Mechanisms of transport of H+, Na+ and K+, across the distal gastric caecum of larval Aedes aegypti.

Measured changes in ion fluxes, transepithelial potential (TEP) and basolateral membrane potential (Vb) in response to ion transporter inhibitors were used to assess the mechanisms of transport of H+, Na+ and K+, across the distal gastric caecum of larval Aedes aegypti, a vector of yellow fever. Preparations were stimulated with 5-hydroxytryptamine (5-HT, 10-6 M) in order to maintain stable rates of H+, Na+, and K+ transport across the distal caecum. Transepithelial potential (TEP), basolateral membrane potential (Vb), and H+, Na+ and K+ fluxes all declined after the addition of a vacuolar-type H+-ATPase (VA) inhibitor, n-ethlymaleimide (NEM), consistent with a primary role for VA in energizing ion transport across the distal gastric caecum. Amiloride also inhibited H+, Na+, and K+ fluxes, consistent with an apically expressed VA that is coupled to a cation:H+ antiporter (AeNHE8), analogous to the coupling of apical VA and cation:nH+ antiporter in Malpighian tubules. A working model of transport of H+, Na+ and K+ across the distal gastric caecum proposes that coupling of VA and AeNHE8 in the apical membrane leads to the removal of intracellular Na+ or K+, thus creating favourable ion gradients to promote the activity of two transporters in the basal membrane, cation:H+ antiporter (AeNHE3) and a bumetanide-sensitive cation chloride cotransporter (CCC).

Kir4.1/Kir5.1 Activity Is Essential for Dietary Sodium Intake-Induced Modulation of Na-Cl Cotransporter.

BACKGROUND: Dietary sodium intake regulates the thiazide-sensitive Na-Cl cotransporter (NCC) in the distal convoluted tubule (DCT). Whether the basolateral, inwardly rectifying potassium channel Kir4.1/Kir5.1 (a heterotetramer of Kir4.1/Kir5.1) in the DCT is essential for mediating the effect of dietary sodium intake on NCC activity is unknown. METHODS: We used electrophysiology, renal clearance techniques, and immunoblotting to examine effects of Kir4.1/Kir5.1 in the DCT and NCC in wild-type and kidney-specific Kir4.1 knockout mice. RESULTS: Low sodium intake stimulated basolateral Kir4.1/Kir5.1 activity, increased basolateral K+ conductance, and hyperpolarized the membrane. Conversely, high sodium intake inhibited the potassium channel, decreased basolateral K+ currents, and depolarized the membrane. Low sodium intake increased total and phosphorylated NCC expression and augmented hydrochlorothiazide-induced natriuresis; high sodium intake had opposite effects. Thus, elevated NCC activity induced by low sodium intake was associated with upregulation of Kir4.1/Kir5.1 activity in the DCT, whereas inhibition of NCC activity by high sodium intake was associated with diminished Kir4.1/Kir5.1 activity. In contrast, dietary sodium intake did not affect NCC activity in knockout mice. Further, Kir4.1 deletion not only abolished basolateral K+ conductance and depolarized the DCT membrane, but also abrogated the stimulating effects induced by low sodium intake on basolateral K+ conductance and hyperpolarization. Finally, dietary sodium intake did not alter urinary potassium excretion rate in hypokalemic knockout and wild-type mice. CONCLUSIONS: Stimulation of Kir4.1/Kir5.1 by low intake of dietary sodium is essential for NCC upregulation, and inhibition of Kir4.1/Kir5.1 induced by high sodium intake is a key step for downregulation of NCC.

Elucidation of protective efficacy of Pentahydroxy flavone isolated from Madhuca indica against arsenite-induced cardiomyopathy: Role of Nrf-2, PPAR-γ, c-fos and c-jun.

BACKGROUND: Madhuca indica J. F. Gmel. (Sapotaceae) is widely used ethnobotanically as anti-diabetic, antipyretic, hepatoprotective, anti-inflammatory and analgesic. It was shown to possess potent anti-apoptotic property. THE AIM OF THE STUDY: To evaluate the possible mechanism of action of isolated phytoconstituent from Madhuca indica Leaves methanolic extract (MI-ALC) on arsenic-induced cardiotoxicity in rats. MATERIALS AND METHODS: The 3,5,7,3',4'-Pentahydroxy flavone (QTN) was isolated and characterized by using HPTLC, 1H NMR, and LC-MS from MI-ALC. QTN (5, 10 and 20mg/kg, p.o.) was administered in arsenic intoxicated rats (5mL/kg, p.o.) for 28days and evaluated for various behavioral, biochemical, molecular and ultra-histological changes. RESULTS: Treatment with QTN (10 and 20mg/kg, p.o.) significantly inhibited (p<0.05) arsenic-induced electrocardiographic, hemodynamic and left ventricular function alterations. Elevated levels of cardiac markers (LDH, CK-MB, AST, ALT, and ALP), altered lipid metabolism (total cholesterol, triglyceride, LDL, HDL, and VLDL) was significantly restored (p<0.05) by QTN. It also significantly inhibited (p<0.05) altered cardiac oxido-nitrosative stress, Na-K-ATPase level and mitochondrial enzymes (I-IV) activity after arsenite administration. QTN significantly increased (p<0.05) myocardial Nrf-2, PPAR-γ and significantly decreased (p<0.05) myocardial c-fos and c-jun mRNA expressions. Flow cytometric analysis showed that treatment with QTN (10 and 20mg/kg) significantly inhibited (p<0.05) arsenite-induce ROS and apoptosis. It also reduced ultra-histological aberrations induced by sodium arsenite. CONCLUSION: Administration of 3,5,7,3',4'- Pentahydroxy flavone (i.e. Quercetin (QTN)) isolated from MI-ALC showed significant protection against arsenic-induced oxido-nitrosative stress and myocardial injury via modulation of Nrf2, PPAR-γ, and apoptosis.

Computed determination of the in vitro optimal chemocombinations of sphaeropsidin A with chemotherapeutic agents to combat melanomas.

PURPOSE: Evasion to new treatments of advanced melanoma is still associated with a poor prognosis. Choosing the best combination of agents that can bypass resistance mechanisms remains a challenge. Sphaeropsidin A (Sph A) is a fungal bioactive secondary metabolite previously shown to force melanoma cells to undergo apoptosis via cell volume dysregulation. This work studied its in vitro combination with cytotoxic chemotherapeutics in a rational manner. METHODS: Four melanoma cell lines harboring different sensitivity levels to pro-apoptotic stimuli were used to build a predictive response surface model allowing the determination of the optimal in vitro combinations of Sph A with two drugs, i.e., cisplatin or temozolomide, owing to a limited set of experimentations. RESULTS: Testing 12 experimental combinations allowed us to build an accurate predictive model that considers the complexity of the drug interaction and determines the optimal combinations according to the endpoint chosen, i.e., the maximal cytotoxic effects. Therefore, combining 4 µM Sph A with 75 µM cisplatin concomitantly for 72 h improved its cytotoxic effects on melanoma cells in a synergistic manner. An optimal in vitro treatment schedule was also obtained for temozolomide. CONCLUSIONS: The use of a response surface model offers the possibility of reducing the experiments while determining accurately the optimal combinations. We herein highlighted that combining the Na+/K+/2Cl- cotransporter and/or anion exchanger inhibitor Sph A with chemotherapeutic agents could improve the therapeutic benefits of conventional chemotherapies against advanced melanomas, particularly because Sph A exerts cytotoxic effects regardless of the genetic BRAF and NRAS status.

Altered ion transport in normal human bronchial epithelial cells following exposure to chemically distinct metal welding fume particles.

Welding fume inhalation causes pulmonary toxicity, including susceptibility to infection. We hypothesized that airway epithelial ion transport is a target of fume toxicity, and investigated the effects of fume particulates from manual metal arc-stainless steel (MMA-SS) and gas metal arc-mild steel (GMA-MS) on ion transport in normal human bronchial epithelium (NHBE) cultured in air-interface. MMA-SS particles, more soluble than GMA-MS particles, contain Cr, Ni, Fe and Mn; GMA-MS particles contain Fe and Mn. MMA-SS or GMA-MS particles (0.0167-166.7µg/cm2) were applied apically to NHBEs. After 18h transepithelial potential difference (Vt), resistance (Rt), and short circuit current (Isc) were measured. Particle effects on Na+ and Cl¯ channels and the Na+,K+,2Cl¯-cotransporter were evaluated using amiloride (apical), 5-nitro-2-[(3-phenylpropyl)amino]benzoic acid (NPPB, apical), and bumetanide (basolateral), respectively. MMA-SS (0.0167-16.7µg/cm2) increased basal Vt. Only 16.7µg/cm2 GMA-MS increased basal Vt significantly. MMA-SS or GMA-MS exposure potentiated Isc responses (decreases) to amiloride and bumetanide, while not affecting those to NPPB, GMA-MS to a lesser degree than MMA-SS. Variable effects on Rt were observed in response to amiloride, and bumetanide. Generally, MMA-SS was more potent in altering responses to amiloride and bumetanide than GMA-MS. Hyperpolarization occurred in the absence of LDH release, but decreases in Vt, Rt, and Isc at higher fume particulate doses accompanied LDH release, to a greater extent for MMA-SS. Thus, Na+ transport and Na+,K+,2Cl¯-cotransport are affected by fume exposure; MMA-MS is more potent than GMA-MS. Enhanced Na+ absorption and decreased airway surface liquid could compromise defenses against infection.

Heightened stress response and cognitive impairment after repeated neonatal sevoflurane exposures might be linked to excessive GABAAR-mediated depolarization.

OBJECTIVE: Children with repeated exposures to anesthesia at an early age are at an increased risk of cognitive impairment. Data in the literature link increased developmental depolarizing γ-aminobutyric acid (GABA) type A receptor (GABAAR) at younger age to neurodevelopmental disorders. Here we investigated the involvement of GABAergic signaling during development in mediating the adverse effects of repeated sevoflurane exposures. METHODS: Sprague-Dawley male rats received repeated exposures to 3 % sevoflurane for 2 h daily for 3 consecutive days on postnatal days (P) 4, 5, and 6; maternally separated and unseparated rats served as controls. A subgroup of rats received three injections of the Na(+)-K(+)-2Cl(-) cotransporter inhibitor, bumetanide (1.82 mg/kg, intraperitoneally) 15 min prior to initiation of each sevoflurane exposure. RESULTS: The results showed that repeated neonatal sevoflurane exposures contribute to learning and memory impairment in the Morris water maze (MWM) at P60. The corticosterone level was significantly increased immediately after repeated neonatal sevoflurane exposures. Repeated neonatal sevoflurane exposures heightened the secretion of corticosterone in response to stress in P7 and P60 rats. Pretreatment of male rats prior to each sevoflurane exposure with bumetanide attenuated the corticosterone level immediately after repeated neonatal sevoflurane exposures, normalized endocrine response to stress at P7 and P60, and attenuated the sevoflurane-induced learning and memory impairment in the MWM. CONCLUSION: These data suggested that the heightened stress response and cognitive impairment after repeated neonatal sevoflurane exposures might be linked to excessive GABAAR-mediated depolarization.

Regulating effect of TongXie-YaoFang on colonic epithelial secretion via Cl- and HCO3- channel.

AIM: To investigate the pharmacological effect of TongXie-YaoFang (TXYF) formula, a Chinese herbal formula, on Diarrhea-predominant irritable bowel syndrome (D-IBS) rats. METHODS: In a neonatal maternal separation plus restraint stress (NMS + RS) model of D-IBS, male Sprague Dawley rats were randomly divided into two groups (NMS + RS group and TXYF-formula group) with no handlings were used as controls (NH group). Starting from postnatal day 60, rats in TXYF-formula group were administered TXYF-formula (4.92 g/100 g bodyweight) orally twice a day for 14 consecutive days while NH group and NMS + RS group were given distilled water. Using short-circuit current technology, we observed 5-HT-induced changes of current across ion channels, such as cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel, epithelial Na+ channel (ENaC), Ca2+-dependent Cl- channel (CACC), Na+-K+-2Cl- co-transporter (NKCC), and Na+-HCO3- co-transporter (NBC), in the colonic epithelium of three groups after exposure to drugs and specific blockers with a Power Lab System (AD Instruments International). RESULTS: Under basal conditions, the changes of short-circuit current (∆Isc, µA/cm2) induced by 5-HT were similar in NH group and TXYF-formula group, and both higher than NMS + RS group (70.86 µA/cm2 ± 12.32 µA/cm2, 67.67 µA/cm2 ± 11.68 µA/cm2vs 38.8 µA/cm2 ± 7.25 µA/cm2, P < 0.01, respectively). When CACC was blocked by 4,4'-diisothiocyanato-stilbene-2,2'-disulfonic acid, 5-HT-induced ∆Isc was smaller in NMS + RS group than in NH group and TXYF-formula group, respectively (48.41 µA/cm2 ± 13.15 µA/cm2vs 74.62 µA/cm2 ± 10.73 µA/cm2, 69.22 µA/cm2 ± 11.7 µA/cm2, P < 0.05, respectively). The similar result could be obtained when ENaC was blocked by Amiloride (44.69 µA/cm2 ± 12.58 µA/cm2vs 62.05 µA/cm2 ± 11.26 µA/cm2, 62.11 µA/cm2 ± 12.01 µA/cm2, P < 0.05, respectively). However, when CFTR Cl- channel was blocked by 1,1-dimethyl piperidinium chloride (DPC), 5-HT-induced ∆Isc did not significantly differ in three groups (42.28 µA/cm2 ± 10.61 µA/cm2vs 51.48 µA/cm2 ± 6.56 µA/cm2vs 47.75 µA/cm2 ± 7.99 µA/cm2, P > 0.05, respectively). The similar results could also be obtained in three groups when NBC and NKCC were respectively blocked by their blockers. CONCLUSION: TXYF-formula can regulate the Cl- and HCO3- secretion of colonic mucosa via CFTR Cl- channel, Cl-/HCO3- exchanger, NBC and NKCC co-transporters.

Red blood cell sodium transport in patients with cirrhosis.

Patients with advanced cirrhosis have abnormal sodium homoeostasis. The study was undertaken to quantify the sodium transport across the plasma membrane of red blood cells (RBC) in patients with cirrhosis. RBC efflux and influx of sodium were studied in vitro with tracer (22) Na(+) according to linear kinetics in 24 patients with cirrhosis and 14 healthy controls. The sodium efflux was modified by ouabain (O), furosemide (F) and a combination of O and F (O + F). RBC sodium was significantly decreased (4·6 versus control 6·3 mmol l(-1) , P<0·001) and directly related to serum sodium (r = 0·57, P<0·05). The RBC fractional sodium efflux was higher in patients with cirrhosis (+46%, P<0·01) compared to controls. Inhibition in both high (145 mmol l(-1) )- and low (120 mmol l(-1) )-sodium buffers showed that the F-insensitive sodium efflux was twice as high in cirrhosis as in controls (P = 0·03-0·007), especially the O-sensitive, F-insensitive efflux was increased (+ 225%, P = 0·01-0·006). Fractional F-sensitive transport was normal in cirrhosis. RBC sodium influx was largely normal in cirrhosis. In conclusion, RBC sodium content is reduced in patients with cirrhosis with a direct relation to serum sodium. Increased RBC sodium efflux is especially related to ouabain-sensitive, furosemide-insensitive transport and thus most likely due to upregulated activity of the sodium-potassium pump. The study gives no evidence to an altered intracellular/extracellular sodium ratio or to a reduced fractional furosemide-sensitive sodium transport in cirrhosis.

Pharmacotherapeutic targeting of cation-chloride cotransporters in neonatal seizures.

Seizures are a common manifestation of acute neurologic insults in neonates and are often resistant to the standard antiepileptic drugs that are efficacious in children and adults. The paucity of evidence-based treatment guidelines, coupled with a rudimentary understanding of disease pathogenesis, has made the current treatment of neonatal seizures empiric and often ineffective, highlighting the need for novel therapies. Key developmental differences in γ-aminobutyric acid (GABA)ergic neurotransmission between the immature and mature brain, and trauma-induced alterations in the function of the cation-chloride cotransporters (CCCs) NKCC1 and KCC2, probably contribute to the poor efficacy of standard antiepileptic drugs used in the treatment of neonatal seizures. Although CCCs are attractive drug targets, bumetanide and other existing CCC inhibitors are suboptimal because of pharmacokinetic constraints and lack of target specificity. Newer approaches including isoform-specific NKCC1 inhibitors with increased central nervous system penetration, and direct and indirect strategies to enhance KCC2-mediated neuronal chloride extrusion, might allow therapeutic modulation of the GABAergic system for neonatal seizure treatment. A PowerPoint slide summarizing this article is available for download in the Supporting Information section here.

Gastric inhibitory peptide, serotonin, and glucagon are unexpected chloride secretagogues in the rectal gland of the skate (Leucoraja erinacea).

Since the discovery of the rectal gland of the dogfish shark 50 years ago, experiments with this tissue have greatly aided our understanding of secondary active chloride secretion and the secretagogues responsible for this function. In contrast, very little is known about the rectal gland of skates. In the present experiments, we performed the first studies in the perfused rectal gland of the little skate (Leucoraja erinacea), an organ weighing less than one-tenth of the shark rectal gland. Our results indicate that the skate gland can be studied by modified perfusion techniques and in primary culture monolayers, and that secretion is blocked by the inhibitors of membrane proteins required for secondary active chloride secretion. Our major finding is that three G protein-coupled receptor agonists, the incretin gastric inhibitory polypeptide (GIP), also known as glucose-dependent insulinotropic peptide, as well as glucagon and serotonin, are unexpected potent chloride secretagogues in the skate but not the shark. Glucagon stimulated chloride secretion to a mean value of 1,661 ± 587 µeq·h(-1)·g(-1) and serotonin stimulated to 2,893 ± 699 µeq·h(-1)·g(-1). GIP stimulated chloride secretion to 3,733 ± 679 µeq·h(-1)·g(-1) and significantly increased tissue cAMP content compared with basal conditions. This is the first report of GIP functioning as a chloride secretagogue in any species or tissue.

Effect of co-transporter blockers on non-synaptic epileptiform activity-computational simulation.

The important role of cation-chloride co-transporters in epilepsy is being supported by an increasing number of investigations. However, enormous complexity is involved since the action of these co-transporters has effects on the ionic homeostasis influencing directly the neuronal excitability and the tissue propensity to sustain seizure. To unravel the complex mechanisms involving the co-transporters action during seizure, this paper shows simulations of non-synaptic epileptiform activity and the effect of the blockage of the two different types of cation-chloride co-transporters present in the brain: Na, K and 2Cl co-transporter (NKCC) and K and Cl co-transporter (KCC). The simulations were performed with an electrochemical model representing the non-synaptic structure of the granule cell layer of the dentate gyrus (DG) of the rat hippocampus. The simulations suggest: (i) the potassium clearance is based on the systemic interplay between the Na/K pump and the NKCC co-transporters; (ii) the simultaneous blockage of the NKCC of the neurons and KCC of glial cells acts efficiently suppressing the epileptiform activities; and (iii) the simulations show that depending on the combined blockage of the co-transporters, the epileptiform activities may be suppressed or enhanced.

Na+/H+ exchanger inhibitor augments hyperosmolarity-induced vasoconstriction by enhancing actin polymerization.

Vascular smooth muscle cells (VSMCs) exhibit shrinkage-induced activation of Na(+)/H(+) exchanger isoform 1 (NHE-1) and Na(+), K(+), 2Cl(-) cotransporter (NKCC) under hyperosmotic conditions. To investigate the roles of these ion transporters in vascular smooth muscle force induced by hyperosmotic stress, we tested the effects of 5-(N, N-dimethyl)-amiloride (DMA; NHE inhibitor), cariporide (a selective NHE-1 inhibitor), and bumetanide (NKCC inhibitor) on the contractile response of rat aortic rings to hyperosmolar solutions. NHE inhibitors significantly augmented the maximum force response and contractile sensitivity to hyperosmolar sucrose, NaCl, and glucose in endothelium-denuded rings. Bumetanide elicited a comparatively modest increase in sensitivity. NHE inhibitors blocked the increase in intracellular pH and enhanced the cell volume decrease of cultured VSMCs after exposure to hyperosmolar sucrose. However, DMA had no effect on the increase in cytosolic free Ca(2+) concentration ([Ca(2+)]i) in rat VSMCs and on the increases in phosphorylation of myosin phosphatase target subunit 1 and myosin light chain (MLC) in aortic rings in response to hyperosmolar sucrose. Hyperosmolar sucrose-induced force was significantly attenuated by cytochalasin B in the presence or absence of DMA. Exposure to hyperosmolar sucrose increased the ratio of F- to G-actin; the ratio was further elevated by DMA. These results suggest that the potentiation of hyperosmotic shrinkage by NHE inhibition promotes actin polymerization in VSMCs and augments force production independent of changes in [Ca(2+)]i and MLC phosphorylation.

Increased excitability and molecular changes in adult rats after a febrile seizure.

Both early life inflammation and prolonged febrile seizures have been associated with increased excitation in the adult brain. We hypothesized this may be due in part to changes in the cation-chloride cotransporter system. Rat pups received saline or lipopolysaccharide/kainic acid (LPS/KA) resulting in inflammation, followed by a behavioral febrile seizure (FS) in approximately 50% of rats. Adult animals from the saline, inflammation, or inflammation + FS groups underwent the following: (1) in vitro electrophysiologic studies; (2) Western blotting or polymerase chain reaction; or (3) application of the Na-K-Cl cotransporter 1 (NKCC1) blocker bumetanide to determine its effect on reversing increased excitability in vitro. The inflammation and inflammation + FS groups demonstrated increased excitability in vitro and increased hippocampal protein expression of NR2B and GABAA α5 receptor subunits and mRNA expression of NKCC1. The inflammation + FS group also had decreased protein expression of GluR2 and GABAA α1 receptor subunits and mRNA and protein expression of KCC2. Bumetanide decreased in vitro 4-aminopyridine-induced inter-ictal activity in the inflammation and inflammation + FS groups. The results demonstrate early-life inflammation with or without a behavioral FS can lead to long-lasting molecular changes and increased excitability in the adult rat hippocampus, although some changes are more extensive when inflammation is accompanied by behavioral seizure activity. Bumetanide is effective in reversing increased excitability in vitro, providing evidence for a causal role for cation-chloride cotransporters and suggesting this drug may prove useful for treating epilepsy that develops after a FS.

Cation-chloride cotransporters NKCC1 and KCC2 as potential targets for novel antiepileptic and antiepileptogenic treatments.

In cortical and hippocampal neurons, cation-chloride cotransporters (CCCs) control the reversal potential (EGABA) of GABAA receptor-mediated current and voltage responses and, consequently, they modulate the efficacy of GABAergic inhibition. Two members of the CCC family, KCC2 (the major neuron-specific K-Cl cotransporter; KCC isoform 2) and NKCC1 (the Na-K-2Cl cotransporter isoform 1 which is expressed in both neurons and glial cells) have attracted much interest in studies on GABAergic signaling under both normal and pathophysiological conditions, such as epilepsy. There is tentative evidence that loop diuretic compounds such as furosemide and bumetanide may have clinically relevant antiepileptic actions, especially when administered in combination with conventional GABA-mimetic drugs such as phenobarbital. Furosemide is a non-selective inhibitor of CCCs while at low concentrations bumetanide is selective for NKCCs. Search for novel antiepileptic drugs (AEDs) is highly motivated especially for the treatment of neonatal seizures which are often resistant to, or even aggravated by conventional AEDs. This review shows that the antiepileptic effects of loop diuretics described in the pertinent literature are based on widely heterogeneous mechanisms ranging from actions on both neuronal NKCC1 and KCC2 to modulation of the brain extracellular volume fraction. A promising strategy for the development of novel CCC-blocking AEDs is based on prodrugs that are activated following their passage across the blood-brain barrier. This article is part of the Special Issue entitled 'New Targets and Approaches to the Treatment of Epilepsy'.

Blocking retinal chloride co-transporters KCC2 and NKCC: impact on direction selective ON and OFF responses in the rat's nucleus of the optic tract.

In the present study we investigated in vivo the effects of pharmacological manipulation of retinal processing on the response properties of direction selective retinal slip cells in the nucleus of the optic tract and dorsal terminal nucleus (NOT-DTN), the key visuomotor interface in the pathway underlying the optokinetic reflex. Employing a moving visual stimulus consisting of either a large dark or light edge we could differentiate direction selective ON and OFF responses in retinal slip cells. To disclose the origin of the retinal slip cells' unexpected OFF response we selectively blocked the retinal ON channels and inactivated the visual cortex by cooling. Cortical cooling had no effect on the direction selectivity of the ON or the OFF response in NOT-DTN retinal slip cells. Blockade of the retinal ON channel with APB led to a loss of the ON and, to a lesser degree, of the OFF response and a reduction in direction selectivity. Subsequent blocking of GABA receptors in the retina with picrotoxin unmasked a vigorous albeit direction unselective OFF response in the NOT-DTN. Disturbing the retinal chloride homeostasis by intraocular injections of bumetanide or furosemide led to a loss of direction selectivity in both the NOT-DTN's ON and the OFF response due to a reduced response in the neuron's preferred direction under bumetanide as well as under furosemide and a slightly increased response in the null direction under bumetanide. Our results indicate that the direction specificity of retinal slip cells in the NOT-DTN of the rat strongly depends on direction selective retinal input which depends on intraretinal chloride homeostasis. On top of the well established input from ON center direction selective ganglion cells we could demonstrate an equally effective input from the retinal OFF system to the NOT-DTN.

ß-Adrenergic receptor stimulation increases surface NKCC2 expression in rat thick ascending limbs in a process inhibited by phosphodiesterase 4.

The thick ascending limb of the loop of Henle (THAL) reabsorbs ∼30% of the filtered NaCl in a process mediated by the apical Na-K-2Cl cotransporter NKCC2. Stimulation of ß-adrenergic receptors in the THAL enhances NaCl reabsorption and increases intracellular cAMP. We found that intracellular cAMP stimulates NKCC2 trafficking to the apical membrane via protein kinase A (PKA). Several cAMP-specific phosphodiesterases (PDE) have been identified in rat THALs, and PDE4 decreases cAMP generated by ß-adrenergic stimulation in other cells. However, it is not known whether ß-adrenergic receptors activation stimulates NKCC2 trafficking. Thus we hypothesized that ß-adrenergic receptor stimulation enhances THAL apical membrane NKCC2 expression via the PKA pathway and PDE4 blunts this effect. THAL suspensions were obtained from Sprague-Dawley rats, and surface NKCC2 expression was measured by surface biotinylation and Western blot. Incubation of THALs with the ß-adrenergic receptor agonist isoproterenol at 0.5 and 1.0 µM increased surface NKCC2 by 17 ± 1 and 29 ± 5% respectively (P < 0.05). Preventing cAMP degradation with 3-isobutyl-methylxanthine (IBMX; a nonselective phosphodiesterase inhibitor) enhanced isoproterenol-stimulated surface NKCC2 expression to 51 ± 7% (P < 0.05 vs. isoproterenol). The ß-adrenergic receptor antagonist propranolol or the PKA inhibitor H-89 completely blocked isoproterenol + IBMX-induced increase on surface NKCC2, while propranolol or H-89 alone had no effect. Selective inhibition of PDE4 with rolipram (20 µM) potentiated the effect of isoproterenol on surface NKCC2 and increased cAMP levels. We concluded that ß-adrenergic receptor stimulation enhances surface NKCC2 expression in the THALs via PKA and PDE4 blunts this effect.

Regulation of epithelial Na+ channels by adrenal steroids: mineralocorticoid and glucocorticoid effects.

Epithelial Na+ channels (ENaC) can be regulated by both mineralocorticoid and glucocorticoid hormones. In the mammalian kidney, effects of mineralocorticoids have been extensively studied, but those of glucocorticoids are complicated by metabolism of the hormones and cross-occupancy of mineralocorticoid receptors. Here, we report effects of dexamethasone, a synthetic glucocorticoid, on ENaC in the rat kidney. Infusion of dexamethasone (24 µg/day) for 1 wk increased the abundance of αENaC 2.26 ± 0.04-fold. This was not accompanied by an induction of Na+ currents (I(Na)) measured in isolated split-open collecting ducts. In addition, hormone treatment did not increase the abundance of the cleaved forms of either αENaC or γENaC or the expression of ßENaC or γENaC protein at the cell surface. The absence of hypokalemia also indicated the lack of ENaC activation in vivo. Dexamethasone increased the abundance of the Na+ transporters Na+/H+ exchanger 3 (NHE3; 1.36 ± 0.07-fold), Na(+)-K(+)-2Cl(-) cotransporter 2 (NKCC2; 1.49 ± 0.07-fold), and Na-Cl cotransporter (NCC; 1.72 ± 0.08-fold). Surface expression of NHE3 and NCC also increased with dexamethasone treatment. To examine whether glucocorticoids could either augment or inhibit the effects of mineralocorticoids, we infused dexamethasone (60 µg/day) together with aldosterone (12 µg/day). Dexamethasone further increased the abundance of αENaC in the presence of aldosterone, suggesting independent effects of the two hormones on this subunit. However, I(Na) was similar in animals treated with dexamethasone+aldosterone and with aldosterone alone. We conclude that dexamethasone can occupy glucocorticoid receptors in cortical collecting duct and induce the synthesis of αENaC. However, this induction is not sufficient to produce an increase in functional Na+ channels in the apical membrane, implying that the abundance of αENaC is not rate limiting for channel formation in the kidney.

Cellular localization of NKCC2 and its possible role in the Cl- absorption in the rat and human distal colonic epithelia.

Recently, we demonstrated the expression of NKCC2, an absorptive isoform of NKCC specifically expressed in the kidney, in the rat gastrointestinal tract including the distal colonic mucosa. This study aims to investigate its localization in colonic epithelia and possible role in the colonic ion transport. Reverse transcription polymerase chain reaction (RT-PCR), Western blotting, and immunohistochemistry were used to investigate the expression and localization of NKCC2. The role of NKCC2 on the colonic ion transport was examined by mean of short-circuit current (I(SC)) monitoring. The results indicated that NKCC2 was expressed in the apical region of the epithelia in rat distal colon and human sigmoid colon. NKCC1, which is a secretive NKCC isoform, was localized predominantly in the basolateral membrane, which has been well documented. Serosal (basolateral) administration of bumetanide, an inhibitor of both NKCC1 and NKCC2, inhibited serosal forskolin-induced I(SC) increase by 66% but enhanced the luminal (apical) forskolin-induced I(SC) response by 63%. Furthermore, the blocking of epithelial Na(+) channels by apical addition of amiloride (10 µmol/L), K(+) channels by tetraethylammoniumion (TEA) (5 mmol/L), or glibenclimide (0.1 mmol/L) did not affect apical forskolin-induced I(SC) increase, excluding the involvement of cations, Na(+) and K(+), in the I(SC) response. The luminal forskolin-induced I(SC) increase was enhanced markedly by the apical pretreatment with bumetanide or the reduction of apical Cl(-) concentration by 114% and 198%, respectively, which were inhibited by apical addition of glibenclimide (1 mmol/L) by more than 60%. This finding suggests the involvement of an anion. Furthermore, the removal of basolateral HCO(3)(-) reduced apical forskolin-induced I(SC) by more than 75% indicated that the apical forskolin-induced I(SC) increase in rat distal colon was mediated by Cl(-) absorption and HCO(3)(-) secretion. In conclusion, NKCC2 is expressed widely in the colonic epithelium in rat distal colon and human sigmoid colon, especially in the apical membrane. It involves the process of colonic Cl(-) absorption coupled with HCO(3)(-) secretion.

Expression of Na+-K+ -2Cl- cotransporter 1 is epigenetically regulated during postnatal development of hypertension.

BACKGROUND: The expression of Na(+)-K(+)-2Cl(-) cotransporter 1 (NKCC1) is upregulated in spontaneously hypertensive rat (SHR). We investigated whether expression of NKCC1 is epigenetically regulated during postnatal development of hypertension. METHODS: The mesenteric arteries from 5-, 10-, and 18-week-old Wistar-Kyoto rats (WKY) and SHRs were subjected to vascular contraction. We determined expression levels of Nkcc1 mRNA and protein, methylation status, and histone modification of Nkcc1 promoter, and DNA methyltransferase (DNMT) activity. RESULTS: The inhibition of dose-response curves by bumetanide, an inhibitor of NKCC1, as well as the expression of Nkcc1 mRNA and protein was comparable between 5-week-old SHR and age-matched WKY, but greater in 18-week-old SHR than in age-matched WKY. Nkcc1 promoter in WKY was getting methylated with age whereas that in SHR mostly remained hypomethylated after development of hypertension. DNMT3B was highly associated with the promoter of WKY, whereas the CXXC finger protein 1 (Cfp1) was highly bound to the promoter of SHR. At the age of 18 weeks, the DNMT activity in aorta of WKY was about threefold higher than that of SHR. The transcription-activating histone code acetyl H3 was higher in SHR than in WKY, whereas suppressive histone code dimethyl H3K9 was greater in WKY than in SHR. CONCLUSION: It is concluded that expression of NKCC1 is epigenetically upregulated during postnatal development of hypertension. Our data indicate that maintenance of hypomethylation in Nkcc1 promoter of SHR resulting from low DNMT activity plays an important role in the upregulation of NKCC1 during development of spontaneous hypertension.

Capsaicinoids regulate airway anion transporters through Rho kinase- and cyclic AMP-dependent mechanisms.

To investigate the effects of capsaicinoids on airway anion transporters, we recorded and analyzed transepithelial currents in human airway epithelial Calu-3 cells. Application of capsaicin (100 µM) attenuated vectorial anion transport, estimated as short-circuit currents (I(SC)), before and after stimulation by forskolin (10 µM) with concomitant reduction of cytosolic cyclic AMP (cAMP) levels. The capsaicin-induced inhibition of I(SC) was also observed in the response to 8-bromo-cAMP (1 mM, a cell-permeable cAMP analog) and 3-isobutyl-1-methylxanthine (1 mM, an inhibitor of phosphodiesterases). The capsaicin-induced inhibition of I(SC) was attributed to suppression of bumetanide (an inhibitor of the basolateral Na(+)-K(+)-2 Cl(-) cotransporter 1)- and 4,4'-dinitrostilbene-2,2'-disulfonic acid (an inhibitor of basolateral HCO(3)(-)-dependent anion transporters)-sensitive components, which reflect anion uptake via basolateral cAMP-dependent anion transporters. In contrast, capsaicin potentiated apical Cl(-) conductance, which reflects conductivity through the cystic fibrosis transmembrane conductance regulator, a cAMP-regulated Cl(-) channel. All these paradoxical effects of capsaicin were mimicked by capsazepine. Forskolin application also increased phosphorylated myosin phosphatase target subunit 1, and the phosphorylation was prevented by capsaicin and capsazepine, suggesting that these capsaicinoids assume aspects of Rho kinase inhibitors. We also found that the increments in apical Cl(-) conductance were caused by conventional Rho kinase inhibitors, Y-27632 (20 µM) and HA-1077 (20 µM), with selective inhibition of basolateral Na(+)-K(+)-2 Cl(-) cotransporter 1. Collectively, capsaicinoids inhibit cAMP-mediated anion transport through down-regulation of basolateral anion uptake, paradoxically accompanied by up-regulation of apical cystic fibrosis transmembrane conductance regulator-mediated anion conductance. The latter is mediated by inhibition of Rho-kinase, which is believed to interact with actin cytoskeleton.