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 NG-nitro-L-arginine methyl ester (L-NAME, 100 microM) by either carbachol (pEC50 [negative logarithm of the EC50] = 6.19 +/- 0.01, Rmax [maximum response] = 93.2 +/- 0.4%; n = 14) or calcium ionophore A23187 (pEC50 = 6.46 +/- 0.02, Rmax = 83.6 +/- 3.6%; n = 8). Anandamide responses were independent of the presence of endothelium or L-NAME (control with endothelium: pEC50 = 6.31 +/- 0.06, Rmax = 94.7 +/- 4.6%; n = 10; with L-NAME: pEC50 = 6.33 +/- 0.04, Rmax = 93.4 +/- 6.0%; n = 4). 2. The selective cannabinoid receptor antagonist, SR 141716A (1 microM) 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 microM) greatly reduced the Rmax 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 Rmax. 3. Precontraction with 60 mM KCl significantly reduced (P < 0.01; n = 4 for all) relaxations to 1 microM 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 mM 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 microM 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 microM) significantly potentiated (P < 0.01), to a similar extent (approximately 2 fold), relaxation to A23187 (pEC50: control, 6.45 +/- 0.04; PMSF, 6.74 +/- 0.10; n = 4) and anandamide (pEC50: control, 6.31 +/- 0.02; PMSF, 6.61 +/- 0.08; n = 8). PMSF also potentiated carbachol responses both in the presence (pEC50: control, 6.25 +/- 0.01; PMSF, 7.00 +/- 0.01; n = 4; P < 0.01) and absence (pEC50: 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 (pEC50: 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 mM) (pEC50: control, 6.19 +/- 0.01; TEA, 5.61 +/- 0.01; n = 6; P < 0.01). In contrast, TEA (1 mM) had no effect on EDHF-mediated relaxation to A23187 (pEC50: control, 6.47 +/- 0.04; TEA, 6.41 +/- 0.02, n = 4) or on anandamide (pEC50: control, 6.28 +/- 0.06; TEA, 6.09 +/- 0.02; n = 5). TEA (10 mM) significantly (P < 0.01) reduced the Rmax for anandamide (control, 94.3 +/- 4.0%; 10 mM TEA, 60.7 +/- 4.4%; n = 5) but had no effect on the Rmax to carbachol or A23187. 6. BaCl2 (100 microM), 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 (pEC50: control, 6.39 +/- 0.01; Ba2+, 6.20 +/- 0.01; n = 4; P < 0.01). BaCl2 (1 mM; which causes non-selective block of K+ channels) significantly (P < 0.01) attenuated relaxations to all three agents (pEC50 values: carbachol, 5.65 +/- 0.02; A23187, 5.84 +/- 0.04; anandamide, 5.95 +/- 0.02; n = 4 for each). 7. Apamin (1mu M), a selective blocker of small conductance, Ca2+-activated, K+ channels (SKCa), 4-aminopyridine (1mM), a blocker of delayed rectifier, voltage-dependent, K+ channels (Kv), and ciclazindol (10mu M), an inhibitor of Kv and adenosine 5'-triphosphate (ATP)-sensitive K+ channels (KATP), significantly reduced EDHF-mediated relaxations to carbachol, but had no significant effects on A23187 or anandamide responses. 8. Glibenclamide (10mu M), a KATP inhibitor and charybdotoxin (100 or 300nM), a blocker of several K+ channel subtypes, had no significant effect on relaxations to any of the agents. Iberiotoxin (50nM), an inhibitor of large conductance, Ca2+-activated, K+ channels (BKCa), had no significant effect on the relaxation responses, either alone or in combination with apamin (1muM). Also, a combination of apamin (1muM) with either glibenclamide (10muM) or 4-aminopyridine (1mM) 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 (1muM) with charybdotoxin (100nM) abolished EDHF-mediated relaxation to carbachol, but had no significant effect on that to A23187. Apamin (1muM) and charybdotoxin (300nM) together consistently inhibited the response to A23187, while apamin (1muM) and ciclazindol (10muM) 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.
摘要
比较了内皮源性超极化因子（EDHF）对甲氧明预收缩的大鼠小肠系膜动脉的舒张作用与大麻素花生四烯乙醇胺（anandamide）的舒张作用。在存在NG-硝基-L-精氨酸甲酯（L-NAME，100μM）的情况下，通过卡巴胆碱（pEC50[EC50的负对数]=6.19±0.01，Rmax[最大反应]=93.2±0.4%；n=14）或钙离子载体A23187（pEC50=6.46±0.02，Rmax=83.6±3.6%；n=8）以浓度和内皮依赖性方式产生EDHF。花生四烯乙醇胺的反应与内皮或L-NAME的存在无关（有内皮的对照：pEC50=6.31±0.06，Rmax=94.7±4.6%；n=10；有L-NAME时：pEC50=6.33±0.04，Rmax=93.4±6.0%；n=4）。2. 选择性大麻素受体拮抗剂SR 141716A（1μM）使卡巴胆碱（2.5倍）和A23187（3.3倍）的浓度-反应曲线向右移动。在有或无内皮的情况下，它也拮抗花生四烯乙醇胺的舒张作用，每种情况下均产生2倍的移动。SR 141716A（10μM）极大地降低了EDHF介导的对卡巴胆碱舒张的Rmax值（对照，93.2±0.4%；SR 141716A，10.7±2.5%；n=5；P<0.001）和对A23187舒张的Rmax值（对照，84.8±2.1%；SR 141716A，3.5±2.3%；n=6；P<0.001），但使花生四烯乙醇胺的浓度-舒张曲线平行移动10倍而不影响Rmax。3. 用60 mM KCl预收缩显著降低（P<0.01；所有n=4）对1μM卡巴胆碱的舒张作用（对照68.8±5.6%对17.8±7.1%）、对A23187的舒张作用（对照71.4±6.1%对3.9±0.45%）和对花生四烯乙醇胺的舒张作用（对照71.1±7.0%对5.2±3.6%）。在25 mM K+存在时也观察到类似效应。用百日咳毒素（PTX；400 ng/ml，2小时）孵育血管也降低（P<0.01；所有n=4）对1μM卡巴胆碱的舒张作用（对照63.5±7.5%对9.0±3.2%）、对A23187的舒张作用（对照77.0±5.8%对16.2±7.1%）和对花生四烯乙醇胺的舒张作用（对照89.8±2.2%对17.6±8.7%）。4. 用蛋白酶抑制剂苯甲基磺酰氟（PMSF；200μM）孵育血管显著增强（P<0.01）对A23187（pEC50：对照，6.45±0.04；PMSF，6.74±0.10；n=4）和花生四烯乙醇胺（pEC50：对照，
