Bicarbonate-sensing soluble adenylyl cyclase is an essential sensor for acid/base homeostasis

Abstract

pH homeostasis is essential for life, yet it remains unclear how animals sense their systemic acid/base (A/B) status. Soluble adenylyl cyclase (sAC) is an evolutionary conserved signaling enzyme that produces the second messenger cAMP in response to bicarbonate ions (HCO(3)(-)). We cloned the sAC ortholog from the dogfish, a shark that regulates blood A/B by absorbing and secreting protons (H(+)) and HCO(3)(-) at its gills. Similar to mammalian sAC, dogfish soluble adenylyl cyclase (dfsAC) is activated by HCO(3)(-) and can be inhibited by two structurally and mechanistically distinct small molecule inhibitors. dfsAC is expressed in the gill epithelium, where the subset of base-secreting cells resides. Injection of inhibitors into animals under alkaline stress confirmed that dfsAC is essential for maintaining systemic pH and HCO(3)(-) levels in the whole organism. One of the downstream effects of dfsAC is to promote the insertion of vacuolar proton pumps into the basolateral membrane to absorb H(+) into the blood. sAC orthologs are present throughout metazoans, and mammalian sAC is expressed in A/B regulatory organs, suggesting that systemic A/B sensing via sAC is widespread in the animal kingdom.

Fig. 1.

Phylogenetic relation of dfsAC to other nucleotidyl cyclases. The GenBank accession numbers are as follows: (i) sACs: dogfish: S. acanthias, ACA52542.1; mouse: Mus musculus, NP_766617.1; rat: Rattus norvegicus, NP_067716.1; human: Homo sapiens, NP_060887.2; Trichoplax: Trichoplax adhaerens, XP_002117857.1 (hypothetical); Starlet sea anemone: Nematostella vectensis, XP_001623318.1 (predicted); mosquito (1): Aedes aegypti, XP_001661592.1 (hypothetical); mosquito (2): Culex quinquefasciatus, XP_001842661.1 (hypothetical); sea urchin: Strongylocentrotus purpuratus, NP_001020380.1; sea squirt: Ciona intestinalis, XP_002121952.1 (predicted); lancet: Branchiostoma floridae, XP_002214797.1 (hypothetical); (ii) guanylyl cyclases: Caenorhabditis elegans: guanylyl cyclase protein 28, isoform D AAC78238.2; zebrafish: Danio rerio, XP_001337045.2 (predicted); mouse: NP_001124165.1 guanylate cyclase 2d; human: H. sapiens, AAA60547.1 soluble retinal isoform; and (iii) tmACs: zebrafish (VI): XP_001922749.1 (predicted, isoform VI); human (I): H. sapiens, Q08828.2 (ADCY1); human (IX): H. sapiens O60503.4 (ADCY9); E. coli cya: AAA67602.1 (adenylate cyclase). Interestingly, sAC orthologs have not been identified in the genomes of some model organisms (e.g., Drosophila melanogaster, D. rerio, C. elegans), suggesting that they may use fundamentally distinct mechanisms for A/B sensing.

Fig. 2.

Activity of recombinant dfsAC. (A) Coomassie blue-stained SDS/PAGE gel of His-tagged dfsAC (53 KDa expected) purified from E. coli. (B) dfsAC activity assayed at a constant pH of 7.75 in the presence of 2.5 mM ATP, 20 mM MgCl₂, 0.5 mM MnCl₂, and the indicated concentrations of NaHCO₃. EC₅₀ = 4.9 mM NaHCO₃. (C) dfsAC activity at the indicated pH. (D) dfsAC activity in the presence of 2.5 mM ATP, 5 mM MnCl₂, and the indicated concentrations of KH7 or 4CE. IC₅₀ for KH7 = 9.6 μM and IC₅₀ for 4CE = 46.9 μM. Values are expressed as fold stimulation relative to the baseline in each experiment (0 mM HCO₃⁻, pH 7.75, or no drug, respectively). Data points reflect the averages of four independent assays for each condition.

Fig. 3.

dfsAC is present in the gill, and cAMP-producing activity in the gill matches recombinant dfsAC. (A) Adenylyl cyclase activity in gill homogenates measured in the presence 50 mM NaCl or NaHCO₃ in the presence or absence of 50 μM KH7. Shown are the averages of quadruple determinations with SEM. Equivalent results were obtained with 4CE. Lowercase letters denote levels of activity that are not significantly different from each other by repeated-measures two-way ANOVA (P < 0.05 for treatment, time, and interaction), Bonferroni’s posttest. (B) KH7-sensitive cAMP activity in gill homogenates measured as indicated in A. EC₅₀ = 2.9 mM NaHCO₃. (C) Western blot of dogfish shark gill homogenate (crude) and high-speed supernatant (sup) using anti-dfsAC in the absence or presence of immunizing peptide. (D) Immunofluorescence using anti-dfsAC (green) in dogfish gill. Nuclei were labeled with DAPI (blue). (E) Staining using anti-dfsAC antisera preabsorbed with excess immunizing peptide; similar results were obtained with preimmune serum. (F and G) Higher magnification pictures of dfsAC immunolabeling. (G) Immunolabeling in pillar cells. (Scale bars = 10 μm.)

Fig. 4.

sAC-dependent VHA translocation during systemic alkalosis in whole fish. Anti-VHA immunolabeling in dogfish gills. Low-power (A) and high-power (B) magnification of gills from untreated dogfish sharks. Low-power (C) and high-power (D) magnification of gills taken from dogfish shark after 12 h of continuous NaHCO₃ infusion. Low-power (E) and high-power (F) magnification of gills taken from dogfish shark after 12 h of continuous NaHCO₃ infusion and injected with KH7 (5 μmol·kg⁻¹ bolus at t = −0.5 and 6 h). (Scale bars = 10 μm.) (G) Percentage of cells showing VHA translocation in gill fragments (n = 4). (H) VHA abundance in membrane-enriched gill samples (n = 5–8). (Top) Bands are representative of each treatment. In G and H, letters indicate different levels of statistical significance by one-way ANOVA, Bonferroni’s multiple comparison test.

Fig. 5.

sAC-dependent VHA translocation during localized alkalosis in isolated gill. (A) Percentage of cells showing VHA translocation in gill fragments internally perfused and incubated in alkaline saline with or without 100 μM 4CE (n = 5). A repeated-measures two-way ANOVA (P < 0.05 for treatment, time, and interaction) or the Tukey–Kramer planned least square means test was used. Letters indicate different levels of statistical significance. Bicarb, bicarbonate. Representative VHA immunostaining of gills internally perfused and incubated in alkaline saline and DMSO (B) or 4CE (C).