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A R Cossins

Publications and source records attributed to A R Cossins.

At least 37 records · Page 2Linked to original sources
PubMed1996

Cold-induced expression of delta 9-desaturase in carp by transcriptional and posttranslational mechanisms.

Poikilothermic animals respond to chronic cold by increasing phosphoglyceride unsaturation to restore the fluidity of cold-rigidified membranes. Despite the importance of this compensatory response, the enzymes involved have not been clearly identified, and the mechanisms that control their activity are unknown. In carp liver, cold induces an 8- to 10-fold increase in specific activity of the microsomal stearoyl coenzyme A desaturase. Cold-induced up-regulation of gene transcription resulted in a 10-fold increase in desaturase transcript amounts after 48 to 60 hours. However, this increase was preceded by the activation of latent desaturase, probably by a posttranslational mechanism. These two mechanisms may act sequentially to match desaturase expression to the demands imposed by a progressive decrease in temperature.

Acclimatization↗
PubMed1996

The effects of oxygenation upon the Cl-dependent K flux pathway in equine red cells.

The effects of oxygen tension (PO2) upon the K influx pathways of equine red cells have been studied using 86Rb+ as congener for K. Equilibration of cells in 100% nitrogen led to a low and Cl-independent K flux. Change to an atmosphere of 100% air led to a rapid sixfold increase in K flux. The oxygen-activated flux was entirely Cl dependent and was maintained for up to 3 h. Oxygenation-evoked activation was dependent upon PO2 over the physiological range with little effect up to 70% saturation of haemoglobin with oxygen but significant effects between 70 and 100%. K flux at low PO2 was unaffected by acidification to pH 7 or by hypotonic cell swelling. By contrast, at high PO2 both manipulations caused a substantial increase in Cl-dependent K flux. N-Ethylmaleimide (NEM; 1 mM) caused a progressive activation of KCl cotransport in cells held under nitrogen. The protein phosphatase inhibitor, calyculin A (100 nM), applied during NEM-evoked activation caused a "clamping" of K influx at that level. This "clamped" activity was unaffected by subsequent oxygenation. We conclude that oxygenation exerts a primary control over cotransport activity and that acidification and cell swelling are secondary modulators. It appears that oxygenation-evoked activation of the Cl-dependent K flux involves a serine/threonine phosphorylation event. Regulating the PO2 of the solution before and during experiments is important in controlling the activity of the KCl cotransporter and cell volume.

Acids↗
PubMed1996

Protein tyrosine phosphorylation and the regulation of KCl cotransport in trout erythrocytes.

Electroneutral salt transporters are activated and deactivated by changes to the phosphorylation status either of the transporter itself or of other, as yet unidentified, regulatory proteins. We have studied the effects of an inhibitor of protein tyrosine kinase (PTK), genistein, upon KCl cotransport in trout erythrocytes. We show that Cl-dependent K fluxes activated by physiological stimuli, i.e. oxygenation and beta-adrenergic agonists, are rapidly and completely blocked by genistein, whilst the inactive analogue of genistein, daidzein, had no effect. By contrast, the protein tyrosine phosphatase (PTP) inhibitor, vanadate (V), caused a slow but strong activation of an inactive cotransporter. This vanadate (V) activated flux was inhibited by genistein as well as by the serine/threonine phosphatase (PSP) inhibitor, calyculin A. However, genistein had no effect upon the activation of the cotransporter by the protein (serine/threonine) kinase (PSK) inhibitor, staurosporine, or by N-ethylmaleimide, which also appears to act by inhibiting a PSK. These results are consistent with a sequential scheme of at least two tyrosine phosphorylation events which lie upstream to the serine/threonine phosphorylation sites in the signal transduction pathway leading from stimulus to transporter activation. The regulation of the activity of KCl cotransporter appears to involve a complex series of phosphorylation reactions.

Animals↗
PubMed1994

Modulation of K(+)-Cl- cotransport in equine red blood cells.

Potassium transport was measured in equine red blood cells, using 86Rb+ influx as a convenient assay. A significant component of volume- and pH-sensitive K(+)-Cl- cotransport to the overall K+ flux was observed in all blood samples studied, although fluxes were variable between animals, and within individuals when measured at intervals over a period of weeks. The aryloxyacetic acid [(dihydroindenyl)oxy]alkanoic acid (DIOA), at a final concentration of 100 microM, inhibited most (> 95%) of the Cl(-)-dependent K+ flux, and DIOA sensitivity was therefore used to define the activity of the K(+)-Cl- cotransport. K(+)-Cl- cotransport was also sensitive to protein phosphatase inhibition with calyculin A or okadaic acid, with inhibition constants of 9 +/- 1 nM for calyculin and about 100 nM for okadaic acid. Peak fluxes were observed at an external pH of 6.7-7.0, with inhibition at higher and lower values. Volume-sensitive K+ fluxes assayed in autologous plasma, controlled for osmolaity, pH and potassium concentration, were significantly lower (28 +/- 8% of control values, n = 6) than those measured in saline. This inhibition was mimicked by the culture medium RPMI, but disappeared following dialysis of the plasma. Phosphate (5.6 mM) inhibited volume-sensitive K+ fluxes by 48 +/- 2%, n = 3; no significant effect was observed by increasing external magnesium concentrations to 0.5 or 2 mM. Thus, inhibition by RPMI, but not that by plasma, may be due to phosphate. Finally, volume- and pH-sensitive K+ fluxes were sensitive to oxygen tension and were abolished reversibly by equilibrating solutions with nitrogen, as opposed to air. Use of solutions equilibrated with different values of Po2 may account for some of the variability in equine red blood cell KCl fluxes. The importance of these observations to equine red blood cell homeostasis and haemodynamics is discussed.

Animals↗
PubMed1994

Role of protein phosphorylation in control of K flux pathways of trout red blood cells.

The role of protein phosphatases in the regulation of K flux pathways of the trout red blood cell has been investigated using the phosphatase inhibitors calyculin A and okadaic acid. Both inhibitors completely blocked an oxygenation-activated Cl-dependent K flux with a 50% inhibitory concentration of 17 and 675 nmol/l, respectively, but not the hypotonically activated Cl-independent K uptake. N-ethylmaleimide (NEM) and staurosporine caused an increase in the Cl-dependent flux. In both cases preincubation with calyculin A blocked activation but, when added during activation, it prevented any further increase with NEM but abolished the staurosporine-induced uptake. K uptake that was activated by NEM and "clamped" by calyculin A was volume sensitive, indicating a dual influence on this pathway. Chelerythrine, a protein kinase inhibitor, activated a Cl-independent K uptake that was unaffected by calyculin A. It is concluded that activation and deactivation of both Cl-dependent and Cl-independent pathways require changes in the phosphorylation of an as yet unidentified target protein(s), although with different sets of protein kinase and/or phosphatases. These observations also suggest a complex model of kinase-phosphatase regulation and provide drugs for the pharmacological definition and manipulation of Cl-dependent and Cl-independent K flux pathways.

Alkaloids↗
PubMed1993

A near perfect temperature adaptation of bilayer order in vertebrate brain membranes.

The bilayer order of a brain synaptic membrane fraction from a number of fish, mammalian and avian species have been compared in relation to their respective body temperatures using steady-state and time-resolved fluorescence anisotropy techniques. Fluorescence anisotropy for both 1,6-diphenyl-1,3,5-hexatriene and trans-parinaric acid increased in the order: antarctic Notothenia, trout, perch, cichlid, rat and starling, this also being the order of increasing body temperature. This suggests that cold-adapted fish species possess more disordered brain membranes than warm-adapted fish species, and mammals and birds membranes were more ordered than fish membranes. Comparison of temperature profiles for both fluorescence probes showed that fish species display similar anisotropies, and by inference bilayer order, to mammals and birds when measured at their respective body temperatures. Time-resolved analysis showed that the interspecific differences in (P2) order parameter was consistently related to body temperature whilst the rotational diffusion coefficient was not. These results suggest that brain membrane order is highly conserved within the vertebrates despite large differences in thermal habits and phylogenetic position. Polar fish species have by far the lowest bilayer order indicating that invasion of extreme cold habitats involved an adaptive decrease in bilayer order and conversely adoption of a high body temperature by mammals involved an adaptive increase in bilayer order. The conservation of membrane static order for these species at their respective body temperatures indicates a regulatory control of this aspect of membrane hydrocarbon structure and the functional importance of this structure.

Adaptation, Physiological↗
PubMed1992

Homeoviscous adaptation under pressure: the pressure dependence of membrane order in brain myelin membranes of deep-sea fish.

Steady-state and time-resolved anistropy of 1,6-diphenyl-1,3,5-hexatriene (DPH) fluorescence have been used to compare the hydrocarbon order of brain myelin membranes from a shallow water (plaice) and two deep-sea fish species (Coryphenoides rupestris and Coryphenoides armatus). At atmospheric pressure the deep sea fish displayed lower steady-state anisotropies than shallow water species although the pressure dependence of anisotropy was similar in all species. Time-resolved measurements allowed the separate determination of the rate of probe motion from the amplitude of that motion. Anisotropy decays were analysed in terms of two correlation times and a constant (r infinity). The r infinity and mean value of P2 order parameter for all species increased with pressure, the graphs for deep-sea species being translated to higher pressures relative to shallow-water species. The resulting pressure coefficients for C. armatus was distinctly less than for the two shallower species. These time-resolved studies show that the interspecific differences provide for similar order parameters in all three species when corrected to their respective habitat conditions of pressure and temperature. This indicates that myelin order is highly conserved despite the profound ordering effects of high hydrostatic pressure.

Adaptation, Physiological↗
PubMed1992

Oxygenation-activated K fluxes in trout red blood cells.

The effect of oxygenation on the dissipative fluxes of K in trout red blood cells has been determined. Unidirectional influx under low oxygen tension (PO2 = 1 kPa) was 0.56 +/- 0.07 mmol.l-1 packed cells.h-1. Within a few minutes of equilibration with high oxygen tension (PO2 = 120 kPa), influx was increased 14-fold, and this was associated with a progressive loss of KCl and a cell shrinkage. K influx progressively declined over the following 3 h to levels close to those characteristic of cells at low oxygen tension. Replacement of medium Cl by NO3- or methane sulfonate inhibited the stimulation due to high oxygen as did furosemide and low extracellular pH. The oxygenation-stimulated influx was highly volume sensitive, being increased by up to 100% by osmotic swelling and decreased by osmotic shrinkage. By contrast, the small influx under low oxygen tension was unaffected by either Cl replacement or by shrinkage and increased only with extreme swelling. Thus high oxygen tension activated a Cl-dependent and furosemide-sensitive K flux. Once activated, the mechanism was rapidly deactivated on transfer back to low oxygen tension but slowly deactivated when maintained at high PO2. The oxygenation-stimulated flux mechanism promotes a rapid and more complete volume regulatory decrease than in cells at low oxygen tension.

Animals↗
PubMed1991

Rapid cold-induced changes of membrane order and delta 9-desaturase activity in endoplasmic reticulum of carp liver: a time-course study of thermal acclimation.

The membrane order of liver endoplasmic reticulum (ER) membranes of 10 degrees C- and 30 degrees C-acclimated carp has been compared using the fluorescence polarization technique with DPH as probe. Membranes from cold-acclimated fish displayed lower polarizations than corresponding membranes from warm-acclimated fish, the difference compensating for 34-50% of the direct effects of temperature upon polarization. The changes in delta 9-desaturase activity and fluorescence polarization of DPH in ER membranes have been monitored as a function of time during cold acclimation of 30 degrees C-acclimated carp. Cooling was achieved in three stages over 48 h. Desaturase activity in both rough and smooth ER showed a rapid increase in activity for the first three days followed by a decline on day 4 and a second increase up to day 10. Polarization of DPH (measured at 10 degrees C) was rapidly reduced on cooling with no further change after day 4. The halftime for change in polarization and for the first desaturase induction were both approx. 2 days although large changes in polarization were evident within 24 h after the onset of cooling. During the cooling phases the daily changes in DPH polarization were quantitatively related to increments in desaturase capacity. The second desaturase induction had no effect upon membrane structure, at least as indicated by the polarization technique.

Animals↗
PubMed1991

Cell volume regulation by trout erythrocytes: characteristics of the transport systems activated by hypotonic swelling.

1. An osmolality reduction of the suspending medium leads to osmotic swelling of trout erythrocytes, which is followed by a volume readjustment towards the original level. The regulatory volume decrease (RVD) was not complete after 1 h. 2. During RVD the cells lost K+ and Cl- but gained Na+. This entry of Na+, which is about half the K+ loss, explains the incomplete volume recovery (it was complete when Na+ was replaced by impermeant N-methyl-D-glucamine). The cells also lose large quantities of taurine, which accounts for about 53% of the volume recovery. In addition RVD is accompanied by the activation of a pathway allowing some large organic cations which are normally impermeant, such as choline or tetramethyl-ammonium, to rapidly penetrate the cells. 3. The swelling-activated K+ loss is not significantly affected by replacement of Cl- by NO3-, indicating that K+ moves through a Cl(-)-independent K+ pathway. Furosemide, DIDS (4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid) and niflumic acid inhibit the K+ loss. From experiments performed in high-K(+)-containing media, it appears that these compounds block the K+ flux, not by inhibiting Cl- movements but by interfering with the K+ pathway. 4. All the volume-activated pathways (K+, Na+, taurine, choline) are fully inhibited by furosemide and by inhibitors of the anion exchanger such as DIDS and niflumic acid. The concentration required for 50% inhibition (IC50) of both inorganic cations and taurine appears to be similar. It is proposed that DIDS interacts with a unique target which controls all the volume-sensitive transport systems.

Amino Acids↗
PubMed1991

Homeoviscous adaptation and thermal compensation of sodium pump of trout erythrocytes.

The effects of thermal acclimation of trout on the transport activity and turnover number of the erythrocyte Na+ pump have been determined. Na+ pump activity was estimated by measuring the ouabain-sensitive K+ influx in Na(+)-loaded cells and the number of active pumps determined by Scatchard analysis of [3H]ouabain binding and by correlation of ouabain binding with pump inhibition. Cold acclimation was associated with an increase in pump activity of up to 60%, although the furosemide-sensitive and residual fluxes were unaffected. The number of ouabain binding sites was similar in both acclimation groups at approximately 21,000-23,000 sites/cell. This means that cold acclimation induced an increase in the transport turnover number of pump molecules from approximately 6 to 9 s-1. Cold acclimation was also associated with a decrease in membrane order as indicated by steady-state fluorescence polarization of the membrane probe, 1,3-diphenyl-1,3,5-hexatriene, with a homeoviscous efficacy of 25-41%. That membrane order may influence pump transport activity is supported by experiments on cholesterol supplementation, which caused both an increase in membrane order and a decrease in pump turnover number. The degree of pump compensation was dependent on the season, with greatest responses in the late spring and declining responses through to winter. By contrast, changes in membrane order were observed throughout the year. Expression of pump activity and erythropoiesis may vary throughout the seasonal cycle in complex ways that confuse the direct comparison study of cellular properties in a heterogeneous population of cells.

Adaptation, Physiological↗
PubMed1990

Temperature adaptation of biological membranes: differential homoeoviscous responses in brush-border and basolateral membranes of carp intestinal mucosa.

The effects of temperature acclimation of carp upon the hydrocarbon order of intestinal membranes has been determined. A fractionation technique has been developed for the simultaneous purification of brush-border and basolateral membrane fractions from the intestinal mucosa. The specific activity of alkaline phosphatase in the brush-border fraction was enhanced 6.4-fold over that of the initial homogenate, whilst the (Na(+)-K+)-stimulated ATPase was enhanced 5.8-fold in the basolateral fraction. The specific activities of NADPH-cytochrome-c reductase, succinate-cytochrome-c reductase and acid phosphatase were not increased in these two fractions. Membrane hydrocarbon order in membranes from 10 and 30 degrees C-acclimated carp has been compared by measuring the steady-state fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene over a range of temperatures. In the brush-border fraction, polarization was identical in both cold- and warm-acclimated groups, whilst large differences were observed in the basolateral fraction sufficient to offset approx. 75% of the temperature-induced ordering effects of cold. The fatty acid composition of the major phosphoglyceride fractions in the brush-border fraction was also largely unaffected by thermal acclimation, whilst the basolateral fraction showed significant increases in the proportion of unsaturated fatty acids in the cold. It is concluded that whilst the basolateral membrane of intestinal mucosa displays a large homoeoviscous response that correlates with a shift in lipid composition, the brush-border membrane does not. These findings are consistent with evidence of functional adaptations of the basolateral membrane during thermal acclimation (Gibson, J.S., Ellory, J.C. and Cossins, A.R. (1985) J. Exp. Biol. 114, 355-364).

Adaptation, Physiological↗
PubMed1990

The temperature dependence of the adrenergic Na+/H+ exchanger of trout erythrocytes.

The effects of temperature upon the adrenergic Na+/H+ exchange of rainbow trout erythrocytes have been studied in vitro. The initial rates of H+ ejection and of increase of intracellular Na+ [( Na+]i) in adrenergically stimulated cells were highly temperature-dependent, with apparent Arrhenius activation energies of 112.8 +/- 10.0 (mean +/- S.D., N = 4) and 84.1 +/- 3.0 kJ mol-1 (N = 3), respectively. The steady-state [Na+]i following stimulation decreased progressively with cooling, whilst the time required for [Na+]i to return to control values after removal of agonist was greatly increased. The change in intracellular pH resulting from adrenergic stimulation was reduced by cooling, such that at 4 degrees C adrenergic responses were barely measurable. The effect of temperature upon the steady-state [Na+]i and pHi was probably caused by a disparity in the temperature dependence of the transport mechanisms that contribute to the respective steady states.

Animals↗
PubMed1989

The adaptation of biological membranes to temperature and pressure: fish from the deep and cold.

The homeostatic regulation of bilayer order is a property of functional importance. Arguably, it is best studied in those organisms which experience and must overcome disturbances in bilayer order which may be imposed by variations in temperature of hydrostatic pressure. This article reviews our recent work on the adaptations of order in brain membranes of those fish which acclimate to seasonal changes in temperature or which have evolved in extreme thermal or abyssal habitats. The effects of temperature and pressure upon hydrocarbon order and phase state are reviewed to indicate the magnitude of the disturbances experienced by animals in their environments over the seasonal or evolutionary timescale. Acclimation of fish to altered temperature leads to a partial correction of order, while comparison of fish from extreme cold environments with those from temperate or tropical waters reveals a more complete adaptation. Fish from the deep sea also display adaptations of bilayer order which largely overcome the ordering effects of pressure.

Adaptation, Physiological↗
PubMed1988

Calorimetric and spectroscopic studies of lipid thermotropic phase behavior in liver inner mitochondrial membranes from a mammalian hibernator.

Arrhenius plots of various enzyme and transport systems associated with the liver mitochondrial inner membranes of ground squirrels exhibit changes in slope at temperatures of 20-25 degrees C in nonhibernating but not in hibernating animals. It has been proposed that the Arrhenius breaks observed in nonhibernating animals are the result of a gel to liquid-crystalline phase transition of the mitochondrial membrane lipids, which also occurs at 20-25 degrees C, and that the absence of such breaks in hibernating animals is due to a major depression of this lipid phase transition to temperatures below 4 degrees C. In order to test this hypothesis, we have examined the thermotropic phase behavior of liver inner mitochondrial membranes from hibernating and nonhibernating Richardson's ground squirrels, Spermophilus richardsonii, by differential scanning calorimetry and by 19F nuclear magnetic resonance and fluorescence polarization spectroscopy. Each of these techniques indicates that no lipid phase transition occurs in the membranes of either hibernating or nonhibernating ground squirrels within the physiological temperature range of this animal (4-37 degrees C). Moreover, differential scanning calorimetric measurements indicate that only a small depression of the lipid gel to liquid-crystalline phase transition, which is centered at about -5 degrees C in nonhibernating animals and at about -9 degrees C in hibernators, occurs. We thus conclude that the Arrhenius plot breaks observed in some membrane-associated enzymatic and transport activities of nonhibernating animals are not the result of a lipid phase transition and that a major shift in the gel to liquid-crystalline lipid phase transition temperature is not responsible for seasonal changes in the thermal behavior of these inner mitochondrial membrane proteins.

Acclimatization↗