Characterization of ionotrophic purinergic receptors in hepatocytes.

UNLABELLED: Ionotrophic purinergic (P2X) receptors function as receptor-gated cation channels, where agonist binding leads to opening of a nonselective cation pore permeable to both Na(+) and Ca(2+). Based on evidence that extracellular adenosine 5'-triphosphate (ATP) stimulates glucose release from liver, these studies evaluate whether P2X receptors are expressed by hepatocytes and contribute to ATP-dependent calcium signaling and glucose release. Studies were performed in isolated hepatocytes from rats and mice and hepatoma cells from humans and rats. Transcripts and protein for both P2X4 and P2X7 were detectable, and immunohistochemistry of intact liver revealed P2X4 in the basolateral and canalicular domains. In whole cell patch clamp studies, exposure to the P2X4/P2X7 receptor agonist 2'3'-O-(4-benzoyl-benzoyl)-adenosine 5'-triphosphate (BzATP; 10 microM) caused a rapid increase in membrane Na(+) conductance. Similarly, with Fluo-3 fluorescence, BzATP induced an increase in intracellular [Ca(2+)]. P2X4 receptors are likely involved because the calcium response to BzATP was inhibited by Cu(2+), and the P2X4 modulators Zn(2+) and ivermectin (0.3-3 microM) each increased intracellular [Ca(2+)]. Exposure to BzATP decreased cellular glycogen content; and P2X4 receptor messenger RNA increased in glycogen-rich liver samples. CONCLUSION: These studies provide evidence that P2X4 receptors are functionally important in hepatocyte Na(+) and Ca(2+) transport, are regulated by extracellular ATP and divalent cation concentrations, and may constitute a mechanism for autocrine regulation of hepatic glycogen metabolism.

Evidence for AMPK-dependent regulation of exocytosis of lipoproteins in a model liver cell line.

5'-AMP-activated kinase (AMPK) plays a key role in the regulation of cellular lipid metabolism. The contribution of vesicular exocytosis to this regulation is not known. Accordingly, we studied the effects of AMPK on exocytosis and intracellular lipid content in a model liver cell line. Activation of AMPK by metformin or 5-aminoimidazole-4-carboxamide-1-beta-d-ribofuranoside (AICAR) increased the rates of constitutive exocytosis by about 2-fold. Stimulation of exocytosis by AMPK occurred within minutes, and persisted after overnight exposure to metformin or AICAR. Activation of AMPK also increased the amount of triacylglycerol (TG) and apolipoprotein B (apoB) secreted from lipid-loaded cells. These effects were accompanied by a decrease in the intracellular lipid content indicating that exocytosis of lipoproteins was involved in these lipid-lowering effects. While AMPK increased the rates of fatty acid oxidation (FAO), the lipid-lowering effects were quantitatively significant even after inhibition of FAO with R-etomoxir. These results suggest that hepatic AMPK stimulates constitutive exocytosis of lipoproteins, which may function in parallel with FAO to regulate intracellular lipid content.

Double-balloon ERCP in patients who have undergone Roux-en-Y surgery: a case series.

BACKGROUND: ERCP is technically challenging in patients who have had a long-limb Roux-en-Y surgical procedure. The recent introduction of the double-balloon endoscope permits the examination of a much longer segment of the small bowel compared with a standard endoscope and may be used to perform ERCP in these patients. OBJECTIVE: To report successful use of double-balloon ERCP in patients who have had a Roux-en-Y surgical procedure. PATIENTS: Fourteen patients with a history of either Roux-en-Y gastric bypass weight-reduction surgery or Roux-en-Y pancreatobiliary surgery required diagnostic and/or therapeutic pancreatobiliary intervention. DESIGN: Case report. INTERVENTION: Double-balloon ERCP. RESULTS: Fourteen patients underwent a total of 20 ERCPs with the double-balloon endoscopy system. The ampulla was successfully reached in 85% of total cases (100% of patients who have had Roux-en-Y weight reduction surgery), with adequate cannulation of either the biliary or pancreatic duct in 80% (88% of patients for weight reduction). Therapeutic intervention, including stone removal, pancreatobiliary-duct dilation, sphincterotomy, stent placement, and removal of previously placed stents, was performed successfully in 6 cases. The mean age was 47 years old. The mean (+/- standard deviation) total duration of the procedure was 99 +/- 48 minutes. There were no immediate or short-term complications. CONCLUSIONS: The double-balloon endoscopy system permits diagnostic and therapeutic ERCP in patients who have had long-limb surgical procedures. Our experience demonstrated that this procedure is well tolerated, safe, and has a high success rate.