Hemopressin is an inverse agonist of CB1 cannabinoid receptors

Abstract

To date, the endogenous ligands described for cannabinoid receptors have been derived from membrane lipids. To identify a peptide ligand for CB(1) cannabinoid receptors, we used the recently described conformation-state sensitive antibodies and screened a panel of endogenous peptides from rodent brain or adipose tissue. This led to the identification of hemopressin (PVNFKFLSH) as a peptide ligand that selectively binds CB(1) cannabinoid receptors. We find that hemopressin is a CB(1) receptor-selective antagonist, because it is able to efficiently block signaling by CB(1) receptors but not by other members of family A G protein-coupled receptors (including the closely related CB(2) receptors). Hemopressin also behaves as an inverse agonist of CB(1) receptors, because it is able to block the constitutive activity of these receptors to the same extent as its well characterized antagonist, rimonabant. Finally, we examine the activity of hemopressin in vivo using different models of pain and find that it exhibits antinociceptive effects when administered by either intrathecal, intraplantar, or oral routes, underscoring hemopressin's therapeutic potential. These results represent a demonstration of a peptide ligand for CB(1) cannabinoid receptors that also exhibits analgesic properties. These findings are likely to have a profound impact on the development of novel therapeutics targeting CB(1) receptors.

Fig. 1.

Identification of hemopressin as a CB₁ receptor-modulating peptide. SKNSH cells (≈1 × 10⁵ cells per well) were treated with (A) 1 μM various peptides (B) 1 μM Hu-210 (Hu) without or with hemopressin (HP), SR141716 (SR), a scrambled peptide (SP), or (C) C-terminally truncated hemopressin peptides and probed with anti-CB₁ receptor antibody by ELISA, as described in Methods. (D) HEK-293 cells (30,000 cells per well) coexpressing pCRE-SeAP and individual receptors were treated with 10 μM forskolin (except for β₂ adrenergic receptors) and its corresponding agonists (100 nM) in the absence or presence of 1 μM hemopressin and SeAP levels determined as described in Methods. Results are the mean ± SEM (n = 6). Statistically significant differences vs. control (*) and vs. agonist alone (+) are indicated; **, P < 0.01; ++, P < 0.01; one-way ANOVA and Dunnett's test.

Fig. 2.

Hemopressin is an antagonist of the CB₁ cannabinoid receptor. (A) Striatal membranes (10 μg) were incubated with 3 nM [³H]SR141716 in the absence or presence of increasing concentrations (0–1 μM) of hemopressin (HP), SR141716 (SR), or scrambled peptide (SP) and the ligand-binding analysis carried out as described (36). (B and C) Striatal membranes (10 μg) were subjected to a GTPγS-binding assay (B) or an adenylyl cyclase assay (C) using increasing concentrations (0–1 μM) of Hu-210 (Hu), HP, or SR or increasing concentrations of Hu in the absence or presence of 10 μM HP or SR, as described (36, 37). (D) HEK-293 cells expressing Flag-tagged CB₁ receptors were treated for 5 min with 100 nM Hu-210 in the absence of presence of 10 μM SR or HP and levels of phosphorylated MAP kinase determined as described (36). Values obtained in the absence of drug treatment were taken as 100%. Results are mean ± SE of triplicate experiments. *, P < 0.05; **, P < 0.01; one-way ANOVA and Dunnett's test.

Fig. 3.

Hemopressin functions as an inverse agonist of CB₁ but not CB₂ receptors. Membranes (10 μg) from striatum (CB₁ receptors) or spleen (CB₂ receptors) were subjected to a GTPγS-binding assay (A) or an adenylyl cyclase assay (B) using 1 μM of Hu-210 (Hu), hemopressin (HP), SR141716 (SR), Hu + SR or Hu + HP, as described (36, 37). Results are mean ± SE of triplicate experiments. *, P < 0.05; **, P < 0.01; one-way ANOVA and Dunnett's test. (C) Neuro 2A cells (30,000 cells per well) coexpressing pCRE-SeAP and CB₁ or CB₂ cannabinoid receptors were treated with 10 μM forskolin and 100 nM Hu-210 (cannabinoid receptor agonist) in the absence or presence of 1 μM hemopressin and probed for SeAP levels, as described in Methods. Results are the mean ± SEM of sextuplicate determinations (n = 2). Statistically significant differences from control (*) and from agonist alone (+) are indicated, *, P < 0.05; **, P < 0.01; ++, P < 0.01; one-way ANOVA and Dunnett's test. (D) Neuro 2A cells expressing CB₁ or CB₂ receptors were treated for 16 h with 100 nM Hu, 10 μM SR or HP, 100 nM Hu + 10 μM SR, or 100 nM Hu + 10 μM HP, and percentage of cells extending neurites was determined as described (19). Values obtained in the absence of drug treatment were taken as 100%. Results are mean ± SE of triplicate experiments. *, P < 0.05; **, P < 0.01; one-way ANOVA and Dunnett's test.

Fig. 4.

In vivo hemopressin antihyperalgesic activity. (A) Comparative analyses of intraplantar (i.pl.) administration of hemopressin and AM251 on the hyperalgesia induced by carrageenan. Rats were treated i.pl. with hemopressin (HP, 10 μg per paw) or AM251 (AM, 10 μg per paw) immediately before the i.pl. injection of carrageenan (Cg, 200 μg per paw) and were evaluated before (0 h, empty bars) and 3 h after Cg injection (black bars). Rats administered i.pl. with vehicle (saline) were subjected to the same protocol (control group). Results are presented as mean ± SEM, n = 6–8 (Ψ, P < 0.001 vs. initial measurement; *, P < 0.05 vs. control group; and ***, P < 0.001 vs. control group, ANOVA with Bonferroni post hoc test). (B) Effect of intrathecal (i.t.) administration of hemopressin. Rats received HP i.t. (0.5 or 5 μg/kg) immediately before i.pl. injection of Cg (200 μg per paw) and were evaluated before (0 h, empty bars) and 3 h after treatment (black bars). Rats administered i.t. with saline were submitted to the same protocol (control group). Results are presented as mean ± SEM, n = 6–8 [Ψ, P < 0.001 vs. initial measurement, *, P < 0.05 vs. control group (3 h) and ***, P < 0.001 vs. control group (3 h); ANOVA with Bonferroni post hoc test]. (C) Effect of oral administration of hemopressin on carrageenan-induced hyperalgesia. Rats were administered with saline (control group) or hemopressin (HP, 50 or 100 μg/kg) per os (p.o.) immediately before the i.pl. injection of carrageenan (Cg, 200 μg per paw), and the nociceptive threshold measured by using an Ugo Basile pressure apparatus was evaluated before (0 h, empty bars) and 3 h after Cg injection (black bars), as described in detail in Methods. Results are presented as mean ± SEM, n = 6–8 [Ψ, P < 0.001 vs. initial measurement; *, P < 0.05 vs. control group (3 h) and ***, P < 0.001 vs. control group (3 h), ANOVA with Bonferroni post hoc test]. (D) Effect of i.p. administration of hemopressin on writhing test. Abdominal contortions resulting from i.p. injection of 0.6% (vol/vol) acetic acid, at a dosage of 60 mg/kg body weight, are contractions of the abdominal muscles with a stretching of hind limbs. The number of abdominal contortions was counted cumulatively over a period of 20 min after acetic acid injection. Hemopressin (HP, 50 or 500 μg/kg) was injected i.p. 1 h before the acetic acid administration. Antinociceptive activity was expressed as the reduction in the number of abdominal contortions between hemopressin- and vehicle-treated animals. Results are presented as mean ± SEM, n = 6–8 (***, P < 0.001 vs. control group; ANOVA with Bonferroni post hoc test).