A role for thromboxaneA2 in constiction to an inhibitor of NO synthase in rat middle cerebral artery

Alister J. McNeish, Kim A. Dora & Christopher J. Garland, Department of Pharmacy & Pharmacology, The University of Bath, Claverton Down, Bath, BA2 7AY

ThromboxaneA2 (TxA2) appears to be involved in vasoconstriction and vasomotion associated with inhibition of NO synthase (NOS) in rat middle cerebral arteries (Benyo et al., 1998, Lacza et al., 2001). We recently reported that in addition to constriction, inhibition of NOS results in a loss of SKCa function in rat middle cerebral arteries (McNeish et al 2005). Furthermore repeated stimulation of TxA2 receptors (TP) inhibits SKCa in rat mesenteric arteries (Crane et al., 2004). Therefore we wished to assess whether stimulation of TP receptors can contribute to both constriction and loss of SKCa activity associated with inhibition of NOS in rat middle cerebral arteries. Male Wistar rats (200-300g) were killed by cervical dislocation and exsanguination. The brain was removed and placed immediately in ice-cold Krebs solution. Segments of the middle cerebral artery (length, ~2mm; diameter, ~150 m m) were mounted in a Mulveny-Halpern myograph containing Krebs solution at 37ºC, gassed with 95% O 2 and 5% CO 2. Smooth muscle membrane potential was recorded with sharp glass microelectrodes (tip resistances of 80-120 MΩ) filled with 2M KCl. All experiments were conducted in the presence of L-NAME (100 μM), the effect of blocking TP receptors, was assessed using the selective antagonist ICI 192,605 (50 μM). The PAR2 agonist SLIGRL (20 μM)-induced, EDHF-mediated hyperpolarization and relaxation was also assessed in the presence and absence of ICI 192,605 and selective blockers of SKCa, (apamin, 50 nM) and IKCa (TRAM-34, 1 μM). Data are mean ± s.e.mean of 4 or more animals. Statistical comparisons were made using one way ANOVA with Bonferroni’s post test. Inhibition of NOS with L-NAME caused a marked vasoconstriction, which was reversed upon inhibition of TP receptors with ICI 192,605. In the presence of L-NAME, SLIGRL-induced EDHF-mediated relaxation was blocked by TRAM-34 alone. However, with additional inhibition of TP receptors with ICI 192,605 the EDHF mediated hyperpolarization and relaxation was not significantly affected by either TRAM-34 or apamin. Only the combined application of TRAM-34 and apamin significantly attenuated EDHF-mediated hyperpolarization and relaxation. In the rat middle cerebral artery the vasoconstriction and vasomotion induced by inhibition of NOS appears to involve the stimulation of TP receptors. Normally, EDHF mediated responses in this artery are dependent upon IKCa alone, as they are inhibited by TRAM-34 alone. However, when TP receptors are also inhibited SKCa contributes to the EDHF response, as SLIGRL-induced hyperpolarization and relaxation were only inhibited by the combined application of TRAM-34 and apamin. Therefore a loss of SKCa activity associated with NOS inhibition may reflect simulation of TP receptors.

Benyo et al., (1998) J. Cereb. Blood Flow Metab. 18, 616-618.
Crane et al., (2004) Br. J. Pharmacol. 142, 43-50.
Lacza et al., (2001) Stroke. 32, 2609-2614.
McNeish et al., (2005) J. Vasc. Res.42, 46-S5041.

Supported by the British Heart Foundation