Changes in the concentration and fatty acid composition of phosphoinositides induced by hormones in hepatocytes.
The hormonal regulation of phosphoinositide levels in isolated hepatocytes was studied using chemical means. Extracted inositol phospholipids were adsorbed to neomycin-coated glass beads and then eluted and quantitated by charring after separation by thin layer chromatography on silica gel. The amounts (in nanograms/mg wet weight) of phosphatidylinositol 4,5-bisphosphate (PIP2), phosphatidylinositol 4-phosphate (PIP), and phosphatidylinositol (PI) were 20 +/- 1, 16 +/- 1, and 1790 +/- 140, respectively). Incubation of the cells with 100 nM vasopressin decreased the value for PIP2 to 10 +/- 0.2 at 15 s, 12 +/- 1.5 at 1 min, and 14 +/- 2.1 at 5 and 30 min. In contrast, the hormone increased 1,2-diacylglycerol plus phosphatidate by over 200 ng/mg wet weight at 5 min under similar conditions (Bocckino, S. B., Blackmore, P. F., Wilson, P. B., and Exton, J. H. (1987) J. Biol. Chem. 262, 15309-15315). PIP2 was also significantly decreased at 15 s by angiotensin II (100 nM), ATP (100 microM), and epinephrine (1 microM). In contrast, PIP was not significantly changed, and PI was significantly decreased (by approximately 15%) at later times (15 and 30 min). The changes in phosphoinositide mass were well correlated with changes in labeled phosphoinositides in hepatocytes previously incubated with [3H]inositol for 90 min. The amounts of inositol phospholipids in liver plasma membranes (in micrograms/mg protein) were 2.1 +/- 0.2 for PIP2, 0.24 +/- 0.03 for PIP, and 23 +/- 4 for PI. Comparison of these values with those for whole cells suggests that PIP2 is enriched in the plasma membrane, whereas PIP is present elsewhere in the cell. The fatty acid composition of whole cell PIP2 showed significant differences from that of PI. The percentages of palmitic, stearic, linoleic, and arachidonic acids were, respectively, 14, 41, 10, and 25 for PIP2 and 10, 34, 7, and 37 for PI. Vasopressin treatment for 15 s did not alter the fatty acid composition of PIP2. The corresponding fatty acid percentages for liver plasma membranes were 13, 41, 11, and 21 for PIP2 and 8, 34, 0, and 40 for PI. The fatty acid composition of PIP in whole cells and plasma membranes resembled that of PIP2.(ABSTRACT TRUNCATED AT 400 WORDS)
Augert
G
G
Howard Hughes Medical Institute, Vanderbilt University School of Medicine, Nashville, Tennessee 37232.
Blackmore
P F
PF
Exton
J H
JH
D000255:Adenosine Triphosphate / Q000494:pharmacology; D000804:Angiotensin II / Q000494:pharmacology*; D000818:Animals; D001127:Arginine Vasopressin / Q000494:pharmacology*; D002462:Cell Membrane / Q000378:metabolism; D002478:Cells, Cultured; D004837:Epinephrine / Q000494:pharmacology*; D005227:Fatty Acids / Q000032:analysis; D007700:Kinetics; D008099:Liver / Q000187:drug effects / Q000378:metabolism*; D008297:Male; D008563:Membrane Lipids / Q000096:biosynthesis / Q000302:isolation & purification; D010716:Phosphatidylinositols / Q000096:biosynthesis* / Q000302:isolation & purification; D051381:Rats; D012016:Reference Values
D005227:Fatty Acids; D008563:Membrane Lipids; D010716:Phosphatidylinositols; D000804:Angiotensin II; D001127:Arginine Vasopressin; D000255:Adenosine Triphosphate; D004837:Epinephrine