RESUMO
ABSTRACT: Diabetic neuropathy, often associated with diabetes mellitus, is a painful condition with no known effective treatment except glycemic control. Studies with neuropathic pain models report alterations in cannabinoid and opioid receptor expression levels; receptors whose activation induces analgesia. We examined whether interactions between CB1R and opioid receptors could be targeted for the treatment of diabetic neuropathy. For this, we generated antibodies that selectively recognize native CB1R-MOR and CB1R-DOR heteromers using a subtractive immunization strategy. We assessed the levels of CB1R, MOR, DOR, and interacting complexes using a model of streptozotocin-induced diabetic neuropathy and detected increased levels of CB1R, MOR, DOR, and CB1R-MOR complexes compared with those in controls. An examination of G-protein signaling revealed that activity induced by the MOR, but not the DOR agonist, was potentiated by low nanomolar doses of CB1R ligands, including antagonists, suggesting an allosteric modulation of MOR signaling by CB1R ligands within CB1R-MOR complexes. Because the peptide endocannabinoid, hemopressin, caused a significant potentiation of MOR activity, we examined its effect on mechanical allodynia and found that it blocked allodynia in wild-type mice and mice with diabetic neuropathy lacking DOR (but have CB1R-MOR complexes). However, hemopressin does not alter the levels of CB1R-MOR complexes in diabetic mice lacking DOR but increases the levels of CB1R-DOR complexes in diabetic mice lacking MOR. Together, these results suggest the involvement of CB1R-MOR and CB1R-DOR complexes in diabetic neuropathy and that hemopressin could be developed as a potential therapeutic for the treatment of this painful condition.
Assuntos
Canabinoides , Diabetes Mellitus Experimental , Neuropatias Diabéticas , Neuralgia , Animais , Diabetes Mellitus Experimental/complicações , Neuropatias Diabéticas/tratamento farmacológico , Modelos Animais de Doenças , Hiperalgesia/tratamento farmacológico , Hiperalgesia/etiologia , Ligantes , Camundongos , Neuralgia/tratamento farmacológico , Receptores Opioides , Receptores Opioides mu/metabolismoRESUMO
The N-terminal region of G protein-coupled receptors can be efficiently targeted for the generation of receptor-selective antibodies. These antibodies are useful for the biochemical characterization of the receptors. In this study, we developed a set of criteria to select the optimal epitope and applied them to generate antibodies to the N-terminal region of 34 different G protein-coupled receptors. The antibody characterization revealed that a subset of antibodies exhibited increased recognition of the receptor following agonist treatment and this increase could be blocked by treatment with the receptor antagonist. An analysis of the epitopes showed that those antibodies that exhibit increased recognition are on average twelve residues long, have an overall net negative charge and are enriched in aspartic and glutamic acids. These antibodies are useful since they facilitate studies examining dose dependent increases in recognition of receptors in heterologous cells as well as in native tissue. Another interesting use of these antibodies is that they facilitate measuring changes in receptor recognition in brain following peripheral drug administration; for example, systemic administration of cocaine, a blocker of dopamine transporter that increases local dopamine levels at the synapse, was found to lead to increases in antibody recognition of dopamine receptors in the brain. Taken together these studies, in addition to describing novel tools to study native receptors, provide a framework for the generation of antibodies to G protein-coupled receptors that can detect ligand-induced conformational changes.
Assuntos
Anticorpos/imunologia , Receptores Acoplados a Proteínas G/imunologia , Animais , Ensaio de Imunoadsorção Enzimática , Epitopos/química , Epitopos/imunologia , Células HEK293 , Humanos , Masculino , Conformação Proteica , Coelhos , Ratos , Ratos Wistar , Receptores Acoplados a Proteínas G/agonistas , Receptores Acoplados a Proteínas G/antagonistas & inibidores , Receptores Acoplados a Proteínas G/químicaRESUMO
Intracellular peptides generated by limited proteolysis are likely to function inside and outside cells and could represent new possibilities for drug development. Here, we used several conformational-sensitive antibodies targeting G-protein coupled receptors to screen for novel pharmacological active peptides. We find that one of these peptides, DITADDEPLT activates cannabinoid type 1 receptors. Single amino acid modifications identified a novel peptide, DIIADDEPLT (Pep19), with slightly better inverse agonist activity at cannabinoid type 1 receptors. Pep19 induced uncoupling protein 1 expression in both white adipose tissue and 3T3-L1 differentiated adipocytes; in the latter, Pep19 activates pERK1/2 and AKT signaling pathways. Uncoupling protein 1 expression induced by Pep19 in 3T3-L1 differentiated adipocytes is blocked by AM251, a cannabinoid type 1 receptors antagonist. Oral administration of Pep19 into diet-induced obese Wistar rats significantly reduces adiposity index, whole body weight, glucose, triacylglycerol, cholesterol and blood pressure, without altering heart rate; changes in the number and size of adipocytes were also observed. Pep19 has no central nervous system effects as suggested by the lack of brain c-Fos expression, cell toxicity, induction of the cannabinoid tetrad, depressive- and anxiety-like behaviors. Therefore, Pep19 has several advantages over previously identified peripherally active cannabinoid compounds, and could have clinical applications.
Assuntos
Adipócitos/metabolismo , Tecido Adiposo Branco/metabolismo , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Obesidade/tratamento farmacológico , Peptídeos , Receptor CB1 de Canabinoide/antagonistas & inibidores , Células 3T3-L1 , Adipócitos/patologia , Tecido Adiposo Branco/patologia , Animais , Encéfalo/metabolismo , Encéfalo/patologia , Dieta Hiperlipídica/efeitos adversos , Masculino , Camundongos , Obesidade/induzido quimicamente , Obesidade/metabolismo , Obesidade/patologia , Peptídeos/química , Peptídeos/farmacologia , Ratos , Ratos Wistar , Receptor CB1 de Canabinoide/metabolismoRESUMO
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.
Assuntos
Agonismo Inverso de Drogas , Hemoglobinas/farmacologia , Fragmentos de Peptídeos/farmacologia , Receptor CB1 de Canabinoide/agonistas , Linhagem Celular , Humanos , LigantesRESUMO
To date, the endogenous ligands described for cannabinoid receptors have been derived from membrane lipids. To identify a peptide ligand for CB1 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 CB1 cannabinoid receptors. We find that hemopressin is a CB1 receptor-selective antagonist, because it is able to efficiently block signaling by CB1 receptors but not by other members of family A G protein-coupled receptors (including the closely related CB2 receptors). Hemopressin also behaves as an inverse agonist of CB1 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 CB1 cannabinoid receptors that also exhibits analgesic properties. These findings are likely to have a profound impact on the development of novel therapeutics targeting CB1 receptors.