Department of Medicine, University of Colorado Health Sciences Center, Denver, Colorado 80262
Submitted 18 August 2003 ; accepted in final form 29 October 2003
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ABSTRACT |
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cell volume; purinergic signaling; cholangiocyte; phosphoinositide 3-kinase; FM143
Two broad models for epithelial ATP release have been considered (22). In one model, it is hypothesized that rapid exocytosis of ATP-containing vesicles accounts for local ATP release. This is analogous to the exocytic pathways seen in endothelial and chromaffin cells (3, 5, 11). Indirect evidence for such a mechanism is further supported by findings in pancreatic acinar cells in which quinacrine staining of cellular ATP stores reveals a punctate distribution of fluorescence consistent with the presence of ATP-containing vesicles (28). An alternative model for epithelial ATP release is derived from patch-clamp studies that have revealed currents carried by ATP and atomic-force microscopy and imaging studies that have documented the presence of cellular microdomains in which ATP is concentrated, suggesting the presence of ATP-permeable channels (1, 20, 29). These ATP channels are associated with members of the ATP-binding cassette family of proteins, including CFTR and multidrug resistant p-glycoprotein (4, 20.) However, the direct evidence suggests that CFTR and multidrug resistant p-glycoprotein are not themselves ATP-permeable channels, but rather facilitate ATP release through undefined mechanisms (4, 30, 33). Thus the molecular basis for ATP release in epithelia has not been identified.
In cholangiocytes, the epithelial cells responsible for biliary secretion, ATP is released across the apical membrane into bile, resulting in autocrine activation of P2Y2 receptors and subsequent stimulation of transepithelial Cl- secretion (2, 8, 12, 13). To date, increases in cell volume represent the most potent stimulus for ATP release and Cl- conductance in cholangiocytes and other epithelia (1416). Specifically, physiological increases in cell volume increase membrane ATP conductance and increase extracellular ATP concentrations 5- to 10-fold within minutes. Cellular response to increasing cell volume is complex and involves activation of both PKC and phosphoinositide 3-kinase (PI3-kinase) (12, 17). Moreover, intracellular dialysis with the lipid products of PI3-kinase simulates ATP release in the absence of changes in cell volume (9). Because PI3-kinase functions as a key regulator of vesicular trafficking (17), these findings suggest that volume-sensitive ATP release might be functionally coupled to vesicular exocytosis (18). Interestingly, cholangiocytes contain a dense population of subapical vesicles
140 nm in diameter that undergo constitutive release at a rate sufficient to replace 12% of the plasma membrane each minute (7).
Accordingly, the purposes of these studies were to assess whether cholangiocytes possess a "volume-sensitive" population of vesicles and to determine whether volume-sensitive vesicular exocytosis and ATP release are functionally related. These experiments document that increases in cholangiocyte cell volume stimulate parallel changes in both vesicular exocytosis and ATP release through a PKC- and PI3-kinase-dependent mechanism. The findings are consistent with a model in which increases in cell volume activate PKC to prime a pool of vesicles for exocytosis that are under subsequent regulatory control by PI3-kinase. Inhibition of vesicular exocytosis at either the PKC- or the PI3-kinase-dependent regulatory step dramatically inhibits volume-sensitive ATP release, suggesting that the two processes are functionally related. These studies provide support for the concept that volume-sensitive ATP release in biliary epithelia occurs through vesicular exocytosis.
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EXPERIMENTAL METHODS |
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Measurement of membrane turnover. The fluorescent probe FM143 (Molecular Probes) was used to measure the rate of exocytosis in isolated cells (13). FM143 is a useful marker for measurement of membrane trafficking because it is weakly fluorescent in aqueous solutions, but its fluorescence intensity increases >300-fold when it partitions into plasma membranes. In addition, FM143 does not cross lipid bilayers, and consequently, measurement of total cellular fluorescence provides a real-time measure of exocytosis. As vesicular membranes fuse with the plasma membrane, they incorporate previously nonfluorescent FM143 in the extracellular media and increase cellular fluorescence (20). Cells were plated on coverslips in imaging chambers and bathed with a standard extracellular solution containing (in mM) 142 NaCl, 4 KCl, 1 KH2PO4, 2 MgCl2, 2 CaCl2, and 10 HEPES/NaOH (pH 7.25). The measured osmolarity of the extracellular solution was between 295 and 305 mosM. The concentration of FM143 was 4 µM. Cells in the imaging chamber were perfused at a rate allowing for complete exchange of the chamber volume in 1 min. Cells were visualized by using an Olympus IMT2 microscope with a x60 oil immersion objective (numerical aperture = 1.4). FM143 was excited with bandpass filters (peak 480 nm) and collected with an emission filter (peak 535 nm). Images were acquired by using 300-ms exposures at 30-s intervals with a Sensicam QE camera, and protocols were designed, executed, and captured with the SlideBook 3.0 software package (Intelligent Imaging Innovations) (21). Quantitative analysis of fluorescent images was performed by using IgorPro3 software (Wavemetrics). Total cellular FM143 fluorescence was measured for individual cells. Background fluorescence was measured from regions absent of cells and subtracted from the total cellular fluorescence. Quantification of exocytosis was performed after initial equilibration of FM143 into the plasma membrane establishing a 100% baseline. Change in total membrane fluorescence (F) was calculated by measuring the difference in peak fluorescence intensities over time from this 100% baseline. Changes in cell size were measured by using video planimetry on SlideBook 3.0 software. Video planimetry is a method for measuring cell surface area (in pixels) that is proportional to cell volume. Cell surface area was utilized instead of cell volume to facilitate the use of real-time measurements of changes in cell size simultaneously with exocytosis.
Measurement of ATP release. ATP release from cells in culture was measured using a luciferase-luciferin (L/L) assay (Calbiochem) (16). Because the luciferase enzyme is not membrane permeable only, extracellular ATP is available for the reaction and the measured fluorescence is proportional to the amount of ATP released (18). For the assay, cells cultured on 35-mm plates were washed with 2 ml PBS. PBS was removed, and 600 µl of Opti-MEM media plus L/L (2 mg/ml) were added to the chamber. Cells were allowed to equilibrate for 10 min before a basal reading to minimize the effects of mechanical stress. Hypotonic exposures (1040%) were created by adding aliquots of distilled H2O plus L/L (2 mg/ml) to the cell plates. Mechanical controls (no change in osmolality) were obtained by adding equal aliquots of Opti-MEM media plus L/L (2 mg/ml). Subsequent to a hypotonic exposure, measurements were taken every 15 s using a Turner luminometer and reported as arbitrary light units (ALU). The peak ALU value over baseline was measured (reported as ALU) and used for determining statistical differences between study interventions.
Cell treatments and experimental reagents. For pharmacological interventions, cells were either incubated with the reagent in medium at 37°C or exposed acutely to the reagent in buffer in the imaging chambers as indicated. To inhibit PI3-kinase activity, cholangiocytes were incubated for 15 min with LY-294002 (100 µM) (22). Inhibition of PKA was accomplished by a 60-min incubation in the cell-permeable triethylammonium salt, Rp-cAMPS (5 µM) (7). To inhibit PKC, cholangiocytes were incubated for 15 min in either chelerythrine chloride (50 µM) or calphostin C (1 µM). Activation of PKC was performed by using an acute exposure to PMA (1 µM) or a 5-min preincubation (1 µM) as described (12). All pharmacological reagents were purchased from either Sigma (St. Louis, MO) or Calbiochem, (San Diego, CA). FM143 was obtained from Molecular Probes (Eugene, OR). Experiments were performed with paired same-day controls at 24°C.
Statistics. Results are expressed as the means ± SE with n representing the number of cells in fluorescence studies and the number of culture plates in luminometry studies. Student's paired and unpaired t-tests were used to determine statistical significance as indicated; P < 0.05 used.
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RESULTS |
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Identification of a volume-sensitive pool of exocytic vesicles. To assess whether increases in cell volume are also associated with an exocytic response, plasma membrane fluorescence was measured by using FM143 (18, 20). After diffusion of FM143 into the extracellular solution, there is a rapid saturation of the plasma membrane with dye. A representative cell is illustrated in Fig. 2B1. If the plasma membrane were static, then fluorescence intensity would remain stable at 100% of initial values. However, cholangiocytes maintained in isotonic solution (0%) exhibited a steady increase in fluorescence at a rate of 1.6 ± 0.01%/min (Fig. 2A, n = 22 cells). This rate of dye accumulation represents constitutive insertion of new plasma membrane from exocytic vesicles (22). The imaging characteristics of a cell exposed to a 30% hypotonic solution are shown in Fig. 2B2. Exposure to hypotonic solution resulted in a rapid increase in cell size (as measured by video planimetry) and fluorescence (Fig. 2A). Mean (F) after exposure to a variety of hypotonic solutions is depicted in Fig. 2C. Exposure to a 30% hypotonic solution increased
F to 15.6 ± 0.9% (n = 42, P < 0.001). Maximal values were obtained after a 40% hypotonic exposure resulting in a mean
F of 32.0 ± 7.5% (n = 5, P < 0.006). Note that the exocytic response was proportional to the decrease in osmolality. Thus cholangiocytes exhibit a constitutive level of membrane turnover, and increases in cholangiocyte cell volume result in rapid increases in exocytosis sufficient to replace 1530% of plasma membrane area within 1 min. These results indicate that increases in cholangiocyte cell volume stimulate vesicular exocytosis.
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Increases in cholangiocyte cell volume precede increases in plasma membrane fluorescence. To evaluate the temporal relationship between changes in cell volume and the exocytic response, cell size was estimated by video planimetry and compared with plasma membrane fluorescence. To measure cell size, cell surface area was estimated in pixels from the two-dimensional images taken during experiments before and after exposure to hypotonic challenge. Fluorescence was evaluated simultaneously in the same cell. A representative study of a single cell (Fig. 3A), demonstrates that after hypotonic exposure, increases in cell size precede increases in plasma membrane fluorescence. Moreover, partial recovery of cell volume (regulatory volume decrease) occurs at a time when fluorescence is still increasing. Similar results were obtained in six cells. Although video planimetry measures surface area and not cell volume directly, the results are consistent with electronic cell sizing using a Coulter counter (23). Figure 3B is a summary plot of four cells illustrating that the magnitude of change in exocytosis (relative fluorescence) is proportional to changes in cell size (relative size). The larger decreases in osmolarity result in greater increases in cell size and a more robust exocytic response. The temporal dissociation between FM143 fluorescence and increases in cell size is consistent with the requirement for activation of intracellular signaling cascades necessary to translate changes in cell volume into an exocytic response. The delay between increases in cell size and increases in fluorescence also suggests that changes in fluorescence are not solely the result of membrane tension, but rather are due to exocytosis and the addition of new plasma membrane. A similar pattern has been identified in liver cells in which cell swelling precedes volume-dependent opening of chloride channels by several minutes (12).
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cAMP-independent regulation of volume-sensitive exocytosis. Previous studies (7) have identified a cAMP-regulated pool of vesicles in biliary cells. To evaluate whether the volume-sensitive pool of vesicles in cholangiocytes is regulated by cAMP, the effects of a cell-permeable inhibitor of PKA, Rp-cAMPS (5 µM), on FM143 signaling was assessed. A representative experiment is shown in Fig. 4A, and quantitative measures of F are summarized in Fig. 4B. Mz-ChA-1 cholangiocytes incubated for 60 min with Rp-cAMPS and subsequently exposed to a 30% hypotonic solution exhibited a mean
F of 11.9 ± 0.3% (n = 7). These values were similar to control studies performed without Rp-cAMPS that yielded a mean
F of 12.7 ± 1.6% (n = 6, P > 0.5). The failure of PKA inhibition to effect volume-stimulated vesicular exocytosis in cholangiocytes is consistent with a cAMP-independent regulatory pathway.
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Regulatory role of PI3-kinase. PI3-kinase is activated within minutes by increases in cholangiocyte cell volume, and inhibition of PI3-kinase by LY-294002 impairs cell volume recovery from swelling (18). To assess whether PI3-kinase modulates volume-stimulated exocytosis, the effect of PI3-kinase inhibition on FM143 fluorescence was evaluated as shown in Fig. 5, A and B. The fluorescence response to a 30% hypotonic challenge decreased from 16.7 ± 1.7% in control cells to 8.9 ± 1.2% (n = 8, P < 0.02) in cells incubated with LY-294002 (100 µM). Previous studies (18) in normal rat cholangiocytes have demonstrated that LY-294002 also inhibits volume-sensitive ATP release. Taken together, the fact that PI3-kinase is involved in both volume-stimulated exocytosis, and volume-sensitive ATP release suggests that ATP release may be coupled to exocytosis.
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PKC regulation of volume-stimulated exocytosis. To assess whether ATP release and exocytosis are PKC regulated, cells were incubated with the broad-spectrum PKC inhibitor chelerythrine. PKC was selected as a candidate regulatory kinase, because the -isoform translocates to the plasma membrane within minutes of an increase in cholangiocyte cell volume, and inhibition of PKC by chelerythrine prevents both activation of volume-activated chloride channels and regulatory volume decrease (12). Incubation with chelerythrine (50 µM) was found to inhibit volume-sensitive increases in both ATP release and FM 143 fluorescence (Fig. 6, A and B). Chelerythrine-treated cells exhibited a mean
F of 7.4 ± 1.4% (n = 15) and a mean ALU of 145.8 ± 86.2 (n = 7). Control cells displayed a mean
F of 14.6 ± 1.3% (n = 14, P < 0.001) and a mean ALU of 3072.5 ± 791.6 (n = 6, P < 0.03). Similar results were observed for cells incubated with calphostin C (1 µM), an unrelated PKC inhibitor. Calphostin C decreased ATP release from 1,454.7 ± 540.0 to 192.9 ± 96.5 ALU (n = 4, P < 0.04). Results for chelerythrine and calphostin C-treated cholangiocytes are summarized in Fig. 6C, and suggest that PKC activation exerts potent regulatory control over both exocytosis and ATP release. These findings provide additional evidence that vesicular exocytosis and ATP release are functionally linked.
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To further assess the role of PKC, the cellular response to acute PKC activation was assessed by exposing cholangiocytes to PMA (1 µM). If activation of typical PKC isoforms alone is sufficient to stimulate exocytosis, then acute exposure to PMA would be anticipated to increase both F and ATP release in the absence of a volume stimulus. However, acute exposure to PMA alone had no effect on either plasma membrane fluorescence or ATP release. In contrast, prior exposure to PMA (1 µM for 5 min) potentiated the response to a subsequent hypotonic exposure (Fig. 7). After incubation with PMA, a 30% decrease in osmolality resulted in a mean
F of 35.1 ± 3.9% (n = 6) and a mean ALU of 3,140.6 ± 686.1 (n = 13). Control cells exposed to a 30% hypotonic solution without PMA resulted in a mean
F of 17.5 ± 0.4% (n = 3, P < 0.01) and a mean ALU of 1,697.8 ± 398.4 (n = 16, P < 0.008). These findings indicate that PKC potentiates the cellular response to increases in cell volume but alone is not sufficient to stimulate exocytosis and ATP release.
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DISCUSSION |
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Despite the emerging importance of purinergic signaling in cholangiocytes and other epithelia, the cellular mechanisms involved in ATP release are not well defined. In liver and biliary cells, volume recovery involves the local release of extracellular ATP, autocrine stimulation of P2 purinergic receptors, and subsequent activation of K+ and Cl- channels in the plasma membrane (18, 25). Activation of this signaling cascade through cell volume increases or exposure to exogenous ATP elicits robust Cl- secretory responses (4), whereas elimination of extracellular ATP or inhibition of P2 purinergic receptors impairs cell volume recovery from swelling.
Patch-clamp studies (1, 26, 27, 29) and atomic force microscopy provide support for a conductive, channel-mediated pathway for ATP release. Alternatively, in chromaffin, pancreatic acinar, and endothelial cells, ATP functions as a neurotransmitter, and vesicular pools containing high concentrations of ATP have been identified (3, 11, 28). Stimulation of exocytosis in these cells results in the quantal release of ATP. These findings are of interest in cholangiocytes, because vesicular exocytosis is emerging as an important site of regulation for the secretory response. Specifically, cholangiocytes possess a dense population of subapical vesicles averaging 140 nm in diameter that undergo a high rate of constitutive exocytosis sufficient to replace 1.33 ± 0.16% of the plasma membrane per minute (7). Evidence in normal rat cholangiocyte monolayers has demonstrated polarity in constitutive ATP release, with apical concentrations of ATP (250 nM) being fivefold higher than basolateral concentrations of ATP (
50 nM) (24). These findings are consistent with the role of cholangiocytes as secretory cells in which polarization of the membrane allows for apically directed secretion into the bile duct lumen. Furthermore, increases in cAMP increase the rate of exocytosis
70% and lead to translocation of aquaporins and other secretory proteins into the plasma membrane (7, 9). However, cAMP does not appear to be directly involved in the control of cell volume or ATP release, implying that other regulatory pathways including both PI3-kinase and PKC are involved (4). Intracellular dialysis with the lipid products of PI3-kinase or with recombinant PKC is capable of activating Cl- channels in the absence of a volume stimulus (12, 18), whereas inhibition of PI3-kinase impairs volume-sensitive ATP release and current activation, providing evidence that these processes are functionally linked (18).
Collectively, these findings suggest that exocytosis plays a role in volume-sensitive ATP release and chloride secretion. To evaluate this more directly, FM143 fluorescence was used to monitor the rate of exocytosis in response to increases in cell volume. Hypotonic exposure resulted in rapid mobilization of a vesicular pool sufficient in size to replace 1540% of the plasma membrane within minutes. Assuming that a vesicle is spherical in shape, then the membrane surface area of a 140-nm vesicle is 0.06 µm2. From capacitance measurements (assuming that the specific capacitance of biological membranes is 1 fF/µm2), the surface area of an Mz-ChA-1 cell is 3,650 µm2 (7). Therefore, a 1540% increase in plasma membrane fluorescence corresponds to the recruitment of
9,00024,000 vesicles per cell. This response is unaffected by inhibition of cAMP and has a distinct kinetic pattern from cAMP-regulated exocytosis. In contrast to the rapid and nonsustained exocytic burst of the volume-sensitive vesicular pool, cAMP-regulated exocytosis is defined by a slower but more sustained increase in vesicular exocytosis (7). Furthermore, inhibition of volume-sensitive vesicular exocytosis prevents ATP release, suggesting that purinergic signaling and cholangiocyte secretion are regulated, in part, by controlling vesicular insertion into the plasma membrane.
Several observations suggest that volume-sensitive vesicles are recruited, in part, by PKC. First, previous studies of Mz-ChA-1 cells indicate that cell volume regulation is mediated by conventional PKC isoforms and that PKC
is the predominant PKC isoform present. Second, increases in cell volume result in rapid translocation of PKC
to the plasma membrane in a time course similar to the increase in FM143 fluorescence and ATP release (12). Third, inhibition of PKC signaling by chelerythrine or calphostin C significantly attenuates cellular responses to volume increase, whereas activation of PKC by exposure to PMA does not affect constitutive membrane turnover, ATP release, or volume-sensitive exocytosis. However, activation of PKC significantly potentiates the cellular response to subsequent hypotonic challenge without affecting the rate of constitutive membrane turnover. These findings are consistent with a role for PKC in priming vesicles for subsequent release. Similar observations have been made in bovine adrenal chromaffin cells in which PMA results in enhanced vesicular exocytosis (9), and in intestinal epithelial cells in which PKC regulates the insertion of vesicles containing GLUT transporters (32).
Assuming that these observations are relevant to cholangiocytes in vivo, several points regarding the interpretation of these data are worth noting. Inhibition of PI3-kinase results in near total inhibition of ATP release, but only 50% of volume-stimulated exocytosis. We are uncertain as to the mechanisms behind this discrepancy but anticipate both technical and biological explanations. As there is no independent measure of the degree of PI3-kinase inhibition in these studies, it is possible that complete enzymatic inhibition did not occur. Indeed, this could account for the variability that is observed in the exocytic responses to volume-stimulus that ranged from near complete inhibition in some individual cells to only partial inhibition in others. The mean change in fluorescence (
F) and same day controls is presented to minimize these considerations.
An alternative and perhaps biologically more attractive explanation is that cell volume increases may result in exocytosis of several distinct populations of vesicles to cope with the changing osmotic environment. These alternate vesicular populations likely contain different cargo, with only a portion of these vesicles containing ATP. Increases in liver cell volume activate multiple signaling pathways in parallel including PI3-kinase, tyrosine kinase, and mitogen-activated protein kinases (10, 32, 34). Therefore, it is possible that the mobilization of a volume-sensitive pool of vesicles involves several kinases targeting various steps in the exocytic pathway. It is also plausible that volume increases trigger the mobilization of these various populations by alternate regulatory paradigms and that PI3-kinase may have a more limited role in exocytosis of other vesicular populations. However, previous work has demonstrated a pivotal role for PI3-kinase in volume-stimulated ATP release and regulation of cell volume (8). Despite the lack of understanding in the defined sequence of events from volume stimulus to cholangiocyte exocytosis, these studies indicate the presence of a cAMP-independent, PKC-sensitive, PI3-kinase-regulated population of vesicles. The implication is that the cholangiocyte secretory response to cell volume increase is controlled through targeting specific vesicular pools to shape membrane transport.
A second limitation results from the fact that the molecular targets of PKC and PI3-kinase in volume-sensitive signaling have not been identified. Therefore, the specific steps in the exocytic pathway in which these kinases act remain speculative. Several observations provide insight into the potential role for PKC in volume-sensitive exocytosis. First, as previously stated, PKC
is the predominant Ca2+ and phorbol-sensitive PKC isoform present in biliary cells (19). Second, inhibition of PKC
before a hypotonic exposure prevents the activation of IClswell, but inhibition of PKC
subsequent to a hypotonic exposure fails to inhibit IClswell (12). Third, PMA in the absence of a hypotonic stimulus has no direct effect on exocytosis or ATP release, but preincubation of cells with PMA potentiates the response to subsequent volume challenge. The observation that acute PMA exposure alone does not result in either exocytosis or ATP release suggests that volume-sensitive signals other than PKC activation are required. The kinetic pattern of the response is consistent with the concept that PKC
serves to prime a pool of vesicles involved in ATP release, converting them from a nonreleasable to a ready-releasable state. In this fashion, PKC acts to target vesicles for incorporation into the volume-sensitive vesicular pool. This meets the functional definition of vesicle docking and priming observed in epithelial cells, but to date, there is no published morphological evidence available in cholangiocytes to directly support this conclusion.
Finally, the nature of the relationship among vesicular exocytosis, ATP release, and cholangiocyte secretion remains to be defined. From these studies, it is clear that exocytosis and ATP release share parallel modes of regulation, indicating that exocytosis is necessary for ATP release. However, the content of volume-sensitive vesicles is unknown. In regard to ATP release, similar results would be obtained regardless of whether the vesicles contained high (mM) concentrations of ATP, as is observed in chromaffin cells, or whether the vesicles contained ATP-permeable channels consistent with a conductive-mediated pathway (11, 15, 26, 27, 30). Clarification of the relationship between vesicular exocytosis and ATP release requires molecular identification of the ATP channels and transporters involved.
In conclusion, these experiments provide further support for regulated vesicular exocytosis as a mechanism for modulating cholangiocyte secretion by producing rapid and dynamic changes in the transport properties of the plasma membrane. The findings are consistent with a model in which volume-sensitive activation of PKC leads to the rapid recruitment of a sizable pool of 10,000 or more vesicles per cell. Moreover, it is clear that PI3-kinase activation plays a key role in regulating subsequent vesicular release. Taken together, these regulatory proteins play a critical role in modulating volume-sensitive changes in cellular ATP release. It is attractive to speculate that cholangiocytes possess multiple vesicular populations that can be mobilized in response to specific physiologic signals, permitting versatility and selectivity in shaping transport properties to meet rapidly changing physiological demands. The identification of these multiple vesicular pools may provide insight into the mechanisms underlying cholangiocyte secretion and bile formation.
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ACKNOWLEDGMENTS |
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GRANTS
This research was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-43278 and DK-46082 (to J. G. Fitz). G. Kilic is a recipient of the American Liycr Scholar Award.
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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REFERENCES |
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