Helen Chasiotis

Tight junctions, tight junction proteins and paracellular permeability across the gill epithelium of fishes: A review

Paracellular permeability characteristics of the fish gill epithelium are broadly accepted to play a key role in piscine salt and water balance. This is typically associated with differences between gill epithelia of teleost fishes residing in seawater versus those in freshwater. In the former, the gill is leaky to facilitate Na(+) secretion and in the latter, the gill is tight to limit passive ion loss. However, studies in freshwater fishes also suggest that varying epithelial tightness can impact ionoregulatory homeostasis. Paracellular permeability of vertebrate epithelia is largely controlled by the tight junction (TJ) complex, and the fish gill is no exception. In turn, the TJ complex is composed of TJ proteins, the abundance and properties of which determine the magnitude of paracellular solute movement. This review provides consolidated information on TJs in fish gills and summarizes recent progress in research that seeks to understand the molecular composition of fish gill TJ complexes and what environmental and systemic factors influence those components.

Effect of cortisol on permeability and tight junction protein transcript abundance in primary cultured gill epithelia from stenohaline goldfish and euryhaline trout

Primary cultured gill epithelia from goldfish and rainbow trout were used to investigate a role for cortisol in the regulation of paracellular permeability and tight junction (TJ) protein transcript abundance in representative stenohaline versus euryhaline freshwater (FW) fish gills. Glucocorticoid and mineralocorticoid receptors are expressed in cultured goldfish gill preparations and cortisol treatment (100, 500 and 1000 ng/mL) dose-dependently elevated transepithelial resistance (TER) and reduced paracellular [(3)H]PEG-4000 flux across cultured goldfish gill epithelia. Despite these dose-dependent tightening effects of cortisol, the response of goldfish TJ protein transcripts (i.e. occludin, claudin b, c, d, e, h, 7, 8d and 12, and ZO-1) were surprisingly small, with only claudin c and h, and ZO-1 transcript levels significantly decreasing at a dose of 1000 ng/mL. Extending the duration of cortisol exposure from 24 to 48 or 96 h (at 500 ng/mL) did little to alter this phenomenon. By comparison, exposing primary cultured trout gill epithelia (i.e. a euryhaline fish gill model) to 500 ng/mL cortisol resulted in a qualitatively similar, but quantitatively stronger epithelial tightening response. Furthermore, transcript abundance of orthologous trout TJ proteins (i.e. occludin, and claudin 30, 28b, 3a, 7, 8d and 12) significantly elevated as would be expected in a tighter epithelium. Taken together, data suggest a conservative role for cortisol in the endocrine regulation of paracellular permeability across the goldfish gill that may relate to stenohalinity.

Glucocorticoid and mineralocorticoid receptors regulate paracellular permeability in a primary cultured gill epithelium

SUMMARY The role of corticosteroid receptors (CRs) in the regulation of gill permeability was examined using a primary cultured trout gill epithelium. The epithelium expressed both glucocorticoid receptors (GR1 and GR2) and a mineralocorticoid receptor (MR), and cortisol treatment significantly increased transepithelial resistance (TER) and decreased paracellular [3H]PEG-4000 flux. Epithelial permeability was unaffected by deoxycorticosterone or aldosterone. The GR antagonist RU486 as well as MR antagonists spironolactone and RU26752 significantly reduced, but did not completely block, the effects of cortisol. The MR antagonist eplerenone was without effect. Only RU486 + spironolactone or RU486 + RU26752 treatment completely suppressed the effects of cortisol. On its own, RU486 had cortisol-like effects which could be blocked by spironolactone, suggesting that although RU486 is a GR antagonist, in this system it may also have agonistic properties that are mediated through the MR. The GR agonist dexamethasone increased TER and reduced [3H]PEG-4000 flux across cultured epithelia and was unaffected by MR antagonists. Therefore, alterations in transcript abundance of select tight junction (TJ) proteins were examined in response to cortisol, dexamethasone (a GR agonist) and RU486 (as a MR agonist). Occludin and claudin-7, -8d, -12 and -31 mRNA were significantly elevated in response to cortisol, dexamethasone or RU486 treatment. Claudin-3a mRNA was significantly elevated in response to cortisol or dexamethasone only, and claudin-28b and -30 mRNA were significantly altered following cortisol or RU486 treatment only. The data support a role for the GRs and MR in regulating gill permeability and suggest that TJ proteins are responsive to cortisol through both or individual CR types.

Occludin immunolocalization and protein expression in goldfish

SUMMARY Tight junctions (TJs) are an integral component of models illustrating ion transport mechanisms across fish epithelia; however, little is known about TJ proteins in fishes. Using immunohistochemical methods and Western blot analysis, we examined the localization and expression of occludin, a transmembrane TJ protein, in goldfish tissues. In goldfish gills, discontinuous occludin immunostaining was detected along the edges of secondary gill lamellae and within parts of the interlamellar region that line the lateral walls of the central venous sinus. In the goldfish intestine, occludin immunolocalized in a TJ-specific distribution pattern to apical regions of columnar epithelial cells lining the intestinal lumen. In the goldfish kidney, occludin was differentially expressed in discrete regions of the nephron. Occludin immunostaining was strongest in the distal segment of the nephron, moderate in the collecting duct and absent in the proximal segment. To investigate a potential role for occludin in the maintenance of the hydromineral balance of fishes, we subjected goldfish to 1, 2 and 4 weeks of food deprivation, and then examined the endpoints of hydromineral status, Na+,K+-ATPase activity and occludin protein expression in the gills, intestine and kidney. Occludin expression altered in response to hydromineral imbalance in a tissue-specific manner suggesting a dynamic role for this TJ protein in the regulation of epithelial permeability in fishes.

Cortisol reduces paracellular permeability and increases occludin abundance in cultured trout gill epithelia

A role for the tight junction (TJ) protein occludin in the regulation of gill paracellular permeability was investigated using primary cultured reconstructed freshwater (FW) rainbow trout gill epithelia composed solely of pavement cells. Cortisol treatment reduced epithelial permeability characteristics, measured as changes in transepithelial resistance (TER) and paracellular [3H]PEG-4000 flux. Cortisol also reduced net Na+ flux rates when epithelia were exposed to apical FW. cDNA encoding for the TJ protein occludin was cloned from rainbow trout and found to be particularly abundant in gill tissue. In cultured gill preparations, occludin immunolocalized to the TJ complex and transcript abundance dose-dependently increased in response to cortisol treatment in association with reduced paracellular permeability. Occludin protein abundance also increased in response to cortisol treatment. However, occludin mRNA levels did not change in response to apical FW exposure, and [3H]PEG-4000 permeability did not decrease. These data support a role for occludin in the endocrine regulation of paracellular permeability across gill epithelia of fishes.

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.

Occludin expression in goldfish held in ion-poor water

With an emphasis on the tight junction protein occludin, the response of goldfish following abrupt exposure (0–120xa0h) as well as long-term acclimation (14 and 28xa0days) to ion-poor water (IPW) was examined. Both abrupt and long-term exposure to IPW lowered serum osmolality, [Na+] and [Cl−], and elevated serum glucose. After abrupt exposure to IPW, gill tissue exhibited a prompt and sustained decrease in Na+–K+–ATPase activity, and a transient increase in occludin expression that returned to control levels by 6xa0h. Following 14 and 28xa0days in IPW, gill occludin expression was markedly elevated, while Na+–K+–ATPase activity was only significantly different (elevated) at day 14. Kidney tissue exhibited an elevation in both Na+–K+–ATPase activity and occludin expression after 28xa0days; however, in the intestine, occludin expression declined at day 14 but did not differ from FW fish at day 28. These studies demonstrate that goldfish can tolerate abrupt as well as sustained exposure to ion-poor surroundings. Data also suggests that occludin may play an adaptive role in fishes acclimated to ion-poor conditions by contributing to the modulation of epithelial barrier properties in ionoregulatory tissues.

Effects of elevated circulating cortisol levels on hydromineral status and gill tight junction protein abundance in the stenohaline goldfish.

A role for cortisol in the regulation of hydromineral balance and gill tight junction (TJ) protein transcript abundance in the stenohaline freshwater goldfish was investigated. Intraperitoneal cortisol implants (50, 100, 200, 400 μg cortisol/g body weight) were used to dose-dependently elevate circulating cortisol levels over a 4 day period. Elevated cortisol did not significantly alter serum osmolality, serum Na(+) or muscle water content, however serum glucose and gill Na(+)-K(+)-ATPase activity were significantly increased and serum Cl(-) levels were significantly reduced when compared to control groups. Transcript levels for glucocorticoid receptor 1 (GR1) and mineralocorticoid receptor (MR) in the gill remained unchanged by cortisol treatment, however glucocorticoid receptor 2 (GR2) mRNA abundance was significantly down-regulated. Conversely, cortisol treatment significantly increased transcript and protein abundance of the TJ protein occludin in goldfish gill tissue, as well as mRNA abundance for claudin e, 7 and 8d. Goldfish tissue expression profiles demonstrated that transcripts encoding for these claudins are particularly abundant in the gill. Overall, results suggest a tightened gill epithelium in response to elevated cortisol levels in goldfish. However, negative autoregulation of gill GR2 transcript suggests a lessened capacity to respond to cortisol and thus a potentially dampened corticosteroid-mediated effect in the gill. Reduced systemic Cl(-) levels also suggest that sustained cortisol elevation in goldfish may have a detrimental effect on other ionoregulatory tissues.

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.