alpha(1B) adrenergic receptors in gonadotrophin-releasing hormone neurones: relation to Transport-P

Abstract

1. Peptidergic neurones accumulate amines via an unusual uptake process, designated Transport-P. [(3)H]-prazosin binds to alpha(1) adrenoceptors on these cells and is displaceable by unlabelled prazosin in concentrations up to 10(-7) 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(-10) M prazosin concentration, is linear up to 10(-7) M and at greater concentrations becomes non-linear. In contrast, in noradrenergic neurones, noradrenaline uptake is linear and saturates above 10(-7) M. In noradrenergic neurones and in non-neuronal cells, there is no uptake of prazosin in concentrations up to 10(-6) 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 alpha(1) adrenoceptors with Transport-P. Polymerase chain reactions and DNA sequencing of the products demonstrated that only the alpha(1B) sub-type of adrenoceptors is present in GnRH cells. 3. In COS cells transfected with alpha(1b) adrenoceptor cDNA and in DDT(1) MF-2 cells which express native alpha(1B) adrenoceptors, [(3)H]-prazosin was displaced by unlabelled prazosin in a normal equilibrium process, with no prazosin paradox in concentrations up to 10(-6) M. In DDT(1) MF-2 cells, [(3)H]-prazosin was displaced likewise by a series of alpha(1) adrenergic agonists, none of which increased the binding of [(3)H]-prazosin. Hence, the prazosin paradox is not due to some function of alpha(1) adrenoceptors, such as internalization of ligand-receptor complexes. 4. In neurones which possess Transport-P, transfection with alpha(1b) adrenoceptor cDNA resulted in over-expression of alpha(1B) adrenoceptors, but the prazosin paradox was unaltered. Thus, alpha(1) adrenoceptors and Transport-P mediate distinct functions in peptidergic neurones.

Figure 1

Uptake of prazosin in peptidergic neurones (GT1-1 GnRH cells). The lower panel demonstrates the uptake of radiolabelled ligand; the upper panel demonstrates uptake of total ligand (labelled and unlabelled), ie, it is corrected for the fall in specific activity of [³H]-prazosin due to isotope dilution. Non-specific uptake was defined as uptake at 0°C. In this and in subsequent Figures, standard error bars are not shown where they are smaller than the sizes of the symbols. ‘B₀' is an abbreviation for binding of the radiolabelled ligand in the absence of unlabelled ligand.

Figure 2

Uptake of noradrenaline in SK-N-SH noradrenergic neurones. As in Figure 1, the lower panel demonstrates uptake of the radiolabelled ligand and the upper panel demonstrates uptake of total ligand (labelled and unlabelled).

Figure 3

The products (shown by arrows) of polymerase chain reactions, using as templates reverse-transcribed (RT) RNA from GnRH neurones or a cDNA library which we constructed from these GnRH neurones. The primers used are described in the text. In (A) (RT-RNA as template), the first PCR employed the A1BF2/A1BB9 primer pair and this was followed by a second PCR which employed the nested primer pair A1BF1/A1BB1. In (B – D) the template was the cDNA library. The primers were A1BF2/A1BB9 in lane B, A1BF4/A1BB4 in lane C and A1BF5/A1BB5 in lane D. Specific products of the predicted sizes were detected in all these reactions. Size markers are shown in parallel lanes in each panel.

Figure 4

(A) Binding of prazosin in COS-7 cells transfected with α_1b adrenoceptor cDNA: [³H]-prazosin is displaced by unlabelled prazosin (IC₅₀ 2×10⁻⁹ M, K_D 5×10⁻¹⁰ M) and there is no increase in the binding of [³H]-prazosin at concentrations of unlabelled prazosin up to 10⁻⁶ M. In the control transfection, COS cells were electroporated in the absence of DNA. (B) Binding of prazosin in DDT₁ MF-2 cells which express native α_1B adrenoceptors, in SK-N-SH noradrenergic neurones and in COS-7 kidney cells. The concentration dependence of the binding of [³H]-prazosin and its displacement by unlabelled prazosin (K_D 4.8×10⁻¹⁰ M) is seen in the DDT₁ MF-2 cells. Binding in the other two cell lines is at very low levels and is unaffected by desipramine (not shown). There is no increase in the binding of [³H]-prazosin at concentrations of unlabelled prazosin up to 10⁻⁶ M in any of the cell lines.

Figure 5

(A) Over-expression of α_1B adrenoceptors in peptidergic neurones which possess Transport-P: GT1-1 GnRH cells were transfected with α_1b adrenoceptor cDNA. In the control transfection, GT1-1 cells were electroporated in the absence of DNA. There was no difference in the prazosin paradox between control and transfected cells. (B) Effect of the α₂ adrenergic agonist clonidine on the binding of [³H]-prazosin in GT1-1 GnRH cells.