Virodhamine and CP55,940 modulate cAMP production and IL-8 release in human bronchial epithelial cells

Abstract

Background and purpose: We investigated expression of cannabinoid receptors and the effects of the endogenous cannabinoid virodhamine and the synthetic agonist CP55,940 on cAMP accumulation and interleukin-8 (IL-8) release in human bronchial epithelial cells.

Experimental approach: Human bronchial epithelial (16HBE14o(-)) cells were used. Total mRNA was isolated and cannabinoid receptor mRNAs were detected by RT-PCR. Expression of CB(1) and CB(2) receptor proteins was detected with Western blotting using receptor-specific antibodies. cAMP accumulation was measured by competitive radioligand binding assay. IL-8 release was measured by ELISA.

Key results: CB(1) and CB(2) receptor mRNAs and proteins were found. Both agonists concentration-dependently decreased forskolin-induced cAMP accumulation. This effect was inhibited by the CB(2) receptor antagonist SR144528, and was sensitive to Pertussis toxin (PTX), suggesting the involvement of CB(2) receptors and G(i/o)-proteins. Cell pretreatment with PTX unmasked a stimulatory component, which was blocked by the CB(1) receptor antagonist SR141716A. CB(2) receptor-mediated inhibition of cAMP production by virodhamine and CP55,940 was paralleled by inhibition of tumor necrosis factor-alpha (TNF-alpha) induced IL-8 release. This inhibition was insensitive to SR141716A. In the absence of agonist, SR144528 by itself reduced TNF-alpha induced IL-8 release.

Conclusions and implications: Our results show for the first time that 16HBE14o(-) cells respond to virodhamine and CP55,940. CB(1) and CB(2) receptor subtypes mediated activation and inhibition of adenylyl cyclase, respectively. Stimulation of the dominant CB(2) receptor signalling pathway diminished cAMP accumulation and TNF-alpha-induced IL-8 release. These observations may imply that cannabinoids exert anti-inflammatory properties in airways by modulating cytokine release.

Figure 1

mRNA and protein expression of CB₁ and CB₂ receptors in 16HBE14o⁻ cells. (a) Total RNA was reverse-transcribed into cDNA and specific fragments amplified by PCR, separated on 2% agarose gel and visualized with ethidium bromide. Restriction analysis was performed with BstEII. (b) Western blot analysis of CB₁ and CB₂ receptor expression (20 μg protein per lane) using receptor-specific antibodies.

Figure 2

Concentration–effect curves of virodhamine (a) and CP55,940 (b) on forskolin-induced cAMP accumulation. Data are presented as mean values±s.e.m. from six experiments. ^*P<0.05 vs control. (c) The lack of effect of the CB receptor antagonists SR141716A (1 μM) and SR144528 (1 μM) on basal cAMP accumulation and that induced by forskolin (0.1 μM). Values of pIC₅₀/pEC₅₀ and ΔE_max are summarized in Table 1.

Figure 3

Effect of CB receptor antagonists on virodhamine and CP55,940-induced inhibition of forskolin induced cAMP accumulation. (a) Virodhamine-induced inhibition in the presence of SR141716A (1 μM) or SR144528 (1 μM). (b) CP55,940-induced inhibition in the presence of SR141716A (1 μM) or SR144528 (1 μM). Virodhamine- and CP55,940-induced inhibition in the absence of SR141716A or SR144528, see Figure 2. Data are presented as mean values±s.e.m. from six experiments. ^*P<0.05 vs control. Values for pIC₅₀/pEC₅₀ and ΔE_max are summarized in Table 1.

Figure 4

Effect of PTX pretreatment on virodhamine and CP55,940-induced modulation of forskolin induced cAMP accumulation. Cells were pretreated with PTX (100 ng ml⁻¹; 20 h). Effect on concentration–response curve of virodhamine alone (a), in the presence of SR141716A (1 μM; b) or in the presence of SR144528 (1 μM; d). Effect on concentration–response curve of CP55,940 alone (c). Data are presented as mean values±s.e.m. from six (a–c) or three experiments (d). ^*P<0.05 vs control. Values of pIC₅₀/pEC₅₀ and ΔE_max are summarized in Table 1.

Figure 5

Effect of CB receptor stimulation on IL-8 release induced by TNF-α. Accumulation of IL-8 was measured 24 h after stimulation with 100 ng ml⁻¹ TNF-α in the presence of virodhamine (a) or CP55,940 (b). Effect of virodhamine in the presence of SR141716A (1 μM, c). Lack of effect of SR141716A (1 μM) on inhibition induced by virodhamine (10 μM) or CP55,940 (10 μM) (d). Data are presented as mean values±s.e.m. from three to five experiments. ^#P<0.10 and ^*P<0.05 vs control, respectively. NS, not significantly different. Values for pIC₅₀ and ΔE_max are summarized in Table 2.

Figure 6

Effect of selective CB receptor antagonists on IL-8 release. Basal and 100 ng ml⁻¹ TNF-α-induced accumulation of IL-8 was measured after 24 h in the absence or presence of SR141716A (1 μM) or SR144528 (1 μM). Data are presented as mean values±s.e.m. from 13 experiments. ^*P<0.05 vs control.

Figure 7

Hypothetical scheme of virodhamine- and CP55,940-induced cannabinoid signalling in 16HBE14o⁻ cells. Stimulation of CB receptors with the non-selective endocannabinoid virodhamine or the non-selective synthetic agonist CP55,940 predominantly inhibits cAMP formation via CB₂ receptor coupling to PTX-sensitive G_i/o-proteins. Stimulation of cAMP formation is mediated by CB₁ receptors and only observed after removing the G_i/o response by PTX pretreatment. The CB₂ receptor-induced decrease in cAMP formation might be responsible for the observed inhibition of TNF-α-induced IL-8 release.