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First published online May 21, 2007
Journal of Experimental Biology 210, 1944-1959 (2007)
Published by The Company of Biologists 2007
doi: 10.1242/jeb.02776

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Mechanisms of acid–base regulation in the African lungfish Protopterus annectens

K. M. Gilmour^1,*, R. M. Euverman¹, A. J. Esbaugh¹, L. Kenney¹, S. F. Chew², Y. K. Ip³ and S. F. Perry¹

¹ Department of Biology and Centre for Advanced Research in Environmental Genomics, University of Ottawa, Ottawa, ON, Canada
² Natural Sciences and Science Education, National Institute of Education, Nanyang Technological University, Republic of Singapore
³ Department of Biological Sciences, National University of Singapore, Republic of Singapore

View larger version (32K): [in this window] [in a new window]   Fig. 1. The effect on the blood acid–base status of African lungfish Protopterus annectens of 1 h of (A–D) acid (3 mmol kg–1 NH4Cl) or (E–H) base (3 mmol kg–1 NaHCO3) infusion. Acid–base status prior to and following the infusion period (marked by the grey bar) was assessed by determining the pH (A,E), HCO3– ion concentration ([HCO3–]) (B,F) and CO2 tension (PaCO2) (C,G) of arterial blood. (D,H) pH–HCO3– diagrams are used to summarize the effect of acid or base infusion on acid–base status. Values are means ± s.e.m.; N=11 for acid-infused fish and N=6–8 for base-infused fish. An asterisk (*) indicates a significant difference from the pre-infusion value (one-way repeated measures ANOVA; P values are indicated on the figure). For pH–HCO3– diagrams, the PCO2 for a given combination of pH and [HCO –3] was calculated using the Henderson–Hasselbalch equation and the appropriate values for pK' and PaCO2 (Boutilier et al., 1984). The buffer line was constructed using data for Protopterus aethiopicus (Lenfant and Johansen, 1968). Pre, period prior to acid or base infusion.

View larger version (12K): [in this window] [in a new window]   Fig. 2. The effect on (A,B) the frequency of breathing air (fair; A) and water (fwater; B), and (C,D) CO2 excretion into air (airCO2; C) and water (waterCO2; D), of a 1 h acid (3 mmol kg–1 NH4Cl) infusion (marked by the grey bar) in African lungfish Protopterus annectens. Values are means ± s.e.m. for the period prior to acid infusion (`pre'), and for measurement periods 0–1, 2–3, 5–6 and 17–18 h post-infusion; N=9–11 for breathing frequencies and waterCO2, and 8 for airCO2. An asterisk (*) indicates a significant increase from the pre-infusion value (one-way RM-ANOVA; P values are indicated on the figure); note that in A, post hoc multiple comparisons tests were unable to detect the origin of the significant difference indicated by the P value.

View larger version (13K): [in this window] [in a new window]   Fig. 3. The effect on (A,B) the frequency of breathing air (fair; A) and water (fwater; B), and (C,D) on CO2 excretion into air (airCO2; C) and water (waterCO2; D), of a 1 h base (3 mmol kg–1 NaCO3) infusion (marked by the grey bar) in African lungfish Protopterus annectens. Values are means ± s.e.m. for the period prior to base infusion (`pre'), and for measurement periods 0–1, 2–3, 5–6 and 17–18 h post-infusion; N=8 throughout. An asterisk (*) indicates a significant difference from the pre-infusion value (one-way RM-ANOVA; P values are indicated on the figure).

View larger version (18K): [in this window] [in a new window]   Fig. 4. The effect in African lungfish Protopterus annectens of 1 h of (A–C) acid (3 mmol kg–1 NH4Cl) or (D–F) base (3 mmol kg–1 NaHCO3) infusion (marked by the grey bar) on net excretion of acidic equivalents into the water (JnetH+; A,D), titratable net acid flux (JnetTA; B,E), and net ammonia excretion (JnetNH3; C,F). JnetH+ was calculated as the sum of JnetTA and JnetNH3, signs considered. Values are means ± s.e.m. for the period prior to acid or base infusion (`pre'), and for measurement periods 0–1, 2–3, 5–6 and 17–18 h post-infusion; N=11 and 8 for, respectively, acid- and base-infused lungfish. An asterisk (*) indicates a significant difference from the pre-infusion value (one-way RM-ANOVA; P values are indicated on the figure); note that in E and F, post hoc multiple comparisons tests were unable to detect the origin of the significant differences indicated by the P values.

View larger version (9K): [in this window] [in a new window]   Fig. 5. The effect of 1 h of base (3 mmol kg–1 NaHCO3) infusion (A) on net excretion of acidic equivalents (JnetH+) into the water (branchial) or urine (kidney), as well as total excretion, and (B) on the relative contributions of branchial and renal excretion to total net excretion of acidic equivalents in African lungfish Protopterus annectens. Values are means ± s.e.m. for the period prior to base infusion (`pre'), and for measurement periods immediately following base infusion (0–4 h) and 18 h post-infusion (17–21 h); N=5–7. In A, an asterisk (*) indicates a significant difference from the pre-infusion value (one-way RM-ANOVA with P values of 0.03, 0.003 and 0.008 for branchial, renal and total JnetH+, respectively). Changes in the relative contributions of branchial and renal excretion to total JnetH+ were not statistically significant in B.

View larger version (7K): [in this window] [in a new window]   Fig. 6. The effect of a 1 h base (3 mmol kg–1 NaHCO3) infusion in African lungfish Protopterus annectens on urine (A) pH and (B) HCO –3 ion concentration ([HCO –3]). Values are means ± s.e.m. for the period prior to base infusion (`pre'), and for measurement periods immediately following base infusion (0–4 h) and 18 h post-infusion (17–21 h); N=5 or 6. An asterisk (*) indicates a significant difference from the pre-infusion value [one-way RM-ANOVA with P values of 0.002 (A) and 0.001 (B)].

View larger version (72K): [in this window] [in a new window]   Fig. 7. The deduced amino acid sequences of the cloned fragments of African lungfish Protopterus annectens (A) Na+/HCO –3 cotransporter (NBC) and (B) V-type H+-ATPase aligned with corresponding regions of NBC and H+ V-ATPase sequences (from GenBank databases) for selected vertebrates. Grey shading indicates amino acid residues that are conserved across all four species, whereas black shading indicates residues that are conserved across three species. lf, lungfish. GenBank protein accession numbers for the sequences used were as follows: (A) NBC: rainbow trout AAN52239, human AAC39840, salamander (Ambystoma tigrinum) AAB61339; (B) H+ V-ATPase: rainbow trout AAD33861, human AAH30640, Xenopus AAH46738.

View larger version (27K): [in this window] [in a new window]   Fig. 8. (A) The nucleotide and deduced amino acid sequences of the carbonic anhydrase (CA) fragment cloned from African lungfish Protopterus annectens blood, together with (B) a phylogenetic tree to illustrate the relationship between this lungfish CA (highlighted in black) and selected vertebrate cytoplasmic CA isoforms. The phylogenetic tree was constructed using neighbour-joining analysis (see Materials and methods for more detail), and was ordered using Drosophila CA (GenBank protein accession AAY56645) as a monophyletic outgroup. Horizontal branch lengths are scaled to represent the relative number of amino acid substitutions occurring along a branch, and support values at the nodes are indicated as a percentage from bootstrap analysis using 100 pseudoreplicates. GenBank protein accession numbers for the sequences used in the tree were as follows. CA I: mouse AAH11223, rat XP_226922, human AAH27890; CA II: mouse AAA37357, rat CAA41227, human AAA51909, Xenopus CAJ83242; CA III: mouse NP_031632, rat AAA40846, human AAA52293, CA XIII: mouse NP_078771, rat XP_222295, human NP_940986; fish cytoplasmic CAs: lamprey AAZ83742, gar AAM94169, tilapia AAQ89896, rainbow trout blood isoform (CAb) AAP73748, rainbow trout cytoplasmic isoform (CAc) AAR99329, zebrafish1 NP_571185, zebrafish2 NP_954685, Japanese dace BAB83090, and carp AAZ83743. Sequences obtained from GenBank were truncated appropriately so as to use only the region that overlapped with the lungfish CAb sequence.

View larger version (11K): [in this window] [in a new window]   Fig. 9. Relative mRNA expression as determined by real-time PCR for (A) Na+/HCO –3 cotransporter (NBC), (B) V-type H+-ATPase and (C) lungfish carbonic anhydrase b isoform (CAb) in gill and kidney tissue sampled from lungfish infused with saline (control), acid (3 mmol kg–1 NH4Cl) or base (3 mmol kg–1 NaHCO3) for 1 h and sampled 4 h or 8 h post-infusion (control fish were sampled only at 8 h). Values are means ± 1 s.e.m.; N=3 or 4. No statistically significant differences in the expression of a gene within a tissue were detected as a result of acid or base infusion (one-way ANOVA, P>0.05 in all cases).

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