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  • 1
    Electronic Resource
    Electronic Resource
    Coupled Na+/H+ exchange in rat parotid basolateral membrane vesicles (1988)
    Springer
    The journal of membrane biology 102 (1988), S. 247-254 
    Details
    ISSN: 1432-1424
    Keywords: exocrine gland ; parotid ; acinar cell ; ion transport ; fluid secretion ; pH regulation ; antiport
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Summary pH gradient-dependent sodium transport in highly purified rat parotid basolateral membrane vesicles was studied under voltage-clamped conditions. In the presence of an outwardly directed H+ gradient (pHin=6.0, pHout=8.0)22Na uptake was approximately ten times greater than uptake measured at pH equilibrium (pHin=pHout=6.0). More than 90% of this sodium flux was inhibited by the potassium-sparing diuretic drug amiloride (K 1 =1.6 μm) while the transport inhibitors furosemide (1mm), bumetanide (1mm) SITS (0.5mm) and DIDS (0.1mm) were without effect. This transport activity copurified with the basolateral membrane marker K+-stimulatedp-nitrophenyl phosphatase. In addition22Na uptake into the vesicles could be driven against a concentration gradient by an outwardly directed H+ gradient. pH gradient-dependent sodium flux exhibited a simple Michaelis-Menten-type dependence on sodium concentration cosistent with the existence of a single transport system withK M =8.0mm at 23°C. A component of pH gradient-dependent, amiloride-sensitive sodium flux was also observed in rabbit parotid basolateral membrane vesicles. These results provide strong evidence for the existence of a Na+/H+ antiport in rat and rabbit parotid acinar basolateral membranes and extend earlier less direct studies which suggested that such a transporter was present in salivary acinar cells and might play a significant role in salivary fluid secretion.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF01925718
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  • 2
    Electronic Resource
    Electronic Resource
    Ionic dependence of bumetanide binding to the rabbit parotid Na/K/Cl cotransporter (1988)
    Springer
    The journal of membrane biology 102 (1988), S. 71-77 
    Details
    ISSN: 1432-1424
    Keywords: loop diuretics ; exocrine gland ; fluid secretion ; parotid ; acinar cell ; ion transport
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Summary The Na/K/Cl-dependent component of the binding of the loop diuretic bumetanide to basolateral membrane vesicles from the rabbit parotid is studied. A Scatchard analysis indicates that this binding is due to a single high-affinity site withK D =3.2±0.3 μm (n=9) at 100mm sodium, 100mm potassium and 5mm chloride. When KCl-dependent22Na transport and tracer [3H]-bumetanide binding are monitored simultaneously as a function of (unlabeled) bumetanide concentration it is found that theK 0.5 for bumetanide inhibition of both processes are identical indicating that the high-affinity bumetanide binding site studied here is identical with a bumetanide-inhibitory site on the Na/K/Cl cotransport system previously identified in this preparation (R.J. Turner, J.N. George and B.J. Baum,J. Membrane Biol. 94:143–152, 1986). High-affinity bumetanide binding exhibits a hyperbolic dependence on both [Na] and [K] consistent with Na/bumetanide and K/bumetanide binding stoichiometries of 1∶1 andK 0.5 values of approximately 33mm for sodium and 23mm for potassium. In contrast, the dependence on [Cl] is biphasic, with bumetanide binding increasing from 0 to 5mm chloride and decreasing toward baseline levels thereafter. Scatchard analysis of this latter inhibitory effect of chloride indicates a competitive interaction with bumetanide in agreement with earlier indications that bumetanide inhibits Na/K/Cl cotransport at a chloride site. However, studies of the effects of various anions on bumetanide binding and22Na transport show a poor correlation between the specificities of these two processes, suggesting that the inhibitory chloride site is not a chloride transport site.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF01875354
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  • 3
    Electronic Resource
    Electronic Resource
    Inactivation of the rabbit parotid Na/K/Cl cotransporter by N-ethylmaleimide (1989)
    Springer
    The journal of membrane biology 112 (1989), S. 51-58 
    Details
    ISSN: 1432-1424
    Keywords: loop diuretics ; exocrine gland ; fluid secretion ; parotid ; acinar cell ; ion transport ; chloride secretion
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Summary The inactivation of the rabbit parotid Na/K/Cl cotransporter by the irreversible sulfhydryl reagent N-ethylmaleimide (NEM) is studied by monitoring its effect on high affinity bumetanide binding to the carrier. NEM reduces the number of bumetanide binding sites with no significant change in the affinity of those remaining. NEM also reduces KCl-dependent22Na flux via the cotransporter by the same factor as the reduction in bumetanide binding sites. Both bumetanide and its analogue furosemide can protect against the effect of NEM. The concentration range over which this protection occurs is in good agreement with affinities of these two compounds for the high affinity bumetanide binding site (2.6 and 85 μm, respectively), indicating an association of this site with the site of action of NEM. Also consistent with this hypothesis are the observations that (i) sodium and potassium, both of which are required for high affinity bumetanide binding, increase the rate of inactivation of binding by NEM and (ii) chloride, at concentrations previously shown to competitively inhibit bumetanide binding, protects the cotransporter against NEM. The effects of NEM on bumetanide binding are mimicked by another highly specific sulfhydryl reagent, methyl methanethiolsulfonate. The apparent rate constant for inactivation of high affinity bumetanide binding by NEM is a hyperbolic function of NEM concentration consistent with a model in which the inactivation reaction is first order in [NEM] and proceeds through an intermediate adsorptive complex. The data indicate that the presence of a reduced sulfhydryl group at or closely related to the bumetanide binding site is essential for the operation of the parotid Na/K/Cl cotransporter.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF01871163
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  • 4
    Electronic Resource
    Electronic Resource
    Solubilization and partial purification of the rabbit parotid Na/K/Cl-dependent bumetanide binding site (1990)
    Springer
    The journal of membrane biology 113 (1990), S. 203-210 
    Details
    ISSN: 1432-1424
    Keywords: loop diuretics ; exocrine gland ; fluid secretion ; parotid ; acinar cell ; ion transport ; chloride secretion ; detergent
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Summary We demonstrate that the high affinity bumetanide binding site of the rabbit parotid acinar cell can be extracted from a basolateral membrane fraction using relatively low concentrations (0.07%, wt/vol; 1 mg membrane protein/ml) of the nonionic detergent Triton X-100. This extracted site cannot be sedimented by ultracentrifugation at 100,000 ×g × 1 hr. Bumetanide binding to this site retains the ionic characteristics of bumetanide binding to native membranes but shows a fivefold increase in binding affinity (K d=0.57±0.15 μm vs.K d=3.3±0.7 μm for native membranes). Inactivation of the extracted bumetanide binding site observed at detergent/protein ratios〉1 can be prevented or (partially) reversed by the addition of exogenous lipid (0.2% soybean phosphatidylcholine). When the 0.07% Triton extract is fractionated by sucrose density gradient centrifugation in 0.24% Triton X-100, 0.2% exogenous lipid and 200mm salt, the high affinity bumetanide binding site sediments as a single band withS 20,w =8.8±0.8 S. This corresponds to a molecular weight ∼200 kDa for the bumetanide binding protein-detergent-lipid complex and represents a sevenfold purification of this site relative to the starting membrane fraction. In contrast to previous attempts to purify Na/K/Cl cotransport proteins and their associated bumetanide binding sites, the present method avoids harsh detergent treatment as well as direct covalent modification (inactivation) of the transporter itself. As a consequence, one can follow the still active protein through a series of extraction and purification steps by directly monitoring its bumetanide binding properties.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF01870072
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