Altered expression of Na(+)/K(+)-ATPase and other osmoregulatory genes in the gills of euryhaline animals in response to salinity transfer: a meta-analysis of 59 quantitative PCR studies over 10 years.

Recent advances in molecular techniques have allowed gene expression in euryhaline animals to be quantified during salinity transfers. As these investigations transition from studying single genes to utilizing genomics-based methodologies, it is an appropriate time to summarize single gene studies. Therefore, a meta-analysis was performed on 59 published studies that used quantitative polymerase chain reaction (qPCR) to examine expression of osmoregulatory genes (the Na(+)/K(+)-ATPase, NKA; the Na(+)/K(+)/2Cl(-) cotransporter, NKCC; carbonic anhydrase, CA; the cystic fibrosis transmembrane regulator, CFTR; and the H(+)-ATPase, HAT) in response to salinity transfer. Based on 887 calculated effect sizes, NKA, NKCC, CA, and HAT are up-regulated after salinity transfer, while surprisingly, CFTR is unchanged. Meta-analysis also identified influential factors contributing to these changes. For example, expression was highest: 1) during transfers from higher to lower salinities comprising a physiological transition from osmoconformity to osmoregulation, 2) 1-3 days following transfer, 3) during dissimilar transfers, and 4) in crustaceans rather than teleosts. Methodological characteristics (e.g., types of controls) were not important. Experiments lacking in the current literature were also identified. Meta-analyses are powerful tools for quantitatively synthesizing a large body of literature, and this report serves as a template for their application in other areas of comparative physiology.

Na-K-2Cl cotransporter and aquaporin 1 in arachnoid granulations, meningiomas, and meningiomas invading dura.

Meningioma invasion of the dura may contribute to the high rate of recurrence. Recently, ion channels that affect cell shape and movement have been implicated in cancer invasion. Combined Na-K-2Cl cotransporter (NKCC1) and aquaporin 1 (AQP1) expression in arachnoid granulations and meningiomas with and without dural invasion has not been characterized. Arachnoid granulations associated with dura were collected from 10 adult formalin-fixed dura/leptomeninges. Thirty-four frozen meningiomas were evaluated by Western blot. An additional 58 formalin-fixed, paraffin-embedded meningiomas including 36 World Health Organization grade I, 15 grade II, and 7 grade III meningiomas were evaluated by immunohistochemistry. By Western blot, NKCC1 was found in 17 (100%) of 17 World Health Organization grade I, 13 (87%) of 15 grade II, and both grade III meningiomas. Distinct AQP1 was not detected in the meningioma lysates tested. By immunohistochemistry, extensive NKCC1 but no AQP1 immunoreactivity was detected in the arachnoid granulation cells. Extensive NKCC1 was detected in meningioma cells in 56 and in capillaries in 43 by immunohistochemistry. In those tumors with dural or bone/soft tissue invasion, NKCC1 immunoreactivity was seen in invading cells in all cases and in their capillaries in the majority. AQP1 was detected in meningioma cells in 29 and in capillaries in all. AQP1 was also detected in cells and capillaries invading the dura or bone in 10 and 18 of 18, respectively. This was extensive in all subtypes of meningiomas studied. These findings suggest that NKCC1 and AQP1 participate in meningioma biology and invasion. NKCC1 might be targeted by FDA-approved NKCC1 inhibitors.

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.

Expression of the Na+-K+-2Cl(-)-cotransporter 2 in the normal and pressure-induced ischemic rat retina.

PURPOSE: To evaluate the expression of the Na(+)-K(+)-2Cl(-)-cotransporter 2 (NKCC2) in the ischemic rat retina. METHODS: Retinal ischemia was induced by pressures 90 to 120 mmHg, above systemic systolic pressure. Immunohistochemistry and western blot analysis were performed. RESULTS: NKCC2 is expressed in the normal retina and its expression is increased by ischemia caused by intraocular pressure elevation. NKCC2 immunoreactivity was observed mainly in axon bundles of ganglion cells and horizontal cell processes in the retina. NKCC2 expression continuously increased with a peak value 3 days (to 415% of normal levels) after ischemic injury, and then gradually decreased to 314% of controls until 2 weeks post injury. The mean density of NKCC2-labeled ganglion cells per mm(2) changed from 1,255 ± 109 in normal retinas to 391 ± 49 and 185 ± 37 at 3 days and 2 weeks after ischemia, respectively (p < 0.05), implying cell death of ganglion cells labeled with NKCC2. CONCLUSIONS: Taken together, these results suggest that NKCC2, which is expressed in retinal ganglion and horizontal cells, may contribute to cell death by ischemic injury in the retina, although the molecular mechanisms involved remain to be clarified.

Effects of third trimester-equivalent ethanol exposure on Cl(-) co-transporter expression, network activity, and GABAergic transmission in the CA3 hippocampal region of neonatal rats.

Fetal alcohol spectrum disorders are often associated with structural and functional hippocampal abnormalities, leading to long-lasting learning and memory deficits. The mechanisms underlying these abnormalities are not fully understood. Here, we investigated whether ethanol exposure during the 3rd trimester-equivalent period alters spontaneous network activity that is involved in neuronal circuit development in the CA3 hippocampal region. This activity is driven by GABA(A) receptors, which can have excitatory actions in developing neurons as a consequence of greater expression of the Cl(-) importer, NKCC1, with respect to expression of the Cl(-) exporter, KCC2, resulting in high [Cl(-)](i). Rat pups were exposed to ethanol vapor from postnatal day (P) 2-16 (4 h/day). Weight gain was significantly reduced in pups exposed to ethanol compared to control at P15 and 16. Brain slices were prepared immediately after the end of the 4-h exposure on P4-16 and experiments were also performed under ethanol-free conditions at the end of the exposure paradigm (P17-22). Ethanol exposure did not significantly affect expression of KCC2 or NKCC1, nor did it affect network activity in the CA3 hippocampal region. Ethanol exposure significantly decreased the frequency (at P9-11) and increased the amplitude (at P5-8 and P17-21) of GABA(A) receptor-mediated miniature postsynaptic currents. These data suggest that repeated in vivo exposure to ethanol during the 3rd trimester-equivalent period alters GABAergic transmission in the CA3 hippocampal region, an effect that could lead to abnormal circuit maturation and perhaps contribute to the pathophysiology of fetal alcohol spectrum disorders.

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.

Postnatal development of Na(+)-K(+)-2Cl(-) co-transporter 1 and K(+)-Cl(-) co-transporter 2 immunoreactivity in multiple brain stem respiratory nuclei of the rat.

Previously, we reported that in rats, GABA(A) and glycine receptor immunoreactivity increased markedly in multiple brain stem respiratory nuclei around postnatal days (P) 12-13, a critical period when abrupt neurochemical, metabolic, ventilatory, and electrophysiological changes occur in the respiratory network and when the system is under greater inhibition than excitation. Since Na(+)-K(+)-2Cl(-) co-transporter 1 (NKCC1) and K(+)-Cl(-) co-transporter 2 (KCC2) play pivotal roles in determining the responses of GABA(A) and glycine receptors, we hypothesized that NKCC1 and KCC2 undergo significant changes during the critical period. An in-depth immunohistochemical and single neuron optical densitometric study of neurons in seven respiratory-related nuclei (the pre-Bötzinger complex [PBC], nucleus ambiguus [Amb], hypoglossal nucleus [XII], ventrolateral subnucleus of solitary tract nucleus [NTS(VL)], retrotrapezoid nucleus/parafacial respiratory group [retrotrapezoid nucleus/parafacial respiratory group (RTN/pFRG)], dorsal motor nucleus of the vagus nerve [dorsal motor nucleus of the vagus nerve (DMNX)], and inferior olivary nucleus [IO]) and a non-respiratory cuneate nucleus (CN, an internal control) was undertaken in P0-P21 rats. Our data revealed that (1) NKCC1 immunoreactivity exhibited a developmental decrease from P0 to P21 in all eight nuclei examined, being relatively high during the first 1½ postnatal weeks and decreased thereafter. The decrease was abrupt and statistically significant at P12 in the PBC, Amb, and XII; (2) KCC2 immunoreactivity in these eight nuclei showed a developmental increase from P0 to P21; and (3) the significant reduction in NKCC1 and the greater dominance of KCC2 around P12 in multiple respiratory nuclei of the brain stem may form the basis of an enhanced inhibition in the respiratory network during the critical period before the system stabilizes to a more mature state.

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.

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.

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.

Expression of the Na(+)-K(+)-2Cl(-)-Cotransporter 2 in the Normal and Pressure-Induced Ischemic Rat Retina

PURPOSE: To evaluate the expression of the Na(+)-K(+)-2Cl(-)-cotransporter 2 (NKCC2) in the ischemic rat retina. METHODS: Retinal ischemia was induced by pressures 90 to 120 mmHg, above systemic systolic pressure. Immunohistochemistry and western blot analysis were performed. RESULTS: NKCC2 is expressed in the normal retina and its expression is increased by ischemia caused by intraocular pressure elevation. NKCC2 immunoreactivity was observed mainly in axon bundles of ganglion cells and horizontal cell processes in the retina. NKCC2 expression continuously increased with a peak value 3 days (to 415% of normal levels) after ischemic injury, and then gradually decreased to 314% of controls until 2 weeks post injury. The mean density of NKCC2-labeled ganglion cells per mm2 changed from 1,255 +/- 109 in normal retinas to 391 +/- 49 and 185 +/- 37 at 3 days and 2 weeks after ischemia, respectively (p < 0.05), implying cell death of ganglion cells labeled with NKCC2. CONCLUSIONS: Taken together, these results suggest that NKCC2, which is expressed in retinal ganglion and horizontal cells, may contribute to cell death by ischemic injury in the retina, although the molecular mechanisms involved remain to be clarified.

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.

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.

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.

KCC2 was downregulated in small neurons localized in epileptogenic human focal cortical dysplasia.

Focal cortical dysplasia (FCD), which is characterized histologically by disorganized cortical lamination and large abnormal cells, is one of the major causes of intractable epilepsies. γ-aminobutyric acid (GABA)(A) receptor-mediated synchronous depolarizing potentials have been observed in FCD tissue. Since alterations in Cl(-) homeostasis might underlie these depolarizing actions of GABA, cation-Cl(-) cotransporters could play critical roles in the generation of these abnormal actions. We examined the expression patterns of NKCC1 and KCC2 by in situ hybridization histochemistry and immunohistochemistry in FCD tissue obtained by surgery from patients with intractable epilepsy. KCC2 mRNA and protein were expressed not only in non-dysplastic neurons in histologically normal portions located in the periphery of the excised cortex, but also in dysplastic cells in FCD tissue. The levels of KCC2 mRNA and protein were significantly decreased in the neurons around large abnormal neurons (giant neurons), but not in giant neurons, compared with non-dysplastic neurons. The neurons localized only around giant neurons significantly smaller than non-dysplastic neurons. However NKCC1 expression did not differ among these cell types. These results suggest that the intracellular Cl(-) concentration ([Cl(-)](i)) of small neurons might increase, so that depolarizing GABA actions could occur in the FCD tissue of epileptic foci.

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.

Simultaneously reduced NKCC1 and Na,K-ATPase expression in murine cochlear lateral wall contribute to conservation of endocochlear potential following a sensorineural hearing loss.

The mechanisms of the response in the murine cochlear lateral wall following sensorineural hearing loss (SNHL) are poorly understood. We focused on comparing the endocochlear potential (EP) with morphological changes in the lateral wall and expression of four important potassium (K(+)) transporters in a mouse model of SNHL induced by co-administration of aminoglycoside and loop diuretic. The expression of the α1 and α2 isoforms of Na,K-ATPase, Na-K-2Cl-Cotransporter-1 (NKCC1) and potassium channel KCNQ1 was assessed. The EP showed a significant decline at 12h post-treatment followed by complete recovery by 2 days post-treatment. The EP was maintained at near normal levels in animals deafened for periods up to 112 days. Despite this recovery, there was a significant and progressive decrease in the thickness of the stria vascularis, which was predominantly due to atrophy of marginal cells. Both protein and mRNA expression of α1 and α2 isoforms of Na,K-ATPase and NKCC1 in the lateral wall were dramatically reduced following a long-term deafening. KCNQ1 expression remained unchanged. These observations provide insight into the detailed mechanisms of EP modulation following SNHL and may have crucial implications in the future treatment of aminoglycoside-induced hearing loss.