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. 2009 Sep;23(9):3020–3029. doi: 10.1096/fj.09-132142

Novel endogenous peptide agonists of cannabinoid receptors

Ivone Gomes *, Julia S Grushko †, Urszula Golebiewska ‡, Sascha Hoogendoorn *, Achla Gupta *, Andrea S Heimann §, Emer S Ferro ‖, Suzanne Scarlata ‡, Lloyd D Fricker †, Lakshmi A Devi *,1
PMCID: PMC2735371  PMID: 19380512

Abstract

Hemopressin (Hp), a 9-residue α-hemoglobin-derived peptide, was previously reported to function as a CB1 cannabinoid receptor antagonist (1). In this study, we report that mass spectrometry (MS) data from peptidomics analyses of mouse brain extracts identified N-terminally extended forms of Hp containing either three (RVD-Hpα) or two (VD-Hpα) additional amino acids, as well as a β-hemoglobin-derived peptide with sequence similarity to that of hemopressin (VD-Hpβ). Characterization of the α-hemoglobin-derived peptides using binding and functional assays shows that in contrast to Hp, which functions as a CB1 cannabinoid receptor antagonist, both RVD-Hpα and VD-Hpα function as agonists. Studies examining the increase in the phosphorylation of ERK1/2 levels or release of intracellular Ca2+ indicate that these peptides activate a signal transduction pathway distinct from that activated by the endocannabinoid, 2-arachidonoylglycerol, or the classic CB1 agonist, Hu-210. This finding suggests an additional mode of regulation of endogenous cannabinoid receptor activity. Taken together, these results suggest that the CB1 receptor is involved in the integration of signals from both lipid- and peptide-derived signaling molecules.—Gomes, I., Grushko, J. S., Golebiewska, U., Hoogendoorn, S., Gupta, A., Heimann, A. S., Ferro, E. S., Scarlata, S., Fricker, L. D., Devi, L. A. Novel endogenous peptide agonists of cannabinoid receptors.

Keywords: G-protein-coupled receptors, pain, analgesia, drug abuse

Cannabinoid receptors are members of the superfamily of Gi/Go-coupled receptors. The major psychoactive component of Cannabis sativa, Δ9-tetrahydrocannabinol, binds to at least two types of cannabinoid receptors, CB1 and CB2 (2). The CB1 receptor is expressed primarily in the central nervous system (3), whereas the CB2 receptor is predominantly expressed in immune cells (4) and is also detectable in brainstem neurons (5) and spinal cord (6). More recently, GPR55, which is highly expressed in large dorsal root ganglion neurons, has also been shown to function as a cannabinoid receptor (7). A number of studies have shown that cannabinoid receptor activation leads to the inhibition of adenylyl cyclase activity, inhibition of calcium channels and D-type potassium channels, increases in the phosphorylation of mitogen-activated protein kinases, and activation of A-type and inwardly rectifying potassium channels (8,9,10). To date, two endogenous ligands of cannabinoid receptors, anandamide and 2-arachidonoylglycerol (2-AG), which are derived from membrane lipids, have been well characterized (11,12,13). Several studies have proposed important roles for the endocannabinoid system (consisting of the receptors and endogenous ligands) in many pathophysiological processes including Parkinson’s disease, Alzheimer’s disease, depression, inflammation, neuropathic pain, and obesity (11, 13,14).

The recent finding that hemopressin (Hp), a 9-residue peptide derived from the α1 chain of hemoglobin, functions as an antagonist of the CB1 receptor and exhibits antinociceptive activity (1) suggests the possibility that cannabinoid receptor activity could be modulated by peptides derived from larger precursor proteins, which would significantly increase the complexity of the endocannabinoid system. Many neuropeptides function in cell-cell communication, and most of these neuropeptides are produced within the secretory pathway by selective proteases that cleave the peptide precursors at specific, well-defined sites. After cleavage, the peptides are stored within vesicles and secreted upon cell stimulation. In addition to these “classic” neuropeptides, a number of bioactive peptides have been identified that appear to be derived from cellular proteins. However, these peptides do not appear to be produced by selective cleavages at specific sites or to be stored within vesicles and released by stimulation. Hp seems to be an example of this “nonclassic” group of neuropeptides. In addition to Hp, the α and β chains of hemoglobin are precursors of peptides named hemorphins and neokyotorphin; these peptides interact with opiate receptors and angiotensin receptors (15,16,17,18,19). Interestingly, Hp functions as an antagonist of the CB1 receptor, is orally active, and causes antinociception (1).

Hp was previously identified in extracts of rat brain using an affinity purification method involving binding to endopeptidase 24.15. In the present study, we investigated whether Hp or Hp-like peptides were major peptides in mouse brain extracts, without using an affinity purification approach. Although the 9-residue Hp was not found, two N-terminally extended forms of Hp were found, one of which was highly represented in a meta-analysis of peptidomics analyses. These longer forms of Hp were then characterized for activity toward CB receptors; interestingly, although the nine-residue Hp is an antagonist of the CB1 receptor, the longer forms are agonists of this receptor. Furthermore, the signaling pathways for the peptide-based agonists appear to be distinct from the lipid-based and synthetic agonists of the CB1 receptor, implying that this receptor has a complex function of integration of a wide variety of signaling molecules.

MATERIALS AND METHODS

Materials

HEK-293 cells, Neuro 2A cells, and CHO cells were obtained from American Type Culture Collection (Manassas, VA, USA). DMEM, F12 medium, penicillin-streptomycin, lipofectamine, and Fluo-4 NW calcium dye were obtained from Invitrogen (Carlsbad, CA, USA). Chimeric G16/Gi3 construct was a kind gift from Dr. R. Margolskee (Mount Sinai School of Medicine, New York, NY, USA). Plasmid cDNAs for CB1 cannabinoid and CB2 cannabinoid receptors and for GPR55 were a kind gift from Dr. K. Mackie (Indiana University, Bloomington, IN, USA). Plasmid cDNAs for μ and δ opioid receptors were a kind gift from Dr. Chris Evans (University of California, Los Angeles, CA, USA). [3H]CP55,940 and SR141716 were obtained from the National Institute on Drug Abuse Drug Supply Program (Bethesda, MD, USA). Antibodies to phosphorylated and total ERK1/2 were obtained from Cell Signaling Technology Inc. (Danvers, MA, USA). The secondary antibodies IRDye680-labeled anti-rabbit and IRDye 800-labeled anti-mouse antibodies were from Li-COR (Lincoln, NE, USA). The anti-rabbit antibody conjugated to horseradish peroxidase (HRP) was from Amersham Biosciences Corp. (Piscataway, NJ, USA). RVD-Hpα, VD-Hpα, VD-Hpβ, and plasmid cDNA for angiotensin 1 (AT1) receptors were from Proteimax (São Paulo, Brazil). All other reagents were obtained from Sigma-Aldrich Corp. (St. Louis, MO, USA).

Cell culture and transfection

HEK-293 cells were grown in DMEM containing 10% FBS and 1% penicillin-streptomycin. Neuro 2A cells were grown in DMEM and F12 medium (50:50) containing 10% FBS and 1% penicillin-streptomycin. CHO cells were grown in F12 medium containing 10% FBS and 1% penicillin-streptomycin. For receptor internalization studies CHO cells were transfected with myc-tagged CB1 cannabinoid receptors using Lipofectamine as per the manufacturer’s protocol (Invitrogen). For experiments measuring increases in intracellular Ca2+ levels, HEK-293 cells were cotransfected with a chimeric G16/Gi3 and either CB1 cannabinoid, CB2 cannabinoid, α2A adrenergic, β2 adrenergic, μ opioid, or δ opioid receptors. In the case of AT1 receptors that signal through Gαq, cells were not cotransfected with the chimeric G16/Gi3.

Mass spectrometric identification of Hp-based peptides

Our composite database consists of MS data from peptidomics experiments (both published and unpublished) from >100 HPLC-MS runs using extracts from various regions of mouse brain (20,21,22,23,24,25,26,27,28,29,30,31). Brain tissues were processed as described previously. Briefly, brain areas were pooled from groups of animals and frozen. For peptide extraction, the tissue pool was sonicated in ice-cold H2O and incubated in a 70°C water bath for 20 min. The homogenate was then cooled on ice and acidified with 0.1 M HCl to a final concentration of 10 mM HCl. The homogenates were kept on ice for 15 min and centrifuged at 15,000 g for 30 min at 4°C. The pH of the supernatant was adjusted to 9.5 by addition of 0.2 M phosphate buffer. The samples were then labeled with differential isotopic trimethylammonium butyrate (TMAB) labels and analyzed by liquid chromatography (LC)-tandem mass spectrometry (MS/MS), as described previously (20,21,22,23,24,25,26,27,28,29,30,31).

Ligand binding and signaling studies

Membranes were prepared from the cerebellum and striatum of C57BL/6 mice brains as described previously (32). For ligand binding, membranes from mice cerebellum (50 μg) were incubated for 2 h at 30°C with 0.5 nM [3H]CP55,940 in the absence or presence of increasing concentrations (0–1 μM) of SR141716, RVD-Hpα, VD-Hpα, or VD-Hpβ as described previously (32) in a buffer containing 50 mM Tris-Cl (pH 7.4), 5 mg/ml BSA (fatty acid free), 1 mM EDTA, 3 mM MgCl2, and protease inhibitor cocktail. At the end of the incubation period, membranes were filtered using GF/B filters (presoaked for 2 h at room temperature with 0.1% polyethyleneimine containing 0.2% fatty acid free BSA) and washed 3 times with ice-cold 50 mM Tris-Cl (pH 7.4). For guanosine 5′-O-(3-thio)triphosphate (GTPγS) binding, membranes from striatum or cerebellum (10 μg) were incubated with the indicated doses of Hu-210, SR141716, RVD-Hpα, or VD-Hpα as described previously (32).

Gαi16-facilitated Ca2+ release

HEK-293 cells coexpressing a chimeric G16/Gi3 with either CB1 cannabinoid, CB2 cannabinoid, α2A adrenergic, β2 adrenergic, μ opioid, or δ opioid receptors or HEK-293 cells expressing AT1 receptors were plated into poly-l-lysine-coated 96-well clear-bottom plates (40,000 cells/well). On the next day, the growth medium was removed, and cells were washed twice in HBSS buffer containing 20 mM HEPES. Cells were incubated with Fluo-4 NW calcium dye (3 μM in 100 μl) for 1 h at 37°C. The different compounds (1 μM) were added to the wells by the robotic arm of the FLEX Station, and intracellular Ca2+ levels were measured for 300 s at excitation 494 nm and emission 516 nm. In experiments examining whether cannabinoid receptor-mediated increases in intracellular Ca2+ levels were blocked by the antagonist, cells were pretreated with SR141716 (10 μM) 30 min before addition of the different ligands.

Phosphorylation of MAP kinase (ERK)

HEK-293 cells expressing myc-tagged CB1 receptors, HA-tagged CB2 receptors, or GPR55 (2×105 cells/well) were treated for 5 min with 1 μM Hu-210, RVD-Hpα, VD-Hpα, or VD-Hpβ in the absence or presence of 10 μM SR141716, and levels of phosphorylated ERK1/2 were determined as described previously (1, 32). For dose-response studies, HEK-293 cells expressing CB1 receptors were treated for 5 min with 0–10 μM Hu-210, RVD-Hpα, or VD-Hpα. For time course studies, HEK-293 cells expressing CB1 receptors were treated with 1 μM Hu-210, RVD-Hpα, or VD-Hpα for 0–20 min. To observe the effect of pertussis toxin (PTX) on phosphorylated ERK1/2 levels, HEK-293 cells expressing CB1 receptors were pretreated for 2 h at 37°C with 15 ng/ml (PTX), followed by a 5-min treatment with 1 μM Hu-210, RVD-Hpα, or VD-Hpα.

Neurite outgrowth assays

Neuro 2A cells endogenously expressing CB1 receptors were treated for 16 h in medium containing 0.1% FBS with 1 μM Hu-210, RVD-Hpα, VD-Hpα, or VD-Hpβ in the absence or presence of 10 μM SR141716, and neurite length was determined as described previously (33, 34).

Receptor internalization studies

CHO cells expressing myc-tagged CB1 receptors (1–2×105 cells /well) were plated onto a poly-l-lysine-coated 24-well plate. On the next day, the growth medium was removed, and cells were treated with 1 μM Hu-210, RVD-Hpα, VD-Hpα, or VD-Hpβ in the absence or presence of 10 μM SR141716 in DMEM for 60 min at 37°C. The plate was then placed on ice, and wells were washed 3 times with 500 μl of ice-cold PBS and subjected to ELISA using 1:1000 of anti-myc polyclonal antibody and 1:1000 of HRP-conjugated anti-rabbit secondary antibody as described previously (32).

Intracellular Ca2+ release in individual cells

Neuro 2A, HEK-293, or HEK-293 cells expressing HA-CB1 were plated in Mattek chambers for 24 h to achieve 50–80% confluence. Before the experiment, cells were starved in serum-free medium from 6 h to overnight. Cells were incubated in HBSS medium with 1 μM Calcium Orange for 45 min, washed 3 times in medium, and subsequently incubated in medium without Calcium Orange for 30 min, followed by 3 additional washes with HBSS medium. Prolonged washing is needed to remove dye nonspecifically bound to the plasma membrane. Cells were imaged on a Zeiss LSM510 laser scanning confocal microscope (Carl Zeiss, Thornwood, NY, USA). From one dish, several cells in one field of view were recorded. Calcium Orange was excited with a 543-nm HeNe laser line, and the emission spectrum was recorded using a long-pass 560 filter. Images were recorded before treatment (to measure Fmin). Cells were treated with 1 μM ligand; after treatment, a time course of fluorescence intensity was recorded for 30 min. For Fmax estimation cells were treated with 5 μg/ml calcimycin (A23187).

Free calcium concentration was calculated from the following equation:

graphic file with name M1.gif

where Kd is the dissociation constant of the calcium indicator (185 nM for Calcium Orange), F is fluorescence intensity at given time point, Fmin is the fluorescence intensity in the absence of stimulator, and Fmax is the intensity in the presence of calcium ionophore. Average intensity of many pixels inside a given cell was calculated using Zeiss software. Data points were averaged and se was calculated using Sigma Plot (Systat Software, Inc., San Jose, CA, USA).

Statistical analysis

The results are expressed as the mean ± se. Statistical comparisons were done using analysis of variance (ANOVA) followed by the Bonferroni multiple comparison test. P < 0.05 indicated a significant difference. Statistical analyses of data were generated using GraphPad Prism 4.02 (GraphPad Software Inc., San Diego, CA, USA).

RESULTS

Identification of endogenous forms of Hp

We have previously shown that Hp, a nonapeptide derived from the α chain of hemoglobin, functions as an antagonist of CB1 cannabinoid receptors (1). A central question is whether Hp represents an endogenous peptide that is normally present in the brain. To address this question, we searched for the Hp sequence in a database consisting of MS results from >100 peptidomics analyses of mouse brain extracts. We did not find Hp, but we did identify longer N-terminally extended Hp peptides containing 11- and 12-aa residues, VDPVNFKLLSH (VD-Hpα) and RVDPVNFKLLSH (RVD-Hpα), respectively, by MS/MS (Fig. 1). RVD-Hpα was detected in many of the brain regions examined, such as hypothalamus, nucleus accumbens, olfactory bulb, cerebellum, prefrontal cortex, and striatum, and VD-Hpα was detected in olfactory bulb, hypothalamus, and cerebellum (data not shown). Supplemental Table 1 summarizes all of the neuropeptides, hemoglobin-derived peptides, and other representative peptides that were detected in at least 5 distinct samples. Whereas VD-Hpα was found in only 8 of the samples, RVD-Hpα was identified in 47 different samples, representing one of the more frequently detected mouse brain peptides (Supplemental Table 1). In addition to these α-hemoglobin-derived forms of Hp, we also found a number of other fragments of α-hemoglobin, although none were found as many times as RVD-Hpα (Supplemental Table 1). Two β-hemoglobin-derived peptides were also detected in our analysis; one corresponds to the previously identified bioactive peptide named VV-hemorphin, and the other has substantial sequence homology to Hp (Supplemental Table 1). Based on the nomenclature system for hemorphins and Hp, we have named this β-hemoglobin-derived Hp-like peptide VD-hemopressinβ (VD-Hpβ).

Figure 1.

Figure 1.

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Mass spectrometric identification of endogenous RVD-Hpα. Peptides were extracted from microwave-irradiated mouse brain, labeled with TMAB isotopic labels, purified by microfiltration using a 10-kDa exclusion unit, and analyzed by LC/MS. In this example, the tandem mass spectrum is shown for the quadruply charged ion with an m/z of 420.5 and a monoisotopic mass of 1423.79 Da (after subtraction of the mass of the hydrogenated form of the TMAB isotopic tag). Inset: an expanded y axis for the indicated m/z range. The observed ions represent b-series, y-series, and internal ions formed secondary to the cleavage of the D-P bond. Fragmentation ions usually lose the trimethylamine moiety. However, the doubly charged b3 ion shows a substantial peak for the fragment that retained the trimethylamine; this is indicated as b32+ + 59. Conversely, the parent ion that lost the trimethylamine moiety is indicated as MH4+ − 59. amu, atomic mass units.

Agonistic activity of RVD-Hpα, VD-Hpα, and VD-Hpβ at CB1 cannabinoid receptors

Because it was previously shown that Hp functions as an antagonist/inverse agonist of CB1 cannabinoid receptors, the effect of RVD-Hpα, VD-Hpα, and VD-Hpβ on signaling by cannabinoid receptors was evaluated by examining their effect on receptor-mediated increases in intracellular Ca2+ levels in heterologous cells expressing a variety of family A G-protein-coupled receptors (GPCRs) along with a promiscuous G protein (Fig. 2). Treatment with RVD-Hpα and VD-Hpα alone caused an increase in intracellular Ca2+ levels in cells expressing CB1 cannabinoid receptors and to a lesser extent in cells expressing CB2 cannabinoid receptors, whereas VD-Hpβ was equally effective at CB1 and CB2 cannabinoid receptors (Fig. 2). None of the three peptides induced increases in intracellular Ca2+ levels in cells expressing μ and δ opioid, α2A and β2 adrenergic, or AT1 receptors (Fig. 2). Moreover, RVD-Hpα , VD-Hpα, and VD-Hpβ-mediated increases in intracellular Ca2+ levels in CB1-expressing cells could be blocked by pretreating cells with the cannabinoid antagonist SR141716 (VD-Hpβ-mediated increases in intracellular Ca2+ levels were only partially blocked by SR141716) (Fig. 3). We also examined the ability of extended Hp peptides to bind to cannabinoid receptors in cerebellar membranes. We found that RVD-Hpα can displace the binding of the cannabinoid ligand, [3H]CP55,940, with nanomolar affinity although to a lesser extent than the classic CB1 antagonist, SR141716 (Supplemental Fig. 1). Similar results were obtained with VD-Hpα and VD-Hpβ (not shown). Taken together, these results suggest that, in contrast to Hp that functions as an antagonist (1), RVD-Hpα and VD-Hpα function as agonists of cannabinoid receptors.

Figure 2.

Figure 2.

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Longer Hp peptides are able to increase CB receptor-mediated intracellular Ca2+ levels. HEK-293 cells (40,000 cells/well) coexpressing chimeric G16/Gi3 and individual receptors (except in the case of AT1 receptors) were treated with 1 μM concentrations of receptor agonists, RVD-Hpα, VD-Hpα, or VD-Hpβ, and intracellular Ca2+ levels were determined. RFU, relative fluorescence units.

Figure 3.

Figure 3.

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Longer Hp peptide-mediated calcium release is blocked by the CB1 receptor antagonist. HEK-293 cells (40,000 cells/well) coexpressing chimeric G16/Gi3 and CB1 receptors were treated with 1 μM HU-210 (Hu), RVD-Hpα, VD-Hpα, or VD-Hpβ in the absence or presence of 10 μM SR141716 (SR), and intracellular Ca2+ levels were determined. Results are means ± se; n = 3. RFU, relative fluorescence units.

Previous studies have shown that treatment with agonists of CB1 receptors can induce neurite outgrowth in cells endogenously expressing these receptors (33, 34). We used this functional assay to measure the agonistic activity of RVD-Hpα, VD-Hpα, and VD-Hpβ. As expected, these peptides increased the number of cells with neurites in a manner similar to that of the cannabinoid agonist, Hu-210 (Fig. 4 and Supplemental Fig. 2). We found that the RVD-Hpα- and VD-Hpα-mediated increases in neurite outgrowth could be blocked by pretreatment with the antagonist, SR141716 (Fig. 4). Activation of CB1 receptors has also been shown to lead to rapid receptor endocytosis (35, 36). We examined the ability of RVD-Hpα, VD-Hpα, and VD-Hpβ to induce receptor endocytosis in CHO cells expressing myc-tagged CB1 receptors. The classic agonist, Hu-210, as well as RVD-Hpα, VD-Hpα, and VD-Hpβ induce CB1 receptor internalization; however, only the RVD-Hpα- and VD-Hpα-induced CB1 receptor internalization can be blocked by the cannabinoid receptor antagonist, SR141716 (Fig. 5). Taken together, these results indicate that RVD-Hpα and VD-Hpα behave as selective agonists of CB1 cannabinoid receptors.

Figure 4.

Figure 4.

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Longer Hpα peptides induce neurite outgrowth in Neuro 2A cells. Neuro2A cells were treated for 16 h with 1 μM Hu-210 (Hu), RVD-Hpα, or VD-Hpα in the absence or presence of 10 μM SR141716 (SR), and the percentage of cells extending neurites was determined. Results are means ± se; n = 3. **P < 0.01, ***P < 0.001; 1-way ANOVA.

Figure 5.

Figure 5.

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Longer Hp peptides induce CB1 receptor internalization. CHO cells expressing myc-tagged CB1 receptors (1–2×105 cells/well) were treated with 1 μM Hu-210 (Hu), RVD-Hpα, VD-Hpα, or VD-Hpβ in the absence or presence of 10 μM SR141716 (SR) and subjected to ELISA using 1:1000 of anti-myc polyclonal antibody and 1:1000 of HRP-conjugated anti-rabbit secondary antibody, as described in Materials and Methods. Results are means ± se of 3 experiments in triplicate. *P < 0.05, **P < 0.01, ***P < 0.001; 1-way ANOVA.

The ability of RVD-Hpα, VD-Hpα, and VD-Hpβ to mediate phosphorylation of ERK1/2 in heterologous cells expressing CB1 receptors was examined. We found that all three peptides enhance phospho-ERK (pERK) levels in cells expressing CB1 receptors; the enhancement of pERK levels by RVD-Hpα and VD-Hpα but not by VD-Hpβ is significantly blocked by SR141716 (Fig. 6A and Supplemental Fig. 3). Next, we focused our studies on RVD-Hpα and VD-Hpα because they exhibited CB1 receptor selectivity. To explore this selectivity further, the ability of these peptides to transduce signals via CB2 or GPR55 receptors was examined. Treatment of cells expressing either CB2 or GPR55 did not lead to substantial increases in pERK levels by RVD-Hpα (increases were found with VD-Hpα in cells expressing CB2 receptors, although they were not statistically significant) (Fig. 6A). Next, we focused on RVD-Hpα and compared the time course of ERK phosphorylation by the peptide ligand with that of the classic CB1 ligand, Hu-210 (Fig. 6B). Both the level and rate of increase in pERK were lower in the case of RVD-Hpα compared with that of Hu-210 (Fig. 6B). The increase in pERK with Hu-210 at early time points (3 and 5 min) is greatly reduced by PTX, whereas the increase in pERK due to RVD-Hpα at 5 min is decreased by only 30% (Fig. 6B). This result, together with the different time course, suggests that stimulation of CB1 receptors by the peptide ligand leads to activation of a signaling pathway distinct from the classic G-protein-mediated pathway. The differences in signaling by the peptide ligands and Hu-210 were further explored using a G-protein activation assay. In both cerebellar and striatal membranes, although Hu-210 treatment caused a robust increase in [35S]GTPγS binding, treatment with the peptide ligands did not (Fig. 7). Taken together, these results suggest that the peptide ligands stimulate CB1 receptors via a mechanism distinct from Hu-210.

Figure 6.

Figure 6.

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Longer Hpα peptides mediate ERK phosphorylation. A) HEK-293 cells expressing myc-tagged CB1 receptors, HA-tagged CB2 receptors, or GPR55 were treated with 1 μM Hu-210 (Hu), RVD-Hpα, or VD-Hpα in the absence or presence of 10 μM SR141716 (SR), and levels of pERK1/2 were determined. Results are means ± se; n = 3. *P < 0.01, **P < 0.001; 1-way ANOVA. B) HEK-293 cells expressing CB1 receptors were treated for 0–20 min with 1 μM Hu-210 or RVD-Hpα in the absence or presence of PTX, and levels of phosphorylated ERK1/2 were determined as described in Materials and Methods. Results are means ± se of 3 experiments in triplicate. **P < 0.01; ***P < 0.001. Representative blots from 3 independent experiments are shown.

Figure 7.

Figure 7.

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Longer Hpα peptides activate a signal transduction pathway distinct from the pathway activated by classic CB1 receptor agonists: [35S]GTPγS binding. Striatal or cerebellar membranes (10 μg) were subjected to a [35S]GTPγS binding assay using the indicated concentrations of Hu-210 (Hu), RVD-Hpα, or VD-Hpα, as described in Materials and Methods. Results are means ± se; n = 3.

To explore the peptide agonist-mediated CB1 receptor signaling, we examined the dynamics of Ca2+ release in the absence of promiscuous G protein. In Neuro 2A cells that endogenously express CB1 receptors as well as in HEK-293 cells expressing recombinant receptors, treatment with the peptide ligands leads to a sustained increase in Ca2+ release that is faster and more robust than treatment with either the endocannabinoid 2-AG, or the classic agonist Hu-210 (Fig. 8A). Under the same conditions, activation of the endogenous adenosine receptors in Neuro 2A cells with 1 μM ATP leads to a rapid (within seconds) and transient increase in Ca2+ release (data not shown). Furthermore, the RVD-Hpα-mediated Ca2+ release is blocked by SR141716 (Fig. 8B). In addition, we found that PTX pretreatment does not block RVD-Hpα-mediated Ca2+ release at early time points (Fig. 8C), although partial blockade is observed at later time points (Supplemental Fig. 4A). This finding suggests that the CB1 receptor-mediated Ca2+ release is not via the Gαi-mediated pathway. Similar results were obtained when HEK cells transfected with CB1 receptors were examined; RVD-Hpα caused a more robust increase in free intracellular Ca2+ than Hu-210 (Supplemental Fig. 4B). The peptide-mediated Ca2+ release in the Neuro 2A cells is seen in the absence of extracellular calcium (Fig. 8D), indicating that the Ca2+ release is from intracellular stores. These results are exciting and indicate that the peptide agonists activate a signal transduction pathway that results in a sustained increase in Ca2+ release, which is distinct from that seen with the endocannabiniod 2-AG. Furthermore, the peptide-mediated Ca2+ signaling is distinct from that of the classic Gαq-coupled receptors that tends to be rapid and transient.

Figure 8.

Figure 8.

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Longer Hpα peptides activate a signal transduction pathway distinct from the pathway activated by classic CB1 receptor agonists: calcium release. A) Neuro 2A cells (7–18 cells) were treated 1 min before data recording with 1 μM Hu-210, RVD-Hpα, VD-Hpα, or 2-AG. The free Ca2+ concentration was calculated as described in Materials and Methods. Data represent means ± se of 7–18 cells. B) Neuro 2A cells were treated 1 min before data recording with 1 μM Hu-210, RVD-Hpα, or Hpα in the absence or presence of 10 μM SR141716. Data represent means ± se of 7–18 cells. C) Neuro 2A cells were treated in the presence or absence of PTX before data recording with 1 μM RVD-Hpα. The free Ca2+ concentration was calculated as described in Materials and Methods. Data represent means ± se of 7–18 cells. D) Neuro 2A cells were treated in the presence or absence of Ca2+-free medium 1 min before data recording with 1 μM RVD-Hpα. The free Ca2+ concentration was calculated as described in Materials and Methods. Data represent means ± se of 18–22 cells.

DISCUSSION

In this study, we show that N-terminally extended forms of Hp function as agonists of CB1 cannabinoid receptors. Whereas previous studies have reported that CB1 receptors can couple to different G proteins (37) and that the same GPCR can activate distinct signaling on being activated by different ligands (38), the present study, for the first time, demonstrates that two distinct “endogenous” ligands that differ in their chemical nature (lipid vs. peptide) activate the same receptor to initiate distinct signaling pathways.

The extended Hp peptides represent fairly abundant peptides, based on the numbers of times each of these peptides were found in LC-MS analysis. Our composite database of MS data represents >100 LC-mass spectrometry runs from different samples of extracts of various mouse brain regions; this database contains ∼1000 peptides that have been identified at least once by MS/MS sequencing and that arise from either secretory pathway “neuropeptide” precursors or cytosolic proteins, such as the hemoglobin-derived Hps. The most frequently found peptides were derived from precursors that are known to be expressed at relatively high levels in mouse brain (Supplemental Table 1). RVD-Hpα was the most frequently detected α-hemoglobin-derived peptide, being found as many times as some of the more abundant neuropeptides. The 9-aa Hp peptide originally found by substrate capture of rat brain extracts was not found in the analysis of mouse brain peptides. It is possible that this difference reflects differential processing in rats vs. mice or the difference between the substrate capture approach and the peptide isolation methods used in the present study. It is also possible that the original 9-residue Hp peptide was formed from RVD-Hpα and VD-Hpα by the hot acid extraction method used in the previous study; D-P bonds are especially sensitive to hot acid, and cleavage of RVD-Hpα and VD-Hpα at the D-P bond would give rise to the shorter nine-residue Hp.

It will be interesting to examine the mechanisms involved in the generation of RVD-Hpα and VD-Hpα. It is likely that proteasomes, a protease complex that degrades proteins into peptides of 4-25 aa, are involved in the formation of the hemoglobin-derived peptides found in the present study; the Hp peptides are within the appropriate size range for proteasome products, and the cleavage sites needed to produce these peptides are consistent with the specificity of the various proteasome catalytic units (39). The finding that bioactive peptide fragments are produced from cytosolic proteins is not novel: as mentioned in the introduction, a number of other hemoglobin-derived peptides have been found to have bioactivities in various systems (15,16,17,18,19). In addition, other bioactive peptides have been identified and found to represent fragments of cytosolic proteins; examples include diazepam-binding inhibitor, a fragment of acyl-CoA-binding protein that binds to GABAA receptors; hippocampal cholinergic neurostimulating peptide (HCNP), a fragment of phosphatidylethanolamine-binding protein that enhances the differentiation of hippocampal neurons; microcryptide-1, a fragment of cytochrome c oxidase subunit 8 that activates neutrophils; and fragments of β-thymosin that affect wound healing and mast cells (40,41,42,43). Little is known about the enzymatic pathways required for the processing of these bioactive peptides or whether this process is regulated as would be expected for endogenous signaling molecules. Preliminary data from our laboratory suggest a possible role of global ischemia in the up-regulation of VD-Hpα and RVD-Hpα (data not shown). A recent study reported an increase in a large number of hemoglobin-derived peptides that was induced by a 3- or 10-min delay between death and microwave irradiation (44) and a previous article from our laboratory listed VD-Hpα as one of the protein-derived peptides that is much more abundant in brains that were not subjected to microwave irradiation as opposed to those that were irradiated (20). Because a delay in processing of brain tissue after decapitation is a well-accepted model of global ischemia (45), this result provides further support to the idea that ischemia may regulate the breakdown of hemoglobin into hemoglobin-derived peptides such as VD-Hpα and RVD-Hpα.

Additional areas for further studies are to identify cell types that produce the hemoglobin and also to identify the pathway by which intracellular peptides and proteins are secreted. Although hemoglobin is traditionally viewed as being specific to erythrocytes, other cell types produce α and/or β hemoglobin. Both α and β hemoglobin mRNA and protein were found in human endometrium, with all epithelial cells and most stromal cells being positive for immunoreactive protein (46). A microarray screen detected α and β hemoglobin mRNA in mouse lens (47). Within the lung, alveolar epithelial type II cells and Clara cells were found to express α and β hemoglobin mRNA and protein (48). Hemoglobin expression was found to decrease when alveolar epithelial type II cells differentiated into type I cells, illustrating specificity of expression in type II but not in type I alveolar epithelial cells (49). βminor hemoglobin expression was induced in macrophages when the cells were stimulated to produce nitric oxide (50). Expression of both chains of hemoglobin was found in the mesangial region of rat glomeruli in kidney and in mesangial cells in vitro; this renal hemoglobin expression was increased with hypoxia (51). Furthermore, there has recently been a report of α and β hemoglobin in neurons and glia (52). Thus, it is possible that the hemoglobin-derived peptides detected in our peptidomics analysis of mouse brain extracts arise from neurons or glia and not from erythrocytes present in the brain tissue. Because hemoglobin lacks a signal peptide, any cell type producing this protein will express it in the cytosol or related compartments and not in the classic secretory pathway. For the hemoglobin-derived peptides to interact with receptors on distant cells, the peptides need to be secreted. The secretion of cytosolic proteins has been well documented; examples include the various interleukins as well as thymosin, HCNP and its precursor protein, and intracellular enzymes such as endopeptidases 24.15 and 24.16 (53,54,55,56). Despite the large number of cytosolic proteins/peptides known to be secreted, the precise mechanisms remain elusive.

Taken together, the data in this study are consistent with the notion that RVD-Hpα and VD-Hpα represent endogenous neuromodulatory peptides with CB1 receptor activity. The amino acid sequence of RVD-Hpα is highly conserved from reptiles to mammals, and only one conservative difference is found among mammals (RVDPVNFKLLSH vs. RVD PVNFKFLSH); both of these peptides function similarly in their binding to CB1 receptors (data not shown). In contrast to the previously identified Hp peptide, which is an antagonist (or inverse agonist) at the CB1 cannabinoid receptors, the peptides identified in the present study that contain 2 or 3 additional N-terminal amino acids are agonists. Another example of bioactive peptides being either agonists or antagonists of the same receptor, depending on peptide length, is that of β-endorphin (57). Another finding from these studies is that longer Hp-derived peptides activate signaling pathways distinct from the classic CB1 receptor agonist, Hu-210, in that 1) the peptides do not induce increases in [35S]GTPγS binding, whereas Hu-210 does; 2) the level and rate of increase in phosphorylation of ERK1/2 by these peptides are different from those of Hu-210; 3) the levels of intracellular Ca2+ release by these peptides are much more robust than those induced by Hu-210; and 4) the effects of the peptides on signaling are partially blocked by PTX in contrast to Hu-210, which is completely blocked. The Ca2+ release generated by longer Hp-derived peptides involves a longer time scale (30 min) than generally seen after activation of Gαq receptors (seconds). The observations that peptide-mediated Ca2+ release is blocked by the CB1 antagonist, is partially blocked by PTX, and involves release of Ca2+ from intracellular stores suggest that the longer Hp peptides activate a Gα pathway. Because the classic CB1 agonist, Hu-210, and the endogenous agonist, 2-AG, only cause a small increase in Ca2+ release, the robust and sustained level observed with longer Hp peptides suggests an unexpected lack of receptor down-regulation that is currently under investigation. The finding that RVD-Hpα and VD-Hpα possess unique agonistic activity at CB1 receptors provides additional tools to understand how the endocannabinoid system is modulated as well as novel candidates to be developed as therapeutic agents in the treatment of pathological conditions involving cannabinoid receptors.

Acknowledgments

This work was supported by NIH grants (DA019521 to L.A.D., GM05313 to S.S., GM071558 to L.A.D. and S.S., and DA04494 and DK51271 to L.D.F.), by a Zwanenberg Foundation Fellowship (to S.H.), by the Fundacao de Amparo a Pesquisa do Estado de São Paulo (Grants 04/04933-2 to E.S.F. and 04/14258-0 to A.S.H.), and by Conselho Nacional de Desenvolvimento Científico e Tecnológico (E.S.F.). Mass spectrometry was performed through Rede de Proteoma do Estado de São Paulo in the Laboratório Nacional de Luz Sincrotron, Campinas, São Paulo, Brazil. We thank Dr. K. Mackie (Indiana University, Bloomington, IN, USA) for the plasmid cDNAs for CB1 cannabinoid and CB2 cannabinoid receptors and for GPR55. We also thank Emeline Maillet and Robert Margolskee for help with analysis using the FLEX Station.

References

  1. Heimann A S, Gomes I, Dale C S, Pagano R L, Gupta A, de Souza L L, Luchessi A D, Castro L M, Giorgi R, Rioli V, Ferro E S, Devi L A. Hemopressin in an inverse agonist of CB1 cannabinoid receptors. Proc Natl Acad Sci U S A. 2007;104:20588–20593. doi: 10.1073/pnas.0706980105. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Begg M, Pacher P, Bátkai S, Osei-Hyiaman D, Offertáler L, Mo F M, Liu J, Kunos G. Evidence for novel cannabinoid receptors. Pharmacol Ther. 2005;106:133–145. doi: 10.1016/j.pharmthera.2004.11.005. [DOI] [PubMed] [Google Scholar]
  3. Matsuda L A, Lolait S J, Brownstein M J, Young A C, Bonner T I. Structure of a cannabinoid receptor and functional expression of the cloned cDNA. Nature. 1990;346:561–564. doi: 10.1038/346561a0. [DOI] [PubMed] [Google Scholar]
  4. Munro S, Thomas K L, Abu-Shaar M. Molecular characterization of a peripheral receptor for cannabinoids. Nature. 1993;365:61–65. doi: 10.1038/365061a0. [DOI] [PubMed] [Google Scholar]
  5. Van Sickle M D, Duncan M, Kingsley P J, Mouihate A, Urbani P, Mackie K, Stella N, Makriyannis A, Piomelli D, Davison J S, Marnett L J, Di Marzo V, Pittman Q J, Patel K D, Sharkey K A. Identification and functional characterization of brainstem cannabinoid CB2 receptors. Science. 2005;310:329–332. doi: 10.1126/science.1115740. [DOI] [PubMed] [Google Scholar]
  6. Zhang J, Hoffert C, Vu H K, Groblewski T, Ahmad S, O'Donnell D. Induction of CB2 receptor expression in the rat spinal cord of neuropathic but not inflammatory chronic pain models. Eur J Neurosci. 2003;17:2750–2754. doi: 10.1046/j.1460-9568.2003.02704.x. [DOI] [PubMed] [Google Scholar]
  7. Lauckner J E, Jensen J B, Chen H Y, Lu H C, Hille B, Mackie K. GPR55 is a cannabinoid receptor that increases intracellular calcium and inhibits M current. Proc Natl Acad Sci U S A. 2008;105:2699–2704. doi: 10.1073/pnas.0711278105. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Pertwee R G. Pharmacology of cannabinoid CB1 and CB2 receptors. Pharmacol Ther. 1997;74:129–180. doi: 10.1016/s0163-7258(97)82001-3. [DOI] [PubMed] [Google Scholar]
  9. Pertwee R G, Ross R A. Cannabinoid receptors and their ligands. Prostaglandins Leukot Essent Fatty Acids. 2002;66:101–121. doi: 10.1054/plef.2001.0341. [DOI] [PubMed] [Google Scholar]
  10. Howlet A C, Mukhopadhyay S. Cellular signal transduction by anandamide and 2-arachidonoylglycerol. Chem Phys Lipids. 2000;108:53–70. doi: 10.1016/s0009-3084(00)00187-0. [DOI] [PubMed] [Google Scholar]
  11. Di Marzo V, Petrosino S. Endocannabinoids and the regulation of their levels in health and disease. Curr Opin Lipidol. 2007;18:129–140. doi: 10.1097/MOL.0b013e32803dbdec. [DOI] [PubMed] [Google Scholar]
  12. Battista N, Fezza F, Finazzi-Agro A, Maccarrone M. The endocannabinoid system in neurodegeneration. Ital J Biochem. 2006;55:283–289. [PubMed] [Google Scholar]
  13. Boyd S T. The endocannabinoid system. Pharmacotherapy. 2006;26:218S–221S. doi: 10.1592/phco.26.12part2.218S. [DOI] [PubMed] [Google Scholar]
  14. Iversen L, Chapman V. Cannabinoids: a real prospect for pain relief. Curr Opin Pharmacol. 2002;2:50–55. doi: 10.1016/s1471-4892(01)00120-5. [DOI] [PubMed] [Google Scholar]
  15. Brantl V, Gramsch C, Lottspeich F, Mertz R, Jaeger K H, Herz A. Novel opioid peptides derived from hemoglobin: hemorphins. Eur J Pharmacol. 1986;125:309–310. doi: 10.1016/0014-2999(86)90044-0. [DOI] [PubMed] [Google Scholar]
  16. Liebmann C, Schrader U, Brantl V. Opioid receptor affinities of the blood-derived tetrapeptides hemorphin and cytochrophin. Eur J Pharmacol. 1997;166:523–526. doi: 10.1016/0014-2999(89)90368-3. [DOI] [PubMed] [Google Scholar]
  17. Moeller I, Lew R A, Mendelsohn F A, Smith A I, Brennan M E, Tetaz T J, Chai S Y. The globin fragment LVV-hemorphin-7 is an endogenous ligand for the AT4 receptor in the brain. J Neurochem. 1997;68:2530–2537. doi: 10.1046/j.1471-4159.1997.68062530.x. [DOI] [PubMed] [Google Scholar]
  18. Lee J, Mustafa T, McDowall S G, Mendelsohn F A, Brennan M, Lew R A, Albiston A L, Chai S Y. Structure-activity study of LVV-hemorphin-7: angiotensin AT4 receptor ligand and inhibitor of insulin-regulated aminopeptidase. J Pharmacol Exp Ther. 2003;305:205–211. doi: 10.1124/jpet.102.045492. [DOI] [PubMed] [Google Scholar]
  19. Fukui K, Shiomi H, Takagi H, Hayashi K, Kiso Y, Kitagawa K. Isolation from bovine brain of a novel analgesic peptapeptide, neo-kyotorphin, containing the Tyr-Arg (kyotorphin) unit. Neuropharmacology. 1983;22:191–196. doi: 10.1016/0028-3908(83)90008-4. [DOI] [PubMed] [Google Scholar]
  20. Che F Y, Lim J, Pan H, Biswas R, Fricker L D. Quantitative neuropeptidomics of microwave-irradiated mouse brain and pituitary. Mol Cell Proteomics. 2005;4:1391–1405. doi: 10.1074/mcp.T500010-MCP200. [DOI] [PubMed] [Google Scholar]
  21. Che F Y, Zhang X, Berezniuk I, Callaway M, Lim J, Fricker L D. Optimization of neuropeptide extraction from the mouse hypothalamus. J Proteome Res. 2007;6:4667–4676. doi: 10.1021/pr060690r. [DOI] [PubMed] [Google Scholar]
  22. Fricker L D. Neuropeptidomics to study peptide processing in animal models of obesity. Endocrinology. 2007;148:4185–4190. doi: 10.1210/en.2007-0123. [DOI] [PubMed] [Google Scholar]
  23. Pan H, Che F Y, Peng B, Steiner D F, Pintar J E, Fricker L D. The role of prohormone convertase-2 in hypothalamic neuropeptide processing: a quantitative neuropeptidomic study. J Neurochem. 2006;98:1763–1777. doi: 10.1111/j.1471-4159.2006.04067.x. [DOI] [PubMed] [Google Scholar]
  24. Decaillot F M, Che F Y, Fricker L D, Devi L A. Peptidomics of Cpefat/fat mouse hypothalamus and striatum: effect of chronic morphine administration. J Mol Neurosci. 2006;28:277–284. doi: 10.1385/JMN:28:3:277. [DOI] [PubMed] [Google Scholar]
  25. Che F Y, Vathy I, Fricker L D. Quantitative peptidomics in mice: effect of cocaine treatment. J Mol Neurosci. 2006;28:265–275. doi: 10.1385/JMN:28:3:265. [DOI] [PubMed] [Google Scholar]
  26. Lim J, Berezniuk I, Che F Y, Parikh R, Biswas R, Pan H, Fricker L D. Altered neuropeptide processing in prefrontal cortex of Cpefat/fat mice: implications for neuropeptide discovery. J Neurochem. 2006;96:1169–1181. doi: 10.1111/j.1471-4159.2005.03614.x. [DOI] [PubMed] [Google Scholar]
  27. Fricker L D, Lim J, Pan H, Che F Y. Peptidomics: identification and quantitation of endogenous peptides in neuroendocrine tissues. Mass Spectrom Rev. 2006;25:327–344. doi: 10.1002/mas.20079. [DOI] [PubMed] [Google Scholar]
  28. Pan H, Nanno D, Che F Y, Zhu X, Salton S R, Steiner D F, Fricker L D, Devi L A. Neuropeptide processing profile in mice lacking prohormone convertase-1. Biochemistry. 2005;44:4939–4948. doi: 10.1021/bi047852m. [DOI] [PubMed] [Google Scholar]
  29. Che F Y, Biswas R, Fricker L D. Relative quantitation of peptides in wild-type and Cpefat/fat mouse pituitary using stable isotopic tags and mass spectrometry. J Mass Spectrom. 2005;40:227–237. doi: 10.1002/jms.742. [DOI] [PubMed] [Google Scholar]
  30. Che F Y, Fricker L D. Quantitative peptidomics of mouse pituitary: comparison of different stable isotopic tags. J Mass Spectrom. 2005;40:238–249. doi: 10.1002/jms.743. [DOI] [PubMed] [Google Scholar]
  31. Che F Y, Yuan Q, Kalinina E, Fricker L D. Peptidomics of Cpefat/fat mouse hypothalamus: effects of food deprivation and exercise on peptide levels. J Biol Chem. 2005;280:4451–4461. doi: 10.1074/jbc.M411178200. [DOI] [PubMed] [Google Scholar]
  32. Gomes I, Filipovska J, Devi L A. Opioid receptor oligomerization: detection and functional characterization of interacting receptors. Methods Mol Med. 2003;84:157–183. doi: 10.1385/1-59259-379-8:157. [DOI] [PubMed] [Google Scholar]
  33. Jordan J D, He J C, Eungdamrong N J, Gomes I, Ali W, Nguyen T, Bivona T G, Philips M R, Devi L A, Iyengar R. Cannabinoid receptor-induced neurite outgrowth is mediated by Rap1 activation through Gαo/i-triggered proteasomal degradation of Rap1GAPII. J Biol Chem. 2005;280:11413–11421. doi: 10.1074/jbc.M411521200. [DOI] [PubMed] [Google Scholar]
  34. He J C, Gomes I, Nguyen T, Jayaram G, Ram P T, Devi L A, Iyengar R. The Gαo/i-coupled cannabinoid receptor-mediated neurite outgrowth involves Rap regulation of Src and Stat3. J Biol Chem. 2005;280:33426–33434. doi: 10.1074/jbc.M502812200. [DOI] [PubMed] [Google Scholar]
  35. Hsieh C, Brown S, Derleth C, Mackie K J. Internalization and recycling of the CB1 cannabinoid receptor. J Neurochem. 1999;73:493–501. doi: 10.1046/j.1471-4159.1999.0730493.x. [DOI] [PubMed] [Google Scholar]
  36. Coutts A A, Anavi-Goffer S, Ross R A, MacEwan D J, Mackie K, Pertwee R G, Irving A J. Agonist-induced internalization and trafficking of cannabinoid CB1 receptors in hippocampal neurons. J Neurosci. 2001;21:2425–2433. doi: 10.1523/JNEUROSCI.21-07-02425.2001. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Lauckner J E, Hille B, Mackie K. The cannabinoid agonist WIN55,2-12-2 increases intracellular calcium via CB1 receptor coupling to Gq/11 G proteins. Proc Natl Acad Sci U S A. 2005;102:19144–19149. doi: 10.1073/pnas.0509588102. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Drake M T, Violin J D, Whalen E J, Wisler J W, Shenoy S K, Lefkowitz R J. β-Arrestin-biased agonism and β2-adrenergic receptor. J Biol Chem. 2008;283:5669–5676. doi: 10.1074/jbc.M708118200. [DOI] [PubMed] [Google Scholar]
  39. Goldberg A L. Protein degradation and protection against misfolded or damaged proteins. Nature. 2003;426:895–899. doi: 10.1038/nature02263. [DOI] [PubMed] [Google Scholar]
  40. Costa E, Guidotti A. Diazepam binding inhibitor (DBI): a peptide with multiple biological actions. Life Sci. 1991;49:325–344. doi: 10.1016/0024-3205(91)90440-m. [DOI] [PubMed] [Google Scholar]
  41. Goldstein A L, Hannappel E, Kleinman H K. Thymosin β4: actin-sequestering protein moonlights to repair injured tissues. Trends Mol Med. 2005;11:421–429. doi: 10.1016/j.molmed.2005.07.004. [DOI] [PubMed] [Google Scholar]
  42. Ojika K, Mitake S, Tohdoh N, Appel S H, Otsuka Y, Katada E, Matsukawa N. Hippocampal cholinergic neurostimulating peptides (HCNP) Prog Neurobiol. 2000;60:37–83. doi: 10.1016/s0301-0082(99)00021-0. [DOI] [PubMed] [Google Scholar]
  43. Mukai H, Hokari Y, Seki T, Takao T, Kubota M, Matsuo Y, Tsukagoshi H, Kato M, Kimura H, Shimonishi Y, Kiso Y, Nishi Y, Wakamatsu K, Munekata E. Discovery of mitocryptide-1, a neutrophil-activating cryptide from healthy porcine heart. J Biol Chem. 2008;283:30596–30605. doi: 10.1074/jbc.M803913200. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Sköld K, Svensson M, Norrman M, Sjögren B, Svenningsson P, Andrén P E. The significance of biochemical and molecular sample integrity in brain proteomics and peptidomics: stathmin 2-20 and peptides as sample quality indicators. Proteomics. 2007;7:4445–4456. doi: 10.1002/pmic.200700142. [DOI] [PubMed] [Google Scholar]
  45. Bazinet R P, Lee H J, Felder C C, Porter A C, Rapoport S I, Rosenberger T A. Rapid high energy microwave fixation is required to determine the anandamide (N-arachidonoylethanolamine) concentration of rat brain. Neurochem Res. 2005;30:597–601. doi: 10.1007/s11064-005-2746-5. [DOI] [PubMed] [Google Scholar]
  46. Dassen H, Kamps R, Punyadeera C, Dijcks F, de Goeij A, Ederveen A, Dunselman G, Groothuis P. Haemoglobin expression in human endometrium. Hum Reprod. 2008;23:635–641. doi: 10.1093/humrep/dem430. [DOI] [PubMed] [Google Scholar]
  47. Wride M A, Mansergh F C, Adams S, Everitt R, Minnema S E, Rancourt D E, Evans M J. Expression profiling and gene discovery in the mouse lens. Mol Vis. 2003;9:360–396. [PubMed] [Google Scholar]
  48. Newton D A, Rao K M, Dluhy R A, Baatz J E. Hemoglobin is expressed by alveolar epithelial cells. J Biol Chem. 2006;281:5668–5676. doi: 10.1074/jbc.M509314200. [DOI] [PubMed] [Google Scholar]
  49. Bhaskaran M, Chen H, Chen Z, Liu L. Hemoglobin is expressed in alveolar epithelial type II cells. Biochem Biophys Res Commun. 2005;333:1348–1352. doi: 10.1016/j.bbrc.2005.06.042. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Liu L, Zeng M, Stamler J S. Hemoglobin induction in mouse macrophages. Proc Natl Acad Sci U S A. 1999;96:6643–6647. doi: 10.1073/pnas.96.12.6643. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Nishi H, Inagi R, Kato H, Tanemoto M, Kojima I, Son D, Fujita T, Nangaku M. Hemoglobin is expressed by mesangial cells and reduces oxidant stress. J Am Soc Nephrol. 2008;19:1500–1508. doi: 10.1681/ASN.2007101085. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Richter F, Meurer B H, Zhu C, Madvedeva V P, Chesselet M F. Neurons express hemoglobin α and β chains in rat and human brains. [E-pub ahead of print] J Comp Neurol. 2009 doi: 10.1002/cne.22062. doi:10.1002/cne.22062. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Slobodyansky E, Guidotti A, Wambebe C, Berkovich A, Costa E. Isolation and characterization of a rat brain triakontatetraneuropeptide, a posttranslational product of diazepam binding inhibitor: specific action at the Ro 5-4864 recognition site. J Neurochem. 1989;53:1276–1284. doi: 10.1111/j.1471-4159.1989.tb07425.x. [DOI] [PubMed] [Google Scholar]
  54. Goumon Y, Angelone T, Schoentgen F, Chasserot-Golaz S, Almas B, Fukami M M, Langley K, Welters I D, Tota B, Aunis D, Metz-Boutigue M H. The hippocampal cholinergic neurostimulating peptide, the N-terminal fragment of the secreted phosphatidylethanolamine-binding protein, possesses a new biological activity on cardiac physiology. J Biol Chem. 2004;279:13054–13064. doi: 10.1074/jbc.M308533200. [DOI] [PubMed] [Google Scholar]
  55. Badamchian M, Damavandy A A, Damavandy H, Wadhwa S D, Katz B, Goldstein A L. Identification and quantification of thymosin β4 in human saliva and tears. Ann N Y Acad Sci. 2007;1112:458–465. doi: 10.1196/annals.1415.046. [DOI] [PubMed] [Google Scholar]
  56. Ferro E S, Carreno F R, Goni C, Garrido P A, Guimaraes A O, Castro L M, Oliveira V, Araujo M C, Rioli V, Gomes M D, Fontenele-Neto J D, Hyslop S. The intracellular distribution and secretion of endopeptidases 24.15 (EC 3.4.24.15) and 24.16 (EC 3.4.24.16) Protein Pept Lett. 2004;11:415–421. doi: 10.2174/0929866043406706. [DOI] [PubMed] [Google Scholar]
  57. Millington W R, Smith D L. The posttranslational processing of β-endorphin in human hypothalamus. J Neurochem. 1991;57:775–781. doi: 10.1111/j.1471-4159.1991.tb08219.x. [DOI] [PubMed] [Google Scholar]

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