Regional Distribution of Cytochrome P‐450 in the Rat Brain: Spectral Quantitation and Contribution of P‐450b,e and P‐450c,d

The cytochrome P-450 (P-450) content of different regions of the rat brain was measured after partial purification of the enzyme from homogenates, and the quantitative contribution of P-450b,e and P-450c,d to brain P-450 was assessed by Western immunoblotting and immunohistochemistry using rabbit antibodies raised against purified hepatic P-450b and P-450c (anti-P-450b and anti-P-450c, respectively). P-450 could be quantitated by its reduced CO difference spectrum after chromatography of homogenates on p-chloroamphetamine-coupled Sepharose. The yield of P-450 from whole brain was 90 ± 19 pmol/g of tissue, which is ~ 1% of the level in liver microsomes from control rats. The amount of P-450 recovered from homogenates of olfactory lobes, hypothalamus, thalamus, striatum, cerebral cortex, and brainstem varied between 40 and 100 pmol/g of tissue. The cerebellum was a region of exceptionally high P-450 content, with yields of up to 400 pmol/g, whereas the substantia nigra yielded only 16-20 pmol/g. Immunohistochemical studies with anti-P-450b and anti-P-450c revealed intense staining of a limited number of cells in the cerebellum with both antibodies and in the thalamus only with anti-P-450c. In the cerebellum, both anti-P-450b and anti-P-450c stained the Bergmann glial cells together with their radial processes. Individual glial cells in the granular cell layer wer also stained. There was no staining of Purkinje cells. In the thalamus, anti-P-450b gave weak staining of certain astroglia, but with anti-P-450c, there was intense staining of neuronal somata. Western immunoblots with P-450 isolated from different brain regions confirmed the distribution of P-450b,e and P-450c,d observed with immunohistochemistry. Of all the brain regions examined, P-450b,e was detected only in P-450 obtained from the cerebellum and P-450c only in the cerebellum and thalamus. However, quantitation of the P-450b,e and P-450c bands on the immunoblots by ¹²⁵I-labeled protein A revealed that these forms of P-450 account for <1% of the P-450 in the cerebellum and thalamus. This low content of P-450b and P-450c was also reflected in a low level of ethoxycoumarin O-deethylase activity in the cerebellum and thalamus. From these studies, it is concluded that there are multiple forms of P-450 in the brain and these different forms of P-450 are highly selectively localized to certain cells. Furthermore, most of the P-450 in the brain remains uncharacterized.