RESUMEN
This study focused on characterizing the potential mechanism of valvular toxicity caused by TGFß receptor inhibitors (TGFßRis) using rat valvular interstitial cells (VICs) to evaluate early biological responses to TGFßR inhibition. Three TGFßRis that achieved similar exposures in the rat were assessed. Two dual TGFßRI/-RII inhibitors caused valvulopathy, whereas a selective TGFßRI inhibitor did not, leading to a hypothesis that TGFß receptor selectivity may influence the potency of valvular toxicity. The dual valvular toxic inhibitors had the most profound effect on altering VIC phenotype including altered morphology, migration, and extracellular matrix production. Reduction of TGFß expression demonstrated that combined TGFß2/ß3 inhibition by small interfering RNA or neutralizing antibodies caused similar alterations as TGFßRis. Inhibition of TGFß3 transcription was only associated with the dual TGFßRis, suggesting that TGFßRII inhibition impacts TGFß3 transcriptional regulation, and that the potency of valvular toxicity may relate to alteration of TGFß2/ß3-mediated processes involved in maintaining proper balance of VIC phenotypes in the heart valve.
RESUMEN
Many in vitro gastrointestinal models have been developed with the hope that they will continue to improve in their similarity to the organs from which they were isolated. Intestinal organoids isolated from various species are now being used to investigate physiology and pathophysiology. In this study, intestinal stem cells were isolated from adult rat duodenum and culture conditions were optimized to promote the growth, differentiation and development of 3D organoids. We optimized and characterized rat duodenal organoids with light and electron microscopy, immunofluorescence and notably, global mRNA expression. The metabolic capacity of these cultures was investigated using probe substrates for multiple phase I and phase II drug metabolizing enzymes and found to be in line with previous results from intestinal primary cultures and a significant improvement over immortalized cell lines. Over the course of differentiation, the gene expression profiles of the rat duodenal organoids were consistent with expected trends in differentiation to various cell lineages reflecting the duodenum in vivo. Further, incubations of these cultures with naproxen and celecoxib resulted in cytotoxicity consistent with the direct cytotoxic effects of these drugs to duodenum in vivo. Based on these characteristics, the rat duodenal organoids described herein will provide a novel platform for investigating a wide variety of mechanistic questions.
Asunto(s)
Antiinflamatorios no Esteroideos/toxicidad , Diferenciación Celular/efectos de los fármacos , Duodeno/efectos de los fármacos , Mucosa Intestinal/efectos de los fármacos , Organoides/efectos de los fármacos , Animales , Antiinflamatorios no Esteroideos/metabolismo , Diferenciación Celular/fisiología , Células Cultivadas , Duodeno/citología , Duodeno/metabolismo , Femenino , Mucosa Intestinal/citología , Mucosa Intestinal/metabolismo , Organoides/metabolismo , Ratas , Ratas Sprague-Dawley , Células Madre/efectos de los fármacos , Células Madre/metabolismoRESUMEN
Neuropathic pain resulting from nerve lesions or dysfunction represents one of the most challenging neurological diseases to treat. A better understanding of the molecular mechanisms responsible for causing these maladaptive responses can help develop novel therapeutic strategies and biomarkers for neuropathic pain. We performed a miRNA expression profiling study of dorsal root ganglion (DRG) tissue from rats four weeks post spinal nerve ligation (SNL), a model of neuropathic pain. TaqMan low density arrays identified 63 miRNAs whose level of expression was significantly altered following SNL surgery. Of these, 59 were downregulated and the ipsilateral L4 DRG, not the injured L5 DRG, showed the most significant downregulation suggesting that miRNA changes in the uninjured afferents may underlie the development and maintenance of neuropathic pain. TargetScan was used to predict mRNA targets for these miRNAs and it was found that the transcripts with multiple predicted target sites belong to neurologically important pathways. By employing different bioinformatic approaches we identified neurite remodeling as a significantly regulated biological pathway, and some of these predictions were confirmed by siRNA knockdown for genes that regulate neurite growth in differentiated Neuro2A cells. In vitro validation for predicted target sites in the 3'-UTR of voltage-gated sodium channel Scn11a, alpha 2/delta1 subunit of voltage-dependent Ca-channel, and purinergic receptor P2rx ligand-gated ion channel 4 using luciferase reporter assays showed that identified miRNAs modulated gene expression significantly. Our results suggest the potential for miRNAs to play a direct role in neuropathic pain.
Asunto(s)
Perfilación de la Expresión Génica , Regulación de la Expresión Génica , MicroARNs/genética , Neuralgia/genética , Nervios Espinales/metabolismo , Nervios Espinales/patología , Animales , Minería de Datos , Modelos Animales de Enfermedad , Pruebas de Enzimas , Ganglios Espinales/metabolismo , Ganglios Espinales/patología , Técnicas de Silenciamiento del Gen , Genes Reporteros , Ligadura , Luciferasas/metabolismo , Masculino , Ratones , MicroARNs/metabolismo , MicroARNs/normas , Neuralgia/patología , Control de Calidad , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN Interferente Pequeño/metabolismo , Ratas , Ratas Sprague-Dawley , Estándares de Referencia , Reproducibilidad de los ResultadosRESUMEN
Endocannabinoids are lipid molecules that serve as natural ligands for the cannabinoid receptors CB1 and CB2. They modulate a diverse set of physiological processes such as pain, cognition, appetite, and emotional states, and their levels and functions are tightly regulated by enzymatic biosynthesis and degradation. 2-Arachidonoylglycerol (2-AG) is the most abundant endocannabinoid in the brain and is believed to be hydrolyzed primarily by the serine hydrolase monoacylglycerol lipase (MAGL). Although 2-AG binds and activates cannabinoid receptors in vitro, when administered in vivo, it induces only transient cannabimimetic effects as a result of its rapid catabolism. Here we show using a mouse model with a targeted disruption of the MAGL gene that MAGL is the major modulator of 2-AG hydrolysis in vivo. Mice lacking MAGL exhibit dramatically reduced 2-AG hydrolase activity and highly elevated 2-AG levels in the nervous system. A lack of MAGL activity and subsequent long-term elevation of 2-AG levels lead to desensitization of brain CB1 receptors with a significant reduction of cannabimimetic effects of CB1 agonists. Also consistent with CB1 desensitization, MAGL-deficient mice do not show alterations in neuropathic and inflammatory pain sensitivity. These findings provide the first genetic in vivo evidence that MAGL is the major regulator of 2-AG levels and signaling and reveal a pivotal role for 2-AG in modulating CB1 receptor sensitization and endocannabinoid tone.
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Moduladores de Receptores de Cannabinoides/fisiología , Endocannabinoides , Monoacilglicerol Lipasas/metabolismo , Receptor Cannabinoide CB1/fisiología , Animales , Activación Enzimática/genética , Activación Enzimática/fisiología , Hidrólisis , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Monoacilglicerol Lipasas/deficiencia , Monoacilglicerol Lipasas/fisiología , Dimensión del Dolor/métodosRESUMEN
Endocannabinoids (eCBs) function as retrograde signaling molecules at synapses throughout the brain, regulate axonal growth and guidance during development, and drive adult neurogenesis. There remains a lack of genetic evidence as to the identity of the enzyme(s) responsible for the synthesis of eCBs in the brain. Diacylglycerol lipase-alpha (DAGLalpha) and -beta (DAGLbeta) synthesize 2-arachidonoyl-glycerol (2-AG), the most abundant eCB in the brain. However, their respective contribution to this and to eCB signaling has not been tested. In the present study, we show approximately 80% reductions in 2-AG levels in the brain and spinal cord in DAGLalpha(-/-) mice and a 50% reduction in the brain in DAGLbeta(-/-) mice. In contrast, DAGLbeta plays a more important role than DAGLalpha in regulating 2-AG levels in the liver, with a 90% reduction seen in DAGLbeta(-/-) mice. Levels of arachidonic acid decrease in parallel with 2-AG, suggesting that DAGL activity controls the steady-state levels of both lipids. In the hippocampus, the postsynaptic release of an eCB results in the transient suppression of GABA-mediated transmission at inhibitory synapses; we now show that this form of synaptic plasticity is completely lost in DAGLalpha(-/-) animals and relatively unaffected in DAGLbeta(-/-) animals. Finally, we show that the control of adult neurogenesis in the hippocampus and subventricular zone is compromised in the DAGLalpha(-/-) and/or DAGLbeta(-/-) mice. These findings provide the first evidence that DAGLalpha is the major biosynthetic enzyme for 2-AG in the nervous system and reveal an essential role for this enzyme in regulating retrograde synaptic plasticity and adult neurogenesis.
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Encéfalo/metabolismo , Moduladores de Receptores de Cannabinoides/fisiología , Endocannabinoides , Lipoproteína Lipasa/genética , Animales , Ácidos Araquidónicos/metabolismo , Encéfalo/citología , Glicéridos/metabolismo , Hipocampo/citología , Hipocampo/metabolismo , Hígado/metabolismo , Ratones , Ratones Noqueados , Neurogénesis , Plasticidad Neuronal , Transducción de Señal , Médula Espinal/metabolismo , Sinapsis/fisiologíaRESUMEN
Validation of gait analysis has the potential to bridge the gap between data from animal pain models and clinical observations. The goal of these studies was to compare alterations in gait due to inflammation or nerve injury to traditional pain measurements in animals. Pharmacological experiments determined whether gait alterations were related to enhanced nociception, edema, or motor nerve dysfunction. Gait was analyzed using an automated system (DigiGait) after injection of an inflammatory agent (carrageenan; CARR or FCA; Freund's complete adjuvant) or nerve injury (axotomy; AXO, partial sciatic nerve ligation; PSNL, spinal nerve ligation; SNL or chronic constriction injury; CCI). All models caused significant alterations in gait and thermal (inflammatory) or mechanical (nerve injury) hyperalgesia. Both indomethacin and morphine were able to block or reverse thermal hyperalgesia and normalize gait in the CARR model. Indomethacin partially blocked and did not reverse paw edema, suggesting that gait alterations must be primarily driven by enhanced nociception. In nerve injury models, AXO, PSNL, CCI, and SNL caused changes to the largest number of gait indices with the rank order being AXO>PSNL=CCI >> SNL. Gabapentin and duloxetine reversed mechanical hyperalgesia but did not normalize gait in any nerve injury model. Collectively, these data suggest that pain is the primary driver of abnormal gait in models of inflammatory but not nerve injury-related pain and suggests that, in the latter, disruption in gait is due to perturbation to the motor system. Gait may therefore constitute an alternative and potentially clinically relevant measure of pain due to inflammation.
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Marcha/efectos de los fármacos , Inflamación/fisiopatología , Dolor/fisiopatología , Nervio Ciático/lesiones , Aminas/farmacología , Analgésicos Opioides/farmacología , Animales , Antiinflamatorios no Esteroideos/farmacología , Axotomía , Carragenina , Ácidos Ciclohexanocarboxílicos/farmacología , Clorhidrato de Duloxetina , Edema/inducido químicamente , Edema/fisiopatología , Adyuvante de Freund , Gabapentina , Marcha/fisiología , Calor , Hiperalgesia/inducido químicamente , Hiperalgesia/fisiopatología , Indometacina/farmacología , Inflamación/inducido químicamente , Masculino , Morfina/farmacología , Neuralgia/fisiopatología , Dolor/inducido químicamente , Dimensión del Dolor/efectos de los fármacos , Umbral del Dolor/efectos de los fármacos , Estimulación Física/efectos adversos , Ratas , Ratas Sprague-Dawley , Nervio Ciático/fisiopatología , Tiofenos/farmacología , Ácido gamma-Aminobutírico/farmacologíaRESUMEN
Interstitial cystitis (IC) is a chronic pelvic-perineal pain syndrome of unknown etiology that mainly targets the lower urinary tract. It is characterized by urinary frequency and urgency, inflammation, stiffening of the bladder wall, and visceral pain. Pain is the most prominent feature of IC and current treatments provide limited relief. To facilitate the identification of novel therapeutic options for IC, efforts are being made to identify more predictive rodent models. A well-characterized rat model is based on administration of cyclophosphamide (CP). The protocol described in this unit replicates this model in mice, demonstrating that CP increases both spontaneous and evoked pain behaviors in this species. This mouse model can be used for understanding disease etiology as well as for the pharmacological evaluation of novel therapeutics for the treatment of IC pain.
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Analgésicos/farmacología , Cistitis Intersticial/fisiopatología , Modelos Animales de Enfermedad , Dolor Pélvico/prevención & control , Animales , Conducta Animal/efectos de los fármacos , Recolección de Datos/métodos , Masculino , Ratones , Umbral del Dolor/efectos de los fármacos , Dolor Pélvico/etiologíaRESUMEN
Here, we have investigated the in vitro pharmacology of a muscarinic agonist, (3R,4R)-3-(3-hexylsulfanyl-pyrazin-2-yloxy)-1-aza-bicyclo[2.2.1]heptane (WAY-132983), and we demonstrated its activity in several models of pain. WAY-132983 had a similar affinity for the five muscarinic receptors (9.4-29.0 nM); however, in calcium mobilization studies it demonstrated moderate selectivity for M(1) (IC(50) = 6.6 nM; E(max) = 65% of 10 muM carbachol-stimulation) over the M(3) (IC(50) = 23 nM; E(max) = 41%) and M(5) receptors (IC(50) = 300 nM; E(max) = 18%). WAY-132983 also activated the M(4) receptor, fully inhibiting forskolin-induced increase in cAMP levels (IC(50) = 10.5 nM); at the M(2) receptor its potency was reduced by 5-fold (IC(50) = 49.8 nM). In vivo, WAY-132983 demonstrated good systemic bioavailability and high brain penetration (>20-fold over plasma levels). In addition, WAY-1329823 produced potent and efficacious antihyperalgesic and antiallodynic effects in rodent models of chemical irritant, chronic inflammatory, neuropathic, and incisional pain. It is noteworthy that efficacy in these models was observed at doses that did not produce analgesia or ataxia. Furthermore, a series of antagonist studies demonstrated that the in vivo activity of WAY-132983 is mediated through activation of muscarinic receptors primarily through the M(4) receptor. The data presented herein suggest that muscarinic agonists, such as WAY-132983, may have a broad therapeutic efficacy for the treatment of pain.
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Hidrocarburos Aromáticos con Puentes/farmacocinética , Agonistas Muscarínicos/farmacología , Dolor/prevención & control , Pirazinas/farmacocinética , Animales , Disponibilidad Biológica , Hidrocarburos Aromáticos con Puentes/farmacología , Enfermedad Crónica , Modelos Animales de Enfermedad , Inflamación , Concentración 50 Inhibidora , Pirazinas/farmacología , Ratas , Receptores MuscarínicosRESUMEN
Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are responsible for the functional hyperpolarization-activated current (I(h)) in dorsal root ganglion (DRG) neurons, playing an important role in pain processing. We found that the known analgesic loperamide inhibited I(h) channels in rat DRG neurons. Loperamide blocked I(h) in a concentration-dependent manner, with an IC(50) = 4.9 +/- 0.6 and 11.0 +/- 0.5 microM for large- and small-diameter neurons, respectively. Loperamide-induced I(h) inhibition was unrelated to the activation of opioid receptors and was reversible, voltage-dependent, use-independent, and was associated with a negative shift of V(1/2) for I(h) steady-state activation. Loperamide block of I(h) was voltage-dependent, gradually decreasing at more hyperpolarized membrane voltages from 89% at -60 mV to 4% at -120 mV in the presence of 3.7 microM loperamide. The voltage sensitivity of block can be explained by a loperamide-induced shift in the steady-state activation of I(h). Inclusion of 10 microM loperamide into the recording pipette did not affect I(h) voltage for half-maximal activation, activation kinetics, and the peak current amplitude, whereas concurrent application of equimolar external loperamide produced a rapid, reversible I(h) inhibition. The observed loperamide-induced I(h) inhibition was not caused by the activation of peripheral opioid receptors because the broad-spectrum opioid receptor antagonist naloxone did not reverse I(h) inhibition. Therefore we suggest that loperamide inhibits I(h) by direct binding to the extracellular region of the channel. Because I(h) channels are involved in pain processing, loperamide-induced inhibition of I(h) channels could provide an additional molecular mechanism for its analgesic action.
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Analgésicos/farmacología , Ganglios Espinales/citología , Loperamida/farmacología , Inhibición Neural/efectos de los fármacos , Neuronas/efectos de los fármacos , Canales de Potasio/metabolismo , Animales , Células Cultivadas , Canales Catiónicos Regulados por Nucleótidos Cíclicos , Relación Dosis-Respuesta a Droga , Relación Dosis-Respuesta en la Radiación , Interacciones Farmacológicas , Estimulación Eléctrica , Canales Regulados por Nucleótidos Cíclicos Activados por Hiperpolarización , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/fisiología , Potenciales de la Membrana/efectos de la radiación , Naloxona/farmacología , Antagonistas de Narcóticos/farmacología , Técnicas de Placa-Clamp/métodos , Ratas , Ratas WistarRESUMEN
Potassium channels are key determinants of neuronal excitability. We recently identified KChIPs as a family of calcium binding proteins that coassociate and colocalize with Kv4 family potassium channels in mammalian brain (An et al. [2000] Nature 403:553). Here, we used light microscopic immunohistochemistry and multilabel immunofluorescence labeling, together with transmission electron microscopic immunohistochemistry, to examine the subcellular distribution of KChIPs and Kv4 channels in adult rat cerebellum. Light microscopic immunohistochemistry was performed on 40-microm free-floating sections using a diaminobenzidine labeling procedure. Multilabel immunofluorescence staining was performed on free-floating sections and on 1-microm ultrathin cryosections. Electron microscopic immunohistochemistry was performed using an immunoperoxidase pre-embedding labeling procedure. By light microscopy, immunoperoxidase labeling showed that Kv4.2, Kv4.3, and KChIPs 1, 3, and 4 (but not KChIP2) were expressed at high levels in cerebellar granule cells (GCs). Kv4.2 and KChIP1 were highly expressed in GCs in rostral cerebellum, whereas Kv4.3 was more highly expressed in GCs in caudal cerebellum. Immunofluorescence labeling revealed that KChIP1 and Kv4.2 are concentrated in somata of cerebellar granule cells and in synaptic glomeruli that surround synaptophysin-positive mossy fiber axon terminals. Electron microscopic analysis revealed that KChIP1 and Kv4.2 immunoreactivity is concentrated along the plasma membrane of cerebellar granule cell somata and dendrites. In synaptic glomeruli, KChIP1 and Kv4.2 immunoreactivity is concentrated along the granule cell dendritic membrane, but is not concentrated at postsynaptic densities. Taken together, these data suggest that A-type potassium channels containing Kv4.2 and KChIP1, and perhaps also KChIP3 and 4, play a critical role in regulating postsynaptic excitability at the cerebellar mossy-fiber/granule cell synapse.
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Proteínas de Unión al Calcio/ultraestructura , Cerebelo/química , Cerebelo/metabolismo , Cerebelo/ultraestructura , Canales de Potasio con Entrada de Voltaje/ultraestructura , Animales , Proteínas de Unión al Calcio/análisis , Proteínas de Unión al Calcio/biosíntesis , Proteínas de Unión al Calcio/metabolismo , Proteínas de Interacción con los Canales Kv , Microscopía de Polarización/métodos , Canales de Potasio con Entrada de Voltaje/biosíntesis , Canales de Potasio con Entrada de Voltaje/metabolismo , Ratas , Canales de Potasio ShalRESUMEN
Voltage-gated potassium (Kv) channels from the Kv4, or Shal-related, gene family underlie a major component of the A-type potassium current in mammalian central neurons. We recently identified a family of calcium-binding proteins, termed KChIPs (Kv channel interacting proteins), that bind to the cytoplasmic N termini of Kv4 family alpha subunits and modulate their surface density, inactivation kinetics, and rate of recovery from inactivation (An et al., 2000). Here, we used single and double-label immunohistochemistry, together with circumscribed lesions and coimmunoprecipitation analyses, to examine the regional and subcellular distribution of KChIPs1-4 and Kv4 family alpha subunits in adult rat brain. Immunohistochemical staining using KChIP-specific monoclonal antibodies revealed that the KChIP polypeptides are concentrated in neuronal somata and dendrites where their cellular and subcellular distribution overlaps, in an isoform-specific manner, with that of Kv4.2 and Kv4.3. For example, immunoreactivity for KChIP1 and Kv4.3 is concentrated in the somata and dendrites of hippocampal, striatal, and neocortical interneurons. Immunoreactivity for KChIP2, KChIP4, and Kv4.2 is concentrated in the apical and basal dendrites of hippocampal and neocortical pyramidal cells. Double-label immunofluorescence labeling revealed that throughout the forebrain, KChIP2 and KChIP4 are frequently colocalized with Kv4.2, whereas in cortical, hippocampal, and striatal interneurons, KChIP1 is frequently colocalized with Kv4.3. Coimmunoprecipitation analyses confirmed that all KChIPs coassociate with Kv4 alpha subunits in brain membranes, indicating that KChIPs 1-4 are integral components of native A-type Kv channel complexes and are likely to play a major role as modulators of somatodendritic excitability.