Production of phosphoinositide-derived messengers

The responses of cells to external stimuli are mediated by a variety of messenger molecules. Three such molecules produced as a result of phosphoinositide metabolism are diglyceride, inositol triphosphate, and arachidonic acid. The first two of these substances have properties common to second messengers such as CAMP. Their production is initiated by specific agonists, they are potent activators of other cellular reactions, and they are rapidly degraded to inactive compounds. Arachidonate is the polyunsaturated fatty acid that is oxygenated to produce a family of compounds including prostaglandins, thromboxane, and leukotrienes that are collectively called icosanoids (Samuelsson, Harvey Lect. 7.5, l-40, 1981). Many icosanoids have the same properties as second messengers listed above, although the icosanoids tend to act upon neighboring cells rather than intracellularly. Phosphoinositide Metabolism Phosphoinositides are ubiquitous components of eucaryotic membranes. In mammals phosphatidylinositol (PI) comprises about 5%-7% of phospholipids, phosphatidylinositol-4 phosphate (PI-4P) I%, and phosphotidylinositol45 diphosphate (PI-4,5P2) 0.4%. The phosphoinositides are in equilibrium with each other through a “futile cycle” of kinases and phosphatases, as shown in the figure. Phosphoinositides break down rapidly in response to receptor-mediated cell activation (Downes and Michell, Cell Calcium 3, 467-502, 1982). Originally only PI was shown to be hydrolyzed; more recently it was discovered that all three phosphoinositides are degraded by a phospholipase C to form diglyceride and one of the inositol phosphates. The inositol phosphates are rapidly degraded to inositol, which is utilized for resynthesis of phosphoinositides. Diglyceride either is hydrolyzed by lipases to monoglyceride (MG) and then to free arachidonate and glycerol or is phosphorylated by diglyceride kinase to form phosphatidic acid (PA). PA is then converted to PI, thus completing the “PI cycle.” Inositol-7,4,5 Triphosphate Formation and Calcium Mobilization Polyphosphoinositides undergo agonist-induced hydrolysis as evidenced by the transient appearance in stimulated cells of inositol-I ,4,5 triphosphate (IPs) and inositol-I ,4 diphosphate (IP& which are products of phospholipase C action on polyphosphoinositides. In blowfly salivary glands (Berridge, Biochem. J. 272, 849-858, 1983) platelets (Agranoff et al., JBC 258, 2076-2078, 1983), and parotid cells (Downes et al., Biochem. J., in press, 1984) IPs and IPn accumulate within a few seconds of stimulation, before changes are seen in IP or inositol. These investigators propose that the majority of PI breakdown occurs by conversion of PI to polyphosphoinositides prior to hydrolysis by phospholipase C. A role for IP3 in calcium mobilization is suggested by a study of permeabilized pancreatic cells (Streb et al., Nature 306,67-69,1983). These workers added IP3 to the medium and showed a rise in extracellular Ca*+, indicating liberation from some intracellular site. IP:! and IP were ineffective in stimulating Ca” mobilization in this assay. These results have been confirmed in a study of saponin-permeabilized liver cells (Joseph et al., JBC 259, 3077-3081, 1984). IPs also specifically promotes Ca’+ release from isolated microsomes of rat liver (Dawson and Irvine, BBRC 120, 858-864, 1984) and rat insulinoma cells (Perentki et al., Nature 309, 562-564, 1984). Hydrolysis of PI by phospholipase C in vitro requires Ca*‘. If IPs is the messenger for Ca’+ mobilization, then hydrolysis of PI-4,5P2 should be calcium-independent (Billah and Lapetina, BBRC 109, 217-222, 1982). Our recent experiments with pure phospholipase C suggest that this may be the case. We have isolated two distinct PI-specific phospholipase C enzymes from seminal vesicles (Hofmann et al., JBC 257, 6461-6469, 1982) and have shown that both enzymes are able to hydrolyze all three phosphoinositides nearly equally. However, in the presence of EGTA these enzymes cleave only PI-4P and PI-4,5P2, indicating that hydrolysis of these substances may occur prior to Ca” flux, as suggested by the physiological experiments. Once intracellular Ca2’ rises, PI breakdown may predominate since it is quantitatively the major potential substrate for phospholipase C. The fraction of each phosphoinositide that is hydrolyzed directly by phospholipase C versus that which undergoes interconversion followed by hydrolysis is unknown and can only be answered by detailed kinetic study of all of the intermediates shown in the figure. The mechanism of initiation of phosphoinositide breakdown remains to be elucidated. Phospholipase C appears to be equally active in extracts from stimulated and unstimulated cells. Studies with small unilamellar vesicles of defined phospholipid composition as substrates indicate that the composition and distribution of lipids in a membrane have enormous effects on enzyme activity. It is possible that activation of phosphoinositide breakdown is initiated by a redistribution or segregation of these lipids in the membrane. The intracellular site of phosphoinositide breakdown is unknown. Most of the biosynthetic enzymes are in the endoplasmic reticulum. If breakdown also takes place in endoplasmic reticulum and the process is initiated by