Al-Damluji, Saad; (2001) Molecular mechanisms of Transport-P. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

The alpha-1 adrenergic ligand [3H]prazosin binds to peptidergic neurones and is displaced by unlabelled prazosin in concentrations up to 10-7 M. However, at greater concentrations of unlabelled prazosin, there was a paradoxical increase in accumulation of [3H]prazosin. This work investigated the causes of this new observation. The conclusions were as follows: 1. Alpha-1 adrenoceptors are not involved in the prazosin paradox. A cDNA library was constructed from the peptidergic neurones and a clone encoding an alpha-1 adrenoceptor was isolated. The affinity of prazosin for the binding sites in the peptidergic neurones was similar to its affinity for alpha-1 adrenoceptors expressed in cultured cells transfected with the alpha-1 adrenoceptor cDNA. However, there was no prazosin paradox in these transfected cells. 2. The prazosin paradox is due to an unusual uptake process which is activated by its ligand. The increase in accumulation of [3H]prazosin is inhibited competitively by antidepressants which inhibit neuronal transporters. Specific binding sites for antidepressants are present in the peptidergic neurones. The prazosin paradox was not seen in membrane preparations, indicating that it requires intact cells or storage organelles. Antidepressant-sensitive prazosin uptake was not seen in aminergic neurones or in non-neuronal cells, indicating that it is a specialised feature of peptidergic neurones. The prazosin paradox is dependent on the energy of the electrochemical proton gradient which is generated by V-ATPase. A fluorescent analogue of prazosin accumulated in a granular distribution, indicating a vesicular location. Accumulation was predominantly in neurones rather than in glial cells. 3. There are specific structural requirements for accumulation via the uptake process. Ligands which have the greatest affinity for the uptake process consist of a basic amine and a skeleton of 18-20 carbons which are in a condensed cyclic structure. Halogens increase affinity. A furan group is essential for activation of the uptake process. 4. Release of amines: following uptake by the peptidergic neurones, the amines can be released from acidified cellular stores by an energy-dependent process. This novel uptake process is designated Transport-P. It is likely to be encoded by an allosteric transport protein. The peptidergic neurone cDNA library was screened for known and unknown members of the families of amine transporters. Two cDNAs were isolated but neither accounts for Transport-P. This is consistent with data which indicate that Transport-P differs radically from known transport processes. An expression-cloning strategy is required to isolate a cDNA which encodes Transport-P, and techniques for this are proposed.

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