Abstract
Intracellular Ca2+ mobilization events were assessed in mouse L cells, which contain native prostaglandin E1 receptors and transfected human β2 adrenergic receptors. Both Fura2 (single cell measurements) and Quin 2, (cuvette assays) were used to determine [Ca2+]i levels. Our results demonstrate that in the transfected cells there is a dose-dependent increase in [Ca2+]i in response to isoproterenol (0.1 nM–100 nM), which is inhibited by the β-adrenergic antagonist, propranolol, and is a result of intracellular Ca2+ release. [Ca2+]1 in these cells was also increased by prostaglandin E1, 8 bromo cyclic AMP, and aluminum fluoride. Both 8 bromo cAMP and isoproterenol induced a rapid increase in the levels of IP1, IP2, and IP3. The data presented demonstrate that the elevation of intracellular cyclic AMP induces an increase in IP3 production which leads to an elevation in [Ca2+];. We propose that this cyclic AMP dependent activation of the IP3 generating system occurs at a post-receptor site.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
Abbreviations
- cAMP:
-
Adenosine Cyclic 3′-5′-Monophosphate
- [Ca2+]i :
-
intracellular [Ca2+]i
- 8 Br cAMP:
-
8 Bromo Adenosine Cyclic 3′-5′-Monophosphate
- DAG:
-
Diacylglycerol
- EGTA]:
-
[Ethylene Bis (oxyethylenenitrilo)] Tetracetic acid
- BSA:
-
Bovine Serum Albumin
- HBSS-H:
-
Hanks' Balanced Salt Solution buffered with HEPES to pH 7.4
- HEPES:
-
4-(2-Hydroxyethyl)-1-piperazineethanesulfonic acid
- PIP2 :
-
Phosphatidylinositol 4,5-bisphosphate
- IP2 :
-
Inositol 4 Phosphate
- IP2 :
-
Inositol 4,5 Bisphosphate
- IP3 :
-
Inositol Trisphosphate
- PGE1 :
-
Prostaglandin E1
- PBS:
-
Phosphate Buffered Saline Solution
References
Berridge MJ: Inositol trisphosphate and diacylglycerol: two interacting second messengers. Ann Rev Biochem 56: 159–193, 1987.
Exton J: Mechanisms of action of calcium mobilizing agonists: some variations on a young theme. Faseb J 2: 2670–2676, 1988
Gilman AG: G proteins: Transducers of receptor-generated signals. Ann Rev Biochem 56: 615–650, 1987
Yoshimasa T, Sibley DR, Bovier M, Lefkowitz RJ, Caron MJ: Cross-talk between cellular signalling pathways suggested by phorbol-ester-induced adenylate cyclase phosphorylation. Nature 327: 67–70, 1987
Hill SJ, Kendal DA: Cross-talk between different receptor-effector systems in the mammalian CNS. In: Cell Signalling, vol. 1, Pergamon Press, 1989, 135–141
Horn VJ, Baum BJ, Ambudkar IS: R adrenergic receptor stimulation induces inositol trisphosphate production and Ca2− mobilization in rat parotid acinar cells. J Biol Chem 263: 12454–12460, 1988
Laufer R, Changeux JP: Calcitonin gene-related peptide and cyclic AMP stimulate phosphoinositide turnover in skeletal muscle cells. J Biol Chem 264: 2683–2689, 1989
Fraser CM, Chung F-Z, Venter CJ: Continuous high density expression of human β2-adrenergic receptors in a mouse cell line previously lacking R-receptors. J Biol Chem 262: 14843–14846, 1987
Chung F-Z, Wang C-D, Potter PC, Venter JC, Fraser CM: Site directed mutagenesis and continuous expression of human β-adrenergic receptors. J Biol Chem 263: 4052–4055, 1988
Steinberg TH, Newman AS, Swanson JA, Silverstein SC: Macrophages possess probenecid-inhibitable organic anion transporters that remove fluorescent dyes from the cytoplasmic matrix. J Cell Biol 105: 2695–2702, 1987
Berridge MJ, Dawson RMC, Downes CP, Heslop JP, Irvine RF: Changes in the level of inositol phosphates following agonist-dependent hydrolysis of membrane phosphoinositides. Biochem J 212: 473–482, 1983
Blackmore PE, Exton JH. Studies on the hepatic calciummobilizing activity of aluminum fluoride and glucagon. J Biol Chem 25: 11056–11063, 1986
Mertz LM, Horn VJ, Baum BJ, Ambudkar IS: Calcium entry in rat parotid acini: activation by carbachol and aluminum fluoride. Am J Physiol 258: C654–661, 1990
Putney JW Jr, Weiss SJ, Leslie BA, Marier SH: Is calcium the final mediator of exocytosis in the rat parotid gland. J Pharm and Exp Therap 203: 144–155, 1977
Putney JW Jr: On the role of cellular calcium in the response of the parotid to dibutyryl and monobutyryl cyclic AMP. Life Sciences 22: 631–638, 1978
Helman J, Ambudkar IS, Baum BJ: Adrenoreceptor mobilization of calcium in rat submandibular cells. Eur J Pharmacol 143: 65–72, 1987
Kim U-H, Kim JW, Rhee SG: Phosphorylation of phospholipase C- by cAMP-dependent protein kinase. J Biol Chem 264: 20167–20170, 1980
Deeg MA, Graeff RM, Walseth TF, Goldberg ND: A Ca 21 linked increase in coupled cAMP synthesis and hydrolysis is an early event in cholinergic and β-adrenergic stimulation of parotid secretion. Proc Natl Acad Sci 85: 7867–7871, 1988
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Horn, V.J., Sheehy, P.A., Goodman, M.B. et al. Activation of the inositol trisphosphate second messenger system by cAMP in a mouse fibroblast cell line. Mol Cell Biochem 101, 43–49 (1991). https://doi.org/10.1007/BF00238436
Received:
Accepted:
Issue Date:
DOI: https://doi.org/10.1007/BF00238436