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Background/aims Silver nanoparticles (AgNPs) are the most frequently used nanomaterials in industrial and biomedical applications. Their functionalization significantly impacts their properties and potential applications. Despite the need to produce, investigate and apply them, not much is known about the toxicity of silver nanoparticles to and their interaction with blood components, such as erythrocytes. Here, we report on the effect of two negatively charged AgNPs (Creighton, and Lee-Meisel) on ion transport in human red blood cells (HRBCs). Methods HRBCs were obtained from blood of adult donors, which was either expired, fresh or refrigerated for variable lengths of time, and from fresh or refrigerated cord blood. Rb+ and K+ ions were measured by atomic emission and absorption spectrophotometry, respectively. Erythrocyte hemoglobin optical density (Hbc OD), was determined at 527 nm to estimate RBC volume in the same tubes in which Rb+ and K+ were measured. Cellular Rb+ uptake and intracellular K+ concentrations, [K]i, were calculated in mmol/L of original cells (LOC) per time. Rubidium, a potassium ion (K+) congener used to measure K+ influx, [K]i, and Hbc ODs were determined in the presence and absence of several concentrations (0-150 µg mL-1) of spherical AgNPs of an average diameter of 10 nm, at different time points (0-60 min). Results Creighton AgNPs inhibited Rb+ influx and depleted the cells of K+ independently of the source and in a time and dose-dependent manner. In contrast, Lee-Meisel AgNPs caused ~ 50 % Rb+ influx inhibition and ~ 15 % K+ loss with larger interindividual variability than Creighton AgNPs. The loss of K+ from HRBCs was entirely accounted for by an increase in extracellular K+ concentration, [K]o. Enhanced dark field optical microscopy in conjunction with CytoViva® hyperspectral imaging helped visualize AgNPs internalized by HRBCs, thus pointing to a potential cause for their cytotoxic effects. Conclusion These findings indicate that HRBC K+ homeostasis is an early and sensitive biomarker for AgNPs toxicity and is a function of their surface functionalization.

Background/Aims: Chelerythrine [CET], a protein kinase C [PKC] inhibitor, is a prop-apoptotic BH3-mimetic binding to BH1-like motifs of Bcl-2 proteins. CET action was examined on PKC phosphorylation-dependent membrane transporters (Na+/K+ pump/ATPase [NKP, NKA], Na+-K+-2Cl+ [NKCC] and K+-Cl- [KCC] cotransporters, and channel-supported K+ loss) in human lens epithelial cells [LECs]. Methods: K+ loss and K+ uptake, using Rb+ as congener, were measured by atomic absorption/emission spectrophotometry with NKP and NKCC inhibitors, and Cl- replacement by NO3ˉ to determine KCC. 3H-Ouabain binding was performed on a pig renal NKA in the presence and absence of CET. Bcl-2 protein and NKA sequences were aligned and motifs identified and mapped using PROSITE in conjunction with BLAST alignments and analysis of conservation and structural similarity based on prediction of secondary and crystal structures. Results: CET inhibited NKP and NKCC by >90% (IC50 values ∼35 and ∼15 µM, respectively) without significant KCC activity change, and stimulated K+ loss by ∼35% at 10-30 µM. Neither ATP levels nor phosphorylation of the NKA α1 subunit changed. 3H-ouabain was displaced from pig renal NKA only at 100 fold higher CET concentrations than the ligand. Sequence alignments of NKA with BH1- and BH3-like motifs containing pro-survival Bcl-2 and BclXl proteins showed more than one BH1-like motif within NKA for interaction with CET or with BH3 motifs. One NKA BH1-like motif (ARAAEILARDGPN) was also found in all P-type ATPases. Also, NKA possessed a second motif similar to that near the BH3 region of Bcl-2. Conclusion: Findings support the hypothesis that CET inhibits NKP by binding to BH1-like motifs and disrupting the α1 subunit catalytic activity through conformational changes. By interacting with Bcl-2 proteins through their complementary BH1- or BH3-like-motifs, NKP proteins may be sensors of normal and pathological cell functions, becoming important yet unrecognized signal transducers in the initial phases of apoptosis. CET action on NKCC1 and K+ channels may involve PKC-regulated mechanisms; however, limited sequence homologies to BH1-like motifs cannot exclude direct effects.

Background/Aims: The quaternary benzo-phenanthridine alkaloid (QBA) chelerythrine (CET) is a pro-apoptotic drug and Na+/K+ pump (NKP) inhibitor in human lens epithelial cells (HLECs). In order to obtain further insight into the mechanism of NKP inhibition by CET, its sub-cellular distribution was quantified in cytosolic and membrane fractions of HLEC cultures by surface-enhanced Raman spectroscopy (SERS). Methods: Silver nanoparticles (AgNPs) prepared by the Creighton method were concentrated, and size-selected using a one-step tangential flow filtration approach. HLECs cultures were exposed to 50 μM CET in 300 mOsM phosphate-buffered NaCl for 30 min. A variety of cytosolic extracts, crude and purified membranes, prepared in lysing solutions in the presence and absence of a non-ionic detergent, were incubated with AgNPs and subjected to SERS analysis. Determinations of CET were based on a linear calibration plot of the integrated CET SERS intensity at its 659 cm-1 marker band as a function of CET concentration. Results: SERS detected chemically unaltered CET in both cytosol and plasma membrane fractions. Normalized for protein, the CET content was some 100 fold higher in the crude and purified plasma membrane fraction than in the soluble cytosolic extract. The total free CET concentration in the cytosol, free of membranes or containing detergent-solubilized membrane material, approached that of the incubation medium of HLECs. Conclusion: Given a negative membrane potential of HLECs the data suggest, but do not prove, that CET may traverse the plasma membrane as a positively charged monomer (CET+) accumulating near or above passive equilibrium distribution. These findings may contribute to a recently proposed hypothesis that CET binds to and inhibits the NKP through its cytosolic aspect.

d Pathology, and e Abstract Background: Increased acidification/P CO2 of sea water is a threat to the environment and affects the homeostasis of marine animals. In this study, the effect of sea water pH changes on the osmotic phase (OP), regulatory volume decrease (RVD) and discharge of the jellyfish Pelagia noctiluca (Cnidaria, Scyphozoa) nematocytes, collected from the Strait of Messina (Italy), was assessed. Methods: Isolated nematocytes, suspended in artificial sea water (ASW) with pH 7.65, 6.5 and 4.5, were exposed to hyposmotic ASW of the same pH values and their osmotic response and RVD measured optically in a special flow through chamber. Nematocyte discharge was analyzed in situ in ASW at all three pH values. Results: At normal pH (7.65), nematocytes subjected to hyposmotic shock first expanded osmotically and then regulated their cell volume within 15 min. Exposure to hyposmotic ASW pH 6.5 and 4.5 compromised the OP and reduced or totally abrogated the ensuing RVD, respectively. Acidic pH also significantly reduced the nematocyte discharge response. Conclusion: Data indicate that the homeostasis and function of Cnidarians may be altered by environmental changes such as sea water acidification, thereby validating their use as novel bioindicators for the quality of the marine environment.

Nematocytes, the stinging cells of Cnidarians, have a cytoplasm confined to a thin rim. The main cell body is occupied by an organoid, the nematocyst, containing the stinging tubule and venom. Exposed to hypotonic shock, nematocytes initially swell during an osmotic phase (OP) and then undergo regulatory volume decrease (RVD) driven by K+, Cl- and obligatory water extrusion mechanisms. The purpose of this report is to characterize the OP. Nematocytes were isolated by the NaSCN/Ca2+ method from tentacles of the jellyfish Pelagia noctiluca, collected in the Strait of Messina, Italy. Isolated nematocytes were subjected to hyposmotic shock in 65% artificial seawater (ASW) for 15 min. The selective aquaporin water channel inhibitor HgCl2 (0.1-25 µM) applied prior to osmotic shock prevented the OP and thus RVD. These effects were attenuated in the presence of 1mM dithiothreitol (DTT), a mercaptide bond reducing agent. AgNO3 (1 µM) and TEA (tetraethylammonium, 100 µM), also reported to inhibit water transport, did not alter the OP but significantly diminished RVD, suggesting different modes of action for the inhibitors tested. Based on estimates of the nematocyte surface area and volume, and OP duration, a relative water permeability of ∼10-7 cm/sec was calculated and the number of putative aquaporin molecules mediating the OP was estimated. This water permeability is 3-4 orders of magnitude lower in comparison to higher order animals and may constitute an evolutionary advantage for Cnidarian survival.

The nature and role of potassium (K) and water transport mediating hyposmotically-induced regulatory volume decrease (RVD) were studied in nematocytes dissociated with 605 mM thiocyanate from acontia

This study explores the nature of K fluxes in human lens epithelial cells (LECs) in hyposmotic solutions. Total ion fluxes, Na-K pump, Cl-dependent Na-K-2Cl (NKCC), K-Cl (KCC) cotransport, and K channels were determined by 85Rb uptake and cell K (Kc) by atomic absorption spectrophotometry, and cell water gravimetrically after exposure to ouabain ± bumetanide (Na-K pump and NKCC inhibitors), and ion channel inhibitors in varying osmolalities with Na, K, or methyl-d-glucamine and Cl, sulfamate, or nitrate. Reverse transcriptase polymerase chain reaction (RT-PCR), Western blot analyses, and immunochemistry were also performed. In isosmotic (300 mosM) media ∼90% of the total Rb influx occurred through the Na-K pump and NKCC and ∼10% through KCC and a residual leak. Hyposmotic media (150 mosM) decreased Kc by a 16-fold higher K permeability and cell water, but failed to inactivate NKCC and activate KCC. Sucrose replacement or extracellular K to >57 mM, but not Rb or Cs, in hyposmotic media prevented Kc and water loss. Rb influx equaled Kc loss, both blocked by clotrimazole (IC50 ∼25 μM) and partially by 1-[(2-chlorophenyl) diphenylmethyl]-1H-pyrazole (TRAM-34) inhibitors of the IK channel KCa3.1 but not by other K channel or connexin hemichannel blockers. Of several anion channel blockers (dihydro-indenyl)oxy]alkanoic acid (DIOA), 4-2(butyl-6,7-dichloro-2-cyclopentylindan-1-on-5-yl)oxybutyric acid (DCPIB), and phloretin totally or partially inhibited Kc loss and Rb influx, respectively. RT-PCR and immunochemistry confirmed the presence of KCa3.1 channels, aside of the KCC1, KCC2, KCC3 and KCC4 isoforms. Apparently, IK channels, possibly in parallel with volume-sensitive outwardly rectifying Cl channels, effect regulatory volume decrease in LECs.

The defense of cell volume against excessive shrinkage or swelling is a requirement for cell function and organismal survival. Cell swelling triggers a coordinated homeostatic response termed regulatory volume decrease (RVD), resulting in K(+) and Cl(-) efflux via activation of K(+) channels, volume-regulated anion channels (VRACs), and the K(+)-Cl(-) cotransporters, including KCC3. Here, we show genetic alanine (Ala) substitution at threonines (Thr) 991 and 1048 in the KCC3a isoform carboxyl-terminus, preventing inhibitory phosphorylation at these sites, not only significantly up-regulates KCC3a activity up to 25-fold in normally inhibitory isotonic conditions, but is also accompanied by reversal of activity of the related bumetanide-sensitive Na(+)-K(+)-2Cl(-) cotransporter isoform 1 (NKCC1). This results in a rapid (10 min) and significant (90%) reduction in intracellular K(+) content (Ki) via both Cl-dependent (KCC3a + NKCC1) and Cl-independent [DCPIB (VRAC inhibitor)-sensitive] pathways, which collectively renders cells less prone to acute swelling in hypotonic osmotic stress. Together, these data demonstrate the phosphorylation state of Thr991/Thr1048 in KCC3a encodes a potent switch of transporter activity, Ki homeostasis, and cell volume regulation, and reveal novel observations into the functional interaction among ion transport molecules involved in RVD.

The defense of cell volume against excessive shrinkage or swelling is a requirement for cell function and organismal survival. Cell swelling triggers a coordinated homeostatic response termed regulatory volume decrease (RVD), resulting in K+ and Cl– efflux via the activation of K+ channels, volume-regulated anion channels (VRACs), and the K+-Cl– cotransporters, including KCC3. Here, we show genetic alanine (Ala) substitution at threonines (Thr) 991 and 1048 in the KCC3a isoform carboxyl-terminus, preventing inhibitory phosphorylation at these sites, not only significantly up-regulates KCC3a activity up to 25-fold in normally inhibitory isotonic conditions, but is also accompanied by reversal of activity of the related bumetanide-sensitive Na+-K+-2Cl– cotransporter isoform 1 (NKCC1). This results in a rapid (90 %) reduction in intracellular K+ content (Ki) via both Cl-dependent (KCC3a + NKCC1) and Cl-independent (DCPIB [VRAC inhibitor]-sensitive) pathways, which collectively renders cells less prone to acute swelling in hypotonic osmotic stress. Together, these data demonstrate the phosphorylation state of Thr991/Thr1048 in the KCC3a encodes a potent switch of transporter activity, Ki homeostasis, and cell volume regulation, and reveal novel observations into the functional interaction among ion transport molecules involved in RVD.

Defense of cell volume against excessive shrinkage or swelling is a requirement for multiple essential cellular functions and organismal survival. Cell swelling triggers a homeostatic counter-respo...

We recently reported potassium-chloride cotransporter activity in human lens epithelial B3 (HLE-B3) cells. The purpose of the present study was to demonstrate in these cells as well as in human lens tissue the potassium-chloride cotransport (KCC) isoforms by reverse transcriptase-polymerase chain reaction (RT-PCR), Western blotting and immunofluorescence microscopy. Of the four KCC genes known to encode the respective proteins and their spliced variants, RT-PCR with both rat and human primers revealed the predicted cDNA fragments of KCC1, KCC3a, KCC3b, and KCC4 but not KCC2 in both HLE-B3 cells and in human lens tissue extracts from cataractous patients. Polyclonal rabbit (rb) anti-rat (rt) and anti-human (hm) antibodies against rtKCC1 and hmKCC3, respectively, and a commercially available rb-anti-mouse (ms) KCC4 antibody were used. Rb anti-rtKCC1-ECL3 [against epitopes within the large extracellular loop 3 (ECL3)] revealed a 150kDa band in HLE-B3 cells consistent with the known molecular weight of KCC1. Rb anti-hmKCC3-ECL3 yielded three bands of 150, 122 and 105kDa, evidence for the presence of KCC3a, KCC3b and possibly KCC3c isoforms. The 122 and 112kDa bands were also demonstrated by rb anti-hmKCC3-CTD [the C-terminal domain (CTD)]. Rb anti-msKCC4 antibody only showed a 100kDa band in HLE-B3 cells. In the human lens tissues, a 115kDa protein was detected with rb anti-rtKCC1-ECL3 and a 100kDa band with rb anti-msKCC4, however, no bands with rb anti-hmKCC3-ECL3 or rb anti-hmKCC3-CTD. Fluorescence microscopy revealed immunocytochemical cytoplasmic and membrane labeling of HLE-B3 cells with anti-KCC1, -KCC3 (laser confocal microscopy) and -KCC4 antibodies and a Cy3-tagged secondary antibody. Hence HLE-B3 cells expressed proteins of the KCC1, KCC3a, b, and KCC4 isoforms, whereas surgically removed cataractous lens tissue expressed only those of KCC1 and KCC4.

K-Cl cotransport (K-Cl COT, KCC) is an electroneutrally coupled movement of K and Cl present in most cells. In this work, we studied the pathways of regulation of K-Cl COT by platelet-derived growth factor (PDGF) in primary cultures of vascular smooth muscle cells (VSMCs). Wortmannin and LY 294002 blocked the PDGF-induced K-Cl COT activation, indicating that the phosphoinositide 3-kinase (PI 3-K) pathway is involved. However, PD 98059 had no effect on K-Cl COT activation by PDGF, suggesting that the mitogen-activated protein kinase pathway is not involved under the experimental conditions tested. Involvement of phosphatases was also examined. Sodium orthovanadate, cyclosporin A and okadaic acid had no effect on PDGF-stimulated K-Cl COT. Calyculin A blocked the PDGF-stimulated K-Cl COT by 60%, suggesting that protein phosphatase-1 (PP-1) is a mediator in the PDGF signaling pathway/s. In conclusion, our results indicate that the PDGF-mediated pathways of K-Cl COT regulation involve the signaling molecules PI 3-K and PP-1.

Sheep K-Cl cotransporter-1(shKCC1) cDNA was cloned from kidney by RT-PCR with an open reading frame of 3258 base pairs exhibiting 92%, 90%, 88% and 87% identity with pig, rabbit and human, rat and mouse KCC1 cDNAs, respectively, encoding an approximately 122 kDa polypeptide of 1086-amino acids. Hydropathy analysis reveals the familiar KCC1 topology with 12 transmembrane domains (TMDs) and the hydrophilic NH2-terminal (NTD) and COOH-terminal (CTD) domains both at the cytoplasmic membrane face. However, shKCC1 has two rather than one large extracellular loops (ECL): ECL3 between TMDs 5 and 6, and ECL6, between TMDs 11 and 12. The translated shKCC1 protein differs in 12 amino acid residues from other KCC1s, mainly within the NTD, ECL3, ICL4, ECL6, and CTD. Notably, a tyrosine residue at position 996 replaces aspartic acid conserved in all other species. Human embryonic kidney (HEK293) cells and mouse NIH/3T3 fibroblasts, transiently transfected with shKCCI-cDNA, revealed the glycosylated approximately 150 kDa proteins by Western blots and positive immunofluorescence-staining with polyclonal rabbit anti-ratKCC1 antibodies. ShKCC1 was functionally expressed in NIH/3T3 cells by an elevated basal Cl-dependent K influx measured with Rb as K-congener that was stimulated three-fold by the KCC-activator N-ethylmaleimide.

Platelet-derived growth factor (PDGF), a potent serum mitogen for vascular smooth muscle cells (VSMCs), plays an important role in membrane transport regulation and in atherosclerosis. K-Cl cotransport (K-Cl COT/KCC), the coupled-movement of K and Cl, is involved in ion homeostasis. VSMCs possess K-Cl COT activity and the KCC1 and KCC3 isoforms. Here, we report on the effect of PDGF on K-Cl COT activity and mRNA expression in primary cultures of rat VSMCs. K-Cl COT was determined as the Cl-dependent Rb influx and mRNA expression by semiquantitative RT-PCR. Twenty four-hour serum deprivation inhibited basal K-Cl COT activity. Addition of PDGF increased total protein content and K-Cl COT activity in a time-dependent manner. PDGF activated K-Cl COT in a dose-dependent manner, both acutely (10 min) and chronically (12 h). AG-1296, a selective inhibitor of the PDGF receptor tyrosine kinase, abolished these effects. Actinomycin D and cycloheximide had no effect on the acute PDGF activation of K-Cl COT, suggesting posttranslational regulation by the drug. Furthermore, PDGF increased KCC1 and decreased KCC3 mRNA expression in a time-dependent manner. These results indicate that chronic activation of K-Cl COT activity by PDGF may involve regulation of the two KCC mRNA isoforms, with KCC1 playing a dominant role in the mechanism of PDGF-mediated activation.

K-Cl cotransport is activated by vasodilators in erythrocytes and vascular smooth muscle cells and its regulation involves putative kinase/phosphatase cascades. N-ethylmaleimide (NEM) activates the system presumably by inhibiting a protein kinase. Nitrovasodilators relax smooth muscle via cGMP-dependent activation of protein kinase G (PKG), a regulator of membrane channels and transporters. We investigated whether PKG regulates K-Cl cotransport activity or mRNA expression in normal, PKG-deficient-vector-only-transfected (PKG-) and PKG-catalytic-domain-transfected (PKG+) rat aortic smooth muscle cells. K-Cl cotransport was calculated as the Cl-dependent Rb influx, and mRNA was determined by semiquantitative RT-PCR. Baseline K-Cl cotransport was higher in PKG+ than in PKG- cells (p

Thiol reagents activate K-Cl cotransport (K-Cl COT), the Cl-dependent and Na-independent ouabain-resistant K flux, in red blood cells (RBCs) of several species, upon depletion of cellular glutathione (GSH). K-Cl COT is physiologically active in high potassium (HK), high GSH (HG) dog RBCs. In this unique model, we studied whether the same inverse relationship exists between GSH levels and K-Cl COT activity found in other species. The effects of GSH depletion by three different chemical reactions [nitrite (NO2)-mediated oxidation, diazene dicarboxylic acid bis- N, N-dimethylamide (diamide)-induced dithiol formation, and glutathione S-transferase (GST)-catalyzed conjugation of GSH with 1-chloro-2,4-dinitrobenzene (CDNB)] were tested on K-Cl COT and regulatory volume decrease (RVD). After 85% GSH depletion, all three interventions stimulated K-Cl COT half-maximally with the following order of potency: diamide > NO2 > CDNB. Repletion of GSH reversed K-Cl COT stimulation by 50%. Cl-dependent RVD accompanied K-Cl COT activation by NO2 and diamide. K-Cl COT activation at concentration ratios of oxidant/GSH greater than unity was irreversible, suggesting either nitrosothiolation, heterodithiol formation, or GST-mediated dinitrophenylation of protein thiols. The data support the hypothesis that an intact redox system, rather than the absolute GSH levels, protects K-Cl COT activity and cell volume regulation from thiol modification.

K-Cl co-transport (COT) is significantly higher in low K (LK), L-antigen (L) positive, than in high K (HK), M-antigen (M) positive, sheep red blood cells (SRBCs) and is inhibited by sheep allo-anti-L1 antibody. To answer the question of whether this difference in K-Cl co-transport activity resides at the level of the transporter or its regulation, a combined immunocytochemical and functional approach was taken. At least four isoforms of K-Cl COT encoded by different KCC genes are known, with 12 transmembrane domains and cytoplasmic C- and N-terminal domains (Ctd and Ntd, respectively). Polyclonal anti-rat (rt)KCC1 antibodies against a fusion peptide with 77 amino acids from the Ctd of rtKCC1 and anti-human (h)KCC3 against an 18-aa peptide from the Ntd of hKCC3, were prepared in rabbits (rb). Two distinctly separate protein bands of 180 and 145 kDa molecular mass were detected in hemoglobin-free ghosts from RBCs of two LK (one homozygous LL and one heterozygous LM) and one HK (homozygous MM) sheep by Western blots with rb anti-rtKCC1 and rb anti-hKCC3. Confocal microscopy showed specific immunostaining of KCC1 with rb anti-rtKCC1, and of KCC3 with rb anti-hKCC3, in white ghosts from both LK and HK SRBCs. To test the functional heterogeneity of K-Cl COT, the effect of the anti-L1 antibody was assessed on K-Cl COT activated by the kinase inhibitor staurosporine. Incubation of LK SRBCs with anti-L1 serum inhibited by 30% staurosporine-stimulated K-Cl COT suggesting that approximately two-thirds of the transport activity is independent of the L1 antigen. That staurosporine altered the L1 antigen/antibody reaction is unlikely since the action of another antibody, anti-Lp, stimulating the Na/K pump flux, was not modified. The present results, in conjunction with earlier work, lead to the hypothesis that the partial anti-L1 inhibition of K-Cl COT may be related to the molecular KCC dimorphism, seen in these cells with anti-KCC1 and anti-KCC3 antibodies.

K-Cl cotransport (KCC) is activated by nitric oxide donors and appears to be regulated by the cGMP signaling pathway. Expression of KCC mRNAs (KCC1–KCC4) in rat vascular smooth muscle cells (VSMCs) is unknown. We have reported the presence of KCC1 and KCC3 mRNAs in primary cultures of VSMCs by specific reverse transcription-polymerase chain reaction. KCC2 mRNA appeared at extremely low levels. KCC4 mRNA was undetectable. Semiquantitative reverse transcription-polymerase chain reaction revealed a 2:1 KCC1/KCC3 mRNA ratio in VSMCs. Depletion of protein kinase G (PKG)-1 from VSMCs did not change KCC3 mRNA expression. Analogous results were obtained with PKG-1-catalytic domain- and vector only-transfected VSMCs lacking endogenous PKG, suggesting no involvement of PKG-1 in the maintenance of basal KCC3 mRNA expression. However, 8-bromo-cGMP, a PKG stimulator, acutely increased KCC3 mRNA expression in a concentration- and time-dependent fashion; this effect was blocked by the PKG inhibitor KT5823 but not by actinomycin D. These findings show that VSMCs express mainly two mRNA isoforms, KCC1 and KCC3, and suggest that PKG participates post-transcriptionally in the acute KCC3 mRNA regulation. The role of KCC3 on cell volume and electrolyte homeostasis in response to PKG modulators remains to be determined.

This study examines the relative contributions of K-Cl cotransport and K+ channels to swelling-induced K+ fluxes in vascular smooth muscle cells (VSMC). DIOA known as a potent in

K-Cl cotransport (COT), a ouabain-insensitive, Cl-dependent bidirectional K flux, is ubiquitously present in all cells, plays a major role in ion and volume homeostasis, and is activated by cell swelling and a variety of chemical interventions. Lithium modulates several cation transport pathways and inhibits phospholipid turnover in red blood cells (RBCs). Lithium also inhibits K-Cl COT by an unknown mechanism. To test the hypothesis whereby Li inhibits swelling-activated K-Cl COT by altering either its osmotic response, its regulation, or by competing with K for binding sites, low K (LK) sheep (S) RBCs were loaded with Li by Na/Li exchange or the cation ionophore nystatin. K-Cl COT was measured as the Cl-dependent, ouabain-insensitive K efflux or Rb influx. The results show that Li altered the cell morphology, and increased both cell volume and diameter. Internal (Li i ) but not external (Li o ) Li inhibited swelling-activated K-Cl COT by 85% with an apparent K i of ∼7 mm. In Cl, Li i decreased K efflux at relative cell volumes between 0.9 and 1.2, and at external pHs between 7.2 and 7.4. Li i reduced the V max and increased the K m for K efflux in Cl. Furthermore, Li i increased the production of diacylglycerol in a bimodal fashion, without significant effects on the phosphatidylinositol concentration, and revealed the presence of a complete PI cycle in LK SRBCs. Finally, phorbol ester treatment and PD89059, an inhibitor of mitogen-activated protein kinase (ERK2) kinase, caused a time-dependent inhibition of K-Cl COT. Hence, Li i appears to inhibit K-Cl COT by acting at an allosteric site on the transporter or its putative regulators, and by modulation of the cellular phospholipid metabolism and a PKC-dependent regulatory pathway, causes an altered response of K-Cl COT to pH and volume.

We examined the effects of pH, internal ionized Ca (Ca2+i), cellular ATP, external divalent cations and quinine on Cl-independent ouabain-resistant K+ efflux in volume-clamped sheep red blood cells (SRBCs) of normal high (HK) and low (LK) intracellular K+ phenotypes. In LK SRBCs the K+ efflux was higher at pH 9.0 (350%) than at pHs 7. 4 and 6.5, and was inhibited by external divalent cations, quinine, and cellular ATP depletion. The above findings suggest that the increased K+ efflux at alkaline pH is due to the opening of ion channels or specific transporters in the cell membrane. In addition, K+ efflux was activated (100%) when Ca2+i was increased (+A23187, +Ca2+o) into the microm range. However, in comparison to human red blood cells, the Ca2+i-induced increase in K+ efflux in LK SRBCs was fourfold smaller and insensitive to quinine and charybdotoxin. The Na+ efflux was also higher at pH 9.0 than at pH 7.4, and activated (about 40%) by increasing Ca2+i. In contrast, in HK SRBCs the K+ efflux at pH 9.0 was neither inhibited by quinine nor activated by Ca2+i. These studies suggest the presence in LK SRBCs, of at least two pathways for Cl--independent K+ and Na+ transport, of which one is unmasked by alkalinization, and the other by a rise in Ca2+i.

Red blood cells (RBCs) from different mammalian species were investigated for the presence of diamide-induced oxidative activation of K-Cl cotransport reported to be present in sheep but absent in human RBCs. K efflux was measured in RBCs from human with hemoglobin (Hb) A or S, glucose-phosphate dehydrogenase (G6PDH) and a cytoskeletal deficiency, and from rat, mouse and rabbit. RBCs were incubated with diamide (0-1.0 mm) in K-free Cl or NO3 media of variable osmolalities (200-450 mOsM). Cl-dependent K efflux or K-Cl cotransport (estimated as the difference between K efflux rate constants in Cl and NO3) was activated by diamide in a sigmoidal fashion. Relative maximum K-Cl cotransport followed the sequence: human HbA (1)rabbit (1.8)sheep (6.9)human HbS (9.5) approximately rat (9.7). Relative diamide concentrations for half maximal activation of K-Cl cotransport followed the sequence: sheep (1.9)human Hb A (1)rabbit (0.75)human HbS and rat (0.67). Cell swelling in 200 mOsM doubled K-Cl cotransport in diamide, both in human HbA and S cells but reduced that in rat RBCs. In contrast, cell shrinkage at 450 mOsM obliterated K-Cl cotransport in human HbA and S but not in rat RBCs. Human RBCs with G6PDH and a cytoskeleton deficiency behaved like HbA RBCs. In mouse RBCs, diamide-activated K-Cl cotransport was 30% higher in isotonic than in hypotonic medium. In human HbA and S, and in low or high K sheep RBCs fractionated by Percoll density gradient, diamide increased the activity of K-Cl cotransport, an effect inversely correlated with cell density. Analysis of pooled data reveals that K-Cl cotransport accounted for about 80% of all K flux in Cl. There was a statistically significant correlation between K-Cl cotransport and K efflux in Cl (P0. 00001) and in NO3 (P0.00001). In conclusion, a diamide-activated K-Cl cotransport was present in human RBCs and in all other mammalian RBCs tested, with a large inter-, and for human and sheep, intraspecies variability for its maximum activity.

The present study further characterizes the two operational modes of K-Cl cotransport in low K+ sheep red blood cells previously described and proposed by us: (i) a membrane-bound cotransport entity devoid of modulation by putative kinases/phosphatases, and (ii) an ATP- and Mg-modulated activity. We now report that quinine, spermine, and 300 µM Cai2+ inhibit (40-100%) K-Cl efflux in ATP- and Mgi2+-depleted cells and hence interact directly with the cotransporter moiety. The sensitivity of K-Cl cotransport to calyculin, however, was modulated by the ATP content of the cells: after depletion of both ATP and Mgi2+ the flux was insensitive to the inhibitor, whereas at about 120 µmol ATP/L cells calyculine inhibited K-Cl efflux by 95-100% in Mgi2+-free cells. The K-Cl cotransporter itself, free of modulation by kinases/phosphatases, ‘senses’ changes in cell volume perhaps at the membrane/cytoskeleton level. Cellular ATP then switches the co-transporter’s operational mode so that under physiological conditions the rate of K-Cl efflux is under the control of a putative kinases/phosphatases pathway.

Chelerythrine [CET], a protein kinase C [PKC] inhibitor, is a prop-apoptotic BH3-mimetic binding to BH1-like motifs of Bcl-2 proteins. CET action was examined on PKC phosphorylation-dependent membrane transporters (Na+/K+ pump/ATPase [NKP, NKA], Na+-K+-2Cl+ [NKCC] and K+-Cl- [KCC] cotransporters, and channel-supported K+ loss) in human lens epithelial cells [LECs].K+ loss and K+ uptake, using Rb+ as congener, were measured by atomic absorption/emission spectrophotometry with NKP and NKCC inhibitors, and Cl- replacement by NO3ˉ to determine KCC. 3H-Ouabain binding was performed on a pig renal NKA in the presence and absence of CET. Bcl-2 protein and NKA sequences were aligned and motifs identified and mapped using PROSITE in conjunction with BLAST alignments and analysis of conservation and structural similarity based on prediction of secondary and crystal structures.CET inhibited NKP and NKCC by90% (IC50 values ~35 and ~15 μM, respectively) without significant KCC activity change, and stimulated K+ loss by ~35% at 10-30 μM. Neither ATP levels nor phosphorylation of the NKA α1 subunit changed. 3H-ouabain was displaced from pig renal NKA only at 100 fold higher CET concentrations than the ligand. Sequence alignments of NKA with BH1- and BH3-like motifs containing pro-survival Bcl-2 and BclXl proteins showed more than one BH1-like motif within NKA for interaction with CET or with BH3 motifs. One NKA BH1-like motif (ARAAEILARDGPN) was also found in all P-type ATPases. Also, NKA possessed a second motif similar to that near the BH3 region of Bcl-2.Findings support the hypothesis that CET inhibits NKP by binding to BH1-like motifs and disrupting the α1 subunit catalytic activity through conformational changes. By interacting with Bcl-2 proteins through their complementary BH1- or BH3-like-motifs, NKP proteins may be sensors of normal and pathological cell functions, becoming important yet unrecognized signal transducers in the initial phases of apoptosis. CET action on NKCC1 and K+ channels may involve PKC-regulated mechanisms; however, limited sequence homologies to BH1-like motifs cannot exclude direct effects.

Swelling-induced human erythrocyte K-Cl cotransport is membrane potential independent and capable of uphill transport. However, a complete thermodynamic analysis of basal and stimulated K-Cl cotransport, at constant cell volume, is missing. This study was performed in low K sheep red blood cells before and after reducing cellular free Mg into the nanomolar range with the divalent cation ionophore A23187 and a chelator, an intervention known to stimulate K-Cl cotransport. The anion exchange inhibitor 4,4'diisothiocyanato-2,2'disulfonic stilbene was used to clamp intracellular pH and Cl or NO3 concentrations. Cell volume was maintained constant as external and internal pH differed by more than two units. K-Cl cotransport was calculated from the K effluxes and Rb (as K congener) influxes measured in Cl and NO3, at constant internal K and external anions, and variable concentrations of extracellular Rb and internal anions, respectively. The external Rb concentration at which net K-Cl cotransport is zero was defined as flux reversal point which changed with internal pH and hence Cl. Plots of the ratio of external Rb concentrations corresponding to the flux reversal points and the internal K concentration versus the ratio of the internal and external Cl concentrations (i.e., the Donnan ratio of the transported ions) yielded slopes near unity for both control and low internal Mg cells. Thus, basal as well as low internal Mg-stimulated net K-Cl cotransport depends on the electrochemical potential gradient of KCl.

Cellular pH, ionized Mg (Mgi2+), and MgATP concentration of red blood cells, concomitantly with cell volume, change transiently during circulation. The action of these three effectors on Cl-dependent K efflux was examined in low-K sheep red blood cells with constant cell volume. Activation of K-Cl efflux by Mgi2+ extraction required ATP, suggesting that phosphorylation of a putative component occurred before Mgi2+ extraction. Conversely, Mg and ATP were synergistic inhibitors of K-Cl cotransport, since maximal inhibition was observed only in cells containing both ATP and > 300 microM Mgi2+. Both findings suggest dual roles for Mg and ATP. At 300-600 microM Mgi2+, lowering the pH from approximately 7.4 to approximately 6.5 stimulated K-Cl efflux only in fed cells, suggesting that protons oppose or release the inhibition by Mgi2+ and ATP. A direct effect of both protons and Mgi2+ on the cotransporter is suggested by their inhibition of K-Cl efflux in ATP-depleted cells. These findings are discussed in light of the current phosphorylation/dephosphorylation hypothesis.

The fetal human lens epithelial cell (LEC) line (FHL124) possesses all four K(+)Cl(-) (KCC) cotransporter isoforms, KCC1-4, despite KCC2 being typically considered a neuronal isoform. Since at least two spliced variants, KCC2a and KCC2b, are co-expressed in cells of the central nervous system, this study sought to define the KCC2 expression profile in FHL124 cells. KCC2a, but not KCC2b transcripts were detected by reverse transcriptase polymerase chain reaction (RT-PCR). Proteins of molecular weights ranging from 95 to 135 kDa were found by Western blotting using non-variant specific anti-KCC2 antibodies directed against two different regions of the KCC2 proteins, and by biotinylation suggesting membrane expression. Immunofluorescence revealed membrane and punctate cytoplasmic staining for KCC2. Low levels of cytosolic αA and αB crystallines, and neuron-specific enolase were also detected contrasting with the strong membrane immunofluorescence staining for the Na/K ATPase α1 subunit. Since the lack of neuron-specific expression of the KCC2b variant in non-neuronal tissues has been proposed under control of a neuron-restrictive silencing element in the KCC2 gene, we hypothesize that this control may be lifted for the KCC2a variant in the FHL124 epithelial cell culture, a non-neuronal tissue of ectodermal origin.

Hydroxyurea (HU) significantly increases fetal hemoglobin (Hb) production and concomitantly affects passive erythrocyte K transport and cell volume in patients homozygous for Hb S, thus decreasing disease severity. Red blood cells (RBCs) with Hb S display a greater adherence to vascular endothelial cells (VECs) than do Hb A cells, thus increasing the probability of vaso-occlusive crisis. The effect of HU on the structure and function of VECs is still unknown. In the present study, HU significantly changed, in a dose-dependent manner, the morphology and monovalent cation composition of cultured VECs after incubation in normal culture medium for up to 10 days in the absence and presence of 0.3 (therapeutic dose) and 3.0 (toxic dose) mmol/L HU. Treated cells showed significant morphologic changes such as an increase in apparent cell size and the formation of multinucleated giant cells. The protein content per dish decreased by 50% and 80% at 0.3 and 3.0 mmol/L HU, respectively, accompanied by an increase in cell Na (maximum, approximately 200%) and cell K (maximum, approximately 50%) contents at about days 4 to 6 and 8 to 10, respectively. In addition, HU decreased RBC adherence to VECs in experiments with 51Cr- loaded Hb A or Hb S RBCs. The HU-induced changes in VEC morphology, cation composition, and RBC adherence may be caused or accompanied by alterations in cell membrane permeability, transformation of endothelial cells, or decreased number/density of VEC adhesion molecules. Precise mechanisms of the HU effects warrant further investigation in light of the reported beneficial effects of HU in the treatment of sickle cell anemia.

THE STUDY BY LANG AND COLLEAGUES (2), published in this issue, shows that red blood cells (RBCs), undergoing suicidal death or “eryptosis,” attach to human umbilical vascular endothelial cells (HUVECs) under experimental flow conditions via their CXC ligand 16 (CXCL16)/SR-PSOX transmembrane ligands with the potential to greatly impact the microvascular circulation. CXCL16, a chemokine with a cysteine-X-cysteine motif, occurs either in soluble form or as a transmembrane receptor (16) that can be tyrosine phosphorylated and bind Src homology 2-containing proteins; it is synonymous with SR-PSOX, the scavenger receptor for phosphatidylserine (PS) and oxidized low-density lipoproteins (oxLDL) (19). Here, I review developments that underlie this discovery (2), emphasizing the complex cellular mechanisms of erythrocyte-augmented/induced vascular occlusion in conjunction with their suicidal death (eryptosis). After 120 days in the circulation, 3 10 9 or one-third milliliter human RBCs/day undergo demise within the reticuloendothelial system by macrophage-mediated phagocytosis. Macrophages, which trap and digest old (but not intact mature) erythrocytes, recognize “senescent” antigens (SCA) of 62 kDa suggested to be related to band-3 proteins involved in Cl/HCO3 exchange (7). An alternate hypothesis considers autoantibodies formed against SCA epitopes derived from band-3 aggregation (13). Simultaneously, another concept emerged: reduction of the number of sialic acid residues (10 9 /cell) would uncover the presence of Thomsen (T)

Nematocytes, the stinging cells of Cnidarians, have a cytoplasm confined to a thin rim. The main cell body is occupied by an organoid, the nematocyst, containing the stinging tubule and venom. Exposed to hypotonic shock, nematocytes initially swell during an osmotic phase (OP) and then undergo regulatory volume decrease (RVD) driven by K(+), Cl(-) and obligatory water extrusion mechanisms. The purpose of this report is to characterize the OP. Nematocytes were isolated by the NaSCN/Ca(2+) method from tentacles of the jellyfish Pelagia noctiluca, collected in the Strait of Messina, Italy. Isolated nematocytes were subjected to hyposmotic shock in 65% artificial seawater (ASW) for 15 min. The selective aquaporin water channel inhibitor HgCl(2) (0.1-25 μM) applied prior to osmotic shock prevented the OP and thus RVD. These effects were attenuated in the presence of 1mM dithiothreitol (DTT), a mercaptide bond reducing agent. AgNO(3) (1 μM) and TEA (tetraethylammonium, 100 μM), also reported to inhibit water transport, did not alter the OP but significantly diminished RVD, suggesting different modes of action for the inhibitors tested. Based on estimates of the nematocyte surface area and volume, and OP duration, a relative water permeability of ~10(-7) cm/sec was calculated and the number of putative aquaporin molecules mediating the OP was estimated. This water permeability is 3-4 orders of magnitude lower in comparison to higher order animals and may constitute an evolutionary advantage for Cnidarian survival.

This chapter defines the emergence of K + –Clˉ cotransport as a functional transport entity. Secondary active transport, where the flux of one of the transported species moving downhill provides the driving force for movement of another ion or non-electrolyte, is a common membrane transport mechanism. Ionic species providing the driving force include Na + , H + and K + . Coupled substrates include different sugars, amino acids and other organic molecules translocated by discrete families of transporters. The family of cation-chloride cotransporters (CCCs) represents a series of membrane proteins, the SLC12 superfamily, capable of the electroneutral coupled transport of Clˉ with Na + (NCC), K + (KCCs) or both Na + and K + (NKCCs). These transporters were recognized and studied in RBCs and epithelia, but more recent work has broadened their known distribution to include neuronal tissue, endothelial and vascular smooth muscle cells, and other cell types, including certain neoplasias. Molecular biology has also revealed a variety of CCC isoforms, with differing tissue distribution and physiological functions.

The nature and role of potassium (K) and water transport mediating hyposmotically-induced regulatory volume decrease (RVD) were studied in nematocytes dissociated with 605 mM thiocyanate from acontia of the Anthozoan Aiptasia diaphana. Cell volume and hence RVD were calculated from the inverse ratios of the cross sectional areas of nematocytes (A/A(o)) measured before (A(o)) and after (A) challenge with 65% artificial sea water (ASW). To distinguish between K channels and K-Cl cotransport (KCC), external sodium (Na) and chloride (Cl) were replaced by K and nitrate (NO(3)), respectively. Inhibitors were added to identify K channels (barium, Ba), and putative kinase (N-ethylmaleimide, NEM) and phosphatase (okadaic acid, OA) regulation of KCC. In 65% NaCl ASW, nematocytes displayed a biphasic change in A/A(o), peaking within 4 min due to osmotic water entry and thereafter declining within 6 min due to RVD. Changing NaCl to KCl or NaNO(3) ASW did not affect the osmotic phase but attenuated RVD, consistent with K channel and KCC mechanisms. Ba (3 mM) inhibited RVD. NEM and OA, applied separately, inhibited the osmotic phase and muted RVD suggesting primary action on water transport (aquaporins). NEM and OA together reduced the peak A/A(o) ratio during the osmotic phase whereas RVD was inhibited when OA preceded NEM. Thus, both K channels and KCC partake in the nematocyte RVD, the extent of which is determined by functional thiols and dephosphorylation of putative aquaporins facilitating the preceding osmotic water shifts.

Erythrocytes possess a Cl-dependent, Na-independent K transport system cotransporting K and Cl in a 1:1 stoichiometry that is membrane potential independent. This K-Cl cotransporter is stimulated by cell swelling, acidification, Mg depletion, and thiol modification. Cell shrinkage, elevation of cellular divalent ions, thiol alkylation, phosphatase inhibitors, and derivatives of certain loop diuretics and stilbenes are inhibitory. Thus regulation of K-Cl cotransport at the membrane and cytoplasmic levels is highly complex. Basal K-Cl cotransport decreases with cellular maturation, whereas its modes of stimulation and inhibition are variable between species. The physiological inactivation appears to be prevented in low-K animal erythrocytes. In certain human hemoglobinopathies, K-Cl cotransport may be the cause of cellular dehydration and volume decrease. K-Cl cotransport occurs also in nonerythroid cells, such as in epithelial and liver cells of other species. At the threshold of molecular characterization, this comprehensive review places our present understanding of the mechanisms modulating K-Cl cotransport physiologically and pathophysiologically into kinetic and thermodynamic perspectives.