Evolution of extreme stomach pH in bilateria inferred from gastric alkalization mechanisms in basal deuterostomes

Abstract

The stomachs of most vertebrates operate at an acidic pH of 2 generated by the gastric H(+)/K(+)-ATPase located in parietal cells. The acidic pH in stomachs of vertebrates is believed to aid digestion and to protect against environmental pathogens. Little attention has been placed on whether acidic gastric pH regulation is a vertebrate character or a deuterostome ancestral trait. Here, we report alkaline conditions up to pH 10.5 in the larval digestive systems of ambulacraria (echinoderm + hemichordate), the closest relative of the chordate. Microelectrode measurements in combination with specific inhibitors for acid-base transporters and ion pumps demonstrated that the gastric alkalization machinery in sea urchin larvae is mainly based on direct H(+) secretion from the stomach lumen and involves a conserved set of ion pumps and transporters. Hemichordate larvae additionally utilized HCO3(-) transport pathways to generate even more alkaline digestive conditions. Molecular analyses in combination with acidification experiments supported these findings and identified genes coding for ion pumps energizing gastric alkalization. Given that insect larval guts were also reported to be alkaline, our discovery raises the hypothesis that the bilaterian ancestor utilized alkaline digestive system while the vertebrate lineage has evolved a strategy to strongly acidify their stomachs.

Figure 1

Characterization of the gastric pH regulatory machinery in ambulacrarian larvae. (A) Microelectrode (el) pH measurements in larval digestive systems of the three species indicating highly alkaline digestive systems pluteus (7-15 dpf) and tornaria (10 -20 dpf) larvae and less alkaline conditions in B. floridae larvae (feeding stage 3 dpf). (B) Dose response curves for the inhibition of gastric alkalization in pluteus larvae were determined for the inhibitors ouabain (OUA), bafilomycin (BAF) and ethyl-isopropyl amiloride (EIPA) with respective IC₅₀ values. (C) Real time traces of gastric pH during application of inhibitors and 5 mM Na⁺ solutions (Supplemental material Figure S1) and washout. (D) Effects of inhibitors including OUA, BAF, EIPA, omeprazole (OMZ), 4,4“-diisothiocyanatostilbene-2,2′-disulphonic acid (DIDS) and acetazolamide (ACZM) as well as 0 mM HCO₃⁻, 5 mM Na⁺ and 0 mM K⁺ seawater solutions (for raw values including control experiments see Supplemental material Figure S1; n = 5-7) on the gastric alkalization machinery. (E) Immunocytochemical analyses in sea urchin pluteus larvae demonstrate the sub cellular localization of Na⁺/K⁺-ATPase (NKA), V-type H⁺-ATPase (VHA) and Na⁺/H⁺-exchanger (NHE3) immunoreactivity in the stomach epithelium of Strongylocentrotus purpuratus plutei. Dotted lines indicate the position of stomach cells. Values are presented as mean ± SE and asterisks denote significant differences (* p < 0.05, ** p < 0.001). holding pipette: hp; lumen:lu; primary body cavity: pbc; stomach: st; oesophagus: oes; intestine: int.

Figure 2

Identification of acid-base transporters in Strongylocentrotus purpuratus and Ptychodera flava using acidification experiments. Relative change in transcript abundance of putative acid-base transporters determined by qPCR in S. purpuratus (A) and P. flava (B) larvae exposed to control (pH 8.1) and acidified conditions (pH7). Time series over 24 h demonstrating relative changes in gastric pH in response to acidified seawater (pH 7) in sea urchin larvae (A; insert). The grey area indicates the sampling time point (3 h) for gene expression studies. Values are presented as mean ± SE and asterisks denote significant differences (* p < 0.05, ** p < 0.001). (C) In situ hybridization of selected acid-base transporters including Na⁺/K⁺-ATPase (NKA), V-type H⁺-ATPase (VHA), K⁺/H⁺-exchanger (KHE) and Na⁺/H⁺-exchanger 3 (NHE3) expressed in the digestive system of S. purpuratus and P. flava larvae raised under control conditions (insert: negative control using sense probe). Arrows indicate the location of the larval mouth. Stomach: st.

Figure 3

A) Hypothetical models of gastric alkalization in echinoid pluteus larvae (A) and hemichordate tornaria larvae (B). Gastric alkalization in S. purpuratus larvae is cation based to achieve net export of protons from the stomach lumen whereas P. flava larvae additionally employ a luminal import of HCO₃⁻. Gastric alkalization is energized by NKA and VHA in sea urchin and NKA and a putative HKA (light grey) in hemichordate larvae. Bars indicate the inhibition of ion pumps and transporters by specific inhibitors. C) Phylogenetic tree depicting the different sections of the digestive systems in duterostome larval stages and a dipteran larva. S. purpuratus and Ptychodera flava representing the ambulacraria have alkaline conditions (pH 9-10) in their stomachs. Neutral to acidic conditions are a character of chordate larvae including the cephalochordate B. floridae, teleost fish and amphibians. Alkaline digestive systems were also found in the anterior midgut of some insect larvae. The differential importance of the three major ion pumps including NKA, VHA and HKA responsible for gastric pH regulation are highlighted for the different groups. A: Ambulacraria ancestor; B: bilateralia ancestor; C: Chordate ancestor; Deuterostome ancestor; V: Vertebrate ancestor.