A comparison of EDHF‐mediated and anandamide‐induced relaxations in the rat isolated mesenteric artery

1 Relaxation of the methoxamine‐precontracted rat small mesenteric artery by endothelium‐derived hyperpolarizing factor (EDHF) was compared with relaxation to the cannabinoid, anandamide (arachidonylethanolamide). EDHF was produced in a concentration‐ and endothelium‐dependent fashion in the presence of N G ‐nitro‐ l ‐arginine methyl ester ( l ‐NAME, 100 μ m ) by either carbachol (pEC 50 [negative logarithm of the EC 50 ]=6.19±0.01, R max [maximum response]=93.2±0.4%; n =14) or calcium ionophore A23187 (pEC 50 =6.46±0.02, R max =83.6±3.6%; n =8). Anandamide responses were independent of the presence of endothelium or l ‐NAME (control with endothelium: pEC 50 =6.31±0.06, R max =94.7±4.6%; n =10; with l ‐NAME: pEC 50 =6.33±0.04, R max =93.4±6.0%; n =4). 2 The selective cannabinoid receptor antagonist, SR 141716A (1 μ m ) caused rightward shifts of the concentration‐response curves to both carbachol (2.5 fold) and A23187 (3.3 fold). It also antagonized anandamide relaxations in the presence or absence of endothelium giving a 2 fold shift in each case. SR 141716A (10 μ m ) greatly reduced the R max values for EDHF‐mediated relaxations to carbachol (control, 93.2±0.4%; SR 141716A, 10.7±2.5%; n =5; P <0.001) and A23187 (control, 84.8±2.1%; SR 141716A, 3.5±2.3%; n =6; P <0.001) but caused a 10 fold parallel shift in the concentration‐relaxation curve for anandamide without affecting R max . 3 Precontraction with 60 m m KCl significantly reduced ( P <0.01; n =4 for all) relaxations to 1 μ m carbachol (control 68.8±5.6% versus 17.8±7.1%), A23187 (control 71.4±6.1% versus 3.9±0.45%) and anandamide (control 71.1±7.0% versus 5.2±3.6%). Similar effects were seen in the presence of 25 m m K + . Incubation of vessels with pertussis toxin (PTX; 400 ng ml −1 , 2 h) also reduced ( P <0.01; n =4 for all) relaxations to 1 μ m carbachol (control 63.5±7.5% versus 9.0±3.2%), A23187 (control 77.0±5.8% versus 16.2±7.1%) and anandamide (control 89.8±2.2% versus 17.6±8.7%). 4 Incubation of vessels with the protease inhibitor phenylmethylsulphonyl fluoride (PMSF; 200 μ m ) significantly potentiated ( P <0.01), to a similar extent (∼2 fold), relaxation to A23187 (pEC 50 : control, 6.45±0.04; PMSF, 6.74±0.10; n =4) and anandamide (pEC 50 : control, 6.31±0.02; PMSF, 6.61±0.08; n =8). PMSF also potentiated carbachol responses both in the presence (pEC 50 : control, 6.25±0.01; PMSF, 7.00±0.01; n =4; P <0.01) and absence (pEC 50 : control, 6.41±0.04; PMSF, 6.88±0.04; n =4; P <0.001) of l ‐NAME. Responses to the nitric oxide donor S‐nitroso‐N‐acetylpenicillamine (SNAP) were also potentiated by PMSF (pEC 50 : control, 7.51±0.06; PMSF, 8.00±0.05, n =4, P <0.001). 5 EDHF‐mediated relaxation to carbachol was significantly attenuated by the K + channel blocker tetraethylammonium (TEA; 1 m m ) (pEC 50 : control, 6.19±0.01; TEA, 5.61±0.01; n =6; P <0.01). In contrast, TEA (1 m m ) had no effect on EDHF‐mediated relaxation to A23187 (pEC 50 : control, 6.47±0.04; TEA, 6.41±0.02, n =4) or on anandamide (pEC 50 : control, 6.28±0.06; TEA, 6.09±0.02; n =5). TEA (10 m m ) significantly ( P <0.01) reduced the R max for anandamide (control, 94.3±4.0%; 10 m m TEA, 60.7±4.4%; n =5) but had no effect on the R max to carbachol or A23187. 6 BaCl 2 (100 μ m ), considered to be selective for blockade of inward rectifier K + channels, had no significant effect on relaxations to carbachol or A23187, but caused a small shift in the anandamide concentration‐response curve (pEC 50 : control, 6.39±0.01; Ba 2+ , 6.20±0.01; n =4; P <0.01). BaCl 2 (1 m m ; which causes non‐selective block of K + channels) significantly ( P <0.01) attenuated relaxations to all three agents (pEC 50 values: carbachol, 5.65±0.02; A23187, 5.84±0.04; anandamide, 5.95±0.02; n =4 for each). 7 Apamin (1 μ m ), a selective blocker of small conductance, Ca 2+ ‐activated, K + channels (SK Ca ), 4‐aminopyridine (1 m m ), a blocker of delayed rectifier, voltage‐dependent, K + channels (K v ), and ciclazindol (10 μ m ), an inhibitor of K v and adenosine 5′‐triphosphate (ATP)‐sensitive K + channels (K ATP ), significantly reduced EDHF‐mediated relaxations to carbachol, but had no significant effects on A23187 or anandamide responses. 8 Glibenclamide (10 μ m ), a K ATP inhibitor and charybdotoxin (100 or 300 n m ), a blocker of several K + channel subtypes, had no significant effect on relaxations to any of the agents. Iberiotoxin (50 n m ), an inhibitor of large conductance, Ca 2+ ‐activated, K + channels (BK Ca ), had no significant effect on the relaxation responses, either alone or in combination with apamin (1 μ m ). Also, a combination of apamin (1 μ m ) with either glibenclamide (10 μ m ) or 4‐aminopyridine (1 m m ) did not inhibit relaxation to carbachol significantly more than apamin alone. Neither combination had any significant effect on relaxation to A23187 or anandamide. 9 A combination of apamin (1 μ m ) with charybdotoxin (100 n m ) abolished EDHF‐mediated relaxation to carbachol, but had no significant effect on that to A23187. Apamin (1 μ m ) and charybdotoxin (300 n m ) together consistently inhibited the response to A23187, while apamin (1 μ m ) and ciclazindol (10 μ m ) together inhibited relaxations to both carbachol and A23187. None of these toxin combinations had any significant effect on relaxation to anandamide. 10 It was concluded that the differential sensitivity to K + channel blockers of EDHF‐mediated responses to carbachol and A23187 might be due to actions on endothelial generation of EDHF, as well as its actions on the vascular smooth muscle, and suggests care must be taken in choosing the means of generating EDHF when making comparative studies. Also, the relaxations to EDHF and anandamide may involve activation of cannabinoid receptors, coupled via PTX‐sensitive G‐proteins to activation of K + conductances. The results support the hypothesis that EDHF is an endocannabinoid but relaxations to EDHF and anandamide show differential sensitivity to K + channel blockers, therefore it is likely that anandamide is not identical to EDHF in the small rat mesenteric artery.