Biomedical subjects

J Vanecek

Publications and source records attributed to J Vanecek.

At least 19 recordsLinked to original sources

PubMed2001

Effects of melatonin on ischemia and reperfusion injury of the rat heart.

Effects of melatonin on various manifestations of ischemia/reperfusion injury of the isolated perfused rat heart were examined. Ischemia- and reperfusion-induced ventricular arrhythmias were studied under constant flow in hearts subjected to 10, 15 or 25 min of regional ischemia (induced by LAD coronary artery occlusion) and 10-min reperfusion. Melatonin was added to the perfusion medium 5 min before ischemia at concentrations of 10 micromol/l or 10 nmol/l and was present throughout the experiment. Recovery of the contractile function was evaluated under constant perfusion pressure after 20-min global ischemia followed by 40-min reperfusion. Hearts were treated with melatonin at a high concentration (10 micromol/l) either 5 min before ischemia only (M1) or 5 min before ischemia and during reperfusion (M2) or only during reperfusion (M3). At the high concentration, melatonin significantly reduced the incidence of reperfusion-induced ventricular fibrillation and decreased arrhythmia score (10% and 2.2+/-0.3, respectively) as compared with the corresponding untreated group (62% and 4.1+/-0.3, respectively); the low concentration had no effect. This substance did not affect the incidence and severity of ischemic arrhythmias. Melatonin (M2, M3) significantly improved the recovery of the contractile function as compared with the untreated group; this protection did not appear if melatonin was absent in the medium during reperfusion (Ml). Our results show that melatonin, in accordance with its potent antioxidant properties, effectively protects the rat heart against injury associated with reperfusion. It appears unlikely that melatonin is cardioprotective at physiological concentrations.

Animals↗

PubMed2001

Dual effect of melatonin on gonadotropin-releasing-hormone-induced Ca(2+) signaling in neonatal rat gonadotropes.

In neonatal rat gonadotropes, melatonin inhibits gonadotropin-releasing-hormone (GnRH)-stimulated increase in intracellular Ca(2+) concentration ([Ca(2+)](i)); in cells transfected with the Mel1a melatonin receptor, however, melatonin has been shown to potentiate agonist-stimulated [Ca(2+)](i) increase. To elucidate this discrepancy, we investigated the effects of melatonin in neonatal gonadotropes over a wide range of melatonin concentrations. Nystatin perforated patch recording of Ca(2+)-dependent potassium currents was used to monitor GnRH-induced [Ca(2+)](i) changes. In 32% of cells, increasing melatonin concentrations in the range of 1 pM to 100 nM prolonged the latency of, and inhibited GnRH (10 nM)-stimulated [Ca(2+)](i) increases in a concentration-dependent manner. In the remaining 68% of cells, the Ca(2+) increase elicited by exposure to 10 nM GnRH was also inhibited by picomolar concentrations of melatonin, but at nanomolar concentrations the inhibitory effect disappeared and melatonin was only able to prolong the latency of the response. This dual effect of melatonin however was not observed in cells stimulated with lower (2 nM) GnRH concentrations; in that case, melatonin was inhibitory at all concentrations tested with an IC(50) of about 30 pM. In contrast, application of nanomolar concentrations of melatonin resulted in potentiation of the GnRH-induced Ca(2+) increase in a small population of gonadotropes which did not respond by inhibition or prolonged latency. These results indicate that in neonatal gonadotropes, melatonin has both inhibitory and potentiating effects on GnRH-stimulated [Ca(2+)](i) increases. Ranges of concentrations needed to produce either effect suggest that two distinct G proteins may be involved, as already observed in transfected cells.

Animals↗

PubMed2000

In vitro entrainment of the circadian rhythm of vasopressin-releasing cells in suprachiasmatic nucleus by vasoactive intestinal polypeptide.

Mammalian circadian pacemaker is located in suprachiasmatic nuclei (SCN) of the hypothalamus. The pacemaker is entrained by light-dark cycle; the photic information is transmitted primarily via the retino-hypothalamic tract (RHT). The main neurotransmitter of the tract is glutamate. RHT fibers end on the ventrolateral part of the nucleus, where vasoactive intestinal peptide (VIP)-immunopositive neurons are localized. They send their axons into dorsomedial SCN, where most of the vasopressinergic (AVP) neurones are located. The AVP neurons retain the clock-like properties in vitro. Vasopressin release from the cultured neurons shows circadian rhythm peaking in the middle of subjective day. VIP induces phase-shifts of the rhythm, magnitude and direction of the shift depending on timing of the application. VIP applied 6-12 h before the peak of vasopressin rhythm induces advances, application 4-8 h after the peak induces delays. The lowest concentration required to induce the phase-shift is 30 nM, further increase of the concentration does not affect the magnitude of the shift. In contrast, glutamate has no effect on the phase of vasopressin rhythm, although in high concentrations it transiently stimulates vasopressin release. The data indicate that the vasopressinergic cells in the SCN contain circadian oscillators, whose rhythms run mutually synchronized in our cultures. VIP acts directly on the vasopressinergic cells to shift the phase of their pacemakers; glutamate has no such effect presumably because in vivo it acts through the VIP-ergic cells but the neuronal network is altered after the dissociation of the cells.

Animals↗

PubMed2000

Melatonin inhibits pituitary adenylyl cyclase-activating polypeptide-induced increase of cyclic AMP accumulation and [Ca2+]i in cultured cells of neonatal rat pituitary.

The effects of melatonin on pituitary adenylyl cyclase-activating polypeptide-induced increase of cyclic AMP and [Ca2+]i were studied in neonatal rat pituitary cells. The polypeptide increased cyclic AMP accumulation. In the presence of melatonin the increase of cyclic AMP was inhibited in a dose-dependent manner, the maximal inhibition was achieved with 1-10 nM melatonin. Pituitary adenylyl cyclase-activating polypeptide also increased [Ca2+]i in 30% of the pituitary cells and melatonin inhibited the effect. Most of the cells sensitive to adenylyl cyclase-activating polypeptide (77%) were also sensitive to GnRH, suggesting they are gonadotrophs. The remaining cells were not identified. The polypeptide-induced [Ca2+]i increase was inhibited in Ca2+-free medium in 2/3 of the cells indicating that Ca2+ influx was involved. To examine causal relationship between cyclic AMP and [Ca2+]i increase, we have studied the effect of adenylyl cyclase activation by forskolin on intracellular Ca2+ concentration. Forskolin had similar effects as adenylyl cyclase-activating polypeptide: it increased [Ca2+]i in the pituitary cells and the increase was dependent on presence of Ca2+ in the medium. Melatonin inhibited the forskolin induced [Ca2+]i increase. Our observations indicate that increase of cyclic AMP stimulates Ca2+ influx in the pituitary cells of neonatal rat and that this mechanism is involved in [Ca2+]i increase induced by the pituitary adenylyl cyclase-activating polypeptide. Because melatonin inhibits increase of cyclic AMP induced by pituitary adenylyl cyclase-activating polypeptide or forskolin, the inhibitory effect of melatonin on Ca2+-influx may be mediated by the decrease of cyclic AMP concentration. This mechanism of melatonin action has not been described previously. Because melatonin inhibits the polypeptide- or forskolin-induced [Ca2+]i also in the cells not sensitive to GnRH, melatonin receptors seem to be present on both gonadotrophs and non-gonadotrophic pituitary cells.

Animals↗

PubMed2000

Different effects of melatonin pretreatment on cAMP and LH responses of the neonatal rat pituitary cells.

The rhythm of the pineal hormone melatonin transduces the effect of photoperiod on seasonal functions. Duration of the melatonin pulse provides information about season and the long melatonin pulse induces reproductive involution in the long day breeders such as photoperiodic rodents. The length of melatonin pulse thus carries photoperiodic information, which regulates the function of target cells. Therefore, we have studied the effects of melatonin pretreatment of various lengths on responsiveness of the neonatal rat pituitary cells cultured in vitro to GnRH or forskolin. In these cells, melatonin treatment inhibits the GnRH-induced LH release as well as the forskolin-induced cAMP accumulation. However, long preincubation with melatonin has a paradoxical stimulatory effect on the cellular responsiveness. When the cells are pretreated with melatonin for 16 hr or more, then rinsed thoroughly and treated with forskolin for 30 min, the increase of cAMP is potentiated. Moreover, in the melatonin-pretreated cells. the subsequent melatonin treatment inhibits the forskolin-induced cAMP accumulation relatively more than in the non-pretreated cells. Although melatonin pretreatment does not potentiate the GnRH-induced LH release, it protects the gonadotrophs against the GnRH-induced desensitization: pretreatment with GnRH for 12 hr or more renders the cells insensitive to subsequent GnRH stimulation, while after pretreatment with GnRH and melatonin, the subsequent GnRH treatment induces significant increase of LH release. These observations indicate that long pretreatment with melatonin improves responsiveness of the pituitary cells to the subsequent stimulation, but its effects on cAMP accumulation and LH release are different.

Animals↗

PubMed2000

Differences in gonadotropin-releasing hormone-induced calcium signaling between melatonin-sensitive and melatonin-insensitive neonatal rat gonadotrophs.

The sensitivity of GnRH-stimulated calcium signaling to melatonin, in a subpopulation of neonatal gonadotrophs, is supposed to be attributable to melatonin receptors. However, it is not yet known whether the intracellular pathway for GnRH action in melatonin-sensitive cells is the same as in melatonin-insensitive cells. By monitoring intracellular Ca2+ changes as an outward current carried through apamin-sensitive Ca2+-activated K+ channels, we compared GnRH-induced calcium responses in these two subpopulations of neonatal gonadotrophs. GnRH induced various oscillatory, as well as nonoscillatory, responses in both cell types that was not related to melatonin sensitivity. Melatonin-sensitive GnRH-induced responses could be clearly distinguished according to the pharmacological properties of their latency. The latency increased in zero extracellular Ca2+ or with the addition of nifedipine, staurosporine, and ryanodine. This effect was only rarely observed in melatonin-insensitive cells. This indicates that there are two pathways for initiation of GnRH-induced calcium signaling in neonatal gonadotrophs. The first pathway is mediated by inositol 1,4,5,-trisphosphate production, whereas the second involves extracellular calcium entry through voltage-dependent L-type Ca2+ channels, protein kinase C activation, and Ca2+ release from a ryanodine-sensitive store, which may coactivate Ca2+ release from an inositol 1,4,5,-trisphosphate-sensitive store. Only the second mechanism is accessible to inhibition by melatonin.

Animals↗

PubMed1999

Pineal nitric oxide synthase, but not heme oxygenase, mRNA is suppressed by continuous exposure to light.

We have previously shown that exposure of rats to constant light (LL) induced a decrease in NO synthase (NOS) activity in the pineal gland. We report here that the use of the sensitive technique of RT-PCR has demonstrated that mRNA for neuronal NOS is present in the pineal, and that it is photoneurally regulated. There was a marked decrease in pineal neuronal NOS mRNA levels in continuous light conditions, similar to the changes seen in NOS enzyme activity. Inducible NOS was not present in the pineal, and there was evidence that the photoregulatable form was not endothelial NOS. The mRNA for two isoforms of heme oxygenase, the enzyme responsible for the generation of the putative neuromodulator carbon monoxide, was also present in the pineal, but neither isoform was photoregulated. Using immunodetection, it was not possible to identify the presence of NOS protein, other than to a minimal extent, even though NOS activity was clearly present. NADPH-diaphorase staining and in situ hybridization were carried out in an attempt to identify the precise location of neuronal NOS message. A strong NADPH-diaphorase reaction was present in sympathetic nerve fibers of the pineal, but pinealocytes showed no or only very weak labelling. In situ hybridization was also unable to identify neuronal NOS message in pinealocytes. These data thus also suggest the possible presence of a pineal-specific NOS isoenzyme.

Animals↗

PubMed1999

Mechanisms of melatonin action in the pituitary and SCN.

We have compared melatonin effects in two different cell types in order to determine general intracellular mechanisms of its action. In neonatal rat pituitary, melatonin acts via the specific membrane receptors to inhibit GnRH-induced LH release. The melatonin effect disappears in adulthood due to the disappearance of the receptors. The mechanism of the melatonin action involves inhibition of the GnRH induced increase of intracellular calcium ([Ca2+])i. Our observations indicate that melatonin has dual inhibitory effect on GnRH-induced [Ca2+]i: it inhibits mobilisation of Ca2+ from endoplasmic reticulum as well as Ca2+ influx through voltage sensitive channels. Besides, melatonin also decreases basal and GnRH- or forskolin-induced increase of cAMP concentration in the pituitary. Although cAMP is not of primary importance for regulation of LH release, the cAMP decrease may participate in the mechanism of inhibitory melatonin action on LH release. Rat suprachiasmatic nuclei (SCN) have a high density of the melatonin receptors throughout the postnatal life. Cultures of dispersed SCN cells show circadian rhythm of vasopressin (AVP) release, with several fold increase in the middle of the day and decrease during night. Melatonin inhibits the spontaneous AVP release. Melatonin also inhibits the AVP release induced by vasoactive intestinal peptide (VIP). Intracellular mechanisms of the melatonin effect may involve cAMP, because melatonin inhibits the VIP-induced increase of cAMP and increase of cAMP formation by forskolin stimulates AVP release from the cultures. On the other hand, involvement of intracellular calcium in the regulation of AVP release may not be excluded. VIP induces [Ca2+]i increase in 14% of the SCN cells and AVP release is stimulated by Ca2+ ionophore ionomycin. Our observations indicate that some of the mechanisms of melatonin action are similar in the pituitary and SCN.

Animals↗

PubMed1999

Inhibitory effect of melatonin on GnRH-induced LH release.

Melatonin inhibits GnRH-induced release of LH and FSH from the neonatal, but not the adult, rat anterior pituitary gland. This action of melatonin is mediated by the specific high-affinity membrane-bound receptors that are absent in adult rats. The intracellular mechanism of melatonin action involves a decrease in intracellular calcium [Ca2+]i in the gonadotrophs; melatonin inhibits GnRH-induced Ca2+ release from endoplasmic reticulum as well as Ca2+ influx through voltage-sensitive channels. Melatonin also inhibits GnRH-induced accumulation of cAMP, which may result in the decreased influx of Ca2+, because cAMP, acting through protein kinase A, stimulates Ca2+ influx into the gonadotrophs. This age-dependent effect of melatonin on gonadotrophin release from the pituitary may be involved in the timing of puberty.

Animals↗

PubMed1998

Melatonin inhibits spontaneous and VIP-induced vasopressin release from suprachiasmatic neurons.

We have studied melatonin effects on vasopressin release from dispersed cells of the rat suprachiasmatic nuclei (SCN). The release follows a circadian rhythm peaking during the day and decreasing at night. Melatonin inhibits the spontaneous increase and accelerates the decrease of vasopressin release. Melatonin also inhibits vasopressin release induced by vasoactive intestinal peptide (EC50=0.4 nM). The inhibition of vasopressin release correlates with the known inhibitory effect of melatonin on spontaneous neuronal activity in SCN.

Animals↗

PubMed1998

Melatonin inhibits the increase of cyclic AMP in rat suprachiasmatic neurons induced by vasoactive intestinal peptide.

The effects of melatonin on basal and vasoactive intestinal peptide (VIP)-induced cAMP concentration was studied in dispersed cells of the rat suprachiasmatic nuclei (SCN). Our data indicate, that VIP induces a rapid increase of cAMP concentration in the cells followed by a slow and prolonged increase in the medium. The VIP-induced increase was dose-dependent in the range of 1-100 nM. Melatonin had no effect on basal cAMP but inhibited the cAMP increase induced by VIP in a dose-dependent manner (EC50 = 0.21 nM). Our observations indicate that melatonin acts through the inhibition of cAMP in the SCN cells similar as shown in other tissues.

Animals↗

PubMed1998

Cellular mechanisms of melatonin action.

The pineal hormone melatonin is involved in photic regulations of various kinds, including adaptation to light intensity, daily changes of light and darkness, and seasonal changes of photoperiod lengths. The melatonin effects are mediated by the specific high-affinity receptors localized on plasma membrane and coupled to GTP-binding protein. Two different G proteins coupled to the melatonin receptors have been described, one sensitive to pertussis toxin and the other sensitive to cholera toxin. On the basis of the molecular structure, three subtypes of the melatonin receptors have been described: Mel1A, Mel1B, and Mel1C. The first two subtypes are found in mammals and may be distinguished pharmacologically using selective antagonists. Melatonin receptor regulates several second messengers: cAMP, cGMP, diacylglycerol, inositol trisphosphate, arachidonic acid, and intracellular Ca2+ concentration ([Ca2+]i). In many cases, its effect is inhibitory and requires previous activation of the cell by a stimulatory agent. Melatonin inhibits cAMP accumulation in most of the cells examined, but the indole effects on other messengers have been often observed only in one type of the cells or tissue, until now. Melatonin also regulates the transcription factors, namely, phosphorylation of cAMP-responsive element binding protein and expression of c-Fos. Molecular mechanisms of the melatonin effects are not clear but may involve at least two parallel transduction pathways, one inhibiting adenylyl cyclase and the other regulating phospholipide metabolism and [Ca2+]i.

Animals↗

PubMed1998

Melatonin inhibits release of luteinizing hormone (LH) via decrease of [Ca2+]i and cyclic AMP.

The role of [Ca2+]i and cAMP in transduction of the melatonin inhibitory effect on GnRH-induced LH release from neonatal rat gonadotrophs has been studied, because melatonin inhibits the increase of both intracellular messengers. Treatments increasing Ca2+ influx (S(-) Bay K8644 or KCI) or cAMP concentration (8-bromo-cAMP or 3-isobutyl-1-methylxanthine) potentiated the GnRH-induced LH release and partially diminished the inhibitory effect of melatonin. Combination of the treatments increasing cAMP and calcium concentrations blocked completely the melatonin inhibition of LH release. The combined treatment with 8-bromo-cAMP and S(-) Bay K8644 also blocked the melatonin inhibition of GnRH-induced [Ca2+]i increase in 89 % of the gonadotrophs, while any of the treatments alone blocked the melatonin effect in about 25 % of these cells. These observations suggest that a cAMP-dependent pathway is involved in regulation of Ca2+ influx by melatonin and melatonin inhibition of LH release may be mediated by the decrease of both messengers.

1-Methyl-3-isobutylxanthine↗

PubMed1997

Melatonin inhibits GnRH-induced increase of cFOS immunoreactivity in neonatal rat pituitary.

In neonatal rat gonadotrophs, melatonin inhibits several GnRH-induced effects: stimulation of LH release as well as the increase of several second messengers as cAMP, diacylglycerol and [Ca2+]i . Recently, GnRH has been shown to induce expression of immediate early genes of fos and jun family in adult rat gonadotrophs. The purpose of this study was to determine, whether melatonin inhibits the GnRH-induced induction of cFos in neonatal rat pituitary cells. The effects of GnRH and/or melatonin on cFos immunoreactivity was determined in primary cultures of neonatal rat pituitary cells attached to the coverslip. GnRH (3 nM) induced a time-dependent increase of cFos immunoreactivity in about 10 to 15% of the cultured cells. Significant increase was observed already 30 min after GnRH administration, the maximal increase occurred after about 60 min and then gradually decreased. Melatonin (100 nM) markedly attenuated the GnRH-induced increase. GnRH increased cFos immunoreactivity in the cells in a dose-dependent manner (EC50=36 pM) and melatonin (100 nM) attenuated the response at all GnRH concentrations tested. Melatonin had no effect on basal cFos immunoreactivity, but it inhibited the GnRH-induced (10 nM) increase of cFos in dose-dependent manner (EC50=12 pM). In conclusion, this is the first report showing the inhibitory effect of melatonin on gene transcriptional activity in gonadotrophs.

Animals↗

PubMed1995

Mechanism of melatonin signal transduction in the neonatal rat pituitary.

Melatonin inhibits GnRH-induced LH release from anterior pituitary of the neonatal rat. It acts via specific high affinity receptors and decreases concentrations of intracellular calcium ([Ca2+]i) and cyclic AMP. To determine which of these second messengers transduces the melatonin inhibition of LH release, we have tested the effect of melatonin in the presence of specific drugs affecting either of these second messengers. Calcium channel antagonist nifedipine inhibited LH release from cultured pituitary to a similar degree as did melatonin and prevented the inhibitory effect of melatonin on LH release. Calcium channel agonist. Bay K potentiated the LH release and reduced the inhibitory effect of melatonin. This observation constitutes strong evidence that melatonin inhibits LH release via inhibition of calcium influx through voltage sensitive channels. The cyclic AMP derivative 8-bromo-cAMP potentiated GnRH-stimulation of LH release but did not prevent the melatonin-induced inhibition of the release. However, when used in combination with low concentration of Bay K, which alone reduced the melatonin effect only partially, 8-bromo-cAMP completely blocked the melatonin effect. This observation suggests that both cAMP and [Ca2+]i may be involved in the effect of melatonin on LH release.

1-Methyl-3-isobutylxanthine↗

PubMed1995

Melatonin inhibits increase of intracellular calcium and cyclic AMP in neonatal rat pituitary via independent pathways.

In neonatal rat pituitary, melatonin inhibits GnRH-induced increase of cAMP and [Ca2+]i. Both effects are transduced by specific high-affinity melatonin receptors coupled with pertussis toxin-sensitive G-protein. We have attempted to determine whether melatonin acts via independent pathways on both messengers or whether the indole directly inhibits only one of the messengers and the second is decreased as a secondary consequence. Melatonin inhibition of cAMP accumulation was not prevented by agents known to block melatonin effect on [Ca2+]i such as Na(+)- or Ca2(+)-free medium, Bay K, nifedipine, KCl or gramicidin. Melatonin effect on [Ca2+]i was not prevented by forskolin or 8-bromo-cAMP. We therefore conclude that melatonin inhibits cAMP accumulation and [Ca2+]i increase independently by separate pathways.

8-Bromo Cyclic Adenosine Monophosphate↗

PubMed1995

Adrenergic control of rat pineal NO synthase.

We have previously shown that exposure of rats to constant light (LL) induced a decrease in NO synthase (NOS) activity in the pineal gland. We present here the evidence that chronic (5 days) norepinephrine (NE) or isoproterenol treatment prevents the effect of LL and enhances pineal NOS activity in LL animals. This effect of NE appears to be mediated by beta-adrenoceptors, because it was not mimicked by the alpha-agonist phenylephrine. Pineal NOS activity was reduced in 16-h light/8-h dark animals treated for 4 days with the beta-adrenergic antagonist propranolol but not with the alpha 1-antagonist prazosin, indicating again an involvement of beta-adrenergic receptor in the control of NOS. Treatment with adrenergic antagonists did not affect cortical NOS activity, suggesting that the control of NOS is different in these two tissues or that the pineal expresses a specific isoform of the enzyme. Taken together, these data suggest that NE controls NOS in the pineal gland through beta-adrenergic receptors. To our knowledge, this represent the first demonstration of a regulation of NOS by a neurotransmitter in the CNS, as assayed under Vmax conditions.

Adrenergic Antagonists↗

PubMed1995

Melatonin inhibition of GnRH-induced LH release from neonatal rat gonadotroph: involvement of Ca2+ not cAMP.

Melatonin inhibits gonadotropin-releasing hormone-induced release of luteinizing hormone (LH) from the neonatal rat gonadotrophs. The second messenger involved is not known, although there are several candidates, including adenosine 3',5'-cyclic monophosphate (cAMP) and intracellular free Ca2+. The present study addresses the question of which second messenger mediates melatonin inhibition of LH release. We found that the effect of melatonin was not prevented by cAMP protagonists, including 8-bromo-cAMP, dibutyryl cAMP, 3-isobutyl-1-methylxanthine, and forskolin. However, treatments that enhanced Ca2+ influx masked the effects of melatonin, and treatments that blocked Ca2+ influx mimicked the effects of melatonin. Moreover, melatonin decreased K(+)-induced LH release, which is dependent on Ca2+ influx but did not block release of LH due to thapsigargin-induced mobilization of Ca2+ from intracellular stores. These findings indicate that melatonin inhibits gonadotropin-releasing hormone-induced LH release, primarily through an action involving inhibition of Ca2+ influx, and that cAMP does not seem to be involved in this effect of melatonin.

Animals↗