A2B adenosine receptors inhibit superoxide production from mitochondrial complex I in rabbit cardiomyocytes via a mechanism sensitive to Pertussis toxin.

Abstract

BACKGROUND AND PURPOSE:

A(2B) adenosine receptors protect against ischaemia/reperfusion injury by activating survival kinases including extracellular signal-regulated kinase (ERK) and phosphatidylinositol 3-kinase (PI3K). However, the underlying mechanism(s) and signalling pathway(s) remain undefined.

EXPERIMENTAL APPROACH:

HEK 293 cells stably transfected with human A(2B) adenosine receptors (HEK-A(2B) ) and isolated adult rabbit cardiomyocytes were used to assay phosphorylation of ERK by Western blot and cation flux through cAMP-gated channels by patch clamp methods. Generation of reactive oxygen species (ROS) by mitochondria was measured with a fluorescent dye.

KEY RESULTS:

In HEK-A(2B) cells, the selective A(2B) receptor agonist Bay 60-6583 (Bay 60) increased ERK phosphorylation and cAMP levels, detected by current through cAMP-gated ion channels. However, increased cAMP or its downstream target protein kinase A was not involved in ERK phosphorylation. Pertussis toxin (PTX) blocked ERK phosphorylation, suggesting receptor coupling to G(i) or G(o) proteins. Phosphorylation was also blocked by inhibition of PI3K (with wortmannin) or of ERK kinase (MEK1/2, with PD 98059) but not by inhibition of NO synthase (NOS). In cardiomyocytes, Bay 60 did not affect cAMP levels but did block the increased superoxide generation induced by rotenone, a mitochondrial complex I inhibitor. This effect of Bay 60 was inhibited by PD 98059, wortmannin or PTX. Inhibition of NOS blocked superoxide production because NOS is downstream of ERK.

CONCLUSION AND IMPLICATIONS:

Activation of A(2B) adenosine receptors reduced superoxide generation from mitochondrial complex I through G(i/o) , ERK, PI3K, and NOS, all of which have been implicated in ischaemic preconditioning.

• Authors: Xiulan Yang, Wenkuan Xin, Xi-Ming Yang, Atsushi Kuno, Thomas C Rich, Michael V Cohen, James M Downey
• Journal: British journal of pharmacology (Br J Pharmacol) Vol. 163 Issue 5 Pg. 995-1006 (Jul 2011) ISSN: 1476-5381 [Electronic] England
• PMID: 21366548 (Publication Type: Journal Article, Research Support, N.I.H., Extramural)
• Copyright: © 2011 The Authors. British Journal of Pharmacology © 2011 The British Pharmacological Society.

Chemical References

• Receptor, Adenosine A2B
• Superoxides
• Nitric Oxide
• Cyclic AMP
• Pertussis Toxin
• Extracellular Signal-Regulated MAP Kinases
• Electron Transport Complex I

Topics

• Animals
• Blotting, Western
• Cyclic AMP (metabolism)
• Electron Transport Complex I (metabolism)
• Extracellular Signal-Regulated MAP Kinases (metabolism)
• Female
• HEK293 Cells
• Humans
• Male
• Membrane Potentials (drug effects)
• Mitochondria, Heart (drug effects, enzymology, metabolism)
• Myocytes, Cardiac (drug effects, enzymology, metabolism)
• Nitric Oxide (metabolism)
• Patch-Clamp Techniques
• Pertussis Toxin (pharmacology)
• Phosphatidylinositol 3-Kinases (metabolism)
• Rabbits
• Receptor, Adenosine A2B (genetics, physiology)
• Superoxides (metabolism)
• Transfection