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.

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.

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.

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.

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.

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.

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.

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.

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.

High salt differentially regulates surface NKCC2 expression in thick ascending limbs of Dahl salt-sensitive and salt-resistant rats.

NaCl reabsorption by the thick ascending limb of the loop of Henle (THAL) occurs via the apical Na-K-2Cl cotransporter, NKCC2. Overall, NKCC2 activity and NaCl reabsorption are regulated by the amount of NKCC2 at the apical surface, and also by phosphorylation. Dahl salt-sensitive rats (SS) exhibit higher NaCl reabsorption by the THAL compared with Dahl salt-resistant rats (SR), and they become hypertensive during high-salt (HS) intake. However, the effect of HS on THAL transport, surface NKCC2 expression, and NKCC2 NH(2)-terminus phosphorylation has not been studied. We hypothesized that HS enhances surface NKCC2 and its phosphorylation in THALs from Dahl SS. THAL suspensions were obtained from a group of SS and SR rats on normal-salt (NS) or HS intake. In SR rats THAL NaCl transport measured as furosemide-sensitive oxygen consumption was decreased by HS (-34%, P < 0.05). In contrast, HS did not affect THAL transport in SS rats. As expected, HS increased systolic blood pressure only in SS rats (Δ 23 ± 2 mmHg, P < 0.002) but not in SR rats (Δ 5 ± 3 mmHg). We next tested the effect of HS intake on apical surface NKCC2 and its NH(2)-terminus threonine phosphorylation (P-NKCC2) in SS and SR rats. HS intake decreased surface NKCC2 by 15 ± 2% (P < 0.03) in THALs from SR without affecting total NKCC2 or NH(2)-terminus P-NKCC2. In contrast, in SS rats HS intake increased surface NKCC2 by 54 ± 6% (P < 0.01) without affecting total NKCC2 expression or P-NKCC2. We conclude that HS intake causes different effects on surface NKCC2 in SS and SR rats. Our data suggest that enhanced surface NKCC2 in SS rats might contribute to enhanced NaCl reabsorption in SS rats during HS intake.

Mechanisms of chloride uptake in frog olfactory receptor neurons.

Odorant stimulation of olfactory receptor neurons (ORNs) leads to the activation of a Ca(2+) permeable cyclic nucleotide-gated (CNG) channel followed by opening of an excitatory Ca(2+)-activated Cl(-) channel, which carries about 70% of the odorant-induced receptor current. This requires ORNs to have a [Cl(-)](i) above the electrochemical equilibrium to render this anionic current excitatory. In mammalian ORNs, the Na(+)-K(+)-2Cl(-) co-transporter 1 (NKCC1) has been characterized as the principal mechanism by which these neurons actively accumulate Cl(-). To determine if NKCC activity is needed in amphibian olfactory transduction, and to characterize its cellular location, we used the suction pipette technique to record from Rana pipiens ORNs. Application of bumetanide, an NKCC blocker, produced a 50% decrease of the odorant-induced current. Similar effects were observed when [Cl(-)](i) was decreased by bathing ORNs in low Cl(-) solution. Both manipulations reduced only the Cl(-) component of the current. Application of bumetanide only to the ORN cell body and not to the cilia decreased the current by again about 50%. The results show that NKCC is required for amphibian olfactory transduction, and suggest that the co-transporter is located basolaterally at the cell body although its presence at the cilia could not be discarded.

The reverse operation of Na(+)/Cl(-)-coupled neurotransmitter transporters--why amphetamines take two to tango.

Sodium-chloride coupled neurotransmitter transporters achieve reuptake of their physiological substrate by exploiting the pre-existing sodium-gradient across the cellular membrane. This terminates the action of previously released substrate in the synaptic cleft. However, a change of the transmembrane ionic gradients or specific binding of some psychostimulant drugs to these proteins, like amphetamine and its derivatives, induce reverse operation of neurotransmitter:sodium symporters. This effect eventually leads to an increase in the synaptic concentration of non-exocytotically released neurotransmitters [and - in the case of the norepinephrine transporters, underlies the well-known indirect sympathomimetic activity]. While this action has long been appreciated, the underlying mechanistic details have been surprisingly difficult to understand. Some aspects can be resolved by incorporating insights into the oligomeric nature of transporters, into the nature of the accompanying ion fluxes, and changes in protein kinase activities.

AE2 Cl-/HCO3- exchanger is required for normal cAMP-stimulated anion secretion in murine proximal colon.

Anion secretion by colonic epithelium is dependent on apical CFTR-mediated anion conductance and basolateral ion transport. In many tissues, the NKCC1 Na(+)-K(+)-2Cl(-) cotransporter mediates basolateral Cl(-) uptake. However, additional evidence suggests that the AE2 Cl(-)/HCO(3)(-) exchanger, when coupled with the NHE1 Na(+)/H(+) exchanger or a Na(+)-HCO(3)(-) cotransporter (NBC), contributes to HCO(3)(-) and/or Cl(-) uptake. To analyze the secretory functions of AE2 in proximal colon, short-circuit current (I(sc)) responses to cAMP and inhibitors of basolateral anion transporters were measured in muscle-stripped wild-type (WT) and AE2-null (AE2(-/-)) proximal colon. In physiological Ringer, the magnitude of cAMP-stimulated I(sc) was the same in WT and AE2(-/-) colon. However, the I(sc) response in AE2(-/-) colon exhibited increased sensitivity to the NKCC1 inhibitor bumetanide and decreased sensitivity to the distilbene derivative SITS (which inhibits AE2 and some NBCs), indicating that loss of AE2 results in a switch to increased NKCC1-supported anion secretion. Removal of HCO(3)(-) resulted in robust cAMP-stimulated I(sc) in both AE2(-/-) and WT colon that was largely mediated by NKCC1, whereas removal of Cl(-) resulted in sharply decreased cAMP-stimulated I(sc) in AE2(-/-) colon relative to WT controls. Inhibition of NHE1 had no effect on cAMP-stimulated I(sc) in AE2(-/-) colon but caused a switch to NKCC1-supported secretion in WT colon. Thus, in AE2(-/-) colon, Cl(-) secretion supported by basolateral NKCC1 is enhanced, whereas HCO(3)(-) secretion is diminished. These results show that AE2 is a component of the basolateral ion transport mechanisms that support anion secretion in the proximal colon.

Expression of the sodium potassium chloride cotransporter (NKCC1) and sodium chloride cotransporter (NCC) and their effects on rat lens transparency.

PURPOSE: To characterize the expression patterns of the Na+-K+-Cl(-) cotransporter (NKCC) 1 and NKCC2, and the Na+-Cl(-) cotransporter (NCC) in the rat lens and to determine if they play a role in regulating lens volume and transparency. METHODS: RT-PCR was performed on RNA extracted from fiber cells to identify sodium dependent cotransporters expressed in the rat lens. Western blotting and immunohistochemistry, using NKCC1, NKCC2, and NCC antibodies, were used to verify expression at the protein level and to localize transporter expression. Organ cultured rat lenses were incubated in Artificial Aqueous Humor (AAH) of varying osmolarities or isotonic AAH that contained either the NKCC specific inhibitor bumetanide, or the NCC specific inhibitor thiazide for up to 18 h. Lens transparency was monitored with dark field microscopy, while tissue morphology and antibody labeling patterns were recorded using a confocal microscope. RESULTS: Molecular experiments showed that NKCC1 and NCC were expressed in the lens at both the transcript and protein levels, but NKCC2 was not. Immunohistochemistry showed that both NKCC1 and NCC were expressed in the lens cortex, but NCC expression was also found in the lens core. In the lens cortex the majority of labeling for both transporters was cytoplasmic in nature, while in the lens core, NCC labeling was associated with the membrane. Exposure of lenses to either hypotonic or hypertonic AAH had no noticeable effects on the predominantly cytoplasmic location of either transporter in the lens cortex. Incubation of lenses in isotonic AAH plus the NKCC inhibitor bumetanide for 18 h induced a cortical opacity that was initiated by a shrinkage of peripheral fiber cells and the dilation of the extracellular space between fiber cells in a deeper zone located some approximately 150 microm in from the capsule. In contrast, lenses incubated in isotonic AAH and the NCC inhibitor thiazide maintained both their transparency and their regular fiber cell morphology. CONCLUSIONS: We have confirmed the expression of NKCC1 in the rat lens and report for the first time the expression of NCC in lens fiber cells. The expression patterns of the two transporters and the differential effects of their specific inhibitors on fiber cell morphology indicate that these transporters play distinct roles in the lens. NKCC1 appears to mediate ion influx in the lens cortex while NCC may play a role in the lens nucleus.

Strain-dependent differences in electrogenic secretion of electrolytes across mouse colon epithelium.

Mice have proven to be powerful models for the study of human physiology and pathophysiology. With the advent of techniques for genomic manipulation, the possibilities for studying inherited diseases in this convenient laboratory mammal are increasing by the day. It has been reported that when knocking out or otherwise modifying genes of interest in mice, the phenotype obtained can vary markedly depending on the genetic background of the animals used in the study. The aim of this work was to study whether the genetic background can influence the characteristics of fluid and electrolyte transepithelial transport in the distal colon of three mouse strains most in use in our and other laboratories. Ussing chamber recordings revealed that the colons of C57Bl/6J, Sv 129 and Black Swiss animals have distinctive responses to the calcium agonists carbachol and histamine that are not explained by the presence of different types of muscarinic and histaminergic receptors in these tissues. We have also found differences in the cAMP-activated, KCNMA1-channel-dependent potassium secretion between the strains. We interpret this to indicate a unique distribution of KCNMA1 channels in lower parts of the crypt of Sv 129 colonic epithelium compared with that of C57Bl/6J and Black Swiss animals. The reported differences should be taken into account when choosing the genetic background of animals to be used for genetic modification.

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).

GABA action in immature neocortical neurons directly depends on the availability of ketone bodies.

In the early postnatal period, energy metabolism in the suckling rodent brain relies to a large extent on metabolic pathways alternate to glucose such as the utilization of ketone bodies (KBs). However, how KBs affect neuronal excitability is not known. Using recordings of single NMDA and GABA-activated channels in neocortical pyramidal cells we studied the effects of KBs on the resting membrane potential (E(m)) and reversal potential of GABA-induced anionic currents (E(GABA)), respectively. We show that during postnatal development (P3-P19) if neocortical brain slices are adequately supplied with KBs, E(m) and E(GABA) are both maintained at negative levels of about -83 and -80 mV, respectively. Conversely, a KB deficiency causes a significant depolarization of both E(m) (>5 mV) and E(GABA) (>15 mV). The KB-mediated shift in E(GABA) is largely determined by the interaction of the NKCC1 cotransporter and Cl(-)/HCO3 transporter(s). Therefore, by inducing a hyperpolarizing shift in E(m) and modulating GABA signaling mode, KBs can efficiently control the excitability of neonatal cortical neurons.

Bumetanide enhances phenobarbital efficacy in a neonatal seizure model.

OBJECTIVES: High levels of expression of the Na+-K+-2Cl- (NKCC1) cotransporter in immature neurons cause the accumulation of intracellular chloride and, therefore, a depolarized Cl- equilibrium potential (E(Cl)). This results in the outward flux of Cl- through GABA(A) channels, the opposite direction compared with mature neurons, in which GABA(A) receptor activation is inhibitory because Cl- flows into the cell. This outward flow of Cl- in neonatal neurons is excitatory and contributes to a greater seizure propensity and poor electroencephalographic response to GABAergic anticonvulsants such as phenobarbital and benzodiazepines. Blocking the NKCC1 transporter with bumetanide prevents outward Cl- flux and causes a more negative GABA equilibrium potential (E(GABA)) in immature neurons. We therefore tested whether bumetanide enhances the anticonvulsant action of phenobarbital in the neonatal brain METHODS: Recurrent seizures were induced in the intact hippocampal preparation in vitro by continuous 5-hour exposure to low-Mg2+ solution. The anticonvulsant efficacy of phenobarbital, bumetanide, and the combination of these drugs was studied RESULTS: Phenobarbital failed to abolish or depress recurrent seizures in 70% of hippocampi. In contrast, phenobarbital in combination with bumetanide abolished seizures in 70% of hippocampi and significantly reduced the frequency, duration, and power of seizures in the remaining 30% INTERPRETATION: Thus, alteration of Cl- transport by bumetanide enables the anticonvulsant action of phenobarbital in immature brain. This is a mechanistic demonstration of rational anticonvulsant polypharmacy. The combination of these agents may comprise an effective therapy for early-life seizures.

Age- and dose-specific anticonvulsant action of bumetanide in immature rats.

GABA exhibits depolarizing action in the immature neurons due to high intracellular activity of chloride ions. It is maintained by cation-chloride cotransporter NKCC1 which is present in immature brain. Bumetanide is a specific inhibitor of this cotransporter. We studied possible anticonvulsant activity of bumetanide in pentylenetetrazol-induced seizures in three age groups of rat pups (7, 12, and 18 days old). Pretreatment with bumetanide (0.2-1 mg/kg i.p.) resulted in dose-dependent decrease of incidence of the tonic phase of generalized tonic-clonic seizures in 12-day-old rats only. No effect was observed in younger and older animals. Higher dose of bumetanide (2.5 mg/kg) did not affect tonic convulsions but, on the contrary, decreased latencies of generalized seizures in 12-day-old animals. Lack of marked anticonvulsant effect is probably due to relative maturity of neurons in the brainstem where the generator of generalized seizures is localized. Age- and dose-specific suppression of the tonic phase needs further analysis.