Vasodilator actions of abnormal-cannabidiol in rat isolated small mesenteric artery

Abstract

1. The nonpsychoactive cannabinoid abnormal-cannabidiol (trans-4-[3-methyl-6-(1-methylethenyl)-2-cyclohexen-1-yl]-5-pentyl-1,3-benzenediol) (abn-cbd) produced concentration-dependent relaxation of methoxamine-precontracted rat small mesenteric artery. Endothelial removal reduced abn-cbd potency six-fold without affecting the maximum relaxation. 2. In endothelium-intact vessels, abn-cbd was less potent under 60 mM KCl-induced tone and inhibited by combination of L-N(G)-nitroarginine methyl ester (L-NAME) (nitric oxide synthase inhibitor; 300 micro M), apamin (small conductance Ca(2+)-activated K(+) channels inhibitor; 50 nM) and charybdotoxin (inhibitor of intermediate conductance Ca(2+)-activated K(+) channels and large conductance Ca(2+)-activated K(+) channels BK(Ca); 50 nM). L-NAME alone or in combination with either toxin alone had little effect. 3. In intact vessels, relaxations to abn-cbd were inhibited by SR 141716A (cannabinoid receptor antagonist; 1 or 3 micro M). Concomitant addition of L-NAME, apamin and charybdotoxin had no further effect. Other cannabinoid receptor antagonists either had little (SR 144528; 1 micro M and AM 251; 1 micro M) or no effect (AM 630; 10 micro M and AM 281; 1 micro M). Inhibition of gap junctions, G(i/o) protein coupling and protein kinase A also had no effect. 4. Endothelium-independent relaxation to abn-cbd was unaffected by L-NAME, apamin plus charybdotoxin or capsaicin (10 micro M). Abn-cbd inhibited CaCl(2)-induced contractions in vessels with depleted intracellular Ca(2+) stores and stimulated with methoxamine or KCl. This was insensitive to SR 141716A (3 micro M) but greatly reduced in vessels stimulated with ionomycin (Ca(2+) ionophore; 1 micro M). 5. We conclude that abn-cbd relaxes the rat small mesenteric artery by endothelium-dependent activation of K(+) channels via SR 141716A-sensitive pathways, which do not involve CB(1) and CB(2) receptors. It also causes endothelium-independent, SR 141716A-insensitive, relaxation by inhibiting Ca(2+) entry through voltage-gated Ca(2+) channels.

Figure 1

Concentration–response curves for abnormal-cannabidiol (abn-cbd) induced relaxation of methoxamine-induced tone in rat isolated small mesenteric artery in the presence (n=4) or absence (n=4) of a functional endothelium. Also shown are the effects of the appropriate amounts of vehicle, 0.01–0.3% v v⁻¹ ethanol (n=6). Values are shown as means and vertical lines represent s.e. mean.

Figure 2

(a) Concentration–response curves for abnormal-cannabidiol (abn-cbd) relaxation of methoxamine-induced tone in endothelium-intact rat isolated small mesenteric artery. Relaxation was elicited by abn-cbd alone, and in the presence of 200 nM, 1 μM or 3 μM SR 141716A; n=4 for all. Values are shown as means and vertical lines represent s.e. mean. (b) Original traces of relaxation to abn-cbd in the absence and presence of 3 μM SR 141716A in separate endothelium-intact vessels are shown. Vertical lines denote addition of drugs at the concentration indicated. (c) Concentration–response curves for abn-cbd relaxation of methoxamine-induced tone in endothelium-denuded rat isolated mesenteric artery. Relaxation was elicited by abn-cbd alone, and in the presence of 3 μM SR 141716A n=4 for both. Values are shown as means and vertical lines represent s.e.mean.

Figure 3

Concentration–response curves for abnormal-cannabidiol (abn-cbd) relaxation of methoxamine-induced tone in endothelium-intact rat isolated small mesenteric artery. (a) Relaxation was elicited by abn-cbd alone (n=8), and in the presence of 1 μM AM 251 (n=4) or 1 μM AM 281 (n=4). (b) Relaxation was elicited by abn-cbd alone and in the presence of 1 μM SR 144528; n=6 for both. Values are shown as means and vertical lines represent s.e.mean.

Figure 4

(a) Concentration–response curves for abnormal-cannabidiol (abn-cbd) relaxation of either methoxamine- or KCl-induced tone in rat isolated small mesenteric artery. Abn-cbd relaxed methoxamine-precontracted vessels in the presence (n=7) or absence (n=7) of endothelium. Relaxation to abn-cbd was also observed in 60 mM KCl-precontracted vessels in the presence (n=7) or absence (n=4) of endothelium. Values are shown as means and vertical lines represent s.e. mean. (b) Original recording of the relaxation to abn-cbd in KCl-precontracted, endothelium-intact rat isolated mesenteric artery is shown. Vertical lines denote addition of drugs at the concentration indicated.

Figure 5

Concentration–response curves for abnormal-cannabidiol (abn-cbd) relaxation of methoxamine-induced tone in endothelium-intact rat isolated mesenteric artery. (a) Relaxation was elicited by abn-cbd alone (n=9), or in the presence of either 300 μM L-NAME alone (n=4) or in the presence of 300 μM L-NAME, 50 nM apamin and 50 nM charybdotoxin (n=6). (b) Relaxation was elicited by abn-cbd alone (n=5), or in the presence of 300 μM L-NAME in combination with 50 nM apamin (n=4) or 50 nM apamin and 50 nM iberiotoxin (n=4). Values are shown as means and vertical lines represent s.e.mean.

Figure 6

Concentration–response curves for abnormal-cannabidiol (abn-cbd) relaxation of methoxamine-induced tone in endothelium-intact rat isolated small mesenteric artery. Relaxation was elicited by abn-cbd alone or in the presence of 400 ng ml⁻¹ pertussis toxin (with 2 h preincubation); n=6 for both. Values are shown as means and vertical lines represent s.e.mean.

Figure 7

(a) Concentration–response curves for CaCl₂-induced contractions of methoxamine (10 μM)-stimulated rat isolated small mesenteric arteries depleted of intracellular Ca²⁺ stores with EGTA as described in Methods. All vessels were denuded of endothelium. Contractions to CaCl₂ were determined in the absence (n=12) or presence of 3 μM (n=4), 10 μM (n=4) or 30 μM (n=4) abnormal-cannabidiol (abn-cbd) in a given vessel. Control response curves were pooled for clarity. Values are shown as means and vertical lines represent s.e. mean. Statistical comparisons were made by paired t-test at individual concentrations. ^*P<0.05, ^**P<0.01, ^***P<0.001 indicate significant differences from control values. (b) Shows an original recording of CaCl₂-induced contractions in the absence and presence of 10 μM abn-cbd. Vertical lines denote addition of drugs at the concentration indicated. WO denotes wash out with Ca²⁺-free Krebs–Henseleit solution.

Figure 8

Abnormal-cannabidiol induced relaxation of contractions to 2.5 mM CaCl₂ in ionomycin-(1 μM; n=9) or methoxamine-(10 μM; n=4) stimulated rat isolated small mesenteric artery in the absence of extracellular Ca²⁺. All vessels were denuded of endothelium and depleted of intracellular Ca²⁺ stores with EGTA as described in Methods. Values are shown as means and vertical lines represent s.e. mean. Statistical comparisons were made by unpaired t-test at individual concentrations. ^*P<0.05, ^**P<0.01, ^***P<0.001 indicate significant differences from the corresponding values using methoxamine.