ISOLATED RAT HEPATOCYTES CAN SIGNAL TO OTHER HEPATOCYTES AND BILE-DUCT CELLS BY RELEASE OF NUCLEOTIDES

Intercellular communication among certain cell types can occur via ATP secretion, which leads to stimulation of nucleotide receptors on targ et cells. In epithelial cells, however, intercellular communication is thought to occur instead via gap junctions. Here we examined whether one epithelial cell type, hepatocytes, can also communicate via nucleo tide secretion. The effects on cytosolic Ca2+ ([Ca2+](i)) of mechanica l stimulation, including microinjection, were examined in isolated rat hepatocytes and in isolated bile duct units using confocal fluorescen ce,ideo microscopy. Mechanical stimulation of a single hepatocyte evok ed an increase in [Ca2+](i) in the stimulated cell plus an unexpected [Ca2+](i) rise in neighboring noncontacting hepatocytes. Perifusion wi th ATP before mechanical stimulation suppressed the [Ca2+](i) increase , but pretreatment with phenylephrine did not. The P-2 receptor antago nist suramin inhibited these intercellular [Ca2+](i) signals. The ATP/ ADPase apyrase reversibly inhibited the [Ca2+](i) rise induced by mech anical stimulation, and did not block vasopressin-induced [Ca2+](i) si gnals. Mechanical stimulation of hepatocytes also induced a [Ca2+](i) increase in cocultured isolated bile duct units, and this [Ca2+](i) in crease was inhibited by apyrase as well. Finally, this form of [Ca2+]( i) signaling could be elicited in the presence of propidium iodide wit hout nuclear labeling by that dye, indicating that this phenomenon doe s not depend on disruption of the stimulated cell. Thus, mechanical st imulation of isolated hepatocytes, including by microinjection, can ev oke [Ca2+](i) signals in the stimulated cell as well as in neighboring noncontacting hepatocytes and bile duct epithelia, This signaling is mediated by release of ATP or other nucleotides into the extracellular space. This is an important technical consideration given the widespr ead use of microinjection techniques for examining mechanisms of signa l transduction. Moreover, the evidence provided suggests a novel parac rine signaling pathway for epithelia, which previously were thought to communicate exclusively via gap junctions.