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, 7 (1), 9560

Pepcan-12 (RVD-hemopressin) Is a CB2 Receptor Positive Allosteric Modulator Constitutively Secreted by Adrenals and in Liver Upon Tissue Damage

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Pepcan-12 (RVD-hemopressin) Is a CB2 Receptor Positive Allosteric Modulator Constitutively Secreted by Adrenals and in Liver Upon Tissue Damage

Vanessa Petrucci et al. Sci Rep.

Abstract

Pepcan-12 (RVD-hemopressin; RVDPVNFKLLSH) is the major peptide of a family of endogenous peptide endocannabinoids (pepcans) shown to act as negative allosteric modulators (NAM) of cannabinoid CB1 receptors. Noradrenergic neurons have been identified to be a specific site of pepcan production. However, it remains unknown whether pepcans occur in the periphery and interact with peripheral CB2 cannabinoid receptors. Here, it is shown that pepcan-12 acts as a potent (K i value ~50 nM) hCB2 receptor positive allosteric modulator (PAM). It significantly potentiated the effects of CB2 receptor agonists, including the endocannabinoid 2-arachidonoyl glycerol (2-AG), for [35S]GTPγS binding and cAMP inhibition (5-10 fold). In mice, the putative precursor pepcan-23 (SALSDLHAHKLRVDPVNFKLLSH) was identified with pepcan-12 in brain, liver and kidney. Pepcan-12 was increased upon endotoxemia and ischemia reperfusion damage where CB2 receptors play a protective role. The adrenals are a major endocrine site of production/secretion of constitutive pepcan-12, as shown by its marked loss after adrenalectomy. However, upon I/R damage pepcan-12 was strongly increased in the liver (from ~100 pmol/g to ~500 pmol/g) independent of adrenals. The wide occurrence of this endogenous hormone-like CB2 receptor PAM, with unforeseen opposite allosteric effects on cannabinoid receptors, suggests its potential role in peripheral pathophysiological processes.

Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Figure 1
Figure 1
Pepcan binding to hCB2 receptors increases binding of orthosteric ligands and positive cooperativity. (A) Pepcan-mediated enhancement of [3H]CP55940 and [3H]WIN55212-2 binding, respectively, to stably transfected hCB2 receptor CHO-K1 cell membranes at 100 nM and 1 µM. (B) Concentration-dependent potentiation of radioligand binding by pepcan-12 (PC-12) versus mutual competition by the orthosteric ligands showing pKB values and the cooperativity factor α. (C) Isotopic dilution in which 1 μM of CP55,940 was added to the CB1 receptor-bound [3H]CP55,940 in the presence of 300 nM pepcan-12 or vehicle. Data show mean values ± SEM of at least six independent experiment. *p < 0.05 vs. vehicle (two-tailed student t-test).
Figure 2
Figure 2
Displacement of fluorescent pepcan-F4 by endogenous pepcans. (A) Specific binding of pepcan-F4 to hCB2 expressing CHO-K1 cells measured by fluorescence, showing nonspecific binding in CHO-K1 WT cells. (BF) Differential dose-dependent displacement of pepcan-F4 by pepcan-12, -14, -15, -17 and -20. Data show mean values of at least three independent experiments each performed in triplicates.
Figure 3
Figure 3
Pepcan-12 potentiates the signaling of CP55,940 and 2-AG. (A) Concentration-dependent increase of CB2 receptor agonist [35S]GTPγS binding by pepcan-12 in CHO-K1-hCB2 cell membranes. (B) Concentration-dependent potentiation of cAMP inhibition of CB2 receptor agonists by pepcan-12 in CHO-K1-hCB2 cells Data show mean values ± SEM of at least six independent experiments. *p < 0.05 vs. vehicle (two-tailed student t-test).
Figure 4
Figure 4
LC-MS/MS quantification of pepcan-12 and pepcan-23 in peripheral tissues upon LPS challenge (6 h) in Swiss albino female mice. Quantification of (A) PC-12 and (B) PC-23 in tissues of wild type Swiss albino female mice in normal, saline (empty circles) and endotoxemic, 5 mg/kg LPS E.coli 055:B5 serotype (closed circles) conditions after 2 h. Data show means and SD. Groups were compared using two-tailed t-Student’s; *p < 0.05; **p < 0.001; ***p < 0.0001; ns = not significant. < LOD, below limit of detection.
Figure 5
Figure 5
LC-MS/MS measurements of pepcan-12 and pepcan-23 in kidney and liver upon ischemia-reperfusion (I/R) damage and associated pathophysiology. (A) Time-dependent increase of PC-12 and PC-23 in kidney upon I/R damage in C57BL6 male mice. Bars show mean values ± SD., ***p < 0.001 vs. sham. < LOD, below limit of detection. (B) Time-course of renal I/R injury related blood urea nitrogen (BUN) levels, data show mean values ± SEM, n = 5, one-way ANOVA, *p < 0.05, **p < 0.01 vs. sham operated. (C) Representative micrographs of kidney samples after renal I/R injury at different time points after reperfusion (6 h, 12 h, 24 h). Arrowheads show Ly6G positive neutrophil granulocytes infiltrating the kidney following ischemic insult. Scale bar: 200 µm. (D) Time-dependent increase of PC-12 and PC-23 in liver upon I/R damage. Bars show mean ± SD. *p < 0.05, **p < 0.01 vs. sham. < LOD, below limit of detection. (E) Time-course of hepatic I/R injury related alanine aminotransferase (ALT) levels (units/liter), data show mean values ± SEM, n = 5–6, one-way ANOVA, **p < 0.01 vs. sham operated. (F) Representative micrographs of liver samples after hepatic I/R injury at different time points after reperfusion (2 h, 6 h, 24 h). Arrowheads show Ly6G positive neutrophil granulocytes infiltrating the liver following ischemic insult. Scale bar is 200 µm.
Figure 6
Figure 6
LC-MS/MS quantification of pepcan-12 in different mouse tissues in wild type and adrenalectomized mice. Levels of PC-12 quantified in wild type (WT) and adrenalectomized (ADX) female Swiss albino mice (A) and female C57/BL6 mice (B). Tissue levels of pepcan-12 quantified in an animal model of renal ischemia reperfusion (renal I/R) after (C) 6 h and (D) 24 h of reperfusion in ADX C57/BL6 male mice. Data shows means and SD. Groups were compared using two-tailed t-Student’s; *p < 0.05; **p < 0.001; ***p < 0.000. < LOQ = below limit of quantification (i.e., detectable but not quantifiable).
Figure 7
Figure 7
Illustration showing the opposite effects of pepcan-12 on CB1 and CB2 receptor G protein signaling. In presence of the endocannabinoid 2-AG, pepcan-12 acts as a negative allosteric modulator (NAM) of CB1 and as a positive allosteric modulator (PAM) of CB2. This illustration was drawn using Microsoft Power Point 2010.

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