Ammonium-dependent sodium uptake in mitochondrion-rich cells of medaka (Oryzias latipes) larvae

2010 ◽  
Vol 298 (2) ◽  
pp. C237-C250 ◽  
Author(s):  
Shu-Chen Wu ◽  
Jiun-Lin Horng ◽  
Sian-Tai Liu ◽  
Pung-Pung Hwang ◽  
Zhi-Hong Wen ◽  
...  
Keyword(s):  
In Situ Hybridization ◽  
Real Time Pcr ◽  
Skin Surface ◽  
Selective Electrode ◽  
Trapping Mechanism ◽  
Water Ph ◽  
Acid Secreting ◽  
Apical Membranes ◽  
Electrode Technique

In this study, a scanning ion-selective electrode technique (SIET) was applied to measure H+, Na+, and NH4+ gradients and apparent fluxes at specific cells on the skin of medaka larvae. Na+ uptake and NH3/NH4+ excretion were detected at most mitochondrion-rich cells (MRCs). H+ probing at MRCs revealed two group of MRCs, i.e., acid-secreting and base-secreting MRCs. Treatment with EIPA (100 μM) blocked 35% of the NH3/NH4+ secretion and 54% of the Na+ uptake, suggesting that the Na+/H+ exchanger (NHE) is involved in Na+ and NH3/NH4+ transport. Low-Na+ water (<0.001 mM) or high-NH4+ (5 mM) acclimation simultaneously increased Na+ uptake and NH3/NH4+ excretion but decreased or even reversed the H+ gradient at the skin and MRCs. The correlation between NH4+ production and H+ consumption at the skin surface suggests that MRCs excrete nonionic NH3 (base) by an acid-trapping mechanism. Raising the external NH4+ significantly blocked NH3/NH4+ excretion and Na+ uptake. In contrast, raising the acidity of the water (pH 7 to pH 6) enhanced NH3/NH4+ excretion and Na+ uptake by MRCs. In situ hybridization and real-time PCR showed that the mRNAs of the Na+/H+ exchanger ( slc9a3) and Rhesus glycoproteins ( Rhcg1 and Rhbg) were colocalized in MRCs of medaka, and their expressions were induced by low-Na+ acclimation. This study suggests a novel Na+/NH4+ exchange pathway in apical membranes of MRCs, in which a coupled NHE and Rh glycoprotein is involved and the Rh glycoprotein may drive the NHE by generating H+ gradients across apical membranes of MRCs.

scholarly journals Acid secretion by mitochondrion-rich cells of medaka (Oryzias latipes) acclimated to acidic freshwater

2012 ◽  
Vol 302 (2) ◽  
pp. R283-R291 ◽  
Author(s):  
Chia-Cheng Lin ◽  
Li-Yih Lin ◽  
Hao-Hsuan Hsu ◽  
Violette Thermes ◽  
Patrick Prunet ◽  
...  
Keyword(s):  
Acid Secretion ◽  
Mrna Level ◽  
Critical Role ◽  
Mrna Levels ◽  
Selective Electrode ◽  
Embryonic Skin ◽  
Apical Membranes ◽  
Acid Acclimation ◽  
Electrode Technique

In the present study, medaka embryos were exposed to acidified freshwater (pH 5) to investigate the mechanism of acid secretion by mitochondrion-rich (MR) cells in embryonic skin. With double or triple in situ hybridization/immunocytochemistry, the Na+/H+ exchanger 3 (NHE3) and H+-ATPase were localized in two distinct subtypes of MR cells. NHE3 was expressed in apical membranes of a major proportion of MR cells, whereas H+-ATPase was expressed in basolateral membranes of a much smaller proportion of MR cells. Gill mRNA levels of NHE3 and H+-ATPase and the two subtypes of MR cells in yolk sac skin were increased by acid acclimation; however, the mRNA level of NHE3 was remarkably higher than that of H+-ATPase. A scanning ion-selective electrode technique was used to measure H+, Na+, and NH4+ transport by individual MR cells in larval skin. Results showed that Na+ uptake and NH4+ excretion by MR cells increased after acid acclimation. These findings suggested that the NHE3/Rh glycoprotein-mediated Na+ uptake/NH4+ excretion mechanism plays a critical role in acidic equivalent (H+/NH4+) excretion by MR cells of the freshwater medaka.

Ammonia excretion by the skin of zebrafish (Danio rerio) larvae

2008 ◽  
Vol 295 (6) ◽  
pp. C1625-C1632 ◽  
Author(s):  
Tin-Han Shih ◽  
Jiun-Lin Horng ◽  
Pung-Pung Hwang ◽  
Li-Yih Lin
Keyword(s):  
Direct Evidence ◽  
Ammonia Excretion ◽  
Bafilomycin A1 ◽  
Selective Electrode ◽  
Zebrafish Larvae ◽  
Trapping Mechanism ◽  
Morpholino Oligonucleotides ◽  
Electrode Technique ◽  
External Ph ◽  
Freshwater Teleosts

The mechanism of ammonia excretion in freshwater teleosts is not well understood. In this study, scanning ion-selective electrode technique was applied to measure H+ and NH4+ fluxes in specific cells on the skin of zebrafish larvae. NH4+ extrusion was relatively high in H+ pump-rich cells, which were identified as the H+-secreting ionocyte in zebrafish. Minor NH4+ extrusion was also detected in keratinocytes and other types of ionocytes in larval skin. NH4+ extrusion from the skin was tightly linked to acid secretion. Increases in the external pH and buffer concentration (5 mM MOPS) diminished H+ and NH4+ gradients at the larval surface. Moreover, coupled decreases in NH4+ and H+ extrusion were found in larvae treated with an H+-pump inhibitor (bafilomycin A1) or H+-pump gene ( atp6v1a) knockdown. Knockdown of Rhcg1 with morpholino-oligonucleotides also decreased NH4+ excretion. This study demonstrates ammonia excretion in epithelial cells of larval skin through an acid-trapping mechanism, and it provides direct evidence for the involvement of the H+ pump and an Rh glycoprotein (Rhcg1) in ammonia excretion.

Quantification of an Eikelboom type 021N bulking event with fluorescence in situ hybridization and real-time PCR

2005 ◽  
Vol 68 (5) ◽  
pp. 695-704 ◽  
Author(s):  
Han Vervaeren ◽  
Kurt De Wilde ◽  
Jorg Matthys ◽  
Nico Boon ◽  
Lutgarde Raskin ◽  
...  
Keyword(s):  
In Situ Hybridization ◽  
Fluorescence In Situ Hybridization ◽  
Real Time ◽  
Real Time Pcr

scholarly journals OCT4B1 Regulates the Cellular Stress Response of Human Dental Pulp Cells with Inflammation

2017 ◽  
Vol 2017 ◽  
pp. 1-10 ◽  
Author(s):  
Lu Liu ◽  
Rong Huang ◽  
Ruiqi Yang ◽  
Xi Wei
Keyword(s):  
In Situ Hybridization ◽  
Survival Rate ◽  
Western Blot ◽  
Real Time ◽  
Real Time Pcr ◽  
Dental Pulp ◽  
Immunofluorescence Staining ◽  
Dental Pulp Cells ◽  
Pulp Cells

Introduction. Infection and apoptosis are combined triggers for inflammation in dental tissues. Octamer-binding transcription factor 4-B1 (OCT4B1), a novel spliced variant of OCT4 family, could respond to the cellular stress and possess antiapoptotic property. However, its specific role in dental pulpitis remains unknown. Methods. To investigate the effect of OCT4B1 on inflammation of dental pulp cells (DPCs), its expression in inflamed dental pulp tissues and DPCs was examined by in situ hybridization, real-time PCR, and FISH assay. OCT4B1 overexpressed DPCs model was established, confirmed by western blot and immunofluorescence staining, and then stimulated with Lipopolysaccharide (LPS). Apoptotic rate was determined by Hoechst/PI staining and FACS. Cell survival rate was calculated by CCK8 assay. Results. In situ hybridization, real-time PCR, and FISH assay revealed that OCT4B1 was extensively expressed in inflamed dental pulp tissues and DPCs with LPS stimulation. Western blot and immunofluorescence staining showed the expression of OCT4B1 and OCT4B increased after OCT4B1 transfection. Hoechst/PI staining and FACS demonstrated that less red/blue fluorescence was detected and apoptotic percentage decreased (3.45%) after transfection. CCK8 demonstrated that the survival rate of pCDH-OCT4B1-flag cells increased. Conclusions. OCT4B1 plays an essential role in inflammation and apoptosis of DPCs. OCT4B might operate synergistically with OCT4B1 to reduce apoptosis.

scholarly journals CircStrn3 Targeting microRNA-9-5p Is Involved in the Regulation of Cartilage Degeneration and Subchondral Bone Remodeling in Osteoarthritis

2021 ◽  
Author(s):  
Bin Li ◽  
Tao Ding ◽  
Haoyi Chen ◽  
Changwei Li ◽  
Bo Chen ◽  
...  
Keyword(s):  
In Situ Hybridization ◽  
Real Time Pcr ◽  
Tensile Strain ◽  
Protective Factor ◽  
Medial Meniscus ◽  
Joint Disease ◽  
Quantitative Real Time Pcr ◽  
Mechanical Instability ◽  
Human And Mouse

Abstract Background: Osteoarthritis (OA) is the most frequent chronic degenerative joint disease, which is a “whole joint” disease including the pathological changes in the cartilage, subchondral bone and the synovium. Mechanical instability is the initiation of the development of OA. Methods: Minus RNA sequencing, fluorescence in situ hybridization and quantitative real-time PCR were used to detect the expression of circStrn3 in human and mouse OA cartilage tissues and chondrocytes. Stimulate chondrocytes to secrete exosomes miR-9-5p by stretching strain. Intra-articular injection of exosomes miR-9-5p into the OA model induced by the operation of instability of the medial meniscus in mice.Results: In the present study, minus RNA sequencing data showed that tensile strain could decrease the expression of circStrn3 in chondrocytes. The results of fluorescence in situ hybridization and quantitative Real-time PCR showed that circStrn3 expression was significantly decreased in human and mouse OA cartilage tissues and chondrocytes. CircStrn3 could inhibit matrix metabolism of chondrocytes through competitively 'sponging' miRNA-9-5p targeting kruppel-like factor 5 (KLF5), indicating that the decreasing of circStrn3 might be a protective factor in mechanical instability-induced OA. Further studies showed that the tensile strain stimulated chondrocytes to secrete exosomes miR-9-5p. Exosomes with high miR-9-5p expression from chondrocytes could inhibit osteoblasts differentiation by targeting KLF5. In addition, intra-articular injection of exosomal miR-9-5p obviously alleviated the progression of OA induced by destabilized medial meniscus surgery in mice. Conclusions: Taken together, these results demonstrated that the reduction of circStrn3 caused the increasing of miR-9-5p, which acted as a protective factor in mechanical instability-induced OA and provided a novel mechanism of communication among joint components and a potential application for the treatment of OA.

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