Biomedical subjects

J Vanĕcek

Publications and source records attributed to J Vanĕcek.

At least 19 recordsLinked to original sources

PubMed1997

Inhibitory effect of melatonin on gonadotropin-releasing hormone-induced Ca2+ oscillations in pituitary cells of newborn rats.

The effect of melatonin on the gonadotropin-releasing-hormone (GnRH)-induced oscillatory rises in intracellular calcium concentration, [Ca2+]i, was studied in cultured cells from the anterior pituitary gland of 6- to 8-day-old rats. GnRH-induced [Ca2+]i oscillations were recorded indirectly by monitoring the activity of apamin-sensitive Ca(2+)-activated K+ channels using the perforated patch-clamp technique and fast microperfusion system. Melatonin (1 nM) inhibited the initiation or attenuated the amplitude of oscillatory current responses induced by 10 nM GnRH in 72% of GnRH-sensitive cells. Analysis of the melatonin dose-inhibition relationship showed that melatonin inhibited the initiation of [Ca2+]i oscillations with IC50 = 0.35 nM. In partially inhibited cells, melatonin reduced the GnRH-induced current amplitude by 55% on the average, prolonged the delay in onset of response to GnRH and decreased the frequency of oscillations. Once initiated by GnRH, the amplitude and frequency of oscillatory currents was inhibited by melatonin after a latency of 10-30 s. These effects of melatonin were fully reversible. After pretreatment of neonatal gonadotropes with pertussis toxin, no inhibition by melatonin was observed. The inhibitory effect of melatonin on initiation, amplitude and frequency of GnRH-induced oscillatory current persisted in the absence of external Ca2+. Melatonin alone did not induce any transmembrane current or membrane potential changes. These observations suggest that melatonin reduces GnRH-induced calcium mobilization from intracellular stores.

Animals↗

PubMed1995

Electrophysiological characterization of GABAA receptors in anterior pituitary cells of newborn rats.

The gamma-aminobutyric acid (GABA)-ergic communication between the CNS and the anterior pituitary gland has been documented in numerous histochemical and biochemical studies but electrophysiological studies characterizing the GABAA receptor in the anterior pituitary are still lacking. In the present report we studied the GABA-induced current responses in cultured cells from the anterior pituitary gland of 6- to 10-day-old rats using the patch-clamp technique in the whole cell configuration. Fast application of GABA (100 microM) induced membrane currents in 90% of cells in 2-day-old cultures. The EC50 for GABA was 22.9 microM and the Hill coefficient was 1.8. The responses to GABA (10 microM) were inhibited by bicuculline (2 microM) to 14%, by picrotoxin (5 microM) to 21% and by zinc (10 microM) to 33%. Inhibition to 56% was observed with 6 microM strychnine. The GABA responses were sensitive to diazepam and pentobarbital. Half-maximal potentiation of responses to GABA (10 microM) was found with 1.0 microM diazepam and with 14.4 microM pentobarbital. The maximal potentiation of GABA responses was 222% for diazepam and 195% for pentobarbital. Pentobarbital (100 microM) did not induce any response in anterior pituitary cells in the absence of GABA. The application of GABA at concentrations 10 microM or higher, induced membrane currents that desensitized. Desensitization proceeded as a biexponential process with estimated fast and slow time constants which decreased with concentration. The responses to GABA (300 microM) desensitized to 93% with time constants of 1.4 and 5.3 s. Half-maximal desensitization was found with 13.4 microM GABA.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1994

Effect of photoperiod on the pineal melatonin rhythm in neonatal rats.

Photoperiod already modulates pineal melatonin rhythm in neonatal rats. Pineal melatonin content was about 500 fmol during day and increased up to 2000 and 3000 fmol at night in 8- and 12-day-old rats, respectively. On long photoperiods (LD 14:10) melatonin was increased above 1000 fmol for about 8 h while on short photoperiods (LD 8:16) for 12 to 14 h. Melatonin pattern may thus transduce photoperiodic effects in neonatal rats. However, no differences in plasma LH were found in the rats kept on long and short photoperiods.

Age Factors↗

PubMed1994

Ontogenesis of melatonin receptors in anterior pituitary and pars tuberalis of golden hamsters.

The ontogenesis of melatonin receptors in the anterior pituitary and pars tuberalis of the Golden hamster was studied using [125I]iodomelatonin as a ligand. The affinity of the binding site to the ligand (Kd) was in the range 21 to 54 pM and it did not change significantly during development. The concentration of the [125I]iodomelatonin binding sites in the anterior pituitary was highest in one-day-old hamsters (Bmax = 14 fmol/mg protein) and thereafter gradually decreased. In adults it reached to about 6% of the neonatal values. In contrast, the concentration of the binding sites in pars tuberalis did not change significantly during ontogenesis and it was in the range of 3 to 5 fmol/mg protein.

Aging↗

PubMed1994

Chemical modifications of melatonin receptors in chicken brain.

The membrane-bound or solubilized melatonin receptors were treated with protein-modifying agents under specific conditions and then assayed for 125I-melatonin binding in order to obtain information on amino acids present in the ligand binding domain. The reagents specific for sulfhydryl (N-ethylmaleimide and p-chloromercuribenzoate), guanidyl (phenylglyoxal), and amino groups (4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid and 1-fluoro-2,4-dinitrobenzene) inhibited 125I-melatonin binding in a dose-dependent manner, and their effects were prevented by pretreatment with cold melatonin. These results suggest the presence of cysteine, arginine, and lysine residues in the melatonin binding domain. Decreased sensitivity of 125I-melatonin binding to guanine nucleotides after N-ethylmaleimide pretreatment suggests the presence of another sulfhydryl group within the coupling domain between the receptor and G protein. Tyrosine reagents tetranitromethane, 7-chloro-4-nitrobenz-2-oxa-1,3-diazole, N-acetylimidazole, and p-nitrobenzenesulfonyl fluoride also inhibited 125I-melatonin binding, and their effects were prevented by cold melatonin pretreatment; however, they were effective only at concentrations when cross-reaction with a sulfhydryl group may occur. Histidine reagent diethyl pyrocarbonate inhibited 125I-melatonin binding in a dose-dependent manner, and its action was reversed by cold melatonin. However, diethyl pyrocarbonate had a smaller effect in a solubilized receptor preparation and, therefore, it could have modified a site remote from the ligand binding site. Our data do not suggest the presence of tryptophanyl, aspartic, or glutamic residues at the ligand binding domain.

4-Chloro-7-nitrobenzofurazan↗

PubMed1992

Localization of 2-[125I]iodomelatonin binding sites in the brain of the Atlantic salmon, Salmo salar L.

The photosensory pineal organ of teleost fish shows a circadian rhythm in melatonin synthesis, and melatonin is known to influence a number of physiological functions. However, the target sites for melatonin are not known. We have investigated the distribution of melatonin binding sites in the brain of the salmon, Salmo salar. Brains were collected for receptor binding assay and autoradiography at each of three time points: just after lights on, just before lights off, and in the dark at midnight (photoperiod light-dark 12:12, lights on at 08.00 h, lights off at 20.00 h). Specific binding of 2-[125I]iodomelatonin was observed in several brain areas. High densities were associated with (1) the optic tectum, (2) the preoptic area, (3) an area encompassing the magnocellular superficial pretectal nucleus ('nucleus rotundus') and the glomerular complex, (4) the inferior lobes of the hypothalamus, (5) the lateral mesencephalic tegmentum including the torus semicircularis, and (6) the molecular layer of the cerebellum. No binding was observed in the pineal organ or in the pituitary. We observed no differences in labeling between brains collected at different time points, except in the preoptic area where binding was high at 20.00 and 24.00 h, but low at 08.00 h, and in the corpus cerebelli, where labeling in the molecular layer was higher at 24.00 and 08.00 h than at 20.00 h.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1991

Melatonin inhibitory effect on luteinizing hormone release is potentiated after long pretreatment with the indole.

The effect of melatonin on luteinizing-hormone releasing-hormone stimulation of cyclic AMP accumulation and luteinizing hormone (LH) release from neonatal rat hemipituitaries was studied in vitro. Melatonin inhibited LH-release from pituitaries of animals kept previously on long but not on short photoperiods; cyclic AMP accumulation was, however, inhibited on both photoperiods. There were no daily changes in the melatonin effect. Inhibition of LH-release was strongly potentiated after 6 h preincubation with melatonin as compared with 20 min preincubation; cyclic AMP was not significantly affected by the length of preincubation.

Animals↗

PubMed1991

Mechanism of melatonin action.

Melatonin transduces the effect of photoperiod on the neuroendocrine system. Synthesis of melatonin in the pineal gland is well described, but the location of its target(s) and the mechanism of its action are little known. In attempt to localize melatonin target(s), the presence of high affinity binding sites in rat brain was determined. Such sites were detected in discrete brain areas, including the hypothalamus and anterior pituitary. Subcellular analysis indicated these binding sites were on plasma membranes, which suggests that melatonin modulates cell functions through intracellular second messengers. The effects of melatonin on second messengers were studied using the neonatal anterior pituitary, in which melatonin is known to inhibit the LHRH-induced release of LH. Studies on the effects of melatonin on second messenger indicated [corrected] that melatonin inhibits accumulation of cAMP and cGMP as well as synthesis of diacylglycerol and release of arachidonic acid. Time-course analysis indicates that inhibition by melatonin of the LHRH-induced release of LH increases following long preincubation. Since the effect of melatonin on LHRH-induced release of LH is prevented by dibutyryl cAMP, we conclude that melatonin might act by inhibiting production of cAMP.

Animals↗

PubMed1990

Melatonin modulates diacylglycerol and arachidonic acid metabolism in the anterior pituitary of immature rats.

In pituitary glands of immature rats prelabeled in vitro with [3H]arachidonic acid, melatonin diminished the luteinizing hormone-releasing hormone (LHRH)-induced increase in [3H]diacylglycerol accumulation as well as [3H]arachidonic acid release from the tissue. Melatonin reduced also LHRH-stimulated incorporation of [3H]glycerol into pituitary [3H]diacylglycerol. The effect was day-time dependent: in the evening experiment melatonin was effective at 0.1 nM concentration while in the morning it had no effect even at 10 nM concentration. The effect of melatonin was also abolished by pretreatment with pertussis toxin. Diacylglycerol and/or arachidonic acid might serve as 2nd messengers transducing the effect of melatonin at the cellular level.

Animals↗

PubMed1989

Melatonin inhibits cyclic AMP and cyclic GMP accumulation in the rat pituitary.

Subnanomolar concentrations of melatonin inhibit cyclic AMP and cyclic GMP accumulation in neonatal rat anterior pituitary stimulated in vitro with luteinizing-hormone releasing-hormone. Melatonin also inhibited forskolin-stimulated cyclic AMP accumulation in pars tuberalis. Inhibition of cyclic AMP accumulation is specific for melatonin, since its analogs N-acetylserotonin and 5-methoxytryptamine are 1000 times less potent. Cyclic nucleotides may thus serve as second messengers transducing the effect of melatonin on cellular level.

Animals↗

PubMed1989

Short days induce changes in specific melatonin binding in hamster median eminence and anterior pituitary.

Autoradiography of 125I-melatonin binding to hamster brain sections revealed a competitive 125I-melatonin binding in median eminence only. Saturation studies on crude membrane fraction revealed high-affinity melatonin binding sites in median eminence (Kd = 59 pM) and anterior pituitary (Kd = 97 pM). In the hamsters maintained on LD 14:10, the concentration of the binding sites was 10.4 and 6.0 fmol/mg protein in median eminence and anterior pituitary respectively; long-term exposure to LD 8:16 decreased the concentration to less than a half.

Animals↗

PubMed1989

Different mechanisms of phase delays and phase advances of the circadian rhythm in rat pineal N-acetyltransferase activity.

The circadian rhythm in rat pineal N-acetyltransferase (NAT) activity, which drives the rhythm in melatonin production, is controlled by a pacemaker located in the suprachiasmatic nucleus of the hypothalamus. As the NAT rhythm has two well-defined phase markers--namely, the time of the evening activity rise and of the morning decline--it is suitable for studies of the entrainment of the pacemaker by environmental light. Phase delays of the NAT rhythm proceed more rapidly than phase advances. One day after a brief light pulse applied before midnight, or after a delay in evening lights-off, or a delay of a light-dark (LD) cycle, phase delays of the evening NAT rise result in almost corresponding delays of the morning NAT decline. Consequently, the NAT rhythm is phase-shifted, but its pattern does not change. One day after a brief light pulse applied past midnight, or after bringing forward morning lights-on, or after an advance of an LD cycle, the morning NAT decline is phase-advanced, but the evening rise is not phase-advanced at all or may even by phase-delayed. Consequently, the phase relationship between the evening NAT activity onset and the morning offset may be compressed considerably, and it may take several transient cycles before phase advances of the morning NAT decline are followed by corresponding advances of the evening NAT rise. Due to the phase-delaying effect of evening light on the NAT rise and to the phase-advancing effect of morning light on the NAT decline, the phase relationship between the NAT rise and the decline is compressed on long days and decompressed on short days. Different phase shifts of the evening NAT rise and of the morning decline, even in opposite directions, are consistent with the hypothesis of a complex, two-component (evening-morning, or E-M) pacemaker controlling the NAT rhythm. As the E-M phase relationship determines duration of the high night melatonin production, and the duration of the nocturnal melatonin pulse may convey information on daylength, the data are consistent with the internal coincidence model for photoperiodic time measurement.

Animals↗

PubMed1988

The effects of peptidoglycan, a pyrogenic constituent of gram-positive microorganisms, on the pharmacokinetics of rifampicin.

Pharmacokinetics of rifampicin (20 mg/kg orally or i.v.) was determined in calves and rabbits. Seven days later a model pyrogen was administered i.v. to the same animals and 1 hr later the rifampicin administration was repeated. The pharmacokinetic analysis of oral rifampicin was performed using a one-compartment open model with absorption. Intravenously administered rifampicin was analysed by a two-compartment intravascular model. Injection of peptidoglycan in pyrogenic doses led to a significant increase of orally applied rifampicin serum levels in both animal species. The i.v. administration of rifampicin had the same parameters in the control and peptidoglycan experiments. Daily pretreatment of rabbits with small doses of peptidoglycan induced tolerance to the pyrogenic effect. In tolerant animals we did not observe any changes of rifampicin serum levels. Elevated temperature alone was not responsible for observed pharmacokinetic changes leading to the increase of bioavailability of oral rifampicin since another pyrogenic substance (endotoxin) had an opposite effect on pharmacokinetics of previously tested drugs.

Administration, Oral↗

PubMed1988

Entrainment of the rat pineal rhythm in melatonin production by light.

Environmental light entrains the rat pineal N-acetyltransferase rhythm which controls melatonin production. One day after 1 min light pulses applied before midnight, or after delays in the evening switch off of light, or after a delay of the light-dark cycle, the evening N-acetyltransferase rise and the morning decline are phase delayed almost to the same extent. Consequently, the pattern of the rhythm does not change. One day after 1 min light pulses applied past midnight, or after bringing forward the morning light onset, or after an advance of the light dark-cycle, the morning N-acetyltransferase decline is phase advanced, but the evening rise is either not phase shifted or it may be even phase delayed. Consequently, the pattern of the rhythm may be changed considerably or the rhythm may be abolished. The data are consistent with an hypothesis of a two-component pacemaker controlling the N-acetyltransferase rhythm. Under all photoperiods which we encounter in nature, the pattern of the N-acetyltransferase rhythm is determined by the entraining effect of light on the pacemaker, but not by the suppressant effect of light.

Acetyltransferases↗

PubMed1988

Melatonin binding sites.

The distribution and characterization of specific melatonin binding sites were studied using 125I-melatonin. Autoradiography revealed only three sites of specific melatonin binding in brain: the suprachiasmatic nuclei, the median eminence, and the small part of choroid plexus at the caudal end of the fourth ventricle. Two other sites were detected outside the CNS: the anterior pituitary and the retina. The specific binding of 125I-melatonin was saturable and reversible. The dissociation constant (KD) of the binding sites was 60 pM. The concentration of the binding sites (Bmax) in the median eminence was 26 fmol/mg protein, and in the pituitary 3 fmol/mg protein. Specificity of the binding sites was tested by displacement of 125I-melatonin. The order of potency--melatonin much less than N-acetyl-5-hydroxytryptamine less than 5-methoxytryptamine much less than 5-hydroxytryptamine = 3,4-dihydroxyphenylethylamine = noradrenaline--shows high specificity of the binding sites for melatonin.

Animals↗

PubMed1988

The melatonin receptors in rat ontogenesis.

The development of melatonin receptors in the rat pituitary and median eminence was studied using [125I]melatonin as a ligand. The specific binding was detected in pituitaries of 20-day-old fetuses already. The affinity of the receptor to the ligand (Kd) was in the range 63-133 pM and it did not change significantly during development. The pituitary concentration of [125I]melatonin binding sites was highest in 20-day-old fetuses (Bmax = 31 fmol/mg protein) and then it gradually decreased in the course of postnatal development, until it reached 10% of that value in 29-day-old males. In contrast, the concentration of melatonin receptors in median eminence did not change markedly in the course of development and it was about 15 fmol/mg protein. The marked decrease in the number of the pituitary receptors may be the cause of the reported developmental loss of the melatonin inhibitory effect on LHRH-induced LH release from anterior pituitary.

Aging↗

PubMed1987

Hypothalamic melatonin receptor sites revealed by autoradiography.

125I-Melatonin was used to localize and characterize the melatonin receptor sites in the rat hypothalamus. Autoradiography revealed that displaceable 125I-melatonin binding occurred in suprachiasmatic nuclei and median eminence only. Further studies performed on crude membrane fractions from median eminences revealed high affinity (Kd = 21 pM) melatonin binding sites (Bmax = 8.5 fmol/mg protein). The order of potency of various indole amines to inhibit 125I-melatonin binding was melatonin much greater than N-acetyl-5-hydroxytryptamine greater than 5-methoxytryptamine greater than 5-hydroxytryptamine.

Animals↗

PubMed1987

Adjustment of the rat pineal N-acetyltransferase rhythm to eight-hour shifts of the light-dark cycle: advance of the cycle disturbs the rhythm more than delay.

After an 8-h delay of a light-dark (LD) cycle by lengthening of one light period, the rat pineal N-acetyltransferase (NAT) rhythm adjusted to the delay shift almost within one cycle. After an 8-h advance of the LD cycle by shortening of one dark period, the NAT rhythm adjusted to the advance shift within 5 cycles only; during the first 2-3 cycles the rhythm was abolished.

Acetyltransferases↗