Na+/H+ exchangers differentially contribute to midgut fluid sodium and proton concentration in the sea urchin larva

Abstract

Regulation of ionic composition and pH is a requisite of all digestive systems in the animal kingdom. Larval stages of the marine superphylum ambulacraria, including echinoderms and hemichordates, were demonstrated to have highly alkaline conditions in their midgut with the underlying epithelial transport mechanisms being largely unknown.Using ion-selective microelectrodes, the present study demonstrated that pluteus larvae of the purple sea urchin have highly alkaline pH (pH ∼9) and low [Na+] ( ̴120 mM) in their midgut fluids, compared to the ionic composition of the surrounding sea water. We pharmacologically investigated the role of Na+/H+-exchangers in intracellular pH regulation and midgut proton and sodium maintenance using the NHE inhibitor 5-(n-ethyl-n-isopropyl)amiloride (EIPA). Basolateral EIPA application decreased midgut pH while luminal application, via micro-injections increased midgut [Na+], without affecting pH. Immunohistochemical analysis demonstrated a luminal localization of NHE-2 (SpSlc9a2) in the midgut epithelium. Specific knockdown of spslc9a2 using vivo morpholinos led to an increase in midgut [Na+] without affecting pH. Acute acidification experiments in combination with qPCR analysis and measurements of midgut pH and [Na+] identified two other NHE isoforms, Spslc9a7 and SpSlc9a8 that potentially contribute to the regulation of [Na+] and pH in midgut fluids.This work provides new insights to ion regulatory mechanisms in the midgut epithelium of sea urchin larvae. The involvement of NHEs in regulating pH and Na+ balance in midgut fluids shows conserved features to insect and vertebrate digestive systems and may contribute to the ability of sea urchin larvae to cope with changes in seawater pH.

Extracellular carbonic anhydrase activity promotes a carbon concentration mechanism in metazoan calcifying cells

“…three times a week. Spawning of males and females was induced by shaking and larval cultures were maintained at a concentration of 25,000 larvae/L at 15 °C with continuous aeration, as previously described ( 62 ).…”

“…H + -selective microelectrodes were used for the in vivo measurement of eCA activity in PMCs. The construction of H + -selective microelectrodes has been previously described ( 5 , 33 , 62 ). Briefly, borosilicate glass capillaries were pulled on a DMZ-Universal puller (Zeiss Instruments) into micropipettes with tip diameters of 1 to 2 μm.…”

Ion Pathways in Biomineralization: Perspectives on Uptake, Transport, and Deposition of Calcium, Carbonate, and Phosphate

“…36b Recently, a new transporter was identified, which mediates the exit of protons from the cell. 47 It is not clear whether the same transporter also mediates exit of protons from the mineralizing vesicles. The enzyme carbonic anhydrase catalyzes the transformation of carbonic acid into protons and bicarbonate ions.…”

“…PMCs use Na + /H + exchange mechanisms to control cellular pH homeostasis during maintenance of the skeleton . Recently, a new transporter was identified, which mediates the exit of protons from the cell . It is not clear whether the same transporter also mediates exit of protons from the mineralizing vesicles.…”

Carbonic anhydrases in development: morphological observations and gene expression profiling in sea urchin embryos exposed to acetazolamide

“…A functional complex or ‘transport metabolon’, composed of CAII and bicarbonate transporters, has been hypothesized [45], possibly involved in the regulation of bicarbonate metabolism and transport, although a real physical contact between the two molecules is still controversial and is a topic of discussion [44,46,47]. Nevertheless, a number of ion transporters/exchangers has been annotated in the sea urchin genome and some have even been described and characterized in various species and different tissues and with varied functions [16,48,49]. In particular, Sp SLC4a10, belonging to the SLC4 bicarbonate transporter family, has been recently identified in PMCs of S. purpuratus embryo and its role in biomineralization via pH i regulatory and HCO3− concentration mechanisms has been hypothesized [16].…”