RESUMEN

Over the past decade, boldine, a naturally occurring alkaloid found in several plant species including the Chilean Boldo tree, has garnered attention for its efficacy in rodent models of human disease. Some of the properties that have been attributed to boldine include antioxidant activities, neuroprotective and analgesic actions, hepatoprotective effects, anti-inflammatory actions, cardioprotective effects and anticancer potential. Compelling data now indicates that boldine blocks connexin (Cx) hemichannels (HCs) and that many if not all of its effects in rodent models of injury and disease are due to CxHC blockade. Here we provide an overview of boldine's pharmacological properties, including its efficacy in rodent models of common human injuries and diseases, and of its absorption, distribution, pharmacokinetics, and metabolism.

RESUMEN

Chronic pain is a major health issue, and the search for new analgesics has become increasingly important because of the addictive properties and unwanted side effects of opioids. To explore potentially new drug targets, we investigated mutations in the NTRK1 gene found in individuals with congenital insensitivity to pain with anhidrosis (CIPA). NTRK1 encodes tropomyosin receptor kinase A (TrkA), the receptor for nerve growth factor (NGF) and that contributes to nociception. Molecular modeling and biochemical analysis identified mutations that decreased the interaction between TrkA and one of its substrates and signaling effectors, phospholipase Cγ (PLCγ). We developed a cell-permeable phosphopeptide derived from TrkA (TAT-pQYP) that bound the Src homology domain 2 (SH2) of PLCγ. In HEK-293T cells, TAT-pQYP inhibited the binding of heterologously expressed TrkA to PLCγ and decreased NGF-induced, TrkA-mediated PLCγ activation and signaling. In mice, intraplantar administration of TAT-pQYP decreased mechanical sensitivity in an inflammatory pain model, suggesting that targeting this interaction may be analgesic. The findings demonstrate a strategy to identify new targets for pain relief by analyzing the signaling pathways that are perturbed in CIPA.

Asunto(s)

Hipohidrosis , Mutación , Insensibilidad Congénita al Dolor , Fosfolipasa C gamma , Receptor trkA , Analgésicos/farmacología , Animales , Canalopatías/genética , Canalopatías/metabolismo , Células HEK293 , Humanos , Hipohidrosis/genética , Hipohidrosis/metabolismo , Ratones , Factor de Crecimiento Nervioso/genética , Factor de Crecimiento Nervioso/farmacología , Dolor/genética , Dolor/metabolismo , Insensibilidad Congénita al Dolor/genética , Insensibilidad Congénita al Dolor/metabolismo , Fosfolipasa C gamma/genética , Fosfolipasa C gamma/metabolismo , Receptor trkA/genética , Receptor trkA/metabolismo

RESUMEN

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.

Asunto(s)

Cannabinoides , Diabetes Mellitus Experimental , Neuropatías Diabéticas , Neuralgia , Animales , Diabetes Mellitus Experimental/complicaciones , Neuropatías Diabéticas/tratamiento farmacológico , Modelos Animales de Enfermedad , Hiperalgesia/tratamiento farmacológico , Hiperalgesia/etiología , Ligandos , Ratones , Neuralgia/tratamiento farmacológico , Receptores Opioides , Receptores Opioides mu/metabolismo

RESUMEN

Oxytocin (OT) and vasopressin (AVP) are endogenous ligands for OT and AVP receptors in the brain and in the peripheral system. Several studies demonstrate that OT and AVP have opposite roles in modulating stress, anxiety and social behaviours. Interestingly, both peptides and their receptors exhibit high sequence homology which could account for the biased signalling interaction of the peptides with OT and AVP receptors. However, how and under which conditions this crosstalk occurs in vivo remains unclear. In this review we shed light on the complexity of the roles of OT and AVP, by focusing on their signalling and behavioural differences and exploring the crosstalk between the receptor systems. Moreover, we discuss the potential of OT and AVP receptors as therapeutic targets to treat human disorders, such as autism, schizophrenia and drug abuse. LINKED ARTICLES: This article is part of a themed issue on Building Bridges in Neuropharmacology. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v179.8/issuetoc.

Asunto(s)

Oxitocina , Vasopresinas , Encéfalo/metabolismo , Humanos , Ligandos , Oxitocina/farmacología , Oxitocina/uso terapéutico , Receptores de Oxitocina/metabolismo , Receptores de Vasopresinas/metabolismo , Conducta Social , Vasopresinas/farmacología

RESUMEN

It is well accepted that treatment of chronic pain with morphine leads to µ opioid receptor (MOR) desensitization and the development of morphine tolerance. MOR activation by the selective peptide agonist, D-Ala2, N-MePhe4, Gly-ol]-enkephalin(DAMGO), leads to robust G protein receptor kinase activation, ß-arrestin recruitment, and subsequent receptor endocytosis, which does not occur in an activation by morphine. However, MOR activation by morphine induces receptor desensitization, in a Protein kinase C (PKC) dependent manner. PKC inhibitors have been reported to decrease receptor desensitization, reduce opiate tolerance, and increase analgesia. However, the exact role of PKC in these processes is not clearly delineated. The difficulties in establishing a particular role for PKC have been, in part, due to the lack of reagents that allow the selective identification of PKC targets. Recently, we generated a conformation state-specific anti-PKC antibody that preferentially recognizes the active state of this kinase. Using this antibody to selectively isolate PKC substrates and a proteomics strategy to establish the identity of the proteins, we examined the effect of morphine treatment on the PKC targets. We found an enhanced interaction of a number of proteins with active PKC, in the presence of morphine. In this article, we discuss the role of these proteins in PKC-mediated MOR desensitization and analgesia. In addition, we posit a role for some of these proteins in mediating pain by TrKA activation, via the activation of transient receptor potential cation channel subfamily V member 1 (TRPV1). Finally, we discuss how these new PKC interacting proteins and pathways could be targeted for the treatment of pain.

RESUMEN

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.

Asunto(s)

Anticuerpos/inmunología , Receptores Acoplados a Proteínas G/inmunología , Animales , Ensayo de Inmunoadsorción Enzimática , Epítopos/química , Epítopos/inmunología , Células HEK293 , Humanos , Masculino , Conformación Proteica , Conejos , Ratas , Ratas Wistar , Receptores Acoplados a Proteínas G/agonistas , Receptores Acoplados a Proteínas G/antagonistas & inhibidores , Receptores Acoplados a Proteínas G/química

RESUMEN

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.

Asunto(s)

Adipocitos/metabolismo , Tejido Adiposo Blanco/metabolismo , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Obesidad/tratamiento farmacológico , Péptidos , Receptor Cannabinoide CB1/antagonistas & inhibidores , Células 3T3-L1 , Adipocitos/patología , Tejido Adiposo Blanco/patología , Animales , Encéfalo/metabolismo , Encéfalo/patología , Dieta Alta en Grasa/efectos adversos , Masculino , Ratones , Obesidad/inducido químicamente , Obesidad/metabolismo , Obesidad/patología , Péptidos/química , Péptidos/farmacología , Ratas , Ratas Wistar , Receptor Cannabinoide CB1/metabolismo

RESUMEN

Protein kinase C (PKC) plays a regulatory role in key pathways in cancer. However, since phosphorylation is a step for classical PKC (cPKC) maturation and does not correlate with activation, there is a lack of tools to detect active PKC in tissue samples. Here, a structure-based rational approach was used to select a peptide to generate an antibody that distinguishes active from inactive cPKC. A peptide conserved in all cPKCs, C2Cat, was chosen since modeling studies based on a crystal structure of PKCß showed that it is localized at the interface between the C2 and catalytic domains of cPKCs in an inactive kinase. Anti-C2Cat recognizes active cPKCs at least two-fold better than inactive kinase in ELISA and immunoprecipitation assays, and detects the temporal dynamics of cPKC activation upon receptor or phorbol stimulation. Furthermore, the antibody is able to detect active PKC in human tissue. Higher levels of active cPKC were observed in the more aggressive triple negative breast cancer tumors as compared to the less aggressive estrogen receptor positive tumors. Thus, this antibody represents a reliable, hitherto unavailable and a valuable tool to study PKC activation in cells and tissues. Similar structure-based rational design strategies can be broadly applied to obtain active-state specific antibodies for other signal transduction molecules.

Asunto(s)

Anticuerpos/metabolismo , Neoplasias de la Mama/metabolismo , Neuroblastoma/metabolismo , Proteína Quinasa C beta/metabolismo , Sitios de Unión/inmunología , Neoplasias de la Mama/inmunología , Neoplasias de la Mama/patología , Carcinogénesis , Línea Celular Tumoral , Activación Enzimática , Femenino , Humanos , Isoenzimas/inmunología , Estadificación de Neoplasias , Neuroblastoma/inmunología , Neuroblastoma/patología , Fragmentos de Péptidos/inmunología , Conformación Proteica , Dominios Proteicos/genética , Proteína Quinasa C beta/genética , Proteína Quinasa C beta/inmunología , Receptores de Estrógenos/metabolismo , Transducción de Señal , Relación Estructura-Actividad

RESUMEN

Direct-acting cannabinoid receptor ligands are well known to reduce hyperalgesic responses after nerve injury, although their psychoactive side effects have damped enthusiasm for their therapeutic development. Hemopressin (Hp) is a nonapeptide that selectively binds CB1 cannabinoid receptors (CB1 receptors) and exerts antinociceptive action in inflammatory pain models. We investigated the effect of Hp on neuropathic pain in rats subjected to chronic constriction injury (CCI) of the sciatic nerve, and explored the mechanisms involved. Oral administration of Hp inhibits mechanical hyperalgesia of CCI-rats up to 6h. Hp treatment also decreases Egr-1 immunoreactivity (Egr-1Ir) in the superficial layer of the dorsal horn of the spinal cord of CCI rats. The antinociceptive effect of Hp seems to be independent of inhibitory descending pain pathway since methysergide (5HT1A receptor antagonist) and yohimbine (α-2 adrenergic receptor antagonist) were unable to prevent Hp antinociceptive effect. Hp decreased calcium flux on DRG neurons from CCI rats, similarly to that observed for AM251, a CB1 receptor antagonist. We also investigated the effect of Hp on potassium channels of CCI rats using UCL 1684 (a blocker of Ca(2+)-activated K(+) channels) which reversed Hp-induced antinociception. Furthermore, concomitant administration of URB-584 (FAAH inhibitor) but not JZL-184 (MAGL inhibitor) potentiates antinociceptive effect of Hp in CCI rats indicating an involvement of anadamide on HP-induced antinociception. Together, these data demonstrate that Hp displays antinociception in pain from neuropathic etiology through local effects. The release of anandamide and the opening of peripheral K(+) channels are involved in the antinociceptive effect.

Asunto(s)

Agonistas de Receptores de Cannabinoides/uso terapéutico , Hemoglobinas/uso terapéutico , Neuralgia/tratamiento farmacológico , Fragmentos de Péptidos/uso terapéutico , Animales , Cannabinoides/metabolismo , Inmunohistoquímica , Masculino , Neuralgia/metabolismo , Canales de Potasio/metabolismo , Ratas , Ratas Wistar

RESUMEN

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.

Asunto(s)

Agonismo Inverso de Drogas , Hemoglobinas/farmacología , Fragmentos de Péptidos/farmacología , Receptor Cannabinoide CB1/agonistas , Línea Celular , Humanos , Ligandos

RESUMEN

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.

Asunto(s)

Animales , Ratas , Endocannabinoides , Inflamación/clasificación

RESUMEN

Several studies have proposed that angiotensin II (Ang II) binds to its receptor AT1 through interactions with residues in helices V and VI, suggesting that the distance between these helices is crucial for ligand binding. Based on a 3D model of AT1 in which the C-terminus of Ang II is docked, we identified the hydrophobic residues of TM V and VI pointing towards the external face of the helices, which may play a role in the structure of the binding pocket and in the structural integrity of the receptor. We performed a systematic mutagenesis study of these residues and examined the binding, localization, maturation, and dimerization of the mutated receptors. We found that mutations of hydrophobic residues to alanine in helix V do not alter binding, whereas mutations to glutamate lead to loss of binding without a loss in cell surface expression, suggesting that the external face of helix V may not directly participate in binding, but may rather contribute to the structure of the binding pocket. In contrast, mutations of hydrophobic residues to glutamate in helix VI lead to a loss in cell surface expression, suggesting that the external surface of helix VI plays a structural role and ensures correct folding of the receptor.

Asunto(s)

Angiotensina II/metabolismo , Proteínas de la Membrana/química , Estructura Secundaria de Proteína , Receptor de Angiotensina Tipo 1/metabolismo , Alanina/química , Alanina/genética , Secuencia de Aminoácidos , Angiotensina II/química , Angiotensina II/genética , Membrana Celular/genética , Membrana Celular/metabolismo , Regulación de la Expresión Génica , Ácido Glutámico/química , Ácido Glutámico/genética , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Ligandos , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Datos de Secuencia Molecular , Mutación , Unión Proteica , Estructura Terciaria de Proteína , Receptor de Angiotensina Tipo 1/química , Receptor de Angiotensina Tipo 1/genética