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  • 1
    Electronic Resource
    Electronic Resource
    Springer
    The journal of membrane biology 138 (1994), S. 65-76 
    ISSN: 1432-1424
    Keywords: Cl- channel ; Na+ channel ; Ion fluxes ; Hormonal regulation ; Epithelial polarity ; cAMP
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Abstract The effect of antidiuretic hormone on transepithelial Na+ and Cl- transport and its modulation by aldosterone (10-6 m) was studied in the Xenopus laevis distal nephron cell line A6-C1 by measuring transepithelial electrophysiological parameters and bidirectional anion fluxes. Vasotocin (or vasopressin) induced a biphasic increase in transepithelial short-circuit current (I sc). Early and late effects were potentiated by aldosterone and could be mimicked by forskolin and BrcAMP, implicating cAMP as a mediator. The early increase in I sc (maximum 1–2 min after hormone addition) was resistant to 50 μm amiloride. Electrophysiological experiments with apical ion substitutions or basolateral bumetanide (0.5 mm), as well as flux studies with 125I- or 36C1-, indicated that this current represented Cl- secretion. The late increase in I sc appeared with a lag of 2–5 min and was maximal after 15–25 min. It corresponded to an increase in Na+ reabsorption, since it was amiloride sensitive. Bidirectional 36C1- flux measurements in aldosterone-treated monolayers maintained under open-circuit conditions showed that the large vasotocin-induced increase in Cl- permeability led, in these conditions, to a threefold increase of a baseline Cl- reabsorption. This study shows that vasotocin induces in A6-C1 cells both a rapid increase in Cl- permeability and a slower increase in Na+ transport. The Cl- permeability, which leads to Cl- secretion under short-circuit conditions, contributes, under the more physiological open-circuit conditions, to the transport of Na+ by allowing its co-reabsorption with Cl-.
    Type of Medium: Electronic Resource
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  • 2
    ISSN: 1432-1424
    Keywords: Key words: Adenosine receptors — A6 cells — Chloride channels — Sodium transport — cAMP — Calcium
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Abstract. The effect of adenosine regulation on sodium and chloride transport was examined in cultured A6 renal epithelial cells. Adenosine and its analogue N6-cyclopentyladenosine (CPA) had different effects on short-circuit current (I sc) depending on the side of addition. Basolateral CPA addition induced an approximately threefold increase of the I sc that reached a maximum effect 20 min after addition and was completely inhibited by preincubation with either an A2 selective antagonist, CSC, or the sodium channel blocker, amiloride. Apical CPA addition induced a biphasic I sc response characterized by a rapid fourfold transient increase over its baseline followed by a decline and a plateau phase that were amiloride insensitive. The A1 adenosine antagonist, CPX, completely prevented this response. This I sc response to apical CPA was also strongly reduced in Cl−-free media and was significantly inhibited either by basolateral bumetanide or apical DPC preincubation. Only basolateral CPA addition was able to induce an increase in cAMP level. CPA, added to cells in suspension, caused a rapid rise in [Ca2+] i that was antagonized by CPX, not affected by CSC and prevented by thapsigargin preincubation. These data suggest that basolateral CPA regulates active sodium transport via A2 adenosine receptors stimulating adenylate cyclase while apical CPA regulates Cl− secretion via A1 receptor-mediated changes in [Ca2+] i .
    Type of Medium: Electronic Resource
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  • 3
    Electronic Resource
    Electronic Resource
    Springer
    The journal of membrane biology 145 (1995), S. 193-204 
    ISSN: 1432-1424
    Keywords: Epithelial cell polarity ; Microfilament ; Microtubule ; Epithelial Na channel ; Cl channel ; Fluid phase endocytosis
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Abstract Antidiuretic hormone (ADH; 2.5 × 10−8 m vasotocin) produces a stimulation of apical fluid phase endocytosis, protein secretion and NaCl reabsorption in Xenopus laevis A6 distal nephron cell epithelia pretreated with aldosterone (10−6 m). The increase of NaCl transport is mediated by a sequential opening of apical Cl and Na conductances. The aim of this study was to characterize the actin and tubulin cytoskeleton of A6 cells and to assess the impact of its disruption on baseline and ADH-induced apical vesicular membrane movements and ion transport to test for possible functional links. The microfilament (MF) and microtubule (MT) networks and their disruption were visualized by confocal laser microscopy. Conditions of depolimerization were selected, by cytochalasin D or cold and nocodazole, respectively. MF disruption produced an increase in baseline apical protein secretion (exocytic movements) (plus 18%) and a decrease of its induction by ADH (minus 35%). MF disruption also increased baseline horseradish peroxidase uptake (endocytic movements) (plus 21%), however, without affecting its ADH-induced increase. In the case of MT disruption, the ADH-induced stimulation of both protein secretion and fluid phase endocytosis was decreased by 70 and 44%, respectively. At the ion transport level, MF and MT disruption only insignificantly affected the ADH-induced Cl conductance, while they decreased the ADH-induced stimulation of Na transport (amiloride-sensitive short-circuit current and conductance) by a factor of 2 to 4. In conclusion, both MT and MF disruption decrease ADH-induced apical protein secretion and Na conductance, while the ADH-induced apical Cl conductance is not significantly affected. Taken together the data support the hypothesis that the modulation of Na channel expression by apical vesicular membrane movements plays a role in Na transport expression and its regulation by ADH.
    Type of Medium: Electronic Resource
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  • 4
    Electronic Resource
    Electronic Resource
    Springer
    The journal of membrane biology 172 (1999), S. 181-192 
    ISSN: 1432-1424
    Keywords: Key words: Cystinuria — Lysinuric protein intolerance — Proximal kidney tubule — Membrane carrier — Permease
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Abstract. The L-type amino acid transporter LAT1 has recently been identified as being a disulfide-linked ``light chain'' of the ubiquitously expressed glycoprotein 4F2hc/CD98. Several LAT1-related transporters have been identified, which share the same putative 12-transmembrane segment topology and also associate with the single transmembrane domain 4F2hc protein. They display differing amino acid substrate specificities, transport kinetics and localizations such as, for instance, y+LAT1 which is localized at the basolateral membrane of transporting epithelia, and the defect of which causes lysinuric protein intolerance. The b0,+AT transporter which associates with the 4F2hc-related rBAT protein to form the luminal high-affinity diamino acid transporter defective in cystinuria, belongs to the same family of glycoprotein-associated amino acid transporters (gpaATs). These glycoprotein-associated transporters function as amino acid exchangers. They extend the specificity range of vectorial amino acid transport when located in the same membrane as carriers that unidirectionally transport one of the exchanged substrates. gpaATs belong to a phylogenetic cluster within the amino acid/polyamine/choline (APC) superfamily of transporters. This cluster, which we designate the LAT family (named after its first vertebrate member), includes some members from nematodes, yeast and bacteria. The latter of these proteins presumably lack association with a second subunit. In this review, we focus on the animal members of the LAT cluster that form, together with some of the nematode members, the family of glycoprotein-associated amino acid transporters (gpaAT family).
    Type of Medium: Electronic Resource
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  • 5
    Electronic Resource
    Electronic Resource
    Springer
    The journal of membrane biology 144 (1995), S. 93-110 
    ISSN: 1432-1424
    Keywords: Aldosterone ; Mineralocorticoid hormone ; Glucocorticoid hormone ; Hormonal regulation ; Na+ channel ; Na+,K+-ATPase
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Conclusions The model for the adrenal steroid action on Na transport in tight epithelia as depicted in Fig. 3A and B dissociates two phases: an early phase during which the pre-existing Na transport machinery is activated and a late phase during which the transport capacity of the machinery is increased. These two sequential phases have been distinguished based on differences in functional aspects of the induced transport, on selective effects of agents interfering with transcriptional regulation and on a correlation of the late response phase with an increase in transport protein synthesis and expression [26, 45, 46, 98, 99, 124]. These observations suggest that a bimodal stimulation of Na transport could involve two different gene networks which are directly (in the physiological meaning) and independently stimulated by the action of the hormone-receptor complex and the following “molecular” cascades (see section Molecular and Physiological Cascades). The relatively clear temporal dissociation of the responses found in experimental situations is probably the consequence of inherent properties of the two networks. Indeed, to generate rapid functional changes, the genes involved in the early response must encode products which have relatively short half-lifes at the mRNA and protein levels. In contrast, the constitutive elements of the Na transport machinery that are increased during the late phase of adrenal steroid action have, as shown for the Na,K-ATPase [82], relatively long half-lifes. Consequently, even though changes in transcription may take place early in the course of the hormonal treatment, they impact on protein synthesis and pools only slowly and after a substantial lag period. On the one hand, ongoing research will soon provide more information on the nature, time course and hormone/receptor specificity of adrenal-steroid-regulated genes. On the other hand, the availability of new technical and molecular tools to study the proteins of the Na transport machinery greatly increases the possibilities for studying its regulation by adrenal steroids. Consequently, it will be a fascinating challenge to relate the data emerging from both approaches, and it appears that only a combination of methods and tools will allow to progressively fill the gap of understanding which still lies between the transcriptional effects and the transport regulation.
    Type of Medium: Electronic Resource
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  • 6
    ISSN: 1432-1424
    Keywords: Key words: Na/Pi-cotransport — Expression cloning — Duodenum — Brush border membrane —Xenopus laevis
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Abstract. In a previous report we documented an increased Na+-dependent transport of inorganic phosphate (P i ) in Xenopus laevis oocytes injected with mRNA isolated from rabbit duodenum (Yagci et al., Pfluegers Arch. 422:211–216, 1992; ref 24). In the present study we have used expression cloning in oocytes to search for the cDNA/mRNA involved in this effect. The identified cDNA (provisionally named PiUS; for P i -uptake stimulator) lead to a 3-4-fold stimulation of Na+-dependent P i -uptake (10ng cRNA injected, 3–5 days of expression). Na+-independent uptake of P i was also affected but transport of sulphate and l-arginine (in the presence or absence of sodium) remained unchanged. The apparent K m -values for the induced Na+-dependent uptake were 0.26 ± 0.04 mm for P i and 14.8 ± 3.0 mm for Na+. The 1796 bp cDNA codes for a protein of 425 amino acids. Hydropathy analysis suggests a lack of transmembrane segments. In vitro translation resulted in a protein of 60 kDa and provided no evidence of glycosylation. In Northern blots a mRNA of ∼2 kb was recognized in various tissues including different intestinal segments, kidney cortex, kidney medulla, liver and heart. Homology searches showed no similarity to proteins involved in membrane transport and its control. In conclusion, we have cloned from a rabbit small intestinal cDNA library a novel cDNA encoding a protein stimulating P i -uptake into Xenopus laevis oocytes, but which is not a P i -transporter itself.
    Type of Medium: Electronic Resource
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  • 7
    Electronic Resource
    Electronic Resource
    Springer
    Journal of neural transmission 38 (1976), S. 249-258 
    ISSN: 1435-1463
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Summary 35 parkinsonian patients have been treated over 3 years with L-Dopa combined with benserazide. After an impressive improvement during the first months of treatment a slow but significant deterioration of the patients' condition was observed. At the end of the observation period however their condition was still significantly better than before starting the treatment. A reduced mean L-Dopa dosage was ruled out as the cause of this deterioration. Withdrawal of the L-Dopa therapy for a few days in 13 patients provided strong evidence that it is due to the progression of the disease and to a partial loss of L-Dopa efficacy.
    Type of Medium: Electronic Resource
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  • 8
    ISSN: 1432-2013
    Keywords: Amiloride cAMP ENaC Epithelial sodium channel Protein kinase A
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract. The rate of Na+ (re)absorption across tight epithelia such as in distal kidney nephron and colon is to a large extent controlled at the level of the epithelial Na+ channel (ENaC). In kidney, antidiuretic hormone (ADH, vasopressin) stimulates the expression/activity of this channel by a cAMP/protein-kinase-A- (PKA-) mediated pathway. However, a clear upregulation of ENaC function by cAMP could not be reproduced with cloned channel subunits in the Xenopus oocyte expression system, suggesting the hypothesis that an additional factor is missing. In contrast, we show here that membrane-permeant cAMP can activate ENaC expressed in Xenopus oocytes (3.8-fold) upon replacement of the rat α-subunit by a new α-subunit cloned from guinea-pig colon (gpα). This α-subunit is 76% identical with its rat orthologue originating from ADH-insensitive rat colon. The biophysical fingerprints of the hybrid ENaC formed by this guinea-pig α-subunit together with rat β- and γ-subunits are indistinguishable from those of rat ENaC (rENaC). Injection of the PKA inhibitor PKI-(6–22)-amide into the oocyte had no effect on the basal activity of rat ENaC but inhibited the activity of gpα-containing hybrid ENaC and greatly decreased its stimulation by cAMP. This suggests that, unlike for rat ENaC, tonic PKA activity is required for basal function of gpα-containing ENaC and that PKA mediates its cAMP-induced activation. This regulatory behaviour is not common to all ENaCs containing an α-subunit cloned from an ADH-responsive tissue since xENaC, which was cloned from the ADH-sensitive Xenopus laevis A6 epithelia, is, when expressed in oocytes, resistant to cAMP, similar to rat ENaC. This study demonstrates that the PKA sensitivity of ENaC can depend on the nature of the ENaC α-subunit and raises the possibility that cAMP can stimulate ENaCs by different mechanisms.
    Type of Medium: Electronic Resource
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