Abstract
Aim:
To investigate the α1B-adrenoceptor (α1B-AR)-mediated cAMP response and underlying mechanisms in HEK293 cells.
Methods:
Full-length cDNA encoding α1B-AR was transfected into HEK293 cells using the calcium phosphate precipitation method, and α1B-AR expression and cAMP accumulation were determined by using the saturation radioligand binding assay and ion-exchange chromatography, respectively.
Results:
Under agonist stimulation, α1B-AR mediated cAMP synthesis in HEK293 cells, and blockade by PLC-PKC or tyrosine kinase did not reduce cAMP accumulation induced by NE. Pretreatment with pertussis toxin (PTX) had little effect on basal cAMP accumulation as well as norepinephrine (NE)-stimulated cAMP accumulation. In addition, pretreatment with cholera toxin (CTX) neither mimicked nor blocked the effect induced by NE. The extracellular Ca2+ chelator egtazic acid (EGTA), nonselective Ca2+ channel blocker CdCl2 and calmodulin (CaM) inhibitor W-7 significantly reduced NE-induced cAMP accumulation from 1.59%±0.47% to 1.00%±0.31%, 0.78%±0.23%, and 0.90%±0.40%, respectively.
Conclusion:
By coupling with a PTX-insensitive G protein, α1BAR promotes Ca2+ influx via receptor-dependent Ca2+ channels, then Ca2+ is linked to CaM to form a Ca2+-CaM complex, which stimulates adenylyl cyclase (AC), thereby increasing the cAMP production in HEK293 cell lines.
Similar content being viewed by others
Log in or create a free account to read this content
Gain free access to this article, as well as selected content from this journal and more on nature.com
or
References
Williams S, Meij JT, Panagia V . Membrane phospholipids and adrenergic receptor function. Mol Cell Biochem 1995; 149/150: 217–21.
Bylund DB, Eikenberg DC, Hieble JP, Langer SZ, Lefkowitz RJ, Minneman KP, et al. International union of pharmacology nomenclature. Pharmacol Rev 1994; 46: 121–36.
Morgan NG, Blackmore PF, Exton JH . Age-related changes in the control of hepatic cyclic AMP levels by α1- and α2-adrenergic receptors in male rats. J Biol Chem 1983; 258: 5103–9.
Morgan NG, Waynick LE, Exton JH . Characterization of the α1-adrenergic control of hepatic cAMP in male rats. Eur J Pharmacol 1983; 96: 1–10.
Chan TM, Exton JH . α1-Adrenergic-mediated accumulation of adenosine 3′:5′-monophosphate in calcium depleted hepatocytes. J Biol Chem 1977; 252: 8645–51.
Blair JB, James ME, Foster JL . Adrenergic control of glucose output and adenosine 3′:5′ monophosphate levels in hepatocytes from juvenile and adult rats. J Biol Chem 1979; 254: 7579–84.
Johnson RD, Minneman KP . Differentiation of α1-adrenergic receptors linked to phosphatidylinositol turnover and cyclic AMP accumulation in rat brain. Mol Pharmacol 1986; 31: 239–46.
Perkins JP, Moore MM . Characterization of the adrenergic receptors mediating a rise in cyclic 3′,5′-adenosine monophosphate in rat cerebral cortex. J Pharmacol Exp Ther 1973; 185: 371–8.
Robinson JP, Kendall DA . Niguldipine discriminates between α1-adrenoceptor-mediated second messenger responses in rat cerebral cortex slices. Br J Pharmacol 1990; 100: 3–4.
Schwinn DA, Page SO, Middleton JP, Lorenz W, Liggett SB, Yamamoto K, et al. The α1C-adrenergic receptor: characterization of signal transduction pathways and mammalian tissue heterogeneity. Mol Pharmacol 1991; 40: 619–26.
Perez DM, DeYoung MB, Graham RM . Coupling of expressed α1B and α1D-adrenergic receptors to multiple signaling pathways is both G protein and cell type specific. Mol Pharmacol 1993; 44: 784–95.
Cotecchia S, Kobilka BK, Daniel KW, Nolan RD, Lapetina EY, Caron MG, et al. Multiple second messenger pathways of α1-adrenergic receptor subtypes expressed in eukaryotic cells. J Biol Chem 1990; 265: 63–9.
Yoshimasa T, Sibley DR, Bouvier M, Lefkowitz JR, Caron MG . Crosstalk between cellular signalling pathways suggested by phorbol-ester-induced adenylate cyclase phosphorylation. Nature (Lond) 1987; 327: 67–70.
Horie K, Itoh H, Tsujimoto G . Hamster α1B-adrenergic receptor directly activates Gs in the transfected Chinese hamster ovary cells. Mol Pharmacol 1995; 48: 394–400.
Filipeanu CM, Brailoiu E, Huhurez G, Slatineanu S, Baltatu O, Branisteanu DD . Multiple effects of tyrosine kinase inhibitors on vascular smooth muscle contraction. Eur J Pharmacol 1995; 281: 29–35.
Abebe W, Edwards JD, Agrawal DK . G-proteins in rat blood vessels-II: assessment of functional involvement. Gen Pharmacol 1995; 26: 75–83.
Thorburn J, Thorburn A . The tyrosine kinase inhibitor, genistein, prevents α-adrenergic-induced cardiac muscle cell hypertrophy by inhibiting activation of the Ras-MAP kinase signaling pathway. Biochem Biophys Res Commun 1994; 202: 1586–91.
Slupsky JR . Raf-dependent signaling pathways in cell growth and differentiation. Cell Growth Oncogenes 1998; 75–95.
Engel G, Hoyer D . 125I-BE2254, a new high affinity radioligand for alpha1-adrenoceptors. Eur J Pharmacol 1981; 73: 221–4.
Weiss BL, Insel PA . Intracellular Ca++ and protein kinase C interact to regulate alpha-1-adrenergic and bradykinin receptor stimulated phospholipase A2 activation in Madin-Darby carby canine kidney cell. J Biol Chem 1991; 266: 2126–33.
Sunahara RK, Dessauer CW, Gilman AG . Complexity and diversity of mammalian adenylyl cyclases. Annu Rev Pharmacol Toxicol 1996; 36: 461–80.
Dolphin AC . G-proteins. Textbook of receptor pharmacology. Boca Raton (USA): CRC Press, Inc; 1996. page 194–5.
Zhu WZ, Gao BB, Li HW, Zhang YY, Han QD . Differential activation of adenylyl cyclase by protein kinase C isoenzymes. Acta Pharmacol Sin 1999; 20: 1025–30.
Shahab A . Transgenic mice with cardiac overexpression of α1B-adrenergic receptors. J Biol Chem 1997; 272: 21253–9.
Zhu X, Gilbert S, Birnbaumer M, Birnbaumer L . Dual signaling potential is common among Gs-coupled receptors and dependent on receptor density. Mol Pharmacol 1994; 46: 460–9.
Minneman KP, Theroux TL, Hollinger S, Han C, Esbenshade TA . Selectivity of agonists for cloned α1-adrenergic receptor subtypes. Mol Pharmacol 1994; 46: 929–36.
Exton JH . Phosphoinositide phospholipase and G proteins in hormone action. Annu Rev Physiol 1994; 56: 349–69.
Taussig R, Tang WJ, Hepler JR, Gilman AG . Distinct patterns of bidirectional regulation of mammalian adenylyl cyclases. J Biol Chem 1994; 269: 6093–100.
Tao L, Guan YY, He H, Han C, Zhang YY, Sun JJ . Comparison of the Ca2+ movement by activation of alpha 1-adrenoceptor subtypes in HEK293 cells. Life Sci 1997; 61: 2127–36.
Author information
Authors and Affiliations
Corresponding author
Additional information
Project supported by the National Natural Science Foundation of China (No 30171083) and the Major State Basic Research Development Program of the People's Republic of China (No G2000056906).
Rights and permissions
About this article
Cite this article
Song, Y., Li, Yf., Dong, Ed. et al. Ca2+ participates in α1B-adrenoceptor-mediated cAMP response in HEK293 cells. Acta Pharmacol Sin 26, 77–84 (2005). https://doi.org/10.1111/j.1745-7254.2005.00018.x
Received:
Accepted:
Issue date:
DOI: https://doi.org/10.1111/j.1745-7254.2005.00018.x
