Rho kinase (ROCK) regulates KCa2.3 channel function in the rat middle cerebral artery

Alister McNeish1, Francesc Jimenez-Altayo2, Christopher Garland2. 1Department of Pharmacy and Pharmacology, University of Bath, Bath, BA27AY, United Kingdom, 2Department of Pharmacology, Oxford University, Oxford, OX13QT, United Kingdom.

Introduction Endothelial calcium activated potassium channels (KCa) are critical in the endothelium dependent hyperpolarizing (EDH) pathway and in regulation of blood pressure (Kohler et al. 2010). Normally combined blockade of KCa2.3 and KCa3.1 is required to block the isolated endothelium derived hyperpolarizing factor (EDHF) response that is independent of NO or PGI2 (Busse et al 2002). In the rat middle cerebral artery inhibition of NOS leads to the loss of KCa2.3 function but subsequent inhibition of TP receptors restores this component of hyperpolarization (McNeish et al 2007). We investigated the TP receptor mediated signalling mechanisms that regulate KCa2.3 function. We have termed hyperpolarization seen in vessels that can synthesise NO as EDH, whereas hyperpolarizations seen in the presence of L-NAME are referred to as EDHF.

Methods: Middle cerebral arteries from male Wistar rats (250-300g) were mounted on a wire myograph for tension recording. Sharp glass microelectrodes were impaled in smooth muscle cells for simultaneous recording of membrane potential as previously described (McNeish et al. 2007)

Results: Resting tension in middle cerebral arteries able to synthesise NO was 0.7 ± 0.1 mN. Subsequent stimulation with the TP agonist U46619 (100 nM) evoked constriction (2.9 ± 0.7 mN n = 7) Under these conditions endothelium dependent hyperpolarization (EDH) evoked by the PAR2 agonist SLIGRL (20 µM; 25.4±2.5 mV) was significantly inhibited by the KCa3.1 inhibitor TRAM-34 (1 µM) alone (11.6 ± 0.9, n = 5, P<0.05), additional inhibition of KCa2.3 with apamin had no additional effect (11.0 ± 1.1mV, n = 5). Inhibition of Rho kinase (ROCK; Y27632, 10 µM) abolished U46619 constriction (0.6 ± 0.3 mN, n = 7, P<0.05) now EDH (21.9±5.3 mV) was not significantly affected by TRAM-34 alone (14.8 ± 2.2) and further addition of apamin was required to significantly inhibit EDH (6.9 ± 1.0 mV n = 6, P<0.05). Following NOS inhibition SLIGRL induced EDHF mediated responses are mediated by KCa3.1 alone, inhibition of Y27632 restored a KCa2.3 component to EDHF responses as TRAM-34 failed to fully block EDHF responses (Control: 21.4 ± 3.9 mV; TRAM-34: 10.6 ± 2.2 mV, n = 4, P<0.05) and further addition of apamin was required to fully block the response (0.2 ± 1.8 mV, n = 4, P<0.05). Statistics were calculated by ANOVA with Bonferroni’s post test.

Discussion: The data demonstrate a novel mechanism for regulation of KCa2.3 channels by ROCK: Stimulation of TP receptors involves ROCK signalling and inhibition of either TP (McNeish et al. 2007) or ROCK restores KCa2.3 function to both EDH and isolated EDHF responses. These findings are likely to have important implications for cardiovascular diseases associated with excess vasoconstrictor activity and endothelial cell dysfunction such as stroke, hypertension and diabetes. The resultant stimulation of ROCK mediated signalling would be expected to exacerbate the effect of the vasoconstrictor activity by inhibition of an important component of endothelium dependent relaxation.

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Kohler et al. (2010) Exp. Op. Thera. Targets.14:143-155

McNeish et al. (2007) Br. J. Pharmacol. 151:441-449