| Research Abstract |
Roles of phospholipid molecular species in cell signaling were studied. Mass changes in the various molecular species of phospholipids were determined after stimulation of human platelets with thrombin and collagen. Upon stimulation, every molecular species of phosphatidylinositol and phosphatidylserine was equally hydrolyzed, whereas the molecular species of phosphatidylcholine and diacyl- and alkenylacyl-phosphatidylethanolazine containing arachidonic acid were selectively hydrolyzed. The Ca^<2+> concentration affects the differential degradation of phospholipid molecular species in activated human platelets.
The thromboxane A_2 antagonist, ONO3708, completely inhibited the increase in cytosolic free Ca^<2+> in human platelets during activation with collagen. Half-maximal Ca^<2+> release and influx required about 3 and 4 nM STA_2, a stable thromboxane A_2 mimetic, respectively. However, half-maximal activation of phospholipase C required about 18 nM STA_2.
The Ca^<2+> channel blocker,
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nifedipine, a dihydropyridine derivative, inhibited the Ca^<2+> influx and release from internal stores caused by collagen or a low concentration of STA_2 (10 nM), but did not inhibit those caused by thrombin or a high concentration of STA_2 (100 nM). These results indicate the presence of two distinct, dihydropyridine-sensitive and insensitive, Ca^<2+> channels dependent on the concentrations and classes of agonists in human platelets.
^<125>I-labeled epidermal growth factor was incorporated into and highly concentrated in endosomes of Chinese hamster V79-UF cells during incubation at 37ﾟC for 8 min after binding to its receptors on the cell surface at 4ﾟC. From the labeled cells, endosomes were isolated by isopyenic centrifugation on a Percoll density gradient and then sucrose density gradients. Endosome membranes were found to differ from plasma membranes in the phospholipid composition. The contents of molecular species of diacylphosphatidyleholine and diacylphosphatidylethanolamine with two monoenoic fatty acids were lower in endosomes than in plasma membranes. The differences in the polar head group and molecular species compositions of phospholipids between endosomes and plasma membranes did not change, regardless of whether or not the proportions of phospholipid molecular species in plasma membranes changed. Less
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