Modulation of tritium-NA uptake by phencyclidine, sigma receptor ligands and histogranin.

Abstract

The effects of PCP and sigma ligands on NA uptake were studied in rat brain synaptosomes and adrenal chromaffin cells. The noradrenaline re-uptake system plays an important role in the termination of synaptic transmission in the CNS, and in the periphery this system lowers circulating levels of catecholamines. Adrenal medullary PCP and sigma binding sites were previously characterized although their function remains unknown. The main focus of the thesis was to determine if PCP and sigma ligands modulated NA uptake and if so, whether this mechanism could be the site of action of the endogenous "PCP-like" peptide histogranin. Thus, the following objectives were examined, (1) the effects of PCP and sigma ligands on noradrenaline uptake in adrenal chromaffin cells and rat brain, (2) the interaction of PCP and sigma ligands with the substrate recognition site on the transporter, (3) the effects of the endogenous "PCP-like peptide" histogranin, on noradrenaline uptake, (4) the identification and characterization the central binding site(s) for histogranin. The characteristics of NA uptake process in both rat brain synaptosomes and adrenal chromaffin cells was first evaluated. It was observed that noradrenaline uptake was dependent on time and temperature, and both tissues displayed similar kinetics and capacity to transport NA. The presence of phencyclidine or sigma ligands was found to inhibit the uptake of NA. A correlation was drawn between the inhibitory potencies of PCP and sigma ligands in rat brain synaptosomes and bovine adrenal chromaffin cells. However, the rank order of potency of these ligands in NA uptake assays did not match that obtained previously in PCP and sigma receptor binding assays. The antidepressant $\rm\lbrack\sp3H\rbrack$desmethylimipramine labelled adrenal medullary sites with similar characteristics to those found in CNS. The sites displayed high affinity, exhibited inverse-temperature and Na$\sp+$ dependency and were potently inhibited by nisoxetine. $\rm\lbrack\sp3H\rbrack$Desmethylimipramine binding revealed that PCP and sigma ligands interacted in a negative allosteric manner with the substrate recognition site on the NA transporter. The effects of PCP and sigma ligands on $\rm\lbrack\sp3H\rbrack$DMI binding did not match that obtained in high affinity PCP and sigma receptor assays. The potency of sigma ligands in NA uptake assays correlated with that observed in $\rm\lbrack\sp3H\rbrack$DMI binding assays (r = 0.84). In contrast, PCP ligands did not correlate, inhibiting $\rm\lbrack\sp3H\rbrack$desmethylimipramine binding with a different order of potency than that observed in $\rm\lbrack\sp3H\rbrack$noradrenaline uptake assays. Scatchard plot analysis of the effects of PCP and sigma ligands on $\rm\lbrack\sp3H\rbrack$desmethylimipramine binding revealed the non-competitive nature of both PCP and sigma ligands at the $\rm\lbrack\sp3H\rbrack$DMI binding site, suggesting that these ligands bound to sites distinct from that of substrates on the transporter.