Cardiac fibroblasts (CF) express adenosine (ADO) receptors, and pharmacological evidence suggests the possible involvement of the A2 (A2a and A2b) receptor (A2aR and A2bR) subtypes in inhibiting cell functions involved in fibrosis. The main objective of this study was to define the contributions of A2a and/or A2b receptors in modulating ADO-induced decreases in CF functions. For this purpose, CF were either treated pharmacologically or had the A2aR or A2bR levels modified through the use of recombinant adenovirus or siRNA. The assessment of mRNA expression in adult rat CF yielded evidence for A1R, A2bR, A2a), and A3R. Endogenously or exogenously enhanced ADO significantly inhibits CF proliferation, collagen, and protein synthesis. A2R and A2aR agonists, although capable of inhibiting CF protein and collagen synthesis, were unable to define the contributions derived from A2aR or A2bR. Overexpression of A2bR in CF yielded significant decreases in basal levels of collagen and protein synthesis and correlated with increases in cAMP levels. However, at higher doses of ADO receptor agonists, significant increases in protein and collagen synthesis were observed. CF with underexpression of A2bR yielded increases in protein and collagen synthesis. In contrast, A2aR underexpression did not modify ADO-induced decreases in CF protein or collagen synthesis. In conclusion, results derived from the molecular manipulation of receptor levels indicate that A2bR are critically involved in ADO-mediated inhibition of CF functions.

Rat cardiac fibroblasts (CF) express multiple adenosine (ADO) receptors. Pharmacological evidence suggests that activation of A(2) receptors may inhibit collagen synthesis via adenylyl cyclase-induced elevation of cellular cAMP. We have characterized the signaling pathways involved in ADO-mediated inhibition of collagen synthesis in primary cultures of adult rat CF. ANG II stimulates collagen production in these cells. Coincubation with agents that elevate cellular cAMP [the ADO agonist, 5'-N-ethylcarboxamidoadensoine (NECA), and forskolin] inhibited the stimulatory effects of ANG II. However, direct stimulators and inhibitors of protein kinase A (PKA) did not alter ANG II-induced collagen synthesis, indicating that PKA does not mediate the inhibitory effects of NECA. Inhibitors of AMP-kinase (AMPK) and extracellular signal-regulated kinase 1/2 (ERK1/2) do not alter NECA-inhibited collagen synthesis. However, activation of exchange factor directly activated by cAMP (Epac) mimicked the effects of NECA on ANG II-stimulated collagen synthesis. Inhibition of phosphoinositol-3 kinase (PI3K) reduced the inhibitory effects of NECA on ANG II-induced collagen synthesis, suggesting that NECA acts via PI3K. Furthermore, inhibition of PI3K also relieved the inhibitory effect of Epac activation on ANG II-stimulated collagen synthesis. Thus it appears that ADO activates the A(2)R-G(s)-adenylyl cyclase pathway and that the resultant cAMP reduces collagen synthesis via a PKA-independent, Epac-dependent pathway that feeds through PI3K.

The ability of adenosine (ADO) to inhibit proliferation and protein synthesis (in particular, collagen synthesis) in cardiac fibroblasts (CF) may ameliorate adverse cardiac remodeling and fibrosis seen in heart failure patients. However, little is known about the signaling pathways that ADO may modulate in CF to alter cell phenotype. Accordingly, this study was designed to identify ADO receptors (AR) and the signaling pathways linked to them in primary cultures of adult rat CF. Quantitative RT-PCR data indicate that the mRNAs for all four known ARs (A(1)R, A(2a)R, A(2b)R, and A(3)R) are present in rat CF, with a greater prevalence of A(2) receptor subtypes. No coupling of AR to the G(q)-phospholipase C signaling pathway or to mobilization of calcium is measurable. Studies using subtype specific agents imply that the A(2a)R and A(2b)R couple to G(s)-adenylyl cyclase and A(1)R couple weakly to G(i)-adenylyl cyclase. 2-Chloroadenosine, 5'-N-ethylcarboxamidoadensoine, and other agents that elevate cellular cAMP stimulate extracellular signal-regulated kinase 1/2 activity in a pertussis toxin-insensitive manner. We conclude that a combination of cAMP-dependent signals generated via A(2a) and A(2b) receptors likely mediate ADO signaling in adult rat CF.

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