RESUMO
The development of opioid tolerance in patients on long-term opioid analgesic treatment is an unsolved matter in clinical practice thus far. Dose escalation is required to restore analgesic efficacy, but at the price of side effects. Intensive research is ongoing to elucidate the underlying mechanisms of opioid analgesic tolerance in the hope of maintaining opioid analgesic efficacy. N-Methyl-D-aspartate receptor (NMDAR) antagonists have shown promising effects regarding opioid analgesic tolerance; however, their use is limited by side effects (memory dysfunction). Nevertheless, the GluN2B receptor remains a future target for the discovery of drugs to restore opioid efficacy. Mechanistically, the long-term activation of µ-opioid receptors (MORs) initiates receptor phosphorylation, which triggers ß-arrestin-MAPKs and NOS-GC-PKG pathway activation, which ultimately ends with GluN2B receptor overactivation and glutamate release. The presence of glutamate and glycine as co-agonists is a prerequisite for GluN2B receptor activation. The extrasynaptic localization of the GluN2B receptor means it is influenced by the glycine level, which is regulated by astrocytic glycine transporter 1 (GlyT1). Enhanced astrocytic glycine release by reverse transporter mechanisms as a consequence of high glutamate levels or unconventional MOR activation on astrocytes could further activate the GluN2B receptor. GlyT1 inhibitors might inhibit this condition, thereby reducing opioid tolerance.
RESUMO
This review summarizes the current understanding of the role of transient receptor potential melastatin-subfamily member 7 (TRPM7) channels in the pathophysiology of neoplastic diseases. The TRPM family represents the largest and most diverse group in the TRP superfamily. Its subtypes are expressed in virtually all human organs playing a central role in (patho)physiological events. The TRPM7 protein (along with TRPM2 and TRPM6) is unique in that it has kinase activity in addition to the channel function. Numerous studies demonstrate the role of TRPM7 chanzyme in tumorigenesis and in other tumor hallmarks such as proliferation, migration, invasion and metastasis. Here we provide an up-to-date overview about the possible role of TRMP7 in a broad range of malignancies such as tumors of the nervous system, head and neck cancers, malignant neoplasms of the upper gastrointestinal tract, colorectal carcinoma, lung cancer, neoplasms of the urinary system, breast cancer, malignant tumors of the female reproductive organs, prostate cancer and other neoplastic pathologies. Experimental data show that the increased expression and/or function of TRPM7 are observed in most malignant tumor types. Thus, TRPM7 chanzyme may be a promising target in tumor therapy.
Assuntos
Neoplasias Pulmonares , Neoplasias da Próstata , Canais de Cátion TRPM , Humanos , Carcinogênese/genética , Transformação Celular Neoplásica , Proteínas Serina-Treonina Quinases/genética , Canais de Cátion TRPM/genéticaRESUMO
ATP, as a paracrine signalling molecule, induces intracellular Ca2+ elevation via the activation of purinergic receptors on the surface of glia-like cochlear supporting cells. These cells, including the Deiters' cells (DCs), are also coupled by gap junctions that allow the propagation of intercellular Ca2+ waves via diffusion of Ca2+ mobilising second messenger IP3 between neighbouring cells. We have compared the ATP-evoked Ca2+ transients and the effect of two different gap junction (GJ) blockers (octanol and carbenoxolone, CBX) on the Ca2+ transients in DCs located in the apical and middle turns of the hemicochlea preparation of BALB/c mice (P14-19). Octanol had no effect on Ca2+ signalling, while CBX inhibited the ATP response, more prominently in the middle turn. Based on astrocyte models and using our experimental results, we successfully simulated the Ca2+ dynamics in DCs in different cochlear regions. The mathematical model reliably described the Ca2+ transients in the DCs and suggested that the tonotopical differences could originate from differences in purinoceptor and Ca2+ pump expressions and in IP3-Ca2+ release mechanisms. The cochlear turn-dependent effect of CBX might be the result of the differing connexin isoform composition of GJs along the tonotopic axis. The contribution of IP3-mediated Ca2+ signalling inhibition by CBX cannot be excluded.
Assuntos
Cálcio , Junções Comunicantes , Camundongos , Animais , Camundongos Endogâmicos BALB C , Cálcio/metabolismo , Junções Comunicantes/metabolismo , Receptores Purinérgicos/metabolismo , Órgão Espiral/metabolismo , Audição , Trifosfato de Adenosina/metabolismoRESUMO
Stress disorders impair sleep and quality of life; however, their pathomechanisms are unknown. Prolactin-releasing peptide (PrRP) is a stress mediator; we therefore hypothesized that PrRP may be involved in the development of stress disorders. PrRP is produced by the medullary A1/A2 noradrenaline (NA) cells, which transmit stress signals to forebrain centers, and by non-NA cells in the hypothalamic dorsomedial nucleus. We found in male rats that both PrRP and PrRP-NA cells innervate melanin-concentrating hormone (MCH) producing neurons in the dorsolateral hypothalamus (DLH). These cells serve as a key hub for regulating sleep and affective states. Ex vivo, PrRP hyperpolarized MCH neurons and further increased the hyperpolarization caused by NA. Following sleep deprivation, intracerebroventricular PrRP injection reduced the number of REM sleep-active MCH cells. PrRP expression in the dorsomedial nucleus was upregulated by sleep deprivation, while downregulated by REM sleep rebound. Both in learned helplessness paradigm and after peripheral inflammation, impaired coping with sustained stress was associated with (1) overactivation of PrRP cells, (2) PrRP protein and receptor depletion in the DLH, and (3) dysregulation of MCH expression. Exposure to stress in the PrRP-insensitive period led to increased passive coping with stress. Normal PrRP signaling, therefore, seems to protect animals against stress-related disorders. PrRP signaling in the DLH is an important component of the PrRP's action, which may be mediated by MCH neurons. Moreover, PrRP receptors were downregulated in the DLH of human suicidal victims. As stress-related mental disorders are the leading cause of suicide, our findings may have particular translational relevance.SIGNIFICANCE STATEMENT Treatment resistance to monoaminergic antidepressants is a major problem. Neuropeptides that modulate the central monoaminergic signaling are promising targets for developing alternative therapeutic strategies. We found that stress-responsive prolactin-releasing peptide (PrRP) cells innervated melanin-concentrating hormone (MCH) neurons that are crucial in the regulation of sleep and mood. PrRP inhibited MCH cell activity and enhanced the inhibitory effect evoked by noradrenaline, a classic monoamine, on MCH neurons. We observed that impaired PrRP signaling led to failure in coping with chronic/repeated stress and was associated with altered MCH expression. We found alterations of the PrRP system also in suicidal human subjects. PrRP dysfunction may underlie stress disorders, and fine-tuning MCH activity by PrRP may be an important part of the mechanism.
Assuntos
Hormônios Hipotalâmicos , Privação do Sono , Ratos , Masculino , Humanos , Animais , Hormônio Liberador de Prolactina/farmacologia , Hormônio Liberador de Prolactina/metabolismo , Privação do Sono/metabolismo , Transtornos do Humor/etiologia , Qualidade de Vida , Ratos Wistar , Hormônios Hipotalâmicos/metabolismo , Sono/fisiologia , Neurônios/fisiologia , Norepinefrina/metabolismoRESUMO
Neurodegenerative-neuroinflammatory disorders of the retina seriously hamper human vision. In searching for key factors that contribute to the development of these pathologies, we considered potential interactions among purinergic neuromodulation, glycinergic neurotransmission, and microglia activity in the retina. Energy deprivation at cellular levels is mainly due to impaired blood circulation leading to increased release of ATP and adenosine as well as glutamate and glycine. Interactions between these modulators and neurotransmitters are manifold. First, P2Y purinoceptor agonists facilitate reuptake of glycine by glycine transporter 1, while its inhibitors reduce reverse-mode operation; these events may lower extracellular glycine levels. The consequential changes in extracellular glycine concentration can lead to parallel changes in the activity of NR1/NR2B type NMDA receptors of which glycine is a mandatory agonist, and thereby may reduce neurodegenerative events in the retina. Second, P2Y purinoceptor agonists and glycine transporter 1 inhibitors may indirectly inhibit microglia activity by decreasing neuronal or glial glycine release in energy-compromised retina. These inhibitions may have a role in microglia activation, which is present during development and progression of neurodegenerative disorders such as glaucomatous and diabetic retinopathies and age-related macular degeneration or loss of retinal neurons caused by thromboembolic events. We have hypothesized that glycine transporter 1 inhibitors and P2Y purinoceptor agonists may have therapeutic importance in neurodegenerative-neuroinflammatory disorders of the retina by decreasing NR1/NR2B NMDA receptor activity and production and release of a series of proinflammatory cytokines from microglial cells.
Assuntos
Glicinérgicos/metabolismo , Inflamação/patologia , Doenças Neurodegenerativas/patologia , Neurônios/patologia , Receptores Purinérgicos/metabolismo , Doenças Retinianas/patologia , Animais , Humanos , Inflamação/complicações , Inflamação/metabolismo , Doenças Neurodegenerativas/complicações , Doenças Neurodegenerativas/metabolismo , Neurônios/metabolismo , Doenças Retinianas/complicações , Doenças Retinianas/metabolismoRESUMO
Age-related hearing loss (ARHL), a sensorineural hearing loss of multifactorial origin, increases its prevalence in aging societies. Besides hearing aids and cochlear implants, there is no FDA approved efficient pharmacotherapy to either cure or prevent ARHL. We hypothesized that selegiline, an antiparkinsonian drug, could be a promising candidate for the treatment due to its complex neuroprotective, antioxidant, antiapoptotic, and dopaminergic neurotransmission enhancing effects. We monitored by repeated Auditory Brainstem Response (ABR) measurements the effect of chronic per os selegiline administration on the hearing function in BALB/c and DBA/2J mice, which strains exhibit moderate and rapid progressive high frequency hearing loss, respectively. The treatments were started at 1 month of age and lasted until almost a year and 5 months of age, respectively. In BALB/c mice, 4 mg/kg selegiline significantly mitigated the progression of ARHL at higher frequencies. Used in a wide dose range (0.15-45 mg/kg), selegiline had no effect in DBA/2J mice. Our results suggest that selegiline can partially preserve the hearing in certain forms of ARHL by alleviating its development. It might also be otoprotective in other mammals or humans.
Assuntos
Envelhecimento/fisiologia , Modelos Animais de Doenças , Perda Auditiva Neurossensorial/tratamento farmacológico , Selegilina/farmacologia , Administração Oral , Animais , Antiparkinsonianos/administração & dosagem , Antiparkinsonianos/farmacologia , Limiar Auditivo/efeitos dos fármacos , Limiar Auditivo/fisiologia , Potenciais Evocados Auditivos do Tronco Encefálico/efeitos dos fármacos , Potenciais Evocados Auditivos do Tronco Encefálico/fisiologia , Perda Auditiva Neurossensorial/fisiopatologia , Humanos , Masculino , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos DBA , Substâncias Protetoras/administração & dosagem , Substâncias Protetoras/farmacologia , Selegilina/administração & dosagem , Transmissão Sináptica/efeitos dos fármacos , Transmissão Sináptica/fisiologiaRESUMO
The limited effect of current medications on neuropathic pain (NP) has initiated large efforts to develop effective treatments. Animal studies showed that glycine transporter (GlyT) inhibitors are promising analgesics in NP, though concerns regarding adverse effects were raised. We aimed to study NFPS and Org-25543, GlyT-1 and GlyT-2 inhibitors, respectively and their combination in rat mononeuropathic pain evoked by partial sciatic nerve ligation. Cerebrospinal fluid (CSF) glycine content was also determined by capillary electrophoresis. Subcutaneous (s.c.) 4 mg/kg NFPS or Org-25543 showed analgesia following acute administration (30-60 min). Small doses of each compound failed to produce antiallodynia up to 180 min after the acute administration. However, NFPS (1 mg/kg) produced antiallodynia after four days of treatment. Co-treatment with subanalgesic doses of NFPS (1 mg/kg) and Org-25543 (2 mg/kg) produced analgesia at 60 min and thereafter meanwhile increased significantly the CSF glycine content. This combination alleviated NP without affecting motor function. Test compounds failed to activate G-proteins in spinal cord. To the best of our knowledge for the first time we demonstrated augmented analgesia by combining GlyT-1 and 2 inhibitors. Increased CSF glycine content supports involvement of glycinergic system. Combining selective GlyT inhibitors or developing non-selective GlyT inhibitors might have therapeutic value in NP.
Assuntos
Proteínas da Membrana Plasmática de Transporte de Glicina/antagonistas & inibidores , Glicina/líquido cefalorraquidiano , Hiperalgesia/prevenção & controle , Neuralgia/tratamento farmacológico , Sarcosina/análogos & derivados , Animais , Hiperalgesia/metabolismo , Hiperalgesia/patologia , Masculino , Atividade Motora , Neuralgia/metabolismo , Neuralgia/patologia , Ratos , Ratos Wistar , Sarcosina/farmacologia , Medula Espinal/efeitos dos fármacos , Medula Espinal/metabolismo , Medula Espinal/patologiaRESUMO
TRPM7 plays an important role in cellular Ca2+, Zn2+ and Mg2+ homeostasis. TRPM7 channels are abundantly expressed in ameloblasts and, in the absence of TRPM7, dental enamel is hypomineralized. The potential role of TRPM7 channels in Ca2+ transport during amelogenesis was investigated in the HAT-7 rat ameloblast cell line. The cells showed strong TRPM7 mRNA and protein expression. Characteristic TRPM7 transmembrane currents were observed, which increased in the absence of intracellular Mg2+ ([Mg2+]i), were reduced by elevated [Mg2+]i, and were inhibited by the TRPM7 inhibitors NS8593 and FTY720. Mibefradil evoked similar currents, which were suppressed by elevated [Mg2+]i, reducing extracellular pH stimulated transmembrane currents, which were inhibited by FTY720. Naltriben and mibefradil both evoked Ca2+ influx, which was further enhanced by the acidic intracellular conditions. The SOCE inhibitor BTP2 blocked Ca2+ entry induced by naltriben but not by mibefradil. Thus, in HAT-7 cells, TRPM7 may serves both as a potential modulator of Orai-dependent Ca2+ uptake and as an independent Ca2+ entry pathway sensitive to pH. Therefore, TRPM7 may contribute directly to transepithelial Ca2+ transport in amelogenesis.
Assuntos
Ameloblastos/metabolismo , Cálcio/metabolismo , Canais de Cátion TRPM/metabolismo , Ameloblastos/citologia , Ameloblastos/efeitos dos fármacos , Anilidas/farmacologia , Animais , Linhagem Celular , Humanos , Concentração de Íons de Hidrogênio , Incisivo/citologia , Ativação do Canal Iônico/efeitos dos fármacos , Transporte de Íons/efeitos dos fármacos , Mibefradil/farmacologia , Camundongos , Modelos Biológicos , Naltrexona/análogos & derivados , Naltrexona/farmacologia , Ratos , Tiadiazóis/farmacologiaRESUMO
The administration of immune checkpoint inhibitors (ICIs) often leads to immune-related adverse events. However, their effect on auditory function is largely unexplored. Thorough preclinical studies have not been published yet, only sporadic cases and pharmacovigilance reports suggest their significance. Here we investigated the effect of anti-PD-1 antibody treatment (4 weeks, intraperitoneally, 200 µg/mouse, 3 times/week) on hearing function and cochlear morphology in C57BL/6J mice. ICI treatment did not influence the hearing thresholds in click or tone burst stimuli at 4-32 kHz frequencies measured by auditory brainstem response. The number and morphology of spiral ganglion neurons were unaltered in all cochlear turns. The apical-middle turns (<32 kHz) showed preservation of the inner and outer hair cells (OHCs), whilst ICI treatment mitigated the age-related loss of OHCs in the basal turn (>32 kHz). The number of Iba1-positive macrophages has also increased moderately in this high frequency region. We conclude that a 4-week long ICI treatment does not affect functional and morphological integrity of the inner ear in the most relevant hearing range (4-32 kHz; apical-middle turns), but a noticeable preservation of OHCs and an increase in macrophage activity appeared in the >32 kHz basal part of the cochlea.
Assuntos
Anticorpos Monoclonais/farmacologia , Limiar Auditivo/efeitos dos fármacos , Cóclea/efeitos dos fármacos , Células Ciliadas Auditivas Externas/efeitos dos fármacos , Receptor de Morte Celular Programada 1/antagonistas & inibidores , Animais , Potenciais Evocados Auditivos do Tronco Encefálico/efeitos dos fármacos , Audição , Inibidores de Checkpoint Imunológico/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Gânglio Espiral da Cóclea/efeitos dos fármacosRESUMO
Exploring the development of the hearing organ helps in the understanding of hearing and hearing impairments and it promotes the development of the regenerative approaches-based therapeutic efforts. The role of supporting cells in the development of the organ of Corti is much less elucidated than that of the cochlear sensory receptor cells. The use of our recently published method of single-cell electroporation loading of a fluorescent Ca2+ probe in the mouse hemicochlea preparation provided an appropriate means to investigate the Deiters' cells at the subcellular level in two different cochlear turns (apical, middle). Deiters' cell's soma and process elongated, and the process became slimmer by maturation without tonotopic preference. The tonotopically heterogeneous spontaneous Ca2+ activity less frequently occurred by maturation and implied subcellular difference. The exogenous ATP- and UTP-evoked Ca2+ responses were maturation-dependent and showed P2Y receptor dominance in the apical turn. By monitoring the basic structural dimensions of this supporting cell type as well as its spontaneous and evoked purinergic Ca2+ signaling in the hemicochlea preparation in different stages in the critical postnatal P5-25 developmental period for the first time, we showed that the soma and the phalangeal process of the Deiters' cells go through age- and tonotopy-dependent changes in the morphometric parameters and purinergic signaling.
Assuntos
Cóclea/metabolismo , Células Ciliadas Auditivas/metabolismo , Trifosfato de Adenosina/farmacologia , Animais , Cálcio/metabolismo , Cóclea/efeitos dos fármacos , Eletroporação , Técnicas In Vitro , Camundongos , Camundongos Endogâmicos BALB C , Modelos Teóricos , Receptores Purinérgicos/metabolismo , Transdução de Sinais/efeitos dos fármacos , Uridina Trifosfato/farmacologiaRESUMO
Interneurons operating with glycine neurotransmitter are involved in the regulation of pain transmission in the dorsal horn of the spinal cord. In addition to interneurons, glycine release also occurs from glial cells neighboring glutamatergic synapses in the spinal cord. Neuronal and glial release of glycine is controlled by glycine transporters (GlyTs). Inhibitors of the two isoforms of GlyTs, the astrocytic type-1 (GlyT-1) and the neuronal type-2 (GlyT-2), decrease pain sensation evoked by injuries of peripheral sensory neurons or inflammation. The function of dorsal horn glycinergic interneurons has been suggested to be reduced in neuropathic pain, which can be reversed by GlyT-2 inhibitors (Org-25543, ALX1393). Several lines of evidence also support that peripheral nerve damage or inflammation may shift glutamatergic neurochemical transmission from N-methyl-D aspartate (NMDA) NR1/NR2A receptor- to NR1/NR2B receptor-mediated events (subunit switch). This pathological overactivation of NR1/NR2B receptors can be reduced by GlyT-1 inhibitors (NFPS, Org-25935), which decrease excessive glycine release from astroglial cells or by selective antagonists of NR2B subunits (ifenprodil, Ro 25-6981). Although several experiments suggest that GlyT inhibitors may represent a novel strategy in the control of neuropathic pain, proving this concept in human beings is hampered by lack of clinically applicable GlyT inhibitors. We also suggest that drugs inhibiting both GlyT-1 and GlyT-2 non-selectively and reversibly, may favorably target neuropathic pain. In this paper we overview inhibitors of the two isoforms of GlyTs as well as the effects of these drugs in experimental models of neuropathic pain. In addition, the possible mechanisms of action of the GlyT inhibitors, i.e. how they affect the neurochemical and pain transmission in the spinal cord, are also discussed. The growing evidence for the possible therapeutic intervention of neuropathic pain by GlyT inhibitors further urges development of drugable compounds, which may beneficially restore impaired pain transmission in various neuropathic conditions.
Assuntos
Proteínas da Membrana Plasmática de Transporte de Glicina/antagonistas & inibidores , Proteínas da Membrana Plasmática de Transporte de Glicina/metabolismo , Neuralgia/tratamento farmacológico , Animais , Glicina/farmacologia , Humanos , Hiperalgesia/tratamento farmacológico , Neuralgia/metabolismo , Neuroglia/metabolismo , Neurônios/metabolismo , Fenóis/farmacologia , Piperidinas/farmacologia , Receptores de N-Metil-D-Aspartato/metabolismo , Serina/análogos & derivados , Serina/farmacologia , Corno Dorsal da Medula Espinal/metabolismo , Sinapses/metabolismo , Transmissão Sináptica/efeitos dos fármacosRESUMO
Hearing impairment is the most common sensory deficit, affecting more than 400 million people worldwide. Sensorineural hearing losses currently lack any specific or efficient pharmacotherapy largely due to the insufficient knowledge of the pathomechanism. Purinergic signaling plays a substantial role in cochlear (patho)physiology. P2 (ionotropic P2X and the metabotropic P2Y) as well as adenosine receptors expressed on cochlear sensory and non-sensory cells are involved mostly in protective mechanisms of the cochlea. They are implicated in the sensitivity adjustment of the receptor cells by a K+ shunt and can attenuate the cochlear amplification by modifying cochlear micromechanics. Cochlear blood flow is also regulated by purines. Here, we propose to comprehend this field with the purine-immune interactions in the cochlea. The role of harmful immune mechanisms in sensorineural hearing losses has been emerging in the horizon of cochlear pathologies. In addition to decreasing hearing sensitivity and increasing cochlear blood supply, influencing the immune system can be the additional avenue for pharmacological targeting of purinergic signaling in the cochlea. Elucidating this complexity of purinergic effects on cochlear functions is necessary and it can result in development of new therapeutic approaches in hearing disabilities, especially in the noise-induced ones.
Assuntos
Cóclea/imunologia , Cóclea/metabolismo , Doenças Cocleares/etiologia , Doenças Cocleares/metabolismo , Transdução de Sinais , Animais , Cálcio/metabolismo , Cóclea/fisiologia , Cóclea/ultraestrutura , Doenças Cocleares/tratamento farmacológico , Doenças Cocleares/fisiopatologia , Expressão Gênica , Perda Auditiva Neurossensorial/etiologia , Perda Auditiva Neurossensorial/metabolismo , Perda Auditiva Neurossensorial/fisiopatologia , Humanos , Sistema Imunitário/imunologia , Sistema Imunitário/metabolismo , Purinérgicos/metabolismo , Receptores Purinérgicos/genética , Receptores Purinérgicos/metabolismo , Receptores Purinérgicos P1/genética , Receptores Purinérgicos P1/metabolismoRESUMO
Purinergic signaling is deeply involved in the development, functions and protective mechanisms of the cochlea. Release of ATP and activation of purinergic receptors on sensory and supporting/epithelial cells play a substantial role in cochlear (patho)physiology. Both the ionotropic P2X and the metabotropic P2Y receptors are widely distributed on the inner and outer hair cells as well as on the different supporting cells in the organ of Corti and on other epithelial cells in the scala media. Among others, they are implicated in the sensitivity adjustment of the receptor cells by a K+ shunt and can attenuate the cochlear amplification by modifying cochlear micromechanics acting on outer hair cells and supporting cells. Cochlear blood flow is also regulated by purines. Sensorineural hearing losses currently lack any specific or efficient pharmacotherapy. Decreasing hearing sensitivity and increasing cochlear blood supply by pharmacological targeting of purinergic signaling in the cochlea are potential new therapeutic approaches in these hearing disabilities, especially in the noise-induced ones.
Assuntos
Perda Auditiva/metabolismo , Órgão Espiral/metabolismo , Receptores Purinérgicos/metabolismo , Trifosfato de Adenosina/farmacologia , Animais , Cóclea/metabolismo , Cóclea/fisiologia , Audição/fisiologia , Perda Auditiva/fisiopatologia , Humanos , Ruído , Órgão Espiral/fisiologia , Purinas/metabolismo , Receptores Purinérgicos/fisiologia , Receptores Purinérgicos P1/metabolismo , Receptores Purinérgicos P1/fisiologia , Receptores Purinérgicos P2/metabolismo , Receptores Purinérgicos P2/fisiologia , Transdução de Sinais/efeitos dos fármacosRESUMO
Ca2+ is an important intracellular messenger and regulator in both physiological and pathophysiological mechanisms in the hearing organ. Investigation of cellular Ca2+ homeostasis in the mature cochlea is hampered by the special anatomy and high vulnerability of the organ. A quick, straightforward and reliable Ca2+ imaging method with high spatial and temporal resolution in the mature organ of Corti is missing. Cell cultures or isolated cells do not preserve the special microenvironment and intercellular communication, while cochlear explants are excised from only a restricted portion of the organ of Corti and usually from neonatal pre-hearing murines. The hemicochlea, prepared from hearing mice allows tonotopic experimental approach on the radial perspective in the basal, middle and apical turns of the organ. We used the preparation recently for functional imaging in supporting cells of the organ of Corti after bulk loading of the Ca2+ indicator. However, bulk loading takes long time, is variable and non-selective, and causes the accumulation of the indicator in the extracellular space. In this study we show the improved labeling of supporting cells of the organ of Corti by targeted single-cell electroporation in mature mouse hemicochlea. Single-cell electroporation proved to be a reliable way of reducing the duration and variability of loading and allowed subcellular Ca2+ imaging by increasing the signal-to-noise ratio, while cell viability was retained during the experiments. We demonstrated the applicability of the method by measuring the effect of purinergic, TRPA1, TRPV1 and ACh receptor stimulation on intracellular Ca2+ concentration at the cellular and subcellular level. In agreement with previous results, ATP evoked reversible and repeatable Ca2+ transients in Deiters', Hensen's and Claudius' cells. TRPA1 and TRPV1 stimulation by AITC and capsaicin, respectively, failed to induce any Ca2+ response in the supporting cells, except in a single Hensen's cell in which AITC evoked transients with smaller amplitude. AITC also caused the displacement of the tissue. Carbachol, agonist of ACh receptors induced Ca2+ transients in about a third of Deiters' and fifth of Hensen's cells. Here we have presented a fast and cell-specific indicator loading method allowing subcellular functional Ca2+ imaging in supporting cells of the organ of Corti in the mature hemicochlea preparation, thus providing a straightforward tool for deciphering the poorly understood regulation of Ca2+ homeostasis in these cells.
Assuntos
Cálcio/metabolismo , Cóclea/citologia , Cóclea/metabolismo , Trifosfato de Adenosina/metabolismo , Compostos de Anilina/administração & dosagem , Animais , Quelantes de Cálcio/administração & dosagem , Sinalização do Cálcio/efeitos dos fármacos , Carbacol/administração & dosagem , Cóclea/efeitos dos fármacos , Eletroporação/métodos , Fluoresceínas/administração & dosagem , Corantes Fluorescentes/administração & dosagem , Fura-2/administração & dosagem , Técnicas In Vitro , Células Labirínticas de Suporte/citologia , Células Labirínticas de Suporte/efeitos dos fármacos , Células Labirínticas de Suporte/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Órgão Espiral/citologia , Órgão Espiral/efeitos dos fármacos , Órgão Espiral/metabolismo , Receptores Colinérgicos/metabolismo , Análise de Célula Única/métodos , Canal de Cátion TRPA1/metabolismo , Canais de Cátion TRPV/metabolismoRESUMO
P2X7 receptors (P2X7Rs) are ligand-gated ion channels sensitive to extracellular ATP. Here we examined for the first time the role of P2X7R in an animal model of schizophrenia. Using the PCP induced schizophrenia model we show that both genetic deletion and pharmacological inhibition of P2X7Rs alleviate schizophrenia-like behavioral alterations. In P2rx7+/+ mice, PCP induced hyperlocomotion, stereotype behavior, ataxia and social withdrawal. In P2X7 receptor deficient mice (P2rx7-/-), the social interactions were increased, whereas the PCP induced hyperlocomotion and stereotype behavior were alleviated. The selective P2X7 receptor antagonist JNJ-47965567 partly replicated the effect of gene deficiency on PCP-induced behavioral changes and counteracted PCP-induced social withdrawal. We also show that PCP treatment upregulates and increases the functional responsiveness of P2X7Rs in the prefrontal cortex of young adult animals. The amplitude of NMDA evoked currents recorded from layer V pyramidal neurons of cortical slices were slightly decreased by both genetic deletion of P2rx7 and by JNJ-47965567. PCP induced alterations in mRNA expression encoding schizophrenia-related genes, such as NR2A, NR2B, neuregulin 1, NR1 and GABA α1 subunit were absent in the PFC of young adult P2rx7-/- animals. Our findings point to P2X7R as a potential therapeutic target in schizophrenia.
Assuntos
Córtex Cerebral/metabolismo , Fenciclidina/efeitos adversos , Células Piramidais/metabolismo , Receptores Purinérgicos P2X7/metabolismo , Esquizofrenia/metabolismo , Animais , Comportamento Animal/efeitos dos fármacos , Córtex Cerebral/patologia , Camundongos , Camundongos Knockout , Niacinamida/análogos & derivados , Niacinamida/farmacologia , Fenciclidina/farmacologia , Piperazinas/farmacologia , Antagonistas do Receptor Purinérgico P2X/farmacologia , Células Piramidais/patologia , Receptores Purinérgicos P2X7/genética , Esquizofrenia/induzido quimicamente , Esquizofrenia/genética , Esquizofrenia/patologiaRESUMO
BACKGROUND: Allyphenyline, a novel α2-adrenoceptor (AR) ligand, has been shown to selectively activate α2C-adrenoceptors (AR) and 5HT1A receptors, but also to behave as a neutral antagonist of α2A-ARs. We exploited this unique pharmacological profile to analyze the role of α2C-ARs and 5HT1A receptors in the regulation of gastric mucosal integrity and gastrointestinal motility. METHODS: Gastric injury was induced by acidified ethanol in Wistar rats. Mucosal catalase and superoxide dismutase levels were measured by assay kits. The effect of allyphenyline on electrical field stimulation (EFS)-induced fundic and colonic contractions was determined in C57BL/6 mice. RESULTS: Intracerebroventricularly injected allyphenyline (3 and 15 nmol/rat) dose dependently inhibited the development of mucosal damage, which was antagonized by ARC 239 (α2B/C-AR and 5HT1A receptor antagonist), (S)-WAY 100135 (selective 5HT1A receptor antagonist), and JP-1302 (selective α2C-AR antagonist). This protection was accompanied by significant elevation of mucosal catalase and superoxide dismutase levels. Allyphenyline (10(-9)-10(-5) M) also inhibited EFS-induced fundic contractions, which was antagonized by ARC 239 and (S)-WAY 100135, but not by JP-1302. Similar inhibition was observed in the colon; however, in this case only ARC 239 reduced this effect, while neither selective inhibition of α2C-ARs and 5HT1A receptors nor genetic deletion of α2A- and α2B-ARs influenced it. CONCLUSIONS: Activation of both central α2C-ARs and 5HT1A receptors contributes to the gastroprotective action of allyphenyline in rats. Its inhibitory effect on fundic contractions is mediated by 5HT1A receptors, but neither α2-ARs nor 5HT1A receptors take part in its inhibitory effect on colonic contractility in mice.
Assuntos
Agonistas de Receptores Adrenérgicos alfa 2/farmacologia , Compostos Alílicos/farmacologia , Motilidade Gastrointestinal/efeitos dos fármacos , Imidazolinas/farmacologia , Receptores Adrenérgicos alfa 2/metabolismo , Agonistas do Receptor de Serotonina/farmacologia , Compostos Alílicos/química , Animais , Colo/efeitos dos fármacos , Colo/fisiologia , Imidazolinas/química , Masculino , Estrutura Molecular , Ratos , Ratos WistarRESUMO
Rat posterior eyecups containing the retina were prepared, loaded with [(3)H]glycine and superfused in order to determine its release originated from glycinergic amacrine cells and/or glial cells. Deprivation of oxygen and glucose from the Krebs-bicarbonate buffer used for superfusion evoked a marked increase of [(3)H]glycine release, an effect that was found to be external Ca(2+)-independent. Whereas oxygen and glucose deprivation increased [(3)H]glycine release, its uptake was reduced suggesting that energy deficiency shifts glycine transporter type-1 operation from normal to reverse mode. The increased release of [(3)H]glycine evoked by oxygen and glucose deprivation was suspended by addition of the non-competitive glycine transporter type-1 inhibitor NFPS and the competitive inhibitor ACPPB further suggesting the involvement of this transporter in the mediation of [(3)H]glycine release. Oxygen and glucose deprivation also evoked [(3)H]glutamate release from rat retina and the concomitantly occurring release of the NMDA receptor agonist glutamate and the coagonist glycine makes NMDA receptor pathological overstimulation possible in hypoxic conditions. [(3)H]Glutamate release was suspended by addition of the excitatory amino acid transporter inhibitor TBOA. Sarcosine, a substrate inhibitor of glycine transporter type-1, also increased [(3)H]glycine release probably by heteroexchange shifting transporter operation into reverse mode. This effect of sarcosine was also external Ca(2+)-independent and could be suspended by NFPS. Energy deficiency in retina induced by ouabain, an inhibitor of the Na(+)-K(+)-dependent ATPase, and by rotenone, a mitochondrial complex I inhibitor added with the glycolytic inhibitor 2-deoxy-D-glucose, led to increase of retinal [(3)H]glycine efflux. These effects of ouabain and rotenone/2-deoxy-D-glucose could also be blocked by NFPS pointed to the preferential reverse mode operation of glycine transporter type-1 as a consequence of impaired cellular energy homeostasis. Immunohistochemical studies revealed that glycine transporter type-1, of which reverse mode operation assures [(3)H]glycine release, is expressed in amacrine cells in the inner nuclear and plexiform layers of the retina and also in Müller macroglia cells. We conclude that disruption of the balanced normal/reverse mode operation of glycine transporter type-1 is likely a significant factor contributing to neurotoxic processes of the retina. The possibility to inhibit glycine transporter type-1 mediated glycine efflux by drugs more potently than glycine uptake might offer some therapeutic potential for the treatment of various neurodegenerative disorders of the retina.
Assuntos
Proteínas da Membrana Plasmática de Transporte de Glicina/metabolismo , Glicina/metabolismo , Isquemia/metabolismo , Retina/metabolismo , Vasos Retinianos/metabolismo , Animais , Cálcio/metabolismo , Glucose/metabolismo , Masculino , Ouabaína/farmacologia , Oxigênio/metabolismo , Ratos , Ratos Wistar , Retina/efeitos dos fármacos , Sarcosina/farmacologiaRESUMO
Glutamate is the main excitatory neurotransmitter of the central nervous system (CNS), released both from neurons and glial cells. Acting via ionotropic (NMDA, AMPA, kainate) and metabotropic glutamate receptors, it is critically involved in essential regulatory functions. Disturbances of glutamatergic neurotransmission can be detected in cognitive and neurodegenerative disorders. This paper summarizes the present knowledge on the modulation of glutamate-mediated responses in the CNS. Emphasis will be put on NMDA receptor channels, which are essential executive and integrative elements of the glutamatergic system. This receptor is crucial for proper functioning of neuronal circuits; its hypofunction or overactivation can result in neuronal disturbances and neurotoxicity. Somewhat surprisingly, NMDA receptors are not widely targeted by pharmacotherapy in clinics; their robust activation or inhibition seems to be desirable only in exceptional cases. However, their fine-tuning might provide a promising manipulation to optimize the activity of the glutamatergic system and to restore proper CNS function. This orchestration utilizes several neuromodulators. Besides the classical ones such as dopamine, novel candidates emerged in the last two decades. The purinergic system is a promising possibility to optimize the activity of the glutamatergic system. It exerts not only direct and indirect influences on NMDA receptors but, by modulating glutamatergic transmission, also plays an important role in glia-neuron communication. These purinergic functions will be illustrated mostly by depicting the modulatory role of the purinergic system on glutamatergic transmission in the prefrontal cortex, a CNS area important for attention, memory and learning.
Assuntos
Neuroglia , Neurônios , Receptores de Glutamato/fisiologia , Receptores Purinérgicos/fisiologia , Transdução de Sinais/fisiologia , Transmissão Sináptica/fisiologia , Animais , Humanos , Neuroglia/metabolismo , Neurônios/metabolismo , Receptores de AMPA/fisiologia , Receptores de N-Metil-D-Aspartato/fisiologiaRESUMO
The therapeutic use of opioids is limited by the development of tolerance to the analgesic effect and the cellular and molecular mechanisms underlying this phenomenon are still not completely understood. For this reason the search for new analgesic derivatives, endowed with lower tolerance, is always an active field. The newly synthesized 14-O-Methylmorphine-6-sulfate (14-O-MeM6SU) shows high efficacy in in vitro assays and a strong analgesic action in the rat tail flick test. The aim of present work was to investigate: the analgesic effect of 14-O-MeM6SU in mouse tail-flick test; the tolerance to analgesic effect of 14-O-MeM6SU compared to morphine in mice, the effects of test compounds on glutamatergic neurotransmission by measuring spontaneous excitatory postsynaptic currents (sEPSCs) of layer V pyramidal cells from rat prefrontal cortices; and the effect of acute and chronic 14-O-MeM6SU treatments on opioid receptor gene expression in SH-SY5Y neuroblastoma cells expressing µ-opioid (MOP) and nociceptin/opioid receptor-like 1 (NOP) receptors. 14-O-MeM6SU was 17 times more potent than morphine in analgesia and had long duration of action in analgesic dose equipotent to morphine. Mice were treated subcutaneously (s.c.) either with 200 µmol/kg morphine or with 14-O-MeM6SU (12 µmol/kg) twice daily for three days. The magnitude of tolerance or cross-tolerance indicated by the shift in antinociceptive ED50 measured was greater for morphine compared to 14-O-MeM6SU. Subsequent to behavioral testing, patch-clamp experiments in layer V pyramidal neurons of rat prefrontal cortical slices in the presence of bicuculline were performed. Both 14-O-MeM6SU (0.1 µM) and morphine (1 µM) decreased the frequency of sEPSCs, indicating reduction of glutamate release. The effect of the novel compound was reversed by the opioid receptor antagonist naloxone, indicating an opioid mediated action. In contrast, the amplitude was not affected. Finally, gene expression data showed a dose dependent down-regulation of MOP receptor after 24h and 48 h exposure to 14-O-MeM6SU. Interestingly, no changes were detected for NOP receptor gene expression. The specific lack of this effect could be related to the lower tolerance development to analgesic effect of 14-O-MeM6SU. Furthermore, 14-O-MeM6SU displayed high intrinsic efficacy possibly an important factor in the observed effects. Further, the observed inhibition of glutamatergic signaling might be attributed also to the reduction of opioid tolerance. Based on our results the development of a new clinically important, safe analgesic agent might be possible.
Assuntos
Analgésicos Opioides/farmacologia , Codeína/análogos & derivados , Morfina/farmacologia , Analgésicos Opioides/efeitos adversos , Animais , Linhagem Celular Tumoral , Codeína/efeitos adversos , Codeína/farmacologia , Relação Dose-Resposta a Droga , Avaliação Pré-Clínica de Medicamentos , Tolerância a Medicamentos , Potenciais Pós-Sinápticos Excitadores/efeitos dos fármacos , Ácido Glutâmico/metabolismo , Humanos , Masculino , Camundongos , Morfina/efeitos adversos , Naloxona/farmacologia , Antagonistas de Entorpecentes/farmacologia , Dor Nociceptiva/tratamento farmacológico , Córtex Pré-Frontal/efeitos dos fármacos , Córtex Pré-Frontal/fisiologia , Células Piramidais/efeitos dos fármacos , Células Piramidais/fisiologia , Ratos Wistar , Receptores Opioides/genética , Receptores Opioides/metabolismo , Receptores Opioides mu/genética , Receptores Opioides mu/metabolismo , Transmissão Sináptica/efeitos dos fármacos , Transmissão Sináptica/fisiologia , Técnicas de Cultura de Tecidos , Receptor de NociceptinaRESUMO
BACKGROUND/AIMS: ATP-gated P2X4 purinergic receptors (P2X4Rs) are cation channels with important roles in diverse cell types. To date, lack of specific inhibitors has hampered investigations on P2X4Rs. Recently, the benzodiazepine derivative, 5-BDBD has been proposed to selectively inhibit P2X4Rs. However, limited evidences are currently available on its inhibitory properties. Thus, we aimed to characterize the inhibitory effects of 5-BDBD on recombinant human P2X4Rs. METHODS: We investigated ATP-induced intracellular Ca(2+) signals and whole cell ion currents in HEK 293 cells that were either transiently or stably transfected with hP2X4Rs. RESULTS: Our data show that ATP (< 1 µM) stimulates P2X4R-mediated Ca(2+) influx while endogenously expressed P2Y receptors are not activated to any significant extent. Both 5-BDBD and TNP-ATP inhibit ATP-induced Ca(2+) signals and inward ion currents in a concentration-dependent manner. Application of two different concentrations of 5-BDBD causes a rightward shift in ATP dose-response curve. Since the magnitude of maximal stimulation does not change, these data suggest that 5-BDBD may competitively inhibit the P2X4Rs. CONCLUSIONS: Our results demonstrate that application of submicromolar ATP concentrations allows reliable assessment of recombinant P2XR functions in HEK 293 cells. Furthermore, 5-BDBD and TNP-ATP have similar inhibitory potencies on the P2X4Rs although their mechanisms of actions are different.