86Rb Influx Research Articles

Vasoconstriction triggered by hydrogen sulfide: Evidence for Na+,K+,2Cl-cotransport and L-type Ca2+ channel-mediated pathway

ObjectivesThis study examined the dose-dependent actions of hydrogen sulfide donor sodium hydrosulphide (NaHS) on isometric contractions and ion transport in rat aorta smooth muscle cells (SMC). MethodsIsometric contraction was measured in ring aortas segments from male Wistar rats. Activity of Na+/K+-pump and Na+,K+,2Cl-cotransport was measured in cultured endothelial and smooth muscle cells from the rat aorta as ouabain-sensitive and ouabain-resistant, bumetanide-sensitive components of the 86Rb influx, respectively. ResultsNaHS exhibited the bimodal action on contractions triggered by modest depolarization ([K+]o=30mM). At 10−4 M, NaHS augmented contractions of intact and endothelium-denuded strips by ~ 15% and 25%, respectively, whereas at concentration of 10−3M it decreased contractile responses by more than two-fold. Contractions evoked by 10−4M NaHS were completely abolished by bumetanide, a potent inhibitor of Na+,K+,2Cl-cotransport, whereas the inhibition seen at 10−3M NaHS was suppressed in the presence of K+ channel blocker TEA. In cultured SMC, 5×10−5M NaHS increased Na+,K+,2Cl- - cotransport without any effect on the activity of this carrier in endothelial cells. In depolarized SMC, 45Ca influx was enhanced in the presence of 10−4M NaHS and suppressed under elevation of [NaHS] up to 10−3M. 45Ca influx triggered by 10−4M NaHS was abolished by bumetanide and L-type Ca2+ channel blocker nicardipine. ConclusionsOur results strongly suggest that contractions of rat aortic rings triggered by low doses of NaHS are mediated by activation of Na+,K+,2Cl-cotransport and Ca2+ influx via L-type channels.

Not all silver nanoparticles are equal: Effect of functionalization on K homeostasis in human red blood cells

Although silver nanoparticles (AgNPs) are one of the most frequently used nanomaterials (NMs) in industrial and biomedical applications, their toxicity and interaction with blood components, such as erythrocytes, has been insufficiently investigated. Here, we report on the effect of AgNPs at the level of cellular function, and particularly on ion transport in human red blood cells (HRBCs). These AgNPs were size‐selected, concentrated and purified by tangential flow filtration before HRBC exposure. Two methods of synthesis were used for the functionalization of AgNPs, Creighton and Lee‐Meisel. HRBCs were obtained from adult donors at a blood bank, expired or fresh or further stored refrigerated in the laboratory for variable lengths of time, and from fresh or stored refrigerated cord blood. Rubidium, used as a K congener to measure K influx, and the K concentration were determined in the presence and absence of several concentrations (0–150 μg/mL) of negatively charged, spherical 10 nm AgNPs at different time points (0–60 min). Our findings indicate that Creighton AgNPs, independently of the source and in a time‐ and dose‐dependent manner, inhibited Rb influx by ~ 80 % (p < 0.001, n=7) and depleted the cells of K by more than 90 % (p < 0.0001, n = 7), although interindividual differences were observed. In comparison to the Creighton, Lee‐Meisel AgNPs caused a maximum Rb inhibition of ~ 50 % and K loss of ~ 15 % with larger interindividual variability than Creighton AgNPs, albeit in a small sample. The loss of K from HRBCs was entirely accounted for by an increase in K concentration in the extracellular medium. These findings indicate that functionalization of AgNPs has a significant effect on HRBC K homeostasis. Further studies are required to identify the mechanisms underlying the observed changes in HRBC ionic composition.Support or Funding InformationFunded by NSF

Effects of Ouabain on Proliferation of Human Endothelial Cells Correlate with Na+,K+-ATPase Activity and Intracellular Ratio of Na+ and K.

Side-by-side with inhibition of the Na+,K+-ATPase ouabain and other cardiotonic steroids (CTS) can affect cell functions by mechanisms other than regulation of the intracellular Na+ and K+ ratio ([Na+]i/[K+]i). Thus, we compared the dose- and time-dependences of the effect of ouabain on intracellular [Na+]i/[K+]i ratio, Na+,K+-ATPase activity, and proliferation of human umbilical vein endothelial cells (HUVEC). Treatment of the cells with 1-3nM ouabain for 24-72h decreased the [Na+]i/[K+]i ratio and increased cell proliferation by 20-50%. We discovered that the same ouabain concentrations increased Na+,K+-ATPase activity by 25-30%, as measured by the rate of (86)Rb(+) influx. Higher ouabain concentrations inhibited Na+,K+-ATPase, increased [Na+]i/[K+]i ratio, suppressed cell growth, and caused cell death. When cells were treated with low ouabain concentrations for 48 or 72h, a negative correlation between [Na+]i/[K+]i ratio and cell growth activation was observed. In cells treated with high ouabain concentrations for 24h, the [Na+]i/[K+]i ratio correlated positively with proliferation inhibition. These data demonstrate that inhibition of HUVEC proliferation at high CTS concentrations correlates with dissipation of the Na+ and K+ concentration gradients, whereas cell growth stimulation by low CTS doses results from activation of Na+,K+-ATPase and decrease in the [Na+]i/[K+]i ratio.

Geochemistry of granitization and magmatic replacement of basic volcanic rocks in the contact aureole of the Yurchik gabbronorite intrusion, Ganal Range, Kamchatka

Geological and geochemical data indicate that the formation of the granulite-like rocks in the contact aureole of the Yurchik gabbronorite intrusion of the Ganal Range, Kamchatka, was caused by the contact metamorphism, metasomatism, and local melting of the initial volcanosedimentary rocks of the Vakhtalka Sequence of the Ganal Group. The temperature in the inner part of the aureole reached 700–800°C and caused the transformation of the basic volcanic rocks of the sequence into two pyroxene-plagioclase, clinopyroxene-amphibole-plagioclase, and amphibole-plagioclase hornfelses, while sedimentary rocks were converted into garnet-biotite ± cordierite hornfelses. The hornfelsed basic volcanic rocks were locally subjected to metasomatic alteration and magmatic replacement with formation of biotite-orthopyroxene-plagioclase metasomatic bodies containing biotite-orthopyroxene-plagioclase ± garnet veinlets and aggregates. During these processes, sedimentary interlayers were converted into garnet enderbites at 700–800°C and 3.2–4.8 kbar. The comparison of the chemical composition of basic volcanic rocks of the Vakhtalka Sequence and their transformation products indicates that the metasomatic alteration and magmatic replacement correspond to siliceous-alkaline metasomatism (granitization) and cause subsequent and uneven influx of SiO2, Al2O3, Na2O, K2O, Rb, Ba, Zr, Nb, and Cl and removal of Fe, Mg, Mn, Ca, Cr, Co, Ti, Y, and S. REE data on basic metavolcanic rocks, hornfelses, and metasomatites suggest that the processes of hornfelsation, metasomatism, and magmatic replacement of the initial volcanic rocks were accompanied by significant increase in LREE and slight decrease in HREE. The Sr and Nd isotope study of the rocks in the aureole showed that the initial basic volcanic rocks of the Vakhtalka Sequence are isotopically close to both mature island arc tholeiites and mid-ocean ridge basalts. The metasomatic alteration and magmatic replacement of volcanic rocks in the aureole lead to the decrease of 143Nd/144Nd and increase of 87Sr/86Sr approximately parallel to mantle array. Pb isotopic ratios in the studied rocks become more radiogenic from initial metavolcanic rocks to metasomatites.

Rb influx in K‐Cl cotransporter 3 (KCC3)‐transfected human embryonic kidney 293 (HEK293) cells and effect of external Na

K‐Cl cotransport (KCC) is encoded by 4 genes (SLC12A4–7) with at least four protein isoforms (KCC1‐KCC4) and electroneutrally extrudes K and Cl under physiological conditions. Major functions of KCC involve cell volume and Cl regulation and thus abnormalities in its activity are associated with numerous pathological conditions. The purpose of this study is to characterize the functional properties of KCC3 in a human cell line. Rb, a K congener, was used to study KCC ± ouabain or bumetanide, inhibitors of other K transport mechanisms i.e. Na/K pump and Na‐K‐2Cl cotransport (NKCC), respectively, ± Cl (sulfamate replacement) ± Na (N‐methyl‐D‐glucamine replacement) in KCC3 transfected HEK293 cells. Rb uptake through KCC was linear up to 10 min whereas Rb uptake through NKCC was not linear even at the earliest time point tested (2.5 min). NKCC was 3 fold >; KCC in Na whereas KCC was about 6 fold >; NKCC in Na‐free medium. NKCC was the largest component of the total Rb flux in Na (Na/K pump 25 %, NKCC 63 %, and KCC 11 %). These findings indicate that KCC3‐transfected HEK293 cells behave like non‐transfected cells from various tissues for KCC, NKCC and Na/K pump activity both in Na or Nafree medium under baseline conditions, thus constituting a promising mammalian experimental model to study the specific role of KCC3 in cell volume regulation and in different signaling pathways. Supported by WSU Foundation and NIH R25

The K‐Cl cotransporter KCC3 is a catalytic switch of cellular K and volume homoestasis

The SLC12A cation cotransporters NKCC1 and KCCs play a crucial role in volume homeostasis, with KCC3 as key mediator of regulatory volume decrease. Dephosphorylation of human KCC3 at T991 & T1048 by protein phosphatase 1is triggered by cell swelling, but its physiological effects have not been systematically explored. By creating isogenic human epithelial cell lines with inducible expression of wild type KCC3 (WT) or a constitutively‐active double alanine KCC3 mutant mimicking the dephosphorylated KCC3 (AA), and measuring KCC3 activity with Rb, a K congener, and cell K (Ki) ± transport inhibitors, we gain insight into the role of KCC3 in cell K and volume homeostasis. In isotonic medium, WT is phosphorylated and of low activity, as assessed by phospho‐specific antibodies against T991 & T1048 and Rb flux. In contrast, induction of AA resulted in a 10 fold greater furosemide‐sensitive (FS) KCC3 activity. In induced AA but not WT cells, Ki loss was >; 90 % in Na and ~ 70 % in Na‐free media, time‐ and external K and Rb concentrations‐dependent and could not be prevented by various Cl, K and Ca channel blockers. In contrast to NKCC and KCC, the induced Ki loss was insensitive to F in the preincubation and flux. Addition of F during induction of AA significantly reduced Ki loss in sulfamate with concomitant inhibition of KCC. Thus, a constitutively active KCC3 promotes an equal FS Ki loss and Rb influx, which may impact certain pathologies. Supported by WSU Foundation and NIH R25.

Low efficiency of functional translation of ouabain-resistant α2-Na+,K+-ATPase mRNA in C7-MDCK epithelial cells

Na(+),K(+)-ATPase is a heterodimer consisting of catalytic α1-α4 and regulatory β1-β3 subunits. Recently, we reported that transfection with ouabain-resistant α1R-Na(+),K(+)-ATPase rescues renal epithelial C7-MDCK cells exclusively expressing the ouabain-sensitive α1S-isoform from the cytotoxic action of ouabain. To explore the role of α2 subunit in ion transport and cytotoxic action of ouabain, we compared the effect of ouabain on K(+) ((86)Rb) influx and the survival of ouabain-treated C7-MDCK cells stably transfected with α1R- and α2R-Na(+),K(+)-ATPase. α2R mRNA in transfected cells was ∼8-fold more abundant than α1R mRNA, whereas immunoreactive α2R protein content was 5-fold lower than endogenous α1S protein. A concentration of 10µmol/L ouabain led to complete inhibition of (86)Rb influx both in mock- and α2R-transfected cells, whereas maximal inhibition of (86)Rb influx in α1R-transfectd cells was observed at 1000µmol/L ouabain. In contrast to the massive death of mock- and α2R-transfected cells exposed to 3µmol/L ouabain , α1R-cells survived after 24h incubation with 1000µmol/L ouabain. Thus, our results show that unlike α1R, the presence of α2R-Na(+),K(+)-ATPase subunit mRNA and immunoreactive protein does not contribute to Na(+)/K(+) pump activity, and does not rescue C7-MDCK cells from the cytotoxic action of ouabain. Our results also suggest that the lack of impact of transfected α2-Na(+),K(+)-ATPase on Na(+)/K(+) pump activity and cell survival can be attributed to the low efficiency of its translation and (or) delivery to the plasma membrane of renal epithelial cells.

Ischemia-induced stimulation of cerebral microvascular endothelial cell Na-K-Cl cotransport involves p38 and JNK MAP kinases.

Previous studies have provided evidence that, in the early hours of ischemic stroke, a luminal membrane blood-brain barrier (BBB) Na-K-Cl cotransporter (NKCC) participates in ischemia-induced cerebral edema formation. Inhibition of BBB NKCC activity by intravenous bumetanide significantly reduces edema and infarct in the rat permanent middle cerebral artery occlusion model of ischemic stroke. We demonstrated previously that the BBB cotransporter is stimulated by hypoxia, aglycemia, and AVP, factors present during cerebral ischemia. However, the underlying mechanisms have not been known. Ischemic conditions have been shown to activate p38 and JNK MAP kinases (MAPKs) in brain, and the p38 and JNK inhibitors SB-239063 and SP-600125, respectively, have been found to reduce brain damage following middle cerebral artery occlusion and subarachnoid hemorrhage, respectively. The present study was conducted to determine whether one or both of these MAPKs participates in ischemic factor stimulation of BBB NKCC activity. Cultured cerebral microvascular endothelial cell NKCC activity was evaluated as bumetanide-sensitive (86)Rb influx. Activities of p38 and JNK were assessed by Western blot and immunofluorescence methods using antibodies that detect total vs. phosphorylated (activated) p38 or JNK. We report that p38 and JNK are present in cultured cerebral microvascular endothelial cells and in BBB endothelial cells in situ and that hypoxia (7% O(2) and 2% O(2)), aglycemia, AVP, and O(2)-glucose deprivation (5- to 120-min exposures) all rapidly activate p38 and JNK in the cells. We also provide evidence that SB-239063 and SP-600125 reduce or abolish ischemic factor stimulation of BBB NKCC activity. These findings support the hypothesis that ischemic factor stimulation of the BBB NKCC involves activation of p38 and JNK MAPKs.

Ischemia-induced stimulation of Na-K-Cl cotransport in cerebral microvascular endothelial cells involves AMP kinase.

Increased blood-brain barrier (BBB) Na-K-Cl cotransporter activity appears to contribute to cerebral edema formation during ischemic stroke. We have shown previously that inhibition of BBB Na-K-Cl cotransporter activity reduces edema and infarct in the rat middle cerebral artery occlusion (MCAO) model of ischemic stroke. We have also shown that the BBB cotransporter is stimulated by the ischemic factors hypoxia, aglycemia, and arginine vasopressin (AVP), although the mechanisms responsible are not well understood. AMP-activated protein kinase (AMPK), a key mediator of cell responses to stress, can be activated by a variety of stresses, including ischemia, hypoxia, and aglycemia. Previous studies have shown that the AMPK inhibitor Compound C significantly reduces infarct in mouse MCAO. The present study was conducted to evaluate the possibility that AMPK participates in ischemic factor-induced stimulation of the BBB Na-K-Cl cotransporter. Cerebral microvascular endothelial cells (CMEC) were assessed for Na-K-Cl cotransporter activity as bumetanide-sensitive (86)Rb influx. AMPK activity was assessed by Western blot analysis and immunofluorescence methods using antibodies that detect total versus phosphorylated (activated) AMPK. We found that hypoxia (7% and 2% O(2)), aglycemia, AVP, and oxygen-glucose deprivation (5- to 120-min exposures) increase activation of AMPK. We also found that Compound C inhibition of AMPK reduces hypoxia-, aglycemia-, and AVP-induced stimulation of CMEC Na-K-Cl cotransporter activity. Confocal immunofluorescence of perfusion-fixed rat brain slices revealed the presence of AMPK, both total and phosphorylated kinase, in BBB in situ of both control and ischemic brain. These findings suggest that ischemic factor stimulation of the BBB Na-K-Cl cotransporter involves activation of AMPK.

Enantioselective Protein-Sterol Interactions Mediate Regulation of Both Prokaryotic and Eukaryotic Inward Rectifier K+ Channels by Cholesterol

Cholesterol is the major sterol component of all mammalian cell plasma membranes and plays a critical role in cell function and growth. Previous studies have shown that cholesterol inhibits inward rectifier K+ (Kir) channels, but have not distinguished whether this is due directly to protein-sterol interactions or indirectly to changes in the physical properties of the lipid bilayer. Using purified bacterial and eukaryotic Kir channels reconstituted into liposomes of controlled lipid composition, we demonstrate by 86Rb+ influx assays that bacterial Kir channels (KirBac1.1 and KirBac3.1) and human Kir2.1 are all inhibited by cholesterol, most likely by locking the channels into prolonged closed states, whereas the enantiomer, ent-cholesterol, does not inhibit these channels. These data indicate that cholesterol regulates Kir channels through direct protein-sterol interactions likely taking advantage of an evolutionarily conserved binding pocket.

K Influx Parameters in Rat Aortic Vascular Smooth Muscle Cells (RASMCs)

Background RASMCs serve as major experimental tool in cardiovascular research. Here we studied further the effect of cell culture passage on K influxes with Rb as congener. Methods Rb uptake was measured by atomic absorption spectrophotometry in RASMCs ± inhibitors of the Na/K pump (ouabain, O), Na-K-2Cl cotransport (NKCC, bumetanide, B), and K-Cl cotransport (KCC, Cl replacement by sulfamate). Rb influx was calculated in nmol/mg protein × time, with O inhibition maximized for NKCC and KCC measurements. RASMC identity was verified immunologically. Results Positive IF anti-αactin staining. Rb uptake nonlinear with time in the absence (total, T) and presence of 1 mM O, and both O and 10 μM B. Rb flux by NKCC {O-insensitive (OIS) – [O+B-insensitive, OBIS]) = > 30% than that by the Na/K pump (T- OIS). Presence of O during the flux alone or flux + preincubation inhibited the Na/K pump, IC50 = 0.14 or 0.17 mM and 45% and 60% of T at 2 mM O, respectively. At 2 mM O, B inhibited 80% of OIS Rb influx with an IC50 = 0.1 μM. OBIS KCC activity (53%) saturated with increasing Cl mol fractions, remainder in 100% sulfamate due to channel Rb influx. KCC Vmax = ~ 1 and 2 nmol/(mg protein × min), and Km = 89 and 59 mM [Cl]o, with Hill n=1, in old vs young RASMCs passages, respectively. Conclusions Na/K pump, NKCC and KCC measurement required inhibitor presence in both preincubation + flux. Apparent Na/K pump O affinity was consistent with the low rodent α1Na/K ATPase isoform O affinity and NKCC B affinity s close to that for renal NKCC2. KCC parameters, except Hill n, were age-dependent, consistent with KCC1, 3 & 4 isoforms present in RASMCs.

Quercetin Stimulates NGF-induced Neurite Outgrowth in PC12 Cells via Activation of Na+/K+/2Cl- Cotransporter

We have recently reported that Na⁺/K⁺/2Cl^- cotransporter isoform 1 (NKCC1) plays an essential role in nerve growth factor (NGF)-induced neurite outgrowth in PC12D cells. On the other hand, it has been reported that dietary flavonoids, such as quercetin, apigenin, and luteolin, stimulate various ion transporters. In the present report, we investigated the effect of quercetin, a flavonoid, on NGF-induced neurite outgrowth in PC12 cells (the parental strain of PC12D cells). Quercetin stimulated the NGF-induced neurite outgrowth in a dose-dependent manner. Knockdown of NKCC1 by RNAi methods abolished the stimulatory effect of flavonoid. Quercetin stimulated NKCC1 activity (measured as bumetanide-sensitive ⁸⁶Rb influx) without any increase in the expression level of NKCC1 protein. The stimulatory effect of quercetin on neurite outgrowth was dependent upon extracellular Cl^-. These observations indicate that quercetin stimulates the NGF-induced neurite outgrowth via an increase in Cl^- incorporation into the intracellular space by activating NKCC1 in PC12 cell.

Decreased NKCC1 Activity in Erythrocytes From African Americans With Hypertension and Dyslipidemia

Recent studies demonstrated a key role of ubiquitous isoform of Na+,K+,2Cl- co-transport (NKCC1) in regulation of myogenic tone and peripheral resistance. We examined the impact of race, gender, and plasma lipid on NKCC1 activity in French Canadians and African Americans with hypertension and dyslipidemia. NKCC and passive erythrocyte membrane permeability to K+, measured as ouabain-resistant, bumetanide-sensitive, and (ouabain+bumetanide)-resistant 86Rb influx, respectively, were compared in 111 French-Canadian men, 107 French-Canadian women, 26 African-American men, and 45 African-American women with essential hypertension and dyslipidemia. The African-American men and women were 7 years younger and presented twofold decreased plasma triglycerides compared to their French-Canadian counterparts (P < 0.01) whereas body mass index (BMI), total cholesterol, low-density lipoprotein, and high-density lipoprotein (HDL) were not different. NKCC was respectively 50 and 38% lower in the African-American men and women than in the French Canadians (P < 0.005) without any differences in passive erythrocyte membrane permeability for K+. We did not observe any impact of age on NKCC in all groups under investigation, whereas plasma triglycerides correlated positively with the activity of this carrier in the French-Canadian men only. NKCC1 activity is lower in erythrocytes of African Americans with essential hypertension and dyslipidemia than in Caucasian counterparts. We suggest that decreased NKCC1 may contribute to the feature of the pathogenesis of salt-sensitive hypertension seen in African Americans.

Cardiotonic steroid-resistant α1-Na+,K+-ATPase rescues renal epithelial cells from the cytotoxic action of ouabain: evidence for a Na i + ,K i + -independent mechanism

Mechanisms underlying the tissue-specific impact of cardiotonic steroids (CTS) on cell survival and death remain poorly understood. This study examines the role of Na(+),K(+)-ATPase alpha subunits in death of Madin-Darby canine kidney (MDCK) cells evoked by 24-h exposure to ouabain. MDCK cells expressing a variant of the alpha1 isoform, CTS-sensitive alpha1S, were stably transfected with a cDNA encoding CTS-resistant alpha1R-Na(+),K(+)-ATPase, whose expression was confirmed by RT-PCR. In mock-transfected and alpha1R-cells, maximal inhibition of (86)Rb influx was observed at 10 and 1000 muM ouabain, respectively, thus confirming high abundance of alpha1R-Na(+),K(+)-ATPase in these cells. Six-hour treatment of alpha1R-cells with 1000 muM ouabain led to the same elevation of the [Na(+)](i)/[K(+)](i) ratio that was detected in mock-transfected cells treated with 3 muM ouabain. However, in contrast to the massive death of mock-transfected cells exposed to 3 muM ouabain, alpha1R-cells survived after 24-h incubation with 1000 muM ouabain. Inversion of the [Na(+)](i)/[K(+)](i) ratio evoked by Na(+),K(+)-ATPase inhibition in K(+)-free medium did not affect survival of alpha1R-cells but increased their sensitivity to ouabain. Our results show that the alpha1R subunit rescues MDCK cells from the cytotoxic action of CTS independently of inhibition of Na(+),K(+)-ATPase-mediated Na(+) and K(+) fluxes and inversion of the [Na(+)](i)/[K(+)](i) ratio.

The effect of long-term treatment with spironolactone on sodium pump abnormalities in the red blood cells of patients with Bartter's syndrome.

To elucidate the possible sodium transport alterations across the cell membranes in Bartter's syndrome and their influencing by spironolactone treatment Na+-K+-ATPase activity was studied by means of radioactive 86Rubidium influx into red blood cells (RBC) of patients with Bartter's syndrome prior to and after a long-term spironolactone administration. As compared with the control subjects and patients with primary aldosteronism the patients with Bartter's syndrome had a more than 5 times higher 86Rb uptake by the RBC, especially in the ouabain-sensitive component. A long-term spironolactone treatment led to the decrease of this high influx. Serum of patients with Bartter's syndrome incubated with healthy RBC distinctly increased their 86Rb influx. The increase nevertheless did not reach the values in the RBC of untreated patients with Bartter's syndrome. Even if our results do not allow to explain fully the mechanism responsible for the Na+-K+-ATPase changes in the RBC of these patients, analysis of the studied parameters demonstrated that none of the known humoral factors as aldosterone, renin, prostaglandins, or changes of the serum potassium were responsible for these abnormalities. The changes of sodium transport in RBC of patients with Bartter's syndrome could be a part of a more general disturbance of the transport mechanism and could significantly participate in the pathogenesis of this disease.

A Conserved Hydrophobic Tetrad near the C Terminus of the Secretory Na+-K+-2Cl- Cotransporter (NKCC1) Is Required for Its Correct Intracellular Processing

Little is known about the intracellular folding and trafficking of integral membrane proteins. Here we identify a hydrophobic amino acid tetrad (ILLV) close to the C terminus of the secretory Na+-K+-2Cl- cotransporter (NKCC1) that is important for the proper intracellular processing of this protein. This tetrad appears in a C-terminal sequence pattern that is conserved across species in a number of members of the NKCC1 gene family (slc12) of electroneutral salt transporters. We studied the effects of various mutations of these amino acids on NKCC1 transiently transfected into HEK-293 cells. Our results show that mutation of two of these residues to alanine leads to a >50% reduction in expression and complex glycosylation levels and that multiple mutations to alanine have cumulative effects. By contrast, scrambling of these amino acids, or mutation of other nearby conserved C-terminal residues, has little effect on these parameters. Mutation of ILLV to AAAA reduces complex glycosylation of NKCC1 by approximately 90% and results in a protein that does not form stable dimers and is retained in the endoplasmic reticulum in a highly aggregated state. Our results are consistent with the hypothesis that mutation of the hydrophobic tetrad ILLV to AAAA leads to the ab initio misfolding and concomitant aggregation of this NKCC1 mutant, resulting in its retention in the endoplasmic reticulum.

Granitization and magmatic replacement in the contact aureole of the Yurchik gabbronorite massif (Ganal Ridge, Kamchatka)

It is shown that formation of high-temperature granulite-like rocks in the contact aureole of the Yurchik gabbronorite intrusion of the Ganal Ridge in East Kamchatka was caused by contact metamorphism, metasomatism, and local melting of the primary sedimentary-volcanogenic rocks of the Vakhtalkinskaya Sequence of the Ganal Group. The temperature in the inner part of the aureole reached 700–800°C and caused transformation of basic rocks into two-pyroxene-plagioclase, clinopyroxene-amphibole-plagioclase, and amphibole-plagioclase rocks, while sedimentary rocks were replaced by garnet-biotite and garnet-cordierite-biotite hornfelses. Locally, basic volcanic hornfelses were subjected to metasomatic alteration with the formation of bodies of biotite-orthopyroxene-plagioclase metasomatites. In the zones of the most intense fluid filtration, the metasomatites experienced local magmatic replacement resulting in the formation of biotite-orthopyroxene-plagioclase ± garnet migmatite veinlets and patches. Bodies of garnet enderbites were formed after sedimentary interlayers at temperatures of 700–800°C and a lithostatic pressure of 3.2–4.8 kbar. The comparison of the chemical composition of the Vakhtalkinskaya basic volcanics and the products of their transformation indicates that, in terms of chemistry, the metasomatic alterations and magmatic replacement correspond to siliceous-alkaline metasomatism (granitization) causing a subsequent and uneven influx of Si, Al, Na, K, Rb, Ba, Zr, Nb, and Cl and removal of Fe, Mg, Mn, Ca, and some trace elements (Cr, Co, Ti, Y, and S). The processes of metamorphism and metasomatism were presumably provoked by highly mineralized mantle fluids that were filtered through magmatic channels that served as pathways for gabbroid magma.

Renal Na+-K+-Cl- cotransporter activity and vasopressin-induced trafficking are lipid raft-dependent.

Apical bumetanide-sensitive Na(+)-K(+)-2Cl(-) cotransporter (NKCC2), the kidney-specific member of a cation-chloride cotransporter superfamily, is an integral membrane protein responsible for the transepithelial reabsorption of NaCl. The role of NKCC2 is essential for renal volume regulation. Vasopressin (AVP) controls NKCC2 surface expression in cells of the thick ascending limb of the loop of Henle (TAL). We found that 40-70% of Triton X-100-insoluble NKCC2 was present in cholesterol-enriched lipid rafts (LR) in rat kidney and cultured TAL cells. The related Na(+)-Cl(-) cotransporter (NCC) from rat kidney was distributed in LR as well. NKCC2-containing LR were detected both intracellularly and in the plasma membrane. Bumetanide-sensitive transport of NKCC2 as analyzed by (86)Rb(+) influx in Xenopus laevis oocytes was markedly reduced by methyl-beta-cyclodextrin (MbetaCD)-induced cholesterol depletion. In TAL, short-term AVP application induced apical vesicular trafficking along with a shift of NKCC2 from non-raft to LR fractions. In parallel, increased colocalization of NKCC2 with the LR ganglioside GM1 and their polar translocation were assessed by confocal analysis. Apical biotinylation showed twofold increases in NKCC2 surface expression. These effects were blunted by mevalonate-lovastatin/MbetaCD-induced cholesterol deprivation. Collectively, these findings demonstrate that a pool of NKCC2 distributes in rafts. Results are consistent with a model in which LR mediate polar insertion, activity, and AVP-induced trafficking of NKCC2 in the control of transepithelial NaCl transport.

Further characteristics of hyposmotically activated K loss in human lens epithelial cells (hLECs)

Regulatory volume decrease of hLECs in hyposmotic (150 mOsM) media involves KCa3.1 (IK) channel activation and cell water loss. High external K and clotrimazole (CTZ), but not a variety of anion channel blockers, were inhibitory. Extending these findings, we studied the selective IK channel blocker (TRAM‐34 [1‐[(2‐chlorophenyl) diphenylmethyl]‐1H‐pyrazole]), and additional anion channel blockers used in other systems, especially DIOA {[(dihydro‐indenyl)oxy]alkanoic acid]}, phloretin and DCPIB [4‐2(butyl‐6,7‐dichloro‐2‐cyclopentylindan‐1‐on‐5‐yl)oxybutyric acid]. Ouabain‐ and bumetanide‐insensitive K loss and Rb influx were measured in hLECs (FHL124) by atomic absorption spectrophometry. TRAM‐34 (&gt;50 μM) reduced K loss by ~ 30% with little effects on Rb influx, an asymmetry contrasting with the total inhibition of both fluxes by CTZ, also an inhibitor of P450, suggesting at least two different mechanisms either at the K channel and/or regulatory levels. Phloretin (0.25–1 mM) reduced Rb influx by ~60%, less affecting K loss. In Cl or NO3, 300 μM DIOA effectively inhibited K loss, but not Rb influx, a concentration tenfold higher than used to block K‐Cl cotransport. DCPIB (50 μM) mainly reduced K‐loss. Hence, hyposmotic K loss, through putative IK channels in hLECs, is associated with and/or rate‐limited by Cl loss through DIOA‐and phloretin‐sensitive anion channels whose nature awaits elucidation.

Li fluxes reveal presence of Na‐Cl Cotransport (NCC) in Human Lens Epithelial Cells (LECs)

Na‐K‐2Cl cotransport (NKCC) moves RbCl/KCl+NaCl/LiCl and, in FHL124 LECs, is not inhibited by hypotonicity. Here, we studied Li fluxes through NKCC in hyposmotic high K media and Rb fluxes in hyposmotic high Na to understand this effect. Li and Rb uptake were measured by atomic absorption spectrophotometry to determine LiKCC or NRbCC through bumetanide‐sensitive (BS) and Cl‐dependent fluxes in Na/K or Na‐free/K‐free (N‐methyl‐D‐glucamine) and Cl or Cl‐free (sulfamate or nitrate) media ± ouabain (O) or [O+B], ± thiazides, at varying Rb, Li or Cl molar fractions (MF). Main findings are: Li influx in isosmotic (300 mOsM) K was 1/3 of Rb influx in Na, and was mainly mediated by a Cl‐dependent Li flux (LiKCC). In 200 mOsM high K, LiKCC was abolished, whereas in high Na, NRbCC remained active. LiKCC and BS‐Li influx (BS‐LiKCC) showed bell‐shape curves for 0.1–1 Li MF, maxing at ∼0.6 MF. BS‐LiKCC was ¼ of LiKCC. The difference, i.e. the B‐insensitive/Cl‐dependent‐Li influx, saturated with Li and Cl MFs. Kms for Li were 11 with and 7–8 mM without external K, respectively; and ∼40 mM for Cl. Neither furosemide (&lt;100 μM) nor thiazide derivatives inhibited LiCC. RTPCR, Western blots and immunochemistry revealed NCC RNA and protein, with NCC‐specific antibodies, besides the expected NKCC1. Thus, a K‐independent/Cl‐dependent Li flux (LiCC) revealed the presence of NCC in FHL124 LECs.