Use of knockout technology to resolve pharmacological problems

Abstract

Knock-out (KO) mouse technology has given pharmacologists a powerful tool to study function in the absence of selective antagonists or inhibitors. Such KO technology can confirm predicted function, serendipitously reveal unrecognized function, or help define the mode of action of a drug. In this issue, Liles et al. demonstrate, employing mice unable to synthesize noradrenaline due to the KO of the dopamine-β-hydroxylase gene, that the sympathomimetic actions of ephedrine are directly, rather than indirectly, mediated. This may end 50 years of debate about the actions of ephedrine.

Knock-out (KO) mouse technology has given pharmacologists a way out of the impasse caused by lack of antagonists or inhibitors of sufficient selectivity. KO studies can confirm what we already suspect. For instance, in the area of adrenergic pharmacology, studies of wild-type (WT) and KO mice have confirmed that the α_2A-adrenoceptor is involved in the hypotensive actions of α₂-adrenoceptor agonists (MacMillan et al., 1996), and that the α_1D-adrenoceptor subtype is involved in pressor responses (Tanoue et al., 2002). KO studies can also throw up serendipitous findings. For instance, postjunctional α_2A-adrenoceptors have been shown to mediate contractions in mouse vas deferens (Cleary et al., 2003). KO technology can confirm tentative conclusions which would have been almost impossible to verify with pharmacological agents alone: it was suggested that α_2A-adrenoceptors were not the only prejunctional α₂-adrenoceptors (Ho et al., 1998), but only KO technology could confirm the additional presence of α_2C- (Altman et al., 1999), and even α_2B-adrenoceptors (Trendelenburg et al., 2003). Such studies confirm predicted function or serendipitously reveal unrecognized function. KO technology can also be used to set up a model system to define the mode of action of a drug.

In this issue of the British Journal of Pharmacology, Liles et al. (2007a) have used KO technology to answer a long-standing question: are responses to l-ephedrine predominantly directly mediated or due indirectly to the release of noradrenaline, or a mixture of both actions. This may not be the first paper to use KO technology in such a way, but is a clear example of the genre. Ephedrine has long been thought of as an indirect sympathomimetic, although debate about its indirect (Fleckenstein and Burn, 1953) or direct (Krogsgaard, 1956) modes of action goes back over 50 years. Ephedrine is a potent substrate for the noradrenaline transporter (Rothman et al., 2003), so that there are cogent reasons for predicting indirect actions.

This study looks at cardiovascular responses in anaesthetized mice (Liles et al., 2007a). Previous studies in anaesthetized rats and isolated tissues have suggested either that the actions of ephedrine were a mixture of direct and indirect actions (Kawasuji et al., 1996; Kobayashi et al., 2003), or that the actions were mainly direct (Liles et al., 2006b). The authors employed mice with KO of the dopamine-β-hydroxylase gene (DBH-KO), unable to synthesize noradrenaline, to answer this question definitively. The pressor and tachycardic responses to tyramine were virtually abolished in anaesthetized DBH-KO mice, demonstrating that tyramine acts virtually exclusively by an indirect mechanism. However, the α-adrenoceptor-mediated pressor response to ephedrine, like those to noradrenaline and phenylephrine, were unaffected by DBH-KO. Likewise, the tachycardia to ephedrine was unaffected by deletion of DBH. Hence, the conclusion is that the actions of ephedrine are directly mediated.

The changes seen in the KO seem so clearly linked to the function of the knocked-out gene that differences in genetic background between KO and WT can presumably be ruled out. Therefore, could adaptive changes due to the KO have confounded the results? This also seems unlikely: there was no evidence of any change in responses to noradrenaline or other directly acting agonists. Indeed, the supersensitivity seen to noradrenaline following chemical sympathectomy was not seen in DBH-KO mice, perhaps because of raised dopamine levels (Liles et al., 2007a).

Of course, the definitive conclusion from this study, that ephedrine (and more correctly l-ephedrine; what of pseudoephedrine?) is predominantly a directly acting sympathomimetic, applies only to the mouse. It is possible that the situation is different in other species. However, the results of KO experiments give us the confidence to re-examine other experimental systems in a new light.

Abbreviations

DBH

dopamine-β-hydroxylase

KO

knockout

WT

wild-type