SNCA and TPPP transcripts increase in oligodendroglial cytoplasmic inclusions in multiple system atrophy.

Multiple system atrophy (MSA) is characterized by glial cytoplasmic inclusions (GCIs) containing aggregated α-synuclein (α-syn) in oligodendrocytes. The origin of α-syn accumulation in GCIs is unclear, in particular whether abnormal α-syn aggregates result from the abnormal elevation of endogenous α-syn expression in MSA or ingested from the neuronal source. Tubulin polymerization promoting protein (TPPP) has been reported to play a crucial role in developing GCI pathology. Here, the total cell body, nucleus, and cytoplasmic area density of SNCA and TPPP transcripts in neurons and oligodendrocytes with and without various α-syn pathologies in the pontine base in autopsy cases of MSA (n = 4) and controls (n = 2) were evaluated using RNAscope with immunofluorescence. Single-nucleus RNA-sequencing data for TPPP was evaluated using control frontal cortex (n = 3). SNCA and TPPP transcripts were present in the nucleus and cytoplasm of oligodendrocytes in both controls and diseased, with higher area density in GCIs and glial nuclear inclusions in MSA. Area densities of SNCA and TPPP transcripts were lower in neurons showing cytoplasmic inclusions in MSA. Indeed, TPPP transcripts were unexpectedly found in neurons, while the anti-TPPP antibody failed to detect immunoreactivity. Single-nucleus RNA-sequencing revealed significant TPPP transcript expression predominantly in oligodendrocytes, but also in excitatory and inhibitory neurons. This study addressed the unclear origin of accumulated α-syn in GCIs, proposing that the elevation of SNCA transcripts may supply templates for misfolded α-syn. In addition, the parallel behavior of TPPP and SNCA transcripts in GCI development highlights their potential synergistic contribution to inclusion formation. In conclusion, this study advances our understanding of MSA pathogenesis, offers insights into the dynamics of SNCA and TPPP transcripts in inclusion formation, and proposes regulating their transcripts for future molecular therapy to MSA.

Aedes aegypti Rhesus glycoproteins contribute to ammonia excretion by larval anal papillae.

In larval Aedes aegypti, transcripts of the Rhesus-like glycoproteins AeRh50-1 and AeRh50-2 have been detected in the anal papillae, sites of ammonia (NH3/NH4+) excretion; however, these putative ammonia transporters have not been previously localized or functionally characterized. In this study, we show that the AeRh50s co-immunolocalize with apical V-type H+-ATPase as well as with basal Na+/K+-ATPase in the epithelium of anal papillae. The double-stranded RNA-mediated knockdown of AeRh50-1 and AeRh50-2 resulted in a significant reduction in AeRh50 protein abundance in the anal papillae, and this was coupled to decreased ammonia excretion. The knockdown of AeRh50-1 resulted in decreased hemolymph [NH4+] and pH whereas knockdown of AeRh50-2 had no effect on these parameters. We conclude that the AeRh50s are important contributors to ammonia excretion at the anal papillae of larval A. aegypti, which may be the basis for their ability to inhabit areas with high ammonia levels.

An animal homolog of plant Mep/Amt transporters promotes ammonia excretion by the anal papillae of the disease vector mosquito Aedes aegypti.

The transcripts of three putative ammonia (NH3/NH4 (+)) transporters, Rhesus-like glycoproteins AeRh50-1, AeRh50-2 and Amt/Mep-like AeAmt1 were detected in the anal papillae of larval Aedes aegypti Quantitative PCR studies revealed 12-fold higher transcript levels of AeAmt1 in anal papillae relative to AeRh50-1, and levels of AeRh50-2 were even lower. Immunoblotting revealed AeAmt1 in anal papillae as a pre-protein with putative monomeric and trimeric forms. AeAmt1 was immunolocalized to the basal side of the anal papillae epithelium where it co-localized with Na(+)/K(+)-ATPase. Ammonium concentration gradients were measured adjacent to anal papillae using the scanning ion-selective electrode technique (SIET) and used to calculate ammonia efflux by the anal papillae. dsRNA-mediated reductions in AeAmt1 decreased ammonia efflux at larval anal papillae and significantly increased ammonia levels in hemolymph, indicating a principal role for AeAmt1 in ammonia excretion. Pharmacological characterization of ammonia transport mechanisms in the anal papillae suggests that, in addition to AeAmt1, the ionomotive pumps V-type H(+)-ATPase and Na(+)/K(+)-ATPase as well as NHE3 are involved in ammonia excretion at the anal papillae.

Adiponectin mediated APPL1-AMPK signaling induces cell migration, MMP activation, and collagen remodeling in cardiac fibroblasts.

Defects in adiponectin action have been implicated in the development of cardiac dysfunction in obesity and diabetes. Cardiac fibroblasts play an important role in regulating extracellular matrix remodeling yet little is known regarding the direct effects of adiponectin on cardiac fibroblasts. In this study, we first demonstrated temporal relocalization of cellular APPL1 in response to adiponectin in primary cardiac fibroblasts and that siRNA-mediated knockdown of APPL1 attenuated stimulation of AMPK by adiponectin. The cell surface content of MT1-MMP and activation of MMP2 were induced by adiponectin and these responses were dependent on AMPK signaling. Enhanced MMP activity facilitated increased fibroblast migration in response to adiponectin which was also prevented by inhibition of AMPK, with no change in cell proliferation observed. Collagen and elastin immunofluorescence demonstrated reorganization of the extracellular matrix in accordance with increased MMP activity, whereas quantitative mRNA analysis, (3) H-proline incorporation and picrosirius red assays showed no change in intracellular or extracellular total collagen levels in response to adiponectin. In summary, these data are the first to report the adiponectin stimulated APPL1-AMPK signaling axis in cardiac fibroblasts and characterize MT1-MMP translocation, MMP2 activity and cell migration as functional outcomes. These effects may be of significance in heart failure associated with obesity and diabetes.

Allatostatin A-like immunoreactivity in the nervous system and gut of the larval midge Chironomus riparius: modulation of hindgut motility, rectal K+ transport and implications for exposure to salinity.

Evidence for the presence of allatostatin (AST) A-like neuropeptides in the larval midge Chironomus riparius is reported. Immunohistochemical studies on the nervous system and gut revealed the presence of AST A-like immunoreactive (AST-IR) cells and processes. The nerve cord contained AST-IR processes that originated from cells in the brain and travelled the length of nerve cord to the terminal ganglion. Within each ganglion, these processes gave rise to varicosities, suggesting that they formed synapses with neurons in the ganglia. Endocrine cells containing AST-IR were present in three regions of the midgut: near the attachment of the Malpighian tubules, between the anterior and posterior midgut, and in the vicinity of the gastric caecae. The terminal ganglion also contained four AST-IR cells that gave rise to axons that projected onto the hindgut and posterior midgut. Application of a cockroach AST to the semi-isolated hindgut of larval C. riparius led to dose-dependent inhibition of muscle contractions with an EC50 of ~10 nmol l(-1) and a decrease in rectal K(+) reabsorption resulting from reduced rectal Na(+)/K(+)-ATPase and vacuolar type H(+)-ATPase activities. The results suggest the presence of endogenous AST-like neuropeptides in larval C. riparius, where these factors play a role in the function of the gut. Furthermore, regulation of ion reabsorption by ASTs at the rectum could serve as an ideal mechanism of ion regulation in the face of abrupt and acute elevated salt levels.

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.

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.

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.

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

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 [(3)H]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 [(3)H]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.

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.

Neocortical inhibitory interneuron subtypes are differentially attuned to synchrony- and rate-coded information.

Neurons can carry information with both the synchrony and rate of their spikes. However, it is unknown whether distinct subtypes of neurons are more sensitive to information carried by synchrony versus rate, or vice versa. Here, we address this question using patterned optical stimulation in slices of somatosensory cortex from mouse lines labelling fast-spiking (FS) and regular-spiking (RS) interneurons. We used optical stimulation in layer 2/3 to encode a 1-bit signal using either the synchrony or rate of activity. We then examined the mutual information between this signal and the interneuron responses. We found that for a synchrony encoding, FS interneurons carried more information in the first five milliseconds, while both interneuron subtypes carried more information than excitatory neurons in later responses. For a rate encoding, we found that RS interneurons carried more information after several milliseconds. These data demonstrate that distinct interneuron subtypes in the neocortex have distinct sensitivities to synchrony versus rate codes.

Transendothelial movement of adiponectin is restricted by glucocorticoids.

Altered permeability of the endothelial barrier in a variety of tissues has implications both in disease pathogenesis and treatment. Glucocorticoids are potent mediators of endothelial permeability, and this forms the basis for their heavily prescribed use as medications to treat ocular disease. However, the effect of glucocorticoids on endothelial barriers elsewhere in the body is less well studied. Here, we investigated glucocorticoid-mediated changes in endothelial flux of Adiponectin (Ad), a hormone with a critical role in diabetes. First, we used monolayers of endothelial cells in vitro and found that the glucocorticoid dexamethasone increased transendothelial electrical resistance and reduced permeability of polyethylene glycol (PEG, molecular weight 4000 Da). Dexamethasone reduced flux of Ad from the apical to basolateral side, measured both by ELISA and Western blotting. We then examined a diabetic rat model induced by treatment with exogenous corticosterone, which was characterized by glucose intolerance and hyperinsulinemia. There was no change in circulating Ad but less Ad protein in skeletal muscle homogenates, despite slightly higher mRNA levels, in diabetic vs control muscles. Dexamethasone-induced changes in Ad flux across endothelial monolayers were associated with alterations in the abundance of select claudin tight junction (TJ) proteins. shRNA-mediated knockdown of one such gene, claudin-7, in HUVEC resulted in decreased TEER and increased adiponectin flux, confirming the functional significance of Dex-induced changes in its expression. In conclusion, our study identifies glucocorticoid-mediated reductions in flux of Ad across endothelial monolayers in vivo and in vitro This suggests that impaired Ad action in target tissues, as a consequence of reduced transendothelial flux, may contribute to the glucocorticoid-induced diabetic phenotype.

Altered transendothelial transport of hormones as a contributor to diabetes.

The vascular endothelium is a dynamic structure responsible for the separation and regulated movement of biological material between circulation and interstitial fluid. Hormones and nutrients can move across the endothelium either via a transcellular or paracellular route. Transcellular endothelial transport is well understood and broadly acknowledged to play an important role in the normal and abnormal physiology of endothelial function. However, less is known about the role of the paracellular route. Although the concept of endothelial dysfunction in diabetes is now widely accepted, we suggest that alterations in paracellular transport should be studied in greater detail and incorporated into this model. In this review we provide an overview of endothelial paracellular permeability and discuss its potential importance in contributing to the development of diabetes and associated complications. Accordingly, we also contend that if better understood, altered endothelial paracellular permeability could be considered as a potential therapeutic target for diabetes.

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 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.

Permeability properties and occludin expression in a primary cultured model gill epithelium from the stenohaline freshwater goldfish.

Techniques for the primary culture of fish gill epithelia on permeable supports have provided 'reconstructed' gill models appropriate for the study of gill permeability characteristics in vitro. Models developed thus far have been derived from euryhaline fish species that can tolerate a wide range of environmental salinity. This study reports on procedures for the primary culture of a model gill epithelium derived from goldfish, a stenohaline freshwater (FW) fish that cannot tolerate high environmental salt concentrations. The reconstructed goldfish gill epithelium was cultured on permeable filter inserts and using electron microscopy and immunocytochemical techniques, was determined to be composed exclusively of gill pavement cells. When cultured under symmetrical conditions (i.e. with culture medium bathing both apical and basolateral surfaces), epithelial preparations generated appreciable transepithelial resistance (TER) (e.g. 1,150 ± 46 Ωcm(2)) within 36-42 h post-seeding in inserts. When apical medium was replaced with FW (asymmetrical conditions to mimic conditions that occur in vivo), epithelia exhibited increased TER and elevated paracellular permeability. Changes in permeability occurred in association with altered occludin-immunoreactive band position by western blot and no change in occludin mRNA abundance. We contend that the goldfish gill model will provide a useful in vitro tool for examining the molecular components of a stenohaline fish gill epithelium that participate in the regulation of gill permeability. The model will allow molecular observations to be made together with assessment of changing physiological properties that relate to permeability. Together, this will allow further insight into mechanisms that regulate gill 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.

Occludin and hydromineral balance in Xenopus laevis.

To investigate the response of the tight junction (TJ) protein occludin to environmental change in an anuran amphibian, we examined occludin tissue distribution, immunolocalization and alterations in mRNA expression in African clawed frogs (Xenopus laevis) acclimated to brackish water (BW) conditions (from freshwater to 2 per thousand, 5 per thousand or 10 per thousand salt water). Occludin mRNA is widely expressed in Xenopus and is abundant in tissues involved in regulating salt and water balance, such as the gastrointestinal (GI) tract, kidney and urinary bladder. Immunohistochemical analyses revealed strong occludin immunolabelling in the apicolateral region of epithelia lining the GI tract and mRNA expression increased along the longitudinal axis of the gut. In kidney tissue, occludin was differentially expressed on the luminal side of the nephron tubule, appearing in the distal tubules and collecting ducts only. In response to BW acclimation, Xenopus exhibited a significant loss of tissue water as well as salinity-dependent elevations in serum osmolality as a result of increased urea levels followed by elevated serum Na(+) and Cl(-) levels. Tissue-specific alterations in the ionomotive enzyme Na(+),K(+)-ATPase were also observed in Xenopus in response to BW acclimation. Most notably, Na(+),K(+)-ATPase activity in the rectum increased in response to elevated environmental salt concentrations while renal activity decreased. Furthermore, acclimation to BW caused tissue-specific and salinity-dependent alterations in occludin mRNA expression within select Xenopus osmoregulatory organs. Taken together, these studies suggest that alterations in occludin, in conjunction with active transport processes, may contribute to amphibian hydromineral homeostasis during environmental change.

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 (0-120 h) as well as long-term acclimation (14 and 28 days) 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 6 h. Following 14 and 28 days 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 28 days; 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.

Occludin immunolocalization and protein expression in goldfish.

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.