The Antidepressant Clomipramine Regulates Cortisol Intracellular Concentrations and Glucocorticoid Receptor Expression in Fibroblasts and Rat Primary Neurones

Abstract

Incubation of LMCAT fibroblasts cells with antidepressants potentiates glucocorticoid receptor (GR)-mediated gene transcription in the presence of cortisol, but not of corticosterone. We have suggested that antidepressants do so by inhibiting the LMCAT cells membrane steroid transporter and thus by increasing cortisol intracellular concentrations. We now confirm and extend this model to primary neuronal cultures. Clomipramine, a tricyclic antidepressant, increased the intracellular accumulation of 3 H-cortisol, but not 3 Hcorticosterone, in LMCAT cells (+80%) and primary rat neurones (+20%). The latter finding is the first demonstration that a membrane steroid transporter is present in neurones. Moreover, verapamil, a membrane steroid transporter inhibitor, reduced the effects of clomipramine on the intracellular accumulation of 3 H-cortisol in LMCAT cells. Finally, clomipramine also decreased GR expression (whole-cell Western blot) in LMCAT cells (50% reduction) and primary rat neurones (80% reduction). This GR downregulation can explain the reduced GR-mediated gene transcription previously described under experimental conditions that do not elicit the effects on the LMCAT cells steroid transporter. This work further supports the hypothesis that membrane steroid transporters regulating the access of glucocorticoids to the brain in vivo are a fundamental target for antidepressant action.

The Antidepressant Desipramine Requires the ABCB1 (Mdr1)-Type p-Glycoprotein to Upregulate the Glucocorticoid Receptor in Mice

“…These findings are remarkably consistent with previous work in cell lines, by us and others, showing that antidepressants are unable to potentiate GR-mediated gene transcription in the presence of an MDR PGP inhibitor or of a glucocorticoid that is not transported by MDR PGP (Pariante et al, 1997(Pariante et al, , 2001(Pariante et al, , 2003a(Pariante et al, , b, 2004bBudziszewska et al, 2000;Miller et al, 2002;Herr et al, 2003). Moreover, we find that desipramine reduces MDR PGP (Mdr1a) expression in the hippocampus of FVB/N control controls, again consistent with previous work in cell lines (Varga et al, 1996;Szabo et al, 1999;Weiss et al, 2003;Pariante et al, 2003b;Weber et al, 2005).…”

“…This confirms the notion that MDR PGP is indeed a barrier to corticosterone access to the brain, and that its absence leads to more corticosterone entering the brain, and thus to an increased negative feedback on the HPA axis. Taken together with previous in vitro work, the present study strongly supports our proposed model that one mechanism through which antidepressants regulate the HPA axis is by reducing the action of glucocorticoid transporters like MDR PGP on the endothelial cells of the BBB (and possibly in neurons), thus leading to enhanced entry of glucocorticoids into the brain and so to facilitated negative feedback (Pariante et al, 2001(Pariante et al, , 2003a(Pariante et al, , b, 2004bPariante, 2006). It is interesting that, in FVB/N controls, there were no effects of desipramine on GR expression in the cortex, and there was a tendency (although not significant) for a GR downregulation in the amygdala.…”

“…Recently, we have described in cell cultures that antidepressants control GR function by increasing the intracellular concentration of glucocorticoids (Pariante et al, 2001(Pariante et al, , 2003a(Pariante et al, , b, 2004b. Glucocorticoids are excreted from fibroblasts, leukocytes, and epithelial cells by the ABCB1-type multiple drug resistance (MDR) p-glycoprotein (PGP) present in two isoforms in rodents (abcb1a and abcb1b) and one isoform only in humans (ABCB1).…”

“…Interestingly, the MDR PGP is also localized at the luminal membrane of the endothelial cells of the blood-brain barrier (BBB), and limits the access of endogenous glucocorticoids to the mouse and human brain Meijer et al, 1998;Karssen et al, 2001Karssen et al, , 2002Uhr et al, 2002;Muller et al, 2003). We have found that antidepressants enhance GR function in mouse fibroblast cells by inhibiting the MDR PGP, and thus increasing intracellular concentrations of glucocorticoids (Pariante et al, 2001(Pariante et al, , 2003a; a similar effect is also present in rat cortical neurones (Pariante et al, 2003a). Consistent with this finding, pretreatment of cells with an MDR PGP inhibitor, or coincubation with a glucocorticoid that is not transported by MDR PGP in vitro, prevents these effects of antidepressants (Pariante et al, 1997(Pariante et al, , 2001(Pariante et al, , 2003a.…”

“…We have found that antidepressants enhance GR function in mouse fibroblast cells by inhibiting the MDR PGP, and thus increasing intracellular concentrations of glucocorticoids (Pariante et al, 2001(Pariante et al, , 2003a; a similar effect is also present in rat cortical neurones (Pariante et al, 2003a). Consistent with this finding, pretreatment of cells with an MDR PGP inhibitor, or coincubation with a glucocorticoid that is not transported by MDR PGP in vitro, prevents these effects of antidepressants (Pariante et al, 1997(Pariante et al, , 2001(Pariante et al, , 2003a. Although other researchers have independently replicated these in vitro findings (Budziszewska et al, 2000;Miller et al, 2002;Herr et al, 2003), this model has never been directly tested in animals.…”