HCO 3 − -dependent soluble adenylyl cyclase activates cystic fibrosis transmembrane conductance regulator in corneal endothelium

Abstract

cAMP-dependent activation of the cystic fibrosis transmembrane conductance regulator (CFTR) regulates fluid transport in many tissues. Secretion by the corneal endothelium is stimulated by cAMP and dependent on HCO[Formula: see text]. We asked whether HCO[Formula: see text] can secondarily increase CFTR permeability in bovine corneal endothelial cells (BCEC) by activating soluble adenylyl cyclase (sAC). Immunofluorescence suggests that sAC is distributed throughout the cytoplasm. HCO[Formula: see text] (40 mM) increased cAMP concentration 42% in the presence of 50 μM rolipram (a phosphodiesterase 4 inhibitor), and a standard HCO[Formula: see text] Ringer solution (28.5 mM) increased apical Cl− permeability by 78% relative to HCO[Formula: see text]-free solution. The HCO[Formula: see text]-dependent increase in Cl−permeability was reduced 60% by 20 mM NaHSO3 (a weak agonist of sAC). NaHSO3 alone increased apical Cl− permeability by only 13%. The HCO[Formula: see text]-dependent increase in Cl−permeability was reduced 57% in the presence of 50 μM Rp-adenosine 3′,5′-cyclic monophosphorothioate, and 86% by 50 μM 5-nitro-2-(3-phenylpropyl-amino)benzoic acid but unaffected by 200 μM apical H2DIDS. CFTR phosphorylation was increased 23, 150, and 32% by 20 mM HSO[Formula: see text], 28.5 mM HCO[Formula: see text], and 28.5 mM HCO[Formula: see text] + 20 mM HSO[Formula: see text], respectively. Activation of apical Cl− permeability by 5 μM genistein was increased synergistically by HCO[Formula: see text] over that due to genistein and HCO[Formula: see text] alone. We conclude that HCO[Formula: see text]-stimulated sAC is a form of autocrine signaling that contributes to baseline cAMP production, thereby affecting baseline CFTR activity in BCEC. This form of autocrine signaling may be important in tissues that express sAC and exhibit robust HCO[Formula: see text] influx (e.g., ocular ciliary epithelium, choroid plexus, and airway epithelium).