CB(1) receptor antagonist SR141716A inhibits Ca(2+)-induced relaxation in CB(1) receptor-deficient mice

Abstract

Mesenteric branch arteries isolated from cannabinoid type 1 receptor knockout (CB(1)(-/-)) mice, their wild-type littermates (CB(1)(+/+) mice), and C57BL/J wild-type mice were studied to test the hypothesis that murine arteries undergo high sensitivity Ca(2+)-induced relaxation that is CB(1) receptor dependent. Confocal microscope analysis of mesenteric branch arteries from wild-type mice showed the presence of Ca(2+) receptor-positive periadventitial nerves. Arterial segments of C57 control mice mounted on wire myographs contracted in response to 5 micromol/L norepinephrine and responded to the cumulative addition of extracellular Ca(2+) with a concentration-dependent relaxation that reached a maximum of 72.0 +/- 6.3% of the prerelaxation tone and had an EC(50) for Ca(2+) of 2.90 +/- 0.54 mmol/L. The relaxation was antagonized by precontraction in buffer containing 100 mmol/L K(+) and by pretreatment with 10 mmol/L tetraethylammonium. Arteries from CB(1)(-/-) and CB(1)(+/+) mice also relaxed in response to extracellular Ca(2+) with no differences being detected between the knockout and their littermate controls. SR141716A, a selective CB(1) antagonist, caused concentration-dependent inhibition of Ca(2+)-induced relaxation in both the knockout and wild-type strains (60% inhibition at 1 micromol/L). O-1918, a cannabidiol analog, had a similar blocking effect in arteries of both wild-type and CB(1)(-/-) mice at 10 micromol/L. In contrast, 1 micromol/L SR144538, a cannabinoid type 2 receptor antagonist, or 50 micromol/L 18alpha-glycyrrhetinic acid, a gap junction blocker, were without effect. SR141716A (1 to 30 micromol/L) was also assessed for nonspecific actions on whole-cell K(+) currents in isolated vascular smooth muscle cells. SR141716A inhibited macroscopic K(+) currents at concentrations higher than those required to inhibit Ca(2+)-induced relaxation, and appeared to have little effect on currents through large conductance Ca(2+)-activated K(+) channels. These data indicate that arteries of the mouse relax in response to cumulative addition of extracellular Ca(2+) in a hyperpolarization-dependent manner and rule out a role for CB(1) or CB(2) receptors in this effect. The possible role of a nonclassical cannabinoid receptor is discussed.

Figure 1

3D confocal images of mesenteric branch arteries of wild-type C57BL/6 mice. Left, Ca²⁺ receptor–positive nerves in the adventitial layer and nuclei of adjacent cells. Middle, Reduction in nerve staining after preadsorption of primary antibody with excess antigen. Right, Absence of staining when primary antibody is omitted from the assay. Note that middle and right panels contain oblong nuclei from smooth muscle cells in the tunica media. Photos were taken with a 63× water immersion objective.

Figure 2

Response of mesenteric branch arteries of wild-type C57BL/6 mice to the cumulative addition of extracellular Ca²⁺ after precontraction with 5 μmol/L NE or 100 mmol/L K⁺ (A) or after precontraction with 5 μmol/L NE and pretreatment with 10 mmol/L TEA or a mixture of 10 mmol/L TEA and 0.3 mmol/L N^G-nitro-l-arginine methyl ester (L-NAME, B). Values are mean±SEM, *P<0.05 vs NE control, n=3 to 5 per group.

Figure 3

A, Response of mesenteric branch arteries isolated from CB₁^+/+ and CB₁^−/− mice to the cumulative addition of extracellular Ca²⁺ after precontraction with 5 μmol/L NE. Values are mean± SEM, n=6; no significant differences were detected. Effect of 0.3 and 1.0 μmol/L SR141716A on Ca²⁺-induced relaxation of mesenteric branch arteries isolated from CB₁^−/− mice (B) and wild-type control mice (C). Values are mean± SEM, n=4 to 6. *P<0.05 between points.

Figure 4

Effect of pretreatment with 3 μmol/L SR144538 on Ca²⁺-induced relaxation of arteries isolated from CB₁^−/− mice (A) or effect of preincubation of the artery with 50 μmol/L 18α-glycyrrhetinic acid on arteries isolated from wild-type C57BL/6 mice (B). Values are mean±SEM, n=3 to 4. No effect of either antagonist was detected.

Figure 5

A, Effect of SR141716A on whole-cell K⁺ currents in isolated vascular smooth muscle cells under control conditions or in the presence of 3 and 10 μmol/L SR141716A. Values are mean current densities±SEM, n=5 to 6. Both concentrations significantly inhibited currents (P<0.05). B, Concentration response data for the inhibitory effect of SR141716A on whole-cell K⁺ currents. Values are mean inhibition±SEM, n=3 to 13, measured by stepping to 60 mV from a holding potential of −60 mV. SR141716A inhibited the currents in a concentration-dependent fashion between 1 and 10 μmol/L.

Figure 6

Inhibition of whole-cell K⁺ currents by SR141716A is additive with TEA. A, Values are mean current density±SEM (n=5) in the absence (control) or presence of either 1 mmol/L TEA or 1 mmol/L TEA and 100 nmol/L iberiotoxin. Note that iberiotoxin produced no additional inhibition to that produced by TEA, indicating that TEA was maximally blocking currents through large conductance K_Ca channels. B, Values are mean current densities±SEM (n=6) in the absence (control) or presence of either 10 μmol/L SR141716A or 10 μmol/L SR141716A and 1 mmol/L TEA. TEA caused additional inhibition of currents above that produced by SR141716A. C, Values are mean current densities±SEM (n=5) in the absence (control) or presence of either 1 mmol/L TEA or 1 mmol/L TEA and 10 μmol/L SR141716A. SR141716A produced additional inhibition in addition to that produced by TEA, indicating that SR141716A is inhibiting currents through channels other than large-conductance K_Ca channels.

Figure 7

Effect of pretreatment with 10 μmol/L of the cannabidiol analog O-1918 on Ca²⁺-induced relaxation of arteries isolated from C57BL/6 mice (A) and CB₁^−/− mice (B). Values are mean±SEM, n=4 or 5 per group, *P <0.05. C, Acetylcholine-induced relaxation in arteries of C57BL/6 mice. Values are mean±SEM, n=3 per group. No differences were detected.