Biomedical subjects

S M Reppert

Publications and source records attributed to S M Reppert.

At least 91 records · Page 5Linked to original sources

PubMed1990

Aluminum fluoride reveals a phosphoinositide system within the suprachiasmatic region of rat hypothalamus.

The phosphoinositide (PI) transduction system has proven to be of major importance in several regions of mammalian brain. In this report, we examined in rats whether a PI system is present in the hypothalamic suprachiasmatic nuclei (SCN), the site of a biological clock that generate circadian rhythms. Autoradiographic localization of phorbol ester binding revealed moderate levels of protein kinase C, a component of the PI system, in the SCN. Hypothalamic explants containing SCN showed substantial incorporation of [3H]myoinositol into lipids. AlF4-, a non-specific activator of G proteins, produced a dose-dependent increase in inositol monophosphate (IP1) levels in the explants in calcium-free medium, with a maximum increase of 216% of control at 50 mM NaF. Medium containing 1.8 mM calcium stimulated a similar increase in IP1 levels, but the stimulatory effects of AlF4- and calcium were not additive, so that the effect of Al4- was obscured in medium containing calcium. AlF4- stimulated accumulation of IP1, as well as inositol bis-, and trisphosphate, over a 40-min time course in the presence and absence of lithium (10 mM LiCl). Lithium, a known inhibitor of phosphatases in the inositol phosphate recycling pathway, raised levels of all 3 inositol phosphates in SCN explants both at baseline (without A1F4-) and after 30 min AlF4- stimulation. The results show the existence of a lithium-sensitive PI system within the suprachiasmatic region of the rat hypothalamus.

Aluminum↗

PubMed1990

Melatonin receptors and signal transduction in melatonin-sensitive and melatonin-insensitive populations of white-footed mice (Peromyscus leucopus).

The pineal hormone melatonin times seasonal alterations in reproductive function in photoperiodic mammals. In white-footed mice, there is variation in responsiveness to the reproductive effects of melatonin between populations originating in different locations; mice from Connecticut (CT) respond normally to melatonin, while mice from Georgia (GA) appear insensitive to melatonin. In the present paper, we compare melatonin receptor distribution and a second messenger response to melatonin in white-footed mice from CT and GA. Specific binding of 125I-labeled melatonin (I-MEL) was observed in a variety of brain regions in each population, but there were no consistent differences in the distribution or intensity of I-MEL binding between the populations. Furthermore, melatonin inhibited forskolin-stimulated cAMP accumulation in median eminence/pars tuberalis explants from both populations. These results suggest that insensitivity to melatonin in GA mice is not due to a gross defect in melatonin receptors or receptor-effector coupling.

Animals↗

PubMed1990

Entrainment of circadian phase in developing gray short-tailed opossums: mother vs. environment.

In a marsupial species, the gray short-tailed opossum (Monodelphis domestica), the suprachiasmatic nuclei (SCN), the site of a circadian clock, are formed postnatally and begin oscillating as a circadian clock on day 20. In this study, we examined how the timing (phase) of the SCN clock in the developing opossum is coordinated to the environmental light-dark cycle. When pups were reared from birth in darkness by intact dams, the circadian phases in SCN metabolic activity (monitored by 2-deoxy-D-[14C]glucose autoradiography) in 27-day-old pups were desynchronized. When pups were reared in a light-dark cycle that was 12 h out of phase with the circadian time of blinded dams, the pattern of SCN metabolic activity on day 20 was rhythmic and in phase with the light-dark cycle but out of phase with the circadian time of the dam. On day 20, retina-mediated light activation of SCN metabolic activity was also demonstrated, and anterograde tract-tracing studies revealed the presence of the retinohypothalamic tract within the SCN. These results show there is no influence of the opossum dam on the timing of the pup's biological clock. Instead, from the inception of the daily rhythm in SCN metabolic activity, its timing is regulated by retina-mediated light-dark entrainment.

Animals↗

PubMed1990

Solubilization and purification of melatonin receptors from lizard brain.

Melatonin receptors in lizard brain were identified and characterized using 125I-labeled melatonin ([125I]MEL) after solubilization with the detergent digitonin. Saturation studies of solubilized material revealed a high affinity binding site, with an apparent equilibrium dissociation constant of 181 +/- 45 pM. Binding was reversible and inhibited by melatonin and closely related analogs, but not by serotonin or norepinephrine. Treatment of solubilized material with the non-hydrolyzable GTP analog, guanosine 5'-(3-O-thiotriphosphate) (GTP-gamma-S), significantly reduced receptor affinity. Gel filtration chromatography of solubilized melatonin receptors revealed a high affinity, large (Mr 400,000) peak of specific binding. Pretreatment with GTP-gamma-S before solubilization resulted in elution of a lower affinity, smaller (Mr 150,000) peak of specific binding. To purify solubilized receptors, a novel affinity chromatography resin was developed by coupling 6-hydroxymelatonin with Epoxy-activated Sepharose 6B. Using this resin, melatonin receptors were purified approximately 10,000-fold. Purified material retained the pharmacologic specificity of melatonin receptors. These results show that melatonin receptors that bind ligand after detergent treatment can be solubilized and substantially purified by affinity chromatography.

Animals↗

PubMed1990

Melatonin receptors are present in the ferret pars tuberalis and pars distalis, but not in brain.

The pineal hormone melatonin regulates reproductive function in seasonally breeding mammals. Recent studies using 125I-labeled 2-iodomelatonin (I-MEL) reveal that the distribution of putative melatonin receptors is species-specific; only the hypophysial pars tuberalis (PT) is a consistent site of I-MEL binding in all photoperiodic species examined. In the present study, we used in vitro autoradiography to examine the distribution of I-MEL binding in the ferret brain and pituitary. We report that I-MEL binding is restricted to the PT and pars distalis (PD) of the pituitary; I-MEL binding is absent from brain. I-MEL binds in the PT and PD with high affinity (Kd values ca. 40 pM) and the rank order of potency for inhibition of I-MEL binding (6-chloromelatonin = melatonin greater than 6-hydroxymelatonin greater than N-acetylserotonin greater than serotonin) is the same as that observed for high-affinity melatonin receptors from other species. The consistent presence of high affinity melatonin receptors in the PT of a variety of photoperiodic species suggests that the PT plays a major role in mediating the effects of melatonin on neuroendocrine function.

Animals↗

PubMed1989

Direct in utero perception of light by the mammalian fetus.

The primary neural pathway for entrainment of circadian rhythms in rodents is the retinohypothalamic tract, which conveys lighting information from the retina to a biological clock in the hypothalamic suprachiasmatic nuclei (SCN). In a precocious rodent species, the spiny mouse, the retinohypothalamic tract is present and functioning within the SCN on the day of birth, as assessed by HRP histochemistry and 14C-labeled 2-deoxyglucose autoradiography, respectively. Furthermore, direct perception of environmental lighting was observed in fetal spiny mice late in gestation. Direct, retina-mediated fetal light perception appears to be a less potent entraining agent than maternal cues communicating time-of-day information to the fetal spiny mouse. Nevertheless, direct fetal light perception may reinforce maternal entraining signals during the prenatal period and therefore be of physiological significance for entrainment of circadian rhythmicity in the fetus.

Animals↗

PubMed1989

Periodic feeding of SCN-lesioned pregnant rats entrains the fetal biological clock.

Destruction of the maternal suprachiasmatic nuclei (SCN) early in gestation disrupts maternal communication of time-of-day information to the rat fetus. In the present study, we demonstrated that periodic feeding (food cue) to SCN-lesioned pregnant rats entrains the fetal biological clock. The phase of the drinking behavior rhythm was examined in pups reared in constant darkness, beginning at weaning. In several control (uncued) litters, pup phases at weaning were scattered. In other control litters where within-litter coordination of phase was observed, the average litter phase was unpredictable. In contrast, drinking rhythms of pups whose dams had received food cue during gestation were synchronized within- and between-litters, suggesting that prenatal food cue entrained the fetuses. The effect of food cue occurs prenatally, as similar results were obtained when offspring of SCN-lesioned, cued dams were fostered to lesioned, uncued dams on the day of birth. The present results, along with data from this and other laboratories, suggest that redundant mechanisms communicate time-of-day information to the fetus.

Animals↗

PubMed1989

Localization and regulation of vasopressin mRNA in human neurons.

Vasopressin (prepropressophysin) mRNA is detected in neurons of the supraoptic, suprachiasmatic, and paraventricular nuclei of human postmortem hypothalamic specimens by quantitated in situ hybridization using 35S-labeled single-stranded cDNA probes directed against exon C of the human vasopressin gene. This hybridization displays the anticipated anatomic distribution, as well as several biochemical features supporting its specificity. Hybridization densities in supraoptic neurons, a measure of vasopressin gene expression, display substantial variability from brain-to-brain. We can attribute much of this brain-to-brain variability to differences in antemortem extracellular volume status. This conclusion is based on a) animal models of the human postmortem process, b) animal models of common agonal events, c) good correlations between antemortem volume status and neuronal vasopressin mRNA hybridization densities in human postmortem specimens matched for age and postmortem interval, and d) our inability to correlate human neuronal vasopressin mRNA hybridization densities with other clinical and postmortem features. These results provide an example of antemortem regulation of a human neuroendocrine gene using postmortem tissue.

Aged↗

PubMed1989

Guanine nucleotide-binding protein regulation of melatonin receptors in lizard brain.

Melatonin receptors were identified and characterized in crude membrane preparations from lizard brain by using 125I-labeled melatonin (125I-Mel), a potent melatonin agonist. 125I-Mel binding sites were saturable; Scatchard analysis revealed high-affinity and lower affinity binding sites, with apparent Kd of 2.3 +/- 1.0 x 10(-11) M and 2.06 +/- 0.43 x 10(-10) M, respectively. Binding was reversible and inhibited by melatonin and closely related analogs but not by serotonin or norepinephrine. Treatment of crude membranes with the nonhydrolyzable GTP analog guanosine 5'-[gamma-thio]triphosphate (GTP[gamma S]), significantly reduced the number of high-affinity receptors and increased the dissociation rate of 125I-Mel from its receptor. Furthermore, GTP[gamma S] treatment of ligand-receptor complexes solubilized by Triton X-100 also led to a rapid dissociation of 125I-Mel from solubilized ligand-receptor complexes. Gel filtration chromatography of solubilized ligand-receptor complexes revealed two major peaks of radio-activity corresponding to Mr greater than 400,000 and Mr ca. 110,000. This elution profile was markedly altered by pretreatment with GTP[gamma S] before solubilization; only the Mr 110,000 peak was present in GTP[gamma S]-pretreated membranes. The results strongly suggest that 125I-Mel binding sites in lizard brain are melatonin receptors, with agonist-promoted guanine nucleotide-binding protein (G protein) coupling and that the apparent molecular size of receptors uncoupled from G proteins is about 110,000.

Amphibians↗

PubMed1989

Melatonin response to exercise training in women.

Previous human studies have indicated that daytime melatonin levels increase when the organism is subjected to the stress of fasting and exercise. Melatonin, epinephrine, and norepinephrine levels were measured during a mock run and in the course of treadmill exercise performed before (T-1), during (T-2), and following (T-3) a progressive conditioning (running) program. Hormonal responses to the training program were determined by comparing values at T-1 and T-3. Plasma melatonin, epinephrine, and norepinephrine levels rose significantly (P less than .01) from baseline values for each exercise intensity during all three treadmill runs. While a dose-response trend was observed in each of the norepinephrine and epinephrine trials, there appeared to be a progressive diminution of this relationship in melatonin between intensities. Further, as training progressed, the peak melatonin concentration was decreased by 52% from T-1 to T-3, while peak epinephrine and norepinephrine values diminished only 19% and 8%, respectively. These results suggest that vigorous exercise training may attenuate rather than augment the secretion of pineal melatonin. Development of a human model of pineal responsiveness to exercise may contribute to the elucidation of exercise-associated reproductive disorders.

Adult↗

PubMed1989

Melatonin receptors in chick brain: characterization and localization.

Melatonin receptors in chick brain were characterized by RRA and localized by in vitro autoradiography, using [125I]melatonin [( 125I]MEL), a biologically active melatonin analog. In membranes from whole brain, radioreceptor studies revealed a high affinity [125I]MEL binding site with an equilibrium dissociation constant of 47.2 +/- 11.5 (mean +/- SEM) pM and a density of 37.8 +/- 8.5 fmol/mg protein. Binding was reversible and competitively inhibited by melatonin and closely related melatonin analogs, but not be norepinephrine or serotonin. In vitro autoradiographic studies of brain revealed a widespread distribution of melatonin receptors. Specific I-MEL binding was observed in retinorecipient and integrative nuclei of the visual system including the avian homolog of the mammalian suprachiasmatic nuclei. Auditory relay nuclei and limbic structures associated with arousal and vocalization were also specifically labeled. This widespread distribution of putative melatonin receptors in chick brain is in marked contrast to the very restricted distribution of melatonin receptors in mammalian brain and suggests that avian sensory systems are affected by melatonin.

Animals↗

PubMed1989

Melatonin signal transduction in hamster brain: inhibition of adenylyl cyclase by a pertussis toxin-sensitive G protein.

Melatonin signal transduction was examined in median eminence/pars tuberalis (ME/PT) explants from Djungarian hamsters. High affinity melatonin receptors in hamster ME/PT were first quantified by in vitro autoradiography using the potent melatonin agonist 125I-labeled melatonin ([125I]MEL). Scatchard analysis of [125I]MEL binding in ME/PT revealed high affinity receptors [dissociation constant (Kd) = 2.75 X 10(-11) M]. [125I]MEL binding was markedly reduced by guanine nucleotides; treatment with the nonhydrolyzable GTP analog guanosine 5'-O-(3-thiotriphosphate) caused a 10-fold decrease in receptor affinity. Melatonin (10 nM) significantly inhibited forskolin-stimulated cAMP accumulation in ME/PT, but not in pituitary or pineal glands. In ME/PT explants, melatonin and 6-chloromelatonin inhibited forskolin-stimulated cAMP accumulation in a dose-dependent manner with similar potency (significant inhibition for each at concentrations greater than or equal to 100 pM). Serotonin significantly inhibited forskolin-stimulated cAMP levels only at doses greater than or equal to 100 microM. Inhibition of [125I]MEL binding in ME/PT by these three indolamines paralleled that determined for inhibition of forskolin-stimulated cAMP accumulation. Pertussis toxin treatment (1 microgram/ml) blocked the ability of melatonin (10 nM) to inhibit forskolin-stimulated cAMP accumulation and significantly reduced [125I]MEL binding. Pertussis toxin ADP-ribosylated the alpha-subunits of at least two guanine nucleotide-binding proteins in ME/PT explants with molecular weights of approximately 40 K. Melatonin did not increase phosphodiesterase activity in ME/PT explants. The results strongly suggest that a signal transduction pathway for melatonin in mammals involves inhibition of adenylyl cyclase by a pertussis toxin-sensitive guanine nucleotide-binding protein.

Adenylate Cyclase Toxin↗

PubMed1989

Localization and characterization of melatonin receptors in rodent brain by in vitro autoradiography.

Little is known of the neural sites of action for the pineal hormone, melatonin. Thus, we developed an in vitro autoradiographic method using 125I-labeled melatonin (I-MEL) to study putative melatonin receptors in rodent brain. We first determined optimal in vitro labeling conditions for autoradiographic detection of I-MEL binding sites in rat median eminence, the most intensely labeled area in the rat brain. We then assessed the pharmacologic and kinetic properties of I-MEL binding sites in rat median eminence by quantitative autoradiography. These sites have high affinity for I-MEL (equilibrium dissociation constant = 43 pM). I-MEL binding was inhibited by nanomolar concentrations of melatonin or 6-chloromelatonin, but was not inhibited by serotonin, dopamine, or norepinephrine (100 microM). These results suggest that I-MEL binding sites identified by in vitro autoradiography represent specific, high-affinity melatonin receptors. Studies of the distribution of I-MEL binding in rat, Syrian hamster, and Djungarian hamster brain confirm that the median eminence and suprachiasmatic nucleus are major sites of I-MEL binding in rodent brain; other brain areas labeled in one or more of these species were the thalamus (paraventricular, anteroventral, and reuniens nuclei, nucleus of the stria medullaris, and medial part of the lateral habenular nucleus), hypothalamus (dorsomedial nucleus), subiculum, and area postrema. The presence of putative melatonin receptors in the suprachiasmatic nuclei and median eminence of these rodent species suggests that these brain regions are important loci for melatonin effects on circadian rhythms and reproduction.

Animals↗

PubMed1988

Putative melatonin receptors in a human biological clock.

In vitro autoradiography with 125I-labeled melatonin was used to examine melatonin binding sites in human hypothalamus. Specific 125I-labeled melatonin binding was localized to the suprachiasmatic nuclei, the site of a putative biological clock, and was not apparent in other hypothalamic regions. Specific 125I-labeled melatonin binding was consistently found in the suprachiasmatic nuclei of hypothalami from adults and fetuses. Densitometric analysis of competition experiments with varying concentrations of melatonin showed monophasic competition curves, with comparable half-maximal inhibition values for the suprachiasmatic nuclei of adults (150 picomolar) and fetuses (110 picomolar). Micromolar concentrations of the melatonin agonist 6-chloromelatonin completely inhibited specific 125I-labeled melatonin binding, whereas the same concentrations of serotonin and norepinephrine caused only a partial reduction in specific binding. The results suggest that putative melatonin receptors are located in a human biological clock.

Autoradiography↗

PubMed1988

The vasopressin gene is expressed prior to regulation in the supraoptic nuclei of fetal rats.

The development of the regulation of vasopressin (prepropressophysin) mRNA in the supraoptic nuclei (SON) of rats was studied using quantitative in situ hybridization. On day 21 of gestation, vasopressin mRNA levels in the SON were increased by osmotic stimulation, with significant positive correlation between vasopressin mRNA levels and plasma osmolality. On day 19 of gestation, vasopressin mRNA levels in the SON were not significantly increased by osmotic stimulation, and no correlation between vasopressin mRNA levels and plasma osmolality was noted. The results suggest that the mechanisms for osmotic regulation of vasopressin mRNA levels develop between days 19 and 21 of gestation, a time course consistent with the initiation of afferent innervation of the SON.

Animals↗

PubMed1988

Iodinated melatonin mimics melatonin action and reveals discrete binding sites in fetal brain.

Iodinated melatonin was used to study melatonin sites of action in brain. Iodomelatonin mimicked the effects of melatonin on reproductive development in Djungarian hamster fetuses. 125I-melatonin injected into the dam was recovered from fetal brain. In vitro autoradiographic studies revealed a remarkably discrete distribution of competitive 125I-melatonin-binding sites in the fetal brain, with binding in median eminence/arcuate nucleus area greater than suprachiasmatic nucleus greater than pineal gland much greater than anterior pituitary gland much greater than preoptic area. 125I-melatonin promises to be a useful tool for understanding the sites and mechanism of action of melatonin.

Animals↗

PubMed1988

Maternal communication of circadian phase to the developing mammal.

In rodents, an entrainable circadian clock begins oscillating prenatally in the hypothalamic suprachiasmatic nuclei (SCN). The maternal circadian system coordinates (entrains) the timing of the developing clock to the prevailing light-dark cycle during both late fetal and early neonatal life. This maternal communication of circadian phase ensures that the developing animal is coordinated to the outside world until maturation of the normal pathway of light-dark entrainment in adults, the retinohypothalamic pathway, permits direct photic entrainment through the neonatal eye. The mechanism of maternal communication of circadian phase remains to be determined, but the necessity of the maternal SCN in this communication has been demonstrated. Indirect lines of evidence suggest that a similar scheme occurs for the development of circadian rhythms in humans.

Animal Communication↗

PubMed1988

Djungarian hamsters exhibit reproductive responses to changes in daylength at extreme photoperiods.

In seasonally breeding species, an animal's photoperiodic history (the daylength or photoperiod previously experienced) influences the reproductive response to new photoperiods. However, this has only been examined over a relatively narrow range of photoperiods. We assessed whether Djungarian hamsters (Phodopus sungorus) respond to daylength changes at extremely long and extremely short photoperiods. To determine the extent and temporal sequence of reproductive responses to photoperiod changes, ultrasonography was employed to determine serial changes in testis weight in individual animals; serial changes in body weight and pelage color were also assessed. Male animals (60-day-old) shifted from 6 h of light/day (6L) to 10L showed partial testicular recrudescence and darkening of the coat. In control animals remaining in 6L, testes remained small, and coat color continued to lighten. Animals shifted from 20L to 16L showed partial and transient testicular regression, whereas testes remained large in control animals remaining in 20L. There were no significant differences in body weight between control and experimental animals in either study. These findings indicate that Djungarian hamsters respond reproductively to changes in photoperiod at extreme daylengths, but the magnitude of the response appears to be dependent on the absolute daylength.

Animals↗