ATPase Immunoreactivity Research Articles

Ontogenetical development of branchial chambers of Litopenaeus vannamei (Boone, 1931) and theirinvolvement in osmoregulation: ionocytes and Na+/K+-ATPase.

Branchial chambers constitute the main osmoregulatory site in almost all decapod crustaceans. However, few studies have been devoted to elucidate the cellular function of specific cells in every osmoregulatory structure of the branchial chambers. In decapod crustaceans, it is well-known that the osmoregulatory function is localized in specific structures that progressively specialize from early developmental stages while specific molecular mechanisms occur. In this study, we found that although the structures developed progressively during the larval and postlarval stages, before reaching juvenile or adult morphology, the osmoregulatory capabilities of Litopenaeus vannamei were gradually established only during the development of branchiostegites and epipodites, but not gills. The cellular structures of the branchial chambers observed during the larval phase do not present the typical ultrastructure of ionocytes, neither Na+/K+-ATPase expression, likely indicating that pleura, branchiostegites, or bud gills do not participate in osmoregulation. During early postlarval stages, the lack of Na+/K+-ATPase immunoreactivity of the ionocytes from the branchiostegites and epipodites suggests that they are immature ionocytes (ionocytes type I). It could be inferred from IIF and TEM results that epipodites and branchiostegites are involved in iono-osmoregulation from PL15, while gills and pleura do not participate in this function.

The Endocannabinoid/Cannabinoid Receptor 2 System Protects Against Cisplatin-Induced Hearing Loss.

Previous studies have demonstrated the presence of cannabinoid 2 receptor (CB2R) in the rat cochlea which was induced by cisplatin. In an organ of Corti-derived cell culture model, it was also shown that an agonist of the CB2R protected these cells against cisplatin-induced apoptosis. In the current study, we determined the distribution of CB2R in the mouse and rat cochleae and examined whether these receptors provide protection against cisplatin-induced hearing loss. In a knock-in mouse model expressing the CB2R tagged with green fluorescent protein, we show distribution of CB2R in the organ of Corti, stria vascularis, spiral ligament and spiral ganglion cells. A similar distribution of CB2R was observed in the rat cochlea using a polyclonal antibody against CB2R. Trans-tympanic administration of (2-methyl-1-propyl-1H-indol-3-yl)-1-naphthalenylmethanone (JWH015), a selective agonist of the CB2R, protected against cisplatin-induced hearing loss which was reversed by blockade of this receptor with 6-iodo-2-methyl-1-[2-(4-morpholinyl)ethyl]-1H-indol-3-yl](4-methoxyphenyl)methanone (AM630), an antagonist of CB2R. JWH015 also reduced the loss of outer hair cells (OHCs) in the organ of Corti, loss of inner hair cell (IHC) ribbon synapses and loss of Na+/K+-ATPase immunoreactivity in the stria vascularis. Administration of AM630 alone produced significant hearing loss (measured by auditory brainstem responses) which was not associated with loss of OHCs, but led to reductions in the levels of IHC ribbon synapses and strial Na+/K+-ATPase immunoreactivity. Furthermore, knock-down of CB2R by trans-tympanic administration of siRNA sensitized the cochlea to cisplatin-induced hearing loss at the low and middle frequencies. Hearing loss induced by cisplatin and AM630 in the rat was associated with increased expression of genes for oxidative stress and inflammatory proteins in the rat cochlea. In vitro studies indicate that JWH015 did not alter cisplatin-induced killing of cancer cells suggesting this agent could be safely used during cisplatin chemotherapy. These data unmask a protective role of the cochlear endocannabinoid/CB2R system which appears tonically active under normal conditions to preserve normal hearing. However, an exogenous agonist is needed to boost the activity of endocannabinoid/CB2R system for protection against a more traumatic cochlear insult, as observed with cisplatin administration.

Vasopressin V1a Receptor of Renal Collecting Duct Intercalated Cells Mediates Urinary Acidification

Vasopressin (AVP) drives urinary concentration via V2 receptors in the distal nephron and collecting duct of the kidney. Parallel activation of V1a receptors (V1aR) has been implicated in renal acid‐base handling, but cell type‐related information on this receptor is still scarce. We hypothesized that activation of V1aR in intercalated cells (IC) contributes to acid‐base homeostasis by the kidney.We generated an anti‐V1aR antibody and verified its specificity using V1aR‐knockout tissues. For localization studies in mice, rat and human tissue, we used immunofluorescence and confocal microscopy or 3D‐structured illumination microscopy (3D‐SIM). For functional studies in vivo, V1aR‐specific agonist, AO‐4‐67, was administered in AVP‐deficient Brattleboro rats and C57/BI6J mice (0.2, 2, or 10μg/kg b.w.; 1 to 4h). Urine was collected in metabolic cages or via ureteral catheter and plasma samples were taken. Isolated, microperfused mouse collecting ducts were analyzed for V1aR‐dependent luminal pH changes and cultured inner medullary collecting duct cells for proton‐secreting vH+‐ATPase regulation.V1aR immunoreactivity of type‐A IC was demonstrated in basolateral membrane and cytoplasmic granules, whereas type B IC revealed perinuclear and subapical granular signal alone. Administration of AO‐4‐67 to Brattleboro rats for 4h resulted in increased urinary proton secretion (+34% after 1h, p<0.05) and decreased bicarbonate excretion (up to −90% after 3h, p<0.001). Similarly, catheterized mice showed rapid urinary acidification in response to AO‐4–67 (pH from 7.18 to 6.78 after 20 min, p<0.05). Basolateral application of V1aR agonist or AVP in isolated perfused collecting ducts resulted in decreased luminal pH values (−5% after 3 min, p<0.05). Cultured collecting duct cells showed significant increases in luminal vH+‐ATPase immunoreactivity upon V1aR stimulation (+90% after 4h, p<0.001).In summary, our results suggest that activation of V1aR in renal type‐A intercalated cells induces proton secretion into the urine and promote urinary acidification.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Gill Histopathologies Following Exposure to Nanosilver or Silver Nitrate

Fish gill is the site for many crucial physiological functions. It is among the first sites of xenobiotic exposure, and gill histopathological alterations may be detected soon after toxicant exposure. Silver (Ag) is one of the most toxic metals to aquatic organisms mainly due to its ability to disrupt ionic regulation. The goal of this study was to determine the effect of ionic and nanoscale Ag on fathead minnow gills by examining gill histology and Na+/K+-ATPase immunoreactivity. Fathead minnows were exposed to two measured concentrations of silver nitrate (AgNO3: 1.3 or 3.7 μg/L as Ag+), citrate silver nanoparticles (citrate-AgNP: 15 or 39 μg/L), and polyvinylpyrrolidone-AgNP (PVP-AgNP) (AgNP: 11 or 50 μg/L). Circulatory disturbances were the most prevalent gill alterations detected and were significantly increased in all Ag treatment groups compared to control. AgNO3 (1.3 μg/L) was the only treatment that significantly elevated the number of total mucous goblet cells present. In all other Ag treatments, the percent of degenerated goblet cells was significantly increased compared to control. When the sum of all histopathological abnormalities (weighted index) was calculated, all Ag groups displayed a significantly higher index, with citrate-AgNP having the highest toxicity (index of 10 ± 0.32 versus 2.4 ± 0.6 in controls). Gill Na+/K+-ATPase immunoreactivity was decreased by Ag. These results indicated that both AgNO3 and AgNP created similar disruptions in gill structure and ionic regulation, possibly due to the ionic Ag portion of each treatment.

Expression of basolateral organic anion and cation transporters in experimental cadmium nephrotoxicity in rat kidney.

Cadmium (Cd)-intoxicated experimental animals exhibit impaired renal secretion of organic anions (OA) and cations (OC), indicating their transporters (Oats and Octs) in the proximal tubule (PT) basolateral membrane as possible targets of Cd. To correlate transport data from the literature with the expression of relevant transporters, we performed immunochemical and RT-PCR studies of renal Oats and Octs in the subchronic (treatment with CdCl2; 2 mg Cd/kg b.m./day, for 2 weeks) and acute (treatment with Cd-metallothionein (CdMT); 0.4 mg Cd/kg b.m., 6 or 12 h before killing) models of Cd nephrotoxicity. In the subchronic model, PT exhibited a minor loss of basolateral invaginations and overall unchanged expression of Na(+)/K(+)-ATPase and GAPDH proteins and mRNAs, while the expression of Oat and Oct proteins and their mRNAs was strongly downregulated. In the acute model, a time-related redistribution of basolateral transporters to the intracellular vesicular compartment was a major finding. However, 6 h following CdMT treatment, the total abundance of Oat and Oct proteins in the renal tissue remained unchanged, the expression of mRNAs decreased only for Oats, while a limited Oat1 and Na(+)/K(+)-ATPase immunoreactivity in the PT apical membrane indicated loss of cell polarity. As tested in rats treated with colchicine, the observed loss/redistribution of basolateral transporters in both models may be independent on microtubules. Therefore, the diminished renal secretion of OA and OC via PT in Cd nephrotoxicity may result from (a) limited loss of secretory surface (basolateral invaginations), (b) selective loss of Oats and Octs, and

Carbaryl exposure and recovery modify the interrenal and thyroidal activities and the mitochondria-rich cell function in the climbing perch Anabas testudineus Bloch

We examined the effects of carbaryl (1-naphthyl methylcarbamate; sevin), a carbamate pesticide, on interrenal and thyroid activities and mitochondrial rich (MR) cell function in climbing perch to understand the physiological basis of toxicity acclimation in this fish to the chemical stressor. Carbaryl exposure (5–20mgL−1) for 48h increased cortisol and glucose, but decreased the T3 level without affecting T4 concentration in the plasma. These responses of the carbaryl-exposed fish were nullified and a rise in plasma T4 occurred in these fish when they were kept for 96h recovery in clean water. A tight plasma mineral control was indicated in the carbaryl-exposed fish as reflected by the unchanged plasma Na, K, Ca and inorganic phosphate levels. The ouabain-sensitive Na+, K+-ATPase activity showed an increase in the gills but the intestinal and renal tissues showed little response to carbaryl treatment. However, substantial increases in the intestinal and renal Na+, K+-ATPase activities occurred in the recovery fish. The MR cells in the branchial epithelia showed a strong Na+, K+-ATPase immunoreactivity to carbaryl treatment indicating an activated MR cell function. The numerical MR cell density remained unchanged, but stretching of secondary gill lamellae as part of gill remodeling occurred during carbaryl exposure. The increased surface of these lamellae with abundant MR cells as a result of its migration into the lamellar surface points to marked structural and functional modifications of these cells in the carbaryl-treated fish which is likely to a target for carbaryl action. The rise in plasma T4 and the restoration of normal branchial epithelia in the recovery fish indicate a thyroidal involvement in the recovery response and survival. Our data thus provide evidence that carbaryl exposure and its recovery evoke interrenal and thyroid disruption in this fish leading to a modified osmotic response including an altered MR cell function.

Ambient salinity modifies the action of triiodothyronine in the air-breathing fish Anabas testudineus Bloch: Effects on mitochondria-rich cell distribution, osmotic and metabolic regulations

The hydromineral and metabolic actions of thyroid hormone on osmotic acclimation in fish is less understood. We, therefore, studied the short-term action of triiodothyronine (T 3), the potent thyroid hormone, on the distribution and the function of gill mitochondria-rich (MR) cells and on the whole body hydromineral and metabolic regulations of air-breathing fish ( Anabas testudineus) adapted to either freshwater (FW) or acclimated to seawater (SA; 30 g L −1). As expected, 24 h T 3 injection (100 ng g −1) elevated ( P < 0.05) plasma T 3 but classically reduced ( P < 0.05) plasma T 4. The higher Na +, K +-ATPase immunoreactivity and the varied distribution pattern of MR cells in the gills of T 3-treated FW and SA fish, suggest an action of T 3 on gill MR cell migration, though the density of these cells remained unchanged after T 3 treatment. The ouabain–sensitive Na +, K +-ATPase activity, a measure of hydromineral competence, showed increases ( P < 0.05) in the gills of both FW and SA fish after T 3 administration, but inhibited ( P < 0.05) in the kidney of the FW fish and not in the SA fish. Exogenous T 3 reduced glucose ( P < 0.05) and urea ( P < 0.05) in the plasma of FW fish, whereas these metabolites were elevated ( P < 0.05) in the SA fish, suggesting a modulatory effect of ambient salinity on the T 3-driven metabolic actions. Our data identify gill MR cell as a target for T 3 action as it promotes the spatial distribution and the osmotic function of these cells in both fresh water and in seawater. The results besides confirming the metabolic and osmotic actions of T 3 in fish support the hypothesis that the differential actions of T 3 may be due to the direct influence of ambient salinity, a major environmental determinant that alters the osmotic and metabolic strategies of fish.

Localization of ion-regulatory epithelia in embryos and hatchlings of two cephalopods

The tissue distribution and ontogeny of Na(+)/K(+)-ATPase has been examined as an indicator for ion-regulatory epithelia in whole animal sections of embryos and hatchlings of two cephalopod species: the squid Loligo vulgaris and the cuttlefish Sepia officinalis. This is the first report of the immunohistochemical localization of cephalopod Na(+)/K(+)-ATPase with the polyclonal antibody alpha (H-300) raised against the human alpha1-subunit of Na(+)/K(+)-ATPase. Na(+)/K(+)-ATPase immunoreactivity was observed in several tissues (gills, pancreatic appendages, nerves), exclusively located in baso-lateral membranes lining blood sinuses. Furthermore, large single cells in the gill of adult L. vulgaris specimens closely resembled Na(+)/K(+)-ATPase-rich cells described in fish. Immunohistochemical observations indicated that the amount and distribution of Na(+)/K(+)-ATPase in late cuttlefish embryos was similar to that found in juvenile and adult stages. The ion-regulatory epithelia (e.g., gills, excretory organs) of the squid embryos and paralarvae exhibited less differentiation than adults. Na(+)/K(+)-ATPase activities for whole animals were higher in hatchlings of S. officinalis (157.0 +/- 32.4 micromol g (FM) (-1) h(-1)) than in those of L. vulgaris (31.8 +/- 3.3 micromol g (FM) (-1) h(-1)). S. officinalis gills and pancreatic appendages achieved activities of 94.8 +/- 18.5 and 421.8 +/- 102.3 micromol(ATP) g (FM) (-1) h(-1), respectively. High concentrations of Na(+)/K(+)-ATPase in late cephalopod embryos might be important in coping with the challenging abiotic conditions (low pH, high pCO(2)) that these organisms encounter inside their eggs. Our results also suggest a higher sensitivity of squid vs. cuttlefish embryos to environmental acid-base disturbances.

Ionoregulatory changes during metamorphosis and salinity exposure of juvenile sea lamprey (Petromyzon marinusL.)

Ammocoetes of the anadromous sea lamprey Petromyzon marinus L. spend many years in freshwater before metamorphosing and migrating to sea. Metamorphosis involves the radical transformation from a substrate-dwelling, filter feeder into a free-swimming, parasitic feeder. In the present work we examined osmoregulatory differences between ammocoetes and transformers (metamorphic juveniles), and the effects of salinity acclimation. We measured the expression of key ion-transporting proteins [Na(+)/K(+)-ATPase, vacuolar (V)-type H(+)-ATPase and carbonic anhydrase (CA)] as well as a number of relevant blood parameters (hematocrit, [Na(+)] and [Cl(-)]). In addition, immunofluorescence microscopy was used to identify and characterize the distributions of Na(+)/K(+)-ATPase, V-type H(+)-ATPase and CA immunoreactive cells in the gill. Ammocoetes did not survive in the experiments with salinities greater than 10 per thousand, whereas survival in high salinity (> or =25-35 per thousand) increased with increased degree of metamorphosis in transformers. Plasma [Na(+)] and [Cl(-)] of ammocoetes in freshwater was lower than transformers and increased markedly at 10 per thousand. In transformers, plasma ions increased only at high salinity (>25 per thousand). Branchial Na(+)/K(+)-ATPase levels were approximately tenfold higher in transformers compared to ammocoetes and salinity did not affect expression in either group. However, branchial H(+)-ATPase expression showed a negative correlation with salinity in both groups. Na(+)/K(+)-ATPase immunoreactivity was strongest in transformers and associated with clusters of cells in the interlamellar spaces. H(+)-ATPase (B subunit) immunoreactivity was localized to epithelial cells not expressing high Na(+)/K(+)-ATPase immunoreactivity and having a similar tissue distribution as carbonic anhydrase. The results indicate that branchial Na(+)/K(+)-ATPase and salinity tolerance increase in metamorphosing lampreys, and that branchial H(+)-ATPase is downregulated by salinity.

Prolonged air-breathing and recovery modify the thyroid and interrenal axes in climbing perch (Anabas testudineus)

To examine the response of thyroid and intrenal axes to hydromineral and metabolic responses in air-breathing fish during their territorial migration, we quantified several physiological markers in the tropical obligate air-breathing perch (Anabas testudineus) during air breathing on land and in water. Plasma T3 and T4 levels remained unaffected after 60 and 120 min of air breathing though T3 had a tendency to increase. Plasma cortisol levels showed a significant 4-fold increase. Plasma cortisol showed a 1.5-fold increase in these recovered fish. Air breathing on land significantly increased plasma glucose (2.8-fold) and plasma urea (5.2-fold) while plasma lactate showed a significant 3.2-fold decrease. Na+,K+-ATPase activity, reflecting the sodium pump activity, showed a 4.4-fold decrease in the gills but produced a 1.8-fold increase in the kidneys after air exposure for 120 min. Significant reversal of the plasma metabolite values and the gill Na+,K+-ATPase activity (15-fold) was obtained during recovery. The immunoreactivity of Na+,K+-ATPase in the branchial chloride cells (CCs) showed little changes. The density of the CCs remained unaffected. A marked increase of the glucocorticoid receptor staining was observed in air-exposed gills. Analyses of the ultrastructure of the branchial epithelia revealed CCs with low Na+, K+-ATPase immunoreactivity, more macrophages and increased intercellular spaces in the air-exposed fish. Our data for the first time provide evidence that the thyroid and interrenals jointly direct adaptive strategies to recover from the stress during prolonged air-breathing thus supporting the hypothesis of a role for thyroid in stress adaptation in fish.

Na+/K+ATPase immunoreactivity in olfactory epithelium of small‐spotted catshark Scyliorhinus canicula (L.): possible presence of ion exchanging cells?

Na+/K+ATPase immunoreactivity in olfactory epithelium of Scyliorhinus canicula was localized in well featured light large cells. It was distributed in the basolateral membranes as reported in the literature for typical ion exchanging cells.

Type IV carbonic anhydrase is present in the gills of spiny dogfish (Squalus acanthias)

Physiological and biochemical studies have provided indirect evidence for a membrane-associated carbonic anhydrase (CA) isoform, similar to mammalian type IV CA, in the gills of dogfish (Squalus acanthias). This CA isoform is linked to the plasma membrane of gill epithelial cells by a glycosylphosphatidylinositol anchor and oriented toward the plasma, such that it can catalyze the dehydration of plasma HCO(3)(-) ions. The present study directly tested the hypothesis that CA IV is present in dogfish gills in a location amenable to catalyzing plasma HCO(3)(-) dehydration. Homology cloning techniques were used to assemble a 1,127 base pair cDNA that coded for a deduced protein of 306 amino acids. Phylogenetic analysis suggested that this protein was a type IV CA. For purposes of comparison, a second cDNA (1,107 base pairs) was cloned from dogfish blood; it encoded a deduced protein of 260 amino acids that was identified as a cytosolic CA through phylogenetic analysis. Using real-time PCR and in situ hybridization, mRNA expression for the dogfish type IV CA was detected in gill tissue and specifically localized to pillar cells and branchial epithelial cells that flanked the pillar cells. Immunohistochemistry using a polyclonal antibody raised against rainbow trout type IV CA revealed a similar pattern of CA IV immunoreactivity and demonstrated a limited degree of colocalization with Na(+)-K(+)-ATPase immunoreactivity. The presence and localization of a type IV CA isoform in the gills of dogfish is consistent with the hypothesis that branchial membrane-bound CA with an extracellular orientation contributes to CO(2) excretion in dogfish by catalyzing the dehydration of plasma HCO(3)(-) ions.

Na+/H+ antiporter, V-H+-ATPase and Na+/K+-ATPase immunolocalization in a marine teleost(Myoxocephalus octodecemspinosus)

Long-term pH compensation in a marine teleost requires the transepithelial excretion of H(+) across the gill epithelium. H(+) efflux in the longhorn sculpin (Myoxocephalus octodecemspinosus) is dependent on external sodium ion concentration and is inhibited by known inhibitors of Na(+)/H(+) exchangers. Our model for proton transport suggests acid-excreting cells in the gill with an apical Na(+)/H(+) antiporter and basolateral Na(+)/K(+)-ATPase. This model is similar to mammalian kidney and elasmobranch gill epithelium in which a basolateral electrogenic-vacuolar proton pump (V-H(+)-ATPase) localizes to base-excreting cells. The objective of this study was to detect the presence and location of membrane transporters in marine fish gills using immunohistochemical staining. Our data indicate the presence of an apical and subapical Na(+)/H(+)-exchanger 2 (NHE2) in the sculpin gill. NHE2 is present in large, ovoid chloride cells and often colocalizes in the same cells as Na(+)/K(+)-ATPase. We also detected V-H(+)-ATPase immunoreactivity, predominantly in cells at the base of the lamellae, with staining patterns indicative of a basolateral location. The 85 kDa protein detected on immunoblots with anti-NHE2 antibodies was found in both control and acid-infused animals and did not change following a large acute acidosis over 8 h.

Expression and changes of Ca 2+-ATPase in neurons and astrocytes in the gerbil hippocampus after transient forebrain ischemia

Ca 2+-ATPase is one of the most powerful modulators of intracellular calcium levels. In this study, we focused on chronological changes in the immunoreactivity and protein levels of Ca 2+-ATPase in the hippocampus after 5 min of transient forebrain ischemia. Ca 2+-ATPase immunoreactivity was significantly altered in the hippocampal CA1 region and in the dentate gyrus, but not in the CA2/3 region after ischemic insult. In the sham-operated group, Ca 2+-ATPase immunoreactivity was detected in the hippocampus. Ca 2+-ATPase immunoreactivity in the CA1 region and in the dentate gyrus, and its protein levels peaked 3 h after ischemic insult. At this time, CA1 pyramidal cells and dentate polymorphic cells showed strong Ca 2+-ATPase immunoreactivity. Thereafter, Ca 2+-ATPase immunoreactivity reduced in the CA1 region and in the dentate gyrus. One day after ischemic insult, Ca 2+-ATPase immunoreactivity was observed in some CA1 non-pyramidal cells, and 4 days after ischemic insult, Ca 2+-ATPase immunoreactivity was detected in astrocytes throughout the CA1 region, but Ca 2+-ATPase immunoreactivity in the dentate gyrus had nearly disappeared. Our results suggest that Ca 2+-ATPase changes may be associated with a response to ischemic damage in hippocampal CA1 pyramidal cells, and that increased Ca 2+-ATPase immunoreactivity in the reactive astrocytes may be associated with the maintenance of intracellular calcium levels.

Altered Na +–K + ATPase immunoreactivity within GABAergic neurons in the gerbil hippocampal complex induced by spontaneous seizure and vigabatrin treatment

In the present study, the expression of Na +–K + ATPase in the gerbil hippocampus associated with various sequelae of spontaneous seizures were investigated in order to identify the roles of Na +–K + ATPase in the epileptogenesis and the recovery mechanisms in these animals. The population of Na +–K + ATPase immunoreactive neurons and Na +–K + ATPase immunodensity were significantly lower in the pre-seizure group of SS gerbils than those in SR gerbils. At 30-min postictal, the Na +–K + ATPase immunoreactivity was significantly elevated in the hippocampal complex. At 3-h postictal, the Na +–K + ATPase immunoreactivity in the hippocampus was declined, as compared to the 30-min postictal. At 12 h after seizure on-set, Na +–K + ATPase expression was re-enhanced in the all regions of the hippocampal complex including the dentate hilus. Following administration of vigabatrin Na +–K + ATPase expression was also increased. The present data suggest that altered Na +–K + ATPase expression may contribute the regulation of the seizure activity in this animal.

Islet transplantation is associated with improvement of renal function among uremic patients with type I diabetes mellitus and kidney transplants.

The potential effects of islet transplantation on the renal function of 36 patients with type I diabetes mellitus and kidney transplants were studied with 4 yr of follow-up monitoring. Kidney-islet recipients were divided into two groups, i.e., patients with successful islet transplants (SI-K group) (n = 24, fasting C-peptide levels of >0.5 ng/ml for >1 yr) and patients with unsuccessful islet transplants (UI-K group) (n = 12, fasting C-peptide levels of <0.5 ng/ml). Kidney graft survival rates and function, urinary albumin excretion rates, and sodium handling were compared. Na(+)/K(+)-ATPase activity in protocol kidney biopsies and in red blood cells was cross-sectionally analyzed. The SI-K group demonstrated better kidney graft survival rates (100, 83, and 83% at 1, 4, and 7 yr, respectively) than did the UI-K group (83, 72, and 51% at 1, 4, and 7 yr, respectively; P = 0.02). The SI-K group demonstrated reductions in exogenous insulin requirements and higher C-peptide levels, compared with the UI-K group, whereas GFR values were similar. Microalbuminuria (urinary albumin index) increased significantly in the UI-K group only (UI-K, from 92.0 +/- 64.9 to 183.8 +/- 83.8, P = 0.05; SI-K, from 108.5 +/- 53.6 to 85.0 +/- 39.0, NS). In the SI-K group, but not in the UI-K group, natriuresis decreased at 2 and 4 yr (P < 0.01). The SI-K group demonstrated greater Na(+)/K(+)-ATPase immunoreactivity in renal tubular cells (P = 0.05) and higher activity in red blood cells (P = 0.03), compared with the UI-K group. The Na(+)/K(+)-ATPase activity in red blood cells was positively correlated with circulating C-peptide levels but not with glycated hemoglobin levels. Successful islet transplantation was associated with improvements in kidney graft survival rates and function among uremic patients with type I diabetes mellitus and kidney grafts.

Stimulation of sodium pump restores membrane potential to neurons excited by glutamate in zebrafish distal retina.

Glutamate either depolarizes or hyperpolarizes retinal neurons. Those are the initial and primary effects. Using a voltage probe (oxonol, DiBaC4 (5)) to study dissociated zebrafish retinal neurons, we find a secondary, longer-term effect: a post-excitatory restoration of membrane potential, termed after-hyperpolarization (AHP). AHP occurs only in neurons that are depolarized by glutamate and typically peaks about 5 min after glutamate application. AHP is seen in dissociated horizontal cells (HCs) and hyperpolarizing, or OFF type, bipolar cells (HBCs). These cells commonly respond with only an AHP component. AHP never occurs in depolarizing, or ON type, bipolar cells (DBCs), which are cell types hyperpolarized by glutamate. AHP is blocked by 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX). It is evoked by kainate, AMPA and the AMPA-selective agonist (S)-5-fluorowillardiine, but not by NMDA, D-aspartate, the kainate-selective agonist SYM 2081 or by DL-2-amino-4-phosphonobutyric acid (DL-AP4). Cells with exclusively AHP responses are tonically depolarized. Resting potentials can be restored by nifedipine, suggesting a tonic, depolarizing action of L-type Ca2+ channels. However AHP is not blocked by nifedipine and is insensitive to [Cl-]o. AHP is blocked by Li+o substitution for Na+o and by ouabain. A mechanism is proposed in which Na+ entering through ionotropic AMPA channels stimulates Na+,K+-ATPase, which, by electrogenic action, restores membrane potential, generating the AHP response. Patterns of ATPase immunoreactivity support localization in the outer plexiform layer (OPL) as cone pedicles, HCs and BCs were positively labelled. Labelling was weaker in the inner plexiform layer (IPL) than in nuclear layers, though two IPL bands of immunoreactive BC terminals could be discerned, one in sublamina a and the other in sublamina b. Persistent stimulation of distal retina by photoreceptor glutamate may induce increased expression and activity of Na+,K+-ATPase, with a consequent impact on distal glutamate responses.

Increased expression of H+-ATPase in inner medullary collecting duct of aquaporin-1-deficient mice.

Phenotype analysis has demonstrated that aquaporin-1 (AQP1) null mice are polyuric and manifest a urinary concentrating defect because of an inability to create a hypertonic medullary interstitium. We report here that deletion of AQP1 is also associated with a decrease in urinary pH from 6.15 +/- (SE) 0.1 to 5.63 +/- 0.07. To explore the mechanism of the decrease in urinary pH, we examined the expression of H+-ATPase in kidneys of AQP1 null mice. There was strong labeling for H+-ATPase in intercalated cells and proximal tubule cells in both AQP1 null and wild-type mice. Strong H+-ATPase immunostaining was also present in the apical plasma membrane of inner medullary collecting duct (IMCD) cells in AQP1 null mice, whereas no H+-ATPase labeling was observed in IMCD cells in wild-type mice. In addition, there was an increase in the prevalence of type A intercalated cells in the IMCD of AQP1 null mice, suggesting that the deletion of intercalated cells from the IMCD, which normally occurs during postnatal kidney development, was impaired. Western blot analysis of H+-ATPase expression in the different regions of the kidney demonstrated a significant increase in H+-ATPase protein in the inner medulla of AQP1 null mice compared with wild-type mice. There were no changes in H+-ATPase expression in the cortex or outer medulla. These results represent the first demonstration of apical H+-ATPase immunoreactivity in IMCD cells in vivo and suggest that the decrease in urinary pH observed in AQP1 null mice is due to upregulation of H+-ATPase in the IMCD. The induction of H+-ATPase expression in IMCD cells of AQP1 null mice may be related to the chronically low interstitial osmolality in these animals. The challenge will be to identify the molecular signal(s) responsible for the de novo H+-ATPase expression.

Elevation of Na +–K + ATPase immunoreactivity in GABAergic neurons in gerbil CA1 region following transient forebrain ischemia

In a previous study, we suggested that GABAergic neurons might be resistant to ischemic insult, because of the maintenance of the GABA shunt, which is one of the ATP synthetic pathways in neurons. In the present study, we identified Na +–K + ATPase immunoreactivity in the gerbil hippocampus in order to determine whether changes in Na +–K + ATPase immunoreactivity correlate with GABA shunt following ischemic insult. At 12 h after ischemia–reperfusion, Na +–K + ATPase immunoreactivity accumulated in some neurons in the CA1 region. However, the protein content of Na +–K + ATPase was not altered. Interestingly, the density of Na +–K + ATPase immunoreactivity in neurons and the protein content in the CA1 region was intensified in the 24 h post-ischemic group. As a result of double immunofluorescence study, Na +–K + ATPase immunoreactive neurons were identified with GABAergic neurons. Therefore, our findings suggest that the increase of Na +–K + ATPase in GABAergic neurons may be able to explain the resistance of these cells to ischemic insult, and support our previous hypothesis that GABA may play an important role as a metabolite in the survival of GABAergic neurons after ischemic insult.

Secretory-defect distal renal tubular acidosis is associated with transporter defect in H(+)-ATPase and anion exchanger-1.

Recent progress in molecular physiology has permitted us to understand pathophysiology of various channelopathies at a molecular level. The secretion of H(+) from alpha-intercalated cells is mediated by apical plasma membrane H(+)-ATPase and basolateral plasma membrane anion exchanger-1 (AE1). Studies have demonstrated the lack of H(+)-ATPase immunostaining in the intercalated cells in a few patients with distal renal tubular acidosis (dRTA). Mutations in H(+)-ATPase and AE1 gene have recently been reported to cause dRTA. This study extends the investigation of the role of transporter defect in dRTA by using immunohistochemical methods. Eleven patients with hyperchloremic metabolic acidosis were diagnosed functionally to have secretory-defect dRTA: urine pH >5.5 during acidemia, normokalemia or hypokalemia, and urine-to-blood pCO(2) <25 mmHg during bicarbonaturia. Renal biopsy tissue was obtained from each patient, and immunohistochemistry was carried out using antibodies to H(+)-ATPase and AE1. For comparison, renal tissues from the patients who had no evidences of distal acidification defect by functional studies were used: four with glomerulopathy or tubulointerstitial nephritis (disease controls) and three from nephrectomized kidneys for renal cell carcinoma (normal controls). The H(+)-ATPase immunoreactivity in alpha-intercalated cells was almost absent in all of the 11 patients with secretory-defect dRTA. In addition, 7 of 11 patients with secretory-defect dRTA were accompanied by negative AE1 immunoreactivity. In both disease controls and normal controls, the immunoreactivity of H(+)-ATPase and AE1 was strong in alpha-intercalated cells. In conclusion, significant defect in acid-base transporters is the major cause of secretory-defect dRTA.