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Biochemical Society Transactions ( 1 991 ) 20
Reversible uncoupling of phosphoinositidase C activation and steroldogenesis In cultured bovine adrenocortical zfr cells. Colin D. Clyne, Eric R.T. Lightly', Mark Dochrell', Ian M. Bird', Simon W. Walker' and Brent C. Williams'.
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Dept. Pharmacology, 1 George Sq., Edinburgh Scotland. *Dept. Clinical Chemistry, Edinburgh Royal Infirmary, Edinburgh EH3 9YW Scotland. 'Dept Medicine, Western General Hospital, Edinburgh EH4 WU Scotland. ACTH and adr stimulate F secretion from primary cultures of bovine zfr cells through increased AC activity, while All and ACh stimulate steroidogenesis through activation of PIC [l ]. Although F secretion to all agonists is maximal after 48 h of culture, cells are consistenly less responsive to All and ACh after 24 h than when freshly isolated. We have investigated whether this reflects changes in second messenger formation, or alterations in the coupling of this signal to steroidogenesis. Bovine zfr cells were isolated and maintained in culture as previously described [2] in 12 well culture plates at a density of 333000 cells/well. Immediately following isolation, and after 24, 48, 72 and 96 h of primary culture, F secretion, cAMP production and PIC activity were determined in response to ACTH (O.lnM), Adr (1uM). All (O.luM), ACh (O.lmM), 8-Br cAMP (1mM) and a combination of A231 87/PMA (both 1OuM). All incubations were performed in triplicate, and experiments repeated at least twice. Details of the agonist incubations and assay procedures can be found in [3]. Mean basal values for F secretion were 6.80, 7.48, 13.10, 11.76 and 15.12 pmol/well/h-/I OOug protein for cells at 0, 24, 48, 72 and 96 h respectively. ACTH and Adr-stimulated F secretion increased over the first 72 h (pcO.Ol), and declined thereafter (n-fold stimulation ratios : 20.4, 32.1, 51.4,69.9 and 35.5 for ACTH, and 1.0, 2.1, 10.9, 17.4 and 9.1 for Adr, for cells at 0, 24, 48, 72 and 96 h respectively). These changes were accompanied by a corresponding increase in cAMP formation over the salile time period, followed by a decrease at 96 h (see fig 1 for ACTH data). The F secretory response to 8-Br cAMP also increased from initial isolation to 72 h, and decreased thereafter (n-fold stimulation ratios : 8.6,28.3,44.2, 69.0, 32.1 at 0, 24, 48, 72 and 96 h respectively). In contrast, the F secretory response to All and ACh was reduced after 24 h (All: pc0.01; ACh pc0.05 in 2 experiments) or unchanged (1 experiment), and rose to a maximum after 48 h before again declining (n-fold stimulation ratios : 43.1, 22.3, 127.1, 68.7 and 21.6 for All, and 4.5, 2.5, 25.1, 11.6 and 2.3 for ACh, for cells at 0, 24, 48, 72 and 96 h respectively).However, the PIC response to All and ACh over this time period did not parallel the changes in F secretion, but was significantly increased (pc0.05) for both agonists at 24 h, rising further at 48 h, before declining by 72 h. (Mean basal [3H]inositol phosphates labelling was 345, 657, 723, 346 and 321 dpm1lOOug protein at 0.24, 48, 72 and 96 h respectively. Corresponding n-fold stimulation Abbreviations used : ACTH, adrenocorticotrophin.Adr. adrenaline. All, angiotensin It. ACh, acetylcholine. F, cortisol. 8-Br CAMP, 8-bromoadenosineB',3qclic monophosphate. PMA, phorbol 12Myristate 13-Acetate. PIC, phosphoinositidase C. AC, adenylate cyclase. PKC, protein kinase C. zfr, zonae fasciculatalreticularis. DAG, diacylglycerol.
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Figure 1 upper panel: Cortisol secretion (1 h) ( 0 )and cAMP formation (1 h) (4) shown over the first 4 days in culture in response to ACTH (0.1nM). Bottom panel: Cortisol secretion (Ih) ( 0 )to All (O.luM), together with PIC activation (15 min) (e)over the first 4 days in culture. Data from 3 combined experiments.
ratios for All were 3.2, 8.9, 16.5, 16.2, 9.6, and 1.4, 2.6, 3.4, 2.5 and 1.3 for ACh). The F secretory response to A231871PMA was significantly reduced (pc0.05) at 24 h, and reached a maximum after 72 h, before declining at 96 h. The protein content of the wells did not vary by more than 20 % over the course of the study. These data demonstrate that while F secretion and AC activation become progressively more tightly coupled over the first 72 h in primary culture, PIC activation and steroidogenesis become uncoupled after 24 h. Thus, the increased PIC activity in response to All and ACh is not accompanied by a corresponding increase in F secretion, even though the steroid synthetic and secretory machinery must be intact, based on the results for ACTH. As the F secretory responseto A23187/PMA, which elevate intracellular calcium and activate PKC independently of receptor activation, was reduced after 24 h, it is unlikely that the defect is due to a failure of the second messengers formed on receptor activation to elevate intracellular calcium. A more likely explanation for the reduced F secretory response to PIC agonists at this time is a failure of DAG formed on receptor activation to stimulate PKC, or a loss of one or more iosoforms of PKC. 1. Bird, I.M., Walker, S.W. & Williams, B.C. (1990) J. Mol. Endocrinol. 5 191-207 2. Williams, B.C., Lightly, E.R.T., Ross, A.R., Bird, I.M. & Walker, S.W. (1989) J. Endocrinol. 121 317-324 3. Walker, S.W., Strachan, M.W.J., Lightly, E.R.T., Williams, B.C., & Bird, I.M. (1990) Mol. Cell. Endocrinol 72 227-238
Biochemical Society Transactions ( 1 991 ) 20
Reversible uncoupling of phosphoinositidase C activation and steroldogenesis In cultured bovine adrenocortical zfr cells. Colin D. Clyne, Eric R.T. Lightly', Mark Dochrell', Ian M. Bird', Simon W. Walker' and Brent C. Williams'.
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Dept. Pharmacology, 1 George Sq., Edinburgh Scotland. *Dept. Clinical Chemistry, Edinburgh Royal Infirmary, Edinburgh EH3 9YW Scotland. 'Dept Medicine, Western General Hospital, Edinburgh EH4 WU Scotland. ACTH and adr stimulate F secretion from primary cultures of bovine zfr cells through increased AC activity, while All and ACh stimulate steroidogenesis through activation of PIC [l ]. Although F secretion to all agonists is maximal after 48 h of culture, cells are consistenly less responsive to All and ACh after 24 h than when freshly isolated. We have investigated whether this reflects changes in second messenger formation, or alterations in the coupling of this signal to steroidogenesis. Bovine zfr cells were isolated and maintained in culture as previously described [2] in 12 well culture plates at a density of 333000 cells/well. Immediately following isolation, and after 24, 48, 72 and 96 h of primary culture, F secretion, cAMP production and PIC activity were determined in response to ACTH (O.lnM), Adr (1uM). All (O.luM), ACh (O.lmM), 8-Br cAMP (1mM) and a combination of A231 87/PMA (both 1OuM). All incubations were performed in triplicate, and experiments repeated at least twice. Details of the agonist incubations and assay procedures can be found in [3]. Mean basal values for F secretion were 6.80, 7.48, 13.10, 11.76 and 15.12 pmol/well/h-/I OOug protein for cells at 0, 24, 48, 72 and 96 h respectively. ACTH and Adr-stimulated F secretion increased over the first 72 h (pcO.Ol), and declined thereafter (n-fold stimulation ratios : 20.4, 32.1, 51.4,69.9 and 35.5 for ACTH, and 1.0, 2.1, 10.9, 17.4 and 9.1 for Adr, for cells at 0, 24, 48, 72 and 96 h respectively). These changes were accompanied by a corresponding increase in cAMP formation over the salile time period, followed by a decrease at 96 h (see fig 1 for ACTH data). The F secretory response to 8-Br cAMP also increased from initial isolation to 72 h, and decreased thereafter (n-fold stimulation ratios : 8.6,28.3,44.2, 69.0, 32.1 at 0, 24, 48, 72 and 96 h respectively). In contrast, the F secretory response to All and ACh was reduced after 24 h (All: pc0.01; ACh pc0.05 in 2 experiments) or unchanged (1 experiment), and rose to a maximum after 48 h before again declining (n-fold stimulation ratios : 43.1, 22.3, 127.1, 68.7 and 21.6 for All, and 4.5, 2.5, 25.1, 11.6 and 2.3 for ACh, for cells at 0, 24, 48, 72 and 96 h respectively).However, the PIC response to All and ACh over this time period did not parallel the changes in F secretion, but was significantly increased (pc0.05) for both agonists at 24 h, rising further at 48 h, before declining by 72 h. (Mean basal [3H]inositol phosphates labelling was 345, 657, 723, 346 and 321 dpm1lOOug protein at 0.24, 48, 72 and 96 h respectively. Corresponding n-fold stimulation Abbreviations used : ACTH, adrenocorticotrophin.Adr. adrenaline. All, angiotensin It. ACh, acetylcholine. F, cortisol. 8-Br CAMP, 8-bromoadenosineB',3qclic monophosphate. PMA, phorbol 12Myristate 13-Acetate. PIC, phosphoinositidase C. AC, adenylate cyclase. PKC, protein kinase C. zfr, zonae fasciculatalreticularis. DAG, diacylglycerol.
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24 48 72 98
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culture time (h)
Figure 1 upper panel: Cortisol secretion (1 h) ( 0 )and cAMP formation (1 h) (4) shown over the first 4 days in culture in response to ACTH (0.1nM). Bottom panel: Cortisol secretion (Ih) ( 0 )to All (O.luM), together with PIC activation (15 min) (e)over the first 4 days in culture. Data from 3 combined experiments.
ratios for All were 3.2, 8.9, 16.5, 16.2, 9.6, and 1.4, 2.6, 3.4, 2.5 and 1.3 for ACh). The F secretory response to A231871PMA was significantly reduced (pc0.05) at 24 h, and reached a maximum after 72 h, before declining at 96 h. The protein content of the wells did not vary by more than 20 % over the course of the study. These data demonstrate that while F secretion and AC activation become progressively more tightly coupled over the first 72 h in primary culture, PIC activation and steroidogenesis become uncoupled after 24 h. Thus, the increased PIC activity in response to All and ACh is not accompanied by a corresponding increase in F secretion, even though the steroid synthetic and secretory machinery must be intact, based on the results for ACTH. As the F secretory responseto A23187/PMA, which elevate intracellular calcium and activate PKC independently of receptor activation, was reduced after 24 h, it is unlikely that the defect is due to a failure of the second messengers formed on receptor activation to elevate intracellular calcium. A more likely explanation for the reduced F secretory response to PIC agonists at this time is a failure of DAG formed on receptor activation to stimulate PKC, or a loss of one or more iosoforms of PKC. 1. Bird, I.M., Walker, S.W. & Williams, B.C. (1990) J. Mol. Endocrinol. 5 191-207 2. Williams, B.C., Lightly, E.R.T., Ross, A.R., Bird, I.M. & Walker, S.W. (1989) J. Endocrinol. 121 317-324 3. Walker, S.W., Strachan, M.W.J., Lightly, E.R.T., Williams, B.C., & Bird, I.M. (1990) Mol. Cell. Endocrinol 72 227-238
