Reduced Efflux Rates Research Articles

Differential responses in ammonia excretion, sodium fluxes and gill permeability explain different sensitivities to acute high environmental ammonia in three freshwater teleosts

We examined the acute physiological responses to high environmental ammonia (HEA), particularly the linkages between branchial ammonia fluxes and unidirectional Na+ fluxes, as well as urea excretion, cortisol, and indicators of gill permeability in three freshwater teleosts differing in their sensitivities to ammonia; the highly sensitive salmonid Oncorhynchus mykiss (rainbow trout), the less sensitive cyprinid Cyprinus carpio (common carp) and the highly resistant cyprinid Carassius auratus (goldfish). Fish were acutely exposed to two sub-lethal ammonia concentrations (as NH4HCO3) at pH 7.9: 1mM for a period of 12h, identical for all species, and 5mM for the cyprinids and 1.4mM for the trout for 3h. Elevation of plasma cortisol at both levels of HEA was apparent in all species. At 1mM, ammonia excretion (Jamm) was inhibited to a greater extent in trout than cyprinids and concurrently a significantly higher plasma ammonia level was evident in trout. However Jamm was reversed in all species at 5 or 1.4mM. Goldfish showed a significant increase in urea excretion rate (Jurea) during HEA exposure. In carp and trout, neither level of HEA elevated Jurea but urea production was increased as evidenced by a considerable elevation of plasma urea. At 1mM HEA, Na+ imbalance became progressively more severe in trout and carp due to a stimulation of unidirectional Na+ efflux (JoutNa) without a concomitant increase in unidirectional Na+ influx (JinNa). Additionally, a transient reduction of JinNa was evident in trout. Goldfish showed an opposite trend for JoutNa with reduced efflux rates and a positive Na+ balance during the first few hours of HEA. However, after 12h of exposure, both JinNa and JoutNa were also increased in both carp and goldfish, whereas only JoutNa was increased in trout, leading to a net Na+ loss. Na+ homeostasis was entirely disrupted in all three species when subjected to the 5 or 1.4mM ammonia for 3h: JinNa was significantly inhibited while considerable activation of JoutNa was observed. Diffusive water efflux rates and net K+ loss rates across the gills were enhanced during HEA only in trout, indicating an increment in gill transcellular permeability. Transepithelial potential was increased in all the species during ammonia exposure, but to the least extent in goldfish. Overall, for several different physiological systems, trout were most disturbed, and goldfish were least disturbed by HEA, helping to explain the differential ammonia tolerance of the three species.

Molecular and biochemical characterization of mechanosensitive channels in Corynebacterium glutamicum

Database searches in the Corynebacterium glutamicum genome sequence revealed homologs of the mechanosensitive channels MscL and YggB of Escherichia coli. To elucidate the physiological role of these putative channels deletion mutants were constructed. Betaine efflux induced by osmotic downshock of the mscL deletion mutant was nearly identical to that of the wild-type, whereas the yggB deletion mutant showed a reduced efflux rate. Interestingly, the double deletion strain, which was expected to have an even more decreased capability of betaine excretion, had only a slightly reduced efflux rate compared to the wild-type and did not show an increased mortality after osmotic downshift. These results led to the hypothesis that C. glutamicum may possess a third type of mechanosensitive channel not related to the MscL and YggB/KefA families. Furthermore it is unlikely that an MscM-like activity is responsible for the betaine efflux, because of the high transport capacity detected in the double deletion mutant.

Molecular and biochemical characterization of mechanosensitive channels in Corynebacterium glutamicum.

Database searches in the Corynebacterium glutamicum genome sequence revealed homologs of the mechanosensitive channels MscL and YggB of Escherichia coli. To elucidate the physiological role of these putative channels deletion mutants were constructed. Betaine efflux induced by osmotic downshock of the mscL deletion mutant was nearly identical to that of the wild-type, whereas the yggB deletion mutant showed a reduced efflux rate. Interestingly, the double deletion strain, which was expected to have an even more decreased capability of betaine excretion, had only a slightly reduced efflux rate compared to the wild-type and did not show an increased mortality after osmotic downshift. These results led to the hypothesis that C. glutamicum may possess a third type of mechanosensitive channel not related to the MscL and YggB/KefA families. Furthermore it is unlikely that an MscM-like activity is responsible for the betaine efflux, because of the high transport capacity detected in the double deletion mutant.

Some aspects of nitrogen metabolism in Mytilus edulis : effects of aerial exposure

The effects of starvation and emersion on the ammonia effluxes of Mytilus edulis L. were studied. Both fed and starved groups showed similar patterns of efflux during re-immersion, indicating no compensation for starvation during anaerobic catabolism; this is taken as evidence that carbohydrate or lipid is the predominant energy source at the outset of anaerobiosis. Instead of an overshoot of ammonia excreted during the 4 h following re-immersion, all groups showed significantly reduced efflux rates independent of emersion duration. Such results suggest some conversion of ammonia during the recovery period, as no significant drop in haemolymph ammonia occurred at this time. The level of ammonia accumulation in haemolymph and mantle cavity fluid decreased exponentially with emersion duration, implying the stabilisation, at a low rate, of ammonia production after a relatively long period of emersion (16 to 24 h). Oxygen levels of the mantle cavity fluid dropped rapidly during emersion but never attained anoxia over 24 h of emersion, which may indicate some oxygen uptake by emersed mussels. It has been concluded that the behaviour and physiological responses shown by M. edulis to emersion appear to reflect a need for respiratory gas exchange more than a response to desiccation – presumably in the interest of energy-conservation. The reductions in ammonia efflux rate during emersion and, to a certain extent, immediately after re-immersion have also been considered here to be energy-saving strategies that illustrate the importance of ammonia in restoring normal levels of some amino acids following re-immersion.

The efflux of a fluorescent probe is catalyzed by an ATP-driven extrusion system in Lactococcus lactis.

Many bacteria, both gram positive and gram negative, extrude in an energy-dependent manner the fluorescent pH indicator 2',7'-bis-(2-carboxyethyl)-5[and -6]-carboxyfluorescein (BCECF) (D. Molenaar, T. Abee, and W. N. Konings, Biochim. Biophys. Acta 1115:75-83, 1991). This efflux was studied in detail in Lactococcus lactis, and several indications that a transport system is involved were found. This transport system is most likely driven by ATP or a related compound. The evidence is that BCECF extrusion (i) occurs against a BCECF gradient, (ii) is strictly correlated with ATP concentration and not with the proton motive force, and (iii) is inhibited by vanadate and to a lesser extent by N,N'-dicyclohexylcarbodiimide. Most convincingly, a UV mutant with a strongly reduced efflux rate was isolated. Such a mutant was isolated from a BCECF-loaded and lactose-energized population by selection of highly fluorescent cells in a flow cytometer-cell sorter. The physiological function of this extrusion system is unknown, but its characteristics classify it among the traffic ATPases.

Evidence on the mechanism of enhanced sucrose uptake at low cell turgor in leaf discs of Phaseolus coccinius

Plants often tolerate water deficits by lowering the osmotic potential of their cell sap. This may be achieved by accumulation of solutes which results in the maintenance of a positive turgor potential. In this study, the effect of water deficit on sugar uptake was investigated in leaf discs of Phaseolus coccinius L. (cv. Scarlet). Evidence is presented that cell turgor affects the kinetics of sugar transport at the membrane level. Uptake kinetics of sucrose, glucose and 3‐O‐methyl glucose by tissues equilibrated in solutions of relatively high (200–400 mOsm) osmotic concentration consisted of a sat‐urable and a linear component. Low external osmotic concentration i.e., high cellular turgor inhibited the saturating component of sucrose uptake, resulting in a linear uptake profile. However, high cell turgor had no effect on glucose or 3‐O‐methyl glucose uptake kinetics. The effect of turgor versus osmotic component of water potential was differentiated by comparing responses to non‐penetrating (manmtol) or polyethylene glycol, (3350) and penetrating (ethylene glycal) osmotica. Changes in sucrose uptake rates and kinetics were due to changes in cellular turgor and not osmotic potential. Furthermore, at low cellular turgor, a net increase in sucrose uptake occurred as a consequence of enhanced influx rates and not as a result of reduced efflux rates. The data are consistent with previous findings that sugar uptake rates are enhanced under low turgor. We present first evidence indicating that the mechanism by which higher rates of sucrose uptake are maintained underwater deficit conditions is by the activation of the saturable transport system. This mechanism supports previous suggestions that changes in cell turgor are sensed and manifested at the membrane level.

Influence of insulin on sodium efflux in barnacle muscle fibers.

The efflux of radiosodium in single muscle fibers from the barnacleBalanus nubilus was irresponsive to internal or external application of insulin. However, this was not the case with fibers isolated from a barnacle specimen pre-exposed overnight to a large dose of insulin. External application of insulin to pre-exposed fibers caused a decrease in the rate of decline of the radiosodium efflux and stopped the decline in the fractional rate constant for Na efflux. Such kinetics were interpreted as indicating that insulin acts either by releasing sequestered Na or abolishing the process of sequestration. Internal application of saline slowed the rate of decline but failed to completely abolish the mechanism of sequestration. Only in the presence of insulin was the fractional loss of Na each second constant. Internal application of insulin caused a prompt step-up in the rate of Na efflux, followed by a reduced efflux rate constant. This meant that injected insulin caused the release of sequestered Na, leading to partial saturation of the efflux. The response of the Na efflux to injected denatured insulin, though resembling that to native insulin was much smaller in size. Internal application of lysozyme produced a transitory step-up in the rate of Na efflux but failed to produce the kinetics observed with native or denatured insulin. Overnight exposure of the barnacle to a dose of denatured insulin failed to render the fiber sensitive to external and internal application of denatured or native insulinin vitro. Experiments with ouabain-poisoned fibers showed that external or internal application of native insulin caused stimulation of the remaining Na efflux. They also showed that a 10-fold increase in the concentration of ouabain failed to further reduce the ouabaininsensitive Na efflux. Microinjection of GTP into ouabain-poisoned fibers pre-exposed to insulin resulted in a striking rise in the remaining Na efflux. The magnitude of this effect was considerably greater than that in unexposed fibers. The response which was dose-dependent could be blunted by prior injection of CaCl2. Similarly, the response to CaCl2 injection could be blunted by prior injection of GTP. The evidence brought forward is compatible with the view that insulin acts by abolishing the mechanism of internal Na sequestration and by increasing the activity of the guanylate cyclase system.

Ontogeny of Amino Acid Metabolism in Mammalian Kidney

Mammalian kidney transports imino-acids and glycine at physiological concentrations on three t ypes of membrane sites: a low-Km proline system, a low-Km glycine system and a ‘common’ high-Km system. Our recent studies (in press) show that absence of the two low-Km systems accounts for postnatal iminoglycinuria in mammals. Further studies with newborn rat kidney reveal additional ontogenetic features. Uptake: The low-Km proline system becomes active 1 week after birth. The low-Km glycine system is not active until the third week, indicating independent genetic contro of these sites. Efflux: Reduced efflux rates explain increased internal accumulation of soluble aminoacids in newborn kidney, at high and low internal concentrations. Efflux rates evolve with age in a manner suggesting that control of this membrane function is segregated from the control of uptake. Catabolism: Rates of oxidation (measured by 14CO2 formation) and conversion of intracellular L-proline and glycine to other soluble metabolites, are lower in newborn kidney compared to mature kidney, while protein incorporation rates are enhanced in the former. These events do not account for the differences between newborn and adult kidney in net transport of substrate. Ontogeny of iminoglycine metabolism in kidney thus involves independent regulation of influx, efflux, incorporation and oxidation. (Supported by grants from MRC, Canada).