Soluble adenylyl cyclase mediates bicarbonate-dependent corneal endothelial cell protection

Abstract

Cyclic AMP produced from membrane receptor complex bound adenylyl cyclases is protective in corneal endothelial cells (CEC). CEC also express soluble adenylyl cyclase (sAC), which is localized throughout the cytoplasm. When activated by HCO(3)(-), cAMP concentration ([cAMP]) increases by ∼50%. Here we ask if cAMP produced from sAC is also protective. We examined the effects of HCO(3)(-), pH, phosphodiesterase 4 inhibition by rolipram, sAC inhibition by 2HE (2-hydroxyestradiol), and sAC small interfering RNA (siRNA) knockdown on basal and staurosporine-mediated apoptosis. HCO(3)(-) (40 mM) or 50 μM rolipram raised [cAMP] to similar levels and protected endothelial cells by 50% relative to a HCO(3)(-)-free control, whereas 2HE, which decreased [cAMP] by 40%, and H89 (PKA inhibitor) doubled the apoptotic rate. sAC expression was reduced by two-thirds in the absence of HCO(3)(-) and was reduced to 15% of control by sAC siRNA. Protection by HCO(3)(-) was eliminated in siRNA-treated cells. Similarly, caspase-3 activity and cytochrome c release were reduced by HCO(3)(-) and enhanced by 2HE or siRNA. Analysis of percent annexin V+ cells as a function of [cAMP] revealed an inverse, nonlinear relation, suggesting a protective threshold [cAMP] of 10 pmol/mg protein. Relative levels of phosphorylated cAMP response element binding protein and phosphorylated Bcl-2 were decreased in CEC treated with 2HE or siRNA, suggesting that HCO(3)(-)-dependent endogenous sAC activity can mobilize antiapoptotic signal transduction. Overall, our data suggest a new role for sAC in endogenous cellular protection.

Fig. 1.

HCO₃⁻ protects corneal endothelial cells. Confluent cultured bovine corneal endothelium cells (BCEC) seeded on coverslips were incubated with either bicarbonate-rich (BR; 40 mM HCO₃⁻) medium or bicarbonate-free (BF; 0 mM HCO₃⁻) DMEM for 48 h. Cells were then treated with 50 nM staurosporine (SP) for 17 h and fixed, and the nuclei was stained with 4,6-diamidino-2-phenylindole. Representative micrographs (×20 objective) show fewer condensed nuclei in BR-incubated cells. Percent condensed nuclei per field were counted. Values are means ± SE; n = 3 separate experiments. *Significantly different from BR or BR + SP, respectively; P < 0.05.

Fig. 2.

Quantitative assessment of apoptosis by flow cytometry. A: representative result from cells incubated in BF media; y-axis, propidium iodide (PI) fluorescence; x-axis, annexin V FITC fluorescence. Bottom right quadrant represents apoptotic portion of population. B: bottom right quadrants from representative experiments of cells incubated in BF or BR media and treated with 50 μM rolipram (Rolip), 10 μM 2-hydroxyestradiol (2HE), or 10 μM N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinollnesulfonamide (H89), ± 10 nM SP. C: summary data. Values are means ± SE; n = 5. *Significantly lower than BF + SP; ⁺ significantly higher than BR; ^ significantly higher than BR + SP: P < 0.05.

Fig. 3.

Quantitative assessment of cyclic AMP (cAMP). Cellular cAMP was extracted from BCEC with 0.1 N HCl. BCEC were cultured either in BF or BR. Increasing concentrations of 2HE were applied for 17 h to the cells that were grown in BR before extracting cAMP. The data were an average of at least three independent experiments and expressed as means ± SE.

Fig. 4.

Effect of soluble adenylyl cyclase (sAC) small interfering RNA (siRNA) on basal and SP-induced apoptosis. A, top: sAC expression and siRNA transfection under BF and BR conditions. Nontransfected cells (NT), cells transfected with scrambled control siRNA sequences (siCon), and cells transfected with sAC-specific siRNA (siRNA) were analyzed for sAC expression (50 kDa) relative to β-actin by Western blot 48 h following transfection. Bottom: densitometric analysis of sAC expression normalized to β-actin and plotted relative to BF no treatment. Values are means ± SE; n = 3. *Significantly less sAC expression (P < 0.05 by paired t-test). B: flow cytometric analysis (annexin V+ cells) of endothelial cells incubated in BF or BR media for 48 h following transfection with siCon or sAC siRNA and treated with SP. Values are means ± SE; n = 7. *Significantly different from NT; ^ significantly different from siCon; ⁺ significantly different from siCon + SP: P < 0.05.

Fig. 5.

Examination of cytchrome c (Cyto C) release and caspase-3 activity. BCECs (5 × 10⁶) were incubated in BF or BR media for 48 h. Cells were treated with 2HE (10 μM) or transfected with 10 nM sAC siRNA ± 0.1 μM SP. A: cytoplasmic extracts were made using a cytosol/mitochondria fractionation kit, as described in materials and methods. Anti-β-actin served as a loading control. Values are means ± SE; n = 3. *Significantly higher than BR; ^ significantly higher than BR + SP: P < 0.05. B: caspase-3 activity (see materials and methods for details). Values are means ± SE; n = 3. *Significantly higher than BR; ^ significantly higher than BR + SP: P < 0.05.

Fig. 6.

Relation between apoptotic rate and cAMP concentration ([cAMP]). Apoptotic rates (shown in Figs. 2, 4, and 5) are plotted against [cAMP] for cells incubated in BR or BF media and treated with or without 50 μM rolipram, 10 μM 2HE, or transfected with siCon or sAC siRNA. [cAMP] is from triplicate experiments. Values are means ± SE.

Fig. 7.

Effect of HCO₃⁻ and sAC inhibition on phosphorylated cAMP response element binding protein (pCREB) and phosphorylated Bcl-2 (pBcl-2) levels. Confluent cells were incubated in either BR or BF for 48 h. Total CREB, pCREB, total Bcl-2, and pBcl-2 were analyzed by Western blot. A: pCREB and pBcl-2 were examined by Western blotting. 30 μg of BXE cell lysate were taken from BF culture (lane 1), BR culture (lane 2), treated with 10 μM 2HE (lane 3), or 10 nM siRNA (lane 4). B: semiquantitative assessment of protein density. The relative level of pCREB and pBcl-2 were normalized to totalized by total CREB and Bcl-2, respectively. The data are representative of three independent experiments and expressed as means ± SE. *P < 0.05.