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.

Calmodulin regulation of gallbladder ion transport becomes dysfunctional during gallstone formation in prairie dogs.

Gallbladder absorption is increased prior to gallstone formation in prairie dogs and may promote cholesterol crystallization. Recent studies indicate that Ca2+-calmodulin (CaM) tonically inhibits gallbladder electrolyte absorption in prairie dogs fed a nonlithogenic diet. We hypothesized that dietary cholesterol alters CaM-dependent regulation of gallbladder ion transport, a possible link between increased gallbladder absorption and gallstone formation. Gallbladders from prairie dogs fed control (N = 24) or 1.2% cholesterol-enriched chow (N = 32) were mounted in Ussing chambers. Electrophysiology and ion flux were measured while exposing the epithelia sequentially to trifluoperazine (TFP), a CaM antagonist, followed by the calcium ionophore A23187. Animals fed the high cholesterol diet developed crystals and gallstones in a time-dependent fashion. Mucosal addition of 50 microM TFP decreased short-circuit current (Isc), transepithelial potential, and tissue conductance in control, crystal, and gallstone animals, but the magnitude of its effect was significantly decreased in animals fed cholesterol. TFP stimulated mucosa-to-serosa Na+ flux by 6.9 +/- 0.9 microeq/cm2/hr in control animals but only 3.1 +/- 0.8 microeq/cm2/hr in gallstone animals. Similarly, TFP increased mucosa-to-serosa Cl- flux by 11.9 +/- 1.4 microeq/cm2/hr in controls but only 4.9 +/- 1.4 microeq/cm2/hr in cholesterol-fed animals. TFP effects were not reversed by A23187, which caused differential effects on Isc and ion transport in cholesterol-fed animals. In conclusion, CaM-mediated inhibition of gallbladder Na+ and Cl- transport is diminished in prairie dogs fed cholesterol. We conclude that gallbladder ion transport is partially released from basal inhibition during gallstone formation and propose that dysfunctional CaM regulation may be a stimulus to increased gallbladder absorption.

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 E2 (PGE2) 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 H20 fluxes were measured before and after inhibition of endogenous prostaglandin synthesis with 10 micromol/L indomethacin. Gallbladders were then exposed to increasing concentrations of PGE2 to a maximal dose of 1 micromol/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 PGE2. PGE2 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.

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.

Elevated biliary calmodulin during gallstone formation: the role of bile acids.

Hepatic bile synthesis is altered during experimental gallstone formation. In response to cholesterol, there is a hydrophobic shift in hepatic bile acid synthesis and hypersecretion of phospholipids. These changes decrease the vesicular capacity for cholesterol and favor crystallization. The mechanism for these changes in hepatic bile formation is unknown. Calmodulin (CaM), a Ca2+ receptor protein involved in cellular secretion, regulates gallbladder transport and may play an important role in alterations of hepatic bile formation during cholelithiasis. We hypothesized that biliary CaM activity is altered during gallstone formation and may be associated with changes in bile acid and phospholipid synthesis. Prairie dogs were fed either control (N = 22) or 1.2% cholesterol-enriched (N = 26) diets for one to six weeks. Cholecystectomy was performed; the common bile duct was cannulated, and hourly bile samples were collected. CaM was measured in bile and gallbladder tissues by radioimmunoassay. Bile samples were analyzed for cholesterol, phospholipids, total bile acids, total protein, calcium, and individual bile acid composition. Compared to controls, gallstone animals had elevated hepatic bile levels of CaM, phospholipids, and cholesterol. Hydrophobic bile acid synthesis was also stimulated, with increased levels of taurochenodeoxycholic acid (TCDCA) and decreased taurocholic acid (TCA). Gallbladder bile demonstrated similar changes. Although gallbladder bile CaM levels were increased, tissue levels were unchanged, suggesting that increased CaM concentration is a hepatic phenomenon. Hepatic bile CaM activity correlated linearly with TCDCA concentration (r = 0.64, P < 0.004) and phospholipid hypersecretion (r = 0.53, P < 0.03). The relationship between biliary CaM and increased concentrations of TCDCA and phospholipids suggests a role for CaM in alterations of hepatocyte secretion that may promote gallstone formation.

Ca2+ calmodulin regulates basal gallbladder absorption.

BACKGROUND: Gallbladder absorption is altered during gallstone formation, a phenomenon that may be partly the result of elevated biliary Ca2+ levels. Recent studies suggest that changes in gallbladder absorption are mediated by intracellular Ca2+ ([Ca2+]ic). However, the mechanisms by which [Ca2+]ic regulates gallbladder ion transport are not known. Calmodulin is a Ca2+ receptor protein in the Ca2+ messenger system that modulates ion transport in the small intestine. We hypothesized that Ca(2+)-calmodulin mediates the effects of [Ca2+]ic on gallbladder absorption. METHODS: Prairie dog gallbladders were mounted in Ussing chambers, and standard electrophysiologic parameters were recorded. Unidirectional Na+, Cl-, and water fluxes were measured before and after mucosal exposure to 5 x 10(-5) mol/L trifluoperazine, a potent calmodulin antagonist. In addition, the ion transport effects of increased extracellular calcium and theophylline were determined in the presence of calmodulin inhibition. RESULTS: Inhibition of calmodulin resulted in an increase in net Na+ and water absorption and converted the gallbladder from a Cl- absorptive state. Similar results were obtained during exposure to two other calmodulin antagonists that differ only in their affinity for calmodulin but not in their hydrophobicity, suggesting that the observed changes were caused by specific calmodulin inhibition. Effects of trifluoperazine were reversed by increasing luminal [Ca2+] or theophylline exposure. CONCLUSIONS: The effects of calmodulin inhibition are directly opposite of the effects of the Ca2+ ionophore. We conclude that Ca(2+)-calmodulin regulates gallbladder absorption at basal [Ca2+]ic. Further studies are needed to determine whether altered calmodulin activity is responsible for increased gallbladder absorption during gallstone formation.