Dennis Kolosov

Permeability properties of the teleost gill epithelium under ion-poor conditions

Permeability properties of the goldfish gill epithelium were examined in vivo and in vitro following exposure to ion-poor water (IPW) conditions. In gill tissue of IPW-acclimated goldfish, transcript abundance of tight junction (TJ) proteins occludin, claudin-b, -d, -e, -h, -7, and -8d increased, whereas ZO-1 and claudin 12 mRNA decreased and claudin-c was unaltered. In association with these changes, TJ depth increased among gill pavement cells (PVCs) and gill PVCs and mitochondria-rich cells (MRCs). PVC and MRC gill cell fractions were isolated using Percoll. Transcripts encoding for occludin, claudin-b, -c, -d, -e, -h, -7, -8d, -12, and ZO-1 were present in both fractions. After IPW acclimation, occludin, claudin-b and -e, and ZO-1 mRNA abundance increased in both fractions. In contrast, claudin-8d mRNA abundance increased in PVCs only while claudin-h decreased in MRCs. Gill permeability was examined using primary cultured goldfish PVC epithelia supplemented with serum derived from IPW-acclimated goldfish. IPW serum supplementation increased transepithelial resistance, reduced [(3)H]PEG-4000 permeability, and enhanced epithelial integrity during in vitro IPW exposure. IPW serum increased mRNA abundance of occludin, claudin-8d and -e in vitro. Using small interfering RNA, we found that occludin abundance was decreased in cultured gill epithelia, resulting in an increase in [(3)H]PEG-4000 flux. As occludin increased in the gills of IPW-acclimated fish as well as cultured gill epithelia exposed to IPW serum, results suggest that occludin is a barrier-forming TJ protein in fish gill epithelia. These studies support the idea that TJ proteins play an important role in regulating gill permeability in IPW.

Claudins in teleost fishes

Teleost fishes are a large and diverse animal group that represent close to 50% of all described vertebrate species. This review consolidates what is known about the claudin (Cldn) family of tight junction (TJ) proteins in teleosts. Cldns are transmembrane proteins of the vertebrate epithelial/endothelial TJ complex that largely determine TJ permeability. Cldns achieve this by expressing barrier or pore forming properties and by exhibiting distinct tissue distribution patterns. So far, ~63 genes encoding for Cldn TJ proteins have been reported in 16 teleost species. Collectively, cldns (or Cldns) are found in a broad array of teleost fish tissues, but select genes exhibit restricted expression patterns. Evidence to date strongly supports the view that Cldns play a vital role in the embryonic development of teleost fishes and in the physiology of tissues and organ systems studied thus far.

Tight junction protein gene expression patterns and changes in transcript abundance during development of model fish gill epithelia

In vertebrates, tight junction (TJ) proteins play an important role in epithelium formation and development, the maintenance of tissue integrity and regulation of TJ permeability. In this study, primary cultured model gill epithelia composed of pavement cells (PVCs) were used to examine TJ protein transcript abundance during the development of epithelium confluence and epithelium resistive properties. Differences in TJ protein expression patterns and transcript abundance between gill models composed of PVCs and models composed of PVCs and mitochondrion-rich cells (MRCs) were also examined. Marked alterations in TJ protein transcript abundance were observed as cells developed to confluence in flask-cultured model gill epithelia. In contrast, during the formation of tissue resistance in insert-cultured epithelia (i.e. epithelia cultured on a permeable substrate), changes in TJ protein mRNA abundance were conservative, despite paracellular marker flux decreasing by orders of magnitude. In both cases significant changes in claudin-8b, -8d, -27b, -28b and -32a transcript abundance were observed, suggesting that temporal alterations in the abundance of these genes are important end points of model gill epithelium integrity. When MRCs were present in cultured gill models, the mRNA abundance of several TJ proteins significantly altered and claudin-10c, -10d and -33b were only detected in preparations that included MRCs. These data provide insight into the role of select TJ proteins in the formation and development of gill epithelia and the maintenance of gill barrier properties. In addition, observations reveal a heterogeneous distribution of claudin TJ proteins in the gill epithelial cells of rainbow trout.

A role for tricellulin in the regulation of gill epithelium permeability

The apical-most region of cell-to-cell contact in a vertebrate epithelium is the tight junction (TJ) complex. It is composed of bicellular TJs (bTJs) that bridge two adjacent epithelial cells and tricellular TJs (tTJs) that are points of contact between three adjoining epithelial cells. Tricellulin (TRIC) is a transmembrane TJ protein of vertebrates that is found in the tTJ complex. Full-length cDNA encoding rainbow trout TRIC was cloned and sequenced. In silico analysis of rainbow trout TRIC revealed a tetraspannin protein with several putative posttranslational modification sites. TRIC mRNA was broadly expressed in rainbow trout tissues and exhibited moderately greater abundance in the gill. In a primary cultured gill epithelium, TRIC localized to tTJs and TRIC protein abundance increased in association with corticosteroid-induced reductions in paracellular permeability. Sodium caprate was used to compromise cultured gill epithelium integrity by disrupting the tTJ complex. Sodium caprate treatment caused a reversible reduction in transepithelial resistance, caused an increase in paracellular permeability (as measured by [³H]PEG-4000 flux), and displaced TRIC from tTJs while leaving bTJs intact. Data from this study support the view that tTJs and the TJ protein TRIC 1) play a role in maintaining gill epithelium integrity and 2) contribute to the regulation of gill epithelium permeability.

Effect of the liquorice root derivatives on salt and water balance in a teleost fish, rainbow trout (Oncorhynchus mykiss).

The effect of liquorice root derivatives (LRDs) glycyrrhizic acid (GL) and glycyrrhetinic acid (18βGA) on salt and water balance and end points of gill ion transport in a freshwater teleost, (rainbow trout) was examined after feeding fish diets containing GL or 18βGA (0, 5, 50 or 500 µg/g diet) for a two week period. Serum cortisol levels and gill 11β-hydroxysteroid dehydrogenase type 2 mRNA abundance decreased in fish fed GL but increased (at select doses) in fish fed 18βGA. At higher doses of GL, gill Na(+)-K(+)-ATPase and H(+)-ATPase activity increased, while cystic fibrosis transmembrane conductance regulator type II mRNA abundance significantly decreased at the lowest dose of GL. End points of gill transcellular ion transport were not significantly altered in fish fed 18βGA, except for a reduction in Na(+)-K(+)-ATPase activity at a 50 µg/g dose. In contrast, high doses of GL and 18βGA increased gill transcript abundance of the tight junction protein claudin-31 (cldn-31). Other end points of gill paracellular transport differed in fishes fed LRDs. Tricellulin mRNA abundance was increased by high dose GL and decreased by high dose 18βGA, and cldn-23a and cldn-27b mRNA abundance significantly decreased in response to GL irrespective of dose. Despite the above observations, systemic end points of salt and water balance (i.e. serum [Na(+)] and [Cl(-)] as well as muscle moisture) were unaffected by LRDs. Therefore data suggest that LRDs can alter end points of ion transport in fishes but that overall salt and water balance need not be perturbed.

Claudin tight junction proteins in rainbow trout (Oncorhynchus mykiss) skin: Spatial response to elevated cortisol levels

This study examined regional distribution and corticosteroid-induced alterations of claudin (cldn) transcript abundance in teleost fish skin. Regional comparison of mRNA encoding 20 Cldns indicated that 12 exhibit differences in abundance along the dorsoventral axis of skin. However, relative abundance of cldns (i.e. most to least abundant) remained similar in different skin regions. Several cldns appear to be present in the epidermis and dermal vasculature whereas others are present only in the epidermis. Increased circulating cortisol levels significantly altered mRNA abundance of 10 cldns in a region specific manner, as well as corticosteroid receptors and 11β-hydroxysteroid dehydrogenase (type 2). Epidermis and epidermal mucous cell morphometrics also altered in response to cortisol, exhibiting changes that appear to enhance skin barrier properties. Taken together, data provide a first look at spatial variation in the molecular physiology of the teleost fish integument TJ complex and region-specific sensitivity to an endocrine factor.

The liquorice root derivative glycyrrhetinic acid can ameliorate ionoregulatory disturbance in rainbow trout (Oncorhynchus mykiss) abruptly exposed to ion-poor water

To consider the idea that a dietary botanical supplement could act as an adaptogen in a teleost fish, the effect of a liquorice root derivative (18β-glycyrrhetinic acid, 18βGA) on rainbow trout following an acute ionoregulatory stressor was examined. Freshwater (FW) trout were fed a control or 18βGA supplemented diet (0, 5, or 50μg 18βGA/g diet) for 2weeks, then abruptly exposed to ion-poor water (IPW) for 24h. Following IPW exposure, muscle moisture content and serum cortisol levels elevated and serum [Na(+)] and/or [Cl(-)] reduced in control and 50μg/g 18βGA-fed fish. However, these endpoints were unaltered in 5μg/g 18βGA-fed fish. Gill tissue was investigated for potential mechanisms of 18βGA action by examining mRNA abundance of genes encoding corticosteroid receptors (CRs), 11β-hydroxysteroid dehydrogenase 2 (11β-hsd2), and tight junction (TJ) proteins, as well as Na(+)-K(+)-ATPase and H(+)-ATPase activity, and mitochondrion-rich cell (MRC) morphometrics. Following IPW exposure, CR and 11β-hsd2 mRNA, MRC fractional surface, Na(+)-K(+)-ATPase and H(+)-ATPase activity were unaltered or decreased in 50μg 18βGA fish, as was mRNA encoding select TJ proteins. In contrast, 5μg 18βGA-fed fish exhibited elevated 11β-hsd2 and CR mRNA abundance versus 50μg 18βGA-fed, and reduced MRC apical area as well as some differences in TJ protein mRNA abundance versus control fish. Data suggest that 18βGA, at low levels, may be adaptogenic in trout and might help to ameliorate ionoregulatory perturbation following IPW exposure. This seems to occur, in part, through 18βGA-induced alterations in the biochemistry and physiology of the gill.

A role for tight junction-associated MARVEL proteins in larval sea lamprey (Petromyzon marinus) osmoregulation

ABSTRACT This study reports on tight junction-associated MARVEL proteins of larval sea lamprey (Petromyzon marinus) and their potential role in ammocoete osmoregulation. Two occludin isoforms (designated Ocln and Ocln-a) and a tricellulin (Tric) were identified. Transcripts encoding ocln, ocln-a and tric were broadly expressed in larval lamprey, with the greatest abundance of ocln in the gut, liver and kidney, ocln-a in the gill and skin, and tric in the kidney. Ocln and Ocln-a resolved as ∼63 kDa and ∼35 kDa MW proteins, respectively, while Tric resolved as a ∼50 kDa protein. Ocln immunolocalized to the gill vasculature and in gill mucous cells while Ocln-a localized to the gill pouch and gill epithelium. Both Ocln and Ocln-a localized in the nephron, the epidermis and the luminal side of the gut. In branchial tissue, Tric exhibited punctate localization, consistent with its presence at regions of tricellular contact. Following ion-poor water (IPW) acclimation of ammocoetes, serum [Na+] and [Cl−] decreased, but not [Ca2+], and carcass moisture content increased. In association, Ocln abundance increased in the skin and kidney, but reduced in the gill of IPW-acclimated ammocoetes while Ocln-a abundance reduced in the kidney only. Tric abundance increased in the gill. Region-specific alterations in ocln, ocln-a and tric mRNA abundance were also observed in the gut. Data support a role for Ocln, Ocln-a and Tric in the osmoregulatory strategies of a basal vertebrate. Summary: A first look at TJ-associated MARVEL proteins in a basal vertebrate (agnathan) supports a role for Ocln, Ocln-a and Tric in the regulation of salt and water balance in freshwater.

Tricellular tight junction-associated angulins in the gill epithelium of rainbow trout

Molecular physiology of the tricellular tight junction (tTJ)-associated proteins lipolysis-stimulated lipoprotein receptor ( lsr, = angulin-1) and an immunoglobulin-like domain-containing receptor ( ildr2, ≈angulin-3) was examined in model trout gill epithelia. Transcripts encoding lsr and ildr2 are broadly expressed in trout organs. A reduction in lsr and ildr2 mRNA abundance was observed during and after confluence in flask-cultured gill cells. In contrast, as high-resistance and low-permeability characteristics developed in a model gill epithelium cultured on permeable polyethylene terephthalate membrane inserts, lsr and ildr2 transcript abundance increased. However, as epithelia entered the developmental plateau phase, lsr abundance returned to initial values, while ildr2 transcript abundance remained elevated. When mitochondrion-rich cells were introduced to model preparations, lsr mRNA abundance was unaltered and ildr2 mRNA abundance significantly increased. Transcript abundance of ildr2 was not altered in association with corticosteroid-induced tightening of the gill epithelium, while lsr mRNA abundance decreased. Transcriptional knockdown of the tTJ protein tricelluin (Tric) reduced Tric abundance, increased gill epithelium permeability, and increased lsr without significantly altering ildr2 transcript abundance. Data suggest that angulins contribute to fish gill epithelium barrier properties but that Lsr and Ildr2 seem likely to play different roles. This is because ildr2 typically exhibited increased abundance in association with decreased model permeability, while lsr abundance changed in a manner that suggested a role in Tric recruitment to the tTJ.