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We applied the whole-cell patch-clamp technique to study the influence of zinc ions (Zn2+) and extracellular protons at acidic pH (pHo) on voltage-gated potassium currents in cultured rat hippocampal neurons. The first goal of the study was to estimate whether Kv1.3 currents significantly contributed to voltage-gated potassium currents in examined cells. Then, the influence of both ions on the activity of other voltage-gated potassium currents in the neurons was examined. We examined both the total current and the delayed - rectifier component. Results obtained in both cases were not significantly different from each other. Available data argued against any significant contribution of Kv1.3 currents to the recorded currents. Nevertheless, application of Zn2+ in the concentration range from 100 µM to 5 mM reversibly modulated the recorded currents. The activation midpoint was shifted by about 40 mV (total current) and 30 mV (delayed-rectifier current) towards positive membrane potentials and the activation kinetics were slowed significantly (2 - 3 fold) upon application of Zn2+. The inactivation midpoint was also shifted towards positive membrane potentials, but less significantly (about 14 mV). The current amplitudes were reduced in a concentration-dependent manner to about 0.5 of the control value. The effects of Zn2+ were saturated at the concentration of 1 mM. Raising extracellular proton concentration by lowering the pHo from 7.35 to 6.4 did not affect significantly the currents. Possible mechanisms underlying the observed phenomena and their possible physiological significance are discussed.
In the present study we applied the whole-cell patch-clamp technique to study the influence of extracellular pH (pHo) on the modulatory effect exerted by zinc ions (Zn2+) on voltage-gated potassium channels Kv1.3 expressed in human lymphocytes. Obtained data provide evidence that lowering of pHo from the 7.35 to 6.4 slowed significantly the current activation rate, shifted the activation midpoint by about 16 mV towards positive membrane potentials and reduced the current amplitude to about 0.55 of the control value. In contrast, raising the pHo from 7.35 to 8.4 did not affect significantly the activation midpoint and current amplitude. Application of Zn2+ in the concentration range from 100 µM to 1 mM at pHo=6.4 slowed additionally the activation rate, shifted the activation midpoint by about 20 mV towards positive membrane potentials and reduced the current amplitude in a concentration-dependent manner. The total effect exerted by Zn2+ and protons at pHo = 6.4 was more significant than the effect exerted by Zn alone. Both the magnitude of the shift and the degree of current inhibition by Zn2+ were independent on pHo in the range from 6.4 to 8.4. The data might suggest that the effects exerted by protons and zinc ions occur independently on each other and probably involve different mechanisms. Changing the holding potential from -90 mV to -60 mV at pHo=7.35 abolished the Zn2+-induced inhibition of the current amplitudes at concentrations below 300 µM. At pHo=6.4 the total inhibition caused by Zn2+ and protons was also diminished, however, a significant reduction was observed at 100 µM concentration. In contrast, changing the holding potential did not change the Zn2+- and proton-induced shift of the activation midpoint. Altogether, obtained data suggest that extracellular protons exert the modulatory effects that are additive to the effects exerted by Zn2+ on the channels. Possible physiological significance of these additive effects is discussed.
The immunotropic properties of calf thymus extract (TFX-Jelfa) is connected with the mimic action of the thymus to modulate the differentiation, maturation and function of prothymocytes and mature thymus dependent (T) cells. The studies were carried out on CFW male mice aged 3 months. The animals were infected per os with 200 larvae of Trichinella spiralis. TFX-Jelfa was administered i.p. at a dose of 10 mg/kg seven times at 24 hour intervals prior to infection. The percentage of CD4+ and CD8+ in suspension of splenocytes and mesenteric lymphonode cells by flow cytometry using monoclonal antibodies coupled with fluorescein isothiocyanate (FITC) or phycoerythrin (PE) were determined. At the same time, cryostat preparations, made from jejunum and muscle samples, were examined by the direct immunofluorescence method using FITC-labeled antibody to mouse CD4+ and CD8+. It has been found that infection with T. spiralis in mice decreases the percentage of CD8+ splenocytes, while the percentage of CD8+ mesenteric lymphonode cells does not change. However, in infected mice the percentage of CD4+ spleen cells and mesenteric lymphonode cells is increased. It has been also found that during the course of infection an increase in the number of CDS+ and CD4+ cells in the basal lamina propria of the intestines was observed. In infected mice, CD4+ lymphocytes were visible in the inflammatory infiltrates of the muscle tissue on the 14lh day, whereas CDS+ lymphocytes were first observed a week later. Pretreatment with TFX does not change the inhibitory effect of infection on the percentage of CD8+ splenocytes, but potentiates the percentage of CD4+ spleen cells and mesenteric lymphonode cells increased by infection. Furthermore, administration of TFX prior to infection also potentiates the stimulatory effect of T. spiralis on the number of CD8+ and CD4+ in the basal lamina propria of the jejunum, and on the number of CDS+ cells in the inflammatory infiltrates of the muscle tissue.
Oxytocin (OXY) has been shown to attenuate some of the physiological and behavioral alterations appearing in stressed rats. Carbetocin (CBT), an oxytocin analog [deamino-1-monocarba-(2-O-methyltyrosine)-oxytocin], was designed to exert prolonged action. In the present study we investigated the impact of these peptides on the behavioral changes in rats exposed repeatedly to restraint stressors. Wistar male rats were exposed to restraint for 1 hour; saline or drugs were administered intraperitoneally immediately after stress termination. Recording of the exploratory activity in the open-field started 60 min later. To explore the possibility of persisting effects of stress and/or drugs, the procedure was repeated for three consecutive days. Restraint moderately suppressed locomotion and rearing, and increased grooming. OXY in 0.3 mg/kg dose showed a tendency to restore the suppressed exploratory activity. In contrast, 1 mg/kg dose potentiated the stress-induced behavioral deficit. Both OXY doses slightly increased grooming. CBT in the same two doses restored the stress-induced deficits in locomotion and rearing but did not influence grooming. The locomotor depression after 1 mg dose of OXY was found also in non-stressed rats in contrast to the increased activity after CBT. The data support the view that post-stress administered CBT exerts a significant effect on the stress-altered spontaneous behavior.
Learning and memory effects of angiotensin II (Ang II) microinjected unilaterally (left or right) and bilaterally into hippocampal CA1 area on the background of the inhibited hippocampal angiotensin 1 receptors type (ATI) of male Wistar rats were studied. It was found that the combination (losartan 100 ^g + Ang II 0.5^g) microinjected bilaterally or into the left CA1 area improved learning and memory in shuttle-box and step through behavioral tests as compared to the respective controls. The effects were more pronounced after injection into the left CA1 area as compared to the right-side. These findings suggest thatAng II infused on the background of the inhibited CA1 hippocampal AT1 receptors ameliorated the cognitive processes. The data show also an asymmetric effect of Ang II on learning and memory processes in the hippocampus. The stronger modulating effect after microinjection of the combination (losartan + Ang II) into the left CA1 hippocampal area suggests a leftward bias in the rat. The results point to a differential distribution of angiotensin II receptors modulating the learning and memory processes in the left and right hippocampal CA1 area.
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Modulatory effect of ghrelin in prepubertal porcine ovarian follicles

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Ghrelin is a novel growth hormone-releasing peptide, originally identified in rat stomach as an endogenous ligand of the growth hormone secretagogue receptor. Ghrelin is an important regulator of growth hormone secretion, food intake, and reproductive function. This study investigates whether or not ghrelin can modulate prepubertal pig ovary function, which was measured as ovarian estradiol secretion, aromatase activity, cell proliferation, and apoptosis. To investigate this, ovarian cells were co-cultured with four different doses of ghrelin (100, 250, 500, and 1000 pg/ml) for 48 h. Culture media samples were collected, and estradiol levels were determined, while aromatase expression was measured in the cultured cells. Cell apoptosis was measured by determination of caspase-3 activity, DNA fragmentation and TUNEL assay. Ghrelin in 250 and 500 pg/ml doses stimulated estradiol secretion. At all doses ghrelin stimulated aromatase activity and protein expression. Moreover, ghrelin increased cell proliferation and decreased apoptosis. This study provides novel evidence that ghrelin has a modulatory effect in the ovary. We suggest two mechanisms that explain how ghrelin acts on estradiol secretion: 1) ghrelin directly influences aromatase activity and protein expression; 2) ghrelin stimulates cell proliferation and antiapoptotic actions.
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