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  • 1
    ISSN: 1432-1424
    Keywords: Key words: Na/Pi-cotransporter — PTH — Endocytosis — Tyrosine-based signals
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Abstract. The rat renal type II Na/Pi-cotransporter (NaPi2), which is regulated by mechanisms involving endocytosis and lysosomal degradation, contains two sequences that show high homology with two tyrosine (Y)-based consensus motifs previously reported to be involved in such intracellular trafficking: GY402FAM matching the consensus sequence GYXXZ, and Y509RWF matching the motif YXXO. Mutations of any of these two Y nearly abolished the NaPi2 mediated 32P i -uptake after cRNA-injection into oocytes. The mechanisms underlying these defects are however different. Mutation of the Y402 results in a lack of glycosylation and reduced surface expression of the cotransporter, that are specific for the Y402 mutation since substitution of the neighboring F403 did not have any effect. The inhibitory effect of the Y509 mutation is related to a functional inactivation of the protein expressed in the plasma membrane; mutation of the neighboring R510 also led to a decrease in the cotransporter activity. Pharmacological activation of the protein kinase C cascade by DOG induced the retrieval of both wild-type (WT) as well as Y509 cotransporters from the oocyte plasma membrane. These data suggest that the Y402 is important for the surface expression whereas Y509 for the function of the type II Na/P i -cotransporter expressed in oocytes. Y509 seems not to be involved in the membrane retrieval of the cotransporter.
    Type of Medium: Electronic Resource
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  • 2
    ISSN: 1432-1424
    Keywords: Key words: Phosphate transport —X. laevis oocytes — TCEP — Disulfide bonds — Degradation
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Abstract. Tris(2-carboxyethyl)phosphine (TCEP) reduces (cleaves) disulfide bonds of the renal proximal tubule type IIa Na/Pi- cotransporter (rat NaPi IIa) and thereby inhibits its function. We tested the effect of TCEP on the murine type IIa Na/P i -cotransporter and the corresponding IIb intestinal isoform both expressed in Xenopus laevis oocytes. After incubation with TCEP the function of NaPi IIa was inhibited and protein amount was decreased. Injection of the lysosomal inhibitor leupeptin prevented degradation of the protein. Exposure of oocytes to TCEP at 0°C led to a reduction in transport function without concomitant loss in Na/Pi IIa protein. In contrast to NaPi type IIa, the type IIb isoform was neither inhibited, nor degraded after incubation with TCEP. These results suggest that cleavage of disulfide bonds led to changes within the confirmation of the type IIa transporter that result in (i) inhibition of the transport activity and (ii) internalization and subsequent lysosomal degradation of transporter protein. Sequence comparisons suggest the involvement/presence of different disulfide bonds in type IIa and type IIb Na/P i -cotransporters.
    Type of Medium: Electronic Resource
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  • 3
    Electronic Resource
    Electronic Resource
    Oxford, UK : Blackwell Science, Ltd
    Journal of neuroendocrinology 14 (2002), S. 0 
    ISSN: 1365-2826
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: Leptin decreases food intake and increases energy expenditure in rodents by inhibiting neurones in the hypothalamic arcuate nucleus. The growth hormone secretagogue (GHS) ghrelin is known to stimulate food intake and to be the endogenous ligand for the GHS-receptor, which is strongly expressed in the arcuate nucleus, like the leptin receptor (Ob-R). In this study, we analysed the effect of systemic ghrelin administration on Fos expression in the arcuate nucleus on neurones expressing Ob-R. Injection of ghrelin (0.2 mg/kg, i.p) significantly increased the number of neurones expressing Fos protein in the ventromedial arcuate nucleus. Fifty-seven percent of all Fos-positive cells in the ventromedial arcuate nucleus were also positive for Ob-R staining. Furthermore, we investigated electrophysiologically the effect of ghrelin and leptin on the activity of arcuate neurones in an in-vitro slice preparation. Ghrelin stimulated the electrical activity dose-dependently in 80% of all cells tested (n=49) with a threshold concentration of 10−11 M; only 8% were inhibited and 12% did not respond. The effect of ghrelin (10−7 M) was weakly antagonized by the peptidic GHS-receptor antagonist (D-Lys3)-GHRP-6 (10−4 M), which also showed a much weaker affinity (IC50, 0.9 × 10−6 M) to the GHS-receptor than ghrelin (IC50, 0.3 × 10−9 M). Ghrelin increased the electrical activity in 76% of all cells which were inhibited by leptin (n=17). These data show that ghrelin interacts with the leptin hypothalamic network in the arcuate nucleus. The opposite effect of leptin and ghrelin on neurones in the arcuate nucleus may serve as a neurophysiological correlate of the orexigenic and anorectic effects of ghrelin and leptin.
    Type of Medium: Electronic Resource
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  • 4
    Electronic Resource
    Electronic Resource
    Springer
    Pflügers Archiv 437 (1999), S. 972-978 
    ISSN: 1432-2013
    Keywords: Key words FLAG ; Phosphate ; Sodium phosphate cotransport ; Topology ; Transport
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract  The rat type II sodium/phosphate cotransporter (NaPi-2) is a 85- to 90-kDa glycosylated protein located at the proximal tubular brush border membrane. Hydropathy predictions suggest eight transmembrane domains (sTM) with a large glycosylated loop between sTM 3 and sTM 4. We have studied the membrane topology of NaPi-2 expressed in oocytes. A 33-amino-acid fragment containing the FLAG epitope was inserted into seven loops connecting the sTMs and into the NH2- and COOH-ends of the protein. FLAG-antibody binding suggested that the loops connecting sTM 1 and sTM 2 as well as sTM 3 and sTM 4 are located extracellularly. Based on the lack of FLAG-antibody binding we suggest intracellular locations for the NH2- and COOH-termini and the region connecting sTM 4 and sTM 5. Immunoprecipitation studies of in vitro translated protein also suggest that the NH2-terminus is sited extracellularly. In immunohistochemical studies with NaPi-2-transfected MDCK cells, an interaction with NH2- and COOH- terminal antipeptide antibodies could only be obtained after membrane permeabilization. The presented data are an experimental documentation of the intracellular location of the NH2- and COOH-termini, and of the extracellular location of extracellular loops 1 and 2.
    Type of Medium: Electronic Resource
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  • 5
    ISSN: 1432-1912
    Keywords: Key words (13R)-spiroforskolin ; Forskolin ; cAMP-activated chloride current ; L-type calcium current Whole-cell recording ; Guinea-pig cardiomyocytes
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract The effects of a new forskolin derivative, (13R)-spiroforskolin, on the ventricular cAMP-activated chloride current (ICl(cAMP)) and the atrial L-type calcium current (ICa,L) were measured by means of whole-cell recording from isolated guinea-pig cardiac myocytes at 30°C and 20–22°C, respectively. In contrast to forskolin, the derivative contains a tetrahydrofuran rather than a tetrahydropyran moiety. (13R)-spiroforskolin activated ICl(cAMP) in 58% of the ventricular myocytes studied. The concentration required for the half maximal effect (EC50 value) amounted to 9.6×10–11 M and was lower than the EC50 value for forskolin (2.4×10–8 M). (13R)-spiroforskolin evoked a smaller maximal ICl(cAMP) amplitude than forskolin. The rundown of the (13R)-spiroforskolin-activated ICl(cAMP) was faster than that of the forskolin-induced current. Neither forskolin nor (13R)-spiroforskolin in maximally effective concentrations increased ICl(cAMP) in cells containing high concentrations of cAMP. Furthermore, as an activator of atrial ICa,L (13R)-spiroforskolin displayed a smaller activation and a lower EC50 value (5.8×10–10 M) than forskolin (EC50 value: 3.7×10–7 M). The effect of (13R)-spiroforskolin was observed in only 30% of the atrial cells studied. None of the drugs exerted a stimulatory effect in atrial cells containing a high [cAMP]. The washout of the drug effect was significantly faster in (13R)-spiroforskolin- than in forskolin-treated atrial myocytes. We conclude that (13R)-spiroforskolin as a forskolin derivative displays unique characteristics. It is a more potent but less efficacious activator of cardiac ionic conductances than the parent compound. The results suggest that (13R)-spiroforskolin, like forskolin, most probably exerts its effects via stimulation of the adenylyl cyclase.
    Type of Medium: Electronic Resource
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