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. 2010 Jul 15;80(2):188-96.
doi: 10.1016/j.bcp.2010.03.020. Epub 2010 Mar 25.

Multivalent dendrimeric and monomeric adenosine agonists attenuate cell death in HL-1 mouse cardiomyocytes expressing the A(3) receptor

Athena M Keene  1 , Ramachandran Balasubramanian, John Lloyd, Asher Shainberg, Kenneth A Jacobson
Affiliations

Multivalent dendrimeric and monomeric adenosine agonists attenuate cell death in HL-1 mouse cardiomyocytes expressing the A(3) receptor

Athena M Keene et al. Biochem Pharmacol. .

Abstract

Multivalent dendrimeric conjugates of GPCR ligands may have increased potency or selectivity in comparison to monomeric ligands, a phenomenon that was tested in a model of cytoprotection in mouse HL-1 cardiomyocytes. Quantitative RT-PCR indicated high expression levels of endogenous A(1) and A(2A) adenosine receptors (ARs), but not of A(2B) and A(3)ARs. Activation of the heterologously expressed human A(3)AR in HL-1 cells by AR agonists significantly attenuated cell damage following 4h exposure to H(2)O(2) (750 microM) but not in untransfected cells. The A(3) agonist IB-MECA (EC(50) 3.8 microM) and the non-selective agonist NECA (EC(50) 3.9 microM) protected A(3) AR-transfected cells against H(2)O(2) in a concentration-dependent manner, as determined by lactate dehydrogenase release. A generation 5.5 PAMAM (polyamidoamine) dendrimeric conjugate of a N(6)-chain-functionalized adenosine agonist was synthesized and its mass indicated an average of 60 amide-linked nucleoside moieties out of 256 theoretical attachment sites. It non-selectively activated the A(3)AR to inhibit forskolin-stimulated cAMP formation (IC(50) 66nM) and, similarly, protected A(3)-transfected HL-1 cells from apoptosis-inducing H(2)O(2) with greater potency (IC(50) 35nM) than monomeric nucleosides. Thus, a PAMAM conjugate retained AR binding affinity and displayed greatly enhanced cardioprotective potency.

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Figures

Figure 1
Figure 1
(A) Structures of a non-selective amine-functionalized AR agonist (4) and an A3 selective dendrimeric conjugate (5) as reported [26]. (B) Synthesis of 8, a G5.5 PAMAM dendrimer with 1 AF488-ED and 60 N-(2-aminoethyl)-ADAC moieties. AF488-ED and N-(2-aminoethyl)-ADAC were conjugated to G5.5 PAMAM dendrimers using carbodiimide coupling.
Figure 2
Figure 2
Gene expression levels of the A1, A2A, and A2B in HL-1 cells compared to A3 ARs measured using qRT-PCR. In three separate experiments, the fold expression of each AR is measured and normalized to the A3AR expression level, which is set to 1, using the ΔΔCT method.
Figure 3
Figure 3
Concentration-dependent protection against H2O2-induced cytotoxicity by A3 selective and non-selective AR agonists (3 nM – 30 μM) in A3AR-transfected (A) and non-transfected (B) HL-1 cells. HL-1 cells were pretreated with NECA (100 nM - 30 μM), IB-MECA (100 nM - 30 μM), or 8 (3 nM - 1 μM) for 1 h prior to the addition of H2O2 (final conc. 750 μM). After 4 h, 100 μl of media was added to 100 μl of the LDH measuring solution provided with the kit and incubated for 10 min. The results were analyzed with a microplate reader. Data shown are mean ± SD from three independent experiments in triplicate.
Figure 4
Figure 4
Effect of the A3 receptor antagonist MRS1191 on the protection by IB-MECA against H2O2-induced cell damage. HL-1 cells were pretreated with MRS1191 (10 μM), an antagonist of the A3 receptor, 1 h before treatment with IB-MECA (30 μM). One h after the addition of the agonist, H2O2 (750 μM) was incubated with the cells for 4 h. 100 μl of media was then added to 100 μl of the LDH measuring solution provided with the kit and incubated for 10 min. The results were analyzed with a microplate reader. Data shown are mean ± SD from three independent experiments in triplicate. Groups labeled * are significantly different from H2O2 control (P < 0.05).
Figure 5
Figure 5
Protection afforded HL-1 cells by 8, a dendrimer-nucleoside conjugate, but not by 7, a control dendrimer, against H2O2-induced apoptosis. Dendrimer compounds (10 μM) were added 1 h prior to the addition of H2O2 (400 μM). The luminescent caspase 3/7 reagent was added 3 h after the addition of H2O2. After 1.5 h, apoptosis was quantified using a luminometer. Data shown are mean ± SD from three independent experiments in triplicate. Groups labeled * are significantly different from control treated with H2O2 alone (P < 0.05). Control cells in the absence of H2O2 gave 182,000 ± 20,000 relative luminescence units.
Figure 6
Figure 6
Schematic description of the mechanism of protection afforded HL-1 cells by multivalent dendrimeric AR agonist 8. The PAMAM dendrimer derivative is shown as a tree-like polymer with covalently attached nucleoside moieties “A”. Mouse HL-1 cells, which express a high basal level of mA1 and mA2AARs, were transfected with hA3AR mRNA in order to increase the expression levels of this subtype. Following transfection, activation of the hA3AR by conjugate 8 afforded significant protection to the HL-1 cells against H2O2-induced apoptosis.
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