Institute of Physiology, Christian-Albrechts University of Kiel, Hermann-Rodewaldstraße 5, 24118 Kiel, Germany.
J Exp Biol. 2021 Apr 1;224(7). doi: 10.1242/jeb.240705. Epub 2021 Apr 15.
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 mmol l-1) in their midgut fluids, compared with the ionic composition of the surrounding seawater. We pharmacologically investigated the role of Na+/H+ exchangers (NHE) 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 quantitative PCR analysis and measurements of midgut pH and [Na+] identified two other NHE isoforms, Spslc9a7 and SpSlc9a8, which potentially contribute to the regulation of [Na+] and pH in midgut fluids. This work provides new insights into 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 of insect and vertebrate digestive systems and may contribute to the ability of sea urchin larvae to cope with changes in seawater pH.
动物王国所有消化系统都需要调节离子组成和 pH 值。海洋超门环节动物和半索动物的幼虫阶段，其中肠呈现高度碱性环境，但其上皮转运机制在很大程度上尚不清楚。本研究使用离子选择性微电极表明，紫海胆的浮游幼虫中肠液具有高度碱性的 pH 值（pH 约为 9）和低的[Na+]（约 120 mmol l-1），与周围海水的离子组成形成对比。我们使用 Na+/H+交换器（NHE）抑制剂 5-（n-乙基-n-异丙基）阿米洛利（EIPA），从药理学角度研究了 NHE 在细胞内 pH 值调节和中肠质子及钠离子维持中的作用。基底外侧 EIPA 应用降低了中肠 pH 值，而通过微注射进行的腔侧应用增加了中肠 [Na+]，而不影响 pH 值。免疫组织化学分析表明，NHE-2（SpSlc9a2）在中肠上皮中呈腔侧定位。使用 Vivo-Morpholinos 特异性敲低 spslc9a2 导致中肠 [Na+]增加而不影响 pH 值。急性酸化实验结合定量 PCR 分析以及中肠 pH 值和[Na+]的测量，鉴定出另外两种 NHE 同工型 Spslc9a7 和 SpSlc9a8，它们可能有助于中肠液中[Na+]和 pH 值的调节。这项工作为海胆幼虫中肠上皮的离子调节机制提供了新的见解。NHE 参与调节中肠液的 pH 值和 Na+平衡，显示出昆虫和脊椎动物消化系统的保守特征，可能有助于海胆幼虫应对海水 pH 值变化的能力。
