Intracellular calcium modulates gallbladder ion transport

JOURNAL

OF SURGICAL

RESEARCH

50, 545-551 (1991)

Intracellular JOE

A. CATES, M.D., KIMBERLY

Research Service, Sepulveda Presented

Calcium Modulates D. SAUNDERS,

VAMC, Sepulveda, California

at the Annual

Meeting

Gallbladder

M.D., MOHAMMED and Department

of the Association

PH.D., AND JOEL J. ROSLYN, M.D.’

of Surgery, UCLA School of Medicine,

for Academic

Although experimentally induced cholesterol gallstone formation has been associated with altered gallbladder (GB) absorption and increased biliary Ca’+, the relationship between these events remains unclear. Recent studies suggest that extracellular Ca” ([Ca2’],) influences GB ion transport. Whether the effects of [Ca2+]- are mediated by changes in intracellular Ca2’ ([Ca2’],) has not been determined. This study was designed to define the effects of altered [Ca2+], on GB ion transport. Prairie dog GBs were mounted in a Ussing chamber and short-circuit current (I,), potential difference (V,), and resistance (R,) were recorded. Mucosal surfaces were exposed to either Dantrolene (Dt) or nickel (Ni2’). Dt “traps” [Ca2+l, within intracellular organelles, thereby lowering cytosolic Ca2+; and Ni2+ prevents influx of [Ca2’],, presumably by binding Ca2+ channels. Although Dt reduced both I, and V, (P < O.Ol), these effects were transient. Transport recovery was probably due to increased [Ca2+],o influx with restoration of [Ca2+] ic. Ni2+ resulted in sustained decreases in I, and V,,,, (P < 0.05) despite subsequent addition of 10 mM Ca 2f . These findings are consistent with the prevention of [Ca2+], influx by Ni2+. We conclude that: (1) [Ca2’],, may be a modulator of GB ion transport and (2) previously reported [Ca’+], effects on ion transport may be mediated through [Ca2’], concentration changes. 0 1991 Academic Press, Inc.

Z. ABEDIN,

Ion Transport

Surgery, Houston,

Los Angeles, California

Texas, November

90024

14-17,199O

Ca2+ has been shown to modulate ion transport in several tissues, including colon [7], small intestine [8-g], and renal tubules [lo]. Donowitz and Asarkof [8] and others [7, 91 have demonstrated that alterations in the concentrations of both intracellular Ca2+ ([C!a2+lic) and extracellular Ca2+ ( [Ca2’],,) are linked to changes in intestinal fluid and electrolyte transport. Furthermore, our laboratory has recently demonstrated in the prairie dog model that changes in [Ca?‘], also affect ion transport across gallbladder epithelia [ 11 J. The importance of Ca2+ in the regulation of gallbladder ion transport is underscored by the finding that biliary Ca2+ concentrations are increased in human [12] and animal [13-141 models of cholesterol gallstone disease. The mechanism by which alterations in [Ca2+], influence gallbladder ion transport has yet to be defined. Specifically, whether Ca2+ exerts its effect directly on gallbladder epithelium or through changes in [Ca2+lie remains unclear. The purpose of the current study, therefore, was to define the effects of altered [Ca2+li, on gallbladder ion transport. The prairie dog gallbladder, a widely used model in cholesterol gallstone research [ 15 171, was selected for its unique transport characteristics which provide the opportunity for evaluation of pharmacologic effects on ion transport [18]. MATERIALS

AND METHODS

Tissues and Solutions

INTRODUCTION Recent studies suggest that altered absorption of sodium and water across gallbladder epithelium occurs during the early stages of experimental gallstone formation [l-2]. Moreover, altered transepithelial absorption has been implicated in the pathogenesis of both cholesterol and pigment gallstones [3-61. Factors which regulate gallbladder absorption, however, remain poorly defined.

r To whom reprint requests and correspondence should be addressed at Division of General Surgery, UCLA School of Medicine, Los Angeles, CA 90024. 545

Adult male prairie dogs, Cynomys ludovicianus, were trapped in the wild and obtained from Otto Marten Locke, New Braunfels, Texas. Animals, weighing approximately 1 kg each, were individually caged in a thermoregulated (23”) room with 12-hr light cycles and fed standard laboratory chow (Purina Laboratory Chow, Ralston-Purina, St. Louis, MO). Following a 16-hr fast (water ad libitum), each animal was anesthetized with ketamine (100 mg/kg body wt, IM) and xylazine (I.5 mg/kg, IM) and underwent midline laparotomy. Each gallbladder was gently lifted out of the abdomen, thus clearly exposing the cystic duct. The cystic duct was clamped proximally and distally and divided, and a meticulous cholecystectomy was then performed. Gallblad0022.4804/91$1.50 Copyright 0 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.

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ders were incised longitudinally and rinsed free of bile with standard Ringer’s solution. Following this procedure, tissues were mounted in a two-piece plexiglass Ussing chamber similar to that described by Schultz and Zalusky [ 191. Ideally, one would like to separate mucosa from the serosa, muscularis, and submucosa. While this has been accomplished in intestinal preparations, great difficulties have been encountered in studies utilizing gallbladder tissues. Because prairie dog gallbladder epithelium is extremely adherent to the thin serosal connective tissue layer, it is not possible to selectively remove and mount isolated mucosal preparations. Gallbladders were minimally stretched over a 0.67-cm2 circular opening and lightly clamped between the two chamber halves. Chamber contact and seal were achieved by use of high-viscosity silicone grease. Both mucosal and serosal surfaces were exposed to equal volumes (10 ml) of bathing solution of the following composition (in m&f): NaCl, 114.2; KCl, 4.8; CaCl,, 1.3; MgCl,, 1.2; NaHCO,, 21.0; Hepes, 5.0; and glucose, 10.0. PH was adjusted and maintained at 7.4, and osmolality ranged from 280 to 300 mosm/kg. Mucosal and serosal perfusions with this physiologic salt solution were achieved by means of a gas-lifting circulating system driven by a water-saturated 95% 0,/5% CO, gaseous mixture. Temperature was maintained at 37°C via water-jacketed reservoirs connected to a constant temperature circulating pump. The reservoirs were capped with glass condensers to minimize water loss due to evaporation. Two agents were utilized which indirectly lower cytosolic Ca2+ via different mechanisms. Dantrolene sodium l-[(5-(p-nitrophenyl)furfurylidenejamino]hy(Dt), dantoin sodium hydrate, is a skeletal muscle relaxant which inhibits the release of intracellular, compartmentalized Ca2+, thereby lowering the concentration of cytosolic Ca2+ [20-231. The second test agent employed was the heavy metal nickel (Ni2+). Described originally for its actions on excitable tissues, Ni2+ prevents the influx of Ca2+ from extracellular sources [24-271. Last, diethylenetriamine pentaacetic acid, DTPA, is an agent which chelates ionized heavy metals [28-311, and in this experiment was used to reverse effects imparted by Ni2+. All chemicals used in this study were obtained from Sigma Chemical Co. (St. Louis, MO). Transepithelial

Electrical

Measurements

All measurements were made with an automatic voltage current clamp (VCC 600, Physiologic Instruments, Houston, TX) following the four-electrode technique. Tips of salt agar bridges were placed within 1 mm of each surface of the membrane and potential difference ( V,) was measured by a pair of matched calomel electrodes (Fischer Scientific, Tustin, CA) immersed in supersaturated KC1 solutions and connected to the VCC. Transepithelial resistance (R,) was determined by recording

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50, NO. 6, JUNE

1991

the transepithelial potential deflection in response to a bipolar current of +lO PA passed across the epithelium by means of a pulse generator via Ag-AgCl electrodes. Compensations for fluid resistance and asymmetry of the calomel electrodes were made prior to mounting tissues in the chamber. Direct current was continually passed through the tissue by means of Ag-AgCl electrodes in sufficient quantity to nullify the spontaneous V,, , except for the time required to measure V,, and R,. Short-circuit current (1,,) was read directly from a digital display on the VCC when the tissue was clamped at 0 mV. R, and I,, measurements were corrected for chamber surface area. I,,, V,, , and Rt were measured at l- to 3-min intervals during the initial equilibration period and over a 30-min period for each tissue exposure. Our laboratory has previously reported that in prairie dog gallbladder tissue net transepithelial fluxes of Na+ and Cl- largely account for 1, [32]. Experimental

Design

After tissues were mounted, both mucosal and serosal surfaces were exposed to a physiologic salt solution for a 1-hr equilibration period. This time interval allowed for tissue stability. Following equilibration, gallbladder mucosal surfaces were randomly exposed to either Dt, 10e7lop5 A!& or Ni2+, 10-6-10-3 M. Because the maximal solubility of Dt in Ringer-HCO, (approximately 2.5 X lop5 M) is difficult to attain without the use of a solubilizing agent, 1 X lop5 M was the highest concentration utilized. To examine the effectiveness of Ni2+ in inhibiting Ca2+ influx from extracellular sources, several tissues were initially exposed to Ni2+ and, following restabilization, underwent additional exposure to excess Ca2+ ( lop2 M). In order to assess reversibility of Ni2+-induced ion transport effects, DTPA was added to Ni’+-containing solutions in eight tissue exposures. Following all tissue exposures, mucosal and serosal surfaces were simultaneously washed with 400 ml of warm bathing solution delivered by gravity and drained through outlets at the bottom of the Ussing Chamber. Following this procedure, repeat measurements of I,,, V,, and R, were obtained. The Ussing chamber, as employed in this series of experiments, has been utilized extensively in the study of various epithelial tissues [7,19,32-341. These investigations have examined thoroughly the integrity and viability of tissues studied using this in vitro model. Histologic and electron microscopic examinations of tissues mounted in the Ussing chamber have previously revealed normal cellular morphology in both intestinal [8] and gallbladder [34] preparations. Moreover, previous studies from this laboratory have demonstrated minimal change in I,, and R,, two accepted indices of tissue viability, despite prolonged (up to 8 hr) tissue exposure to the Ussing chamber preparation. Tissue viability was assessed in the current experiment by: (1) ability of the tissue to maintain active ion transport, (2) return of tis-

547

CATES ET AL.: INTRACELLULAR CALCIUM AND GALLBLADDER ION TRANSPORT

210

a ~~0.01 vs baseline

t /

180. a

I

170. -104

-8

160,rn I””

Baseline

IO-7

Dantrolene

10-6

10-5

Cont. (M)

FIG. 1. Effects of dantrolene (Dt) on gallbladder short-circuit current (I,). Z,, expressed in WA/cm ‘, is represented on the Y axis, and Dt concentration (M) is seen on the X axis. Data are reported as means t SEM from 17 tissue exposures.

sue I,, and V,, to baseline values following drug exposure and subsequent mucosal and serosal washing, and (3) mucosal tissue exposure to lop2 M theophylline prior to termination of experiments. Statistical

Analysis

All data are presented as means + standard error of the mean. Each tissue served as its own control and statistical comparisons were made using Student’s paired t-tests. Regression analysis was performed using the method of least squares. RESULTS

Dantrolene

and Ion Transport

The effects of Dt on gallbladder electrophysiology are summarized in Figs. l-4. Exposure of prairie dog gallbladder epithelium to Dt resulted in a dose;dependent inhibition of ion transport. The effects of Dt on I,, the most sensitive indicator of ion transport among the parameters studied, are summarized in Fig. 1. Maximal inhibition in I,, was observed at lop5 M (1 X 10e5) Dt (P < 0.001 vs baseline). Lesser, although statistically significant, effects were also seen at lop6 and lop7 A4 concentrations. Regression analysis was performed to further define the relationship between I,, and Dt concentration. As shown in Fig. 2, this analysis demonstrated a linear correlation between percentage inhibition of I,, and Dt concentration (P < 0.007). As Dt concentration increased, percentage inhibition of I,, likewise increased. Figure 3 illustrates transepithelial potential difference, V,, , at baseline and at each of the various Dt concentrations studied. Although there is an apparent stepwise decrease in V,, as drug concentration in-

FIG. 2. Scatter percentage inhibition centration [log M].

-4

-5

-6

-7

Dantrolene

Concentration

(logy)

plot demonstrating linear correlation between of short-circuit current (I,) and dantrolene con-

creases, when compared to baseline, only lop6 and 10e5 M Dt met statistical significance (P < 0.01). Tissue exposure to various Dt concentrations, whether mucosal or serosal, did not significantly alter R,. Moreover, when Dt was applied to the serosal surfaces, there were no demonstrable effects on tissue I,, or V,,. Fig. 4 depicts I,, versus time for a typical tissue exposed to lop5 M Dt. Following drug addition, the nadir I,, was reached by approximately 4 min. Continued observation, however, revealed a gradual increase in I,,, and baseline values were reached within 22 min. This “spontaneous reversal” of I= inhibition was consistently observed throughout all Dt concentrations studied. Nickel and Ion Transport The effects of Ni2+ on I, are displayed in Fig. 5. When compared to control values, exposure of tissues to 10e5 M Ni2+ resulted in a 17% decrease in I,. Moreover, successive lo-fold concentration increases in Ni2+ produced

10 a

a 1~0.01 vo bareline

“nls8 7-

8-

Baseline

10-7

10-6

Dantrolene

Concentration

10-5

(tvl)

FIG. 3. Mean transepithelial potential difference (V,) line and each of the dantrolene concentrations studied.

at base-

548

JOURNAL

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RESEARCH:

VOL.

50, NO. 6, JUNE

; z m3’ 50

1991

12.1x

p ( 0.001

40

A

/ ic A A

10

’

’

I

I

’

’

’

’

’

’

I

I

’

Time (2 min intervals) FIG. 4. Time course of short-circuit current (I,) inhibition by dantrolene. Results represent a typical tissue which, following stabilization, was exposed to 10e5 Dt. Nadir Z, is measured approximately 4 min following Ni2+ exposure, and tissue subsequently undergoes spontaneous recovery.

parallel decreases in I,. Maximal I,, inhibition (68%) was recorded at 10e3 M Ni’+. As one would anticipate, regression analysis also demonstrated a linear correlation between Ni2+ concentration and percentage inhibition in I,, (P < 0.001, see Fig. 6). Figure 7 demonstrates the inhibition of transepithelial potential difference at each of the various Ni2+ concentrations studied. Successive lo-fold increases in Ni2+ from lop5 to 10e3 M corresponded to 15, 25, and 36% inhibition in V,, respectively. Ni2+- induced inhibition in V,,,, , although lesser in magnitude, clearly paralleled changes seen in IX. Ni2+ did not alter I,, , V,, , or l& when added to tissue serosal surfaces, nor did it affect R, when applied mucosally. Figure 8 illustrates the time course for a tissue exposed to Ni’+. Although the nadir I, was again observed at approximately 4 min following drug addition, the tis-

’

FIG. 6. short-circuit

A

N

-7

I

A

I

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I

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A

20

I

2

A

30

160’

A

A

t

1

1

L\

43

Linear current

-5

-4

-3

Nickel Concentration

(log@

7 -2

correlation between percentage inhibition (I,) and nickel concentration [log M].

of

sue exhibited no spontaneous recovery. Only after the addition of DTPA did 1 return to baseline. The sustained effects of Ni2+ on I, were not reversed by tissue washing alone, and similar trends were again seen for all concentrations tested. These findings were markedly different than the time course observed for exposure to Dt (see Fig. 4). Nickel, Calcium, and DTPA Figure 9 demonstrates the effects of various combinations of Ni2+, Ca’+, and DTPA on tissue 1,. Exposure of tissues to lop4 M Ni2+ again resulted in a significant decrease in I,,. The inhibitory effect of Ni2+ persisted, however, despite subsequent addition of lo-’ M Ca2+ to the bathing solution. In a separate, but related, set of exposures, lop3 M DTPA was added to bathing solutions both with and without 10e4 M Ni2+ pretreatment. DTPA had no affect on I, in the absence of Ni2+. However, DTPA completely reversed the I, inhibition seen follow-

ia I220

T

2oa

a ptO.001 b ~~0.04 c wo.03

180

vs Baseline “8 10-M “8 IO-4M

9,-

a

a ~‘0.02

vs baseline

b ~‘0.05

VII 10-5

160 140

wnss

i2a ISC

7

100

1

80 60 4a 20

Baseline

a Baseline

IO-5

10-4

Nickel Concentration

FIG. 5.

(M)

Effects of nickel on short-circuit

current.

Nickel

Concentration

FIG. 7. Mean transepithelial potential at each of the various nickel concentrations

(Ml difference studied.

(V,)

measured

CATES ET AL.: INTRACELLULAR

CALCIUM

AND GALLBLADDER

549

ION TRANSPORT

DANTROUNE /

NZCKEL /

240

L \

I Ca++-

ATPtLSe

-Ca++

DAhZROLrTNE

FIG. 10. Major homeostatic mechanisms involved in the regulation of intracellular Ca’+. Points of action of nickel and dantrolene are schematically represented.

Time (2 min intervals) FIG. 8. Time course of short-circuit current (I,) inhibition by nickel. Depicted is a single tissue which, following stabilization, was is sustained, with no evidence of exposed to 1O-4 Ni ‘*+. Z, inhibition spontaneous recovery. Following addition of DTPA, however, Z, promptly returns to baseline.

ing exposure of tissues to Ni2+ (I, returned to within 97% of baseline in all eight tissue exposures). DISCUSSION

The present in vitro study indicates that pharmacologic reduction of cytosolic calcium concentration results in reduced gallbladder epithelial ion transport. Using standard electrophysiologic techniques, we have demonstrated that exposure of prairie dog gallbladder epithelia to both Dt and Ni2+, two agents known to reduce cytosolic Ca” (by different mechanisms), led to a

220 7

I

200

a ptO.01 vs baseline

and Ni+DTPA

I 100

t

i a

Baseline

Ni*Ca**

Ni lo-4M

Ni*DTPA

Exxposure FIG. 9. Tissue short-circuit current (I,) measured upon addition of 10m4M N?, Ni2+ + lo-’ M Ca’+, and Ni” + 10-a M DTPA. Ni” alone produces significant (30%) inhibition of Z,. Subsequent addition of Ca2+ to Ni’+-containing solutions have no effect on Isc inhibition. Addition of DTPA to Ni’+-containing solutions results in 97% reversal of Z,, inhibition.

dose-dependent inhibition of I,, and V,, without affecting Rt. Moreover, the effects induced by both Dt and Ni2’ were fully reversible. Last, neither agent significantly altered I,, V,,,,, or R, when applied to serosal tissue surfaces. Although both Ni2+ and Dt resulted in ion transport inhibition, major differences were observed in the time courses exhibited by each agent respectively. Dt-induced I,, inhibition was transient in nature and spontaneous recovery was observed for all tissue exposures. In contrast, Ni2+ effects were sustained, with I, inhibition lasting for up to 90 min in selected tissues. Possible explanations for these findings must take into consideration the homeostatic mechanisms which collectively regulate cytosolic Ca2+ concentrations in normal tissues (Fig. 10). fCa2+lec diffuses down a steep electrochemical gradient through protein channels to gain entry into the cell. Direct (Ca2+-ATPase) and indirect (Na+-Ca2+ antiport) active transport are responsible for the extrusion of Ca2’ and maintenance of this diffusional gradient. Further control of cytosolic Ca2+ is afforded through uptake into and release from intracellular organelles. Dt, by preventing the release of compartmentalized Ca2+ sources, lowers the concentration of cytosolic Ca2+. In the current experiment, spontaneous recovery of ion transport following Dt exposure is likely due to increased Ca2+ influx from extracellular sources. In contrast, Ni2+ prevents the influx of Ca2+ from extracellular fluids. Under these circumstances, recovery of normal cytosolic Ca2+ is limited to mobilization of finite, compartmentalized sources. Following rapid exhaustion of this available Ca2+, cytosolic-free Ca2+ concentrations remain decreased. In the current experiment, transient and sustained decreases in I, following Dt and Ni2+ exposures, respectively, coincide temporally with proposed cytosolic Ca2+ concentration changes. These findings provide initial evidence that cytosolic Ca2+ may play a role in the regulation of gallbladder ion transport. Dt is a skeletal muscle relaxant used clinically in the treatment of malignant hyperthermia and muscle spas-

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ticity. Its specific actions on cellular Ca2+ homeostasis have been studied extensively in models of skeletal muscle [20-231 and more recently in intestinal epithelium [35]. Desmedt and Hainaut [20,2l], using the aequorin method to measure [Cap+lic, demonstrated in isolated muscle fibers that Dt (1) reversibly decreased cytosolic Ca’+ concentrations, (2) decreased Ca2+ efflux across plasma membrane, and (3) did not alter influx of [Ca2+Jec. Subsequent investigation has demonstrated that these findings are secondary to “trapping” of Ca2+ within intracellular organelles [20-231. In the present study, exposure of Dt to mucosal gallbladder surfaces resulted in transient, yet significant, decreases in I,, and V,,. These findings, although indirect, do suggest that the concentration of cytosolic Ca2’ may directly influence gallbladder ion transport. The divalent cation, Ni2+, in contrast, is utilized primarily for its role as an investigational agent in the study of cellular Ca2+ homeostasis. Early studies, performed by Jones and Heinemann [24, 251, focused primarily on models of nervous tissue. Utilizing direct cellular micropuncture techniques, they demonstrated that Ni2+ blocked both pre- and postsynaptic influx of [Ca2’],,. Ni2+ has subsequently been shown by various independent laboratories to block the influx of [Ca2+],, in both hepatocytes [ 271 and cardiac muscle cells [ 261. In the current experiment, exposure of gallbladder epithelia to Ni2+ resulted in pronounced decreases in I., and V,,. Moreover, these effects appear to be specific in that they were completely reversible upon addition of DTPA. Inhibition of ion transport by Ni2+, coupled with its known mechanism of action, adds further support to the notion of intracellular Ca2+ as a modulator of gallbladder ion transport. Acting through different mechanisms, both Ni2+ and In this study, Dt lower cytosolic Ca2+ concentrations. both agents also reversibly inhibited transepithelial ion transport. Taken together, these findings suggest that there is a direct relationship between cytosolic Ca2+ and transepithelial ion transport in the prairie dog gallbladder model. Due to the indirect nature of these experiments, however, a toxic effect of either Ni2+ or Dt on gallbladder tissues must be considered as a possible explanation for our findings. Several observations presented herein suggest that the inhibition of ion transport reported in this study was not merely a manifestation of tissue toxicity: (1) inhibition of tissue I,, and V, by both Ni2+ and Dt was completely reversible; (2) serosal exposure of Ni2+ and/or Dt had no affect on any of the electrophysiologic parameters studied; (3) despite tissue exposure to maximal concentrations of both agents, gallbladders exhibited sustained ion transport for periods of up to 8 hr; (4) tissue resistance remained unchanged despite mucosal and/or serosal exposure to maximal concentratissues extions of Dt and Ni2’; and (5) post-treatment hibited a normal 1= response to mucosal theophylline exposure (as previously reported in this model [34]).

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Although there has been considerable investigation into the role of Ca2+ as a modulator of intestinal absorption, little has been published about its function in the regulation of gallbladder ion transport. Data from the current experiment, therefore, is best analyzed in light of previous studies, most of which have been carried out in a model of rabbit intestine. Donowitz et al. [8,33,35, 361 and others [ 7,9, 10,371 have demonstrated that (1) decreases in [Ca2+],, and manipulations expected to lower [Ca2+lic (verapamil, dantrolene, or cadmium exposure) resulted in decreases in both I, and V, and (2) elevated [Ca2+jec and the Ca2+ ionophore A23187 produced the opposite effect. Moreover, these changes were correlated with specific alterations in Na+ and Cl- unidirectional fluxes. This study represents the third phase in a similar series of investigations of gallbladder epithelium. Our laboratory has previously published data demonstrating that [Ca2+]= concentration changes are directly proportional to both 1, and V, [ll]. Moreover, we have also shown that gallbladder exposure to verapamil results in a reversible inhibition of I,, and V,, [34]. Data from this experiment are consistent with the aforementioned observations and add further support for the role of [Ca2+], as a regulator of gallbladder ion transport. In summary, the present in vitro study has demonstrated that exposure of prairie dog gallbladder epithelia to Dt and/or Ni2+ results in significant decreases in transepithelial ion transport. Both Dt and Ni2+ are known to predictably decrease cytosolic Ca2+ through different mechanisms. Taken together, these observations suggest that in prairie dog gallbladder epithelium decreases in cytosolic Ca2’ are associated with concomitant decreases in transepithelial ion transport. We therefore conclude that [Ca2+lic may be a modulator of transepithelial ion transport in the prairie dog gallbladder. Furthermore, the modulation of ion transport by [Ca2+],e may, in fact, be secondary to changes in [Ca2+]ic. Future investigation incorporating the direct measurement of [Ca2+lic in gallbladder epithelia will be required to ultimately address these issues. REFERENCES 1.

2.

3.

4. 5.

6.

Roslyn, J. J., Conter, R. L., and DenBesten, L. Enhanced gallbladder absorption and cholesterol gallstone formation. Surg. Forum 36: 221, 1984. Conter, R. L., Roslyn, J. J., Porter-Fink, V., and DenBesten, L. Gallbladder absorption increases during early cholesterol gallstone formation. Am. J. Surg. 151: 184, 1968. Abdou, M. S., Strichartz, S. D., Abedin, M. A., and Roslyn, J. J. Biliary lipids alter ion transport during cholesterol gallstone formation. J. Sug. Res. 44: 672, 1988. Lee, S. P. Enhanced fluid transport across gallbladder mucosa in experimental choielithiasis. Am. J. Physial. 234: E575, 1978. Roslyn, J. J., Doty, J. E., Pitt, H. A., Conter, R. L., and DenBesten, L. Enhanced gallbladder absorption during gallstone formation. Am. J. Med. Sci. 151: 184, 1986. Strichartz, S. Z., Abedin, M. Z., and Roslyn, J. J. Pigment gall-

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