Validación del modelo ANFIS

[^H]Prazosin (3 to 18x10"^® M) bound to both the cultured neonatal cells and the GnRH cell line. Time course studies showed that apparent equilibrium was reached by 60 minutes. Displacement studies at equilibrium showed that [^Hjprazosin was displaced by unlabelled prazosin in concentrations of lO'^® to 10’^ M, suggesting the presence of a i adrenoceptor binding sites in both the neonatal cells and the GnRH cell line (Figure 7.4.1). However, at concentrations of unlabelled prazosin greater than 10'^ M, there was a paradoxical increase in apparent pHjprazosin binding (Figure 7.4.1; Al-Damluji et al, 1993).

Further experiments examined the hypothesis that this paradoxical increase in apparent [^Hjprazosin binding could be due to an uptake process which becomes apparent at greater concentrations of the ligand. Desipramine is a tricyclic antidepressant which inhibits neuronal uptake (Glowinski & Axelrod, 1964). Desipramine (10‘^ M) reduced the total amount of

[^Hjprazosin associated with the cells (Bq) by 40% (Figure 7.4.2A). In the presence of

was seen above lO'^ M. This was consistent with the hypothesis that the paradoxical increase in binding of [^Hjprazosin was due to cellular uptake of the radioligand.

Although desipramine 10'^ M reduced the total amount of [^H]prazosin associated with the cells, it had no effect on the specific binding of [^H]prazosin (Figure 7.4.2B), indicating that at this concentration, desipramine did not interfere with the binding of [^H]prazosin to these a i adrenoceptors.

Figure 7.4.2C demonstrates radioligand binding curves for [^H]prazosin and its

displacement by unlabelled prazosin, in the presence of desipramine 10‘^ M. The density

(Bmax) of the binding sites was 150 fmol/mg in the neonatal cells and 126 fmol/mg in the

GnRH cell line (Al-Damluji et al, 1993). The affinity K<j of prazosin was 4.4x10"^ M in the neonatal cells and 11x10"^ M in the GnRH cell line (Al-Damluji et al, 1993). This is similar to the affinity of prazosin for the cloned a iy adrenoceptors expressed in cultured cells (Ki 2.5x10-9 M; Cotecchia et al, 1988).

As prazosin may bind with lower affinity to adrenoceptors (Kj 41 nM; Regan et al,

1988), we further investigated the identity of the adrenoceptors in the cell cultures. The

selective a i adrenoceptor ligand [^^^IjHEAT bound to the cells with high affinity (at a concentration of 3x10"^^ M, maximum binding [BqI was 26% of total counts and NSB was

1.9%; Al-Damluji et al, 1993). The a i adrenoceptor agonist methoxamine displaced 63% of

the bound [^^^IjHEAT; this confirmed the presence of a i adrenoceptors (Al-Damluji et al,

1993). We also examined the cells for adrenoceptors using [^Hjidazoxan which binds to

0C2B adrenoceptors (K| 17 nM; Regan et al, 1988). In two separate experiments in the neonatal cells, [^H]idazoxan (1.8x10-9 M) binding did not exceed 1% of total counts, and this was not displaced with the a2 agonist clonidine (Al-Damluji et al, 1993). Similarly, in

93 the GnRH cells, binding of [^H]idazoxan (at concentrations up to 10'^ M) did not exceed 1% of total counts and was not displaced by clonidine (Al-Damluji et al, 1993). These

experiments confirmed the identity of the [^Hjprazosin binding sites as a i adrenoceptors.

In broken cell (membrane) preparations, [^Hjprazosin was displaced by unlabelled prazosin in the concentration range 10*^ to 10’^ M, and there was no paradoxical increase in apparent binding of [^Hjprazosin at concentrations of unlabelled prazosin greater than 10'^ M (Figure 7.4.2D; Al-Damluji et al, 1993). This indicated that the paradoxical increase requires the presence of intact cells or storage organelles, and is unlikely to be due to some non-specific association of prazosin with cell membranes.

Further experiments examined the possible contribution of ionic changes to the paradoxical increase in [^Hjprazosin binding at concentrations of unlabelled prazosin greater than 10"^ M. Increasing concentrations of unlabelled prazosin had no effect on the pH of Krebs-Ringer

solutions buffered with either 25 mM HEPES or 25 mM Tris (Figure 7.4.3; Al-Damluji et al, 1993). An identical pattern of displacement of [^Hjprazosin by unlabelled prazosin (lO'l® to 10'^ M) was observed in the two buffers. At greater concentrations of unlabelled prazosin, there was an identical increase in the apparent binding of [^Hjprazosin up to 10’^ M;

saturation was evident above this concentration in both buffers (Figure 7.4.3; Al-Damluji et al, 1993). These experiments demonstrated that the paradoxical increase is not due to some effects of prazosin on ionic strength or buffering capacity.

We postulated that the decline in radioactivity at unlabelled prazosin concentrations up to 10'^ M was due to displacement of [^Hjprazosin by unlabelled prazosin from a i

adrenoceptors in the peptidergic neurones; the paradoxical increase in radioactivity was due to a cellular uptake process which is activated by prazosin (Al-Damluji & Krsmanovic,

1992). The affinity of prazosin for the uptake process is lower than its affinity for the receptors; this would explain why displacement of [^Hjprazosin from the receptors is observed at low concentrations of unlabelled prazosin, whereas uptake is evident at prazosin concentrations greater than 10"^ M (Figure 7.4.1). Uptake of prazosin is demonstrable at low

concentrations of the drug, as desipramine reduced the association of nanomolar

concentrations of prazosin with the cells (Figure 7.4.2A). Prazosin therefore both binds to a i adrenoceptors and is subject to cellular uptake. The proportion of prazosin which is bound to a i adrenoceptors would be expected to be dependent on the concentration of

prazosin in the assay; at low concentrations, a greater proportion of prazosin will presumably be bound to receptors than at high concentrations, when uptake becomes more prominent due to saturation of the receptors and activation of the uptake process (Al-Damluji et al,

1993).