The present study demonstrates the localisation of mdr1a/mdr1b mRNA encoding Pgp around brain capillaries, suggesting that Pgp is expressed at the endothelial cells forming the blood- brain barrier. This finding is consistent with previous studies demonstrating expression of Pgp in endothelial cells at the protein level (Thiebaut et al., 1989; Sugawara, 1990; Cordon-Cardo
et al., 1990; Beaulieu et al., 1995; Lechardeur et al., 1996). By immunoelectron and confocal microscopy of brain sections the presence of Pgp at endothelial cells lining the BBB appeared to be exclusively present at the luminal membrane (Sugawara et al., 1990; Tsuji et al., 1992; Stewart et al., 1996; Virgintino et al., 2002). Convincing evidence also came from a study using luminal membrane preparations from rat brain microvasculature isolated by density modification (Beaulieu et al., 1997). These preparations were strongly enriched of the brain
endothelial membrane marker, the glucose transporter GLUT1, and of Pgp, whereas the presence of the abluminal membrane marker integrin αv was reduced. The strong enrichment of Pgp in the luminal membrane fractions can only be explained by assuming that the luminal membrane of brain capillaries is the major site of Pgp expression.
Contradictory results from studies of Pardridge et al., however, suggest that Pgp is mainly expressed at the astrocyte foot processes (Pardridge et al., 1997; Golden and Pardridge, 1999). They observed that the anti-Pgp antibody MRK16 bound to isolated human brain microvessels with a similar and overlapping punctuated staining pattern as an antibody against the astrocyte marker glial fibrillary acidic protein (GFAP). In contrast, staining for GLUT1 was continuous and showed only minimal overlap. As astrocyte foot processes are tightly associated with the basement membrane of brain capillaries and remain so after isolation of the capillaries, these results are suggestive for astrocyte specific expression (Golden and Pardridge, 2000).
Although Pgp present at astrocytes may affect total brain uptake of Pgp substrates, astrocyte localisation is not likely to affect uptake into neurons. Hence, it would not explain the reduction of neuronal retention of dexamethasone in its neuronal targets like hippocampal and paraventricular neurons due to presence of Pgp (Meijer et al., 1998). Another study examining colocalisation of GFAP and Pgp did not find both proteins colocalised in brain sections of adult rats (Matsuoka et al., 1999). Our data show that mRNA encoding for Pgp is expressed in endothelial cells of adult rat brain suggesting that Pgp is localised at brain endothelial cells. This finding is consistent with a previous study performing in situ hybridisation histochemistry on brain sections of mice at embryonic stages (Qin and Sato, 1995), during which expression of Pgp is very low (Matsuoka et al., 1999; Tsai et al., 2002).
Although it might be possible, though unlikely, that mRNA is expressed in astrocyte foot processes closely attached to the endothelial cells, the discontinuous staining pattern of antibodies against Pgp may satisfactorily be explained by local induction of Pgp due to unknown astrocyte-derived factors. It is known that in in vitro cultures astrocytes induce various BBB properties including expression of Pgp (Tatsuta et al., 1994; Gaillard et al., 2000). Thus, it appears from this and other studies that under normal, healthy conditions Pgp is only expressed by endothelial cells, although it can not be excluded that under certain pathological conditions astrocytes may express Pgp as well.
A remarkable finding of the present study is the mRNA expression found in hippocampal neurons, particularly in the granule cells of the dentate gyrus. No other studies have reported neuronal expression of Pgp. The differential expression of various hippocampal neuronal cell layers and the fact that the sense probe did not give any signal indicate that labelling was specific and not due to the high cell density in the granule cell layer. It is at present unknown whether mdr1a, mdr1b or both are expressed in these neurons. Recently, using reverse transcriptase PCR mdr1a and mdr1b were both found to be expressed in hippocampus, whereas only mdr1a was found in other brain regions (Kwan et al., 2003).
FIGURE 3. Reduced expression of mdr1 mRNA in dentate gyrus after kainic acid. Representative autoradiographs of coronal brain sections showing mdr1 mRNA expression in hippocampus of a rat treated with saline (A) or a rat treated with kainic acid 6 hours before decapitation (B). Sense probe did not result in signal confirming specificity of the mdr1 probe (C). In situ hybridisation was performed with a radioactive-labelled probe.
FIGURE 2. Anti-Pgp (C219) immuno- histochemical staining of hypothalamic paraventricular nucleus illustrating the high vascularisation of this brain area. Bar indicates 100 µm.
FIGURE 4. Presence of P-glycoprotein and mdr mRNA encoding Pgp in rat hippocampus. (A) Immunohistochemical staining shows highest staining in stratum granulosum moleculare (arrowheads), while staining is relatively weak in CA1 (arrows). (B) DIG-labelled in situ hybridisation of mdr1 mRNA in hippocampus demonstrates that mdr1 mRNA is mainly present in the dentate gyrus (arrows). (C) Detail of medial dentate gyrus shows granule cells expressing mdr1 mRNA. Bar in A/B indicates 500 µm, while bar in C indicates 50 µm.
Unfortunately, C219 immunohistochemistry did not reveal protein expression nor in the DG itself nor in its projection area CA3, even though the C219 epitope is encoded by the part of the gene covered by the probe sequence. Protein expression may be low and/or diluted and, thus, below threshold of detection of C219 immunohistochemistry. With respect to this, it is interesting that a recent report using RT-PCR showed presence of mdr1a/1b mRNA in LMCAT fibroblast cells (Webster et al., 2002). These cells are known to express a functional Pgp-like transporter (Kralli and Yamamoto, 1996; Medh et al., 1998; Pariante et al., 2001), that can not be detected at Western blot with the C219 antibody (Kralli and Yamamoto, 1996). Alternatively, our mRNA probe may detect mRNA that encodes a hippocampal protein closely related to Pgp, although blasting the sequence against known sequences in Genbank did not result in any significant hits of other than mdr proteins. Whether granule cells express functional protein, remains therefore unresolved for now.
Dexamethasone did not affect mRNA expression in granule cells, while our methodology did not permit to detect whether dexamethasone has altered the expression at the BBB. The resolution of radioactive in situ hybridisation is not high enough to detect the widespread and scattered distribution throughout the brain of mdr mRNA expression at the BBB. Non- radioactive in situ hybridisation is not suitable to detect group differences due to nonlinearity of the visualisation technique. Several studies have evaluated the effects of dexamethasone on Pgp expression at protein as well as mRNA expression level in various settings (Zhao et al., 1993; Chieli et al., 1994; Sérée et al., 1998; Salphati and Benet, 1998; Demeule et al., 1999; Aquilante et al., 2000). However, contradictory results were obtained which suggests that