J. Gregory Fitz

Localization of the cystic fibrosis transmembrane conductance regulator in human bile duct epithelial cells

BACKGROUND Liver dysfunction is a common manifestation of cystic fibrosis (CF), a disease caused by mutations affecting the CF transmembrane conductance regulator (CFTR). The aim of this study was to examine the distribution and role of CFTR in liver. METHODS CFTR messenger RNA was detected in cryosections of human liver by in situ hybridization. CFTR immunoreactivity was detected using antibodies raised against two CFTR peptides. RESULTS The predominant site of CFTR messenger RNA and immunoreactivity in liver is the intrahepatic bile duct. CFTR is not detected in hepatocytes of normal liver or in livers exhibiting bile duct proliferation. Within bile duct cells, CFTR is localized at or near the apical plasma membrane. CONCLUSIONS The apical localization of CFTR in bile duct cells suggests a model explaining how the CFTR-associated Cl- channel contributes to normal biliary secretion. This model suggests that if CFTR expression could be promoted in intrahepatic duct cells by somatic gene therapy, this might prevent the occurrence of liver disease in CF.

The kindling model of epilepsy: a review.

t Department of Medicine (Neurology), Duke University Medical Center, Director, Epilepsy Center, Veterans Administration Medical Center, Durham, NC 27705, U.S.A. Z Department of Neurology, Albert Einstein College of Medicine, Bronx, N Y, U.S.A. 3Department of Medicine (Neurology), Duke University Medical Center, Durham, N C 27710, U.S.A. *Department of Medicine, University of California, School of Medicine, San Francisco, CA, U.S.A.

GTP-binding proteins regulate high conductance anion channels in rat bile duct epithelial cells.

SummaryEpithelial cells from the intrahepatic bile duct contribute to bile formation, but little is known of the cellular mechanisms responsible. In these studies, we have characterized the endogenous GTP-binding proteins (G proteins) present in these cells and evaluated their role in regulation of high conductance anion channels. G proteins were identified in purified plasma membranes of isolated bile duct epithelial cells using specific antisera on Western blots, and ion channel activity was measured in excised inside-out membrane patches using patch-clamp recording techniques. In patches without spontaneous channel activity, addition of cholera toxin to the cytoplasmic surface had no effect (n=10). Addition of pertussis toxin caused an activation of channels in 13/34 (38%) attempts, as detected by an increase in channel open probability. Activated channels were anion selective (gluconate/Cl− permeability ratio of 0.17±0.04) and had a unitary conductance of ∼380 pS. Channel open probability was also increased by the nonhydrolyzable GDP analogue guanosine 5′-0-(2-thiodiphosphate) in 8/14 (57%) attempts. In contrast, channel open probability was rapidly and reversibly decreased by the nonhydrolyzable analogue of GTP 5′guanylylimidodiphosphate in 7/9 (78%) attempts. Western blotting with specific antisera revealed that both Giα−2 and Giα−3 were present in significant amounts, whereas Giα−1 and Goα were not detected. These studies indicate that in bile duct epithelial cells, high conductance anion channels are inhibited, in a membrane-delimited manner, by PTX-sensitive G proteins.

Metabolic Stress Opens K+ Channels in Hepatoma Cells through a Ca2+- and Protein Kinase Cα-dependent Mechanism

These studies of a model liver cell line evaluate the mechanisms responsible for regulated release of K+ ions during metabolic stress. Metabolic inhibition of HTC hepatoma cells by exposure to 2,4-dinitrophenol (50 μM) and 2-deoxy-D-glucose (10 mM) stimulated outward currents carried by K+ of 974 ± 75 pA at 0 mV (n = 20, p < 0.001). Currents were inhibited by chelation of intracellular Ca2+ or exposure to apamin (50 nM), an inhibitor of SKCa channels. In cell-attached recordings from intact cells, removal of metabolic substrates (25/28 cells) or exposure to metabolic inhibitors (32/40 cells) opened K+-selective channels with a conductance of 6.5 ± 0.2 pS. Channels had an open probability of 0.31 ± 0.08 and opened in bursts averaging 3.55 ± 0.27 ms in duration (n = 6). Metabolic stress was associated with rapid translocation of the α isoform of protein kinase C (PKCα) from cytosol to membrane; and down-regulation of PKCα by phorbol esters or exposure to the PKC inhibitor chelerythrine (10 μM) each inhibited currents. Moreover, intracellular perfusion with purified PKCα activated currents in a Ca2+- and concentration-dependent manner. These findings indicate that metabolic stress leads to opening of apamin-sensitive SKCa channels in hepatoma cells through a Ca2+- and PKC-dependent mechanism and suggest that PKCα may be selectively involved in the response. This mechanism functionally couples the metabolic state of cells to membrane K+ permeability and represents a potential target for modification of liver injury associated with ischemia and preservation.

Sphincter of oddi dysfunction after liver transplantation

To The Editor: We read with great interest the study by Douzdjian et al (1) presenting five patients with sphincter of Oddi dysfunction (SOD) following orthotopic liver transplantation (OLT). Many aspects of this peculiar entity remain obscure including its pathophysiology, incidence, and treatment. The study by Douzdjian et al (1), showing abnormal sphincter of Oddi manometry in patients with clinical evidence of SOD, provides further support for papillary dyskinesia as one cause of biliary problems following OLT. We concur with Douzdjian et al (1) that denervation and devascularization of the papilla as suggested by Stieber et al (2) is only a partial explanation of the disease. The fact that some patients develop SOD and others do not while the same surgical technique is applied is intriguing. Our recent experience suggests that a dilated recipient common bile duct (CBD) represents a significant risk factor for developing posttransplant SOD. Four patients with dilated CBD (>15 mm in diameter) had liver transplantation with choledochocholedochostomy with T-tube drainage. The discrepancy in size between donor and recipient CBD was minimal. Two of these patients had cryptogenic cirrhosis, one had autoimmune hepatitis, and one hepatitis C cirrhosis. At the time of T-tube damping, significant increases in bilirubin and alkaline phosphatase levels were observed in each patient. T-tube cholangiograms showed no evidence for anastomotic strictures or leaks, but increased or persistent diffuse dilatation of the CBD was present in all cases. The liver enzyme levels normalized within one to three days following T-tube reopening. Two patients had repeat attempts at T-tube clamping with similar results. None of these patients had evidence of rejection. Suspecting SOD, an ERCP was performed in each patient with insertion of a stent (8-10 French, 5 cm in length) through the papilla without sphincterotomy. The T tube could be clamped within 24 hr following the endoscopic procedure with no subsequent elevation of serum liver enzymes. Stents were removed endoscopically four to six weeks later without further biliary complications. Use of CC for anastomosis in recipients with dilated CBD is uncommon since hepatojejunostomy is usually preferred, particularly if there is marked discrepancy between donor and recipient CBD. This experience with four cases strongly suggests that CBD dilation is a important risk factor for posttransplant SOD. While the mechanism is unclear, preexisting papilla dyskinesia may be a contributing factor. However, resolution of SOD over time without specific intervention argues for a contribution from peritransplant factors, eg, denervation or devascularization. We are currently selecting hepatojejunostomy reconstruction in cases involving large recipient CBD, even in the absence of size discrepancy. Evaluation of other risk factors is warranted since posttransplant SOD can also occur in the presence of small CBD. We would also stress the importance of early diagnosis of SOD to permit appropriate therapy. Short stents placed endoscopically through the papilla appear to be curative. Endoscopic sphincterotomy has not been necessary in our patients. Recognition of this entity may prevent misdiagnoses, such as interpreting anastomotic narrowing as a significant stricture resulting in unnecessary endoscopic or surgical intervention. The bedside manometry technique through the T tube as described by Sherman et al (3) and proposed by Douzdjian et al (1) as a screening test for SOD may be useful. However, the specificity and sensitivity of this test is still unclear. For instance, in the study by Douzdjian et al (1), one of the five patients with SOD had low basal pressure. Similarly, there is no information about false positive manometry, which may result in unnecessary treatments. We consider the presence of diffuse dilatation of the CBD with elevation of the liver enzymes after T-tube clamping and which resolves promptly after reopening as sufficient to warrant a presumptive diagnosis of SOD and to proceed with treatment. PIERRE-ALAIN CLAVIEN, 1 MD, PHD CARLOS A. CAMARGO, JR, 1 M D JOHN BAILLIE, 2 M D J. GREGORY FITZ, 2 M D Liver Transplantation Program Department of Surgery I and Medicine 2 Duke University Medical Center Durham, North Carolina

Muscarinic cholinergic regulation of epileptic spiking in kindling.

Electroencephalographic monitoring of spontaneous interictal spiking (SIS) following kindling demonstrated that SIS occurs in both amygdalas and that it declines sharply during the days following kindling. Systemically administered muscarinic antagonists which pass the blood-brain barrier (atropine and scopolamine) activated interictal spiking in kindled rats but not in controls. Interictal spiking activated by atropine was reversed by physostigmine. Both physostigmine and choline, agents which increase brain ACh concentration by different mechanisms, caused a reduction in spontaneous (not drug activated) interictal spiking. The results of these pharmacologic studies indicate that the interaction of endogenous ACh with central muscarinic receptors is capable of suppressing SIS in kindled rats. Whether cholinergic suppression of SIS represents a convulsant or an anticonvulsant action is presently unclear.

Paraneoplastic Hepatopathy Associated With Soft Tissue Sarcoma

Paraneoplastic syndromes associated with mesodermal tumors are relatively uncommon. An unusual case manifested by fever, anemia, thrombocytosis, coagulopathy, and idiopathic cholestatic liver dysfunction in association with soft tissue sarcoma is reported. A paraneoplastic syndrome is postulated in the absence of anatomic obstruction of bile flow, evidence of an infectious etiology, or neoplastic hepatic involvement.

HCO3 --coupled Na+ influx is a major determinant of Na+ turnover and Na+/K+ pump activity in rat hepatocytes

SummaryRecent studies in hepatocytes indicate that Na+-coupled HCO3− transport contributes importantly, to regulation of intracellular pH and membrane HCO3− transport. However, the direction of net coupled Na+ and HCO3− movement and the effect of HCO3− on Na+ turnover and Na+/K+ pump activity are not known. In these studies, the effect of HCO3− on Na+ influx and turnover were measured in primary rat hepatocyte cultures with22Na+, and [Na+]i was measured in single hepatocytes using the Na+-sensitive fluorochrome SBFI. Na+/K+ pump activity was measured in intact perfused rat liver and hepatocyte monolayers as Na+-dependent or ouabain-suppressible86Rb uptake, and was measured in single hepatocytes as the effect of transient pump inhibition by removal of extracellular K+ on membrane potential difference (PD) and [Na+]i. In hepatocyte monolayers, HCO3− increased22Na+ entry and turnover rates by 50–65%, without measurably altering22Na+ pool size or cell volume, and HCO3− also increased Na+/K+ pump activity by 70%. In single cells, exposure to HCO3− produced an abrupt and sustained rise in [Na+]i, from ≈8 to 12mm. Na+/K+ pump activity assessed in single cells by PD excursions during transient K+ removal increased ≃2.5-fold in the presence of HCO3−, and the rise in [Na+]i produced by inhibition of the Na+/K+ pump was similarly increased ≃2.5-fold in the presence of HCO3−. In intact perfused rat liver, HCO3− increased both Na+/K+ pump activity and O2 consumption. These findings indicate that, in hepatocytes, net coupled Na+ and HCO3− movement is inward and represents a major determinant of Na+ influx and Na+/K+ pump activity. About half of hepatic Na+/K+ pump activity appears dedicated to recycling Na+ entering in conjunction with HCO3− to maintain [Na+]i within the physiologic range.

Modulation of colonic motility by substance P, cholecystokinin and neuropeptide Y

The effects of substance P, cholecystokinin and neuropeptide Y were examined on rabbit distal colonic motility. All three agents produced increased contractile activity but the mechanisms responsible differed depending on the agent tested. In the intact animal, peptide effects were measured under basal conditions and following exposure to atropine, tetrodotoxin and the alpha-adrenergic antagonist phentolamine. Administration of all three peptides resulted in a stimulation of colonic motility. Phentolamine did not significantly effect substance P-, cholecystokinin- or neuropeptide Y-induced activity. By contrast, the in vivo activity induced by cholecystokinin and neuropeptide Y, but not substance P, was nearly eliminated by tetrodotoxin. Only the neuropeptide Y response was partially atropine sensitive. In isolated colonic strips, cholecystokinin-induced activity, but not that produced by neuropeptide Y or substance P, was blocked by tetrodotoxin. Atropine did not significantly inhibit any of the hormone-induced contractions.

Inhibition of Colonic Motility by Cholecystokinin

Distal colonic motor activity was measured in 12 control subjects and seven constipation-predominant irritable bowel syndrome patients to examine the effects of intravenous administration of cholecystokinin. In the basal state, no significant motility differences were noted between these two groups. Following the intravenous administration of the hormone cholecystokinin, a statistically significant reduction in colonic motility in control subjects and a non-significant decrease in motility in irritable bowel syndrome patients was seen. Our results do not suggest an exaggeration of the colonic motor response to cholecystokinin occurs in irritable bowel syndrome.