Prairie Dog Gallbladder Research Articles

Electrogenic bicarbonate secretion by prairie dog gallbladder

Pathological rates of gallbladder salt and water transport may promote the formation of cholesterol gallstones. Because prairie dogs are widely used as a model of this event, we characterized gallbladder ion transport in animals fed control chow by using electrophysiology, ion substitution, pharmacology, isotopic fluxes, impedance analysis, and molecular biology. In contrast to the electroneutral properties of rabbit and Necturus gallbladders, prairie dog gallbladders generated significant short-circuit current (I(sc); 171 +/- 21 microA/cm(2)) and lumen-negative potential difference (-10.1 +/- 1.2 mV) under basal conditions. Unidirectional radioisotopic fluxes demonstrated electroneutral NaCl absorption, whereas the residual net ion flux corresponded to I(sc). In response to 2 microM forskolin, I(sc) exceeded 270 microA/cm(2), and impedance estimates of the apical membrane resistance decreased from 200 Omega.cm(2) to 13 Omega.cm(2). The forskolin-induced I(sc) was dependent on extracellular HCO(3)(-) and was blocked by serosal 4,4'-dinitrostilben-2,2'-disulfonic acid (DNDS) and acetazolamide, whereas serosal bumetanide and Cl(-) ion substitution had little effect. Serosal trans-6-cyano-4-(N-ethylsulfonyl-N-methylamino)-3-hydroxy-2,2-dimethyl-chroman and Ba(2+) reduced I(sc), consistent with the inhibition of cAMP-dependent K(+) channels. Immunoprecipitation and confocal microscopy localized cystic fibrosis transmembrane conductance regulator protein (CFTR) to the apical membrane and subapical vesicles. Consistent with serosal DNDS sensitivity, pancreatic sodium-bicarbonate cotransporter protein pNBC1 expression was localized to the basolateral membrane. We conclude that prairie dog gallbladders secrete bicarbonate through cAMP-dependent apical CFTR anion channels. Basolateral HCO(3)(-) entry is mediated by DNDS-sensitive pNBC1, and the driving force for apical anion secretion is provided by K(+) channel activation.

Functional characterization of Na(+)/H(+) exchangers in primary cultures of prairie dog gallbladder.

Gallbladder Na(+) absorption is linked to gallstone formation in prairie dogs. We previously reported Na(+)/H(+) exchanger (NHE1-3) expression in native gallbladder tissues. Here we report the functional characterization of NHE1, NHE2 and NHE3 in primary cultures of prairie dog gallbladder epithelial cells (GBECs). Immunohistochemical studies showed that GBECs grown to confluency are homogeneous epithelial cells of gastrointestinal origin. Electron microscopic analysis of GBECs demonstrated that the cells form polarized monolayers characterized by tight junctions and apical microvilli. GBECs grown on Snapwells exhibited polarity and developed transepithelial short-circuit current, I(sc), (11.6 +/- 0.5 microA. cm(-2)), potential differences, V(t) (2.1 +/- 0.2 mV), and resistance, R(t) (169 +/- 12 omega. cm(2)). NHE activity in GBECs assessed by measuring dimethylamiloride-inhibitable (22)Na(+) uptake under a H(+) gradient was the same whether grown on permeable Snapwells or plastic wells. The basal rate of (22)Na(+) uptake was 21.4 +/- 1.3 nmol x mg prot(-1) x min(-1), of which 9.5 +/- 0.7 (approximately 45%) was mediated through apically-restricted NHE. Selective inhibition with HOE-694 revealed that NHE1, NHE2 and NHE3 accounted for approximately 6%, approximately 66% and approximately 28% of GBECs' total NHE activity, respectively. GBECs exhibited saturable NHE kinetics ( V(max) 9.2 +/- 0.3 nmol x mg prot(-1) x min(-1); K(m) 11.4 +/- 1.4 m M Na(+)). Expression of NHE1, NHE2 and NHE3 mRNAs was confirmed by RT-PCR analysis. These results demonstrate that the primary cultures of GBECs exhibit Na(+) transport characteristics similar to native gallbladder tissues, suggesting that these cells can be used as a tool for studying the mechanisms of gallbladder ion transport both under physiologic conditions and during gallstone formation.

Characterization of NA+/H+ exchanger isoform (NHE1, NH32 and NHE3) expression in prairie dog gallbladder.

Gallbladder Na+ absorption is linked to gallstone formation in prairie dogs. Na+/H+ exchange (NHE) is one of the major Na+ absorptive pathways in gallbladder. In this study, we measured gallbladder Na+/H+ exchange and characterized the NHE isoforms expressed in prairie dogs. Na+/H+ exchange activity was assessed by measuring amiloride-inhibitable transepithelial Na+ flux and apical 22Na+ uptake using dimethylamiloride (DMA). HOE-694 was used to determine NHE2 and NHE3 contributions. Basal JNams was higher than JNasm with JNanet absorption. Mucosal DMA inhibited transepithelial Na+ flux in a dose-dependent fashion, causing JNams equal to JNasm and blocking JNanet absorption at 100 microm. Basal 22Na+ uptake rate was 10.9 +/- 1.0 micromol. cm-2. hr-1 which was inhibited by approximately 43% by mucosal DMA and approximately 30% by mucosal HOE-694 at 100 microm. RT-PCR and Northern blot analysis demonstrated expression of mRNAs encoding NHE1, NHE2 and NHE3 in the gallbladder. Expression of NHE1, NHE2 and NHE3 polypeptides was confirmed using isoform-specific anti-NHE antibodies. These data suggest that Na+/H+ exchange accounts for a substantial fraction of gallbladder apical Na+ entry and most of net Na+ absorption in prairie dogs. The NHE2 and NHE3 isoforms, but not NHE1, are involved in gallbladder apical Na+ uptake and transepithelial Na+ absorption.

Functional expression of NA +/H + exchange in primary cultures of prairie dog gallbladder epithelium

Functional expression of NA +/H + exchange in primary cultures of prairie dog gallbladder epithelium

Relative contributions of NHE-2 and NHE-3 to apical NA +/H + exchange in prairie dog gallbladder epithelium

Relative contributions of NHE-2 and NHE-3 to apical NA +/H + exchange in prairie dog gallbladder epithelium

Endogenous prostaglandins modulate chloride secretion by prairie dog gallbladder

In addition to concentrating bile, the gallbladder secretes chloride (Cl -) and mucus into its lumen. We recently observed that gallbladder Cl - secretion is increased in prairie dogs during the formation of cholesterol crystals, a period of altered mucosal prostaglandin synthesis. Pathologic Cl - secretion is characteristic of other epithelial disorders such as cystic fibrosis and hypercalciuric nephrolithiasis and may be important in gallstone pathogenesis. We hypothesized that concentrations of endogenous prostaglandin E 2 (PGE 2) found during experimental gallstone formation may mediate increased Cl - secretion by prairie dog gallbladder. Prairie dog gallbladders were harvested by cholecystectomy and mounted in Ussing chambers. Unidirectional transepithelial Cl -, Na +, and H 2O fluxes were measured before and after inhibition of endogenous prostaglandin synthesis with 10 μmol/L indomethacin. Gallbladders were then exposed to increasing concentrations of PGE 2 to a maximal dose of 1 μmol/L, as found in animals with gallstones. Standard electrophysiologic parameters were recorded simultaneously. Indomethacin increased mucosal resistance and stimulated gallbladder Na + and Cl - absorption. These effects were rapidly reversed by PGE 2. PGE 2 promoted Cl - secretion and decreased mucosal Na + absorption at concentrations found in the gallbladder bile of animals with gallstones. Endogenous prostaglandin metabolism modulates gallbladder Cl - secretion and may promote changes in Cl - transport associated with cholelithiasis. (J Lab Clin Med 2000;135:82-88)

Octreotide stimulates Ca++ secretion by the gallbladder: A risk factor for gallstones

Background: Gallstone formation during octreotide therapy has been linked to elevated levels of gallbladder bile Ca ++, a well-known prolithogenic factor. Although the subcutaneous administration of octreotide raises gallbladder bile Ca ++ in prairie dogs, the mechanism for this effect is unknown. Octreotide has been shown to increase gallbladder Na + and water absorption in Ussing chamber studies. Given the known effects of octreotide on gallbladder ion transport, we hypothesized that octreotide may also promote gallstone formation by stimulating gallbladder Ca ++ secretion, thereby raising the lumenal concentration of biliary Ca ++. Methods: After cholecystectomy, prairie dog gallbladders were mounted in Ussing chambers, and standard electrophysiologic parameters were recorded. Unidirectional fluxes of Ca ++ and Na + were measured before and after serosal exposure to 50 nmol/L octreotide. Results: Under basal conditions normal prairie dog gallbladder absorbed mucosal Ca ++. Serosal octreotide converted the gallbladder from a state of basal Ca ++ absorption to one of net Ca ++ secretion by stimulating serosa to mucosa Ca ++ flux. As anticipated, octreotide increased net Na + absorption by stimulating mucosa to serosa Na + flux and decreased tissue conductance and short-circuit current significantly compared with baseline values. Conclusion: Fifty nanomoles per liter octreotide stimulated Ca ++ secretion by gallbladder epithelium, a possible mechanism for increased biliary Ca ++ in prairie dogs receiving subcutaneous injections. Ca ++ secretion linked to octreotide therapy may induce gallstones by raising biliary levels of Ca ++, a known prolithogenic factor. (Surgery 1999;125:509-13.)

Establishment and Characterization of Primary Gallbladder Epithelial Cell Cultures in the Prairie Dog

Background.The prairie dog has become the established animal gallstone model. This species has a unique propensity to form cholesterol gallstones in response to dietary manipulations. The development of a reliable gallbladder cell culture technique is critical for understanding pathogenic mechanisms of gallstone formation.Materials and methods.Prairie dogs underwent laparotomy and cholecystectomy, followed by initiation of cell cultures. [3H]Thymidine incorporation was used to assess cell growth, and cell lines were assessed using routine histochemical and immunohistochemical staining.Results.Cell yields from prairie dog gallbladders were 4–8 × 106viable cells per animal with viability ranging from 80 to 95%. When plated at 5 × 105cells/cm2, cell clusters, visible within 24 h, coalesced into confluent monolayers within 3–5 days. Cultures remained viable for 6–8 weeks and could be passed for three to four subcultures. Immunohistochemical staining demonstrated a high degree of epithelial purity with immunopositivity for AE1/AE3, and cytokeratin, with no vimentin positivity (mesenchymal antigen). Intracytoplasmic vacuoles demonstrated positive staining for Alcian blue, periodic acid–Schiff, and mucicarmine and an anti-gallbladder mucin antibody confirmed the presence of the glycoprotein mucin.Conclusions.This study demonstrates a reliable method for initiation and maintenance of prairie dog gallbladder epithelial cell cultures with a high degree of purity. This technique should allow further studies into the pathogenesis of cholesterol gallstones in this model.

Chloride channel CLC-3 is expressed by gallbladder epithelium: A potential regulator of Cl− secretion

Gallbladder epithelium exhibits uncoupled Na + absorption and CIsecretion in prairie dogs. Prominent gallbladder C1permeability suggests the presence of an unidentified apical CIchannel. In renal tubules, disorders of epithelial transport function such as Dent's disease have been found to originate in altered C1channel function. We have recently found that gallbladder C1secretion is increased prior to cholesterol-induced gallstone formation. The mechanism for gallbladder CIsecretion, however, is not completely understood. Recent molecular cloning experiments have identified two distinct families of chloride channels: the CIC and CLC channels. The CLC family includes several molecular isoforms, CLC0-7, with tissue-specific distribution and function. CLC-2,-3 and -4 are expressed by several tissues, whereas CLC-KI and CLC-5 are highly kidney-specific. We hypothesized that CLC-3 is expressed by gallbladder epithelium and may regulate gallbladder C1transport with important implications in gallstone formation. Total RNA was extracted from both prairie dog and human gallbladder epithelia, reverse transcribed, and amplified by PCR using CLC-3 specific primers. Molecular cloning and sequence analysis demonstrated the predicted 508 bp eDNA with 94% nucleotide homology between prairie dog and human CLC-3 cDNAs. The deduced 168 amino acid sequences were 99% identical. Subsequent RT-PCR analysis confirmed the expression of CLC-3 by the human gallbladder following gallstone formation. This is the first report that CLC-3 is expressed by human gallbladder during gallstone formation. Alterations in CLC-3 activity may alter epithelial CIpermeability and promote gallstones.

Different pathways mediate cholecystokinin actions in cholelithiasis.

Smooth muscle from gallbladders with cholesterol stones exhibits impaired response to cholecystokinin (CCK). This study investigated whether the impaired response is mediated by different signal-transduction pathways responsible for CCK-induced contraction in prairie dog and human gallbladders with cholesterol stones. Gallbladder muscle cells were isolated enzymatically to study contraction. Protein kinase C (PKC) activity was measured by examining the phosphorylation of a specific substrate peptide from myelin basic protein Ac-MBP-(4-14). Gallbladder muscle cells from high-cholesterol-fed prairie dogs contracted less in response to CCK octapeptide (CCK-8) than those from the control group. However, inositol-1,4,5-trisphosphate (IP3), diacylglycerol, and guanosine 5'-O-(3-thiotriphosphate) induced the same magnitudes of contraction in these two groups. In control prairie dog and human gallbladders, the maximal contraction caused by 10(-8) M CCK-8 was blocked by the calmodulin antagonist CGS9343B but not by the PKC inhibitor H-7. Conversely, in gallbladders with cholesterol stones from prairie dogs or human patients, the maximal contraction induced by 10(-8) M CCK-8 was blocked by H-7 and chelerythrine but not by CGS9343B. In these gallbladders CCK-8 caused a significant PKC translocation from the cytosol to the membrane. High CCK concentrations may activate the calmodulin-dependent pathway in functionally normal gallbladder muscle and the PKC-dependent pathway in muscle from gallbladders with cholesterol stones. The defect of gallbladder muscle after cholesterol feeding and stones might reside in the steps before G protein activation.

Nitric oxide and gallbladder motility in prairie dogs.

In this study we evaluated the role of nitric oxide (NO) on gallbladder motility in the normal prairie dog by 1) immunohistochemistry, 2) an enzymatic assay for NO synthase (NOS), and 3) an in vivo model to measure whole gallbladder tone and contractility. NOS was localized to gallbladder mucosal cells by NADPH-diaphorase and polyclonal antibodies to a constitutive brain NOS. Gallbladder mucosal homogenates demonstrated total NOS activity in the range of 578 +/- 115 pmol x mg protein(-1) x 30 min(-1). Blockade of NOS activity in vivo using N(omega)-nitro-L-arginine methyl ester resulted in an up to 80% increase in gallbladder tone from basal. A 40% increase in tone was seen with methylene blue, suggesting that tone was maintained by both NO activation of guanylate cyclase and possibly direct effects on Ca2+ channels. An exogenous nitrosothiol, S-nitroso-N-acetyl-cysteine, abolished cholecystokinin (CCK) octapeptide and bethanechol-stimulated gallbladder contraction. We conclude that the prairie dog gallbladder contains constitutive NOS and synthesizes NO, which is important for the maintenance of basal gallbladder tone and is an inhibitor of the contractile response of the gallbladder to agonists such as CCK and bethanechol.

Apical and Basolateral Ca2+Channels Modulate Cytosolic Ca2+in Gallbladder Epithelia

Gallstone formation is associated with altered gallbladder (GB) ion transport and increased concentration of GB bile Ca2+. Recent studies show that increased cytosolic Ca2+([Ca2+]i) stimulates GB Cl−secretion. However, the mechanism by which extracellular Ca2+([Ca2+]e) enters the cytosol remains unclear. We tested the hypothesis that entry of [Ca2+]einto cytosol occurs via apical and basolateral membrane Ca2+channels. Prairie dog GBs were mounted in Ussing chambers, standard electrophysiologic parameters were recorded, and unidirectional Cl−fluxes (J, μEq·cm−2·hr−1) were measured using36Cl at various mucosal Ca2+in the absence or presence of mucosal lanthanum (La3+), a non-diffusible Ca2+channel blocker. Serosal [Ca2+]ewas maintained at trace levels. In the absence of mucosal La3+, short circuit current (Isc) showed a positive correlation with mucosal [Ca2+]eas represented by a second order polynomial equation (y= 4.1 + 2.5x− 0.73x2,r= 0.68,P< 0.001). In contrast, unidirectional mucosa to serosa Cl−flux (JClms) was inversely correlated with [Ca2+]e(y= 47.9 − 8.7x+ 0.9x2,r= 0.51,P< .05) Addition of 1 mMmucosal La3+blunted the effects of [Ca2+]eon electrophysiologic parameters and JClms. However, basolateral repletion with 5 mMCa2+reverses the blocking effects of La3+on JClms. These data suggest that [Ca2+]eenters the cytosol via apical and basolateral Ca2+channels. We conclude that GB apical Ca2+channels may represent a pathway for biliary Ca2+entry into the cell and therefore may represent an important regulatory pathway for GB ion transport during gallstone formation.

Octreotide promotes gallbladder absorption in prairie dogs: A potential cause of gallstones

Background/Aims Gallstone formation during octreotide administration has been causally linked to increased biliary concentrations of calcium, protein, and total lipids, all purported prolithogenic factors. These changes may be caused by octreotide-induced gallbladder stasis or a direct effect of octreotide on gallbladder absorption. We tested the hypothesis that octreotide stimulates gallbladder ion and water transport. Methods Prairie dog gallbladders were mounted in Ussing chambers and bathed in oxygenated Ringer's solution. Electrophysiological parameters were recorded, and unidirectional Na +, Cl −, and H 2O fluxes were measured before and after serosal exposure to 50 nmol/L octreotide. Results Octreotide exposure caused a significant decrease in transepithelial short-circuit current and potential difference and an increase in tissue resistance compared with baseline. These alterations in electrophysiological parameters coincided with changes in ion transport. Octreotide stimulated net Na + and H 2O absorption and converted the gallbladder from a state of Cl − secretion to one of Cl − absorption by increasing mucosal to serosal fluxes. Octreotide effects on ion transport were blocked by 4,4′-diisothiocynostilbene2,2′-disulfonic acid and amiloride and reversed by theophylline. Conclusions Octreotide may promote gallstone formation by inducing gallbladder stasis and by directly increasing gallbladder absorption, which may act synergistically to increase the concentration of prolithogenic factors in bile and to facilitate nucleation and stone growth.

Contractile activity and levels of cGMP and cAMP of prairie dog gallbladder and ileal muscle in response to S-nitroso-N-acetylpenicillamine (SNAP)

Contractile activity and levels of cGMP and cAMP of prairie dog gallbladder and ileal muscle in response to S-nitroso-N-acetylpenicillamine (SNAP)

Nitric oxide determines basal tone in the fasting prairie dog gallbladder: H.A. Salomons, R. Henihan, W. LaMorte, G.D. Offner, N.H. Afdhal, Sections of Gastroenterology and Surgery, Boston City Hospital and Boston University, Boston, MA

Nitric oxide determines basal tone in the fasting prairie dog gallbladder: H.A. Salomons, R. Henihan, W. LaMorte, G.D. Offner, N.H. Afdhal, Sections of Gastroenterology and Surgery, Boston City Hospital and Boston University, Boston, MA

Establishment and characterization of a prairie dog gallbladder epithelial cell culture: J.M. Fisk, W.C. Chapman, R. Bluth, D. Pierce, L.F. Williams. Depts. of Surgery & Pathology, Vanderbilt University, Nashville, TN

Establishment and characterization of a prairie dog gallbladder epithelial cell culture: J.M. Fisk, W.C. Chapman, R. Bluth, D. Pierce, L.F. Williams. Depts. of Surgery & Pathology, Vanderbilt University, Nashville, TN

Protein kinase C regulates prairie dog gallbladder ion transport.

Gallstone formation is characterized by increased biliary calcium (Ca2+) level and altered gallbladder absorption. Recent studies suggest that luminal Ca2+ regulates gallbladder ion transport via intracellular calcium ([Ca2+]ic). Ca2+-calmodulin and protein kinase C (PKC) are two major systems through which [Ca2+]ic carries out second-messenger functions in many cell types. We have previously shown that Ca2+-calmodulin regulates basal gallbladder ion transport in prairie dog. The present study tests the hypothesis that PKC is also essential in regulation of gallbladder ion transport in this model. The role of PKC in regulation of gallbladder ion transport was determined by studying the effects of phorbol esters, synthetic analogues of diacylglycerol, which directly activates PKC. Gallbladders were mounted in Ussing chambers, and standard electrophysiologic parameters were recorded after exposing tissues to either 10(-5) mol/L of 4-alpha-phorbol 12,13-didecanoate (PDD), 4-beta-phorbol 12-myristate 13-acetate, 4-beta-phorbol 12,13-dibutyrate (PDB), or 10(-4) mol/L serotonin. Unidirectional Na+, Cl-, and H2O fluxes were measured before and after treatment with only inactive PDD and most active PDB. Mucosal and serosal exposure of tissues to either 4-beta-phorbol 12-myristate 13-acetate or PDB resulted in a decrease in short-circuit current and transepithelial potential difference without any change in tissue resistance. Serotonin induced similar changes in gallbladder electrical properties. PDB caused an inhibition of mucosal to serosal fluxes of Na+, Cl-, and H2O, with a decrease in net Na+ absorption, an increase in net Cl- secretion, and a conversion of net H2O absorption to net H2O secretion. Serosal-to-mucosal fluxes of Na+, Cl-, and H2O did not change. Inactive PDD had no effect on either electrophysiologic parameters or ion and water fluxes. Pretreatment of tissues with PKC antagonist 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine blocked the phorbol ester-induced inhibition of ion transport. PKC regulates gallbladder ion transport in the prairie dog by inhibiting Na+ absorption and stimulating Cl- secretion.

Biliary phospholipases in the prairie dog model for cholesterol cholelithiasis

Lysolecithin has been implicated as a contributing factor in the pathogenesis of cholecystitis and cholesterol cholelithiasis. The phospholipases are key enzymes in the generation of a number of metabolites including lysolecithin, but conflicting reports exist concerning the presence of these enzymes in the biliary tract. In this study, measurement of phosphatidylcholine-specific phospholipase activity by means of the hydrolysis of radiolabeled phosphatidylcholine (100 nmol) by 90 micrograms of homogenate protein during a 60-min incubation demonstrated substantial enzyme activities in gastric fundus and distal ileum (90% and 70% hydrolysis, respectively), whereas activity was virtually undetectable in gallbladder mucosa (0.7% hydrolysis). Additional studies were conducted in prairie dogs fed diets high in cholesterol or with trace amounts of cholesterol using homogenates of gallbladder mucosa, seromuscularis and full-thickness tissue, as well as samples of hepatic and gallbladder bile. The only hydrolytic activity in excess of blank values that was detected was a highly variable phospholipase A2 activity in several gallbladder biles from animals given diets with both low levels and high levels of cholesterol, with the enzyme activities of the two dietary groups being similar. These results demonstrate that prairie dog gallbladder contains extremely low levels of phospholipase activity, in marked contrast to other gastrointestinal tissues. However, there was evidence of a phospholipase A2 activity in gallbladder bile. In light of the low activity in gallbladder tissue, the source of this enzyme appears to be the liver and not the gallbladder.(ABSTRACT TRUNCATED AT 250 WORDS)

Cholesterol nucleates rapidly from mixed micelles in the prairie dog

Current theory suggests that the nucleation of cholesterol in human bile requires the aggregation and fusion of cholesterol-enriched phospholipid vesicles. This theory is based on observations which do not exclude the precipitation of cholesterol from mixed micelles. The present study examines the role of mixed micelles and vesicles in the formation of cholesterol monohydrate crystals in the prairie dog. The intermicellar bile salt concentration of prairie dog gallbladder bile was determined using equilibrium dialysis. Model bile equivalent to gallbladder bile from cholesterol-fed prairie dogs was used as dialysant yielding the intermicellar (dialysate) concentration of 9 mM. Cholesterol carriers in gallbladder bile from 11 cholesterol-fed animals were then separated by Sephacryl S200 gel filtration chromatography using eluant buffer containing the intermicellar bile salt concentration. Gel filtration chromatography of fresh bile demonstrated that 100% of cholesterol was carried in the mixed micellar fraction with no vesicles observed in any of the 11 animals. The gallbladder bile nucleation time was 2.0 ± 0.3 days for the cholesterol-fed animals. Gel filtration chromatography immediately after nucleation again revealed a single mixed micellar peak. These data indicate that cholesterol is carried exclusively in and nucleates rapidly from mixed micelles in the cholesterol-fed prairie dog and that cholesterol-phospholipid vesicles are not required in this process.

Inhibition of prostaglandin synthesis fails to prevent gallbladder mucin hypersecretion in the cholesterol-fed prairie dog

Gallstone formation in the cholesterol-fed prairie dog is preceded by an increase in mucin secretion by the gallbladder epithelium, and mucin hypersecretion is believed to promote cholesterol gallstone formation by accelerating the nucleation of cholesterol monohydrate crystals. Some studies have suggested that gallbladder mucin hypersecretion is mediated by increases in gallbladder prostaglandin synthesis, but other observations are difficult to reconcile with this view. An organ culture technique was used to measure mucin secretion in normal prairie dog gallbladder in response to exogenous prostaglandins and agents that increased or decreased endogenous prostaglandin production. Incubation with indomethacin produced a concentration-dependent inhibition of endogenous prostaglandin synthesis with virtually complete inhibition at 10−5 mol/L indomethacin. However, indomethacin had no effect on gallbladder mucin secretion at concentrations as high as 10−5 mol/L, and significant inhibition of mucin secretion was only found at 10−4 mol/L indomethacin, a concentration that also produced a significant increase in lactate dehydrogenase release from cultured explants. Incubation of gallbladder explants with the calcium ionophore A23187 significantly stimulated endogenous prostaglandin synthesis in a concentration-dependent manner, increasing synthesis of prostaglandins E and F to as much as 278% ± 20% and 335% ± 21% of basal values, respectively; however, the same concentrations of A23187 did not stimulate mucin secretion. Incubation of gallbladder explants in the presence of exogenous prostaglandin E2 or prostaglandin F2a in concentrations as high as 10−6 mol/L also did not stimulate mucin secretion. Prairie dogs fed a lithogenic 1.2% cholesterol diet showed a significant increase in gallbladder mucin secretion after 1 week (117.5 ± 10.2% of control, P < 0.05), and 4 of 5 had formed cholesterol monohydrate crystals after 3 weeks. Long-term treatment with indomethacin, 1.2 mg · kg−1 · day−1, failed to inhibit gallbladder mucin hypersecretion (129.2 ± 10.7% of control after 1 week) or cholesterol monohydrate crystal formation (3/5) in cholesterol-fed prairie dogs. Furthermore, incubation of explants with 10−5 mol/L indomethacin failed to prevent in vitro mucin hypersecretion in cholesterol-fed animals. These findings suggest that prostaglandins do not regulate gallbladder mucin secretion in the prairie dog, and it is unlikely that increases in gallbladder prostaglandin synthesis are responsible for mediating gallbladder mucin hypersecretion during cholelithiasis in the prairie dog.