published_or_final_version / Zoology / Doctoral / Doctor of Philosophy

published_or_final_version / Zoology / Doctoral / Doctor of Philosophy

published_or_final_version / abstract / Biochemistry / Master / Master of Philosophy

published_or_final_version / Zoology / Master / Master of Philosophy

Intracellular recordings were made from neurones of the rat locus coeruleus (lc) contained within a brain slice maintained <i>in vitro</i>. When applied to the slice in known concentrations, K opioid receptor agonists produced a concentration-dependent, naloxone-reversible depression of the electrically evoked excitatory post-synaptic potential (epsp). This effect of K agonists was observed in the absence of changes in the membrane potential or input resistance of the post-synaptic cell. Similarly, the K agonists had no effect on the tetrodotoxin-resistant action potential waveform. Naloxone antagonised the response to U50488 with an apparent dissociation equilibrium constant (K<SUB>d)</SUB> of 28 nM, consistent with the response being mediated via K opioid receptors. In contrast, u opioid receptor agonists caused a membrane hyperpolarisation concomitant with a fall in neuronal input resistance, and depressed the tetrodotoxin-resistant action potential. These effects were concentration-dependent and antagonised by naloxone; the hyperpolarising action of [D-Ala<SUP>2</SUP> , NMePhe<SUP>4</SUP> , Gly-ol<SUP>5</SUP> ] enkephalin (DAGO) was antagonised by naloxone with a K<SUB>d</SUB> of 1.5 nM. These findings are in agreement with previous reports that u receptor activation increases a potassium conductance in lc neurones. The epsp was depressed, but not abolished, by the excitatory amino acid antagonists, 2-amino-5-phosphonovaleric acid (2APV) and kynurenic acid, suggesting that the epsp was at least partly mediated by an excitatory amino acid. U50488 did not depress the depolarisation produced by local application of L-glutamic acid. In addition to the epsp, a noradrenergic inhibitory post-synaptic potential (ipsp) could be evoked in lc neurones. U50488 depressed the ipsp, but this effect was not reversed by naloxone and therefore not mediated via opioid receptors. U50488 had no effect on the all or nothing depolarising potential which could be evoked in a proportion of lc neurones. The effect of U50488 on the epsp was reduced when experiments were performed in the presence of agents - either barium, quinine or 4-aminopyridine - which block potassium conductances. An <i>in vitro</i> autoradiographic study of <SUP>3</SUP> H bremazocine binding within the lc revealed that the majority of binding was displaced by a combination of unlabelled DAGO and [D-Ser<SUP>2</SUP> ] Leu enkephalin Threonine (DLSET) and so represented u sites. A significant proportion however, was displaceable by unlabelled U50488 and thus represented K binding sites. It is concluded that K opioid receptors are situated pre-synaptically within the lc and when activated depress excitatory synaptic transmission.

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The active form of vitamin D is the steroid hormone 1,25-dihydroxyvitamin D₃ [1,25(OH)₂D₃]. Central to the mechanism of action of 1,25(OH)₂D₃ is its specific, high affinity intracellular receptor. This research focused on the participation of this receptor in the biology, biochemistry, and molecular biology of the vitamin D regulatory system. The effects of 1,25(OH)₂D₃ on the differentiation of hematopoietic cells were investigated using the cultured human promyelocytic leukemia cell line, HL-60, as a model. It was observed that 1,25(OH)₂D₃ induced macrophage differentiation in HL-60 cells and that a direct biochemical correlation existed between 1,25(OH)₂D₃ receptor saturation and a 1,25(OH)₂D₃-stimulated bioresponse. These data implicate 1,25(OH)₂D₃ as a natural cell differentiating agent and the 1,25(OH)₂D₃ receptor as the mediator of this hormone's action. Since the most fundamental level of control occurs by the regulation of gene expression, studies were undertaken to define the transcriptional control by 1,25(OH)₂D₃ over a known vitamin D-regulated endpoint protein. This work resulted in the molecular cloning of cDNAs to two avian intestinal calcium binding proteins, vitamin D-dependent calcium binding protein and a novel calmodulin-like protein. To gain further insight into the role of the 1,25(OH)₂D₃ receptor as a transcriptional regulator, avian and mammalian 1,25(OH)₂D₃ receptor mRNAs were characterized extensively by the techniques of in vitro translation and immunoprecipitation. These mRNAs were then utilized to construct cDNA libraries from which avian and human intestinal 1,25(OH)₂D₃ receptor cDNAs were isolated and their identity verified by hybrid-selected translation, sequencing, and Northern analysis. It was concluded that demonstrated 1,25(OH)₂D₃ receptors are polypeptides of 52-60 kDa whose activity is regulated by 1,25(OH)₂D₃ at both an mRNA and posttranslational level. Furthermore, the deduced amino acid sequence of receptor mRNA included a highly conserved cysteine, lysine, and arginine rich region that is homologous to other steroid receptors and the oncogene product v- erbA. Thus, the vitamin D receptor to be a specific trans -acting factor, modulating the pleiotropic effects of vitamin D including calcium homeostasis, and cellular differentiation.