Pharmacological Analysis of Sympathetically-Mediated Constriction in Wild Type and α 1 -adrenoceptor Knock Out Mouse Tail Artery

Claire Stevenson1, Craig Daly1, Elspeth McLachlan2, Ian McGrath1. 1University of Glasgow, Glasgow, UK, 2Neuroscience Research Australia, New South Wales, Australia

Noradrenergic and purinergic transmission from sympathetic nerves produces depolarisation of the rat tail artery (Sneddon and Burnstock, 1984), a blood vessel supplying the skin, but contraction appears to be mediated entirely through activation of α1- and α2-adrenoceptors (ARs) (Yeoh et al., 2004). Separating α1- and α2-ARs pharmacologically is difficult due to a lack of specific agonists and antagonists to distinguish between the α1- and α2-ARs. Development of transgenic mice lacking α1-ARs (α1-null) allows this complex pharmacology to be simplified in the mouse tail artery. Here we report the involvement of α1-ARs, α2-ARs and P2X purinoceptors in transmission from sympathetic nerves to vascular smooth muscle and investigate the effects of genetically deleting the α1-ARs in the α1-null mice.

Male C57/Bl Wild Type (WT) and α1-null mice (aged 4-6 months) were killed by CO2 asphyxiation. Ring segments 2 mm in length were removed from proximal tail artery, mounted on wire myographs under 200 mg resting tension and incubated with capsaicin (1 µM) for 30 minutes to block any possible sensory nerve-mediated relaxation. Contractions were evoked by supramaximal perivascular stimuli (20 V; 0.3 ms pulse width; 20 pulses at 0.5 and 8 Hz) and tension was recorded using Chart. The effects of 100 nM prazosin (α1-AR antagonist), 100 nM rauwolscine (α2-AR antagonist) and 1 mM suramin (P2X receptor antagonist) were tested. There were six mice in each group (n = 6). Data are expressed as mean ± SEM. Paired and un-paired t-tests were carried out as appropriate, with P < 0.05 taken as significant.

The mean peak responses of the α1-null arteries to perivascular nerve stimulation were 42.4% and 52.7% smaller at 0.5 Hz and at 8 Hz, respectively, than those in WT arteries (P < 0.05). Stimulation at both frequencies produced an initial fast response followed by a slow prolonged response. The time taken for the response at 8 Hz to decay to half the peak response was greater in the α1-null vessels compared to WT (un-paired t-test; P < 0.01; WT – 13 seconds ± 3.5; α1-null – 24.1 seconds ± 6.1). Prazosin reduced the peak response in both strains of mice and at both frequencies. α2-AR blockade was greater at 0.5 Hz and contributed mainly towards the 2nd component of the response at both frequencies. The remaining P2X receptor contribution following incubation with both prazosin and rauwolscine is smaller in the α1-null vessels at high frequency (unpaired t-test; P < 0.01; WT – 167mg ± 3; α1-null – 53mg ± 1). Genetic and pharmacological deletion of α1-ARs lead to responses that were similar in amplitude and duration.

Each contribution of the three post-junctional receptors shows a fast and a slower response and the relative contribution of each differs with frequency. These results suggest that α1-AR activation is not crucial in initiating changes in vascular tone with the α2-ARs contributing most in response to nerve stimulation; particularly at the lower stimulation frequency. Prazosin (100 nM) probably has an additional action as well as antagonism of α1-ARs. Synergy between the receptors is probable and this synergy may be altered in the α1-null animals where the α2-AR and purinergic receptor components may compensate for the loss of the α1-ARs.

(1) Sneddon P & Burnstock G, Eur J Pharmacol 106(1): 149-152, 1984. (2) Yeoh M et al, J Physiol 561: 583-596, 2004.