Benoit Dugué

Effects of psychological stress on plasma interleukins-1 beta and 6, C-reactive protein, tumour necrosis factor alpha, anti-diuretic hormone and serum Cortisol

The study was undertaken to determine whether psychological stress influences immunobiological functions and is an important preanalytical factor to be considered in connection with blood specimen collection. Two kinds of stress were applied, the Stroop colour conflict test and the thrill of a novice about to make the first jump with a parachute. In both test situations, the level of the stress indicators cortisol or anti-diuretic hormone rose significantly. The concentrations of the cytokines studied did not change significantly. However, in the parachute test significant positive correlations were found, e.g. between the changes of cortisol and C-reactive protein and between anti-diuretic hormone and interleukin-1 beta. This suggests that there is an interaction between the endocrine and the immune systems in the response to a psychological stress.

Acute and long‐term effects of winter swimming and whole‐body cryotherapy on plasma antioxidative capacity in healthy women

The effects of severe cold stress on total peroxyl radical trapping antioxidant capacity of plasma (TRAP) were studied in two groups of healthy women: a whole‐body cryotherapy group (WBC, n = 10) and a winter swimming group (WS, n = 10). The biovariability of TRAP values was also analysed. The WBC group was exposed to −110°C for 2 min, whereas the exposure for the WS group lasted 20 s in ice‐cold water. Sessions were organized three times per week for 12 weeks. Blood specimens were collected at 2, 4, 8 and 12 weeks at rest, 2 and 35 min after the cold exposures and at the corresponding times without cold exposure on a separate day. Conventional methods were used to determine TRAP values. The between‐subject variation was 13.6% and the within‐subject variation 6.4%. The index of individuality was 0.46, and the index of heterogeneity was 0.079. These results indicate a marked heterogeneity among subjects. During the first 4 weeks, the mean TRAP value significantly increased at 2 min after cold exposure in the WBC group, returning to baseline 35 min after the exposure. Similar changes were observed in the WS group. However, all changes due to cold were relatively mild (<5%). After 4 weeks no changes in TRAP values after the cold exposures were noticed and no long‐term changes in basal TRAP values were observed. In the main, regular WBC and WS do not seem to be harmful as far as plasma antioxidative capacity is concerned.

Purinergic agonist ATP is a comitogen in thyroid FRTL-5 cells

Several growth factors may stimulate proliferation of thyroid cells. This effect has, in part, been dependent on calcium entry. In the present study using FRTL‐5 cells, we show that in addition to its effect on calcium fluxes, ATP acts as a comitogen in these cells. In medium containing 5% serum, but no TSH, ATP stimulated the incorporation of 3H‐thymidine in a dose‐ and time‐dependent manner in the cells. At least a 24‐h incubation with ATP was necessary to observe the enhanced (30–50%) incorporation of 3H‐thymidine and an increased (30%) cell number. The effect of ATP was dependent on insulin in the incubation medium. Furthermore, ATP enhanced the TSH‐mediated incorporation of 3H‐thymidine. The effect of ATP was apparently mediated via a G‐protein dependent mechanism, as no stimulation of thymidine incorporation was observed in cells treated with pertussis toxin. The effect of ATP was not dependent on the activation of protein kinase C (PKC), as ATP was effective in cells with downregulated PKC. ATP rapidly phosphorylated mitogen activated protein (MAP) kinase in FRTL‐5 cells. In addition, ATP stimulated the expression of a 62 kDa c‐fos dependent protein in a dose‐ and time‐dependent manner. Our results thus suggest that extracellular ATP, in the presence of insulin, may be a cofactor in the regulation of thyroid cell proliferation, probably by phosphorylating MAP kinase and stimulating the expression of c‐fos.

Preanalytical factors and the measurement of cytokines in human subjects

Cytokines are widely measured in research. However, cytokine analyses are influenced by a myriad of factors. For instance, a delay in the separation of plasma from cells may lead to a 50% decrease in the concentration of tumor necrosis factor in plasma. Another example is the secretion of interleukin-1β in women which can be twice as high during the follicular phase as in the luteal phase. The factors influencing the outcome of these tests can be divided into in vivo preanalytical factors (e.g., aging chronobiological rhythms, diet, etc), in vitro preanalytical factors (e.g., specimen collection, equipment, transport, storage, etc), and analytical factors. To improve the value of the cytokine tests, factors strongly influencing the results have to be controlled. This can be done by using standardized assays and specimen collection procedures. In general, sufficient attention is not given to the preanalytical factors, especially in the measurement of cytokines. This article reviews the preanalytical factors which may influence the outcome of these tests in human subjects.

Preanalytical factors and standardized specimen collection: Influence of psychological stress

In order to devise and evaluate standardized specimen collection procedures, we studied the influence of psychological stress on the results of commonly analysed blood components: creatine kinase, lactate dehydrogenase, total protein and albumin in serum and blood picture. In addition, serum cortisol was assayed. Two kinds of stress were used: the Stroop test, a colour conflict task, and the thrill caused by the first jump of new parachutists. More changes were observed after the parachutist test than after the Stroop test. There was a difference in the responses of males and females. Females were more sensitive, especially to the parachutist test. Most of the changes observed were interpreted as being caused by haemoconcentration, possibly related to muscular tension. Cortisol, commonly used to indicate the level of stress, did not react much and is therefore not a good index of psychological stress.

Extracellular ATP-mediated phospholipase a2 activation in rat thyroid FRTL-5 cells: Regulation by a Gi/Go protein, Ca2+, and mitogen-activated protein kinase

We investigated the mechanism of phospholipase A2 (PLA2) activation in response to the P2 receptor agonist ATP in rat thyroid FRTL‐5 cells. The PLA2 activity was determined by measuring the release of [3H]‐arachidonic acid (AA) from prelabeled cells. ATP evoked a dose‐ and time‐dependent AA release. This release was totally inhibited by pertussis toxin (PTX) treatment, indicating the involvement of a Gi/Go protein. The AA release was also diminished by chelating extracellular Ca2+ with EGTA or by inhibiting influx of Ca2+ using Ni2+. Although the activation of protein kinase C (PKC) by 12‐phorbol 13‐myristate acetate (PMA) alone did not induce any AA release, the ATP‐evoked AA release was significantly reduced when PKC was inhibited by GF109203X or by a long incubation with PMA to downregulate PKC. Both the ATP‐evoked AA release and the mitogen‐activated protein kinase (MAP kinase) phosphorylation were decreased by the MAP kinase kinase (MEK) inhibitor PD98059. Furthermore, the ATP‐evoked MAP kinase phosphorylation was also inhibited by GF109203X and by downregulation of PKC, suggesting a PKC‐mediated activation of MAP kinase. Inhibiting Src‐like kinases by PP1 attenuated both the MAP kinase phosphorylation and the AA release. These results suggest that these kinases are involved in the regulation of MAP kinase and PLA2 activation. Elevation of intracellular cAMP by TSH or by dBucAMP did not induce a phosphorylation of MAP kinase. Furthermore, neither the ATP‐evoked AA release nor the MAP kinase phosphorylation were attenuated by TSH or dBucAMP. Taken together, our results suggest that ATP regulates the activation of PLA2 by a Gi/Go protein‐dependent mechanism. Moreover, Ca2+, PKC, MAP kinase, and Src‐like kinases are also involved in this regulatory process. J. Cell. Physiol. 183:155–162, 2000.

Redox modulation of intracellular free calcium concentration in thyroid FRTL-5 cells: evidence for an enhanced extrusion of calcium.

Redox modulation is involved in the regulation of the intracellular free calcium concentration ([Ca2+]i) in several cell types. In thyroid cells, including thyroid FRTL-5 cells, changes in [Ca2+]i regulate important functions. In the present study we investigated the effects of the oxidizing compounds thimerosal and t-butyl hydroperoxide on [Ca2+]i in thyroid FRTL-5 cells. Thimerosal mobilized sequestered calcium, and evoked modest store-dependent calcium entry. Both compounds potently attenuated the increase in [Ca2+]i when store-operated calcium entry was evoked with thapsigargin. The entry of barium was not attenuated. Experiments performed with high extracellular pH, in sodium-free buffer and in the presence of vanadate suggested that thimerosal decreased [Ca2+]i by activating a calcium extrusion mechanism, probably a plasma membrane Ca2+-ATPase. All the observed effects were abrogated by the reducing agent beta-mercaptoethanol. The mechanism of action was apparently mediated via activation of protein kinase C, as thimerosal potently stimulated binding of [3H]phorbol 12, 13-dibutyrate, and was without effect on store-operated calcium entry in cells treated with staurosporine or in cells with down-regulated protein kinase C. Thimerosal did not depolarize the membrane potential, as evaluated using patch-clamp in the whole-cell mode. In immunoprecipitates obtained with an antibody against plasma membrane Ca2+-ATPase, we observed several phosphorylated bands in cells stimulated with thimerosal. In conclusion, we have shown that thimerosal attenuates an increase in [Ca2+]i, probably by activating a plasma membrane Ca2+-ATPase.

PROTEIN TYROSINE PHOSPHORYLATION AND CALCIUM SIGNALING IN THYROID FRTL-5 CELLS

We examined the importance of tyrosine kinase(s) on the ATP‐evoked Ca2+ entry and DNA synthesis of thyroid FRTL‐5 cells. ATP rapidly and transiently tyrosine phosphorylated a 72‐kDa protein(s). This phosphorylation was abolished by pertussis toxin and by the tyrosine kinase inhibitor genistein, and was dependent on Ca2+ entry. Pretreatment of the cells with genistein did not affect the release of sequestered Ca2+, but the capacitative Ca2+ or Ba2+ entry evoked by ATP or thapsigargin was attenuated. Pretreatment of the cells with orthovanadate enhanced the increase in intracellular free Ca2+ ([Ca2+]i), whereas the Ba2+ entry was not increased. Phorbol 12‐myristate 13‐acetate (PMA) phosphorylated the same protein(s) as did ATP. Genistein inhibited the ATP‐evoked phosphorylation of MAP kinase and attenuated both the ATP‐ and the PMA‐evoked DNA synthesis. However, genistein did not inhibit the ATP‐evoked expression of c‐fos. Furthermore, genistein enhanced the ATP‐evoked release of arachidonic acid. Thus, ATP activates a tyrosine kinase via a Ca2+‐dependent mechanism. A genistein‐sensitive mechanism participates, in part, in the ATP‐evoked activation of DNA synthesis. Genistein inhibits only modestly capacitative Ca2+ entry in FRTL‐5 cells. J. Cell. Physiol. 175:211–219, 1998.

Ceramide 1-(2-cyanoethyl) phosphate enhances store-operated Ca2+ entry in thyroid FRTL-5 cells.

Sphingolipid derivatives cause diverse effects towards the regulation of intracellular free Ca(2+) concentrations ([Ca(2+)](i)) in a multitude of nonexcitable cells. In the present investigation, the effect of C-8 ceramide-1-(2-cyanoethyl) phosphate (C1CP) on store-operated Ca(2+) (SOC) entry was investigated. C1CP evoked a modest increase in [Ca(2+)](i). The increase was inhibited by the SOC channel antagonist 1-(beta-[3-(4methoxyphenyl)propoxy]-4-methoxyphenethyl)-1H-imidazole (SKF96365) but not by overnight pretreatment of the cells with pertussis toxin. C1CP did not invoke the production of inositol phosphates. When cells were stimulated with both C1CP and thapsigargin, the thapsigargin-invoked increase in [Ca(2+)](i) was enhanced in comparison to control cells. When Ca(2+) was added to cells treated with both C1CP and thapsigargin in a Ca(2+)-free buffer, the increase in [Ca(2+)](i) was enhanced in comparison to control cells. In patch-clamp experiments, C1CP hyperpolarized the membrane potential (E(m)) of the cells and attenuated the thapsigargin-invoked depolarization of the E(m). The effects of C1CP came, in part, as a result of a decreased conductance of the cell membrane towards Cl(-) ions, as C1CP in a Cl(-)-free solution also enhanced Ca(2+) entry. Barium 2-cyanoethylphosphate (Ba2Cy), which also contains the 2-cyanoethyl group, did not modulate thapsigargin-invoked changes in [Ca(2+)](i) nor did it modulate the E(m). In conclusion, C1CP enhances SOC entry, in part, via hyperpolarization of the E(m) and attenuation of the thapsigargin-invoked membrane depolarization, thus increasing the electrochemical gradient for Ca(2+) ions. Hence, C1CP may be a useful reagent for investigating the cellular effects of ceramide derivatives.