No suppresses myogenic tone in rat middle cerebral artery by activating BKca channels

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

The rat middle cerebral artery exhibits a strong myogenic response. This myogenic response is normally suppressed by a basal release of NO as inhibition of nitric oxide synthase (NOS) causes both vasoconstriction and smooth muscle depolarization associated with oscillations in tension and membrane potential (vasomotion; McNeish et al., 2005).We wished to characterise the cellular mechanisms which underpin the vasoconstrictor effect of inhibitors of NOS by simultaneously measuring smooth muscle cell (SMC) membrane potential and tension or SMC Ca2+ and tension

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 μm) were mounted in Krebs solution (or MOPS solution for imaging experiments) in a Mulveny-Halpern myograph. Smooth muscle cell membrane potential (Em) was recorded with sharp glass microelectrodes (tip resistances of 80-120 MΩ) filled with 2M KCl. Changes in SMC Ca2+ were recorded on a confocal microscope (Olympus FV-300) with vessels that had been pre-incubated with a Ca2+-sensitive fluorescent dye (Fluo-4AM). 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 (100 μM) caused SMC depolarization associated with constriction and the development of oscillations in E m and tension. L-NAME also caused an increase in SMC Ca2+ and synchronisation of Ca2+ waves, that appeared to be temporally linked to changes in tension and Em. The effect of L-NAME was mimicked by the guanylyl cyclase blocker, ODQ (10 μM) and the BKCa blocker, iberiotoxin (100nM). Relaxation and hyperpolarization elicited by the NO donor DEA-NONOate (300 nM) was inhibited by iberiotoxin and ODQ but not by glibenclamide (10 μM), apamin (50 nM) or TRAM-34 (1 μM). L-NAME-induced constriction could be fully reversed by nifedipine (1 m M, block of L-type Ca2+ channels), which also abolished oscillations in SMC Ca2+. In the presence of L-NAME, niflumic acid (100 μM, blockade of calcium activated chloride channels; Cl Ca) relaxed and hyperpolarised middle cerebral arteries but had little effect on SMC Ca2+, a structurally distinct ClCa blocker, DIDS (300 μM), failed to elicit relaxation or hyperpolarization.

In rat middle cerebral arteries the basal NO synthase activity results in suppression of myogenic tone. Inhibition of NOS causes vasoconstriction and depolarization involving the entry of Ca2+ through L-type Ca2 channels. The vasomotion appears to be underpinned by oscillations in membrane potential and SMC Ca2+. The peak of oscillations in Em (circa -25mV) and the effect of a blocker of ClCa, niflumic acid, suggest that a chloride conductance may be involved in the L-NAME induced constriction and vasomotion; however a structurally distinct blocker of ClCa, DIDS, had no effect. Blockade of BKCa mimics the effects of L-NAME suggesting that NO normally suppresses myogenic tone by activation of SMC BKCa channels.

Supported by the British Heart Foundation

McNeish et al. (2005) Stroke. 36, 1526-1532.