An Unknown Endogenous Inhibitor of Na/Ca Exchange Can Enhance the Cardiac Muscle Contractility

Hiller, Reuben; Shpak, Chagit; Shavit, Gabriel; Shpak, Beny; Khananshvili, Daniel

doi:10.1006/bbrc.2000.3645

Cited by 10 publications

(19 citation statements)

References 30 publications

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“…What is known is that NCX IF is a small, polar, and uncharged compound that is poorly retained even on polar-embedded RP columns. HILIC on an amide silica column was used alongside gel filtration, cation exchange, and C 30 RP separation a few years ago [151], and recently the same authors revisited the problem [152] with a set of new columns of which two were HILIC phases (poly(succinimide) and poly(2-hydroxyethyl aspartamide) silica) and one a b-cyclodextran silica operated in NP mode in their continued efforts to purify the elusive substance for identification. The HILIC and NP separations were found imperative for the overall purification scheme, but provided a much lower yield of the NCX IF activity (30 -60%) compared to the RP columns.…”

Section: Pharmaceutical Development and Drug Discoverymentioning

confidence: 99%

Hydrophilic interaction chromatography

Hemström

Irgum²

2006

J of Separation Science

1,086

818

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Separation of polar compounds on polar stationary phases with partly aqueous eluents is by no means a new separation mode in LC. The first HPLC applications were published more than 30 years ago, and were for a long time mostly confined to carbohydrate analysis. In the early 1990s new phases started to emerge, and the practice was given a name, hydrophilic interaction chromatography (HILIC). Although the use of this separation mode has been relatively limited, we have seen a sudden increase in popularity over the last few years, promoted by the need to analyze polar compounds in increasingly complex mixtures. Another reason for the increase in popularity is the widespread use of MS coupled to LC. The partly aqueous eluents high in ACN with a limited need of adding salt is almost ideal for ESI. The applications now encompass most categories of polar compounds, charged as well as uncharged, although HILIC is particularly well suited for solutes lacking charge where coulombic interactions cannot be used to mediate retention. The review attempts to summarize the ongoing discussion on the separation mechanism and gives an overview of the stationary phases used and the applications addressed with this separation mode in LC.

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Section: Pharmaceutical Development and Drug Discoverymentioning

confidence: 99%

Hydrophilic interaction chromatography

Hemström

Irgum²

2006

J of Separation Science

1,086

818

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“…Since milligram quantities of purified NCX IF are required for structural studies, here we describe further improvements in the purification procedures. Although our previous work provided accumulating evidence for the NCX IF -induced inhibition of NCX1 activities in vitro (17)(18)(19) and for the modulation of muscle contractility (17,(20)(21)(22)(23), there was no evidence for inhibition of NCX1 in the single-cell system. Therefore, the present work represents the first attempt at measuring the direct actions of NCX IF on the Na + /Ca 2+ exchanger activities in a single-cell system by performing simultaneous measurements of I NCX and cytosolic [Ca 2+ ] i .…”

Section: Discussionmentioning

confidence: 91%

“…1). The new purification scheme provides a final yield of 0.03 mg NCX IF per kg of starting material, which is at least 3-4 times higher than the highest yields obtained in previous preparations (17)(18)(19)(20)(21)(22)(23). A major reason for this improvement is the efficacy of the new HILIC columns, Krumasil-Silica (Fig.…”

Section: Hplc Procedures For Ncx If Purificationmentioning

confidence: 91%

“…The sarcolemma vesicles were obtained according to established procedures (27,28). The Na i -dependent 45 Ca uptake was initiated by rapid dilution of Na-loaded vesicles in an assay medium containing 45 Ca with lipid/protein-free aliquots of NCX IF (17)(18)(19)(20)(21). The inhibitory capacity of NCX IF preparations was quantitatively evaluated in arbitrary inhibitory units (one inhibitory unit represents the amount of NCX IF that inhibits the Na i -dependent 45 Cauptake by 1% under fixed experimental conditions) (17)(18)(19)(20)(21)(22)(23).…”

Section: Effect Of Ncx If On Na I -Dependent 45 Ca Uptake In Sarcolemmentioning

confidence: 99%

“…Mass-spectroscopic and gel-filtration analyses of active substances revealed a small (350-550 Da) polar molecule that is not retained on the regular RP-HPLC columns (17,18). Chemical tests and spectroscopic analyses showed no content for conjugated bonds, aromatic rings, primary amines, or aldehyde groups, thereby resembling the properties of non-reducing "sugar-like" molecules (17,18). This low-abundance compound inhibits NCX1 activites in the range of 10 −6 -10 −7 M (18), but it has no effect on the Na + /K + -ATPase or SR Ca 2+ -ATPase activities (19).…”

mentioning

confidence: 99%

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Direct Loading of the Purified Endogenous Inhibitor into the Cytoplasm of Patched Cardiomyocytes Blocks the Ion Currents and Calcium Transport through the NCX1 Protein

et al. 2008

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The Na(+)-Ca(2+) exchanger in mammalian heart muscle (NCX1) is the central transporter protein that regulates extrusion of Ca(2+) from the heart cell. However, the functional biochemistry and physiology of NCX1 have been severely hampered by the absence of any specific high-affinity inhibitor. Here we describe advanced procedures for purifying a candidate inhibitor, previously called endogenous inhibitor factor (NCX(IF)), and demonstrate its direct actions on NCX1 activities in the single-cell system. A combination of advanced HILIC (hydrophilic interaction liquid chromatography) procedures with analytical tests suggests that the properties of NCX(IF) resemble those of a small (disaccharide size) polar molecule lacking any aromatic rings, conjugated bonds, or a primary amino group. The effects of NCX(IF) on the NCX1-mediated ion currents (I(NCX)) and cytosolic Ca(2+) extrusion were detected by a combination of patch-clamp and confocal microscopy under conditions in which the purified NCX(IF) was directly loaded into the cytoplasm of patched cardiomyocytes. It was demonstrated that cytosolic NCX(IF) blocks the Ca(2+)-activated NCX1 inward current and the accompanying extrusion of Ca(2+) from the cell with high efficacy. A constant fraction of NCX1 inhibition was observed under conditions in which the cytosolic [Ca(2+)](i) was varied at fixed doses of NCX(IF), suggesting that the degree of inhibition is controlled by NCX(IF) dose and not by cytosolic Ca(2+) concentration. NCX(IF) blocks equally well both the Ca(2+) extrusion and Ca(2+) entry modes of NCX1, consistent with thermodynamic principles expected for the functioning of a bidirectional "carrier-type" transport system. We concluded that NCX(IF) interacts with a putative regulatory domain from the cytosolic side and, thus, may play an important regulatory role in controlling Ca(2+) signaling in the heart. This may represent a new potential tool for developing novel treatments for cardiac Ca(2+) signaling dysfunction.

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