1 Peptidergic neurones accumulate amines via an unusual uptake process, designated Transport-P.[ 3 H]-prazosin binds to a 1 adrenoceptors on these cells and is displaceable by unlabelled prazosin in concentrations up to 10 77 M. However, at greater concentrations of prazosin, there is a paradoxical accumulation of [ 3 H]-prazosin which we have attributed to Transport-P. Uptake of prazosin via Transport-P is detectable at 10 710 M prazosin concentration, is linear up to 10 77 M and at greater concentrations becomes non-linear. In contrast, in noradrenergic neurones, noradrenaline uptake is linear and saturates above 10 77 M. In noradrenergic neurones and in non-neuronal cells, there is no uptake of prazosin in concentrations up to 10 76 M, suggesting that Transport-P is a specialised function of peptidergic neurones. 2 Using a mouse peptidergic (gonadotrophin-releasing hormone, GnRH) neuronal cell line which possesses Transport-P, we have studied the interaction of a 1 adrenoceptors with Transport-P. Polymerase chain reactions and DNA sequencing of the products demonstrated that only the a 1B sub-type of adrenoceptors is present in GnRH cells. 3 In COS cells transfected with a 1b adrenoceptor cDNA and in DDT 1 MF-2 cells which express native a 1B adrenoceptors, [ 3 H]-prazosin was displaced by unlabelled prazosin in a normal equilibrium process, with no prazosin paradox in concentrations up to 10 76 M. In DDT 1 MF-2 cells, [ 3 H]-prazosin was displaced likewise by a series of a 1 adrenergic agonists, none of which increased the binding of [ 3 H]-prazosin. Hence, the prazosin paradox is not due to some function of a 1 adrenoceptors, such as internalization of ligand-receptor complexes. 4 In neurones which possess Transport-P, transfection with a 1b adrenoceptor cDNA resulted in over-expression of a 1B adrenoceptors, but the prazosin paradox was unaltered. Thus, a 1 adrenoceptors and Transport-P mediate distinct functions in peptidergic neurones.

The annexins are a major class of calcium-binding proteins with unknown functions. In an attempt to define novel model systems in which to study members of the annexin family, we have investigated the expression of annexins in eggs from the sea urchin Lytechinus pictus. Western blot analysis of L. pictus eggs using antisera raised against human annexins I, V and VI revealed the presence of immunoreactive proteins of approximately 34 kDa, 35 kDa and 68 kDa respectively. The sea urchin annexins behaved similarly to their mammalian counterparts, both during purification and in their ability to bind calcium-dependently to anionic phospholipids. Of the three sea urchin annexins, the 34 kDa form was most abundant, yielding sufficient quantities for peptide microsequencing. The amino acid sequences derived in this way showed the L. pictus annexin to be closely related both to mammalian annexin I and to annexins IX, X and XII from Drosophila and Hydra. However, N-terminal sequence from the L. pictus annexin showed it to be a novel member of the annexin super-gene family. The results are interesting in view of the complex evolution of the annexin gene family, and also point to the potential usefulness of echinoderm eggs as a model system in which to study annexin function.

1 Transport-P is an uptake process for amines in peptidergic neurones of the hypothalamus. It diers from other uptake processes by its anatomical location in post-synaptic neurones, its functional properties and by the structure of its ligands. Transport-P accumulates amines in intracellular vesicles, derives its energy from the electrochemical proton gradient and is linked to vacuolar-type ATPase (V-ATPase). Transport-P is blocked by antidepressants. We have now studied the release of amines following uptake by Transport-P in a cell line of hypothalamic peptidergic neurones. 2 Release of prazosin was not inhibited by the antidepressant desipramine; as Transport-P is blocked by desipramine, this indicated that amines are released by a mechanism which is independent of Transport-P. 3 Release of prazosin was sensitive to temperature and conformed to the Arrhenius equation. Release was minimal in the range 0 ± 258C but accelerated exponentially at higher temperatures up to 338C. The activation energy for the release of prazosin is 83.1 kJ mol 71, corresponding to a temperature quotient (Q 10 ) value of 3. 4 Release was accelerated by the organic base chloroquine, the ionophore monensin, ba®lomycinA1 which inhibits V-ATPase and by increasing extracellular pH. Thus, retention of prazosin requires an intracellular proton gradient which is generated by V-ATPase. 5 Fluorescence microscopy demonstrated that release of BODIPY FL prazosin was temperature dependent and was accelerated by chloroquine and monensin. 6 Thus, following uptake by Transport-P, amines are accumulated in acidi®ed intracellular stores. Their retention in peptidergic neurones requires intracellular acidity. The amines are released by a temperature-dependent process which is resistant to antidepressants.