Intracellular signals and volume regulatory response in skate erythrocytes

The volume regulatory response of erythrocytes (RBCs) of the little skate Raja erinacea subjected to 30% dilution of their medium is mimicked by the application of phorbol ester or calcium ionophore, implicating protein kinase C and phosphoinositide metabolism in that response. To investigate the signaling link between dilution of the medium and cell response, we measured levels of inositol phosphates (associated with intracellular calcium release) and of diacylglycerol (the physiological activator of protein kinase C) in control and hyposmotically treated RBCs. Labeled inositol monophosphate (IP1) was significantly higher in osmotically shocked than in control cells. Inositol bis- and trisphosphate levels (IP2 and IP3) were low and did not alter with dilution treatment. Separation of the isomers in the IP1 fraction indicated that the dilution effect was likely to result from the breakdown of phosphatidylinositol directly, without the involvement of the IP3 and related messenger molecules. Hyposmotic treatment also elevated the diacylglycerol content of the skate RBCs, providing evidence for the activation of protein kinase C as part of the volume regulatory response. The results are interpreted as indicating that the response to hypotonic media in skate erythrocytes is mediated primarily by protein kinase C and any involvement of calcium is associated with that pathway rather than with the production and metabolism of IP3.