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ABSTRACT: The beta-adrenergic stress response in red blood cells (RBCs) of rainbow trout shows seasonal changes in expression. We have explored the mechanisms underpinning this response by following, over a period of 27 months, changes in beta-adrenergic receptor (beta-AR) binding characteristics, beta-adrenergically stimulated RBC Na(+)/H(+) exchanger (betaNHE) activity, together with beta-AR and betaNHE mRNA levels and plasma steroid hormone and lactate levels. These parameters were measured at approximately monthly intervals in a single population of fish held under semi-natural conditions. Membrane-bound, high-affinity beta-ARs were present in RBCs at all sampling times, varying from 668+/-112 receptors cell(-1) to 2654+/-882 receptors cell(-1) (mean +/- S.E.M.; N=8). betaNHE activity, however, was reduced by 57% and 34% in December 1999 and February 2001, respectively, compared with an otherwise sustained influx that averaged 110.4+/-2.3 mmol l(-1) RBCs h(-1) (N=119). Only one reduction coincided with a spawning period but both were preceded by transient increases in circulating testosterone. betaNHE activity measured under standard conditions was not correlated with the number or affinity of beta-ARs nor with water temperature, but both beta-AR numbers and betaNHE activity were positively related to their respective mRNA levels (P=0.005 and 0.038, respectively). Pharmaceutical intervention in the transduction cascade linking the beta-AR and betaNHE failed to indicate any failure of the transduction elements in RBCs displaying low betaNHE activity. Similarly, we failed to demonstrate any link between seasonal cortisol fluctuations and seasonally reduced betaNHE activity. However, the betaNHE activity of age-separated RBC fractions showed that younger RBCs had a significantly higher betaNHE response than older RBCs, consistent with the seasonal reductions in betaNHE being linked to turnover of RBCs and erythropoiesis. Testosterone is known to induce erythropoiesis and we conclude that seasonal reductions in betaNHE are not caused by changes in beta-AR numbers but may be linked to testosterone-induced erythropoiesis.
Journal of Experimental Biology 02/2004; 207(Pt 2):357-67. · 3.00 Impact Factor
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ABSTRACT: Carp respond to cold by the upregulated expression of Delta9-acyl-CoA desaturase. Here we report the cloning and characterization of Cds2, a second Delta9-acyl CoA-desaturase expressed in carp liver. Both Cds1 and Cds2 complemented the ole1 mutation in Saccharomyces cerevisiae, permitting the synthesis of delta9-monounsaturates, confirming their identity as delta9-desaturases. We demonstrate that under a standard feeding regime it is the Cds2, and not Cds1, transcript that is transiently upregulated during the first few days of cooling from 30 degrees C to 10 degrees C, the period when cold-induced membrane restructuring occurs. Cds2 exists as two differentially spliced transcripts, differing by a small segment from the 3'-untranslated region, the ratio of which varies with temperature. Feeding a diet enriched in saturated fats produced a fourfold increase in Cds1 transcript levels, which was blocked by cooling to 15 degrees C. Cds2 transcript levels, however, showed no substantial response to the saturated diet. Thus carp liver uniquely expresses two isoforms of delta9-acyl CoA desaturase, possibly formed by a recent duplication event, that are differentially regulated by cooling and dietary treatment.
AJP Regulatory Integrative and Comparative Physiology 02/2003; 284(1):R41-50. · 3.34 Impact Factor
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ABSTRACT: Cold acclimation of carp from 30 degrees C to 10 degrees C causes a restructuring of liver microsomal phospholipids characterised by increased proportions of monounsaturated fatty acid in phosphatidylcholine (PC) and phosphatidylethanolamine (PE). Here, we have used electrospray ionisation mass spectrometry (ESI-MS) to determine the patterns of alteration to individual molecular species compositions of PC, PE and phosphatidylinositol (PI) in response to gradually decreasing temperature. The results demonstrate that cold induces precise changes to a limited number of phospholipid species, and that these changes are distinct and different for each phospholipid class. The major change for PC was increased 16:1/22:6, but for PE the species that increased was 18:1/22:6. By contrast, the PI species that increased during cold acclimation were characterised by an sn-1 monounsaturated fatty acid in combination with arachidonoyl or eicosapentaenoyl fatty acid at the sn-2 position. Analysis of acyl distribution indicates that cold only caused the accumulation of monounsaturated fatty acids at the sn-1 and not at the sn-2 position of phospholipids. These results highlight the tight and restricted range of modifications that membranes make to their phospholipid composition in response to thermal stress.
Journal of Experimental Biology 01/2003; 205(Pt 24):3989-97. · 3.00 Impact Factor
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ABSTRACT: All organisms respond to environmental challenge by adaptive responses, although, in many cases, the underlying molecular mechanisms are not understood. In the case of membranes, the physical structure of membrane phospholipids is conserved in the face of cold, rigidifying conditions by the elevated proportions of unsaturated fatty acids. We have observed a clear positional specificity in this substitution and head group preferences in carp liver membranes. We have also demonstrated changes in the activity of lipid desaturases that mediate the unsaturation response, caused by both transcriptional and post-translational mechanisms. Another hepatic isoform has recently been discovered with sensitivity, not to cooling, but to dietary variations. Finally, we are testing the importance of desaturase inductions in the inducible cold tolerance of the whole animal.
Biochemical Society Transactions 12/2002; 30(Pt 6):1082-6. · 3.71 Impact Factor
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ABSTRACT: Stereological sampling regimes, in particular volume and number estimation, often require systematic uniformly random sections throughout a specimen. A method has been developed to increase the efficiency of preparing fish larvae for sectioning prior to histological or stereological analysis. Embedding a group of larvae in a resin block using this technique greatly reduces the quantity of sections produced and allows easy assessment of sample groups. Saving time in this way therefore makes stereology a more viable research tool.
Journal of Microscopy 07/2002; 206(Pt 3):179-81. · 1.63 Impact Factor
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ABSTRACT: Hypotonic swelling of teleost erythrocytes activates multiple transport systems leading to the regulatory decrease of cell volume. We have examined using pharmacological manipulation the swelling-induced taurine flux pathway in red blood cells of the rainbow trout and its relationship to swelling-induced K flux pathways. We show that the activation and deactivation of taurine flux is rapid and that the flux is a sigmoidal function of cell volume. N-ethylmaleimide (NEM) and the non-specific protein kinase inhibitor, staurosporine, both inactivated the hypotonically-induced taurine flux with concentrations eliciting half-maximal inhibition (IC50s) of 212 and 17 micromol(-1), respectively. The low taurine fluxes under isotonic conditions were unaffected. By contrast, the tyrosine kinase inhibitor, genistein, partially inhibited taurine flux under both isotonic and hypotonic conditions. The specific phosphatase inhibitor, calyculin A, had no inhibitory or stimulatory effect under either condition whilst the less-specific phosphatase inhibitor, ortho-vanadate, reduced taurine flux only under hypotonic conditions. In these respects the regulatory control of the taurine pathway differs from the Cl-dependent K flux. However, NEM and staurosporine also inhibited the Cl-independent K flux, both with similar IC50s to those observed for taurine fluxes. This supports the idea of the hypotonically-induced taurine flux and the Cl-independent K flux sharing the same transport pathway.
Pflügers Archiv - European Journal of Physiology 08/2000; 440(3):467-75. · 4.46 Impact Factor
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ABSTRACT: Larval Dover sole fed an Artemia diet supplemented with n-3 long-chain (C20 + C22) polyunsaturated fatty acids (PUFA) are known to be more resistant to low-temperature injury. Here we explore the relationship between tissue fatty acid composition and tolerance of stressful environmental conditions over the larval and early juvenile periods. Artemia nauplii supplemented with n-3 long-chain PUFA-deficient and PUFA-enriched oil emulsions were fed to two groups of larvae. Whole body tissue samples from the resulting PUFA-deficient and -enriched juveniles possessed 12.1 and 21.9% n-3 long-chain PUFA, respectively. These differences were at the expense of C18 PUFA, while proportions of saturated fatty acids, monounsaturated fatty acids, and total PUFA were unaffected. Brain and eye tissues from the PUFA-deficient fish contained lower levels of 22:6n-3, known to be important for optimal nervous system function, incorporating instead a range of fatty acids of lower unsaturation. PUFA-deprived juveniles showed substantially greater mortality when exposed to a combination of low temperature and low salinity, as well as to high temperature and to hypoxia. After adaptation to the different diets, both dietary groups were fed a common formulated feed high in n-3 long-chain PUFA. Tissue PUFA in both groups progressively increased to the same high value, with a consequent loss of the differences in cold-susceptibility. These correlated changes support a link between dietary manipulation of n-3 long-chain PUFA and development of a stress-sensitive phenotype. PUFA deprivation had no detectable effect upon static hydrocarbon order of purified brain membranes (as assessed by fluorescence polarization) but was associated with an increase in the whole-body content of prostaglandins. We conclude that susceptibility to environmental stress is responsive to dietary n-3 long-chain PUFA manipulation, possibly due to altered tissue development or the overproduction of eicosanoids.
Lipids 08/2000; 35(7):745-55. · 2.13 Impact Factor
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ABSTRACT: Teleost species from cold environments possess more disordered brain synaptic membranes than species from warm habitats, thereby providing equivalent physical structures at their respective habitat temperatures. We have related this adaptive interspecific biophysical response to the fatty acid composition of brain membranes from 17 teleost species obtained from Antarctic, temperate and semi-tropical waters, as well as from rat and turkey as representative homeotherms. Cold-adaptive increases in membrane disorder (determined by fluorescence anisotropy with diphenylhexatriene as probe) were correlated with large and linear increases in the proportion of unsaturated fatty acids, from 35 to 60 % in phosphatidylcholine (PtdCho) and from 55 to 85 % in phosphatidylethanolamine (PtdEth). For PtdCho, the cold-adaptive increase in unsaturation was associated almost entirely with increased proportions (from 7 to 40 %) of polyunsaturated fatty acids (PUFAs), with mono-unsaturates (MUFAs) providing an approximately constant proportion in all species. Exactly opposite effects were evident for phosphatidylethanolamine (PtdEth). Thus, the compositional adaptation for PtdCho occurred largely by exchange of polyunsaturated and mono-unsaturated fatty acid in the sn-2 position, whilst for PtdEth it involved exchanges between saturates and mono-unsaturates at the sn-1 position. This difference may be related to the different molecular shapes of the two phosphoglycerides and the need to maintain the balance between bilayer-stabilising and -destabilising tendencies. This comparative study provides a more comprehensive view of the compositional adjustments that accompany and perhaps account for temperature-adaptive interspecific differences in membrane physical structure.
Journal of Experimental Biology 08/2000; 203(Pt 14):2105-15. · 3.00 Impact Factor
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ABSTRACT: Oxygen is essential for all higher forms of animal life. It is required for oxidative phosphorylation, which forms the bulk of the energy supply of most animals. In many vertebrates, transport of O(2) from respiratory to other tissues, and of CO(2) in the opposite direction, involves red cells. These are highly specialised, adapted for their respiratory function. Intracellular haemoglobin, carbonic anhydrase and the membrane anion exchanger (AE1) increase the effective O(2)- and CO(2)-carrying capacity of red cells by approximately 100-fold. O(2) also has a pathological role. It is a very reactive species chemically, and oxidation, free radical generation and peroxide formation can be major hazards. Cells that come into contact with potentially damaging levels of O(2) have a variety of systems to protect them against oxidative damage. Those in red cells include catalase, superoxide dismutase and glutathione. In this review, we focus on a third role of O(2), as a regulator of membrane transport systems, a role with important consequences for the homeostasis of the red cell and also the organism as a whole. We show that regulation of red cell transporters by O(2) is widespread throughout the vertebrate kingdom. The effect of O(2) is selective but involves a wide range of transporters, including inorganic and organic systems, and both electroneutral and conductive pathways. Finally, we discuss what is known about the mechanism of the O(2) effect and comment on its physiological and pathological roles.
Journal of Experimental Biology 06/2000; 203(Pt 9):1395-407. · 3.00 Impact Factor
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ABSTRACT: Hypotonic swelling of teleost erythrocytes activates multiple transport systems leading to the regulatory decrease of cell volume. We have examined using pharmacological manipulation the swelling-induced taurine flux pathway in red blood cells of the rainbow trout and its relationship to swelling-induced K flux pathways. We show that the activation and deactivation of taurine flux is rapid and that the flux is a sigmoidal function of cell volume. N-ethylmaleimide (NEM) and the non-specific protein kinase inhibitor, staurosporine, both inactivated the hypotonically-induced taurine flux with concentrations eliciting half-maximal inhibition (IC50s) of 212 and 17 mol l-1, respectively. The low taurine fluxes under isotonic conditions were unaffected. By contrast, the tyrosine kinase inhibitor, genistein, partially inhibited taurine flux under both isotonic and hypotonic conditions. The specific phosphatase inhibitor, calyculin A, had no inhibitory or stimulatory effect under either condition whilst the less-specific phosphatase inhibitor, ortho-vanadate, reduced taurine flux only under hypotonic conditions. In these respects the regulatory control of the taurine pathway differs from the Cl-dependent K flux. However, NEM and staurosporine also inhibited the Cl-independent K flux, both with similar IC50s to those observed for taurine fluxes. This supports the idea of the hypotonically-induced taurine flux and the Cl-independent K flux sharing the same transport pathway.
Pflügers Archiv - European Journal of Physiology 05/2000; 440(3):467-475. · 4.46 Impact Factor
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ABSTRACT: Cold acclimation induces a transient enzymatic activation of the acyl CoA-(&Dgr;)(9)-desaturase in carp liver. We have determined thresholds for two underlying mechanisms; namely, the activation of latent enzyme and the induced synthesis of new desaturase. Carp were progressively cooled from 30 degrees C to 23, 17 and 10 degrees C, where they were held for up to 5 days. Endoplasmic reticulum phospholipids showed substantial changes in fatty acid composition, with linear decreases in the proportion of saturates with temperature over the full range of cooling (11.3 % in phosphatidylcholine and 15.8 % in phosphatidylethanolamine). In the phosphatidyl-ethanolamine fraction, this was linked to increased proportions of monoenes, particularly 20:1(n-9). Modest cooling to 23 degrees C on day 1 induced a 2.5-fold transient increase in delta(9)-desaturase activity without any change in the amount of desaturase protein or transcript. Further cooling to 17 degrees C induced a greater and more sustained increase in desaturase activity, reaching sevenfold on day 5, with a 10- to 20-fold increase in the amount of desaturase transcript. Extreme cooling to 10 degrees C led to a very large, but transient, 40- to 50-fold increase in desaturase transcript amounts, a modest 40-50 % increase in desaturase protein but no further increase in activity over that observed at 17 degrees C. These results distinguish at least three mechanisms involved in cold-induced lipid restructuring; the activation of latent desaturase observed with gentle cooling, the induction of desaturase gene transcription and, finally, a third unidentified lipid compensatory mechanism that occurs with extreme cooling. The complex nature of cold-induced lipid restructuring also involves changes in the activity of other biosynthetic enzymes, including elongase and positional- and phospholipid-specific acyltransferases.
Journal of Experimental Biology 03/2000; 203(Pt 3):641-50. · 3.00 Impact Factor
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ABSTRACT: 1. Na+-K+-2Cl- cotransport (NKCC) was studied in turkey red cells using Na+ dependence or bumetanide sensitivity of 86Rb+ influx to monitor activity of the transporter. 2. Deoxygenation was the major physiological stimulus for NKCC activity: oxygen tensions (PO2) over the physiological range modulated the transporter, with a PO2 for half-maximal activation of about 41 mmHg (n = 3). In air, activity of NKCC was also stimulated by shrinkage and isoproteronol (isoprenaline, 5 microgr;M). By contrast, in deoxygenated cells, although the transporter activity was markedly elevated, it was no longer sensitive to volume or beta-adrenergic stimulation. 3. Calyculin A, a protein phosphatase inhibitor, stimulated cotransport with a lag of about 5 min. N-Ethylmaleimide (NEM) inhibited cotransport and also blocked the stimulatory effect of calyculin A if administered before calyculin A. Stimulation by calyculin A and deoxygenation were not additive. Staurosporine (2 microM) inhibited deoxygenated-stimulated K+ influxes, but not those stimulated by calyculin A. NEM added during calyculin A stimulation, i.e. during the 5 min lag, caused transport activity to be clamped at levels intermediate between maximal (calyculin A alone) and control. Cells treated with calyculin A alone or with calyculin A followed by NEM were no longer sensitive to volume, isoproteronol or PO2. 4. The results have characterized the interaction between deoxygenation and other stimuli of NKCC activity. They have also shown that it is possible to manipulate the transporter in a reciprocal way to that shown previously for K+-Cl- cotransport.
The Journal of Physiology 07/1999; 517 ( Pt 2):421-9. · 4.72 Impact Factor
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ABSTRACT: 1. Cl--dependent K+ (86Rb+) influxes were measured in oxygenated and deoxygenated equine red blood cells, whose free [Mg2+]i had been clamped, to examine the effect on O2 dependency of the K+-Cl- cotransporter. 2. Total [Mg2+]i was 2.55 +/- 0.07 mM (mean +/- s.e.m. , n = 6). Free [Mg2+]i was estimated at 0.45 +/- 0.04 and 0.68 +/- 0. 03 mM (mean +/- s.e.m., n = 4) in oxygenated and deoxygenated red cells, respectively. 3. K+-Cl- cotransport was minimal in deoxygenated cells but substantial in oxygenated ones. Cl--dependent K+ influx, inhibited by calyculin A, consistent with mediation via the K+-Cl- cotransporter, was revealed by depleting deoxygenated cells of Mg2+. 4. Decreasing [Mg2+]i stimulated K+ influx, and increasing [Mg2+]i inhibited it, in both oxygenated and deoxygenated red cells. When free [Mg2+]i was clamped, Cl--dependent K+ influxes were always greater in oxygenated cells than in deoxygenated ones, and changes in free [Mg2+]i of the magnitude occurring during oxygenation-deoxygenation cycles had a minimal effect. Physiological fluctuations in free [Mg2+]i are unlikely to provide the primary link coupling activity of the K+-Cl- cotransporter with O2 tension. 5. Volume and H+ ion sensitivity of K+ influx in Mg2+-clamped red cells were increased in O2 compared with those in deoxygenated cells at the same free [Mg2+]i, by about 6- and 2-fold, respectively, but again these features were not responsible for the higher fluxes in oxygenated cells. 6. Regulation of the K+-Cl- cotransporter by O2 is very similar in equine, sheep and in normal human (HbA) red cells, but altered in human sickle cells. Present results imply that, as in sheep red cells, O2 dependence of K+-Cl- cotransport in equine red cells is not mediated via changes in free [Mg2+]i and that cotransport in Mg2+-clamped red cells is still stimulated by O2. This behaviour is contrary to that reported for human sickle (HbS) cells.
The Journal of Physiology 04/1999; 515 ( Pt 2):431-7. · 4.72 Impact Factor
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ABSTRACT: The relationship between phospholipid saturation and membrane physical structure in a complex, highly polyunsaturated biological membrane (trout liver microsomes) has been studied by the graded and specific hydrogenation of polyunsaturated fatty acids. The homogeneous catalyst Pd(QS)2 caused rapid and effective hydrogenation, increasing the proportion of saturated fatty acids from 20-30% up to 60%, without loss or fragmentation. Long chain, polyunsaturated fatty acids (20:5 omega 3, 22:6 omega 3) were rapidly converted to a large number of partially hydrogenated isomers, and ultimately to the fully saturated C20 or C22 fatty acids. C18 mono- and di-unsaturates showed slower rates of hydrogenation. Increased saturation was closely associated with an increased membrane physical order as determined by the fluorescence anisotropy probe, 1,6-diphenyl-1,3,5-hexatriene. However, extensive hydrogenation led to highly ordered membranes exhibiting a gel-liquid crystalline phase transition between 30 and 60 degrees C. Polyunsaturated membranes can thus be converted into partially or substantially saturated membranes with measurable phase structure without direct alteration of other membrane components. This offers a less equivocal means of assessing the influence of polyunsaturation upon membrane structure and function.
Biochimica et Biophysica Acta 02/1998; 1368(1):41-51. · 4.66 Impact Factor
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ABSTRACT: The volume sensitivity of different K flux pathways has been determined in trout red blood cells subjected to volume perturbation. Gentle hyposmotic swelling induced a K influx in a Cl-containing saline but not in NO3- or methanesulfonate (MeSF)-containing salines, consistent with the activation of a Cl-dependent flux. Extreme hyposmotic swelling led to larger K fluxes in all salines but with reduced anion discrimination of the Cl-dependent flux. In contrast to these graded responses, isosmotic swelling using ammonium chloride or beta-adrenergic stimulation activated only Cl-dependent fluxes in an all-or-none fashion. The relationship between the hyposmotically and isosmotically induced pathways was studied by coactivation using either ammonium chloride or isoproterenol with anisosmotic treatment. Cells in ammonium chloride-containing hyposmotic salines showed no additive K flux over that induced by hyposmotic treatment alone, indicating that the isosmotically induced Cl-dependent flux was identical to the hyposmotically induced Cl-dependent flux. However, cells coactivated by hyposmotic and beta-adrenergic treatment showed a small Cl-dependent flux in addition to that induced by hyposmotic treatment alone. This small third component was unaffected by anisosmotic treatment. We conclude that the major Cl-dependent and Cl-independent K flux pathways are distinct and separate and that the former has an anion dependence that varies with cell volume and a volume sensitivity that varies with ionic strength.
The American journal of physiology 05/1997; 272(4 Pt 1):C1099-111.
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ABSTRACT: Animal cells regulate their volume in the short term by controlling solute movements into and out of the cell. A quite of dissipative transport systems are involved which allow either regulatory volume increase (RVI) or decrease (RVD) responses depending upon the direction of the electrochemical gradients of the solutes. Many of these transporters have been identified at the molecular level and structure-function studies have identified transmembrane transport domains and cytoplasmic regulatory domains. In vertebrate red blood cells, protein phosphorylation appears to be central to the coordinated regulation of transporter activity. Inhibitors of protein phosphatases (PPs) cause inhibition of the K+/Cl- cotransporter (a transporter mediating RVD), whilst some inhibitors of protein kinases (PKs) cause activation. A sequence of potential phosphorylation sites appears to constitute a cascade of reactions leading to transporter regulation. PP and PK inhibitors have opposite effects on transporters mediating RVI responses, which is consistent with the coordinated but reciprocal regulation of transporters activated during both RVI and RVD using some common phosphorylation reactions. The transporters are sensitive to other stimuli including, in red blood cells, changes in PO2 and pH. These responses are also sensitive to PK/PP inhibitors and may involve elements of the volume-sensitive transduction pathway.
Journal of Experimental Biology 02/1997; 200(Pt 2):343-52. · 3.00 Impact Factor
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ABSTRACT: Cold exposure of carp leads to the induced activity of the hepatic delta 9-desaturase (Schünke, M. and Wodtke, E. (1983) Biochim. Biophys. Acta 734, 70-75). We have investigated the controlled expression of this enzyme using isolated carp hepatocytes. Culture at 30 degrees C, of cells isolated from 30 degrees C-acclimated carp. resulted in an 8-13-fold increase in desaturase-specific activity over 4 days, whilst another enzyme of intermediary metabolism, glucose-6-phosphatase, decreased by more than 60%. This desaturase induction was associated with a loss of intracellular lipid vesicles and with increases in the levels of oleic acid of membrane phosphoglycerides and corresponding decreases in 22:6(n - 3). Supplementation of cultures with oleic acid and with polyunsaturated fatty acids did not cause any reduction in the desaturase induction. The level of immunodetectable desaturase protein increased during culture at 30 degrees C and a desaturase mRNA was detected after 2 days of culture by Northern analysis. These results suggest that in vitro culture leads to an increased synthesis of desaturase protein by means of activated gene transcription. Significantly, transfer of cultures of 30 degrees C-acclimated hepatocytes to 10 degrees C resulted in a smaller induction of desaturase activity; thus cold transfer of cells in itself did not induce hepatocyte desaturase activity as does whole animal cooling. This suggests either that cold induction of desaturase activity in vivo involves systemic control or that the conditions imposed by culture prevent cold induction.
Biochimica et Biophysica Acta 09/1996; 1302(3):207-16. · 4.66 Impact Factor
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ABSTRACT: 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.
Pflügers Archiv - European Journal of Physiology 09/1996; 432(4):727-34. · 4.46 Impact Factor
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ABSTRACT: 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.
Pflügers Archiv - European Journal of Physiology 07/1996; 432(2):270-7. · 4.46 Impact Factor
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ABSTRACT: 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.
Science 03/1996; 271(5250):815-8. · 31.20 Impact Factor
