RESUMEN
BACKGROUND: l-amino acids, such as monosodium glutamate (MSG), activate the umami receptor T1R1/T1R3. We previously showed increased peristalsis in response to activation of T1R1/T1R3 by MSG in mouse colon. However, the expression and function of these receptors in the different regions of the stomach are not clear. METHODS: Mouse gastric smooth muscle cells (SMCs) were isolated and cultured in Dulbecco's Modified Eagle Medium. Expression of T1R1 and T1R3 was measured by RT-PCR and Western blot. The effect of MSG with and without inosine monophosphate (IMP, an allosteric activator of T1R1/T1R3) on acetylcholine (ACh)-induced contraction was measured in muscle strips and isolated SMCs by scanning micrometry. The effect of MSG with or without IMP on activation of G proteins and ACh-induced Ca2+ release was measured in SMCs. KEY RESULTS: Monosodium glutamate inhibited ACh-induced contractions in muscle strips from both antrum and fundus and the effect of MSG was augmented by IMP; the effects were concentration-dependent and not affected by the nitric oxide synthase inhibitor, L-NNA, or tetrodotoxin suggesting a direct effect on SMCs. In isolated gastric SMCs, T1R1 and T1R3 transcripts and protein were identified. Addition of MSG with or without IMP inhibited ACh-induced Ca2+ release and muscle contraction; the effect on contraction was blocked by pertussis toxin suggesting activation of Gαi proteins. MSG in the presence of IMP selectively activated Gαi2 . CONCLUSIONS AND INFERENCES: Umami receptors (T1R1/T1R3) are present on SMCs of the stomach, and activation of these receptors induces muscle relaxation by decreasing [Ca2+ ]i via Gαi2 .
Asunto(s)
Músculo Liso/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Estómago , Animales , Femenino , Masculino , Ratones , Ratones Endogámicos C57BLRESUMEN
The insulinotropic effects of the incretin hormone, glucagon-like peptide-1 (GLP-1) are mediated via GLP-1 receptors (GLP-1R) present on pancreatic ß cells. GLP-1 causes a decrease in the motility of stomach and intestine which involves both central and peripheral nervous systems. The expression and function of GLP-1R in gastrointestinal smooth muscle, however, are not clear. Muscle strips and isolated muscle cells were prepared from mouse colon and the effect of GLP-1(7-36) amide on acetylcholine (ACh)-induced contraction was measured. Muscle cells in culture were used to identify the expression of GLP-1R and the signaling pathways activated by GLP-1(7-36) amide. GLP-1R was expressed in the mucosal and non-mucosal tissue preparations derived from colon, and in smooth muscle cell cultures devoid of other cells such as enteric neurons. In colonic muscle strips, the addition of GLP-1(7-36) amide caused dose-dependent inhibition of acetylcholine-induced contractions. The effect of GLP-1(7-36) amide was partly inhibited by the neuronal blocker tetrodotoxin and nitric oxide (NO) synthase inhibitor l-NNA suggesting both NO-dependent neural and NO-independent direct effects on smooth muscle. In isolated colonic smooth muscle cells, GLP-1(7-36) amide caused an increase in Gαs activity, cAMP levels, and PKA activity, and inhibited ACh-induced contraction. The effect of GLP-1(7-36) amide on Gαs activity and cAMP levels was blocked by NF449, an inhibitor of Gαs, and the effect of GLP-1(7-36) amide on contraction was blocked by NF449 and myristoylated PKI, an inhibitor of PKA. We conclude that colonic smooth muscle cells express GLP-1R, and GLP-1(7-36) amide inhibits acetylcholine-induced contraction via GLP-1R coupled to the Gαs/cAMP/PKA pathway.
Asunto(s)
Acetilcolina/metabolismo , Receptor del Péptido 1 Similar al Glucagón/metabolismo , Contracción Muscular , Músculo Liso/metabolismo , Transducción de Señal , Animales , Colon/metabolismo , Colon/fisiología , AMP Cíclico/metabolismo , Expresión Génica , Péptido 1 Similar al Glucagón/metabolismo , Receptor del Péptido 1 Similar al Glucagón/genética , Receptor del Péptido 1 Similar al Glucagón/fisiología , Ratones , Músculo Liso/fisiologíaRESUMEN
BACKGROUND: Isovaleric acid (IVA) is a 5-carbon branched-chain fatty acid present in fermented foods and produced in the colon by bacterial fermentation of leucine. We previously reported that the shorter, straight-chain fatty acids acetate, propionate and butyrate differentially affect colonic motility; however, the effect of branched-chain fatty acids on gut smooth muscle and motility is unknown. AIMS: To determine the effect of IVA on contractility of colonic smooth muscle. METHODS: Murine colonic segments were placed in a longitudinal orientation in organ baths in Krebs buffer and fastened to force transducers. Segments were contracted with acetylcholine (ACh), and the effects of IVA on ACh-induced contraction were measured in the absence and presence of tetrodotoxin (TTx) or inhibitors of nitric oxide synthase [L-N-nitroarginine (L-NNA)] or adenylate cyclase (SQ22536). The effect of IVA on ACh-induced contraction was also measured in isolated muscle cells in the presence or absence of SQ22536 or protein kinase A (PKA) inhibitor (H-89). Direct activation of PKA was measured in isolated muscle cells. RESULTS: In colonic segments, ACh-induced contraction was inhibited by IVA in a concentration-dependent fashion; the IVA response was not affected by TTx or L-NNA but inhibited by SQ22536. Similarly, in isolated colonic muscle cells, ACh-induced contraction was inhibited by IVA in a concentration-dependent fashion and the effect blocked by SQ22536 and H-89. IVA also increased PKA activity in isolated smooth muscle cells. CONCLUSIONS: The branched-chain fatty acid IVA acts directly on colonic smooth muscle and causes muscle relaxation via the PKA pathway.
Asunto(s)
Colon/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , AMP Cíclico/metabolismo , Ácidos Grasos Volátiles/farmacología , Relajación Muscular/fisiología , Músculo Liso/metabolismo , Ácidos Pentanoicos/farmacología , Animales , Colon/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Femenino , Hemiterpenos , Masculino , Ratones , Ratones Endogámicos C57BL , Relajación Muscular/efectos de los fármacos , Músculo Liso/efectos de los fármacos , Técnicas de Cultivo de Órganos , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiologíaRESUMEN
In gastrointestinal smooth muscle, acetylcholine induced muscle contraction is biphasic, initial peak followed by sustained contraction. Contraction is regulated by phosphorylation of 20 kDa myosin light chain (MLC) at Ser19, interaction of actin and myosin, and actin polymerization. The present study characterized the signaling mechanisms involved in actin polymerization during initial and sustained muscle contraction in response to muscarinic M3 receptor activation in gastric smooth muscle cells by targeting the effectors of initial (phospholipase C (PLC)-ß/Ca2+ pathway) and sustained (RhoA/focal adhesion kinase (FAK)/Rho kinase pathway) contraction. The initial Ca2+ dependent contraction and actin polymerization is mediated by sequential activation of PLC-ß1 via Gαq, IP3 formation, Ca2+ release and Ca2+ dependent phosphorylation of proline-rich-tyrosine kinase 2 (Pyk2) at Tyr402. The sustained Ca2+ independent contraction and actin polymerization is mediated by activation of RhoA, and phosphorylation of FAK at Tyr397. Both phosphorylation of Pyk2 and FAK leads to phosphorylation of paxillin at Tyr118 and association of phosphorylated paxillin with the GEF proteins p21-activated kinase (PAK) interacting exchange factor α, ß (α and ß PIX) and DOCK 180. These GEF proteins stimulate Cdc42 leading to the activation of nucleation promoting factor N-WASP (neuronal Wiskott-Aldrich syndrome protein), which interacts with actin related protein complex 2/3 (Arp2/3) to induce actin polymerization and muscle contraction. Acetylcholine induced muscle contraction is inhibited by actin polymerization inhibitors. Thus, our results suggest that a novel mechanism for the regulation of smooth muscle contraction is mediated by actin polymerization in gastrointestinal smooth muscle which is independent of MLC20 phosphorylation.
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Actinas/metabolismo , Contracción Muscular/fisiología , Músculo Liso/fisiología , Multimerización de Proteína/fisiología , Estómago/fisiología , Animales , Calcio/metabolismo , Cationes Bivalentes/metabolismo , Ratones , Ratones Endogámicos C57BL , Cadenas Ligeras de Miosina/metabolismo , Fosforilación/fisiología , Conejos , Receptor Muscarínico M3 , Transducción de Señal/fisiologíaRESUMEN
BACKGROUND: A subset of cannabis users develop some degree of Cannabis Use Disorder (CUD). Although behavioral therapy has some success in treating CUD, many users relapse, often citing altered sleep, mood, and irritability. Preclinical animal tests of cannabinoid withdrawal focus primarily on somatic-related behaviors precipitated by a cannabinoid receptor antagonist. The goal of the present study was to develop novel cannabinoid withdrawal assays that are either antagonist-precipitated or spontaneously induced by abstinence. METHODS: C57BL/6â¯J mice were repeatedly administered the phytocannabinoid Δ9-tetrahydrocannabinol (THC; 1, 10 or 50â¯mg/kg, s.c.), the synthetic cannabinoid receptor agonist JWH-018 (1â¯mg/kg, s.c.), or vehicle (1:1:18 parts ethanol:Kolliphor EL:saline, s.c.) for 6 days. Withdrawal was precipitated with the cannabinoid receptor inverse agonist rimonabant (3â¯mg/kg, i.p.) or elicited via abstinence (i.e., spontaneous withdrawal), and putative stress-related behavior was scored. Classic somatic signs of cannabinoid withdrawal were also quantified. RESULTS: Precipitated THC withdrawal significantly increased plasma corticosterone. Precipitated withdrawal from either THC or JWH-018 suppressed marble burying, increased struggling in the tail suspension test, and elicited somatic withdrawal behaviors. The monoacylglycerol lipase inhibitor JZL184 attenuated somatic precipitated withdrawal but had no effect on marble burying or struggling. Spontaneous THC or JWH-018 withdrawal-induced paw tremors, head twitches, and struggled in the tail suspension test after 24-48â¯h abstinence. JZL184 or THC attenuated these spontaneous withdrawal-induced behaviors. CONCLUSION: Outcomes from tail suspension and marble burying tests reveal that THC withdrawal is multifaceted, eliciting and suppressing behaviors in these tests, in addition to inducing well-documented somatic signs of withdrawal.
Asunto(s)
Conducta Animal/efectos de los fármacos , Agonistas de Receptores de Cannabinoides/efectos adversos , Abuso de Marihuana/etiología , Síndrome de Abstinencia a Sustancias/etiología , Animales , Benzodioxoles/efectos adversos , Dronabinol/efectos adversos , Indoles/efectos adversos , Masculino , Ratones , Ratones Endogámicos C57BL , Naftalenos/efectos adversos , Piperidinas/efectos adversos , Pirazoles/efectos adversos , RimonabantRESUMEN
BACKGROUND AND PURPOSE: Gabapentin is commonly prescribed for nerve pain but may also cause dizziness, sedation and gait disturbances. Similarly, inhibition of the endogenous cannabinoid enzyme monoacylglycerol lipase (MAGL) has antinociceptive and anti-inflammatory properties but also induces sedation in mice at high doses. To limit these side effects, the present study investigated the analgesic effects of coadministering a MAGL inhibitor with gabapentin. EXPERIMENTAL APPROACH: Mice subjected to the chronic constriction injury model of neuropathic pain were administered the MAGL inhibitor KML29 (1-40 mg·kg-1 , i.p.), gabapentin (1-50 mg·kg-1 , i.p.) or both compounds. Mice were tested for mechanical and cold allodynia. The function and expression of cannabinoid CB1 receptors in whole brain homogenates and lipid profile of spinal cords were assessed after repeated drug administration. KEY RESULTS: The combination of low-dose KML29:gabapentin additively attenuated mechanical allodynia and synergistically reduced cold allodynia. The CB1 antagonist, rimonabant, partially reversed the anti-allodynic effects of KML29:gabapentin in mechanical allodynia but not cold allodynia. The anti-allodynic effects of KML29:gabapentin did not undergo tolerance in mechanical allodynia after repeated administration but produced mild tolerance in cold allodynia. High dose KML29 alone reduced CB1 receptor expression and function, but KML29:gabapentin reduced the density of CB1 receptors but did not alter their function. KML29:gabapentin influenced additional signalling pathways (including fatty acids) other than the pathways activated by a higher dose of either drug alone. CONCLUSION AND IMPLICATIONS: These data support the strategy of combining MAGL inhibition with a commonly prescribed analgesic as a therapeutic approach for attenuating neuropathic pain.
Asunto(s)
Aminas/farmacología , Analgésicos/farmacología , Benzodioxoles/farmacología , Ácidos Ciclohexanocarboxílicos/farmacología , Neuralgia/tratamiento farmacológico , Piperidinas/farmacología , Ácido gamma-Aminobutírico/farmacología , Aminas/administración & dosificación , Analgésicos/administración & dosificación , Animales , Benzodioxoles/administración & dosificación , Ácidos Ciclohexanocarboxílicos/administración & dosificación , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Sinergismo Farmacológico , Tolerancia a Medicamentos , Inhibidores Enzimáticos/administración & dosificación , Inhibidores Enzimáticos/farmacología , Gabapentina , Hiperalgesia/tratamiento farmacológico , Masculino , Ratones , Ratones Endogámicos C57BL , Monoacilglicerol Lipasas/antagonistas & inhibidores , Piperidinas/administración & dosificación , Pirazoles/farmacología , Receptor Cannabinoide CB1/efectos de los fármacos , Rimonabant , Transducción de Señal/efectos de los fármacos , Ácido gamma-Aminobutírico/administración & dosificaciónRESUMEN
Non-steroidal anti-inflammatory drugs (NSAIDs) are common analgesic drugs that also cause well-known, negative gastrointestinal (GI) side effects. The physiological mechanism(s) of NSAID-induced GI damage are unclear and are likely due to multiple causes. The most studied contributing mechanisms are increased gastric acid secretion and increased gastric motility. The present study was designed to determine which ulcerogenic effects of the NSAID diclofenac sodium are reversed by blocking the endocannabinoid catabolic enzyme monoacylglycerol lipase (MAGL). Both male and female mice were used to identify possible sex differences. We hypothesized that the MAGL inhibitor JZL184 would attenuate diclofenac-induced increases in both gastric acid secretion and gastric motility. Diclofenac dose-dependently induced gastric hemorrhages to a similar extent in both male and female mice. Gastric hemorrhage severity significantly correlated with gastric levels of myeloperoxidase, an objective measure of neutrophil infiltration. Similarly, JZL184 reduced gastric acidity, in controls as well as mice treated with pentagastrin, which stimulates gastric acid release. As hypothesized, JZL184 decreased gastric motility. Surprisingly, diclofenac also slowed gastric emptying, indicating that diclofenac-induced ulcers most likely occur through increased gastric acid secretion, and not increased gastric motility, as measured in the present study. Thus, MAGL inhibition may proffer gastroprotection through modulating the secretory pathway of gastric hemorrhage. These data underscore the importance of sampling multiple time points and using both sexes in research, in addition to multiple mechanistic targets, and contribute to the basic understanding of NSAID-induced gastric inflammation.
Asunto(s)
Antiinflamatorios no Esteroideos/efectos adversos , Inhibidores Enzimáticos/farmacología , Mucosa Gástrica/metabolismo , Motilidad Gastrointestinal/efectos de los fármacos , Monoacilglicerol Lipasas/antagonistas & inhibidores , Estómago/efectos de los fármacos , Animales , Cannabinoides/farmacología , Femenino , Ácido Gástrico/metabolismo , Hemorragia Gastrointestinal/inducido químicamente , Hemorragia Gastrointestinal/prevención & control , Masculino , Ratones , Ratones Endogámicos ICR , Peroxidasa/metabolismo , Estómago/fisiologíaRESUMEN
Dextromethorphan (DM) is a commonly used antitussive and is currently the only FDA-approved pharmaceutical treatment for pseudobulbar affect. Its safety profile and diverse pharmacologic actions in the central nervous system have stimulated new interest for repurposing it. Numerous preclinical investigations and many open-label or blinded clinical studies have demonstrated its beneficial effects across a variety of neurological and psychiatric disorders. However, the optimal dose and safety of chronic dosing are not fully known. This review summarizes the preclinical and clinical effects of DM and its putative mechanisms of action, focusing on depression, stroke, traumatic brain injury, seizure, pain, methotrexate neurotoxicity, Parkinson's disease and autism. Moreover, we offer suggestions for future research with DM to advance the treatment for these and other neurological and psychiatric disorders.
Asunto(s)
Dextrometorfano , Animales , Antitusígenos/farmacocinética , Antitusígenos/farmacología , Antitusígenos/uso terapéutico , Dextrometorfano/farmacocinética , Dextrometorfano/farmacología , Dextrometorfano/uso terapéutico , HumanosRESUMEN
BACKGROUND AND PURPOSE: Neuropathic pain is commonly treated with GABA analogues, steroids or non-steroidal anti-inflammatory drugs (NSAIDs). NSAIDs inhibit one or more COX isozymes but chronic COX inhibition paradoxically increases gastrointestinal inflammation and risk of unwanted cardiovascular events. The cannabinoids also have analgesic and anti-inflammatory properties and reduce neuropathic pain in animal models. The present study investigated the analgesic effects of inhibiting both monoacylglycerol lipase (MAGL) and COX enzymes, using low doses of both inhibitors. EXPERIMENTAL APPROACH: Mice subjected to chronic constriction injury (CCI) were tested for mechanical and cold allodynia after administration of the MAGL inhibitor, JZL184, or the non-selective COX inhibitor diclofenac. Then, both drugs were co-administered at fixed dose proportions of 1:3, 1:1 and 3:1, based on their ED50 values. PGs, endocannabinoids and related lipids were quantified in lumbar spinal cord. KEY RESULTS: Combining low doses of JZL184 and diclofenac synergistically attenuated mechanical allodynia and additively reduced cold allodynia. The cannabinoid CB1 receptor antagonist, rimonabant, but not the CB2 receptor antagonist, SR144528, blocked the analgesic effects of the JZL184 and diclofenac combination on mechanical allodynia, implying that CB1 receptors were primarily responsible for the anti-allodynia. Diclofenac alone and with JZL184 significantly reduced PGE2 and PGF2α in lumbar spinal cord tissue, whereas JZL184 alone caused significant increases in the endocannabinoid metabolite, N-arachidonoyl glycine. CONCLUSIONS AND IMPLICATIONS: Combining COX and MAGL inhibition is a promising therapeutic approach for reducing neuropathic pain with minimal side effects.
Asunto(s)
Analgésicos/uso terapéutico , Benzodioxoles/uso terapéutico , Inhibidores de la Ciclooxigenasa/uso terapéutico , Diclofenaco/uso terapéutico , Monoacilglicerol Lipasas/antagonistas & inhibidores , Neuralgia/tratamiento farmacológico , Piperidinas/uso terapéutico , Animales , Antagonistas de Receptores de Cannabinoides/farmacología , Sinergismo Farmacológico , Quimioterapia Combinada , Eicosanoides/metabolismo , Endocannabinoides/metabolismo , Ácidos Grasos/metabolismo , Hiperalgesia/tratamiento farmacológico , Hiperalgesia/metabolismo , Masculino , Ratones Endogámicos C57BL , Neuralgia/metabolismo , Piperidinas/farmacología , Prostaglandina-Endoperóxido Sintasas , Pirazoles/farmacología , Rimonabant , Médula Espinal/metabolismoRESUMEN
The physiological and behavioral effects of stress are well characterized. Endocannabinoids are produced on demand and function to attenuate many of the physiological effects of the stress response. The endocannabinoid system is made up of cannabinoid receptors, the fatty acid signaling molecules that bind to and activate these receptors, and the enzymes that synthesize and catabolize these endocannabinoid signaling molecules. Cannabinoid research has recently grown substantially, due in no small part to the development of genetic research models as well as highly selective pharmaceutical tools. The purpose of this minireview is to discuss a subset of the many parallels between cannabinoid and behavioral neuroimmunology research, with specific discussion of interactions between the endocannabinoid system and psychological stress, emotionality, and inflammation.