Modulation by endocannabinoids of whole cell potassium currents in freshly isolated smooth muscle cells of the rat small mesenteric artery

W.-S. Vanessa Ho, Paul A. Smith
School of Biomedical Sciences, University of Nottingham, Nottingham, United Kingdom

In rat aorta smooth muscle cells, the endocannabinoid N-arachidonoylethanolamide (anandamide) inhibits K+ channels by cannabinoid CB1 receptor-independent mechanisms (Van den Bossche I & Vanheel B, 2000). Whether similar effects are observed in resistance arteries or induced by the other major endocannabinoid, 2-arachidonoylglycerol (2-AG) is unknown. Here, we have studied the effects of anandamide and 2-AG on K+ currents in myocytes isolated from the rat small mesenteric artery. On the day of experiment, male Wistar rats (200-300g) were killed by cervical dislocation and smooth muscle cells isolated from secondary and tertiary branches of the superior mesenteric artery using proteolytic digestion. Recordings were made, at 24-26°C, using the perforated-patch whole cell, voltage-clamp technique. The external solution contained (mM): NaCl 138, KCl 5.8, MgCl 2 1.2, CaCl2 2.5, glucose 12, HEPES 10, adjusted to pH 7.4 with NaOH. The pipette solution contained (mM): K2SO4 75, KCl 55, MgSO4 5, HEPES 10, EGTA 0.5; adjusted to pH 7.4 with KOH, and 100 μgml-1 amphotericin B. Currents were evoked from a holding potential of -60mV by test steps (VT) between -80 to +70mV, 500ms in duration. Pilot experiments showed that the K+ ion is the main charge carrier of the whole cell outward current (IK) elicited under the present conditions. IK was obtained from the same cell before, and after a 2-3 min perfusion with test compound. Data are expressed as mean±s.e.m (n≥3 cells from at least 3 rats) using IK elicited by VT’s between -10mV and +70mV and, unless otherwise stated, the whole data set from current-voltage plots were analysed by two-way analysis of variance (P<0.05 was considered statistically significant).

Application of 10μM 2-AG attenuated IK by 27 to 41% (P<0.05) in a voltage-independent manner. To probe the duration of 2-AG induced inhibition, IK was also measured at 6-9 min after the initial exposure to 2-AG. The resultant mean current amplitude was significantly larger than that obtained immediately after 2-AG treatment (P<0.01) and similar to that of control, thus the inhibitory effect of 2-AG was lost over time. On the other hand, anandamide (10μM) significantly reduced IK (P<0.01) but this occurred in a voltage-dependent manner (P<0.01, repeated measure one-way analysis of variance; e.g. at -10mV: 65±17%; at +70mV: 15±15%) and did not change with time. Interestingly, 10μM arachidonic acid, the hydrolysis product of both 2-AG and anandamide, significantly enhanced IK (P<0.05). The potentiation tended to increase with VT and, at +50mV, the mean current amplitude was increased by 153±87%. The vehicle (0.1% ethanol) had no effect on IK. To conclude, 2-AG and anandamide inhibit, whereas arachidonic acid potentiates, whole cell K+ currents in smooth muscle cells of the rat small mesenteric artery. Given that 2-AG is more labile than anandamide in isolated mesenteric arteries (Ho & Randall, 2007), we hypothesize that rapid degradation to arachidonic acid limits the inhibitory effect of 2-AG and contribute to the different characteristics of IK inhibition induced by anandamide and 2-AG.

Van den Bossche I & Vanheel B (2000). Br J Pharmacol 131: 85-93
Ho WS & Randall MD (2007). Br J Pharmacol 150: 641-651



This study was supported by the British Heart Foundation and Anne McLaren Fellowship