Nonsteroid anti-inflammatory drugs (NSAIDs) inhibit the activity of cyclooxygenase (COX) -1 and COX-2. It has been suggested that there may be an additional product of the COX-1 gene, called COX-3, present in the brain (and other tissues) and particularly sensitive to inhibition by paracetamol and related drugs (Chandrasekharan et al., 2002). Here we have characterised COX enzymes present in a number of rat tissues by drug sensitivity and western blot analysis.

Male Wistar rats (220-250 g) were anaesthetised with Inactin™ (120 mg kg^-1, i.p.) and blood removed via a cannula placed into the carotid artery. Some blood was centrifuged to produce plasma and the rest aliquoted into 96-well plates. Animals were then killed and test tissues removed. Tissues were finely chopped, except for aortae which were cut into rings, and placed into 100 µl incubates of rat plasma. The test compounds, naproxen (non-selective), paracetamol, rofecoxib (COX-2-selective) or SC560 (COX-1-selective) (all 10^-6 to 10^-3 M) were then added and incubations continued for 60 min. 50 µl aliquots were then removed ('basal') and calcium ionophore (A23187, 50 µM) added. Incubations then continued for a further 30 min before medium was removed ('stimulated'). As a measure of prostanoid production the amounts of 6-keto-PGF₁ (a marker of PGI₂) or thromboxane (Tx) B₂ (a marker of TxA₂) in samples were determined by radioimmunoassay. The net production of prostanoid by tissues in response to A23187 was determined as 'stimulated' minus 'basal'.

Prostanoid formation in the aorta, heart, lung and whole blood was inhibited by compounds with the order of potency, SC560 > naproxen > paracetamol rofecoxib; in the brain and cerebellum the compounds were approximately equipotent (Figure 1; n=4-6 for all). Western blot analysis demonstrated expression of COX-1 in the aorta, heart, lung and whole blood and COX-1 and COX-2 in the cerebellum and brain (n=3 for all). No evidence was found for expression of COX-3.

Figure 1. Inhibition of prostanoid formation by naproxen (), paracetamol (), rofecoxib () and SC560 () in rat whole blood and cerebellum.

Our data show that in fresh tissues (i.e. avoiding the risk of enzyme induction) prostanoid formation in peripheral tissues is due to the activity of COX-1. In tissues from the CNS, prostanoid production is supported by both COX-1 and COX-2. There is no evidence in the rat for the presence of COX-3.

Chandrasekharan, N.V. et al. (2002). Proc. Natl. Acad. Sci. USA, 99, 13926-13931.

This work was supported by a grant from the William Harvey Research Foundation.