 ~Eq/hr/cm’ was mostly of 3.2 + 0.5nEq/hr/cm2 and net Cl- secretion of accounted for by net Na+ absorption 2.9 k 0.3 pEq/hr/cm2. 3. In HCO; free Ringer’s, net Na+ flux was virtually abolished, net Cl- flux decreased by 50% and I, was reduced by 77%. 4. 10m3M mucosal amiloride and DIDS reduced I, by 28 and 24%, respectively. 5. Mucosal NaCl diffusion potentials indicated that the paracellular pathway was cation selective. 6. Thin section electron micrographs showed a single cell population in this epithelium suggesting that net Na+ absorption and Cl- secretion may emerge from the same cells. 7. We conclude that prairie dog gallbladder epithelium is an electrogenic tissue and, in contrast to gallbladders of most other species, simultaneously but independently absorbs Na+ and secretes Cl-.

INTRODUCTION The prairie dog has emerged as an important animal model for the study of human cholesterol gallstone disease. Prairie dog hepatic and gallbladder bile compositions are similar to those of man (Brenneman et al., 1972). Furthermore, prairie dogs maintained on a cholesterol-enriched diet develop cholesterol gallstones in a manner that recapitulates events known to occur in humans with cholelithiasis (Gurll and DenBesten, 1978; Holzbach et al., 1976). Our previous studies of prairie dog gallbladder suggest that: (I) its epithelium is electrogenic (Roslyn et al., 1989) resembling human (Rose et al., 1973) and porcine (O’Grady and Wolters, 1989) gallbladders; and (2) alterations in gallbladder electrolyte and water transport may have a pathogenic role in cholesterol gallstone formation (Conter et al., 1986). More specifically, our Ussing chamber experiments demonstrated that an increase in the ratio of lecithin to bile acid concentrations, as seen in cholesterol gallstone disease, stimulated transepithelial potential difference (V,,) and short-circuit current (I,) in prairie dog gallbladder (Roslyn et al., 1989). In other words, cholesterol gallstone disease seems to be associated with stimulation of ion (and H,O) transport *Requests for reprints and correspondence should be addressed to: Joel J. Roslyn, Division of General Surgery, UCLA School of Medicine, Los Angeles, CA 90024, U.S.A. Telephone: (213) 825-6156.

across the gallbladder epithelium. Nonetheless, our ability to understand the observed alterations in ion and fluid transport associated with altered bile composition and with cholesterol gallstone formation have been hampered by the paucity of our knowledge of normal ion transport in this model. Studies on the nature of transepithelial ion and water transport across gallbladder epithelia, conducted mostly in Necturus maculosus and rabbit, have produced several mechanistic models that contemplate transepithelial water flow as coupled to either (1) a luminal NaCl symport (Cremaschi and Henin, 1975; Frizzell et al., 1975), (2) a parallel countertransport by which luminal Na+ is exchanged with intracellular H+ and luminal Cl- is exchanged with intracellular HCO; (or OH-) (Baerentsen et al., 1983; Reuss, 1984), (3) a Na-K-2Cl symport (Davis and Finn, 1985), or (4) a combination of these various transport modalities (Cremaschi et al., 1983, 1987). These animal models share a common feature, i.e., the measured V, in both Necturus and rabbit gallbladders is usually less than 2 mV, and no significant I, is generated. Thus, ion transport across these epithelia is said to be nearly electroneutral. Non-electroneutral ion transport across a mammalian gallbladder has been previously described. Rose et al. (1973) reported that isolated human gallbladder epithelium generated a lumen negative V,, of approximately 8 mV and a I, of 5 pEq/hr/cm2. Net Na+ flux from mucosa to serosa accounted for most of the measured I,. Transepithelial resistance, 335

336

JOELJ. RCJ~LYN et al.

R,, was in the order of 50Rcm*. More recently, O’Grady and Wolters (1989) reported that the porcine gallbladder exhibits a lumen negative V,, of 5.5 mV and a I, of 2.5 pEq/hr/cm2. The obvious departure of transepithelial electrophysiological parameters noted in the prairie dog from the range of values described for Necturus and rabbit gallbladders and their close approximation to those measured in human and porcine gallbladder remains unexplained. In an effort to understand the mechanisms of ion transport operational in prairie dog gallbladder, and to identify the origin of V,, and I, in this tissue, studies of unidirectional ion fluxes, ion carrier-specific inhibitors and HCO; substitution experiments were performed. In addition, thin section electron micrographs were examined to determine if the electrogenicity of transport in prairie dog gallbladder originated from a distinct subpopulation of crypt-like epithelial cells.

MATERIALS

AND METHODS

Tissues and solutions

Adult male prairie dogs (Cynomys ludovicianus), trapped in the wild and obtained from Otto Marten Locke of New Braunfels. TX were caged in thermoreaulated (23°C) rooms and maintained on a control labo&tory chow (Purina Laboratory Chow, Ralston Purina, St Louis, MO) for two months prior to study. After a 16 hr fast with water ad lib, each animal was anesthetized with ketamine (100 m&kg of body weight) and diazepam (0.15 mg/kg of body weight). The gallbladders were removed and incised longitudinally under the dissecting microscope to minimize tissue trauma, rinsed free of bile with warm Ringer’s solution, and then mounted in a two piece Plexiglas Ussing type chamber similar to that described by Schultz and Zalusky (1964). Gallbladders were stretched over a 0.67 cm* circular opening and lightly clamped between the two chamber halves. Chamber contact and seal was achieved with highviscosity silicone grease. Both mucosal and serosal surfaces were exposed to equal volumes (10 ml) of bathing solution of the following composition (in mM): 140 Na, 124 Cl, 21HCO,, 5.4K, 1.3Ca, 1.2Mg, 2.4HPO,, 0.6H,PG,, 5.0 HEPES (N-2-Hydroxyethylpiperazine-N’-2-ethanesulfonic acid) and 10 glucose; pH adjusted to 7.4. The solution was maintained at 37°C and gassed with 95% O,-5% CO,. Bicarbonate-free solutions were prepared by replacement of HCO; with equimolar amounts of Na,SO, adjusted to pH 7.4 and gassed with 100% 0,. Amiloride and DIDS (4,4’-diisothiocyanostilbene-2,2’disuifonic acid) were purchased from the Sigma Chemical Co. (St. Louis, MO): Ouabain was purchased from CalBiochem (San Diego, CA). **Na and 36C1were brought from DuPont New England Nuclear (Boston, MA). Eiectrophysiological

measurements

Gpen circuit transepithelial voltage (V,) and short circuit current (I,) were measured following standard 4-electrode techniques using an automatic voltage current/clamp (VCC 600, Physiologic Instruments, Houston, TX). Voltage sensing electrodes (calomel electrode, Fisher Scientific, Tustin, CA) and current electrodes (Ag/AgCl wire) were connected to the chamber by agar-bridges filled with the respective bathing solution. Compensation for the resistance of the bathina fluid between the tips of the voltage sensing agarbridged was performed in all experiments. TransepitheIial resistance CR.) in re. ., was determined from V,.-deflections ..sponse to f IO yA/cm* pulses generated by the clamp device.

Measurement

ofparacelhdarpathway

ionic selectivity

Previous experiments indicated that prairie dog gallbladder epithelium developed a significant, lumen negative V, (Roslyn et al., 1989). To examine the contribution of the paracellular (i.e. tight junctional) ionic selectivity to this parameter, NaCl diffusion (dilution) potentials were created by replacing one half of the NaCl in the mucosal bath isosmotically with sucrose (Barry et al., 1971; Berry and Rector, 1978). It must be noted that since the intracellular Na+ activity, Nar, and the cell membrane potential, V,, of prairie dog gallbladder are unknown, NaCl diffusion potentials are valid only if it is assumed that a mucosal Na+ of 14OmM, will be higher than Nat and that V, remains at - 50 to -60 mV during the development of a mucosal positive electrical potential (Palant et al., 1983). Sodium and chloride fluxes

Unidirectional transepithehal fluxes of Na+ or Cl- were determined using 22Na or 36C1.Briefly, 1.5 pCi of **Na were added to 10 ml of Ringer’s solution in the mucosal reservoir, and 20min later, when steady state could be assumed, unidirectional mucosa to serosa flux of Na+ (Jz) was determined from three 10 min flux periods. After this, both surfaces of the tissue were simultaneously washed with 4OOml of warm bathing solution delivered by gravity and drained through outlets at the bottom of the Ussing chamber. Washing took less than 2min and did not have significant effects on the electrical parameters measured. After washing, the unidirectional serosa to mucosa flux of Na+ (Jg) was determined. Background counts were determined in 1 ml ahquots obtained from the serosal and mucosal chambers prior to addition of radioisotopes. The tissue was washed again, and unidirectional Cl- fluxes (Ji\ and J$) were measured in a similar manner. Sequence of radioisotopes and direction of fluxes was randomized for individual experiments. Measurement of Na+ and Clfluxes was performed under short circuit conditions except for short periods (IO-15 set) required to record open circuit V,. Radioisotopes were assayed in 10 ml of Scintiverse II (Fisher Scientific) using a well type liquid scintillation counter (LS 8000, Beckman Instruments, Fullerton, CA). Unidirectional Na+ and Cl- fluxes were calculated by a standard formula (Schultz and Zalusky, 1964). Net residual flux (Jk,) was defined according to Field and co-workers (1971) and represents the portion of the measured I, unaccounted for by Na+ and Cl- fluxes,

Thus, J,“,,= 0 means that the sum of net fluxes of ions other Na+ and Cl- is zero. In contrast, if Jk, > 0, there is a flux of an unidentified anion from serosa to mucosa, or cation from mucosa to serosa, or both. Electron microscopy

Fresh gallbladder tissue specimens were fixed in 2.5% nlutaraldehvde in 0.1 M sodium Cacodvlate buffer (PH 7.3), postfixed in’ osmium tetroxide, treated-en bloc with tannic acid, dehydrated in ethanol and propylene oxide and embedded in Spurr Epon. Tissues were then cut in cross section (70-80nm) on a Sorval MT 28 ultramicrotome with a diamond knife (DuPont) and examined with a Philips EM 201 transmission microscope. Statistical

analysis

All data are expressed as mean k standard error of the mean (SEM). A two tailed Student’s f-test for paired or unpaired variables was used, as appropriate. A value of P < 0.05 was accepted as significant.

Ion transport in prairie dog gallbladder

337

Table 1. Electrical properties of gallbladder in diliennt species Species

R, (n cm’)

Nectums

100*

Rabbit Guinea pig Porcine Man Prairie doe

25 156 56 52 51

V,,

Reference

I, (dWW~2)

W)

+0.7 f 0.2 + I .4 * 0.4 -1.1 kO.2 f5.5 * 0.5 +7.6 + 0.6 +11.5+0.7

-0.3 * 2.5 f 5.1 f 6.9 +

Fromm et al. (1985) Machen and Diamond (1969) Winterhager et al. (1986) O’Grady and Walters (I 989) Rose et al. (1973) Present studv

0.1 0.2 0.4 0.3

*Tissue epithelial resistance 84 R cm2, subepithelial resistance 16 D cm’.

RESULTS

Electrophysiological measurements

The mean R,, V,, and I, for fifteen gallbladders were 51 f 5 R cm2, 11.5 + 0.7 mV and 6.9 + 0.3 pEq/ hr/cm2, respectively. These values are similar to those previously reported (Roslyn et al., 1989) and correspond to tissues in which control unidirectional ion fluxes were determined. In Table 1, we compare these results with those published by other investigators in Necturus, rabbit, porcine, guinea pig and human gallbladder. Basic electrophysiological properties of prairie dog gallbladder resemble most closely those of man and pig. Inhibitor effects

Table 2 summarizes the effects of ion transport inhibitors on the electrophysiological parameters of the gallbladder. 10m3M mucosal amiloride significantly and reversibly inhibited I, by 28%, V,, by 21%, and increased R, by 14%. Its effects became evident within 1 min with peak effects seen in 4-6 min. 10s6 to 10e4 M mucosal amiloride had little effect on ion transport parameters. Serosal amiloride (10m3M) was without effect on electrophysiological parameters. 10m3M mucosal DIDS significantly inhibited I, by 24%) V,, by 16% and increased R, by 16%. Peak effects were noted within 8-10 min. Lower concentrations of mucosal DIDS (10m6 to low4 M) did not have significant effects on transport parameters. 10e3 M DIDS was also without effect when applied to the serosal chamber. Serosal addition of lo-’ M ouabain completely and irreversibly abolished I, within 20min. NaCl diffusion potentials Replacement of the control mucosal Ringer’s with isosmotic NaCl-sucrose Ringer’s led to a significant decrease in the spontaneous serosal positive V,,,, from 10.1 + 1.1 mV to 7.9 + 0.7mV (P < 0.02, N =4 measurements). This suggests that the tight junctions of prairie dog gallbladder epithelium exhibit higher permeability for cations than for anions, and that a

spontaneous transjunctional diffusion potential could not have contributed to the V, measured under open-circuit conditions. Unidirectional fluxes Table 3 summarizes unidirectional Na+ and Clfluxes in 15 control gallbladders, studied initially in the presence of normal Ringer’s solution containing 21 mM HCO; and gassed with 95% 0,5% C02. There was a net absorption of Na+ (3.2 f 0.5 pEq/hr/cm2) and a net secretion of Cl(2.9 f 0.3 pEq/hr/cm2), and together, these accounted for about 88% of the measured I,. Thus, prairie dog gallbladder epithelium generates substantial I, through a combination of net Na+ absorption and net Cl- secretion. The residual ion flux (&) was not significantly different from zero. A comparison of Jr: (3.2 & 0.5 pEq/hr/cm’) in the present study with Jr: (14.2 + 2.3 p Eq/hr/cm2) recently reported for rabbit gallbladder by Moran et al. (1986) indicates a comparatively smaller rate of Na+ transport in prairie dog gallbladder. Effect of HCd;

on ion flux

The effects of HCO; depletion on Na+ and Cl- fluxes are shown in Table 4. As compared to fluxes among control tissues (Table 3), a virtual absence of Jfz and a reduction in J”,, and I, occurred in six gallbladder tissues bathed in HCO; free bathing solution and gassed with 100% 02. Replacement of HCO; free bathing solution with normal Ringer containing 21 mM HCO; and gassed with 95% O,-5% CO, resulted in a significant stimulation of unidirectional fluxes of both Na+ and Cl-, with a net absorption of Na+ (3.2 + 0.8 pEq/hr/cm2) and a net secretion of Cl(2.1 f 1.8 pEq/hr/cm’) similar to that observed under basal conditions. A three-fold increase in I, was observed during HCO; repletion compared to HCO; depletion (1.6 + 0.2 ~~4.8 + 0.7, P < 0.01). As in basal conditions, J.9, was not significantly different from zero.

Table 2. Effects of ion transport inhibitors on gallbladder electrophysiological Inhibitors

Exposure

N

1, (~Eqlhrkm’) Initial Final % Change

Amiloride: IO-‘M IO-’ M lo-’ M

Mucosa Mucosa Serosa

6 6.8kO.7 13 7.1 kO.4 4 6.6 + 1.0

6.8f0.6 5.1 f 0.4’ 7.0 * I.2

-28

DIDS: 1O-4 M lo-)M

Mucosa Mucosa

3 6.OkO.2 IO 6.6 f0.6 3 5.6kO.9

5.9kO.l 5.0 f0.3t 5.3kO.7

-2 -24 -5

lo-‘M

Serosa

Values are mean f SEM. *P < 0.001, tP < 0.005, $P < 0.01 vs corresponding

0 6

initial value.

Initial

V, (mv) Final

7.2 f 0.7 10.9 * 1.0 8.4 f 1.4

7.2 + 0.7 8.6 * 0.7. 8.9 k 1.8

-21

11.5k3.4 11.3&1.6 8.7k2.9

11.6k3.2 9.5+1.5t 8.2 f 1.9

-I -16 -6

% Change 0 6

parameters Initial

R, (D cm2) Final % Change

38+ 5 49+4 46 + 8

38 * 5 56 f 5* 44*7

0 14 -4

52 f 8 51 f5 48*9

53 * 8 59 f 5i 52: Ii

2 16 8

338

JOELJ. ROSLYN et al. Table 3. Sodium and chloride flux in prairie dog gallbladder epithelium Ion flux (gEq/hr/cm2) R,

8 cm2 5lt5

.I:;

J”B nel

JC’ aI*

JO *Ill

JC’ ncl

3.2kO.5

8.7kO.9

11.6+0.8

-2.9iO.3

JN” sm

12.2k0.8

9.OkO.7

1%

Jf

icWWm’

6.9kO.3

0.8iO.6

Numbers are means i SEM (N = 15).

Electron microscopy Electron microscopic examination of the prairie dog gallbladder showed that the epithelium was composed of a single layer of mo~holo~c~ly similar columnar cells with a well developed brush-border membrane (BBM) and a large number of mitochondria (MC) (Fig. 1). The apical region of the cells contained a large amount of rough endoplasmic reticulum along with numerous vesicles. Magnification of the apical region of the cells showed junctions (TJ) connecting adjacent cells. The basolateral membrane (BLM) showed complex interdigitations with lateral intercellular spaces. DISCUSSION

These data represent the first report of the ion transport characteristics of prairie dog galibladder epithehum. The importance of these studies is underscored by the widespread use of this animal as a model for cholesterol gallstone research (Gurll and DenBesten, 1978; Holzbach et al., 1976) and by the recent observations which link altered gallbladder absorptive function to the pathogenesis of cholesterol gallstones (Conter et al., 1986; Abdou ef ni., 1988). Using standard electrophysiologic parameters of gallbladder epithelial transport, we have demonstrated that prairie dog gallbladder epithelium is an electrogenie tissue which simultaneously but independently absorbs Na+ and secretes Cl-. The prominent electrogenicity demonstrated by this tissue makes it potentially useful in the study of comparative gallbladder physiology. Classic unidirectional ion flux studies indicate that the spontaneous I, exhibited by prairie dog gallbladder epithelium originates through a combination of (1) net mucosal Na+ absorption (46% of measured I,) and (2) net Cl- secretion (42% of measured I,). Electron micrograph studies demonstrate a single cell population in prairie dog gallbladder epithelium. These findings suggest that net Na+ absorption and Cl- secretion may emerge from the same cells as opposed to distinct subpopulations of epithelial cells. The findings that prairie dog gallbladder epithelium is electrogenic distinguishes it from Necturus (Fromm et al., 1985), rabbit (Machen and Diamond, 1969) and guinea pig (Winterhager et al., 1986) gallbladders which exhibit predominently electroneutral NaCl transport. The prairie dog gallbladder epithelium is similar to humans (Rose et al., 1973) and porcine

(O’Grady and Wolters, 1989) gallbladders in that these tissues are all electrogenic and are characterized by lumen negative potential gradients. However, the combination of net Na+ absorption and Clsecretion observed in the prairie dog in the current study, is different than human (Rose et al., 1973) and porcine @‘Grady and Wolters, 1989) tissues in which the measured I, is mostly derived from net Na+ absorption. Since both electroneutral and electrogenic HCO; fluxes have been demons~ated in other g~lbladder epithelia (Diamond, 1964; Heintze et al., 1981; Moran et al., 1986), we examined the role of HCO; flux in the generation of I, across prairie dog gallbladder. Nominal absence of HCO; (and CO,) abolishes JNa net. Subsequent exposure of tissue to normal Ringer’s containing 21 mM HCO, and gassed with 95% O,-5% COz not only stimulated Jf;t", Jz and Jfi but Jzs and JFAas well, and resulted in near-normalization of I,. There are at least four known mechanisms whereby exposure of an epithelium to HCO; may influence the vectorial orientation and flux rates for Na+ and/or Cl-. HCO; induced stimulation of ion fluxes could result from: (1) modi~cation of the cell membrane potential, V, , and electrical driving forces across the cell membrane (Gunter-Smith and Schultz, 1982; Moran et al., 1986); (2) acceleration of membrane-associated ion exchange processes in response to an induced change in intracellular pH (pH,) (Vaughn-Jones, 1982) (3) direct involvement in anion exchange processes such as Na+-inde~ndent and Na+-dependent Cl-/HCO; exchange; and (4) acceleration of electrogenic Na+/HCO; cotransport (Boron and Boulpaep, 1983). In a series of studies, Adler et al. (1965a,b), demonstrated an interaction between pCOz and extracellular HCO;, so as to regulate and m~ntain extracellular pH, although inducing a fall in pH,. Roos and Boron (1981) suggest that even though cellular acid extrusion is stimulated by a fall in pHi, continuous intracellular HCO; efflux (favored by the electrochemical gradient) leads to further fall in pHi. Ultimately the recovery of pH, from the CO,-induced acid load will depend on the HCO; ~~eability of the cell membrane and the proton extruding capacity of the amiloride sensitive Na+/H+ exchange (Deitmer et al., 1980; Johnson et al., 1976; Murer et al., 1976). In the present study, exposure of prairie dog gallbladder to HCO; and CO2 resulted in independent stimulation

Table 4. Effects of bicarbonate on sodium and chloride fluxes Ion flux (~Eq/hr/~2) R, n cm’ HCO; HCO,

depletion repletion

51 f3 56 * 7

P 10.4:1.3 16.2 f 2.0*

Jk

JN” n,

J:s

10.6 rf 2.0 13.0 & 2.6t

-0.2 f 0.8 3.2 f 0.8’

10.3 f 2.5 14.0 f 2.9f

Numbers indicate mean k SEM (N = 6). *P < 0.001, tP < 0.025, $P < 0.01 vs corresponding

HCO< depletion (paired r-test).

JC’ sn,

11.6k3.2 16.1 f 4.6t

JC’ IK(

1,

Jzl

rWWm=

-1.3kO.9 1.6f0.2 0.6 f 0.4 -2.1 + 1.8 4.8 & 0.72 -0.5 k 0.9

Ion transport in prairie dog gallbladder

(4

Fig. 1. (A) Ultrastructure of prairie dog gallbladder epithelium showing uniform columnar cells with a well-developed brush-border membrane (BBM) at luminal surface. Nuclei (N) are basally located and intracytoplasmic organelles including abundant mitochondria (MC) are largely in the supranuclear region. The basolateral membrane (BLM) show complex interdigitations and lateral spaces; (x 3600). (B) Enlargement of apical region of cells showing intercelhdar tight junctions (TJ), mitochondria (MC) and rough endoplasmic reticulum (RER) with occasional mu& granules; (x 6750).

339

JOELJ.ROSLYNet al.

340

of both unidirectional Na+ fluxes of which the absorptive flux increased more than the secretory flux. We hypothesize that CO,/HCO; mediated stimulation of J$ may have resulted from stimulation of a mucosal antiporter. This ass~ption is reinforced by our finding that mucosal amilorlde inhibited basal I, in concentrations well above those required to inhibit Na+ channels but known to decrease Na+/H+ exchange (Johnson ef al., 1976). Na+/H+ antiporters on both epithelial cell membranes that exhibit quantitatively different H+ flux rates (mucosal membrane > serosal membrane) have been recently described in the cells of the outer medullary thin descending limb of the rabbit kidney (Kurtz, 1988), and could explain bilateral CO,/HCO; dependent stimulation of Na+ fluxes. However, lack of an amiloride effect on I, when introduced into the serosal chamber argues against a role for a serosal Na+/H+ antiporter in the C02/HCO;-dependent stimulation of Jz. Alternatively, HCO; may have resulted in stimulation of JE through cell membrane hyperpolarizaton (Deitmer and Ellis, 1980; Moran et al., 1986) or stimulation of serosal Na+/HCO; co-transport. Stimulation of Na+/HCO; inffux can be interpreted as an hom~s~tic process whereby the epithelial cell augments its acid extruding capacity when ambient pCOZ is raised (Boyarsky et al., 1988) and other acid loading exchangers such as the Cl-/HCO; exchanger are activated. Finally, it is worth mentioning that in the rabbit gallbladder, Moran et al. (1986) found that HCO; stimulated Jg but not fg which contrasts with our findings in prairie dog gallbladder. Stimulation of Jz$ by CO,/HCO; may have resulted from an increase in the activity of mucosal Cl-/HCO; exchanger (Roos and Boron, 1981; Thomas, 1984; Vaughan-Jones, 1982) that is sensitive to DIDS. The inability of DIDS to alter I, when introduced into the serosal chamber argues against the involvement of classical Cl-/HCO; exchange in HCO;-dependent stimulation of Jg. Operation of Cl-/HCO; exchange could raise intracellular Clmovement, across the mucosal membrane, in a manner similar to that described in cyclic AMP-stimulated Necrurus gallbladder (Petersen and Reuss, 1983). Sirn~~neo~ serosal Na+ efflux via an electrogenie serosal Na+/K+ pump would then explain the observed ouabain sensitive prominent I,. Clearly, additional studies beyond the intended scope of the present work are required to further clarify the transport characteristics of prairie dog gallbladder, specifically of its serosal membrane and to characterize Cl- conductive Amway on its mucosal membrane whose presence is suggested by our experimental results. Further experiments are required to examine the role of the various forms of Cl-/HCO; exchange in the generation of I, in this tissue. What is the significance of a Cl- secretory pathway in an ~ithelium generally regarded as absorptive? The answer is not forthcoming from the present study. However, tracheal epithelium displays a transepithelial voltage in the order of 10 to 30 mV, and its I, is entirely accounted for by spontaneous CI- secretion and Nat absorption (Welsh, 1986). Welsh

suggests

that

this

property

enables

the

epithelium to undergo changes in the direction and volume of transepithelial fluid transport when stimulated by a variety of neurohumoral agents. In summary, we have documented that the prairie dog ~llbladder epithelium exhibits a prominent I,. in contrast to gallbladders of most other species, prairie dog gallbladder shows a spontaneous absorp tion of Na+ and secretion of Cl- that accounts for most of the observed I,. Further studies are needed to fully characterize the processes responsible for the el~~ophysiolo~~ properties of this ~ithelium. Nevertheless, as a model of human cholesterol gallstone disease, prairie dog gallbladder holds an important advantage. Its electrically prominent V,,,, and I, closely approximate human gallbladder parameters and therefore, this model provides an opportunity to study the effects of luminal and ne~oh~oral factors on gallbladder electrolyte transport. Acknowledgements-We thank Drs Michael E. DutTey and Pamela J. Gunter-Smith for their valuable comments. This work was supported by Veterans Administration Research Career Development Awards (J.R.) and Merit Review

Board grants to J.R. and C.E.P.

REFERENCES

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