Main

The antidepressant properties of selective serotonin reuptake inhibitors (SSRI), such as fluoxetine, as well as other classes of antidepressant agents, involve an increase in the synaptic levels of 5-HT in corticolimbic structures. However, SSRIs also increase 5-HT levels at inhibitory 5-HT1A and 5-HT1B autoreceptors localized on serotoninergic cell bodies and terminals, respectively, thereby attenuating their postsynaptic effects (Artigas 1995; Gobert et al. 1997a). The progressive desensitization of these autoreceptors may, therefore, underlie the delay in onset of antidepressant action (Artigas 1995; Artigas et al. 1996). By mimicking desensitization, blockade of 5-HT1A and/or 5-HT1B autoreceptors enhances the increase in 5-HT levels provoked by SSRIs (Artigas et al. 1996; Gobert et al. 1997a; Hjorth and Auerbach 1996). Correspondingly, blockade of 5-HT1A autoreceptors by the arylalkylamine, (-)pindolol, may accelerate the antidepressant actions of SSRIs in humans (Artigas et al. 1996; Blier and Bergeron 1995). However, (-)pindolol's interaction with human 5-HT1A receptors has not, as yet, been directly characterized (Artigas 1995), complicating the interpretation of its actions. In this light, the present study examined the efficacy of (-)pindolol at recombinant 5-HT1A receptors heterologously expressed in a mammalian (CHO) cell line (Newman-Tancredi et al. 1992). Its actions were compared with those of 5-HT and the selective 5-HT1A receptor antagonist, WAY 100,135 (Fletcher et al. 1993), which also potentiates the increase in 5-HT levels provoked by SSRIs in rats (Hjorth and Auerbach 1996).

METHODS

Affinity at h5-HT1A receptors (Newman-Tancredi et al. 1992) was determined in competition experiments with [3H]-8-OH-DPAT (212 Ci/mmol, Amersham). CHO-h5-HT1A membranes (20 μg protein) were incubated (2.5 h, 22°C) in triplicate with competing ligands in a buffer containing HEPES 20 mmol/L (pH 7.5) and MgSO4 5 mmol/L. Nonspecific binding was defined with 10 μmol/L 5-HT. Efficacy was determined by measuring receptor-linked G-protein activation by [35S]-GTPγS binding as previously described (Newman-Tancredi et al. 1997). Briefly, CHO-h5-HT1A membranes (50 μg protein) were incubated (20 min, 22°C) in triplicate with agonist/antagonist in a buffer containing HEPES 20 mmol/L (pH 7.4), GDP 3 μmol/L, MgSO4 3 mmol/L, [35S]-GTPγS (1100 Ci/mmol, NEN) 0.1 nmol/L. Incubations were terminated by rapid filtration and radioactivity determined by liquid scintillation counting. Binding isotherms were analyzed by nonlinear regression, and results are expressed as mean ± SEM of at least three determinations.

RESULTS

(-)Pindolol, (±)pindolol, WAY 100,135, and 5-HT inhibited [3H]-8-OH-DPAT binding to h5-HT1A receptors with Ki values of 6.4 ± 1.2, 8.2 ± 1.7, 11.5 ± 2.3, and 0.82 ± 0.13 nmol/L respectively (Figure 1A ). In a measure of receptor-mediated G-protein activation, (-)pindolol stimulated [35S]-GTPγS binding with an EC50 of 11.5 ± 4.5 nmol/L and an Emax of 20.3 ± 2.1% relative to 5-HT (Emax = 100%, EC50 = 15 ± 3.8 nmol/L). (-)Pindolol antagonized the stimulation of [35S]-GTPγS binding induced by 5-HT (100 nmol/L), reducing it to the same level of binding as in the presence of (-)pindolol alone (19.8 ± 3.7%, IC50 = 263 ± 42 nmol/L; Figure 1B). WAY 100,135 did not modify [35S]-GTPγS binding but reduced 5-HT (100 nmol/L) stimulated [35S]-GTPγS binding to basal levels with an IC50 of 119 ± 24 nmol/L. In control experiments, (-)pindolol (1 μmol/L)-induced [35S]-GTPγS binding was blocked by the selective 5-HT1A receptor antagonist, WAY 100,635 (10 μmol/L; Newman-Tancredi et al. 1996) reducing it to 3.6 ± 4.2% of that induced by 5-HT. Basal [35S]-GTPγS binding to untransfected CHO cells (9760 ± 850 dpm) was not altered by 10 μmol/L 5-HT (10230 ± 1190 dpm) or by 10 μmol/L (-)pindolol (9780 ± 910 dpm).

Figure 1
figure 1

(A): Competition binding isotherms for [3H]-8-OH-DPAT binding at h5-HT1A receptors stably expressed in CHO cells. (B and C): Effects of (-)pindolol and WAY 100,135 (WAY) alone and in the presence of 5-HT (100 nmol/L) on stimulation of [35S]-GTPγS binding to CHO-h5-HT1A membranes, expressed as a percentage of the maximal binding given by 5-HT (10 μmol/L). Points shown are means of triplicate determinations from representative experiments.

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DISCUSSION

The present data show that (-)pindolol interacts with recombinant 5-HT1A receptors with high affinity (nanomolar Ki value) comparable to that of WAY 100,135. However, in contrast to the endogenous (full) agonist, 5-HT, and the antagonist, WAY 100,135, (-)pindolol elicits a modest (ca. 20%) activation of [35S]-GTPγS binding. (-)Pindolol concentration-dependently reduced 5-HT-induced [35S]-GTPγS binding to a level comparable to that elicited by (-)pindolol alone. It is concluded that (-)pindolol behaves as a weak partial agonist at CHO-h5-HT1A receptors. These in vitro data at recombinant human 5-HT1A receptors are consistent with a report of weak partial agonist properties of (-)pindolol at rat 5-HT1A receptors (De Vivo and Maayani 1990) and results from in vivo studies in humans, where (-)pindolol acted as a weak partial agonist in modulation of core temperature, corticosterone, and prolactin levels (for review, see Meltzer and Maes 1996).

As discussed elsewhere (Newman-Tancredi et al. 1997). CHO-h5-HT1A cell membranes constitute a model of postsynaptic 5-HT1A receptors, with agonist efficacies that correspond to those observed for agonist activation of hippocampal 5-HT1A sites (e.g., Odagaki and Fuxe 1995). In contrast, at presynaptic 5-HT1A receptors, which display high density and receptor reserve, the efficacy of (-)pindolol may be amplified (Meller et al. 1990). Indeed, at high doses, (-)pindolol behaves as a partial agonist in several models thought to reflect activation of autoreceptor-mediated activity in animals and, possibly, in humans (Meltzer and Maes 1996; Hjorth and Auerbach 1996).

Antagonism of postsynaptic 5-HT1A receptors and an agonist action at 5-HT1A autoreceptors would be inconsistent with a potentiation of the activity of serotoninergic neurones by (-)pindolol either alone or in combination with SSRIs (Redrobe et al. 1996; Blier et al. 1997). However, in dialysis studies in conscious animals, (-)pindolol fails to affect, or actually increases, postsynaptic levels of 5-HT, consistent with antagonist actions at 5-HT1A autoreceptors (Hjorth and Auerbach 1996). Further, as mentioned above, (-)pindolol facilitates the influence of SSRIs upon postsynaptic 5-HT levels, whereas the partial agonist, buspirone, inhibits the actions of SSRIs (Gobert et al. 1997b). In addition, it has been suggested, on the basis of electrophysiological studies, that low doses (2.5 mg PO) of (-)pindolol may preferentially antagonize raphe-localized rather than hippocampal 5-HT1A receptors in rats (Blier et al. 1997). Indeed, the induction of corticosterone secretion observed in humans (which may reflect agonist activity at postsynaptic receptors) is seen at high doses of (-)pindolol (30.0 mg PO; Meltzer and Maes 1996). The present data show that the intrinsic activity of (-)pindolol at human receptors is markedly lower than that of 5-HT, a relationship likely to hold true for both pre- and postsynaptic sites. Thus, even if (-)pindolol itself exerts partial agonist actions at 5-HT1A autoreceptors, it will reduce their stimulation by endogenous 5-HT. Hence, (-)pindolol may increase the ability of SSRIs to enhance postsynaptic 5-HT levels and, thereby, their antidepressant actions.

In conclusion, the present data, although globally in accordance with the hypothesis that (-)pindolol increases the antidepressant effects of SSRIs by antagonism of 5-HT at inhibitory 5-HT1A autoreceptors, highlight the need for further investigation. Indeed, (-)pindolol possesses marked affinity at β-adrenergic and 5-HT1B receptors, in addition to weak partial agonist activity at 5-HT1A receptors. Therefore, clinical studies with selective and “neutral” 5-HT1A receptor antagonists will be required to confirm that blockade of 5-HT1A autoreceptors genuinely enhances the therapeutic actions of SSRIs and other antidepressant agents.