Abstract 1024: Glucose-6-Phosphate Dehydrogenase (G6PD) Functions as a Modulator of Contractile Function in Coronary Artery .
Objective: Inhibition of G6PD has recently been demonstrated to relax precontracted coronary and pulmonary arteries. However, the mechanism underlying the modulation of vascular function following G6PD inhibition is unknown. This study was undertaken to elucidate the role of G6PD in modulating smooth muscle contraction evoked by KCl, and to identify the signaling pathway associated with it.
Methods & Results: KCl (30 mM) treatment of bovine coronary artery (CA) and coronary artery smooth muscle cells (CASMCs) for 20 minutes increased (P<0.05; n=20) G6PD activity by 1.9 ± 0.2-fold as compared to respective time-match controls (CA: 0.3 ± 0.1 and CASMCs: 0.5 ± 0.1 nmol/min/mg protein, respectively). Amphoterecin B (50 μM; n = 8), a cation-selective pore forming agent, also increased (P<0.01) the G6PD activity (1.7 ± 0.3-fold) in Ca2+ -independent manner, thus substantiating our findings that KCl-induced depolarization activates G6PD. To elucidate the signaling cascade involved in KCl-induced G6PD activation, we identified five classical PKC phosphorylation sites for G6PD and hypothesized that PKC phosphorylation is required for its activation. PKC activity was estimated following various treatments and we found that  KCl increased (P<0.05) PKC activity by 8-fold from basal level (4.0 ± 0.4 units/ml),  chelerythrine (10 μM) and rotterlin (10 μM); PKC inhibitors, blocked activation of G6PD by KCl, and  phorbol 12, 13-dibutyrate (PDBu; 10 μM), a PKC activator, increased (P<0.01) G6PD phosphorylation detected by 2-D electrophoresis and activity by 2.0 ± 0.4-fold. Interestingly, silencing of G6PD in CASMCs and CA with gene-specific [GS] siRNA (as confirmed by 60 ± 7% decrease in expression and 51 ± 15% in activity compared to that of a non-targeting siRNA [NT]), suppressed (P<0.01) contraction of CA evoked by KCl (GS: 0.6 ± 0.2 g vs. NT: 2.0 ± 0.1 g) and PDBu (GS: 1.0 ± 0.1 g vs. NT: 0.5 ± 0.1 g). Consistently, 6-aminonicotinamide (5 mM), a G6PD inhibitor, decreased (P<0.01) KCl- and PDBu-induced contraction by 45 ± 6% and 56 ± 8%, respectively, as compared to controls.
Conclusion: We have identified a novel role for G6PD as a crucial modulator of smooth muscle contraction evoked by membrane depolarization and PKC pathway. (Supported by AHA Grant #0435070N)