Isolated rat hepatocytes can signal to other hepatocytes and bile duct cells by release of nucleotides.

Abstract

Intercellular communication among certain cell types can occur via ATP secretion, which leads to stimulation of nucleotide receptors on target cells. In epithelial cells, however, intercellular communication is thought to occur instead via gap junctions. Here [Arg8]vasopressin, phenylephrine, and propidium iodide were from Sigma; fluo-3/AM was from Molecular Probes; suramin was from Biomol (Plymouth Meeting, PA); and indodicarbocyanine (Cy5) was from Biological Detection Systems (Pittsburgh). All other chemicals were of the highest quality commercially available.Preparation of Isolated Hepatocytes. Isolated rat hepatocytes were prepared in the Hepatocyte Isolation Core of the Yale Liver Center as described (5, 12). Briefly, rat livers were perfused with Hanks' A medium and then with Hanks' B medium containing 0.05% collagenase (Boehringer Mannheim) and 0.8 unit trypsin inhibitor (Sigma)/U tryptic activity.Livers were then excised, minced, and passed through serial nylon mesh filters, and the resultant cells were washed. These cells were suspended at a concentration of 7.5 x 105 cells per ml in Leibovitz L-15 medium (GIBCO) containing 10% fetal calf serum, 50 units penicillin, and 50 mg streptomycin/ml, and plated onto glass coverslips. Cells were incubated at 37°C and used 2-6 h after plating. When prepared in this fashion, "40% of isolated hepatocytes were not in contact with other hepatocytes, while '60% were in aggregates of two or more. Cell viability by trypan blue exclusion was measured 2 h after plating and exceeded 90%.Preparation of Isolated Bile Duct Units. Bile duct units, which are polarized and physiologically intact segments of bile duct epithelia, also were prepared in the Hepatocyte Isolation Core of the Yale Liver Center as described (13). After livers were perfused with Hanks' B medium as described above, the portal tissue residue was mechanically separated from parenchymal tissue first by shaking and then by forcing the tissue through a syringe to dissociate the remaining hepatocytes. The tissue was then minced in solution C, which contained a-MEM supplemented -with 0.066% collagenase, 0.033% Pronase, 0.006% DNase, 3% fetal calf serum, 0.1% bovine serum albumin, and penicillin/streptomycin at 100,000 units per 100 mg/liter. The minced tissue was then shaken at 37°C for 30 min, minced again, and sequentially filtered. Fragments remaining on the filters were digested for an additional 30 min Abbreviations: [Ca2+]i, cytosolic calcium; Cy5, indodicarbocyanine.

Exogenous Mg-ATP Induces a Large Inhibition of Pyruvate Kinase in Intact Rat Hepatocytes

“…It has been recently demonstrated that isolated rat hepatocytes can signal to other hepatocytes by the release of ATP, suggesting a novel paracrine signaling pathway exists (51). This finding favors a physiological role of exogenous ATP, which may trigger a protective effect.…”

Cyclic AMP Regulates Bicarbonate Secretion in Cholangiocytes Through Release of ATP Into Bile

“…Because forskolin-mediated secretion involves CFTR (Figure 1b) (3)(4)(5), and activation of CFTR in other systems may result in ATP release (19)(20)(21), we investigated whether forskolin-mediated secretion involves autocrine signaling via luminal ATP. IBDU lumens were perfused with either apyrase to hydrolyze luminal ATP or suramin to block apical P2Y receptors (22). Apyrase reduced the forskolin-induced increase in luminal pH by 86%, and suramin reduced the forskolin-induced increase by 90% (n=6, p<0.01; Figure 8a-b).…”

P2 receptor-mediated signaling in mast cell biology

“…Mechanic stimulation of mouse fibroblasts, hepatocytes, or glial cells evokes an increase in Ca 2+ not only in stimulated cells but also in distant cells [100][101][102]. This remote signaling is mediated by ATP or other nucleotides.…”