Inhibition Of NKCC Research Articles

Rapid cold hardening modifies ion regulation to delay anoxia-induced spreading depolarization in the CNS of the locust

Insects experience different kinds of environmental stresses that can impair neural performance, leading to spreading depolarization (SD) of nerve cells and neural shutdown underlying coma. SD is associated with a sudden loss of ion, notably K+, homeostasis in the central nervous system. The sensitivity of an insect's nervous system to stress (e.g., anoxia) can be modulated by acute pre-treatment. Rapid cold hardening (RCH) is a form of preconditioning, in which a brief exposure to low temperature can enhance the stress tolerance of insects. We used a pharmacological approach to investigate whether RCH affects anoxia-induced SD in the locust, Locusta migratoria, via one or more of the following homeostatic mechanisms: (1) Na+/K+-ATPase (NKA), (2) Na+/K+/2Cl− co-transporter (NKCC), and (3) voltage-gated K+ (Kv) channels. We also assessed abundance and phosphorylation of NKCC using immunoblotting. We found that inhibition of NKA or Kv channels delayed the onset of anoxia-induced SD in both control and RCH preparations. However, NKCC inhibition preferentially abrogated the effect of RCH. Additionally, we observed a higher abundance of NKCC in RCH preps but no statistical difference in its phosphorylation level, indicating the involvement of NKCC expression or degradation as part of the RCH mechanism.

ИССЛЕДОВАНИЕ РОЛИ NA+, K+, 2CL- - КОТРАНСПОРТА В РЕГУЛЯЦИИ ТОНУСА ЛЕГОЧНОЙ АРТЕРИИ

Purpose. We studied the role of Na+, K+, 2Cl- cotransport (NKCC) in contractile activity of pulmonary artery seg- ments with intact endothelium induced by incubation in nonisosmotic solutions. Materials and methods. The influence of nonisosmotic solutions and blocker of NKCC on vascular segments mechanical tension was studied in isometric regime with organ bath technique performed with the 4-channel Myobath II and software complex LAB-TRAX-4/16 (Germany). Hyperosmotic shrinkage was induced by adding 50-300 mM su- crose to the Krebs solution, the hypoosmotic environment was created by reducing the concentration of NaCl from 70 mM to 40 mM. Bumetanide was used as an NKCC blocker. The amplitude of the contractile responses was evaluated as a percentage of the control contraction in highpotassium solution. Results. The addition of 50-300 mM sucrose to the Krebs incubation solution caused a development of dose- dependent of sustained contractile responses. The amplitude of hyperosmotic striction increased with inhibition of NKCC. Incubation of segments in hypoosmotic solution caused the development of transient contractile responses, the amplitude of which decreased in the presence of NKCC inhibitor. Conclusion. Iincubation of pulmonary artery vascular segments in hyper- and hyposmotic solutions induces con- tractile responses, the amplitude of which depends on the NKCC activity.

Sodium Dichloroacetate Pharmacological Effect as Related to Na-K-2Cl Cotransporter Inhibition in Rats.

The study objective was to investigate a possible sodium dichloroacetate (DCA) pharmacological mechanism causing an increase in diuresis in rats. The aim was to define characteristics of 24-hour urinary Na+, K+, Cl−, Ca2+, and Mg2+ excretion in Wistar male rats and to evaluate effect of a single-dose DCA and repeated DCA dosage on diuresis. Six control and 6 DCA-treated male rats aged 5 to weeks after a single DCA dose and repeated dosage were tested. The single DCA dose treatment caused a significantly higher 24-hour diuresis when compared to control (P < .05), and it was related to increased Cl−, Na+, and K+ urine excretion and a significant increase in Ca2+ and Mg2+ excretion (P < .05); after the repeated 4-week DCA dosage, the diuresis was not increased, but the excretion of the Na+, Cl−, Ca2+, and Mg2+ ions was significantly higher. Kidney immunohistochemistry has revealed that DCA continuous treatment results in an increase in the size of Henle loop thick ascending limb epithelial cells (P < .001). The study results show a significantly reduced RNA expression of Na-K-2Cl co-transporter (NKCC1) in thymus of 4-week DCA-treated rats (P < .03). The study data have indicated a possible mechanism of such pharmacological effect to be NKCC inhibition.

Everything we always wanted to know about furosemide but were afraid to ask.

Furosemide is a widely used, potent natriuretic drug, which inhibits the Na(+)-K(+)-2Cl(-) cotransporter (NKCC)-2 in the ascending limb of the loop of Henle applied to reduce extracellular fluid volume expansion in heart and kidney disease. Undesirable consequences of furosemide, such as worsening of kidney function and unpredictable effects on sodium balance, led to this critical evaluation of how inhibition of NKCC affects renal and cardiovascular physiology. This evaluation reveals important knowledge gaps, involving furosemide as a drug, the function of NKCC2 (and NKCC1), and renal and systemic indirect effects of NKCC inhibition. Regarding renal effects, renal blood flow and glomerular filtration rate could become compromised by activation of tubuloglomerular feedback or by renin release, particularly if renal function is already compromised. Modulation of the intrarenal renin angiotensin system, however, is ill-defined. Regarding systemic effects, vasodilation followed by nonspecific NKCC inhibition and changes in venous compliance are not well understood. Repetitive administration of furosemide induces short-term (braking phenomenon, acute diuretic resistance) and long-term (chronic diuretic resistance) adaptations, of which the mechanisms are not well known. Modulation of NKCC2 expression and activity in kidney and heart failure is ill-defined. Lastly, furosemide's effects on cutaneous sodium stores and on uric acid levels could be beneficial or detrimental. Concluding, a considerable knowledge gap is identified regarding a potent drug with a relatively specific renal target, NKCC2, and renal and systemic actions. Resolving these questions would increase the understanding of NKCCs and their actions and improve rational use of furosemide in pathophysiology of fluid volume expansion.

Role of sterile 20/sps1-related proline/alanine-rich kinase in mice with endotoxic shock

Background: Na + -K + -2Cl − co-transporters (NKCCs) are involved in the regulation of permeability and tissue edema during sepsis. Inhibition of NKCC can reduce inflammation, edema formation, and bacterial burden. STE20/SPS1-realted proline/alanine-rich kinase (SPAK) is known to phosphorylate and activate NKCCs. However, there is no data regarding the role of SPAK in the pathological responses of sepsis. Therefore, the aim of this study was to examine the changes of systemic responses to endotoxemia in SPAK knockout mice. Materials and Methods: Wild-type and SPAK knockout mice were randomly given with vehicle (saline) or Escherichia coli lipopolysaccharide (LPS, 50 mg/kg) and monitored for 24 h. The alterations of hemodynamics, blood glucose, biochemical variables, plasma nitric oxide (NO) levels, blood flow, superoxide levels, and survival rate were analyzed during the experimental period. Results: In this study, LPS induced circulatory failure, hypoglycemia, multiple organ dysfunction, and mortality in wild-type mice. The NO levels of plasma were augmented and blood flow of the tongue, palm, sole, and abdomen were reduced in wild-type mice with endotoxic shock. However, there were no significant differences in these functional parameters and survival rate between wild-type and SPAK knockout mice with endotoxemia. Conclusions: These results demonstrate that inhibition of SPAK did not improve circulatory failure, hypoglycemia, multiple organ dysfunction, or mortality in mice that treated LPS. Thus, it seems that SPAK may not play an important role in endotoxic shock.

Ammonium‐evoked alterations in intracellular sodium and pH reduce glial glutamate transport activity

The clearance of extracellular glutamate is mainly mediated by pH- and sodium-dependent transport into astrocytes. During hepatic encephalopathy (HE), however, elevated extracellular glutamate concentrations are observed. The primary candidate responsible for the toxic effects observed during HE is ammonium (NH(4) (+)/NH(3)). Here, we examined the effects of NH(4) (+)/NH(3) on steady-state intracellular pH (pH(i)) and sodium concentration ([Na(+)](i)) in cultured astrocytes in two different age groups. Moreover, we assessed the influence of NH(4) (+)/NH(3) on glutamate transporter activity by measuring D-aspartate-induced pH(i) and [Na(+)](i) transients. In 20-34 days in vitro (DIV) astrocytes, NH(4) (+)/NH(3) decreased steady-state pH(i) by 0.19 pH units and increased [Na(+)](i) by 21 mM. D-Aspartate-induced pH(i) and [Na(+)](i) transients were reduced by 80-90% in the presence of NH(4) (+)/NH(3), indicating a dramatic reduction of glutamate uptake activity. In 9-16 DIV astrocytes, in contrast, pH(i) and [Na(+)](i) were minimally affected by NH(4) (+)/NH(3), and D-aspartate-induced pH(i) and [Na(+)](i) transients were reduced by only 30-40%. Next we determined the contribution of Na(+), K(+), Cl(-)-cotransport (NKCC). Immunocytochemical stainings indicated an increased expression of NKCC1 in 20-34 DIV astrocytes. Moreover, inhibition of NKCC with bumetanide prevented NH(4) (+)/NH(3)-evoked changes in steady-state pH(i) and [Na(+)](i) and attenuated the reduction of D-aspartate-induced pH(i) and [Na(+)](i) transients by NH(4) (+)/NH(3) to 30% in 20-34 DIV astrocytes. Our results suggest that NH(4) (+)/NH(3) decreases steady-state pH(i) and increases steady-state [Na(+)](i) in astrocytes by an age-dependent activation of NKCC. These NH(4) (+)/NH(3)-evoked changes in the transmembrane pH and sodium gradients directly reduce glutamate transport activity, and may, thus, contribute to elevated extracellular glutamate levels observed during HE.

Sodium Ion Transporters as New Therapeutic Targets in Heart Failure

Sodium ion transporters in sarcolemma are involved in numerous vital cell functions, such as excitability, excitation-contraction coupling, energy metabolism, pH and volume regulation, development and growth. In a number of cardiac pathologies, the intracellular sodium concentration ([Na+]i) is elevated. Since [Na+]i and intracellular Ca2+ concentration ([Ca2+]i are coupled through the Na+/Ca(2+)-exchanger, these cardiac pathologies display disturbed calcium handling. For instance, [Na+]i is increased in heart failure (HF) leading to Na+/Ca(2+)-exchanger mediated increase in [Ca2+]i, reduced contractility and increased propensity to arrhythmias. Several studies support the contention that an increase in [Na+]i and [Ca2+]i transduces a signal the nucleus, that triggers development of cardiac remodelling and hypertrophy. Pharmacological intervention, which favourably interferes with [Na+]i and [Ca2+]i homeostasis, might prevent hypertrophy, cardiac remodelling, arrhythmias and HF. The most important sodium transport mechanisms that may underlie increased [Na+]i are: Na+/H(+)-exchanger (NHE-1), Na+-HCO(3)(-) co-transporter (NBC), Na(+)-K(+)-Cl(-) co-transporter (NKCC), Na(+)-channel, Na+/K(+)-ATPase and Na+/Ca(2+)-exchanger (NCX). Preclinical studies showed that pharmacological interventions, targeted against sarcolemmal sodium ion transporters, proved effective in ameliorating heart failure. In this respect: 1) NHE-1 inhibition reduces cardiac remodelling, hypertrophy and HF, although, in the patients following coronary artery bypass graft surgery, it was associated with an increase of stroke. 2) The activity of NBC is up-regulated, during the development of hypertrophy and may be a therapeutic strategy to prevent the development of hypertrophy and HF. 3) NKCC is increased in post-infarction HF, and the inhibition of NKCC attenuated post-infarction remodelling. 4) Inactivation of sodium channels is impaired in HF, which may result, in increased Na+ influx and prolongation of the action potential. 5) Blockade of NCX may be useful as a part of a combined therapeutic approach. Inhibition of reversed mode, or activation of forward mode NCX reduce Ca2+ overload. 6) Inhibition of Na+/K(+)-ATPase (digoxin), is used to increase contractility, however, it enhances progression of HF. Oppositely, new drugs which increase activity of Na+/K(+)-ATPase may prevent the development of cardiac remodelling hypertrophy and HF.

Potassium channel and NKCC cotransporter involvement in ocular refractive control mechanisms.

Myopia affects well over 30% of adult humans globally. However, the underlying physiological mechanism is little understood. This study tested the hypothesis that ocular growth and refractive compensation to optical defocus can be controlled by manipulation of potassium and chloride ion-driven transretinal fluid movements to the choroid. Chicks were raised with +/−10D or zero power optical defocus rendering the focal plane of the eye in front of, behind, or at the level of the retinal photoreceptors respectively. Intravitreal injections of barium chloride, a non-specific inhibitor of potassium channels in the retina and RPE or bumetanide, a selective inhibitor of the sodium-potassium-chloride cotransporter were made, targeting fluid control mechanisms. Comparison of refractive compensation to 5mM Ba2+ and 10−5 M bumetanide compared with control saline injected eyes shows significant change for both positive and negative lens defocus for Ba2+ but significant change only for negative lens defocus with bumetanide ; ; ; ; ; ). Vitreous chamber depths showed a main effect for drug conditions with less depth change in response to defocus shown for Ba2+ relative to Saline, while bumetanide injected eyes showed a trend to increased depth without a significant interaction with applied defocus. The results indicate that both K channels and the NKCC cotransporter play a role in refractive compensation with NKCC blockade showing far more specificity for negative, compared with positive, lens defocus. Probable sites of action relevant to refractive control include the apical retinal pigment epithelium membrane and the photoreceptor/ON bipolar synapse. The similarities between the biometric effects of NKCC inhibition and biometric reports of the blockade of the retinal ON response, suggest a possible common mechanism. The selective inhibition of refractive compensation to negative lens in chick by loop diuretics such as bumetanide suggests that these drugs may be effective in the therapeutic management of human myopia.

Purinergic inhibition of Na+,K+,Cl− cotransport in C11-MDCK cells: Role of stress-activated protein kinases

Previously, we observed that sustained activation of P2Y1 leads to inhibition of Na+,K+,Cl− cotransport (NKCC) in C11 cells resembling intercalated cells from collecting ducts of the Madin-Darby canine kidney. This study examined the role of stress-activated protein kinases (SAPK) in NKCC inhibition triggered by purinergic receptors. Treatment of C11 cells with ATP led to sustained phosphorylation of SAPK such as JNK and p38. Activation of these kinases also occurred in anisomycin-treated cells. Surprisingly, we observed that compounds SP600125 and SB202190, known as potent inhibitors of JNK and p38 in cell-free systems, activated rather than inhibited phosphorylation of the kinases in C11 cells. Importantly, similarly to ATP, all the above-listed activators of JNK and p38 phosphorylation inhibited NKCC. Thus, our results suggest that activation of JNK and/or p38 contributes to NKCC suppression detected in intercalated-like cells from distal tubules after their exposure to P2Y1 agonists.

Transient Activation and Delayed Inhibition of Na+,K+,Cl–Cotransport in ATP-treated C11-MDCK Cells Involve Distinct P2Y Receptor Subtypes and Signaling Mechanisms

In C11-MDCK cells, which resemble intercalated cells from collecting ducts of the canine kidney, P2Y agonists promote transient activation of the Na+,K+,Cl- cotransporter (NKCC), followed by its sustained inhibition. We designed this study to identify P2Y receptor subtypes involved in dual regulation of this carrier. Real time polymerase chain reaction analysis demonstrated that C11-MDCK cells express abundant P2Y1 and P2Y2 mRNA compared with that of other P2Y receptor subtypes. The rank order of potency of agents (ATP approximately UTP >> 2-(methylthio)-ATP (2MeSATP); adenosine 5'-[beta-thio]diphosphate (ADPbetaS) inactive) indicated that P2Y2 rather than P2Y1 receptors mediate a 3-4-fold activation of NKCC within the first 5-10 min of nucleotide addition. NKCC activation in ATP-treated cells was abolished by the intracellular calcium chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid, calmodulin (CaM) antagonists trifluoroperazine and W-7, and KN-62, an inhibitor of Ca2+/CaM-dependent protein kinase II. By contrast with the transient activation, 30-min incubation with nucleotides produced up to 4-5-fold inhibition of NKCC, and this inhibition exhibited a rank order of potency (2MeSATP > ADPbetaS > ATP >> UTP) typical of P2Y1 receptors. Unlike the early response, delayed inhibition of NKCC occurred in 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-loaded cells and was completely abolished by the P2Y1 antagonists MRS2179 and MRS2500. Transient activation and delayed inhibition of NKCC in C11 cell monolayers were observed after the addition of ATP to mucosal and serosal solutions, respectively. NKCC inhibition triggered by basolateral application of ADPbetaS was abolished by MRS2500. Our results thus show that transient activation and delayed inhibition of NKCC in ATP-treated C11-MDCK cells is mediated by Ca2+/CaM-dependent protein kinase II- and Ca2+-independent signaling triggered by apical P2Y2 and basolateral P2Y1 receptors, respectively.

Cell-volume-dependent vascular smooth muscle contraction: role of Na+, K+, 2Cl- cotransport, intracellular Cl- and L-type Ca2+ channels.

This study elucidates the role of cell volume in contractions of endothelium-denuded vascular smooth muscle rings (VSMR) from the rat aorta. We observed that hyposmotic swelling as well as hyper- and isosmotic shrinkage led to VSMR contractions. Swelling-induced contractions were accompanied by activation of Ca2+ influx and were abolished by nifedipine and verapamil. In contrast, contractions of shrunken cells were insensitive to the presence of L-type channel inhibitors and occurred in the absence of Ca2+ o. Thirty minutes preincubation with bumetanide, a potent Na+, K+, CI- cotransport (NKCC) inhibitor, decreased Cl(-)i content, nifedipine-sensitive 45Ca uptake and contractions triggered by modest depolarization ([K+]o = 36 mM). Elevation of [K+]o to 66 mM completely abolished the effect of bumetanide on these parameters. Bumetanide almost completely abrogated phenylephrine-induced contraction, partially suppressed contractions triggered by hyperosmotic shrinkage, but potentiated contractions of isosmotically shrunken VSMR. Our results suggest that bumetanide suppresses contraction of modestly depolarized cells via NKCC inhibition and Cl(-)i-mediated membrane hyperpolarization, whereas augmented contraction of isosmotically shrunken VSMR by bumetanide is a consequence of suppression of NKCC-mediated regulatory volume increase. The mechanism of bumetanide inhibition of contraction of phenylephrine-treated and hyperosmotically shrunken VSMR should be examined further.

K+ Transport and Volume Regulatory Response by NKCC in Resting Rat Hindlimb Skeletal Muscle

This study tested the hypothesis that the NKCC is involved in volume regulation, specifically regulatory volume increase (RVI), in resting skeletal muscle. Neurally and vascularly isolated rat hindlimbs were perfused with a bovine erythrocyte perfusate containing ⁴²K or ⁸⁶Rb as markers of unidirectional K⁺ flux across the sarcolemma. Compared to controls, perfusion with 120 µM bumetanide (a specific inhibitor of the NKCC) decreased J_inK by 15±2%, indicating the functional presence of the NKCC. Experiments with ouabain (to block active K⁺ transport by the Na,K ATPase) showed that the bumetanide-sensitive component of J_inK comprised 35% of the total ouabain-sensitive J_inK. Inhibition of NKCC resulted in a net loss of water by muscle. When hindlimbs were perfused with hypertonic (380 mOsm/L by addition of sucrose) perfusate for 20 min, after initially blocking K⁺ channels with 1 mM barium, J_inK rapidly (2-3 min) increased 2-fold followed by a rapid decline. This rapid, transient increase in J_inK was abolished with bumetanide, confirming that perfusion with hypertonic perfusate stimulated NKCC activity and RVI. The hypertonic perfusate also resulted in temporally associated decreases in net water uptake by muscle. It is concluded that a functional NKCC is present in mammalian skeletal muscle and that it is involved in cell volume regulation.

Protection of ischemic myocardium in diabetics by inhibition of electroneutral Na+-K+-2Cl- cotransporter.

Diabetes increases both the incidence of cardiovascular disease and complications of myocardial infarction and heart failure. Studies using diabetic animals have shown that changes in myocardial sodium transporters result in alterations in intracellular sodium (Na(i)) homeostasis. Because the changes in sodium homeostasis can be due to increased entry of Na+ via the electroneutral Na+-K+-2Cl- cotransporter (NKCC), we conducted experiments in acute diabetic hearts to determine if 1) net inward cation flux via NKCC is increased, 2) this cotransporter contributes to a greater increase in Na(i) during ischemia, and 3) inhibition of NKCC limits injury and improves function after ischemia-reperfusion. These issues were investigated in perfused type I diabetic and nondiabetic rat hearts subjected to ischemia and 60 min of reperfusion. A group of diabetic and nondiabetic hearts was perfused with 5 microM of bumetanide, an inhibitor of NKCC. Flux via NKCC, Na(i), and ATP was measured in each group with the use of radiotracer 86Rb, 23Na, and 31P nuclear magnetic resonance spectroscopy, respectively, whereas ischemic injury was assessed by measuring creatine kinase release on reperfusion. Cation flux via NKCC, as measured by 86Rb uptake, was significantly increased in diabetic hearts. Inhibition of NKCC significantly reduced ischemic injury in diabetic hearts, improved functional recovery on reperfusion, attenuated the ischemic rise in Na(i), and conserved ATP during ischemia-reperfusion. Parallel studies in nondiabetic hearts showed that NKCC inhibition was not cardioprotective. These findings demonstrate that flux via NKCC is increased in type I diabetic hearts and that inhibition with bumetanide attenuates changes in Na(i) and ATP during ischemia and protects against ischemic injury. The data suggest a therapeutic role for pharmacological agents that inhibit flux via NKCC in diabetic patients with myocardial ischemia.