Inhibition of the cardiac inward rectifier potassium currents by KB-R7943

KB-R7943 (2-[2-[4-(4-nitrobenzyloxy)phenyl]ethyl]isothiourea) was developed as a specific inhibitor of the sarcolemmal sodium–calcium exchanger (NCX) with potential experimental and therapeutic use. However, KB-R7943 is shown to be a potent blocker of several ion currents including inward and delayed rectifier K⁺ currents of cardiomyocytes. To further characterize KB-R7943 as a blocker of the cardiac inward rectifiers we compared KB-R7943 sensitivity of the background inward rectifier (I_K1) and the carbacholine-induced inward rectifier (I_KACh) currents in mammalian (Rattus norvegicus; rat) and fish (Carassius carassius; crucian carp) cardiac myocytes. The basal I_K1 of ventricular myocytes was blocked with apparent IC50-values of 4.6 × 10^− 6 M and 3.5 × 10^− 6 M for rat and fish, respectively. I_KACh was almost an order of magnitude more sensitive to KB-R7943 than I_K1 with IC₅₀-values of 6.2 × 10^− 7 M for rat and 2.5 × 10^− 7 M for fish. The fish cardiac NCX current was half-maximally blocked at the concentration of 1.9–3 × 10^− 6 M in both forward and reversed mode of operation. Thus, the sensitivity of three cardiac currents to KB-R7943 block increases in the order I_K1 ~ I_NCX < I_KACh. Therefore, the ability of KB-R7943 to block inward rectifier potassium currents, in particular I_KACh, should be taken into account when interpreting the data with this inhibitor from in vivo and in vitro experiments in both mammalian and fish models.     

The topology of the C-terminal sections of the NCX1 Na+/Ca2+ exchanger and the NCKX2 Na+/Ca2+-K+ exchanger

Mammalian Na⁺/Ca²⁺ (NCX) and Na⁺/Ca²⁺-K⁺ exchangers (NCKX) are polytopic membrane proteins that play critical roles in calcium homeostasis in many cells. Although hydropathy plots for NCX and NCKX are very similar, reported topological models for NCX1 and NCKX2 differ in the orientation of the three C-terminal transmembrane segments (TMS). NCX1 is thought to have 9 TMS and a re-entrant loop, whereas NCKX2 is thought to have 10 TMS. The current topological model of NCKX2 is very similar to the 10 membrane spanning helices seen in the recently reported crystal structure of NCX_MJ, a distantly related archaebacterial Na⁺/Ca²⁺ exchanger. Here we reinvestigate the orientation of the three C-terminal TMS of NCX1 and NCKX2 using mass-tagging experiments of substituted cysteine residues. Our results suggest that NCX1, NCKX2 and NCX_MJ all share the same 10 TMS topology.     

Roles of transient receptor potential canonical (TRPC) channels and reverse-mode Na/Ca exchanger on cell proliferation in human cardiac fibroblasts: Effects of transforming growth factor β1

Expression of transient receptor potential canonical channels (TRPC) and the effects of transforming growth factor-β1 (TGF-β1) on Ca²⁺ signals and fibroblast proliferation were investigated in human cardiac fibroblasts. The conventional and quantitative real-time RT-PCR, western blot, immunocytochemical analysis, and intracellular Ca²⁺ concentration [Ca²⁺]_i measurement were applied. Cell proliferation and cell cycle progression were assessed using MTT assays and fluorescence activated cell sorting. Human cardiac fibroblasts have the expression of TRPC1,3,4,6 mRNA and proteins. 1-oleoyl-2-acetyl-sn-glycerol (OAG) and thapsigargin induced extracellular Ca²⁺-mediated [Ca²⁺]_i rise. siRNA for knock down of TRPC6 reduced OAG-induced Ca²⁺ entry. Hyperforin as well as angiotensin II (Ang II) induced Ca²⁺ entry. KB-R7943, a reverse-mode Na⁺/Ca²⁺ exchanger (NCX) inhibitor, and/or replacement of Na⁺ with NMDG⁺ inhibited thapsigargin-, OAG- and Ang II-induced Ca²⁺ entry. Treatment with TGF-β1 increased thapsigargin-, OAG- and Ang II-induced Ca²⁺ entry with an enhancement of TRPC1,6 protein expression, suppressed by KB-R7943. TGF-β1 and AngII promoted cell cycle progression from G0/G1 to S/G2/M and cell proliferation. A decrease of the extracellular Ca²⁺ and KB-R7943 suppressed it. Human cardiac fibroblasts contain several TRPC-mediated Ca²⁺ influx pathways, which activate the reverse-mode NCX. TGF-β1 enhances the Ca²⁺ influx pathways requiring Ca²⁺ signals for its effect on fibroblast proliferation.     

A key role for reverse Na+/Ca2+ exchange influenced by the actin cytoskeleton in store-operated Ca2+ entry in human platelets: evidence against the de novo conformational coupling hypothesis

We have previously demonstrated a role for the reorganization of the actin cytoskeleton in store-operated calcium entry (SOCE) in human platelets and interpreted this as evidence for a de novo conformational coupling step in SOCE activation involving the type II IP(3) receptor and the platelet hTRPC1-containing store-operated channel (SOC). Here, we present evidence challenging this model. The actin polymerization inhibitors cytochalasin D or latrunculin A significantly reduced Ca2+ but not Mn2+ or Na+ entry into thapsigargin (TG)-treated platelets. Jasplakinolide, which induces actin polymerization, also inhibited Ca2+ but not Mn2+ or Na+ entry. However, an anti-hTRPC1 antibody inhibited TG-evoked entry of all three cations, indicating that they all permeate an hTRPC1-containing store-operated channel (SOC). These results indicate that the reorganization of the actin cytoskeleton is not involved in SOC activation. The inhibitors of the Na+/Ca2+ exchanger (NCX), KB-R7943 or SN-6, caused a dose-dependent inhibition of Ca2+ but not Mn2+ or Na+ entry into TG-treated platelets. The effects of the NCX inhibitors were not additive with those of actin polymerization inhibitors, suggesting a common point of action. These results indicate a role for two Ca2+ permeable pathways activated following Ca2+ store depletion in human platelets: A Ca2+-permeable, hTRPC1-containing SOC and reverse Na+/Ca2+ exchange, which is activated following Na+ entry through the SOC and requires a functional actin cytoskeleton.     

<Superscript>65</Superscript>Zn<Superscript>2+</Superscript> transport by lobster hepato-pancreatic baso-lateral membrane vesicles

The lobster (Homarus americanus) hepato-pancreatic epithelial baso-lateral cell membrane possesses three transport proteins that transfer calcium between the cytoplasm and hemolymph: an ATP-dependent calcium ATPase, a sodium-calcium exchanger, and a verapamil-sensitive cation channel. We used standard centrifugation methods to prepare purified hepato-pancreatic baso-lateral membrane vesicles and a rapid filtration procedure to investigate whether ⁶⁵Zn²⁺ transfer across this epithelial cell border occurs by any of these previously described transporters for calcium. Baso-lateral membrane vesicles were osmotically reactive and exhibited a time course of uptake that was linear for 10–15 s and approached equilibrium by 120 s. In the absence of sodium, ⁶⁵Zn²⁺ influx was a hyperbolic function of external zinc concentration and followed the Michaelis-Menten equation for carrier transport. This carrier transport was stimulated by the addition of 150 M ATP (increase in K_m and J_max) and inhibited by the simultaneous presence of 150 mol l^–1 ATP+250 mol l^–1 vanadate (decrease in both K_m and J_max). In the absence of ATP, ⁶⁵Zn²⁺ influx was a sigmoidal function of preloaded vesicular sodium concentration (0, 5, 10, 20, 30, 45, and 75 mmol l^–1) and exhibited a Hill Coefficient of 4.03±1.14, consistent with the exchange of 3 Na⁺/1Zn²⁺. Using Dixon analysis, calcium was shown to be a competitive inhibitor of baso-lateral membrane vesicle ⁶⁵Zn²⁺ influx by both the ATP-dependent (K_i=205 nmol l^–1 Ca²⁺) and sodium-dependent (K_i=2.47 mol l^–1 Ca²⁺) transport processes. These results suggest that zinc transport across the lobster hepato-pancreatic baso-lateral membrane largely occurred by the ATP-dependent calcium ATPase and sodium-calcium exchanger carrier proteins.Communicated by: I.D. Hume     

Advanced procedures for separation and analysis of low molecular weight inhibitor (NCXIF) of the cardiac sodium-calcium exchanger

A low molecular weight inhibitor (NCX(IF)) of the cardiac Na/Ca exchanger, isolated from the calf ventricle tissue, is capable of regulating the muscle strip's contractility and relaxation without involving the beta-activation pathway. The structural analysis of NCX(IF) requires highly purified preparations that fulfill the demanding requirements for mass spectra and NMR analyses. No such preparation is yet available. To this end, new HPLC procedures were developed by a combination of the reverse phase, normal phase, and HILIC (hydrophilic liquid chromatography) techniques. The specific activity of NCX(IF) is 10(5) times higher in the purified preparations (as compared to the crude extract) showing a 2-5% yield of total inhibitory activity and 20-100 microg content of final material. The purification yield reveals that 1 kg ventricle muscle contains 0.1-0.2 mg NCX(IF), meaning that the tissue concentrations of NCX(IF) may reach 10(-7)-10(-6) M. The diode-array scanning of purified preparations of NCX(IF) shows a homogeneous 3D peak with a maximal absorption at 202 nm. These spectral properties may represent a five-membered ring (e.g., proline, histidine) and/or simple chemical groups (like amine, carbonyl, ester, etc.), but not an aromatic ring or complex conjugates (alkyne, alkene, aldehyde, etc.). NCX(IF) does not respond to phenol/sulfur reagent, suggesting that it lacks reducing (aldo) sugar. NCX(IF) shows a faint response to fluorescamine, meaning that it may contain an amino group (or its derivative). It is believed that a combination of presently developed procedures with LC/MS and LC/MS/MS may provide a useful tool for structural analysis of NCX(IF).     

Essential Role of the CBD1-CBD2 Linker in Slow Dissociation of Ca2+ from the Regulatory Two-domain Tandem of NCX1

In NCX proteins CBD1 and CBD2 domains are connected through a short linker (3 or 4 amino acids) forming a regulatory tandem (CBD12). Only three of the six CBD12 Ca²⁺-binding sites contribute to NCX regulation. Two of them are located on CBD1 (K_d = ∼0.2 μm), and one is on CBD2 (K_d = ∼5 μm). Here we analyze how the intrinsic properties of individual regulatory sites are affected by linker-dependent interactions in CBD12 (AD splice variant). The three sites of CBD12 and CBD1 + CBD2 have comparable K_d values but differ dramatically in their Ca²⁺ dissociation kinetics. CBD12 exhibits multiphasic kinetics for the dissociation of three Ca²⁺ ions (k_r = 280 s⁻¹, k_f = 7 s⁻¹, and k_s = 0.4 s⁻¹), whereas the dissociation of two Ca²⁺ ions from CBD1 (k_f = 16 s⁻¹) and one Ca²⁺ ion from CBD2 (k_r = 125 s⁻¹) is monophasic. Insertion of seven alanines into the linker (CBD12–7Ala) abolishes slow dissociation of Ca²⁺, whereas the kinetic and equilibrium properties of three Ca²⁺ sites of CBD12–7Ala and CBD1 + CBD2 are similar. Therefore, the linker-dependent interactions in CBD12 decelerate the Ca²⁺ on/off kinetics at a specific CBD1 site by 50–80-fold, thereby representing Ca²⁺ “occlusion” at CBD12. Notably, the kinetic and equilibrium properties of the remaining two sites of CBD12 are “linker-independent,” so their intrinsic properties are preserved in CBD12. In conclusion, the dynamic properties of three sites are specifically modified, conserved, diversified, and integrated by the linker in CBD12, thereby generating a wide range dynamic sensor.     

Molecular characterization of cytotoxic and resistance mechanisms induced by NCX 4040, a novel NO-NSAID, in pancreatic cancer cell lines*

Although non steroidal antiinflammatory drugs (NSAIDs) have been shown to be effective as chemopreventive agents, important side-effects limit their clinical use. A promising novel class of drugs, nitric oxide-donating NSAIDs (NO-NSAIDs), has been found to be more active than classical NSAIDs. This study explored the effect of the NO-donating aspirin derivative, NCX 4040, on three human pancreatic adenocarcinoma cell lines (Capan-2, MIA PaCa-2 and T3M4). NCX 4040 activity was compared with that of NCX 4016 (an NO₂-positional isomer of NCX 4040), SNAP (a standard NO-releasing molecule), NCX 4042 (denitrated analog of NCX 4040), and aspirin. NCX 4040 showed a striking cytocidal activity in all cell lines, already inducing significant percentages of apoptotic cells at 10 μM in Capan-2 cell lines. This study focused on the biological mechanisms of sensitivity and resistance to NCX 4040, highlighting that the cytotoxic action of this drug may be due to the hyperexpression of Bax, its translocation to the mitochondria, the release of Cytochrome C, and the activation of caspases-9 and -3, overall in a p53-independent manner. Moreover, the use of a specific COX-2 inhibitor (NS 398) in the experimental models showed that COX-2 hyperexpression could partially explain the resistance mechanisms to NCX 4040. This study was supported by Istituto Oncologico Romagnolo, Forlì, and by the Italian, Ministry of Health, 2004.     

GABA transporters mediate glycine release from cerebellum nerve endings: roles of Ca(2+)channels, mitochondrial Na(+)/Ca(2+) exchangers, vesicular GABA/glycine transporters and anion channels

GABA transporters accumulate GABA to inactivate or reutilize it. Transporter-mediated GABA release can also occur. Recent findings indicate that GABA transporters can perform additional functions. We investigated how activation of GABA transporters can mediate release of glycine. Nerve endings purified from mouse cerebellum were prelabeled with [(3)H]glycine in presence of the glycine GlyT1 transporter inhibitor NFPS to label selectively GlyT2-bearing terminals. GABA was added under superfusion conditions and the mechanisms of the GABA-evoked [(3)H]glycine release were characterized. GABA stimulated [(3)H]glycine release in a concentration-dependent manner (EC(50) = 8.26 μM). The GABA-evoked release was insensitive to GABA(A) and GABA(B) receptor antagonists, but it was abolished by GABA transporter inhibitors. About 25% of the evoked release was dependent on external Ca(2+) entering the nerve terminals through VSCCs sensitive to ω-conotoxins. The external Ca(2+)-independent release involved mitochondrial Ca(2+), as it was prevented by the Na(+)/Ca(2+) exchanger inhibitor CGP37157. The GABA uptake-mediated increases in cytosolic Ca(2+) did not trigger exocytotic release because the [(3)H]glycine efflux was insensitive to clostridial toxins. Bafilomycin inhibited the evoked release likely because it reduced vesicular storage of [(3)H]glycine so that less [(3)H]glycine can become cytosolic when GABA taken up exchanges with [(3)H]glycine at the vesicular inhibitory amino acid transporters shared by the two amino acids. The GABA-evoked [(3)H]glycine efflux could be prevented by niflumic acid or NPPB indicating that the evoked release occurred essentially by permeation through anion channels. In conclusion, GABA uptake into GlyT2-bearing cerebellar nerve endings triggered glycine release which occurred essentially by permeation through Ca(2+)-dependent anion channels. Glial GABA release mediated by anion channels was proposed to underlie tonic inhibition in the cerebellum; the present results suggest that glycine release by neuronal anion channels also might contribute to tonic inhibition.     

NCX3 is a major functional isoform of the sodium-calcium exchanger in osteoblasts

The calcium phosphate-based skeleton of vertebrates serves as the major reservoir for metabolically available calcium ions. The skeleton is formed by osteoblasts which first secrete a proteinaceous matrix and then provide Ca++ for the calcification process. The two calcium efflux ports found in most cells are the plasma membrane Ca-ATPase (PMCA) and the sodium-calcium exchanger (NCX). In osteoblasts, PMCA and NCX are located on opposing sides of the cell with NCX facing the mineralizing bone surface. Two isoforms of NCX have been identified in osteoblasts NCX1, and NCX3. The purpose of this study was to determine the extent to which each of the two NCX isoforms support delivery of Ca++ into sites of calcification and to discern if one could compensate for the other. SiRNA technology was used to knockdown each isoform separately in MC3T3-E1 osteoblasts. Osteoblasts in which either NCX1 or NCX3 was impaired were tested for Ca++ efflux using the Ca++ specific fluorophore, fluo-4, in a sodium-dependent calcium uptake assay adapted for image analysis. NCX3 was found to serve as a major contributor of Ca++ translocation out of osteoblasts into calcifying bone matrix. NCX1 had little to no involvement.