The complete amino acid sequence of the human neurokinin-3 receptor was deduced by DNA sgqumtco analysis of human genomic fragments. Comparison of the predicted primary structure with those for the human neurokinin receptors 1 and 2 shows a highly conserved pattern of seven hydrophobi¢ regions with maximum divergence oceuring at the amino-and carboxy-terrnini, The position of intron-exon junctions are identical to those in other reported neurokinin genes, Using a chimeric genomic.cDNA gone, the human NK-3 receptor was expressed in Xenopus laevts oooytes where it mediates membrane conductance changes in response to its agonist, neurokinin B. More significantly, expression of the 8ene in mammalian cells resulted in detection of receptor binding as well as neurokinin-stimulated calcium mobilization and araehidonie acid release, all displaying the pharmacological characteristics expected of a nearokinin-3 receptor, By using the polymerase chain reaction we have shown that mRNA for the human neurokinin-3 receptor is expressed predominantly in the egntral nervous system,
Summary. High-pressure liquid chromatography with electrochemical detection was used to identify and measure catecholamines in rat, rabbit, sheep, guinea-pig and human uteri and follow changes with pregnancy. Noradrenaline was consistently the major catecholamine and pregnancy caused a regionally specific fall in its concentration which, in rat, rabbit and guinea-pig, was associated with a decline in total content. Adrenaline was undetectable (<10 pmol/g myometrium) in all species and at all gestational ages studied. Dopamine and its metabolite 3,4-dihydroxyphenylacetic acid (DOPAC) were detected at high concentrations in guinea-pig and particularly sheep uterus. In guinea-pig uterus the dopamine/DOPAC ratio fell dramatically with pregnancy, suggesting that increased quantities of dopamine were released and catabolized. The dopamine/noradrenaline ratios suggested that dopamine is stored with noradrenaline in adrenergic neurones in guinea-pig myometrium and within an additional neuronal or cellular store(s) in sheep uterus.
:Homologues of mammalian Ras conserved in Saccharomyces cerevisiac mediate glucose-stimulated cyclic AMP formation and we used this response to test for regulation of yeast Ras activity by the a-mating factor signal transduction pathway. a-Mating factor suppresses glucose-stimulated cyclic AMP formation by up to 57+ 12.6% (n= 5) and similar inhibition was observed in four different yeast strains (MATa cells). Moreover, this response is potent (IC,, =0.14&O. 19 uM (n= 4)), rapid (maximal within l-2 min), and displays an absolute requirement for both the a-mating factor receptor (STEZ) and associated G-protein b-subunit (STEI). Inhibition appears independent of both phosphodiesterase activation and cl-mating factorstimulated cytoplasmic alkalinization. Also, basal cyclic AMP levels are unaffected by pheromone. This is the first demonstration that a cell-surface receptor linked to a heterotrimeric G-protein can suppress Ras-dependent activity and could provide important insight into mechanisms controlling ~21'~ in man. Inhibition of Rus-dependent cyclic AMP formation could also be a key event facilitating responses characteristic of yeast mating.
The regulatory factors controlling uterine activity during pregnancy remain unclear in many species. Since myometrial relaxants raise intracellular cyclic AMP, modulation of signalling pathways coupling cell-surface receptors to adenylate cyclase activation could be an important site for control. To assess the functional activity of the stimulatory GTP-binding protein Gs we have measured adenylate cyclase activation by GTP, its non-hydrolysable analogue guanosine 5'-(beta-gamma-imido)triphosphate (Gpp(NH)p), fluoride, forskolin and manganese in a 50,000 g membrane fraction prepared from the myometrium of non-pregnant, mid-pregnant (30-32 days) and late-pregnant (62-66 days) guinea-pigs (full term 67 +/- 2 days). While forskolin- and manganese-dependent enzyme activation was unaltered by pregnancy, maximal stimulation by Gpp(NH)p and fluoride was enhanced by up to 200%. Recovery of adenylate cyclase activity in the 50,000 g fraction was essentially constant at 20-24% of the total activity throughout pregnancy, and thus cannot explain the increases observed. Since guanine nucleotides and fluoride stimulate adenylate cyclase through activating Gs, and forskolin and manganese act at the level of the catalytic unit, these data are consistent with a pregnancy-related increase in Gs functional coupling while adenylate cyclase activity is unaltered. These observations suggest a physiological regulation of myometrial Gs activity during pregnancy which could facilitate hormonal stimulation of adenylate cyclase and contribute to uterine quiescence by increasing uterine sensitivity to relaxants.
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