Midgut Na+ and pH homeostasis is differentally regulated by Na+/H+ exchangers in the sea urchin larva

Abstract

Analogous to the situation in several lepidopteran and dipteran insects also the larval stages of some echinoderms and hemichordates (ambulacraria superphylum) evolved highly alkaline midguts up to pH 10.5. Despite its pivotal role in species´ sensitivity to changes in seawater pH, the underlying epithelial transport mechanisms are largely unknown. Using ion-selective microelectrodes we found that pluteus larva of the purple sea urchin not only have highly alkaline midgut fluids (pH ~9) but also a substantial reduced sodium concentration (150 mM) compared to the surrounding sea water (450 mM). We pharmacologically investigated the role of Na+/H+ exchangers in intracellular pH (pHi) regulation and midgut proton and sodium maintenance using the NHE inhibitor 5 (n ethyl n isopropyl)amiloride (EIPA). While life cell imaging demonstrated the importance of NHEs in pHi regulation of midgut epithelia cells, basolateral EIPA application decreased midgut pH whereas luminal application, via micro-injections, increased midgut [Na+], without affecting pH. Gene expression analyses identified putative NHE candidates for midgut pH and Na+ homeostasis. Based on this information an antibody was generated against the sea urchin Slc9a2, which showed that the protein is localized in luminal membranes of the midgut. Additionally the use of specific vivo morpholino knock down of spslc9a2 provoked an increase in midgut [Na+] without affecting pH underlining the role of this transporter in midgut Na+ maintenance. This work provides new insights of NHEs being involved in ion regulatory mechanisms, especially the maintenance of pH and [Na+] in larval midgut fluids and thereby show conserved features to insect and vertebrate digestive systems, which may contribute to the ability of sea urchin larvae to cope with changes in seawater pH.