Obstetrics and Gynaecology - Research Publications

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    Melatonin receptor potentiation of cyclic AMP and the cystic fibrosis transmembrane conductance regulator ion channel
    Nelson, CS ; Marino, JL ; Allen, CN (MUNKSGAARD INT PUBL LTD, 1999-03-01)
    We have used the cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel as a model system to study the cAMP signal transduction pathways coupled to the Xenopus melatonin receptor. During forskolin (Fsk) stimulation, melatonin reduced the amplitude of the CFTR currents in oocytes injected with in vitro transcribed cRNAs for the Xenopus melatonin receptor and CFTR. Pertussis toxin (Ptx) treatment eliminated melatonin inhibition of Fsk stimulated CFTR currents. In oocytes injected with cRNA for melatonin receptors, serotonin receptors (5-HT7), and CFTR Cl- channels, application of melatonin together with serotonin (5-HT) activated an additional inward current showing potentiation of adenylyl cyclases by melatonin receptors. Subthreshold activation of 5-HT7 receptors was sufficient and necessary to permit activation of CFTR channels by melatonin. Preexposure to melatonin desensitized the melatonin receptor mediated response. Therefore, based on this model system, the effects of melatonin in vivo could be either positive or negative modulation of other neuronal inputs, depending on the mode of adenylyl cyclase stimulation.
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    Cloning and characterization of Kir3.1 (GIRK1) C-terminal alternative splice variants
    Nelson, CS ; Marino, JL ; Allen, CN (ELSEVIER SCIENCE BV, 1997-06-01)
    Southern blot analysis of RT-PCR products from brain and heart revealed multiple products for a C-terminal region of Kir3.1. Sequencing yielded clones for wild-type Kir3.1 and three Kir3.1 C-terminal alternative splice variants, including a unique alternative exon. Two of these variants encoded truncated Kir3.1 molecules. Tissue distribution and electrophysiological characterization of a single truncated variant, Kir3.1(00) were then examined. Kir3.1 channels are gated by G-protein beta gamma-subunits binding to the C-terminal domain, thus, the truncation of Kir3.1(00) removes a major functional domain. When incorporated into heteromeric channels with other family members (Kir3.1, 3.2 or 3.4) several functional changes were observed: (1) Kir3.1(00) changes G-protein activation of Kir3 channels; (2) Kir3.1(00) is restricted in its ability to assemble with other channel subunits as heteromers; and (3) incorporation of Kir3.1(00) into heteromeric channel complexes alters the kinetics of channel re-activation.
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    The sensitivity of N-methyl-D-aspartate receptors to lead inhibition is dependent on the receptor subunit composition
    Omelchenko, IA ; Nelson, CS ; Marino, JL ; Allen, CN (WILLIAMS & WILKINS, 1996-07-01)
    Pb+2 is a potent inhibitor of N-methyl-D-aspartate (NMDA) receptors and its action is dependent on neuronal maturation. Developmentally regulated expression of NMDA receptor subunits may underlie the changing sensitivity to Pb+2. In oocytes expressing in vitro transcribed cRNAs for zeta 1 epsilon 1 or zeta 1 epsilon 2 NMDA receptor subunits, Pb+2 inhibited glutamate-activated currents with IC50 values of 0.87 +/- 0.25 and 1.21 +/- 0.22 microM, respectively, and NMDA-activated currents with IC50 values of 1.37 +/- 0.47 and 1.11 +/- 0.33 microM, respectively. In oocytes expressing zeta 1 epsilon 1 epsilon 2 subunits, the IC50 values for Pb+2 blockade of NMDA- or glutamate-activated currents were significantly larger when compared to zeta 1 epsilon 1 or zeta 1 epsilon 2 combinations. Pb+2 concentrations greater than 1 microM inhibited glutamate-activated currents with an IC50 of 6.1 +/- 1.22 microM and NMDA-activated currents with an IC50 of 6.64 +/- 3.34 microM. Pb+2 reduced the maximal current amplitude consistent with a noncompetitive block. zeta 1 epsilon 1 epsilon 2 NMDA receptors were potentiated by low concentrations of Pb+2 ( < 1.0 microM). These data suggest that brain regions with zeta 1 epsilon 1 or zeta 1 epsilon 2 NMDA receptors subunits would be more vulnerable to Pb+2 toxicity than those with zeta 1 epsilon 1 epsilon 2 NMDA-receptors, which are expressed later in development. These data provide a mechanism for the reported changes in the efficacy of block of NMDA receptors by Pb+2 during development.
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    Melatonin receptors activate heteromeric G-protein coupled Kir3 channels
    Nelson, CS ; Marino, JL ; Allen, CN (RAPID SCIENCE PUBLISHERS, 1996-02-29)
    The effects of melatonin on circadian pacemaker activity in the central nervous system may be the result of melatonin receptor activation of G-protein coupled potassium channels which inhibit the action potential firing of neurons. Xenopus laevis and human1a melatonin receptors stimulated heteromeric G-protein activated inwardly rectifying potassium channels (Kir3.1/Kir3.2) when expressed in vitro in oocytes. Pertussis toxin reduced iodo-melatonin (87.1% reduction) and melatonin (90.3% reduction) stimulated currents in a time-dependent manner for cells expressing X. laevis receptors. A similar pertussis toxin inhibition was observed for human melatonin receptors (melatonin, 78.9% reduction). This suggests a potential role for heteromeric Kir3 channels in the receptor-mediated actions of melatonin in vivo.