Skip to main content
PMC home page
Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 1986 Nov;83(22):8540–8544. doi: 10.1073/pnas.83.22.8540

Phosphatidylinositol 4,5-bisphosphate turnover is transient while phosphatidylinositol turnover is persistent in thyrotropin-releasing hormone-stimulated rat pituitary cells.

A Imai, M C Gershengorn
PMCID: PMC386966  PMID: 3022295

Abstract

Stimulated inositolphospholipid turnover has been proposed to be initiated and sustained by hydrolysis of phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2], which may be replenished by an enhanced flux of phosphatidylinositol (PtdIns) to PtdIns 4-phosphate (PtdIns4P) to PtdIns(4,5)P2. To determine whether there is continued hydrolysis and resynthesis of PtdIns(4,5)P2 in rat pituitary cells (GH3 cells) during stimulation by thyrotropin-releasing hormone (TRH), we investigated the turnover kinetics of the inositolphospholipids and of phosphatidic acid (PtdOH). In cells incubated with 32Pi for 1 min, TRH rapidly and persistently (for at least 30 min) enhanced the rate of 32P-labeling of PtdOH. After a lag time of 1 min, TRH markedly and persistently increased 32P-labeling of PtdIns also. In contrast, TRH caused only a transient increase in 32P-labeling of PtdIns(4,5)P2 that lasted less than 2 min. There was no rapid (before 10 min) effect of TRH on 32P-labeling of PtdIns4P. By 2 min of TRH stimulation, specific 32P radioactivity in PtdOH increased from 3.6% (control) of that in the gamma-phosphate of ATP to 15%; in PtdIns, from 0.07% to 1.3%; and in PtdIns(4,5)P2, from 3.8% to 5.4% (specific 32P radioactivity in PtdIns4P was 1.7% of that in ATP in control and TRH-stimulated cells). In cells exposed to TRH for 4 min and then to 32Pi, 32P-labeling of PtdOH and PtdIns increased, but that of PtdIns(4,5)P2 was not affected. Last, persistent turnover of PtdOH and PtdIns was not caused by initial hydrolysis of PtdIns(4,5)P2 because the turnover of PtdOH and PtdIns could be terminated by displacement of TRH from its receptor by chlordiazepoxide and restarted by reoccupying the receptors with TRH. These data demonstrate that turnover of PtdIns(4,5)P2 is stimulated only transiently, whereas turnover of PtdIns and PtdOH is stimulated persistently by TRH in GH3 cells. Hence, inositolphospholipid turnover in GH3 cells does not occur via continued hydrolysis of PtdIns(4,5)P2 accompanied by enhanced flux of PtdIns to PtdIns4P to PtdIns(4,5)P2, but there is direct and persistent hydrolysis of PtdIns. The dissociation of these actions suggests that there are separate mechanisms involved in coupling TRH-receptor complexes to stimulation of PtdIns(4,5)P2 and PtdIns hydrolysis in GH3 cells.

Full text

PDF
8540

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Berridge M. J. Inositol trisphosphate and diacylglycerol as second messengers. Biochem J. 1984 Jun 1;220(2):345–360. doi: 10.1042/bj2200345. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Broekman M. J., Ward J. W., Marcus A. J. Phospholipid metabolism in stimulated human platelets. Changes in phosphatidylinositol, phosphatidic acid, and lysophospholipids. J Clin Invest. 1980 Aug;66(2):275–283. doi: 10.1172/JCI109854. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Drummond A. H., Bushfield M., Macphee C. H. Thyrotropin-releasing hormone-stimulated [3H]inositol metabolism in GH3 pituitary tumor cells. Studies with lithium. Mol Pharmacol. 1984 Mar;25(2):201–208. [PubMed] [Google Scholar]
  4. Drummond A. H. Chlordiazepoxide is a competitive thyrotropin-releasing hormone receptor antagonist in GH3 pituitary tumour cells. Biochem Biophys Res Commun. 1985 Feb 28;127(1):63–70. doi: 10.1016/s0006-291x(85)80126-1. [DOI] [PubMed] [Google Scholar]
  5. Gershengorn M. C., Marcus-Samuels B. E., Geras E. Estrogens increase the number of thyrotropin-releasing hormone receptors on mammotropic cells in culture. Endocrinology. 1979 Jul;105(1):171–176. doi: 10.1210/endo-105-1-171. [DOI] [PubMed] [Google Scholar]
  6. Gershengorn M. C. Mechanism of thyrotropin releasing hormone stimulation of pituitary hormone secretion. Annu Rev Physiol. 1986;48:515–526. doi: 10.1146/annurev.ph.48.030186.002503. [DOI] [PubMed] [Google Scholar]
  7. Griendling K. K., Rittenhouse S. E., Brock T. A., Ekstein L. S., Gimbrone M. A., Jr, Alexander R. W. Sustained diacylglycerol formation from inositol phospholipids in angiotensin II-stimulated vascular smooth muscle cells. J Biol Chem. 1986 May 5;261(13):5901–5906. [PubMed] [Google Scholar]
  8. Hinkle P. M., Tashjian A. H., Jr Thyrotropin-releasing hormone regulates the number of its own receptors in the GH3 strain of pituitary cells in culture. Biochemistry. 1975 Aug 26;14(17):3845–3851. doi: 10.1021/bi00688a017. [DOI] [PubMed] [Google Scholar]
  9. Hokin L. E. Receptors and phosphoinositide-generated second messengers. Annu Rev Biochem. 1985;54:205–235. doi: 10.1146/annurev.bi.54.070185.001225. [DOI] [PubMed] [Google Scholar]
  10. Holmsen H., Dangelmaier C. A., Akkerman J. W. Determination of levels of glycolytic intermediates and nucleotides in platelets by pulse-labeling with [32P]orthophosphate. Anal Biochem. 1983 May;131(1):266–272. doi: 10.1016/0003-2697(83)90165-3. [DOI] [PubMed] [Google Scholar]
  11. Imai A., Gershengorn M. C. Evidence for tight coupling of thyrotropin-releasing hormone receptors to stimulated inositol trisphosphate formation in rat pituitary cells. J Biol Chem. 1985 Sep 5;260(19):10536–10540. [PubMed] [Google Scholar]
  12. Imai A., Ishizuka Y., Nakashima S., Nozawa Y. Differential activation of membrane phospholipid turnover by compound 48/80 and ionophore A23187 in rat mast cells. Arch Biochem Biophys. 1984 Jul;232(1):259–268. doi: 10.1016/0003-9861(84)90542-3. [DOI] [PubMed] [Google Scholar]
  13. Irvine R. F., Letcher A. J., Heslop J. P., Berridge M. J. The inositol tris/tetrakisphosphate pathway--demonstration of Ins(1,4,5)P3 3-kinase activity in animal tissues. Nature. 1986 Apr 17;320(6063):631–634. doi: 10.1038/320631a0. [DOI] [PubMed] [Google Scholar]
  14. Irvine R. F. The enzymology of stimulated inositol lipid turnover. Cell Calcium. 1982 Oct;3(4-5):295–309. doi: 10.1016/0143-4160(82)90018-5. [DOI] [PubMed] [Google Scholar]
  15. Koréh K., Monaco M. E. The relationship of hormone-sensitive and hormone-insensitive phosphatidylinositol to phosphatidylinositol 4,5-bisphosphate in the WRK-1 cell. J Biol Chem. 1986 Jan 5;261(1):88–91. [PubMed] [Google Scholar]
  16. Leiter A. B., Weinberg M., Isohashi F., Utter M. F. Relationshiop between phosphorylation and activity of pyruvate dehydrogenase in rat liver mitochondria and the absence of such a relationship for pyruvate carboxylase. J Biol Chem. 1978 Apr 25;253(8):2716–2723. [PubMed] [Google Scholar]
  17. Majerus P. W., Neufeld E. J., Wilson D. B. Production of phosphoinositide-derived messengers. Cell. 1984 Jul;37(3):701–703. doi: 10.1016/0092-8674(84)90405-7. [DOI] [PubMed] [Google Scholar]
  18. Martin T. F., Lucas D. O., Bajjalieh S. M., Kowalchyk J. A. Thyrotropin-releasing hormone activates a Ca2+-dependent polyphosphoinositide phosphodiesterase in permeable GH3 cells. GTP gamma S potentiation by a cholera and pertussis toxin-insensitive mechanism. J Biol Chem. 1986 Feb 25;261(6):2918–2927. [PubMed] [Google Scholar]
  19. Michell R. H., Kirk C. J., Jones L. M., Downes C. P., Creba J. A. The stimulation of inositol lipid metabolism that accompanies calcium mobilization in stimulated cells: defined characteristics and unanswered questions. Philos Trans R Soc Lond B Biol Sci. 1981 Dec 18;296(1080):123–138. doi: 10.1098/rstb.1981.0177. [DOI] [PubMed] [Google Scholar]
  20. Rebecchi M. J., Gershengorn M. C. Thyroliberin stimulates rapid hydrolysis of phosphatidylinositol 4,5-bisphosphate by a phosphodiesterase in rat mammotropic pituitary cells. Evidence for an early Ca2+-independent action. Biochem J. 1983 Nov 15;216(2):287–294. doi: 10.1042/bj2160287. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Rebecchi M. J., Kolesnick R. N., Gershengorn M. C. Thyrotropin-releasing hormone stimulates rapid loss of phosphatidylinositol and its conversion to 1,2-diacylglycerol and phosphatidic acid in rat mammotropic pituitary cells. Association with calcium mobilization and prolactin secretion. J Biol Chem. 1983 Jan 10;258(1):227–234. [PubMed] [Google Scholar]
  22. Rebecchi M. J., Monaco M. E., Gershengorn M. C. Thyrotropin releasing hormone rapidly enhances [32P]orthophosphate incorporation into phosphatidic acid in cloned GH3 cells. Biochem Biophys Res Commun. 1981 Jul 16;101(1):124–130. doi: 10.1016/s0006-291x(81)80019-8. [DOI] [PubMed] [Google Scholar]
  23. Seyfred M. A., Farrell L. E., Wells W. W. Characterization of D-myo-inositol 1,4,5-trisphosphate phosphatase in rat liver plasma membranes. J Biol Chem. 1984 Nov 10;259(21):13204–13208. [PubMed] [Google Scholar]
  24. Storey D. J., Shears S. B., Kirk C. J., Michell R. H. Stepwise enzymatic dephosphorylation of inositol 1,4,5-trisphosphate to inositol in liver. Nature. 1984 Nov 22;312(5992):374–376. doi: 10.1038/312374a0. [DOI] [PubMed] [Google Scholar]
  25. Straub R. E., Gershengorn M. C. Thyrotropin-releasing hormone and GTP activate inositol trisphosphate formation in membranes isolated from rat pituitary cells. J Biol Chem. 1986 Feb 25;261(6):2712–2717. [PubMed] [Google Scholar]
  26. Wilson D. B., Neufeld E. J., Majerus P. W. Phosphoinositide interconversion in thrombin-stimulated human platelets. J Biol Chem. 1985 Jan 25;260(2):1046–1051. [PubMed] [Google Scholar]

Articles from Proceedings of the National Academy of Sciences of the United States of America are provided here courtesy of National Academy of Sciences

RESOURCES