Adenylyl cyclase 6 enhances NKCC2 expression and mediates vasopressin-induced phosphorylation of NKCC2 and NCC.

Arginine vasopressin (AVP) affects kidney function via vasopressin V2 receptors that are linked to activation of adenylyl cyclase (AC) and an increase in cyclic adenosine monophosphate formation. AVP/cyclic adenosine monophosphate enhance the phosphorylation of the Na-K-2Cl cotransporter (NKCC2) at serine residue 126 (pS126 NKCC2) and of the Na-Cl cotransporter (NCC) at threonine 58 (pT58 NCC). The isoform(s) of AC involved in these responses, however, were unknown. Phosphorylation of S126 NKCC2 and T58 NCC, induced by the V2 receptor agonist (1-desamino-8-D-arginine vasopressin) in wild-type mice, is lacking in knockout mice for AC isoform 6 (AC6). With regard to NKCC2 phosphorylation, the stimulatory effect of 1-desamino-8-D-AVP and the defect in AC6(-/-) mice seem to be restricted to the medullary portion of the thick ascending limb. AC6 is also a stimulator of total renal NKCC2 protein abundance in medullary and cortical thick ascending limb. Consequently, mice lacking AC6 have lower NKCC2 expression and a mild Bartter syndrome-like phenotype, including lower plasma concentrations of K+ and H+ and compensatory upregulation of NCC. Increased AC6-independent phosphorylation of NKCC2 at S126 might help to stabilize NKCC2 activity in the absence of AC6. Renal AC6 determines total NKCC2 expression and mediates vasopressin-induced NKCC2/NCC phosphorylation. These regulatory mechanisms, which are defective in AC knockout mice, are likely responsible for the observed mild Bartter syndrome.

NKCC1 upregulation disrupts chloride homeostasis in the hypothalamus and increases neuronal activity-sympathetic drive in hypertension.

Hypertension is a major risk factor for coronary artery disease, stroke, and kidney failure. However, the etiology of hypertension in most patients is poorly understood. Increased sympathetic drive emanating from the hypothalamic paraventricular nucleus (PVN) plays a major role in the development of hypertension. Na(+)-K(+)-2Cl(-) cotransporter-1 (NKCC1) in the brain is critically involved in maintaining chloride homeostasis and in neuronal responses mediated by GABA(A) receptors. Here we present novel evidence that the GABA reversal potential (E(GABA)) of PVN presympathetic neurons undergoes a depolarizing shift that diminishes GABA inhibition in spontaneously hypertensive rats (SHRs). Inhibition of NKCC1, but not KCC2, normalizes E(GABA) and restores GABA inhibition of PVN neurons in SHRs. The mRNA and protein levels of NKCC1, but not KCC2, in the PVN are significantly increased in SHRs, and the NKCC1 proteins on the plasma membrane are highly glycosylated. Inhibiting NKCC1 N-glycosylation restores E(GABA) and GABAergic inhibition of PVN presympathetic neurons in SHRs. Furthermore, NKCC1 inhibition significantly reduces the sympathetic vasomotor tone and augments the sympathoinhibitory responses to GABA(A) receptor activation in the PVN in SHRs. These findings suggest that increased NKCC1 activity and glycosylation disrupt chloride homeostasis and impair synaptic inhibition in the PVN to augment the sympathetic drive in hypertension. This information greatly improves our understanding of the pathogenesis of hypertension and helps to design better treatment strategies for neurogenic hypertension.

Altered inhibition in tuberous sclerosis and type IIb cortical dysplasia.

OBJECTIVE: The most common neurological symptom of tuberous sclerosis complex (TSC) and focal cortical dysplasia (FCD) is early life refractory epilepsy. As previous studies have shown enhanced excitatory glutamatergic neurotransmission in TSC and FCD brains, we hypothesized that neurons associated with these lesions may also express altered γ-aminobutyric acid (GABA)(A) receptor (GABA(A)R)-mediated inhibition. METHODS: Expression of the GABA(A)R subunits α1 and α4, and the Na(+)-K(+)-2Cl(-) (NKCC1) and the K(+)-Cl(-) (KCC2) transporters, in human TSC and FCD type II specimens were analyzed by Western blot and double label immunocytochemistry. GABA(A) R responses in dysplastic neurons from a single case of TSC were measured by perforated patch recording and compared to normal-appearing cortical neurons from a non-TSC epilepsy case. RESULTS: TSC and FCD type IIb lesions demonstrated decreased expression of GABA(A)R α1, and increased NKCC1 and decreased KCC2 levels. In contrast, FCD type IIa lesions showed decreased α4, and increased expression of both NKCC1 and KCC2 transporters. Patch clamp recordings from dysplastic neurons in acute slices from TSC tubers demonstrated excitatory GABA(A)R responses that were significantly attenuated by the NKCC1 inhibitor bumetanide, in contrast to hyperpolarizing GABA(A)R-mediated currents in normal neurons from non-TSC cortical slices. INTERPRETATION: Expression and function of GABA(A)Rs in TSC and FCD type IIb suggest the relative benzodiazepine insensitivity and more excitatory action of GABA compared to FCD type IIa. These factors may contribute to resistance of seizure activity to anticonvulsants that increase GABAergic function, and may justify add-on trials of the NKCC1 inhibitor bumetanide for the treatment of TSC and FCD type IIb-related epilepsy.

Chronic hyperosmotic stress converts GABAergic inhibition into excitation in vasopressin and oxytocin neurons in the rat.

In mammals, the increased secretion of arginine-vasopressin (AVP) (antidiuretic hormone) and oxytocin (natriuretic hormone) is a key physiological response to hyperosmotic stress. In this study, we examined whether chronic hyperosmotic stress weakens GABA(A) receptor-mediated synaptic inhibition in rat hypothalamic magnocellular neurosecretory cells (MNCs) secreting these hormones. Gramicidin-perforated recordings of MNCs in acute hypothalamic slices prepared from control rats and ones subjected to the chronic hyperosmotic stress revealed that this challenge not only attenuated the GABAergic inhibition but actually converted it into excitation. The hyperosmotic stress caused a profound depolarizing shift in the reversal potential of GABAergic response (E(GABA)) in MNCs. This E(GABA) shift was associated with increased expression of Na(+)-K(+)-2Cl(-) cotransporter 1 (NKCC1) in MNCs and was blocked by the NKCC inhibitor bumetanide as well as by decreasing NKCC activity through a reduction of extracellular sodium. Blocking central oxytocin receptors during the hyperosmotic stress prevented the switch to GABAergic excitation. Finally, intravenous injection of the GABA(A) receptor antagonist bicuculline lowered the plasma levels of AVP and oxytocin in rats under the chronic hyperosmotic stress. We conclude that the GABAergic responses of MNCs switch between inhibition and excitation in response to physiological needs through the regulation of transmembrane Cl(-) gradients.

Sildenafil reduces polyuria in rats with lithium-induced NDI.

Lithium (Li)-treated patients often develop urinary concentrating defect and polyuria, a condition known as nephrogenic diabetes insipidus (NDI). In a rat model of Li-induced NDI, we studied the effect that sildenafil (Sil), a phosphodiesterase 5 (PDE5) inhibitor, has on renal expression of aquaporin-2 (AQP2), urea transporter UT-A1, Na(+)/H(+) exchanger 3 (NHE3), Na(+)-K(+)-2Cl(-) cotransporter (NKCC2), epithelial Na channel (ENaC; α-, ß-, and γ-subunits), endothelial nitric oxide synthase (eNOS), and inducible nitric oxide synthase. We also evaluated cGMP levels in medullary collecting duct cells in suspension. For 4 wk, Wistar rats received Li (40 mmol/kg food) or no treatment (control), some receiving, in weeks 2-4, Sil (200 mg/kg food) or Li and Sil (Li+Sil). In Li+Sil rats, urine output and free water clearance were markedly lower, whereas urinary osmolality was higher, than in Li rats. The cGMP levels in the suspensions of medullary collecting duct cells were markedly higher in the Li+Sil and Sil groups than in the control and Li groups. Semiquantitative immunoblotting revealed the following: in Li+Sil rats, AQP2 expression was partially normalized, whereas that of UT-A1, γ-ENaC, and eNOS was completely normalized; and expression of NKCC2 and NHE3 was significantly higher in Li rats than in controls. Inulin clearance was normal in all groups. Mean arterial pressure and plasma arginine vasopressin did not differ among the groups. Sil completely reversed the Li-induced increase in renal vascular resistance. We conclude that, in experimental Li-induced NDI, Sil reduces polyuria, increases urinary osmolality, and decreases free water clearance via upregulation of renal AQP2 and UT-A1.

Molecular and functional expression of cation-chloride cotransporters in dorsal root ganglion neurons during postnatal maturation.

GABA depolarizes and excites central neurons during early development, becoming inhibitory and hyperpolarizing with maturation. This "developmental shift" occurs abruptly, reflecting a decrease in intracellular Cl(-) concentration ([Cl(-)](i)) and a hyperpolarizing shift in Cl(-) equilibrium potential due to upregulation of the K(+)-Cl(-) cotransporter KCC2b, a neuron-specific Cl(-) extruder. In contrast, primary afferent neurons (PANs) are depolarized by GABA throughout adulthood because of expression of NKCC1, a Na(+)-K(+)-2Cl(-) cotransporter that accumulates Cl(-) above equilibrium. The GABA(A)-mediated depolarization of PANs determines presynaptic inhibition in the spinal cord, a key mechanism gating somatosensory information. Little is known about developmental changes in Cl(-) transporter expression and Cl(-) homeostasis in PANs. Whether NKCC1 is expressed in PANs of all phenotypes or is restricted to subpopulations (e.g., nociceptors) is debatable. Likewise, whether PANs express KCC2s is controversial. We investigated NKCC1 and K(+)-Cl(-) cotransporter expression in rat and mouse dorsal root ganglion (DRG) neurons with molecular methods. Using fluorescence imaging microscopy, we measured [Cl(-)](i) in acutely dissociated rat DRG neurons (P0-P21) loaded with N-(ethoxycarbonylmethyl)-6-methoxyquinolinium bromide and classified with phenotypic markers. DRG neurons of all sizes express two NKCC1 mRNAs, one full-length and a shorter splice variant lacking exon 21. Immunolabeling with validated antibodies revealed ubiquitous expression of NKCC1 in DRG neurons irrespective of postnatal age and phenotype. As maturation progresses [Cl(-)](i) decreases gradually, persisting above equilibrium in >95% mature neurons. DRG neurons express mRNAs for KCC1, KCC3s, and KCC4, but not for KCC2s. Mechanisms underlying PANs' developmental changes in Cl(-) homeostasis are discussed and compared with those of central neurons.

Increased aquaporin-1 and Na+ -K+ -2Cl- cotransporter 1 expression in choroid plexus leads to blood-cerebrospinal fluid barrier disruption and necrosis of hippocampal CA1 cells in acute rat models of hyponatremia.

Hyponatremia is a metabolic disorder characterized by increased cerebrospinal fluid (CSF) volume and pressure, although the site of brain insult is unclear. Specifically, the hippocampus, which is in direct contact with expanding CSF ventricles, may be involved. The present study was undertaken to investigate the possible roles of choroid plexus aquaporin-1 (AQP1) and of cation chloride transporters (Na(+) -K(+) -2Cl(-) cotransporter 1 [NKCC1] and K(+) -Cl(-) cotransporter 4 [KCC4]) in the underlying hippocampal pathophysiology of hyponatremia in acute (6 and 12 hr duration) experimental models. It was found that the expressions of AQP1 and NKCC1 proteins in choroid plexus were significantly increased, whereas the expression of KCC4 protein was unchanged vs. control values after 6 and 12 hr of hyponatremia. Choroid plexuses with increased AQP1 and NKCC1 after 6 hr of hyponatremia showed caspase 3-dependent apoptosis and disruption of the blood-CSF barrier. Furthermore, necrotic changes in CA1 neuronal cells were observed after 6 and 12 hr of hyponatremia. Overall, these data suggest that increases in AQP1 and NKCC1 expression under hyposmotic stress may be one of the molecular mechanisms underlying the pathophysiology of acute hyponatremia, such as the necrotic cell death of hippocampal CA1 region by increasing water transport across the blood-CSF barrier. Furthermore, we suggest that opening of the blood-CSF barrier after acute hyponatremia may be triggered the secondary adverse conditions that are capable of enhancing selective necrosis in hippocampal CA1 cells.

The secretory KCa1.1 channel localises to crypts of distal mouse colon: functional and molecular evidence.

The colonic epithelium absorbs and secretes electrolytes and water. Ion and water absorption occurs primarily in surface cells, whereas crypt cells perform secretion. Ion transport in distal colon is regulated by aldosterone, which stimulates both Na(+) absorption and K(+) secretion. The electrogenic Na(+) absorption is mediated by epithelial Na(+) channel (ENaC) in surface cells. Previously, we identified the large conductance Ca(2+)-activated K(+) channel, K(Ca)1.1 or big potassium (BK) channel, as the only relevant K(+) secretory pathway in mouse distal colon. The exact localisation of K(Ca)1.1 channels along the crypt axis is, however, still controversial. The aim of this project was to further define the localisation of the K(Ca)1.1 channel in mouse distal colonic epithelium. Through quantification of mRNA extracted from micro-dissected surface and crypt cells, we confirmed that Na(+)/K(+)/2Cl(-) (NKCC1) is expressed primarily in the crypts and γ-ENaC primarily in the surface cells. The K(Ca)1.1 α-subunit mRNA was like NKCC1, mainly expressed in the crypts. The crypt to surface expression pattern of the channels and transporters was not altered when plasma aldosterone was elevated. The mRNA levels for NKCC1, γ-ENaC and K(Ca)1.1 α-subunit were, however, under these circumstances substantially augmented (K(Ca)1.1 α-subunit, twofold; NKCC1, twofold and ENaC, tenfold). Functionally, we show that ENaC-mediated Na(+) absorption and BK channel-mediated K(+) secretion are two independent processes. These findings show that K(Ca)1.1-mediated K(+) secretion mainly occurs in the crypts of the murine distal colon. This is in agreement with the general model of ion secretion being preferentially located to the crypt and not surface enterocytes.

Down-regulation of Kir4.1 in the cerebral cortex of rats with liver failure and in cultured astrocytes treated with glutamine: Implications for astrocytic dysfunction in hepatic encephalopathy.

Brain edema in acute hepatic encephalopathy (HE) is due mainly to swelling of astrocytes. Efflux of potassium is implicated in the prevention of glial swelling under hypoosmotic conditions. We investigated whether pathogenic factors of HE, glutamine (Gln) and/or ammonia, induce alterations in the expression of glial potassium channels (Kir4.1, Kir2.1) and Na(+) -K(+) -2Cl(-) cotransporter-1 (NKCC1) in rat cerebral cortex and cultured rat cortical astrocytes and whether these alterations have consequences for potassium efflux and astrocytic swelling. Thioacetamide-induced acute liver failure in rats resulted in significant decreases in the Kir4.1 mRNA and protein contents of cerebral cortex, whereas expression of Kir2.1 and NKCC1 remained unaltered. Incubation of primary cortical astrocytes for 72 hr in the presence of Gln (5 mM), but not of ammonia (5 mM or 10 mM), induced a decrease in the levels of Kir4.1 mRNA and protein. Similarly to incubation with Gln, reduction of Kir4.1 mRNA expression by RNA interference caused swelling of astrocytes as shown by confocal imaging followed by 3D computational analysis. Gln reduced the astrocytic uptake of D-[(3) H]aspartate, but, in contrast to the earlier reported effect of ammonia, this reduction was not accompanied by decreased expression of the astrocytic glutamate transporter GLT-1 mRNA. Both Gln and ammonia decreased hypoosmolarity-induced (86) Rb efflux from the cells, but the effect was more pronounced with Gln. The results indicate that down-regulation of Kir4.1 may mediate distinct aspects of Gln-induced astrocytic dysfunction in HE.

Dynamic gene expression of GH/PRL-family hormone receptors in gill and kidney during freshwater-acclimation of Mozambique tilapia.

In teleosts, prolactin (PRL) and growth hormone (GH) act at key osmoregulatory tissues to regulate hydromineral balance. This study was aimed at characterizing patterns of expression for genes encoding receptors for the GH/PRL-family of hormones in the gill and kidney of Mozambique tilapia (Oreochromis mossambicus) during freshwater (FW)-acclimation. Transfer of seawater (SW)-acclimated tilapia to FW elicited rapid and sustained increases in plasma levels and pituitary gene expression of PRL177 and PRL188; plasma hormone and pituitary mRNA levels of GH were unchanged. In the gill, PRL receptor 1 (PRLR1) mRNA increased markedly after transfer to FW by 6h, while increases in GH receptor (GHR) mRNA were observed 48 h and 14 d after the transfer. By contrast, neither PRLR2 nor the somatolactin receptor (SLR) was responsive to FW transfer. Paralleling these endocrine responses were marked increases in branchial gene expression of a Na+/Cl- cotransporter and a Na+/H+ exchanger, indicators of FW-type mitochondrion-rich cells (MRCs), at 24 and 48 h after FW transfer, respectively. Expression of Na+/K+/2Cl- cotransporter, an indicator of SW-type MRCs, was sharply down-regulated by 6h after transfer to FW. In kidney, PRLR1, PRLR2 and SLR mRNA levels were unchanged, while GHR mRNA was up-regulated from 6h after FW transfer to all points thereafter. Collectively, these results suggest that the modulation of the gene expression for PRL and GH receptors in osmoregulatory tissues represents an important aspect of FW-acclimation of tilapia.

Possible involvement of DNA methylation in NKCC1 gene expression during postnatal development and in response to ischemia.

In CNS, GABA(A) receptor-mediated responses switch from depolarization to hyperpolarization during postnatal development. This switch is mediated by developmental down-regulation of inwardly directed Na(+)-K(+)-2Cl(-) co-transporter type 1 (NKCC1) and up-regulation of outwardly directed K(+)-Cl(-) co-transporter type 2. While several factors have been shown to regulate K(+)-Cl(-) co-transporter type 2 expression, little is known about the mechanisms by which the expression of NKCC1 is regulated during postnatal development. Here, we report a novel epigenetic mechanism underlying the developmental regulation of NKCC1 gene expression in the rat cerebral cortex. In vitro DNA methylation of the NKCC1 promoter region, which contains a high density of cytosine-phosphodiester-guanine islands, significantly decreased the expression of NKCC1 mRNA, and the degree of methylation of the NKCC1 promoter region significantly increased during postnatal development. In addition, treatment with 5-aza-2'-deoxycytidine, a specific DNA methyltransferase inhibitor, elicited an increase in the expression of NKCC1 mRNA and protein in cortical slice cultures. Focal ischemic injury induced by the occlusion of the middle cerebral artery led to the re-expression of NKCC1 mRNA and protein even in the mature rat cortex. The re-expression of NKCC1 mRNA and protein in the injured cerebral cortex was related to a decrease in the methylation status of the NKCC1 promoter region. Our results indicate that epigenetic mechanisms, such as DNA methylation, might be involved in the regulation of NKCC1 gene expression during postnatal development as well as under pathological conditions.

Increased A-to-I RNA editing of the transcript for GABAA receptor subunit α3 during chick retinal development.

Adenosine-to-inosine (A-to-I) RNA editing is a cotranscriptional or posttranscriptional gene regulatory mechanism that increases the diversity of the proteome in the nervous system. Recently, the transcript for GABA type A receptor subunit α3 was found to be subjected to RNA editing. The aim of this study was to determine if editing of the chicken α3 subunit transcript occurs in the retina and if the editing is temporally regulated during development. We also raised the question if editing of the α3 transcript was temporally associated with the suggested developmental shift from excitation to inhibition in the GABA system. The editing frequency was studied by using Sanger and Pyrosequencing, and to monitor the temporal aspects, we studied the messenger RNA expression of the GABAA receptor subunits and chloride pumps, known to be involved in the switch. The results showed that the chick α3 subunit was subjected to RNA editing, and its expression was restricted to cells in the inner nuclear and ganglion cell layer in the retina. The extent of editing increased during development (after embryonic days 8-9) concomitantly with an increase of expression of the chloride pump KCC2. Expression of several GABAA receptor subunits known to mediate synaptic GABA actions was upregulated at this time. We conclude that editing of the chick GABAA subunit α3 transcript in chick retina gives rise to an amino acid change that may be of importance in the switch from excitatory to inhibitory receptors.

Surface expression of sodium channels and transporters in rat kidney: effects of dietary sodium.

The abundance of Na transport proteins in the luminal membrane of the rat kidney was assessed using in situ biotinylation and immunoblotting. When animals were fed an Na-deficient diet for 1 wk, the amounts of epithelial Na channel (ENaC) beta-subunit (beta-ENaC) and gamma-subunit (gamma-ENaC) and Na-Cl cotransporter (NCC) protein in the surface fraction increased relative to controls by 1.9-, 3.5-, and 1.5-fold, respectively. The amounts of the luminal Na/H exchanger (NHE3) and the luminal Na-K-2Cl cotransporter (NKCC2) did not change significantly. The increases in ENaC subunits were mimicked by administration of aldosterone for 1 wk, but the increase in NCC was not. When the animals were fed a high-Na (5% NaCl) diet for 1 wk, the surface expression of beta-ENaC increased by 50%, whereas that of the other membrane proteins did not change, relative to controls. The biochemical parameter most strongly affected by dietary Na was the abundance of the 65-kDa cleaved form of gamma-ENaC at the surface. This increased by 8.5-fold with Na depletion and decreased by 40% with Na loading. The overall 14-fold change reflected regulation of the total abundance of the subunit as well as the fraction of the subunit protein in the cleaved form. We conclude that cleavage of gamma-ENaC and its expression at the apical surface play a major role in the regulation of renal Na reabsorption.

Unilateral ureteral obstruction alters expression of acid-base transporters in rat kidney.

PURPOSE: Unilateral ureteral obstruction is a common clinical problem that is often associated with a urinary acidification defect caused by decreased net H(+) secretion and/or HCO(3)(-) reabsorption. To clarify the molecular mechanisms of these defects we examined expression levels of key acid-base transporters along the renal nephron segments and collecting duct. MATERIALS AND METHODS: Wistar rats (Møllegard Breeding Centre, Eiby, Denmark) underwent 24-hour unilateral ureteral obstruction, unilateral ureteral obstruction release followed for 4 days or unilateral ureteral obstruction release followed for 4 days plus experimental acidosis induced by NH(4)Cl oral administration. After sacrifice kidneys were processed for immunoblotting and immunohistochemistry. RESULTS: Semiquantitative immunoblotting revealed that unilateral ureteral obstruction caused significant mean +/- SE down-regulation of type 3 Na(+)/H(+) exchanger to 53% +/- 9%, electrogenic Na(+)/HCO(3)(-) cotransporter to 60% +/- 9%, type 1 bumetanide sensitive Na(+)-K(+)(NH(4)(+)) -2Cl(-) cotransporter to 64% +/- 7%, electroneutral Na(+)/HCO(3)(-) cotransporter to 43% +/- 4% and anion exchanger (pendrin) to 53% +/- 10% in the obstructed kidney, which was confirmed by immunohistochemistry. After release of unilateral ureteral obstruction down-regulation of these transporters persisted together with marked down-regulation of H(+)-adenosine triphosphatase in the obstructed kidney. In rats with unilateral ureteral obstruction release followed for 4 days with experimental acidosis induced by NH(4)Cl oral administration plasma pH and HCO(3)(-) were dramatically decreased in response to NH(4)Cl for 2 days compared with those in sham operated rats with acid loading, indicating a defect in H(+) excretion and HCO(3)(-) reabsorption after obstruction release. Expression of these transporters did not change in the contralateral nonobstructed kidney of rats with unilateral ureteral obstruction and unilateral ureteral obstruction release followed for 4 days. CONCLUSIONS: The expression of renal acid-base transporters is markedly decreased in the obstructed kidney, which may be responsible for the contribution of impaired renal H(+) excretion and HCO(3)(-) reabsorption to the urinary acidification defect in response to unilateral ureteral obstruction.

Kinetic properties of Cl uptake mediated by Na+-dependent K+-2Cl cotransport in immature rat neocortical neurons.

GABA, the main inhibitory neurotransmitter in the adult nervous system, evokes depolarizing membrane responses in immature neurons, which are crucial for the generation of early network activity. Although it is well accepted that depolarizing GABA actions are caused by an elevated intracellular Cl- concentration ([Cl-]i), the mechanisms of Cl- accumulation in immature neurons are still a matter of debate. Using patch-clamp, microfluorimetric, immunohistochemical, and molecular biological approaches, we studied the mechanism of Cl- uptake in Cajal-Retzius (CR) cells of immature [postnatal day 0 (P0) to P3] rat neocortex. Gramicidin-perforated patch-clamp and 6-methoxy-N-ethylquinolinium-microfluorimetric measurements revealed a steady-state [Cl-]i of approximately 30 mM that was reduced to values close to passive distribution by bumetanide or Na+-free solutions, suggesting a participation of Na+-K+-2Cl- cotransport isoform 1 (NKCC1) in maintaining elevated [Cl-]i. Expression of NKCC1 was found in CR cells on the mRNA and protein levels. To determine the contribution of NKCC1 to [Cl-]i homeostasis in detail, Cl- uptake rates were analyzed after artificial [Cl-]i depletion. Active Cl- uptake was relatively slow (47.2 +/- 5.0 microM/s) and was abolished by bumetanide or Na+-free solution. Accordingly, whole-cell patch-clamp recordings revealed a low Cl- conductance in CR cells. The low capacity of NKCC1-mediated Cl- uptake was sufficient to maintain excitatory GABAergic membrane responses, however, only at low stimulation frequencies. In summary, our results demonstrate that NKCC1 is abundant in CR cells of immature rat neocortex and that the slow Cl- uptake mediated by this transporter is sufficient to maintain high [Cl-]i required to render GABA responses excitatory.

Endoplasmic reticulum Ca2+ dysregulation and endoplasmic reticulum stress following in vitro neuronal ischemia: role of Na+-K+-Cl- cotransporter.

We investigated the role of Na(+)-K(+)-Cl(-) cotransporter (NKCC1) in conjunction with Na(+)/Ca(2+) exchanger (NCX) in disruption of endoplasmic reticulum (ER) Ca(2+) homeostasis and ER stress development in primary cortical neurons following in vitro ischemia. Oxygen-glucose deprivation (OGD) and reoxygenation (REOX) caused a rise in [Na(+)](cyt) which was accompanied by an elevation in [Ca(2+)](cyt). Inhibition of NKCC1 with its potent inhibitor bumetanide abolished the OGD/REOX-induced rise in [Na(+)](cyt) and [Ca(2+)](cyt). Moreover, OGD significantly increased Ca(2+)(ER) accumulation. Following REOX, a biphasic change in Ca(2+)(ER) occurred with an initial release of Ca(2+)(ER) which was sensitive to inositol 1,4,5-trisphosphate receptor (IP(3)R) inhibition and a subsequent refilling of Ca(2+)(ER) stores. Inhibition of NKCC1 activity with its inhibitor or genetic ablation prevented the release of Ca(2+)(ER). A similar result was obtained with inhibition of reversed mode operation of NCX (NCX(rev)). OGD/REOX also triggered a transient increase of glucose regulated protein 78 (GRP78), phospho-form of the alpha subunit of eukaryotic initiation factor 2 (p-eIF2alpha), and cleaved caspase 12 proteins. Pre-treatment of neurons with NKCC1 inhibitor bumetanide inhibited upregulation of GRP78 and attenuated the level of cleaved caspase 12 and p-eIF2alpha. Inhibition of NKCC1 reduced cytochrome C release and neuronal death. Taken together, these results suggest that NKCC1 and NCX(rev) may be involved in ischemic cell damage in part via disrupting ER Ca(2+) homeostasis and ER function.

Roles of the cation-chloride cotransporters in neurological disease.

In the nervous system, the intracellular chloride concentration ([Cl(-)](i)) determines the strength and polarity of gamma-aminobutyric acid (GABA)-mediated neurotransmission. [Cl(-)](i) is determined, in part, by the activities of the SLC12 cation-chloride cotransporters (CCCs). These transporters include the Na-K-2Cl cotransporter NKCC1, which mediates chloride influx, and various K-Cl cotransporters--such as KCC2 and KCC3-that extrude chloride. A precise balance between NKCC1 and KCC2 activity is necessary for inhibitory GABAergic signaling in the adult CNS, and for excitatory GABAergic signaling in the developing CNS and the adult PNS. Altered chloride homeostasis, resulting from mutation or dysfunction of NKCC1 and/or KCC2, causes neuronal hypoexcitability or hyperexcitability; such derangements have been implicated in the pathogenesis of seizures and neuropathic pain. [Cl(-)](i) is also regulated to maintain normal cell volume. Dysfunction of NKCC1 or of swelling-activated K-Cl cotransporters has been implicated in the damaging secondary effects of cerebral edema after ischemic and traumatic brain injury, as well as in swelling-related neurodegeneration. CCCs represent attractive therapeutic targets in neurological disorders the pathogenesis of which involves deranged cellular chloride homoestasis.

Chronic noradrenaline increases renal expression of NHE-3, NBC-1, BSC-1 and aquaporin-2.

1. Because chronic activation of the renal sympathetic nervous system promotes sodium and water retention, it is conceivable that long-term exposure of the kidney to the sympathetic neurotransmitter noradrenaline upregulates the expression of key renal epithelial transport systems. 2. To test this hypothesis, we used immunoblotting of renal cortical and medullary tissue to investigate the abundance of major transport systems expressed along the renal tubule in response to long-term (15 days) infusions of noradrenaline (600 ng/min) in rats. 3. Mean arterial blood pressure and heart rate were significantly elevated in rats receiving chronic infusions of noradrenaline (128 +/- 10 mmHg and 492 +/- 16 b.p.m., respectively) compared with animals treated with saline only (89 +/- 3 mmHg and 376 +/- 14 b.p.m., respectively). 4. Chronic infusions of noradrenaline also increased the protein abundance of the cortical Na(+)/H(+) exchanger isoform 3 (NHE-3; 2.5-fold; P = 0.0142), the cortical sodium-bicarbonate cotransporter NBC-1 (2.5-fold; P = 0.0067), the bumetanide-sensitive sodium-potassium-chloride cotransporter BSC-1/NKCC2 in the inner stripe of outer medulla (threefold; P = 0.0020) and aquaporin-2 in the inner medulla (twofold; P = 0.0039). 5. In contrast, noradrenaline did not significantly affect expression of the thiazide-sensitive Na(+)-Cl(-) cotransporter in the cortex, Na(+)/K(+)-ATPase-alpha(1) in the cortex and inner stripe of the outer or inner medulla, the inwardly rectifying K(+) channel (ROMK-1) in the inner stripe of the outer medulla or aquaporin-1 in the cortex or inner medulla. Noradrenaline did significantly, but modestly (less than twofold), increase aquaporin-1 in the inner stripe of the outer medulla. 6. We conclude that noradrenaline-induced increases in the expression of NHE-3, NBC-1, BSC-1 and aquaporin-2 are likely to play an important role in the regulation of salt and water transport by noradrenaline in the kidney and may explain, at least in part, the altered renal sodium and water handling associated with overactivation of the sympathetic system.

Differential expression of absorptive cation-chloride-cotransporters in the intestinal and renal tissues of the European eel (Anguilla anguilla).

Duplicate pairs of isoforms of each of the NKCC2 and the NCC absorptive cation-chloride-cotransporters have been isolated from the European eel. As with mammalian NKCC2, NKCC2alpha isoform mRNA expression was restricted to renal tissues, whereas NKCC2beta isoform expression was present in intestine and urinary bladder. Similar to mammalian NCC, NCCalpha mRNA expression was also found in the kidney, whereas, expression of NCCbeta mRNA was found at low levels in a number of tissues but particularly in intestine. Following 3 weeks of transfer of yellow or silver (adult life stages) eels from freshwater (FW) to seawater (SW), renal mRNA expression of NKCC2alpha did not change whereas NCCalpha expression was reduced although only significantly in silver eels. This suggests that any changes in renal sodium chloride re-absorption in SW-acclimated fish may be due to decreased NCCalpha cotransporter activity rather than the result of suppression of NKCCalpha cotransporter activity. Intestinal mRNA expression of NKCC2beta generally increased following SW acclimation, although maximal increases occurred later in yellow (7 days) than silver (2 days) eels. Average levels of NKCC2beta mRNA abundance in the middle intestine were 89% of those in the anterior, and this was reduced to 44% (of the level in the anterior intestine) in posterior intestine/rectum. Expression of NCCbeta was only found in the posterior intestine/rectum. Together these results suggest intestinal sodium chloride absorption may switch from occurring via NKCCbeta to NCCbeta as imbibed fluid travels down the intestine and the concentration of luminal potassium decreases.

Reversal of renal tubule transporter downregulation during severe leptospirosis with antimicrobial therapy.

Tubular dysfunction is a hallmark of severe leptospirosis. Antimicrobial therapy is thought to interfere on renal involvement. We evaluated the expression of a proximal tubule type-3 Na+/H+ exchanger (NHE3) and a thick ascending limb Na+-K+-2Cl(-) cotransporter (NKCC2) in controls and treated hamsters. Animals infected by a serovar Copenhageni isolate, were treated or not with ampicillin (AMP) and/or N-acetylcysteine (NAC). Leptospiral antigen(s) and expression of renal transporters were evaluated by immunohistochemistry, and serum thiobarbituric acid (TBARS) was quantified. Infected hamsters had high amounts of detectable leptospiral antigen(s) in target tissues while renal expression of NHE3 and NKCC2 decreased. Ampicillin treatment was associated with minimal or no detection of leptospiral antigens, normal expression of NHE3 and NKCC2 transporters, and reduced levels of TBARS. NAC effect was restricted to lowering TBARS. Early and late AMP treatment rescued tubular defects in severe leptospirosis disease, and there was no evidence of benefit from antioxidant therapy.