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Expression of β2-adrenoceptors mediating cyclic AMP accumulation in a neuronal cell line
2162 cAMP at later stages abolished further rise, but when steady state levels of p-adrenoceptors were achieved, 8-bromo-cAMP did not mimick the acute desensitization of adenylate cyclase caused by isoproterenol or glucagon. The aoquisition of ~-receptors and /~-adrenergic responsiveness was also inhibited (strongly) by forskolin and ( p i n , ally) by glucagon. Preliminary data suggest that cAMP-elevating agents also reduce the level of mRNA for the
~-~renoceptor~ The results suggest that whereas in hepatocytes cAMP appears not to be involved in the short-term homologous or heterologom desensitization of ~adrenoceptor-linked adenylate cyclase, the mechanisms of long-term regulation include a suppressive effect of cAMP on /~.adrenoceptor synthesis, possibly exerted at least in part at the transeriptional level. References Refsnes, M., D. Sandnes, ~. Melien, "I.E. Sand, S. Jacobsen and 2". Christoffersen, 1983, FEBS Letters, 164, 291. Sandnes, D., T.E. Sand, G. Sager, G.O. Bmnstad, M.IL Refsnes, l.P. Gladhaug, S. Jacobsen and T. Christoffersen, 1986, Exp. Cell Res., 165, 117. Refsnes, M., D. Sandnes and T. Christoffersen, 1987, Eur. J. Biochem., 163, 457.
P.fr.052 ]
Expression of jO.~-~oceptors mediating cyclic AMP accumulation in a neuronal cell line Hill, S3., R~¢k, A., Millns, P. a n d Kendall, D.A. of PhysiologFand Pharma~logF, Meth'calSchoo~ Queen's Medical Centre, Nottingham NG7 2UH, U.K.
Rat CNS BS0 cells exhibit a number of properties characteristic of a neuronal cell line (Schubert et al., 1974). e.g. The cells possess an ~xcitable membrane, extend processes in serum-free and dibutyryl cyclic AMP-containing media and express the neurotransmitter synthetic enzymes tyrosine hydroxylase and glutamic acid decarboxylase (Schubert et al., 1974). In this communication we demonstrate that isoprenaline and noradrenaiine produce a marked stimulation of cyclic AMP accumulation in this cell line which is mediated by/~-adrenoeeptor stimulation. B50 neuronal and C6 glioma cells were grown in DMEM medium supplemented with 2mM glutamine and 105 foetal calf serum at 370C under an atmosphere of 10~ CO~. in humidified air. Cyclic AMP accumulation was measured in confluent cells prelabelled with [3HI-adenine as described by Donaldson et al., (1988). Experiments were performed in the presence of the pbosphodiesterase inhibitor rolipram (0.1 mM). Where appropriate antagonists were preincubated with the cells for 30 min prior to addition of isoprenaline (0.1 pM, C6 cells or 0.5/zM, B50 cells). Incubations were terminated after a further 10 rain by addition of I M HCi and H-cyclic AMP was separated from 3H-adenine by sequential Dowex-Alumina chromatography as described previously (Donaldson et al., 1988). Noradrenaline (NA) and isoprenaline (ISO) stimulated cyclic AMP accumulation in both B50 (NA - ECs0 - 11 ± 3 /~M, n --4; I S O - ECso --0.11 ± 0.02/zM, n - 6 ) and C6 (NA - ECso =0.19 ± 0.06/tM, n -- 3; ISO - ECso --0.013 ± 0.001 ~M, n --- 3) ceil lines. Isoprenaline (0.5/zM) produced a rapid 01/2 - 1.8 min) and sustained increase in H-cyclic AMP accumulation which was maintained for up to 120 rain. The response to isoprenaline (0.1 tzM) in C6 cells was somewhat slower (tt/2 circa 5-8 min) but was again maintained at an elevated level for at least 120 min. The response to 0.5/AM isoprenaline was antagonised by both propranolol (ICso = 8.4 ± 1.6 × 10 -9 M; n -- 3) and the/~-selective antagonist ICI 118551 (ICso = 1.8 ± 0.3 × 10 -9 M; n -- 7). However~ no a:',enuation of the response to isoprenaline (0.5 ;zM) was observed at concentrations of the/31-adrenoceptor ant3gonist atenoloi at concentrations up to 10 ~M (n -- 3). In contrast, in C6 cells, which have previously been shown to possess/~l-adrenoceptors (Harden and McCarthy, 1982), atenolol inhibited isoprenaline-induced (0.1/tM) cyclic A M P accumulation (ICso - 1"6 ± 0.8/tM; n = 7). Furthermore the P2-selective antagonist ICI 118551 was much less potent in the C6 cell line (ICso - 2.2 ± 0.5 × 10 M; n = 3) while propranolol was approximately an order of magnitude weaker (IC5o - 7.8 ± 0.2 × 1 0 - ' M; n -- 3) than ih the 1350 cells.
2163 In conclusion, the present data suggest that i~oprenaline mediates cyclic AMP accumulation in BSO cells via activation of flz-adrenoceptors, while in C6 cells the cyclic AMP response is mediated by ~i-adrenoceptors.
References Donaldson, J., Hill, S.J. and Brown, A.M. 1988, Molec. Pharmacol. 33, 626. Harden, T.K. and McCarthy, K.D., 1982, J. Pharmacol. Exp. Ther. 222, 600. Schubert, D., Heinemann, S., Carlisle, W., Tarikas, H., Kimes, B., Patrick, J. Steinbach, J.H., Culp, W. and Brandt, B.L., 1974, Nature, 249, 224.
I P.fr.053 [
Desensitization of the beta-adrenoceptor coupled adenylyl cyclase system by isowenafine in cultured human lymphoblast cells Kay, O. and Elliott, J.M. Dept. of Pharmacologr mid Toxicologg, St. Mary's Hospital Medical School, London W2 I PG. U.K.
Exposure of lymphocytes to isoprenaline leads to a rapid loss in the beta-adrenoceptor coupled adenylyl cyclase activity. The extent and manne," of de~nsitization depends upon the duration of exposure and the isoprenaline concentration (Sandnes et al., 1987). We have previously demonstrated that incubation of huma~ lymphoblast cells with isoprenaline causes down-regulation of beta-adrenceptor number in a concentration and time dependent manner (Kay and Elliott, 1988). We have therefore examined the adenylyl cyclase response to various agonists in cultured lymphoblast cells after chronic coincubation with isoprenaline. Stable lymphoblastoid cell lines from 8 subjects were maintained in RPMI 1640 medium supplemented with 10~ foetal calf serum. Confluent cells were coincubated with isoprenaline (10-7-10 -9 M) for 4 h, 24 h or 7 days. Coincubations were stopped by centrifugafion and the cells washed and then resuspended in a balanced salt solution as described by Ebstein et al., (1985). Adenylyl cyclase was stimulated by isoprenaline (0.1 and 10 ~M), PGE1 (0.1 and 1 ~tM) or forskolin (100 ~tM) and then the cyclic AMP determined by a protein binding assay. Desensitization of beta-adrenoceptor coupled adenylyl cyclase respoase by isoprenaline was concentration and time dependent. The reduction in cyclic AMP accumulation after 4 h coincubation with 10 -s M isoprenaline was 24~ whereas with 10 -7 M it was 98~. After 24 h coincubation with 10 -* M isoprenaline there was a 36~ loss of cyclic AMP which increased to 90~; with 10 -7 M isoprenaline. After 7 day coincubation with 10 -9 M isoprenaline the reduction in cyclic AMP was 87~. The extent of desensit'-tzation broadly paralleled the down-regulation of beta-adrenoceptor number which we have previously reported (Kay and Elliott, 1988). The adenylyl cyclase response to PGE1 and to forskolin was unaffected by coincubation with 10 -9 M isoprenaline at any of the time points but was reduced by up to 70~; following 10 -7 M isoprenaline coincubation for 4 h or 24 h. These data indicate that the loss of beta-adrenoceptors in human iymphoblast cells following isoprenaline coincubation is accompanied by a decrease in receptor function as defined by adenylyl cyclase activity. The extent and manner of desensitization is concentration and time dependent since low concentrations of isoprenaline reduced the beta-adrenoceptor stimulated adenylyl cyclase activity alone (homologous desensitization) whereas higher concentrations or longer duration of coincubation additionally decreased the response to stimulation by PGE1 and forskolin (heterologous desensitization). We conclude that beta-adrenoceptor regulation in the cultured lymphoblast is similar to that in the lymphocyte and that this cell provides a useful model to investigate beta-adrenoceptor function in man.
References Ebstein, R.P. et al., 1985, Experientia, 41, 1552-1554. Kay, G. and Elliott, J.M., 1988, Br. J. Pharmacol., 95, 740P. Sandnes, D. et al., 1987, Biochem. Pharmacol., 36, 1303-1311.
~-~renoceptor~ The results suggest that whereas in hepatocytes cAMP appears not to be involved in the short-term homologous or heterologom desensitization of ~adrenoceptor-linked adenylate cyclase, the mechanisms of long-term regulation include a suppressive effect of cAMP on /~.adrenoceptor synthesis, possibly exerted at least in part at the transeriptional level. References Refsnes, M., D. Sandnes, ~. Melien, "I.E. Sand, S. Jacobsen and 2". Christoffersen, 1983, FEBS Letters, 164, 291. Sandnes, D., T.E. Sand, G. Sager, G.O. Bmnstad, M.IL Refsnes, l.P. Gladhaug, S. Jacobsen and T. Christoffersen, 1986, Exp. Cell Res., 165, 117. Refsnes, M., D. Sandnes and T. Christoffersen, 1987, Eur. J. Biochem., 163, 457.
P.fr.052 ]
Expression of jO.~-~oceptors mediating cyclic AMP accumulation in a neuronal cell line Hill, S3., R~¢k, A., Millns, P. a n d Kendall, D.A. of PhysiologFand Pharma~logF, Meth'calSchoo~ Queen's Medical Centre, Nottingham NG7 2UH, U.K.
Rat CNS BS0 cells exhibit a number of properties characteristic of a neuronal cell line (Schubert et al., 1974). e.g. The cells possess an ~xcitable membrane, extend processes in serum-free and dibutyryl cyclic AMP-containing media and express the neurotransmitter synthetic enzymes tyrosine hydroxylase and glutamic acid decarboxylase (Schubert et al., 1974). In this communication we demonstrate that isoprenaline and noradrenaiine produce a marked stimulation of cyclic AMP accumulation in this cell line which is mediated by/~-adrenoeeptor stimulation. B50 neuronal and C6 glioma cells were grown in DMEM medium supplemented with 2mM glutamine and 105 foetal calf serum at 370C under an atmosphere of 10~ CO~. in humidified air. Cyclic AMP accumulation was measured in confluent cells prelabelled with [3HI-adenine as described by Donaldson et al., (1988). Experiments were performed in the presence of the pbosphodiesterase inhibitor rolipram (0.1 mM). Where appropriate antagonists were preincubated with the cells for 30 min prior to addition of isoprenaline (0.1 pM, C6 cells or 0.5/zM, B50 cells). Incubations were terminated after a further 10 rain by addition of I M HCi and H-cyclic AMP was separated from 3H-adenine by sequential Dowex-Alumina chromatography as described previously (Donaldson et al., 1988). Noradrenaline (NA) and isoprenaline (ISO) stimulated cyclic AMP accumulation in both B50 (NA - ECs0 - 11 ± 3 /~M, n --4; I S O - ECso --0.11 ± 0.02/zM, n - 6 ) and C6 (NA - ECso =0.19 ± 0.06/tM, n -- 3; ISO - ECso --0.013 ± 0.001 ~M, n --- 3) ceil lines. Isoprenaline (0.5/zM) produced a rapid 01/2 - 1.8 min) and sustained increase in H-cyclic AMP accumulation which was maintained for up to 120 rain. The response to isoprenaline (0.1 tzM) in C6 cells was somewhat slower (tt/2 circa 5-8 min) but was again maintained at an elevated level for at least 120 min. The response to 0.5/AM isoprenaline was antagonised by both propranolol (ICso = 8.4 ± 1.6 × 10 -9 M; n -- 3) and the/~-selective antagonist ICI 118551 (ICso = 1.8 ± 0.3 × 10 -9 M; n -- 7). However~ no a:',enuation of the response to isoprenaline (0.5 ;zM) was observed at concentrations of the/31-adrenoceptor ant3gonist atenoloi at concentrations up to 10 ~M (n -- 3). In contrast, in C6 cells, which have previously been shown to possess/~l-adrenoceptors (Harden and McCarthy, 1982), atenolol inhibited isoprenaline-induced (0.1/tM) cyclic A M P accumulation (ICso - 1"6 ± 0.8/tM; n = 7). Furthermore the P2-selective antagonist ICI 118551 was much less potent in the C6 cell line (ICso - 2.2 ± 0.5 × 10 M; n = 3) while propranolol was approximately an order of magnitude weaker (IC5o - 7.8 ± 0.2 × 1 0 - ' M; n -- 3) than ih the 1350 cells.
2163 In conclusion, the present data suggest that i~oprenaline mediates cyclic AMP accumulation in BSO cells via activation of flz-adrenoceptors, while in C6 cells the cyclic AMP response is mediated by ~i-adrenoceptors.
References Donaldson, J., Hill, S.J. and Brown, A.M. 1988, Molec. Pharmacol. 33, 626. Harden, T.K. and McCarthy, K.D., 1982, J. Pharmacol. Exp. Ther. 222, 600. Schubert, D., Heinemann, S., Carlisle, W., Tarikas, H., Kimes, B., Patrick, J. Steinbach, J.H., Culp, W. and Brandt, B.L., 1974, Nature, 249, 224.
I P.fr.053 [
Desensitization of the beta-adrenoceptor coupled adenylyl cyclase system by isowenafine in cultured human lymphoblast cells Kay, O. and Elliott, J.M. Dept. of Pharmacologr mid Toxicologg, St. Mary's Hospital Medical School, London W2 I PG. U.K.
Exposure of lymphocytes to isoprenaline leads to a rapid loss in the beta-adrenoceptor coupled adenylyl cyclase activity. The extent and manne," of de~nsitization depends upon the duration of exposure and the isoprenaline concentration (Sandnes et al., 1987). We have previously demonstrated that incubation of huma~ lymphoblast cells with isoprenaline causes down-regulation of beta-adrenceptor number in a concentration and time dependent manner (Kay and Elliott, 1988). We have therefore examined the adenylyl cyclase response to various agonists in cultured lymphoblast cells after chronic coincubation with isoprenaline. Stable lymphoblastoid cell lines from 8 subjects were maintained in RPMI 1640 medium supplemented with 10~ foetal calf serum. Confluent cells were coincubated with isoprenaline (10-7-10 -9 M) for 4 h, 24 h or 7 days. Coincubations were stopped by centrifugafion and the cells washed and then resuspended in a balanced salt solution as described by Ebstein et al., (1985). Adenylyl cyclase was stimulated by isoprenaline (0.1 and 10 ~M), PGE1 (0.1 and 1 ~tM) or forskolin (100 ~tM) and then the cyclic AMP determined by a protein binding assay. Desensitization of beta-adrenoceptor coupled adenylyl cyclase respoase by isoprenaline was concentration and time dependent. The reduction in cyclic AMP accumulation after 4 h coincubation with 10 -s M isoprenaline was 24~ whereas with 10 -7 M it was 98~. After 24 h coincubation with 10 -* M isoprenaline there was a 36~ loss of cyclic AMP which increased to 90~; with 10 -7 M isoprenaline. After 7 day coincubation with 10 -9 M isoprenaline the reduction in cyclic AMP was 87~. The extent of desensit'-tzation broadly paralleled the down-regulation of beta-adrenoceptor number which we have previously reported (Kay and Elliott, 1988). The adenylyl cyclase response to PGE1 and to forskolin was unaffected by coincubation with 10 -9 M isoprenaline at any of the time points but was reduced by up to 70~; following 10 -7 M isoprenaline coincubation for 4 h or 24 h. These data indicate that the loss of beta-adrenoceptors in human iymphoblast cells following isoprenaline coincubation is accompanied by a decrease in receptor function as defined by adenylyl cyclase activity. The extent and manner of desensitization is concentration and time dependent since low concentrations of isoprenaline reduced the beta-adrenoceptor stimulated adenylyl cyclase activity alone (homologous desensitization) whereas higher concentrations or longer duration of coincubation additionally decreased the response to stimulation by PGE1 and forskolin (heterologous desensitization). We conclude that beta-adrenoceptor regulation in the cultured lymphoblast is similar to that in the lymphocyte and that this cell provides a useful model to investigate beta-adrenoceptor function in man.
References Ebstein, R.P. et al., 1985, Experientia, 41, 1552-1554. Kay, G. and Elliott, J.M., 1988, Br. J. Pharmacol., 95, 740P. Sandnes, D. et al., 1987, Biochem. Pharmacol., 36, 1303-1311.