EDHF relaxations in the rat mid cerebral artery can be blocked by either charybdotoxin or TRAM-34, indicating that the opening of IK_Ca channels is critical to the EDHF response in this artery (Marrelli et al., 2003; McNeish et al., 2004). However, the contribution of IK_Ca and SK_Ca to endothelium dependent hyperpolarization can differ depending on the level of tone and the membrane potential (Crane et al., 2003). Therefore, we investigated if SK_Ca activation may be involved in endothelium dependent hyperpolarization under different levels of tone in the rat mid cerebral artery.

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 mid cerebral artery (length, ~2mm; diameter, ~150µm) were mounted in a Mulvany-Halpern myograph containing Krebs solution at 37ºC, gassed with 95% O₂ and 5% CO₂. Smooth muscle membrane potential was recorded by impaling sharp glass microelectrodes (tip resistances of 80-120 MΩ) filled with 2M KCl. Changes in membrane potential and tension induced by either the PAR2 agonist SLIGRL (20 µM) or K⁺ (15 mM) and their possible blockade by apamin, TRAM-34 or their combination were assessed simultaneously. Experiments were performed under low tone (myogenic tone) or high tone (induced by L-NAME, 100 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.

Under low tone, relaxation and hyperpolarization to SLIGRL (89.0 ± 10.7 % and –18.2 ± 2.7 mV, respectively, n=11) was unaffected by apamin or TRAM-34. Combining apamin and TRAM-34 did not effect SLIGRL-induced relaxation, but almost completely abolished the hyperpolarization (-5.5 ± 1.4 mV, n=8). Addition of L-NAME caused marked vasoconstriction and depolarization (2.8 ± 0.4 mN and 12.8 ± 0.7 mV, respectively, n=4). Under these conditions, SLIGRL-induced relaxation and hyperpolarization (80.9 ± 0.7 % and –19.6 ± 3.0 mV) was almost completely abolished by TRAM-34 alone (6.0 ± 1.8 % and –1.2 ± 0.3 mV), while apamin had no effect. K+ induced relaxation or hyperpolarization at either low tone (100.0 ± 13.5 % and 21.4 ± 1.9 mV) or high tone (70.2 ± 11.1 % and 18.3 ± 4.2 mV) were unaffected by apamin, TRAM-34 or their combination.

With high tone and absence of NO, IK_Ca plays a critical role in EDHF mediated relaxation and hyperpolarization to SLIGRL. Under low tone, blockade of IK_Ca is not sufficient to modify SLIGRL-induced hyperpolarization. However, a combination of apamin and TRAM-34 did inhibit hyperpolarization. This suggests that functional SK_Ca channels are present in rat mid cerebral arteries and can contribute to endothelium dependent hyperpolarization, depending on the degree of tone.

Crane et al. (2003) J. Physiol. 553, 183-189
Marrelli et al. (2003) Am. J. Physiol. 285, H1590-H1597
McNeish et al. (2004) BPS abstract, University of Bath, July.

This work was supported by the British Heart Foundation.