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Perfused Gills Reveal Fundamental Principles of pH Regulation and Ammonia Homeostasis in the Cephalopod Octopus vulgaris.

ABSTRACT: In contrast to terrestrial animals most aquatic species can be characterized by relatively higher blood [Formula: see text] concentrations despite its potential toxicity to the central nervous system. Although many aquatic species excrete [Formula: see text] via specialized epithelia little information is available regarding the mechanistic basis for NH₃/[Formula: see text] homeostasis in molluscs. Using perfused gills of Octopus vulgaris we studied acid-base regulation and ammonia excretion pathways in this cephalopod species. The octopus gill is capable of regulating ammonia (NH₃/[Formula: see text]) homeostasis by the accumulation of ammonia at low blood levels (<260 ?M) and secretion at blood ammonia concentrations exceeding in vivo levels of 300 ?M. [Formula: see text] transport is sensitive to the adenylyl cyclase inhibitor KH7 indicating that this process is mediated through cAMP-dependent pathways. The perfused octopus gill has substantial pH regulatory abilities during an acidosis, accompanied by an increased secretion of [Formula: see text]. Immunohistochemical and qPCR analyses revealed tissue specific expression and localization of Na⁺/K⁺-ATPase, V-type H⁺-ATPase, Na⁺/H⁺-exchanger 3, and Rhesus protein in the gill. Using the octopus gill as a molluscan model, our results highlight the coupling of acid-base regulation and nitrogen excretion, which may represent a conserved pH regulatory mechanism across many marine taxa.

SUBMITTER: Hu MY

PROVIDER: S-EPMC5357659 | biostudies-literature | 2017

REPOSITORIES: biostudies-literature

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Perfused Gills Reveal Fundamental Principles of pH Regulation and Ammonia Homeostasis in the Cephalopod Octopus vulgaris.

Hu Marian Y MY Sung Po-Hsuan PH Guh Ying-Jey YJ Lee Jay-Ron JR Hwang Pung-Pung PP Weihrauch Dirk D Tseng Yung-Che YC

Frontiers in physiology 20170320

In contrast to terrestrial animals most aquatic species can be characterized by relatively higher blood [Formula: see text] concentrations despite its potential toxicity to the central nervous system. Although many aquatic species excrete [Formula: see text] via specialized epithelia little information is available regarding the mechanistic basis for NH3/[Formula: see text] homeostasis in molluscs. Using perfused gills of Octopus vulgaris we studied acid-base regulation and amm ...[more]

PMID: 28373845

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Project description:BACKGROUND: Quantitative real-time polymerase chain reaction (RT-qPCR) is valuable for studying the molecular events underlying physiological and behavioral phenomena. Normalization of real-time PCR data is critical for a reliable mRNA quantification. Here we identify reference genes to be utilized in RT-qPCR experiments to normalize and monitor the expression of target genes in the brain of the cephalopod mollusc Octopus vulgaris, an invertebrate. Such an approach is novel for this taxon and of advantage in future experiments given the complexity of the behavioral repertoire of this species when compared with its relatively simple neural organization. RESULTS: We chose 16S, and 18S rRNA, actB, EEF1A, tubA and ubi as candidate reference genes (housekeeping genes, HKG). The expression of 16S and 18S was highly variable and did not meet the requirements of candidate HKG. The expression of the other genes was almost stable and uniform among samples. We analyzed the expression of HKG into two different set of animals using tissues taken from the central nervous system (brain parts) and mantle (here considered as control tissue) by BestKeeper, geNorm and NormFinder. We found that HKG expressions differed considerably with respect to brain area and octopus samples in an HKG-specific manner. However, when the mantle is treated as control tissue and the entire central nervous system is considered, NormFinder revealed tubA and ubi as the most suitable HKG pair. These two genes were utilized to evaluate the relative expression of the genes FoxP, creb, dat and TH in O. vulgaris. CONCLUSION: We analyzed the expression profiles of some genes here identified for O. vulgaris by applying RT-qPCR analysis for the first time in cephalopods. We validated candidate reference genes and found the expression of ubi and tubA to be the most appropriate to evaluate the expression of target genes in the brain of different octopuses. Our results also underline the importance of choosing a proper normalization strategy when analyzing gene expression by qPCR taking into appropriate account the experimental setting and variability of the sample of animals (and tissues), thus providing a set of HGK which expression appears to be unaffected by the experimental factor(s).

Dataset Information

Perfused Gills Reveal Fundamental Principles of pH Regulation and Ammonia Homeostasis in the Cephalopod Octopus vulgaris.

Publications

Perfused Gills Reveal Fundamental Principles of pH Regulation and Ammonia Homeostasis in the Cephalopod <i>Octopus vulgaris</i>.

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