Thus the role of Ins-(1,4,5) em P /em 3R1 in apparently selectively activating Ca2+ inflow is associated with the location of Ins(1,4,5) em P /em 3R1 close to the plasma membrane. Taken together, the present results with Ins(1,4,5) em P /em 3 analogues selective for either Ins(1,4,5) em P /em 3R1 or Ins(1,4,5) em P /em 3R2, the previous results using an anti-Ins(1,4,5) em P /em 3R1 antibody to inhibit Ins-(1,4,5) em P /em 3 function and adenophostin A to activate Ca2+ inflow [9], and the evidence that some Ins(1,4,5) em P /em 3R1s are closely associated with the plasma membrane [18,19], suggest that, in EMT inhibitor-2 hepatocytes, the activation of SOCs requires the release of Ca2+ from a small region of the ER close to the plasma membrane and enriched in Ins(1,4,5) em P /em 3R1. of the type I (A) and type II (B) Ins(1,4,5)plane) and 3?m below equatorial respectively. Panels m and n are controls in which primary antibody has been omitted. All images in (A) and (B) were obtained with the same confocal gain setting. Freshly isolated hepatocytes exhibit little polarity, but when attached to a collagen-coated glass surface begin to regain polarity (assessed, for example, by formation of the cortical actin cytoskeleton) after about 4?h in culture [15,36]. To determine whether re-polarization affects the intracellular distribution of Ins(1,4,5)-condition, the majority of the ER has a very low density of Ins(1,4,5)(completely polarized) have shown that Ins(1,4,5) em P /em 3R1 is located principally near the plasma membrane, while Ins(1,4,5) em P /em 3R2 is located at the plasma membrane and bile canaliculus [17C19]. Moreover, Ins(1,4,5) em P /em 3R2 located around the bile canaliculus appears to be responsible for the initiation of Ca2+ waves [17]. Studies employing subcellular fractionation and electron microscopy have provided clear EMT inhibitor-2 evidence that, in liver cells, some Ins(1,4,5) em P /em 3R1s are located in regions of the ER that are closely associated with the plasma membrane through F-actin [18,19]. The present immunofluorescence results showing Ins(1,4,5) em P /em 3R1 at the cell periphery are consistent with these results. Thus the role of Ins-(1,4,5) em P /em 3R1 in apparently selectively activating Ca2+ inflow is associated with the location of Ins(1,4,5) em P /em 3R1 close to the plasma membrane. Taken together, the present results with Ins(1,4,5) em P /em 3 analogues selective for either Ins(1,4,5) em P /em 3R1 or Ins(1,4,5) em P /em 3R2, the previous results using an anti-Ins(1,4,5) em P /em 3R1 antibody to inhibit Ins-(1,4,5) em P /em 3 function and adenophostin A to EMT inhibitor-2 activate Ca2+ inflow [9], and the evidence that some Ins(1,4,5) em P /em 3R1s are closely associated with the plasma membrane [18,19], suggest that, in hepatocytes, the activation of SOCs requires the release of Ca2+ from a small region of the ER close to the plasma EMT inhibitor-2 membrane and enriched in Ins(1,4,5) em P /em 3R1. The requirement for Ins(1,4,5) em P /em 3R1 for the activation of SOCs in hepatocytes may simply reflect enrichment of those putative subregions of the ER involved in the activation of SOCs with Ins(1,4,5) em P /em 3R1. It is hypothesized in the present paper that the role of Ins(1,4,5) em P /em 3R1 is solely to mediate release of Ca2+ from the ER. At this stage, there is no evidence for direct interaction between the Ins(1,4,5) em P /em 3R1 protein and the SOC protein in hepatocytes. em In vivo /em , where the activation of SOCs is initiated by Ins(1,4,5) em P /em 3 generated by the hormone-induced activation of phospholipase C, additional factors will likely also influence NOV the relative ability of Ins(1,4,5) em P /em 3 to activate SOCs and release Ca2+from intracellular stores. These include the rate of metabolism of Ins(1,4,5) em P /em 3 [6], the effects of Ca2+ on the affinity of Ins(1,4,5) em P /em 3 for Ins(1,4,5) em P /em 3Rs [45] and the locations of hormone receptors and phospholipase C in relation to that of Ins(1,4,5) em P /em 3Rs [46,47]. Acknowledgments We acknowledge financial support from the Wellcome Trust (Programme Grant 060554 to B.V.L.P.) and NHMRC Grant 102125 (to G.J.B. and R.B.G.). The provision of monoclonal antibodies KM1112 against Ins(1,4,5) em P /em 3R1 and KM1083 against Ins(1,4,5) em P /em 3R2 by Professor K. Mikoshiba, University of Tokyo, Tokyo, Japan, is gratefully acknowledged. We also thank Dr S. J. Mills for gifts of synthetic Ins(1,4,6) em P /em 3 and Ins(1,2,4,5) em P /em 4, and acknowledge Dr Jenny Hiscock for conducting the confocal microscopy, and Ms Lee-Anne Addis for preparation of the typescript..