Large intravenous bilirubin loads increase the cytotoxicity of bile and lower the resistance of the canalicular membrane to cytotoxic injury and cause cholestasis in pigs.

BACKGROUND: Large intravenous bilirubin loads cause loss of hepatic canalicular membrane microvilli and cholestasis. This study examines whether these untoward effects might be due to canalicular membrane injury from cytotoxic bile. METHODS: The cytotoxicity of bile was assayed against pig erythrocytes before and throughout 4.5-h intravenous infusion of 170 microg kg(-1) body weight of bilirubin in anaesthetized pigs. The capacity to generate canalicular bile flow was tested before and after bilirubin infusion by means of short-term intraportal cholic acid infusion. RESULTS: Bilirubin infusion increased the cytotoxicity of hepatic bile, reduced biliary phospholipid secretion by 90%, and caused cholestasis. Cholic acid infusion before bilirubin also increased the cytotoxicity of bile but increased bile flow and doubled biliary phospholipid output. CONCLUSION: Large intravenous bilirubin infusions increase the cytotoxicity of bile, suppress biliary phospholipid secretion, and render hepatic canalicular membrane microvilli susceptible to injury from cytotoxic bile so that cholestasis occurs.

Large intravenous loads of bilirubin photoconversion products, in contrast to bilirubin, do not cause cholestasis in bile acid-depleted pigs.

BACKGROUND: Large intravenous bilirubin infusions in bile acid-depleted pigs (BADP) destroy hepatocyte canalicular membrane microvilli (CMV) and cause cholestasis. This study examines whether bilirubin photoconversion product infusions do the same. METHODS: The effects of systemic infusion of 135 mumol.kg-1 body weight bilirubin photoconversion products on CMV density and choleretic response to intraportal bile acid infusion were studied in BADP. Furthermore, the effects of 135 mumol.kg-1 b.w. bilirubin infusion, either through an arteriovenous bilirubin photoconversion shunt device (PCD) or intravenously, were measured in PCD-connected BADP. RESULTS: Intravenous bilirubin photoconversion product infusions affected neither the CMV density nor the choleretic response to cholic acid infusion, and neither did bilirubin infusion through the PCD. In contrast, intravenous bilirubin infusion caused canalicular injury and cholestasis in four of six PCD-connected BADP. CONCLUSION: Bilirubin photoconversion products do not destroy CMV or cause cholestasis in BADP. A bilirubin photoconversion shunt device can confer cholestasis protection to bilirubin-loaded BADP.

Bile acids protect the liver against the cholestatic effect of large bilirubin loads.

BACKGROUND: This study was undertaken to elucidate why large bilirubin loads cause canalicular cholestasis and whether bile acid infusions protect against bilirubin-induced cholestasis. METHODS: The effects of bilirubin infusion on canalicular bile secretion and canalicular membrane morphology were studied in bile acid-depleted pigs (BADP), bile acid-primed pigs (BAPP), and pigs co-infused with bile acids during bilirubin loading (BACIP). RESULTS: Bilirubin caused complete cholestasis in BADP, 38% bile flow reduction in BAPP, and no effect on bile flow in BACIP. Scanning electron micrographs showed loss of 70% of canalicular microvilli in BADP, 13% loss and pathologic changes in the remaining 75% of microvilli in BAPP, and no canalicular changes in BACIP. Cholestasis was not due to hydromechanical obstruction of bile ductules or bile Ca2+ depletion. CONCLUSION: Bilirubin causes cholestasis in BADP by injuring canalicular microvilli. Intravenous glycocholate infusions fully protect the liver against bilirubin-induced cholestasis and canalicular microvillar injury.

Secretin causes H+/HCO3- secretion from pig pancreatic ductules by vacuolar-type H(+)-adenosine triphosphatase.

BACKGROUND/AIMS: Secretin stimulates pancreatic ductules to secrete HCO3- into pancreatic juice and H+ into interstitial fluid. The aim of the present study was first to examine whether ductular H+ secretion is inhibited by micromolar concentrations of bafilomycin A1, which blocks vacuolar H(+)-adenosine triphosphatase by specific action, and secondly to test for evidence of ductular Na+/HCO3- cotransport. METHODS: Ductular H+ secretion was estimated from the rate of intracellular pH recovery after acid-loading (24 mmol/L NH4Cl) microdissected pancreatic ductules from pig, mounted in a flow-through perfusion chamber on the stage of a fluorescent microscope. Intracellular pH was measured using the fluorescent pH indicator 2'7'-bis (carboxyethyl)-5,6-carboxyfluorescein and dual-wave-length excitation of fluorescence. The ducts were superfused perfused with either HCO3(-)-free HEPES-containing buffers or HCO3(-)-containing buffers. RESULTS: Secretin (10(-8) mol/L) induced a net H+ secretion of 1.87 +/- 0.23 mumol.mL cell vol-1.min-1 that was blocked by 10(-6) mol/L bafilomycin A1 and was unaffected by Na+ substitution with choline using HEPES superfusion buffers. Secretin-stimulated ductules superfused with bicarbonate-containing, Cl(-)-free buffers showed Na(+)-dependent and 4,4'-diisothiocyanostilbene-2, 2'-disulfonic acid-inhibitable alkalinization of intracellular pH. CONCLUSIONS: Secretin causes H+/HCO3- secretion from pancreatic ductules by a mechanism involving vacuolar-type H(+)-adenosine phosphatase. Pancreatic ductules also show Na+/HCO3- cotransport, which may account for a small fraction of secreted bicarbonate.

Secretin causes H+ secretion from intrahepatic bile ductules by vacuolar-type H(+)-ATPase.

Intrahepatic bile duct epithelial cells contribute to bile formation by hormone-dependently secreting HCO3- to bile and H+ to periductular fluid. The present study was undertaken to determine whether the secretin-induced H+ secretion is due to activation of a H(+)-ATPase or Na(+)-H+ exchange. H+ secretion was estimated from the rate of intracellular pH (pHi) recovery after acid loading (24 mM NH4Cl) of microdissected bile ductules from pig liver mounted in a flow-through chamber on the stage of a microscope. pHi was measured from an estimated average of 10-15 epithelial cells using the fluorescent pHi indicator 2',7'-bis(carboxyethyl)-5,6-carboxyfluorescein and dual-wavelength excitation of fluorescence. The ducts were superfused with HCO3(-)-free N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid buffers. We found that secretin induced net H+ secretion of 4.53 +/- 0.7 mumol.ml cell volume-1 x min-1. This H+ secretion was blocked by 10(-6) M bafilomycin A1 but was unaffected by Na+ substitution with choline in the superfusion buffer. The experiments also showed that bafilomycin A1 did not block Na(+)-H+ exchange. The secretin-induced H+ secretion is probably caused by a vacuolar-type H(+)-ATPase and may constitute an important element of the cellular mechanisms causing secretin-dependent ductular HCO3- secretion into bile.

Secretin stimulates bile ductules to secrete both H+ and HCO3(-)-ions.

Secretin-dependent ductular HCO3- secretion into bile may involve secretion of H+ to interstitial fluid and HCO3- to bile by the ductular epithelium. To determine whether secretin causes bile ductules to secrete H+, we have examined the effect of secretin on the elimination of an intracellular acid load from bile ductular epithelium during pharmacological blockade of Na(+)-H+ exchange and in the absence of HCO3-. Microdissected bile ductules from pigs were suspended in HCO3- free HEPES buffer and loaded with acid using an NH4Cl prepulse technique. Intracellular pH was measured using dual-wavelength excitation of BCECF fluorescence. Na(+)-H+ exchange was defined as a Na(+)-dependent and amiloride- and 5-(N,N-hexamethylene)-amiloride-sensitive efflux of H(+)-ions following acid loading. We found that secretin stimulated ductular H+ secretion independent of Na(+)-H+ exchange. Blockade of Na(+)-H+ exchange by hexamethylene-amiloride did not affect secretin-dependent ductular HCO3- choleresis in vivo. We conclude that secretin stimulates bile ductules to secrete H(+)-ions to interstitial fluid as well as HCO3- ions to bile by a mechanism independent of Na(+)-H+ exchange.

Na(+)-H+ exchange is not important for pancreatic HCO3- secretion in the pig.

UNLABELLED: Pancreatic inter- and intralobular duct cells extrude H(+)-ions to interstitial fluid when they secrete HCO3- to pancreatic juice. This study assesses the potential importance of Na(+)-H(+)-ion exchange for H(+)-ion extrusion and secretion of HCO3-, using the Na(+)-H+ exchange blockers amiloride and hexamethylene-amiloride. Intracellular pH (pHi) in inter- and intralobular pancreatic duct epithelium was measured using BCECF fluorescence. H(+)-ion efflux was measured using a NH4Cl prepulse, acid-loading technique. In HCO3(-)-free media, pHi recovery following acid loading was blocked by amiloride (10(-4) M) and hexamethylene-amiloride (10(-6) M), demonstrating amiloride- and hexamethylene-amiloride-sensitive Na(+)-H+ exchange. However, 5 x 10(-6) M hexamethylene-amiloride did not reduce secretin-dependent pancreatic HCO3- secretion in vivo. Maximal H(+)-efflux through Na(+)-H+ exchange was 1.5 +/- 0.2 mumol min-1 ml cell volume-1, i.e. less than 1% of estimated net H(+)-ion efflux during HCO3- secretion. CONCLUSION: amiloride- and hexamethylene amiloride sensitive Na(+)-H+ exchange is not important for secretin-dependent pancreatic HCO3- secretion in the pig. Other mechanisms for H+ extrusion dominate.

Intravenous bilirubin infusion causes vacuolization of the cytoplasm of hepatocytes and canalicular cholestasis.

To examine whether intravenous bilirubin infusion causes cholestasis and impairs liver metabolism, bile secretion and ethanol clearance were measured in 34 anaesthetized pigs before and after intravenous infusion of 0.5 mumol kg-1 min-1 bilirubin for 4.5 hours. Bilirubin infusion increased plasma bilirubin to 556 +/- 76 mumol l-1 and hepatic tissue bilirubin to 3.5 +/- 1.3 mmol kg tissue weight-1. Bilirubin infusion depressed bilirubin secretion and net hepatic uptake of cholate and taurocholate, and caused a 86 +/- 6% reduction of cholate-induced bile secretion. Bilirubin caused formation of large cytoplasmic vacuoles in hepatocytes and dilatation of bile canaliculi. Ethanol clearance and secretin-dependent ductular bile secretion were unaffected by bilirubin. We conclude that intravenous infusion of unconjugated bilirubin causes accumulation of bilirubin in the liver, vacuolization of the hepatocyte cytoplasm and canalicular but not ductular cholestasis. The canalicular cholestasis is not due to impaired hepatic mitochondrial energy metabolism, but may be due to inhibition of a common pathway for lipid, bilirubin and bile salt secretion from hepatocytes.