Visual detection of transport‐P in peptidergic neurones

Abstract

Hypothalamic peptidergic neurones possess an uptake process for amines (transport‐P), for which prazosin is a substrate. It is characterized by a paradoxical increase in the accumulation of [3H]‐prazosin when the concentration of unlabelled prazosin is increased above 10−7 m. This increase is due to activation of a proton‐dependent, vacuolar‐type ATPase‐linked pump that is blocked by tricyclic antidepressants. This study utilized a fluorescence method to detect amine uptake in individual cells. Prazosin is fluorescent but most of its emission spectrum is in the ultraviolet range. We therefore used an analogue of prazosin in which the furan ring had been substituted with a fluorescent group, BODIPY FL. This compound's emission maximum is in the green part of the visible spectrum. BODIPY FL prazosin accumulated in immortalised peptidergic neurones and the characteristic emission spectrum of the compound was evident in these cells. Accumulation of BODIPY FL prazosin was saturable and was inhibited by the tricyclic antidepressant desipramine and by unlabelled prazosin. As previously described for prazosin, uptake of BODIPY FL prazosin was blocked by cold temperature and by the organic base chloroquine. Thus, prazosin and BODIPY FL prazosin were accumulated by the same uptake process. BODIPY FL prazosin accumulated in a granular distribution, which is compatible with storage in intracellular vesicles. Hypothalamic cells from foetal rats in primary culture also accumulated BODIPY FL prazosin by a desipramine‐sensitive process. Uptake was predominantly in neurones and glial cells did not accumulate the amine. Fluorescent detection provides visual evidence for amine uptake in peptidergic neurones and should enable detailed study of the distribution of this process in the brain. British Journal of Pharmacology (1997) 120, 876–882; doi:

α1B adrenergic receptors in gonadotrophin‐releasing hormone neurones: relation to Transport‐P

“…Further, following accumulation by Transport‐P, retention of the amines in peptidergic neurones requires maintenance of intracellular acidity ( Shen & Al‐Damluji, 1997 ). A microscopic method for detection of Transport‐P revealed that a fluorescent analogue of prazosin accumulated in peptidergic neurones in a granular distribution and that this process could be inhibited by chloroquine ( Al‐Damluji et al ., 1997 ). This indicated that these amines accumulate in acidified intracellular vesicles.…”

“…A considerable amount of evidence supported this second part of the hypothesis. Thus: (a) the paradoxical increase in binding of [ 3 H]‐prazosin is inhibited competitively by antidepressants which are known to inhibit the pre‐synaptic re‐uptake of amines ( Al‐Damluji et al ., 1993 ; Al‐Damluji & Kopin, 1996b ); (b) the paradoxical increase in accumulation of [ 3 H]‐prazosin was not seen in membrane preparations, indicating that it requires intact cells or storage organelles ( Al‐Damluji et al ., 1993 ); (c) the prazosin paradox is an energy‐dependent process, the source of energy being the electrochemical proton gradient which is generated by vacuolar‐type ATPase (V‐ATPase; Al‐Damluji & Kopin, 1996a ); (d) ligands for Transport‐P must possess a specific chemical structure, consisting of a hydrophobic phenyl group, an alkyl side chain and a basic amine ( Al‐Damluji & Kopin, 1998 ); (e) a fluorescent analogue of prazosin accumulated in intracellular vesicles, providing visual evidence for uptake ( Al‐Damluji et al ., 1997 ); (f) following accumulation by Transport‐P, the amines can be released by an energy‐dependent process ( Shen & Al‐Damluji, 1997 ).…”

Structural properties of phenylethylamine derivatives which inhibit transport‐P in peptidergic neurones

“…We therefore studied the accumulation of [ 3 H]‐verapamil, which is a phenylethylamine derivative which possesses the structural properties which enable interaction with transport‐P, as identified in this study. [ 3 H]‐verapamil was internalized by GnRH cells in a similar manner to prazosin (Al‐Damluji, 1996). Further, phenylethylamine and its derivatives inhibited the uptake of prazosin competitively (Figure 8), suggesting that these compounds and prazosin act on the same transport‐P carrier molecule in GnRH neurones.…”

“…(3) Uptake 2 accumulates a wide range of compounds, including 5‐HT, histamine and isoprenaline (Grohmann & Trendelenburg, 1984) which do not interact with transport‐P (Table 1), indicating that transport‐P is more selective in its substrate spectrum than Uptake 2 . Thus, transport‐P differs from Uptake 2 in its neuronal location (Al‐Damluji et al , 1997), functional properties (Al‐Damluji & Kopin, 1996a) and in the structure of its ligands.…”

“…Transport‐P differs from Uptake 2 in non‐neuronal cells in that it is insensitive to steroid hormones (Al‐Damluji & Kopin, 1996a). Further, microscopic studies using a fluorescent analogue of prazosin demonstrated that in the hypothalamus, transport‐P exists predominantly in neurones rather than in glial cells (Al‐Damluji et al , 1997).…”