hemopressin and 3-(2-hydroxy-4-(1-1-dimethylheptyl)phenyl)-4-(3-hydroxypropyl)cyclohexanol

The α-hemoglobin-derived dodecapeptide RVD-hemopressin (RVDPVNFKLLSH) has been proposed to be an endogenous agonist for the cannabinoid receptor type 1 (CB(1)). To study this peptide, we have raised mAbs against its C-terminal part. Using an immunoaffinity mass spectrometry approach, a whole family of N-terminally extended peptides in addition to RVD-Hpα were identified in rodent brain extracts and human and mouse plasma. We designated these peptides Pepcan-12 (RVDPVNFKLLSH) to Pepcan-23 (SALSDLHAHKLRVDPVNFKLLSH), referring to peptide length. The most abundant Pepcans found in the brain were tested for CB(1) receptor binding. In the classical radioligand displacement assay, Pepcan-12 was the most efficacious ligand but only partially displaced both [(3)H]CP55,940 and [(3)H]WIN55,212-2. The data were fitted with the allosteric ternary complex model, revealing a cooperativity factor value α < 1, thus indicating a negative allosteric modulation. Dissociation kinetic studies of [(3)H]CP55,940 in the absence and presence of Pepcan-12 confirmed these results by showing increased dissociation rate constants induced by Pepcan-12. A fluorescently labeled Pepcan-12 analog was synthesized to investigate the binding to CB(1) receptors. Competition binding studies revealed K(i) values of several Pepcans in the nanomolar range. Accordingly, using competitive ELISA, we found low nanomolar concentrations of Pepcans in human plasma and ∼100 pmol/g in mouse brain. Surprisingly, Pepcan-12 exhibited potent negative allosteric modulation of the orthosteric agonist-induced cAMP accumulation, [(35)S]GTPγS binding, and CB(1) receptor internalization. Pepcans are the first endogenous allosteric modulators identified for CB(1) receptors. Given their abundance in the brain, Pepcans could play an important physiological role in modulating endocannabinoid signaling.

Topics: Allosteric Regulation; Amino Acid Sequence; Animals; Antibodies, Monoclonal, Murine-Derived; Binding, Competitive; Brain; Cannabinoid Receptor Modulators; CHO Cells; Cricetinae; Cyclohexanols; Epitope Mapping; Female; Hemoglobins; HL-60 Cells; Humans; Male; Mice; Mice, Inbred C57BL; Mice, Inbred NZB; Molecular Sequence Data; Peptide Fragments; Protein Binding; Protein Transport; Rats; Receptor, Cannabinoid, CB1; Signal Transduction; Sus scrofa; Tandem Mass Spectrometry

Hemopressin is a short, nine amino acid peptide (H-Pro-Val-Asn-Phe-Lys-Leu-Leu-Ser-His-OH) isolated from rat brain that behaves as an inverse agonist at the cannabinoid receptor CB(1), and is shown here to inhibit agonist-induced receptor internalization in a heterologous cell model. Since this peptide occurs naturally in the rodent brain, we determined its effect on appetite, an established central target of cannabinoid signaling. Hemopressin dose-dependently decreases night-time food intake in normal male rats and mice, as well as in obese ob/ob male mice, when administered centrally or systemically, without causing any obvious adverse side effects. The normal, behavioral satiety sequence is maintained in male mice fasted overnight, though refeeding is attenuated. The anorectic effect is absent in CB(1) receptor null mutant male mice, and hemopressin can block CB(1) agonist-induced hyperphagia in male rats, providing strong evidence for antagonism of the CB(1) receptor in vivo. We speculate that hemopressin may act as an endogenous functional antagonist at CB(1) receptors and modulate the activity of appetite pathways in the brain.

Topics: Analysis of Variance; Animals; Behavior, Animal; Benzoxazines; Chlorocebus aethiops; Circadian Rhythm; COS Cells; Cyclohexanols; Dose-Response Relationship, Drug; Drinking Behavior; Dronabinol; Drug Administration Routes; Eating; Food Deprivation; Green Fluorescent Proteins; Hemoglobins; Hyperphagia; Leptin; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Morpholines; Naphthalenes; Peptide Fragments; Piperidines; Protein Transport; Psychotropic Drugs; Pyrazoles; Rats; Rats, Sprague-Dawley; Receptor, Cannabinoid, CB1; Rimonabant; Time Factors; Transfection