Extra-vesicular binding of noradrenaline and guanethidine in the adrenergic neurones of the rat heart: a proposed site of action of adrenergic neurone blocking agents.
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1 The binding and efflux characteristics of [14C]-guanethidine and [3H]-noradrenaline were studied in heart slices from rats which were pretreated with reserpine and nialamide. 2 Binding of both compounds occurred at extra-vesicular sites within the adrenergic neurone. After a brief period of rapid washout, the efflux of [14C]-guanethidine and [3H]-noradrenaline proceeded at a steady rate. The efflux of both compounds appeared to occur from a single intraneuronal compartment. 3 (+)-Amphetamine accelerated the efflux of [14C]-noradrenaline; this effect was inhibited by desipramine. 4 Unlabelled guanethidine and amantadine also increased the efflux of labelled compounds. Cocaine in high concentrations increased slightly the efflux of [14C]-guanethidine but not that of [3H]-noradrenaline. 5 Heart slices labelled with [3H]-noradrenaline became refractory to successive exposures to releasing agents although an appreciable amount of labelled compound was still present in in these slices. 6 It is suggested that [14C]-guanethidine and [3H]-noradrenaline are bound at a common extravesicular site within the adrenergic neurone. Binding of guanethidine to the extra-vesicular site may be relevant to its pharmacological action, i.e., the blockade of adrenergic transmission. (+info)
Denitrifying Pseudomonas aeruginosa: some parameters of growth and active transport.
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Optimal cell yield of Pseudomonas aeruginosa grown under denitrifying conditions was obtained with 100 mM nitrate as the terminal electron acceptor, irrespective of the medium used. Nitrite as the terminal electron acceptor supported poor denitrifying growth when concentrations of less than 15 mM, but not higher, were used, apparently owing to toxicity exerted by nitrite. Nitrite accumulated in the medium during early exponential phase when nitrate was the terminal electron acceptor and then decreased to extinction before midexponential phase. The maximal rate of glucose and gluconate transport was supported by 1 mM nitrate or nitrite as the terminal electron acceptor under anaerobic conditions. The transport rate was greater with nitrate than with nitrite as the terminal electron acceptor, but the greatest transport rate was observed under aerobic conditions with oxygen as the terminal electron acceptor. When P. aeruginosa was inoculated into a denitrifying environment, nitrate reductase was detected after 3 h of incubation, nitrite reductase was detected after another 4 h of incubation, and maximal nitrate and nitrite reductase activities peaked together during midexponential phase. The latter coincided with maximal glucose transport activity. (+info)
Lung fluid transport in aquaporin-1 and aquaporin-4 knockout mice.
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The mammalian lung expresses water channel aquaporin-1 (AQP1) in microvascular endothelia and aquaporin-4 (AQP4) in airway epithelia. To test whether these water channels facilitate fluid movement between airspace, interstitial, and capillary compartments, we measured passive and active fluid transport in AQP1 and AQP4 knockout mice. Airspace-capillary osmotic water permeability (Pf) was measured in isolated perfused lungs by a pleural surface fluorescence method. Pf was remarkably reduced in AQP1 (-/-) mice (measured in cm/s x 0.001, SE, n = 5-10: 17 +/- 2 [+/+]; 6.6 +/- 0.6 AQP1 [+/-]; 1.7 +/- 0.3 AQP1 [-/-]; 12 +/- 1 AQP4 [-/-]). Microvascular endothelial water permeability, measured by a related pleural surface fluorescence method in which the airspace was filled with inert perfluorocarbon, was reduced more than 10-fold in AQP1 (-/-) vs. (+/+) mice. Hydrostatically induced lung interstitial and alveolar edema was measured by a gravimetric method and by direct measurement of extravascular lung water. Both approaches indicated a more than twofold reduction in lung water accumulation in AQP1 (-/-) vs. (+/+) mice in response to a 5- to 10-cm H2O increase in pulmonary artery pressure for five minutes. Active, near-isosmolar alveolar fluid absorption (Jv) was measured in in situ perfused lungs using 125I-albumin as an airspace fluid volume marker. Jv (measured in percent fluid uptake at 30 min, n = 5) in (+/+) mice was 6.0 +/- 0.6 (37 degrees C), increased to 16 +/- 1 by beta-agonists, and inhibited to less than 2.0 by amiloride, ouabain, or cooling to 23 degrees C. Jv (with isoproterenol) was not affected by aquaporin deletion (18.9 +/- 2.2 [+/+]; 16.4 +/- 1.5 AQP1 [-/-]; 16.3 +/- 1.7 AQP4 [-/-]). These results indicate that osmotically driven water transport across microvessels in adult lung occurs by a transcellular route through AQP1 water channels and that the microvascular endothelium is a significant barrier for airspace-capillary osmotic water transport. AQP1 facilitates hydrostatically driven lung edema but is not required for active near-isosmolar absorption of alveolar fluid. (+info)
Tyrosine kinase inhibitors and immunosuppressants perturb the myo-inositol but not the betaine cotransporter in isotonic and hypertonic MDCK cells.
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BACKGROUND: The sodium/myo-inositol cotransporter (SMIT) and the betaine cotransporter (BGT1) are essential for the accumulation of myo-inositol and betaine, and hence cell survival in a hypertonic environment. The underlying molecular mechanism involves an increase in transcription of the SMIT and BGT1 genes through binding of a trans-acting factor to enhancer elements in the 5' flanking region of both genes, resulting in increased mRNA abundance and increased activity of the cotransporters. Current evidence regarding transcriptional and post-transcriptional regulation indicates that both cotransporters are regulated in parallel. METHODS: To investigate the signal transduction of hypertonic stress, we examined the effect of tyrosine kinase inhibitors and immunosuppressants on the hypertonicity-induced activity of the two cotransporters in Madin-Darby canine kidney (MDCK) cells. RESULTS: None of the agents studied affected BGT1 activity in isotonic or hypertonic conditions. Treatment of MDCK cells with genistein, a tyrosine kinase inhibitor, increased SMIT activity in hypertonic but not isotonic conditions. The stimulation of SMIT by genistein was accompanied by a parallel increase in mRNA abundance. In contrast, treating cells with tyrphostin A23, another tyrosine kinase inhibitor, or cyclosporine A, an immunosuppressant, inhibited SMIT activity in hypertonic cells. FK506, another immunosuppressant, increased SMIT activity, but only in isotonic conditions. CONCLUSIONS: These results provide the first evidence of divergent regulatory pathways modulating SMIT and BGT activity. (+info)
Active transport of calcium across the isolated midgut of Hyalophora cecropia.
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1. The net flux of 45Ca from lumen to blood side across the isolated and short-circuited Cecropia midgut was 1-9 +/- 0-2 muequiv. cm-2h-1 in 8 mM Ca and the flux ratio was as high as 56 to 1. 2. The calcium influx was depressed by anoxia; 73% after 30 min. 3. The kinetics of Ca transport were anomalous; the apparent Km varied with Ca concentration from less than 0-2 to greater than 5-6 mM Ca and the apparent Vmax varied from less than 1-3 to greater than 3-3 muequiv. cm-2h-1. 4. The calcium influx showed a delay before the tracer steady state was attained, indicating the existence in the transport route of a calcium pool equivalent to 5-7 muequiv/g. wet weight of midgut tissue. 5 High calcium (16 mM) depressed the short-circuit current and potassium transport from blood to lumen side across the midgut. 6. Calcium depressed magnesium transport, from lumen to blood side across the midgut, and magnesium depressed the calcium transport. 7. Ca transport by the midgut does not regulate the Ca level in the haemolymph in vivo; it merely aids the diffusion of calcium down its electrochemical gradient. However, Ca transport may assist the uptake of the nutrients from the midgut contents. (+info)
A monoclonal antibody to the COOH-terminal acidic portion of Ran inhibits both the recycling of Ran and nuclear protein import in living cells.
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A small GTPase Ran is a key regulator for active nuclear transport. In immunoblotting analysis, a monoclonal antibody against recombinant human Ran, designated ARAN1, was found to recognize an epitope in the COOH-terminal domain of Ran. In a solution binding assay, ARAN1 recognized Ran when complexed with importin beta, transportin, and CAS, but not the Ran-GTP or the Ran-GDP alone, indicating that the COOH-terminal domain of Ran is exposed via its interaction with importin beta-related proteins. In addition, ARAN1 suppressed the binding of RanBP1 to the Ran-importin beta complex. When injected into the nucleus of BHK cells, ARAN1 was rapidly exported to the cytoplasm, indicating that the Ran-importin beta-related protein complex is exported as a complex from the nucleus to the cytoplasm in living cells. Moreover, ARAN1, when injected into the cultured cells induces the accumulation of endogenous Ran in the cytoplasm and prevents the nuclear import of SV-40 T-antigen nuclear localization signal substrates. From these findings, we propose that the binding of RanBP1 to the Ran-importin beta complex is required for the dissociation of the complex in the cytoplasm and that the released Ran is recycled to the nucleus, which is essential for the nuclear protein transport. (+info)
Genetic evidence for ATP-dependent endoplasmic reticulum-to-Golgi apparatus trafficking of ceramide for sphingomyelin synthesis in Chinese hamster ovary cells.
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LY-A strain is a Chinese hamster ovary cell mutant resistant to sphingomyelin (SM)-directed cytolysin and has a defect in de novo SM synthesis. Metabolic labeling experiments with radioactive serine, sphingosine, and choline showed that LY-A cells were defective in synthesis of SM from these precursors, but not syntheses of ceramide (Cer), glycosphingolipids, or phosphatidylcholine, indicating a specific defect in the conversion of Cer to SM in LY-A cells. In vitro experiments showed that the specific defect of SM formation in LY-A cells was not due to alterations in enzymatic activities responsible for SM synthesis or degradation. When cells were treated with brefeldin A, which causes fusion of the Golgi apparatus with the endoplasmic reticulum (ER), de novo SM synthesis in LY-A cells was restored to the wild-type level. Pulse-chase experiments with a fluorescent Cer analogue, N-(4,4-difluoro-5,7-dimethyl-4-bora-3a, 4a-diaza-s-indacene-3-pentanoyl)-D-erythro-sphingosine (C5-DMB-Cer), revealed that in wild-type cells C5-DMB-Cer was redistributed from intracellular membranes to the Golgi apparatus in an intracellular ATP-dependent manner, and that LY-A cells were defective in the energy-dependent redistribution of C5-DMB-Cer. Under ATP-depleted conditions, conversion of C5-DMB-Cer to C5-DMB-SM and of [3H]sphingosine to [3H]SM in wild-type cells decreased to the levels in LY-A cells, which were not affected by ATP depletion. ER-to-Golgi apparatus trafficking of glycosylphosphatidylinositol-anchored or membrane-spanning proteins in LY-A cells appeared to be normal. These results indicate that the predominant pathway of ER-to-Golgi apparatus trafficking of Cer for de novo SM synthesis is ATP dependent and that this pathway is almost completely impaired in LY-A cells. In addition, the specific defect of SM synthesis in LY-A cells suggests different pathways of Cer transport for glycosphingolipids versus SM synthesis. (+info)
Kinetic and thermodynamic aspects of lipid translocation in biological membranes.
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A theoretical analysis of the lipid translocation in cellular bilayer membranes is presented. We focus on an integrative model of active and passive transport processes determining the asymmetrical distribution of the major lipid components between the monolayers. The active translocation of the aminophospholipids phosphatidylserine and phosphatidylethanolamine is mathematically described by kinetic equations resulting from a realistic ATP-dependent transport mechanism. Concerning the passive transport of the aminophospholipids as well as of phosphatidylcholine, sphingomyelin, and cholesterol, two different approaches are used. The first treatment makes use of thermodynamic flux-force relationships. Relevant forces are transversal concentration differences of the lipids as well as differences in the mechanical states of the monolayers due to lateral compressions. Both forces, originating primarily from the operation of an aminophospholipid translocase, are expressed as functions of the lipid compositions of the two monolayers. In the case of mechanical forces, lipid-specific parameters such as different molecular surface areas and compression force constants are taken into account. Using invariance principles, it is shown how the phenomenological coefficients depend on the total lipid amounts. In a second approach, passive transport is analyzed in terms of kinetic mechanisms of carrier-mediated translocation, where mechanical effects are incorporated into the translocation rate constants. The thermodynamic as well as the kinetic approach are applied to simulate the time-dependent redistribution of the lipid components in human red blood cells. In the thermodynamic model the steady-state asymmetrical lipid distribution of erythrocyte membranes is simulated well under certain parameter restrictions: 1) the time scales of uncoupled passive transbilayer movement must be different among the lipid species; 2) positive cross-couplings of the passive lipid fluxes are needed, which, however, may be chosen lipid-unspecifically. A comparison of the thermodynamic and the kinetic approaches reveals that antiport mechanisms for passive lipid movements may be excluded. Simulations with kinetic symport mechanisms are in qualitative agreement with experimental data but show discrepancies in the asymmetrical distribution for sphingomyelin. (+info)