PubMed Health⌕ Search

Biomedical subjects

S M Reppert

Publications and source records attributed to S M Reppert.

At least 19 recordsLinked to original sources
PubMed1997

The Mel1a melatonin receptor is coupled to parallel signal transduction pathways.

The recent cloning of a family of high affinity melatonin receptors has provided us with a unique opportunity to define the signal transduction pathways used by these receptors. We have studied signaling through the human Mel1a receptor subtype by stable expression of receptor complementary DNA in NIH 3T3 cells. Our data indicate that the human Mel1a receptor is coupled to inhibition of forskolin-stimulated cAMP accumulation by a pertussis toxin-sensitive G protein. Although melatonin alone is without effect on phosphoinositide hydrolysis, it potentiates the effects of PGF2 alpha stimulation on phospholipase C activation. Melatonin potentiates arachidonate release stimulated by PGF2 alpha and by ionomycin. The effects of melatonin on arachidonate release are sensitive to inhibition of protein kinase C. They are independent of the effects of melatonin on cAMP and do not appear to involve activation of mitogen-activated protein kinase. The effects of melatonin on both phosphoinositide hydrolysis and arachidonate release are sensitive to pertussis toxin treatment. Thus, we show that the melatonin signal is transduced by parallel pathways involving inhibition of adenylyl cyclase and potentiation of phospholipase activation.

3T3 Cells↗
PubMed1996

The Mel1a melatonin receptor gene is expressed in human suprachiasmatic nuclei.

The pineal hormone melatonin influences circadian rhythmicity in many vertebrate species. The circadian effects of melatonin in humans have led to its use to treat jet lag and circadian-based sleep disorders. Melatonin is thought to influence circadian rhythmicity by acting in the suprachiasmatic nuclei (SCN). The recent cloning of two melatonin receptor subtypes with high affinity for melatonin allows molecular analysis of melatonin receptors in human SCN. We report that Mel1a receptor mRNA is detectable in neonatal human SCN by in situ hybridization. Mel1b and melatonin-related receptor mRNAs were not detected. The presence of Mel1a receptor mRNA in human SCN supports the hypothesis that the Mel1a receptor is responsible for the circadian effects of melatonin in humans.

Adult↗
PubMed1996

Molecular characterization of prothoracicotropic hormone (PTTH) from the giant silkmoth Antheraea pernyi: developmental appearance of PTTH-expressing cells and relationship to circadian clock cells in central brain.

Using a PCR strategy, we have cloned the cDNA for prothoracicotropic hormone (PTTH) from the giant silkmoth, Antheraea pernyi. The A. pernyi PTTH cDNA encodes a preprohormone of 221 amino acids that is 51 and 71% identical at the amino acid level with Bombyx mori and Samia cynthia ricini PTTHs, respectively. Bacterially expressed, recombinant A. pernyi PTTH stimulates adult development when injected into debrained pupae. PTTH protein (ca. 30 kDa by Western blot) and mRNA (ca. 0.9 kb by Northern blot) are expressed in brain. Immunocytochemistry and in situ hybridization show that PTTH protein and mRNA are colocalized in L-NSC III from Day 4 of embryogenesis through adult life, with little variation in either protein or mRNA levels at the various ecdyses. A pair of cells expressing immunoreactivity for the circadian clock protein PER is located in the same region as PTTH-expressing L-NSC III in A. pernyi brain. However, double-label immunocytochemical studies show that PTTH and PER are located in different cells. The close anatomical location between PTTH- and PER-expressing cells suggests routes of communication between these two cell populations that may be important for the circadian control of PTTH release.

Amino Acid Sequence↗
PubMed1996

Cloning of a melatonin-related receptor from human pituitary.

We have cloned an orphan G protein-coupled receptor from a human pituitary cDNA library using a probe generated by PCR. The cDNA, designated H9, encodes a protein of 613 amino acids that is 45% identical at the amino acid level to the recently cloned human Mel(1a) and Mel(1b) melatonin receptors. Structural analyses of the encoded protein and its gene, along with phylogenetic analysis, further show that H9 is closely related to the G protein-coupled melatonin receptor family. Unusual features of the protein encoded by H9 include a lack of N-linked glycosylation sites and a carboxyl tail >300 amino acids long. H9 transiently expressed in COS-1 cells did not bind [125I]melatonin or [3H]melatonin. H9 mRNA is expressed in hypothalamus and pituitary, suggesting that the encoded receptor and its natural ligand are involved in neuroendocrine function.

Amino Acid Sequence↗
PubMed1996

Gap junctions couple astrocytes but not neurons in dissociated cultures of rat suprachiasmatic nucleus.

Individual neurons dissociated from rat suprachiasmatic nucleus can express independently phased circadian firing rhythms in culture. The phases of these rhythms are unperturbed by reversible blockade of neuronal firing lasting 2.5 days, indicating that multiple circadian clocks continue to operate in the absence of conventional synaptic transmission. The possibility remains, however, that these circadian rhythms might depend on some other form of intercellular communication. In the present study, a potential role for gap junctional coupling in SCN cultures was evaluated by introduction of the tracer molecule Neurobiotin into both neurons (n = 98) and astrocytes (n = 10), as well as by immunolabeling for specific connexins, the molecular components of gap junctions. Astrocytes were extensively coupled to each other by connexin 43-positive gap junctions, but no evidence was found for coupling of neurons to each other or to astrocytes. These data support the hypothesis that neurons expressing independently phased circadian rhythms in SCN cultures ('clock cells') are autonomous, single cell circadian oscillators, but do not exclude a role for glia in synchronizing neuronal clock cells in vivo.

Animals↗
PubMed1996

Circadian clock neurons in the silkmoth Antheraea pernyi: novel mechanisms of Period protein regulation.

We examined Period (PER) protein regulation in the brain of the silkmoth Antheraea pernyi. PER expression is restricted to the cytoplasm and axons of eight neurons, with no evidence of temporal movement into the nucleus. These neurons appear to be circadian clock cells, because PER and per mRNA are colocalized and their levels oscillate in these cells, Timeless protein immunoreactivity is coexpressed in each PER-positive neuron, and clock protein and mRNA oscillations are all suppressed in these neurons by constant light. A per antisense RNA oscillation was detected that is spatially restricted to PER-expressing cells, suggesting a novel mechanism of PER regulation. PER-positive neurons and their projections are strategically positioned for regulating prothoracicotropic hormone and eclosion hormone, two neurohormones under circadian control. Differences in the molecular details of PER expression and regulation between the brains of silkmoths and fruitflies provide insights into the mechanisms of clock gene regulation.

Animals↗
PubMed1996

Period protein is necessary for circadian control of egg hatching behavior in the silkmoth Antheraea pernyi.

We examined the molecular basis of the circadian control of egg hatching behavior in the silkmoth Antheraea pernyi. Egg hatching is rhythmically gated, persists under constant darkness, and can be entrained by light by midembryogenesis. The time of appearance of photic entrainment by the silkmoth embryo coincides with the appearance of Period (PER) and Timeless (TIM) proteins in eight cells in embryonic brain. Although daily rhythms in PER and/or TIM immunoreactivity in embryonic brain were not detected, a robust circadian oscillation of PER immunoreactivity is present in the nuclei of midgut epithelium. per antisense oligodeoxynucleotide treatment of pharate larvae on the day before hatching consistently abolishes the circadian gate of egg hatching behavior. per antisense treatment also causes a dramatic decrease in PER immunoreactivity in newly hatched larvae. The results provide direct evidence that PER is a necessary element of a circadian system in the silkmoth.

Animals↗
PubMed1996

Structure, characterization, and expression of the gene encoding the mouse Mel1a melatonin receptor.

Recently, a distinct family of G protein-coupled receptors has been cloned that mediates the biological effects of melatonin. Of two sub-types cloned from mammals (Mel1a and Mel1b), the Mel1a receptor appears to mediate the circadian and reproductive effects of the hormone. We now report the cloning, characterization, and expression of the gene encoding the Mel1a receptor in mice. The receptor gene is composed of two exons, separated by an intron of greater than 13 kilobases. Exon 1 encodes the entire 5'-untranslated region and the coding region through the first cytoplasmic loop. Exon 2 encodes the rest of the coding region and the entire 3'-untranslated region. 5'-Rapid amplification of complementary DNA ends and ribonuclease protection analyses show that the major transcription start site is 103 nucleotides upstream of the translation start codon. Sequence analysis of 1.1 kilobases of the 5'-flanking region reveals that it does not contain TATA or CAAT boxes. The 5'-flanking region drives luciferase expression 114-fold over basal levels in a murine retinal cell line that endogenously expresses the Mel1a receptor. The mouse receptor binds 2-[125]iodomelatonin with high affinity (K(d) = 55.6 pM) when expressed transiently in COS-7 cells. In situ hybridization studies establish that Mel1a receptor messenger RNA is expressed in the hypothalamic suprachiasmatic nuclei and hypophyseal pars tuberalis, presumed sites of the circadian and some of reproductive actions of melatonin, respectively. These results provide information on Mel1a receptor gene structure essential for designing transgenic and gene knock-out studies and analyzing the transcriptional regulation of receptor gene expression.

Amino Acid Sequence↗
PubMed1996

Nature's knockout: the Mel1b receptor is not necessary for reproductive and circadian responses to melatonin in Siberian hamsters.

The pineal hormone melatonin regulates seasonal reproduction and influences the timing of circadian rhythms. The Mel1a and Mel1b receptors are the high-affinity melatonin receptors present in mammals. Unexpectedly, the Mel1b receptor gene of the Siberian hamster, Phodopus sungorus, cannot encode a functional receptor; two nonsense mutations are present within the coding region. Southern blot analysis indicates that this is a single copy gene. The Mel1b receptor gene is nonfunctional in outbred populations of P. sungorus and Phodopus campbelli. Siberian hamsters lacking a functional Mel1b receptor nevertheless show seasonal reproductive and circadian responses to melatonin, indicating that the Mel1b receptor is not necessary for these responses. These data support the hypothesis that the Mel1a receptor, which does encode a functional receptor in this species, mediates reproductive and circadian responses to melatonin.

Amino Acid Sequence↗
PubMed1995

Melatonin madness.

Explore the source record for details and available documents.

Amino Acid Sequence↗
PubMed1995

Molecular characterization of a second melatonin receptor expressed in human retina and brain: the Mel1b melatonin receptor.

A G protein-coupled receptor for the pineal hormone melatonin was recently cloned from mammals and designated the Mel1a melatonin receptor. We now report the cloning of a second G protein-coupled melatonin receptor from humans and designate it the Mel1b melatonin receptor. The Mel1b receptor cDNA encodes a protein of 362 amino acids that is 60% identical at the amino acid level to the human Mel1a receptor. Transient expression of the Mel1b receptor in COS-1 cells results in high-affinity 2-[125I]iodomelatonin binding (Kd = 160 +/- 30 pM). In addition, the rank order of inhibition of specific 2-[125I]iodomelatonin binding by eight ligands is similar to that exhibited by the Mel1a melatonin receptor. Functional studies of NIH 3T3 cells stably expressing the Mel1b melatonin receptor indicate that it is coupled to inhibition of adenylyl cyclase. Comparative reverse transcription PCR shows that the Mel1b melatonin receptor is expressed in retina and, to a lesser extent, brain. PCR analysis of human-rodent somatic cell hybrids maps the Mel1b receptor gene (MTNR1B) to human chromosome 11q21-22. The Mel1b melatonin receptor may mediate the reported actions of melatonin in retina and participate in some of the neurobiological effects of melatonin in mammals.

Amino Acid Sequence↗
PubMed1995

Mapping of the gene for the Mel1a-melatonin receptor to human chromosome 4 (MTNR1A) and mouse chromosome 8 (Mtnr1a).

The pineal hormone melatonin elicits potent circadian and reproductive effects in mammals. We report the chromosomal location of the gene for the Mel1a-melatonin receptor that likely mediates these circadian and reproductive actions. PCR analysis of human-rodent somatic cell hybrids showed that the receptor gene (MTNR1A) maps to human chromosome 4q35.1. An interspecific backcross analysis revealed that the mouse gene (Mtnr1a) maps to the proximal portion of chromosome 8. These loci may be involved in genetically based circadian and neuroendocrine disorders.

Animals↗
PubMed1995

c-fos and jun-B mRNAs are transiently expressed in fetal rodent suprachiasmatic nucleus following dopaminergic stimulation.

We examined the time-course of expression of c-fos and jun-B mRNAs in the fetal rat suprachiasmatic nuclei (SCN) following maternal cocaine injection on gestational day 20. Both c-fos and jun-B mRNA levels increased within 20 min, peaked at 40 min and declined to baseline by 120 min after cocaine treatment (30 mg/kg). In mice, the D1-dopamine agonist, SKF 38393, induced c-fos and jun-B mRNAs in the fetal SCN and striatum. Regulated expression of immediate early genes in the fetal SCN may play a role in entrainment of the fetal clock.

2,3,4,5-Tetrahydro-7,8-dihydroxy-1-phenyl-1H-3-ben↗
PubMed1995

The A2b adenosine receptor mediates cAMP responses to adenosine receptor agonists in human intestinal epithelia.

Adenosine is thought to be a major effector in immunological stimulation of Cl- secretion in intestinal epithelia. Previous studies indicate that both apical and basolateral domains of intestinal epithelial cells possess functionally defined adenosine receptors. However, it is unclear whether the same receptor subclass is expressed, what the receptor subclass(es) is, or how the receptors signal the Cl- secretory response. We now characterize the intestinal epithelial adenosine receptor subtype using the model epithelium, T84. Both apical and basolateral adenosine receptor agonist response profiles revealed a hierarchy (ED50) of 5'-(N-ethylcarboxamido)adenosine > adenosine > CGS-21680. Similarly inhibition studies revealed identical ID50 hierarchies for apical and basolateral antagonism by xanthine amine congener > 1,3-diethyl-8-phenylxanthine > aminophylline. Analyses of both agonist and antagonist pharmacological hierarchies in Chinese hamster ovary cells stably expressing the A2b receptor revealed these same hierarchies. Northern blots performed on RNA extracted from polarized T84 monolayers demonstrated no detectable message for A1 or A2a adenosine receptor, but strong hybridization was detected for the A2b adenosine receptor. Subsequent Northern blots of RNA prepared from human alimentary tract revealed that A2b adenosine receptor message was heavily expressed throughout the colon, in the appendix, and more modestly expressed in the small intestine (ileum). Analyses of cAMP generation in T84 cells in response to adenosine indicated that the basolateral A2b receptor elicits Cl- secretion through this signaling pathway. Stimulation of Cl- secretion through the apical A2b receptor exhibited relatively small but significant increases in cAMP compared with basolateral stimulation. The protein kinase A inhibitor H-89, used at concentrations that did not affect short circuit current responses to the Ca(2+)-mediated agonist carbachol, effectively inhibited short circuit current elicited by either apical or basolateral adenosine. These data suggest that the major intestinal epithelial adenosine receptor is the A2b subclass, which is positively coupled to adenylate cyclase. Such observations have potentially important implications for the treatment of diarrheal diseases.

Adenylyl Cyclases↗
PubMed1995

Interaction between the circadian clocks of mother and fetus.

In mammals, a unidirectional communication exists between the biological clocks of the mother and fetus. As a biological clock begins oscillating in the suprachiasmatic nuclei of the fetus, redundant circadian signals entrain the fetal clock to the prevailing light-dark cycle. Recent studies have revealed an activatable dopamine system within the fetal hypothalamus which may serve as a final common pathway by which maternal signals entrain the fetus. An entrained biological clock during fetal life makes the developing mammal better prepared for life in the outside world.

Animals↗
PubMed1995

Definition of the developmental transition from dopaminergic to photic regulation of c-fos gene expression in the rat suprachiasmatic nucleus.

D1-dopamine receptor stimulation induces c-fos gene expression in the fetal suprachiasmatic nucleus (SCN), but not in the adult rat SCN. Light exposure at night induces c-fos gene expression in the adult SCN. The present experiments were designed to define the developmental transition from dopaminergic to photic regulation of c-fos gene expression in the SCN. Treatment with the D1-dopamine receptor agonist SKF 38393 (10 mg/kg) increased SCN c-fos gene expression during both day and night on postnatal day (PD) 0, 1, and at night on PD 2, but the c-fos response disappeared by PD 4. Photic induction of c-fos gene expression was apparent during both day and night at each age examined, from PD 0 through PD 6. The magnitude and distribution of c-fos expression following light during the daytime was distinguishable from the response to light at night beginning on PD 2, indicating that the circadian clock regulates (gates) the c-fos response by PD 2. Orbital enucleation prevents the induction of c-fos by light at night on PD 2, indicating retinal mediation. Developmental loss of the c-fos response to SKF is not precipitated by the arrival of the retinohypothalamic tract; animals enucleated on PD 0 were insensitive to SKF on PD 6, as were visually intact controls. The results demonstrate that both dopaminergic and photic inputs can regulate SCN c-fos gene expression early in the neonatal period, and that the developmental loss of sensitivity to SKF is not due to the arrival of photic input to the SCN. The developmental transition from dopaminergic to photic regulation of c-fos gene expression roughly parallels the developmental transition from maternal to photic entrainment of the developing biological clock.

2,3,4,5-Tetrahydro-7,8-dihydroxy-1-phenyl-1H-3-ben↗
PubMed1995

Period protein from the giant silkmoth Antheraea pernyi functions as a circadian clock element in Drosophila melanogaster.

Homologs of the Drosophila clock gene per have recently been cloned in Lepidopteran and Blattarian insect species. To assess the extent to which clock mechanisms are conserved among phylogenetically distant species, we determined whether PER protein from the silkmoth Antheraea pernyi can function in the Drosophila circadian timing system. When expressed in transgenic Drosophila, the silkmoth PER protein is detected in the expected neural cell types, with diurnal changes in abundance that are similar to those observed in wild-type fruitflies. Behavioral analysis demonstrates that the silkmoth protein can serve as a molecular element of the Drosophila clock system; expression of the protein shortens circadian period in a dose-dependent manner and restores pacemaker functions to arrhythmic per0 mutants. This comparative study also suggests that the involvement of PER in different aspects of circadian timing, such as period determination, strength of rhythmicity, and clock out-put, requires distinct molecular interactions.

Animals↗