Phosphatidylinositol 4,5-bisphosphate hydrolysis in turkey erythrocytes is regulated by P2y purinoceptors.
When intact [3H]inositol-loaded turkey erythrocytes were stimulated with the purinergic agonist ADP, there was a rapid increase (2.5-fold after 30 sec) in the intracellular content of [3H]inositol 1,4,5-trisphosphate, followed by increases in the levels of [3H]inositol bisphosphate and [3H]inositol 1,3,4,5-tetrakisphosphate (4-fold and 5-fold, respectively, after 3 min). [3H]inositol monophosphate levels did not rise in the first 3 min of ADP stimulation but increased slowly thereafter, demonstrating that the primary response of turkey erythrocytes to purinergic stimulation is hydrolysis of phosphatidylinositol 4,5-bisphosphate. Inositol phosphate accumulation was evoked by a P2y purinoceptor, as indicated by the rank order of potencies of a variety of purinergic agonists. 2-Methylthioadenosine 5'-triphosphate was the most potent agonist tested, with an EC50 value of 0.36 microM. High performance liquid chromatography analysis demonstrated the presence of three distinct inositol tetrakisphosphate isomers in [3H]inositol-loaded turkey erythrocytes, inositol 1,3,4,6-tetrakisphosphate [Ins(1,3,4,5)P4], inositol 1,3,4,6-tetrakisphosphate [Ins(1,3,4,6)P4], and inositol 3,4,5,6-tetrakisphosphate. Prolonged stimulation with adenosine 5'-O-(2-thiodiphosphate), a nonhydrolyzable analogue of ADP, resulted in a 60-fold increase in the level of [3H]Ins(1,3,4,5)P4, whereas a substantial rise in the [3H]Ins(1,3,4,6)P4 fraction was also seen. These results indicate that turkey erythrocytes represent a valuable model system for studies of purinoceptor function as well as fundamental aspects of cell surface receptor-regulated phosphoinositide metabolism.
Berrie
C P
CP
Department of Cellular Pharmacology, Smith Kline and French Research Limited, Welwyn, England.
Hawkins
P T
PT
Stephens
L R
LR
Harden
T K
TK
Downes
C P
CP
D000244:Adenosine Diphosphate / Q000494:pharmacology; D000818:Animals; D002851:Chromatography, High Pressure Liquid; D004912:Erythrocytes / Q000378:metabolism*; D007295:Inositol Phosphates / Q000097:blood*; D019269:Phosphatidylinositol 4,5-Diphosphate; D010716:Phosphatidylinositols / Q000097:blood*; D011687:Purines / Q000494:pharmacology*; D011983:Receptors, Purinergic / Q000097:blood* / Q000187:drug effects; D013403:Sugar Phosphates / Q000097:blood*; D014422:Turkeys
D007295:Inositol Phosphates; D019269:Phosphatidylinositol 4,5-Diphosphate; D010716:Phosphatidylinositols; D011687:Purines; D011983:Receptors, Purinergic; D013403:Sugar Phosphates; D000244:Adenosine Diphosphate