ABSTRACT
Several hydrazone, oxime, carbazone and semicarbazone derivatives of 14-alkoxycodeinones and 14-alkoxydihydrocodeinones were synthesised [1] and characterised in in vitro radioligand binding assays in rat brain membrane preparations. The tested compounds show the highest affinity for the mu opioid binding sites and most of them have agonist character. Subtype analysis of the binding shows mu2 specificity. However, some of these ligands are able to block partially (40-60%) the high affinity (putative mu1) opioid binding sites while all of them act as reversible ligands at the low affinity (putative mu2) sites.
Subject(s)
Morphinans/metabolism , Receptors, Opioid/metabolism , Affinity Labels , Animals , Brain/metabolism , In Vitro Techniques , Kinetics , Morphinans/chemical synthesis , Morphinans/chemistry , Naloxone/metabolism , Narcotic Antagonists/metabolism , Oxycodone/analogs & derivatives , Oxycodone/metabolism , Rats , Rats, Wistar , Receptors, Opioid/agonists , Receptors, Opioid, delta/agonists , Receptors, Opioid, delta/antagonists & inhibitors , Receptors, Opioid, delta/metabolism , Receptors, Opioid, kappa/agonists , Receptors, Opioid, kappa/antagonists & inhibitors , Receptors, Opioid, kappa/metabolism , Receptors, Opioid, mu/agonists , Receptors, Opioid, mu/antagonists & inhibitors , Receptors, Opioid, mu/metabolism , Structure-Activity RelationshipSubject(s)
Adenylyl Cyclases/chemistry , Adenylyl Cyclases/metabolism , Protein Structure, Secondary , Adenylyl Cyclases/genetics , Amino Acid Sequence , Animals , Binding Sites , Chromosome Mapping , Circadian Rhythm , Conserved Sequence , Genetic Diseases, Inborn/enzymology , Humans , Mammals , Molecular Sequence Data , Pineal Gland/enzymology , Rats , Sequence Alignment , Sequence Homology, Amino Acid , XenopusABSTRACT
Nocturnal melatonin production in the pineal gland is under the control of norepinephrine released from superior cervical ganglia afferents in a rhythmic manner, and of cyclic AMP. Cyclic AMP increases the expression of serotonin N-acetyltransferase and of inducible cAMP early repressor that undergo circadian oscillations crucial for the maintenance and regulation of the biological clock. In the present study, we demonstrate a circadian pattern of expression of the calcium/calmodulin activated adenylyl cyclase type 1 (AC1) mRNA in the rat pineal gland. In situ hybridization revealed that maximal AC1 mRNA expression occurred at midday (12:00-15:00), with a very low signal at night (0:00-3:00). We established that this rhythmic pattern was controlled by the noradrenergic innervation of the pineal gland and by the environmental light conditions. Finally, we observed a circadian responsiveness of the pineal AC activity to calcium/calmodulin, with a lag due to the processing of the protein. At midday, AC activity was inhibited by calcium (40%) either in the presence or absence of calmodulin, while at night the enzyme was markedly (3-fold) activated by the calcium-calmodulin complex. These findings suggest (i) the involvement of AC1 acting as the center of a gating mechanism, between cyclic AMP and calcium signals, important for the fine tuning of the pineal circadian rhythm; and (ii) a possible regulation of cyclic AMP on the expression of AC1 in the rat pineal gland.
Subject(s)
Adenylyl Cyclases/metabolism , Circadian Rhythm , Membrane Proteins , Nerve Tissue Proteins , Pineal Gland/enzymology , Animals , Calcium/physiology , Calmodulin/physiology , Gene Expression Regulation, Enzymologic/radiation effects , In Situ Hybridization , Light , Male , Norepinephrine/physiology , RNA, Messenger/genetics , Rats , Rats, WistarABSTRACT
A novel mammalian adenylyl cyclase was identified by reverse transcription-polymerase chain reaction amplification using degenerate primers based on a conserved region of previously described adenylyl cyclases (Premont, R. T. (1994) Methods Enzymol. 238, 116-127). The full-length cDNA sequence obtained from mouse brain predicts a 1353-amino acid protein possessing a 12-membrane span topology, and containing two regions of high similarity with the catalytic domains of adenylyl cyclases. Comparison of this novel adenylyl cyclase with the eight previously described mammalian enzymes indicates that this type 9 adenylyl cyclase sequence is the most divergent, defining a sixth distinct subclass of mammalian adenylyl cyclases. The AC9 gene has been localized to human chromosome band 16p13.3-13.2. The 8.5-kb mRNA encoding the type 9 adenylyl cyclase is widely distributed, being readily detected in all tissues tested, and is found at very high levels in skeletal muscle and brain. AC9 mRNA is found throughout rat brain but is particularly abundant in hippocampus, cerebellum, and neocortex. An antiserum directed against the carboxyl terminus of the type 9 adenylyl cyclase detects native and expressed recombinant AC9 protein in tissue and cell membranes. Levels of the AC9 protein are highest in mouse brain membranes. Characterization of expressed recombinant AC9 reveals that the protein is a functional adenylyl cyclase that is stimulated by Mg2+, forskolin, and mutationally activated Gsalpha. AC9 activity is not affected by Ca2+/calmodulin or by G protein betagamma-subunits. Thus AC9 represents a functional G protein-regulated adenylyl cyclase found in brain and in most somatic tissues.
