RESUMEN
BACKGROUND: Long-term use of antiseizure drugs is associated with a low bone mineral density (BMD) and an increased fracture risk. The literature regarding institutionalised children on chronic antiseizure drugs is limited. Therefore, the aim of this cross-sectional study is to evaluate the prevalence of low BMD and the history of fractures in institutionalised children with epilepsy and intellectual disability (ID). METHODS: A dual-energy X-ray absorptiometry of lumbar spine (L1-L4) and hip was performed in 24 children, residing in a long-stay care facility in the Netherlands. Additionally, serum concentrations of albumin, calcium and 25-hydroxyvitamin D were determined. Data on fractures were retrospectively extracted from the medical files. RESULTS: Ages of the children (14 male and 10 female) ranged from 5 to 17 years with a mean age of 13.0 (±3.2). The criteria of the International Society for Clinical Densitometry (ISCD) were used for classification of bone mineral disorders. Eight (33.3%) children had a normal BMD (Z-score > - 2.0). Of the 16 children with a low BMD (Z-score ≤ - 2.0), three were diagnosed as osteoporotic, based on their fracture history. Ten children (41.7%) were reported to have at least one fracture in their medical history. Serum concentrations of albumin-corrected calcium (2.28-2.50 mmol/L) and (supplemented) vitamin D (16-137 nmol/L) were within the normal range. CONCLUSIONS: This study demonstrated that 67% of institutionalised children with epilepsy and ID had low BMD and 42% had a history of at least one fracture, despite supplementation of calcium and vitamin D in accordance with the Dutch guidelines.
Asunto(s)
Epilepsia , Discapacidad Intelectual , Osteoporosis , Adolescente , Densidad Ósea , Niño , Niño Institucionalizado , Preescolar , Estudios Transversales , Epilepsia/tratamiento farmacológico , Epilepsia/epidemiología , Femenino , Humanos , Discapacidad Intelectual/epidemiología , Masculino , Estudios RetrospectivosRESUMEN
Transient receptor potential channel vanilloid type 6 (TRPV6) is a non-selective cation channel with high permeability for Ca²âº ions. So far, the role of TRPV6 in pancreatic beta cells is unknown. In the present study, we characterized the role of TRPV6 in controlling calcium signaling, cell proliferation as well as insulin expression, and secretion in experimental INS-1E beta cell model. TRPV6 protein production was downregulated using siRNA by approx. 70%, as detected by Western blot. Intracellular free Ca²âº ([Ca²âº]i) was measured by fluorescence Ca²âº imaging using fura-2. Calcineurin/NFAT signaling was analyzed using a NFAT reporter assay as well as a calcineurin activity assay. TRPV6 downregulation resulted in impaired cellular calcium influx. Its downregulation also reduced cell proliferation and decreased insulin mRNA expression. These changes were companied by the inhibition of the calcineurin/NFAT signaling. In contrast, insulin exocytosis was not affected by TRPV6 downregulation. In conclusion, this study demonstrates for the first time the expression of TRPV6 in INS-1E cells and rat pancreatic beta cells and describes its role in modulating calcium signaling, beta cell proliferation and insulin mRNA expression. In contrast, TRPV6 fails to influence insulin secretion.
Asunto(s)
Proliferación Celular/fisiología , Insulinoma/metabolismo , Canales Catiónicos TRPV/fisiología , Animales , Calcio/metabolismo , Línea Celular Tumoral , Homeostasis , Insulina/metabolismo , Secreción de Insulina , Insulinoma/patología , Fosforilación , Ratas , Canales Catiónicos TRPV/genética , Canales Catiónicos TRPV/metabolismoRESUMEN
BACKGROUND: Children with severe neurological impairment and intellectual disability (ID) are susceptible for developing low bone mineral density (BMD) and fractures. BMD is generally measured with dual-energy X-ray absorptiometry (DXA). OBJECTIVE: To describe the occurrence of factors that may influence the feasibility of DXA and the accuracy of DXA outcome in children with severe neurological impairment and ID. MATERIALS AND METHODS: Based on literature and expert opinion, a list of disrupting factors was developed. Occurrence of these factors was assessed in 27 children who underwent DXA measurement. RESULTS: Disrupting factors that occurred most frequently were movement during measurement (82%), aberrant body composition (67%), small length for age (56%) and scoliosis (37%). The number of disrupting factors per child was mean 5.3 (range 1-8). No correlation was found between DXA outcomes and the number of disrupting factors. CONCLUSION: Factors that may negatively influence the accuracy of DXA outcome are frequently present in children with severe neurological impairment and ID. No systematic deviation of DXA outcome in coherence with the amount of disrupting factors was found, but physicians should be aware of the possible influence of disrupting factors on the accuracy of DXA.
Asunto(s)
Absorciometría de Fotón , Artefactos , Densidad Ósea , Fracturas Óseas/diagnóstico por imagen , Fracturas Óseas/etiología , Discapacidad Intelectual/complicaciones , Vértebras Lumbares/diagnóstico por imagen , Enfermedades del Sistema Nervioso/complicaciones , Osteoporosis/diagnóstico por imagen , Osteoporosis/etiología , Imagen de Cuerpo Entero , Adolescente , Lista de Verificación , Niño , Preescolar , Humanos , Lactante , Masculino , Proyectos Piloto , Factores de Riesgo , Adulto JovenRESUMEN
PURPOSE: The aim of this longitudinal study was to assess trabecular bone scores (TBS) in institutionalized adults with refractory epilepsy and intellectual disability and to study the association of TBS and incident fractures during seven years of follow-up. METHODS: In 2009 and 2016, all institutionalized adult patients of a long-stay care facility in the Netherlands (n=261) were invited to undergo a dual-energy X-ray absorptiometry (DXA) including vertebral fracture assessment (VFA) and assessment of TBS. Vertebrae T4-L4 were analyzed using quantitative morphometry. New and worsening vertebral fractures (VFs) were considered as incident VFs. Data regarding clinical fractures were extracted from the medical files. Patients were treated with anti-osteoporosis medication according to the Dutch guideline. RESULTS: Baseline and follow-up DXA, VFA and TBS could be obtained in 136 patients (83 male) aged between 18 and 79 years old (44.7±15.5). At baseline, 36 patients (26.5%) were diagnosed with osteoporosis, 68 (50.0%) with osteopenia and 32 patients (23.5%) had a normal bone mineral density (BMD). As for TBS, 26 patients (19.1%) had a partially degraded microarchitecture and 26 patients (19.1%) a degraded microarchitecture. During seven years of follow-up, 80 patients (59%) sustained at least one fracture, of which 28 patients (35%) had one or more major osteoporotic fractures. Thirty-four patients (25.0%) had at least one new or worsening morphometric VF. Compared to baseline, TBS significantly decreased over seven years of follow-up in non-treated patients (-0.039±0.064, p<.001). In patients who were treated with bisphosphonates for more than one year during follow-up, TBS did not change significantly (p=.093). In multivariate analyses, no significant associations were found between TBS at baseline and incident fractures during follow-up. CONCLUSION: In this study, we found a high incidence of fractures and TBS decreased significantly over seven years of follow-up in non-treated institutionalized adult patients with refractory epilepsy and intellectual disability, but TBS was not associated with incident fractures.
Asunto(s)
Epilepsia Refractaria , Discapacidad Intelectual , Fracturas de la Columna Vertebral , Adulto , Humanos , Masculino , Adolescente , Adulto Joven , Persona de Mediana Edad , Anciano , Hueso Esponjoso/diagnóstico por imagen , Estudios de Seguimiento , Epilepsia Refractaria/complicaciones , Discapacidad Intelectual/epidemiología , Discapacidad Intelectual/complicaciones , Estudios Longitudinales , Vértebras Lumbares , Fracturas de la Columna Vertebral/complicaciones , Fracturas de la Columna Vertebral/epidemiología , Densidad ÓseaRESUMEN
Currently, the identification of apoptotic or damaged human corneal endothelial (HCE) cells is limited to a morphological assessment and vital staining. Specific electrophysiological investigations may prospectively help to identify damaged HCE cells at an earlier stage. Besides calcium imaging, the so-called patch-clamp technique is an important test method enabling one to assay the effect of various substances on ion channels and receptors of the cell membrane. First electrophysiological pilot experiments with cultivated and freshly isolated HCE cells have revealed promising results. In this way, the expression of certain transient receptor potential channels (TRPs) could be demonstrated. However, the function of these channels is still not fully elucidated. In humans, TRPs play a crucial role in the sense of taste, pheromones, temperature and pain and are involved in osmolarity. This review summarises the current literature on the electrophysiology of the human corneal endothelium and deduces potential approaches to a sensitive vitality and function test under utilisation of the electrophysiological properties of HCE cells.
Asunto(s)
Células Endoteliales/fisiología , Epitelio Corneal/fisiología , Técnicas de Placa-Clamp/métodos , Canales de Potencial de Receptor Transitorio/fisiología , Animales , Epitelio Corneal/citología , Humanos , Modelos BiológicosRESUMEN
PURPOSE: To determine the incidence of clinical fractures over seven years of follow-up, in adults with epilepsy and intellectual disability, residing in a long-stay care facility. METHODS: In 2009, all institutionalized adult patients (n = 261) were invited to undergo a Dual-energy X-ray Absorptiometry (DXA) measurement and a Vertebral Fracture Assessment (VFA). Participants were followed over seven years or until date of discharge (in case of moving from the care facility) or date of death. The patients' medical files were screened for radiology reports and staff notes, to identify clinical fractures. Fracture incidence rates (IR) were determined and compared for subgroups, by calculating incidence rate ratios. Hazard ratios were calculated to identify factors associated with fracture risk, using Cox Proportional Hazards analyses. RESULTS: A total of 205 patients (124 male, 60.5%) aged between 18 and 88 years (median 48, IQR 34-60) were enrolled. At baseline, 92 patients (44.9%) were diagnosed with osteopenia and 65 (31.7%) with osteoporosis. Between 2009 and 2016, 30 patients (14.6%) deceased and 3 patients (1.5%) left the care facility. During follow-up, 156 clinical fractures were reported in 82 patients (40.0%). Thirty-eight patients (18.5%) had at least one major osteoporotic fracture. Overall, the IR was 11.6 fractures per 100 person-years. Fracture risk was significantly lower in patients who were wheelchair dependent than in patients who were able to walk (p<.001). CONCLUSION: This study demonstrated that 40% of institutionalized adults with epilepsy and intellectual disability had at least one clinical fracture during seven years of follow-up, despite adequate anti-osteoporosis treatment.
Asunto(s)
Epilepsia , Discapacidad Intelectual , Fracturas Osteoporóticas , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Densidad Ósea , Epilepsia/epidemiología , Estudios de Seguimiento , Humanos , Incidencia , Discapacidad Intelectual/complicaciones , Discapacidad Intelectual/epidemiología , Masculino , Persona de Mediana Edad , Fracturas Osteoporóticas/epidemiología , Adulto JovenRESUMEN
PURPOSE: Long-term exposure to anti-epileptic drugs has been shown to decrease bone mineral density (BMD). The aim of this 7-year follow-up study was to explore changes in bone status, using quantitative ultrasound (QUS) and Dual-energy X-ray Absorptiometry (DXA) in adults with refractory epilepsy and intellectual disability (ID) residing at a long-term care facility. Both measurements can be challenging to conduct in this population. METHODS: In 2009 and 2016, a total of 126 patients (18-79 years) underwent QUS of the heel and DXA of lumbar spine (LS) and hip (femoral neck (FN) and total hip (TH)). Subgroup analysis was performed for patients with (group A, n = 53) and without (group B, n = 73) bisphosphonate use during follow-up. RESULTS: Overall, weak to moderate correlations between changes in DXA and QUS parameters were found. For group A, correlations varied from r = .31 to .59, whereas correlations did not exceed r = .40 in group B. Patients in group A showed a larger increase or a smaller decrease in BMD for all DXA regions during follow-up (p < .001 for ΔLS and ΔFN BMD, p = .001 for ΔTH BMD). For change in QUS parameters, no significant difference between groups was found. CONCLUSION: In this study we demonstrated the limited use of QUS in the monitoring of bone status in our study population. Although correlations between changes in QUS parameters and axial DXA are positive and mostly significant, QUS only explains little of the variability in DXA values and is inadequate for measuring treatment response in this population.
Asunto(s)
Absorciometría de Fotón/normas , Anticonvulsivantes/efectos adversos , Densidad Ósea , Epilepsia Refractaria/diagnóstico por imagen , Discapacidad Intelectual , Ultrasonografía/normas , Adolescente , Adulto , Anciano , Comorbilidad , Difosfonatos/uso terapéutico , Epilepsia Refractaria/tratamiento farmacológico , Epilepsia Refractaria/epidemiología , Femenino , Estudios de Seguimiento , Humanos , Discapacidad Intelectual/epidemiología , Masculino , Persona de Mediana Edad , Adulto JovenRESUMEN
TRPV4 is a Ca2+-permeable, nonselective cation channel. Recently, TRPV4 was implicated in controlling peripheral insulin sensitivity, insulin secretion and apoptosis of pancreatic beta cells. Here, we characterize the role and potential mechanisms of TRPV4 in regulating insulin mRNA expression and cell death in insulin producing INS-1E cells and rat pancreatic islets. TRPV4 protein production was downregulated by siRNA. Intracellular calcium level was measured using Fluo-3 AM. Gene expression was studied by real-time PCR. Phosphorylation of extracellular signal-regulated kinase (ERK1 and ERK2) was detected by Western blot. Nitric oxide (NO) production was assessed by chemiluminescent reaction. Reactive oxygen species (ROS) level was analysed using a fluorogenic dye (DCFDA). Cell death was evaluated by determination of cytoplasmic histone-associated DNA fragments. Downregulation of TRPV4 neither affected insulin mRNA expression nor INS-1E cell growth. By contrast, pharmacological TRPV4 activation by 100nmol/l GSK1016790A increased Ca2+ levels in INS-1E cells and enhanced insulin mRNA expression after 1 and 3h, whereas a suppression of insulin mRNA expression was detected after 24h incubation. GSK1016790A increased ERK1/2 phosphorylation and NO production but not ROS production. Pharmacological blockade of ERK1/2 attenuated GSK1016790A-induced insulin mRNA expression. Inhibition of NO synthesis by l-NAME failed to affect insulin mRNA expression in GSK1016790A treated INS-1E cells. Furthermore, inhibition of NO production attenuated GSK1016790A-induced INS-1E cell death. In pancreatic islets, 100nmol/l GSK1016790A increased insulin mRNA levels after 3h without inducing cytotoxicity after 24h. In conclusion, TRPV4 differently regulates insulin mRNA expression in INS-1E cells via ERK1/2 and NO-dependent mechanisms.
Asunto(s)
Calcio/metabolismo , Insulina/genética , Óxido Nítrico/metabolismo , Canales Catiónicos TRPV/genética , Animales , Proliferación Celular/genética , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Insulina/metabolismo , Células Secretoras de Insulina/metabolismo , Células Secretoras de Insulina/patología , Leucina/administración & dosificación , Leucina/análogos & derivados , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Fosforilación , ARN Mensajero/genética , Ratas , Especies Reactivas de Oxígeno/metabolismo , Sulfonamidas/administración & dosificación , Canales Catiónicos TRPV/metabolismoRESUMEN
Orexins A (OXA) and B (OXB) control energy homeostasis by regulating food intake, energy expenditure and sleep-wake cycle. Several studies showed that OXA stimulates insulin secretion and proliferation of beta cells. However, mechanisms of action are still not well understood. Here, we investigated whether ERK and transient receptor potential channels (TRPs) play a role in mediating the effect of OXA on cell growth, insulin production, and secretion using the established INS-1E cell line. Cell proliferation was measured using BrdU assay. Insulin mRNA expression was detected by real-time PCR. Insulin secretion was assessed using ELISA. Intracellular calcium levels were measured using fluorescence calcium imaging (fura-2/AM). Extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation was detected by Western blot. TRP channel activity was blocked by lanthanum (III) chloride (La3+; 100 - 300 µM) or ruthenium red (RuR; 10 µM). OXA (100 nM) stimulated INS-1E cell proliferation, insulin secretion, intracellular Ca2+ concentration and ERK1/2 phosphorylation, without changing insulin mRNA expression. Inhibition of ERK1/2 by 10 µM U0126 attenuated OXA-stimulated INS-1E cell proliferation. Blockade of TRP channel activity by La3+ or RuR rendered OXA ineffective at modulating Ca2+ regulation and insulin release. In contrast, the L-type channel blocker nifedipine (10 µM) failed to affect OXA-stimulated insulin release. Taken together, OXA increases INS-1E cell proliferation via ERK1/2-dependent mechanism. Furthermore, OXA stimulates insulin secretion from INS-1E cells. TRPs are relevant for OXA-stimulated insulin secretion and intracellular calcium regulation.
Asunto(s)
Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Orexinas/farmacología , Canales de Potencial de Receptor Transitorio/metabolismo , Animales , Calcio/metabolismo , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Insulina/metabolismo , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Receptores de Orexina/genética , ARN Mensajero/metabolismo , RatasRESUMEN
Capsaicin (CAP), the pungent ingredient of chili peppers, inhibits growth of various solid cancers via TRPV1 as well as TRPV1-independent mechanisms. Recently, we showed that TRPV1 regulates intracellular calcium level and chromogranin A secretion in pancreatic neuroendocrine tumor (NET) cells. In the present study, we characterize the role of the TRPV1 agonist - CAP - in controlling proliferation and apoptosis of pancreatic BON and QGP-1 NET cells. We demonstrate that CAP reduces viability and proliferation, and stimulates apoptotic death of NET cells. CAP causes mitochondrial membrane potential loss, inhibits ATP synthesis and reduces mitochondrial Bcl-2 protein production. In addition, CAP increases cytochrome c and cleaved caspase 3 levels in cytoplasm. CAP reduces reactive oxygen species (ROS) generation. The antioxidant N-acetyl-l-cysteine (NAC) acts synergistically with CAP to reduce ROS generation, without affecting CAP-induced toxicity. TRPV1 protein reduction by 75% reduction fails to attenuate CAP-induced cytotoxicity. In summary, these results suggest that CAP induces cytotoxicity by disturbing mitochondrial potential, and inhibits ATP synthesis in NET cells. Stimulation of ROS generation by CAP appears to be a secondary effect, not related to CAP-induced cytotoxicity. These results justify further evaluation of CAP in modulating pancreatic NETs in vivo.
Asunto(s)
Capsaicina/farmacología , Mitocondrias/metabolismo , Tumores Neuroendocrinos/patología , Neoplasias Pancreáticas/patología , Adenosina Trifosfato/metabolismo , Apoptosis/efectos de los fármacos , Muerte Celular/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Humanos , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Mitocondrias/efectos de los fármacos , Tumores Neuroendocrinos/metabolismo , Neoplasias Pancreáticas/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Transducción de Señal/efectos de los fármacos , Factores de TiempoRESUMEN
Transient receptor potential channel vanilloid type 4 (TRPV4) is a Ca(2+)- and Mg(2+)-permeable cation channel that influences oxidative metabolism and insulin sensitivity. The role of TRPV4 in pancreatic beta cells is largely unknown. Here, we characterize the role of TRPV4 in controlling intracellular Ca(2+) and insulin secretion in INS-1E beta cells. Osmotic, thermal or pharmacological activation of TRPV4 caused a rapid rise of intracellular Ca(2+) and enhanced glucose-stimulated insulin secretion. In the presence of the TRPV channel blocker ruthenium red (RuR) or after suppression of TRPV4 protein production, TRPV4 activators failed to increase [Ca(2+)]i and insulin secretion in INS-1E cells.
Asunto(s)
Calcio/metabolismo , Glucosa/metabolismo , Insulina/metabolismo , Islotes Pancreáticos/metabolismo , Canales Catiónicos TRPV/metabolismo , Línea Celular , Humanos , Secreción de InsulinaRESUMEN
Cannabinoid receptor type 1 (CB1)-induced suppression of transient receptor potential vanilloid type 1 (TRPV1) activation provides a therapeutic option to reduce inflammation and pain in different animal disease models through mechanisms involving dampening of TRPV1 activation and signaling events. As we found in both mouse corneal epithelium and human corneal epithelial cells (HCEC) that there is CB1 and TRPV1 expression colocalization based on overlap of coimmunostaining, we determined in mouse corneal wound healing models and in human corneal epithelial cells (HCEC) if they interact with one another to reduce TRPV1-induced inflammatory and scarring responses. Corneal epithelial debridement elicited in vivo a more rapid wound healing response in wildtype (WT) than in CB1(-/-) mice suggesting functional interaction between CB1 and TRPV1. CB1 activation by injury is tenable based on the identification in mouse corneas of 2-arachidonylglycerol (2-AG) with tandem LC-MS/MS, a selective endocannabinoid CB1 ligand. Suppression of corneal TRPV1 activation by CB1 is indicated since following alkali burning, CB1 activation with WIN55,212-2 (WIN) reduced immune cell stromal infiltration and scarring. Western blot analysis of coimmunoprecipitates identified protein-protein interaction between CB1 and TRPV1. Other immunocomplexes were also identified containing transforming growth factor kinase 1 (TAK1), TRPV1 and CB1. CB1 siRNA gene silencing prevented suppression by WIN of TRPV1-induced TAK1-JNK1 signaling. WIN reduced TRPV1-induced Ca(2+) transients in fura2-loaded HCEC whereas pertussis toxin (PTX) preincubation obviated suppression by WIN of such rises caused by capsaicin (CAP). Whole cell patch clamp analysis of HCEC showed that WIN blocked subsequent CAP-induced increases in nonselective outward currents. Taken together, CB1 activation by injury-induced release of endocannabinoids such as 2-AG downregulates TRPV1 mediated inflammation and corneal opacification. Such suppression occurs through protein-protein interaction between TRPV1 and CB1 leading to declines in TRPV1 phosphorylation status. CB1 activation of the GTP binding protein, G(i/o) contributes to CB1 mediated TRPV1 dephosphorylation leading to TRPV1 desensitization, declines in TRPV1-induced increases in currents and pro-inflammatory signaling events.
Asunto(s)
Epitelio Corneal/lesiones , Receptor Cannabinoide CB1/metabolismo , Canales Catiónicos TRPV/metabolismo , Animales , Ácidos Araquidónicos/metabolismo , Benzoxazinas/farmacología , Calcio/metabolismo , Bloqueadores de los Canales de Calcio/farmacología , Línea Celular , Modelos Animales de Enfermedad , Endocannabinoides/metabolismo , Células Epiteliales/efectos de los fármacos , Células Epiteliales/fisiología , Epitelio Corneal/citología , Epitelio Corneal/metabolismo , Glicéridos/metabolismo , Humanos , Inmunidad Innata/efectos de los fármacos , Quinasas Quinasa Quinasa PAM/metabolismo , Ratones , Ratones Noqueados , Proteína Quinasa 8 Activada por Mitógenos/metabolismo , Morfolinas/farmacología , Naftalenos/farmacología , Técnicas de Placa-Clamp , Toxina del Pertussis/farmacología , Unión Proteica , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Receptor Cannabinoide CB1/antagonistas & inhibidores , Receptor Cannabinoide CB1/genética , Transducción de Señal , Canales Catiónicos TRPV/antagonistas & inhibidores , Cicatrización de Heridas/efectos de los fármacosRESUMEN
Low bone mineral density (BMD) and fractures are common in people with intellectual disabilities (ID). Reduced mobility in case of motor impairment and the use of anti-epileptic drugs contribute to the development of low BMD. Quantitative ultrasound (QUS) measurement of the heel bone is a non-invasive and radiation-free method for measuring bone status that can be used outside the hospital. QUS might be used for screening purposes to identify people with intellectual disability with poor bone status, who are in need of supplementary examination and treatment. To investigate feasibility of QUS in this group, QUS of the heel bone was performed on-site in 151 people with ID living in residential care. Measurements were successfully performed in at least one foot in 94.7%, were interpretable (resulting in a stiffness index) in 91.6%, and induced barely or no stress in 90.4% of the study population. Measurements generally took less than 10 min. In 93 persons bone status of both feet had been measured. The "mean percentage of the absolute difference" between outcomes of both feet was 15.5% (±15.3% SD, range 0-76.5%). Ultrasound measurement of the heel bone is a feasible and non-stressful method for measuring bone status in people with ID. Since the mean difference between outcomes of the left and right foot were large, measurement of both feet is recommended to prevent inaccurate interpretation.
Asunto(s)
Densidad Ósea , Enfermedades Óseas/complicaciones , Enfermedades Óseas/diagnóstico por imagen , Calcáneo/diagnóstico por imagen , Discapacidad Intelectual/complicaciones , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Niño , Preescolar , Estudios de Factibilidad , Femenino , Humanos , Lactante , Masculino , Persona de Mediana Edad , Aceptación de la Atención de Salud , Sistemas de Atención de Punto , Instituciones Residenciales , Ultrasonografía , Adulto JovenRESUMEN
This electrophysiological study was undertaken to investigate the role of voltage-operated Ca(2+) channels (VOCCs) in cultivated human neuroendocrine tumor (NET) cells. Patch-clamp techniques, measurements of intracellular Ca(2+) ([Ca(2+)](i)), and secretion analysis were performed using cultured human NET BON cells. Ba(2+) inward currents through R-type channels (Ca(V)2.3) were measured and identified by SNX-482 (10 n M), a novel voltage-sensitive R-type Ca(2+) channel antagonist. In the presence of nifedipine (5 micro M), omega-Conotoxin GVIA (100 n M) and omega-Agatoxin IVA (20 n M), R-type channel currents were also detectable. Release of Ca(2+) from intracellular Ca(2+) stores by intracellular application of inositol-1,4,5-trisphosphate (InsP(3); 10 micro M) via the patch pipette during whole-cell configuration as well as induction of capacitative Ca(2+) entry (CCE), a passive maneuver to release Ca(2+) from intracellular Ca(2+) stores, led to an increase in [Ca(2+)](i). This effect could be reduced by SNX-482 (20 n M). In addition, SNX-482 (25 n M) also decreased chromogranin A (CgA) secretion, whereas omega-Conotoxin GVIA (500 n M) and nifedipine (5 micro M) failed to reduce CgA secretion. We conclude that these data reveal neuronal R-type channel activity (Ca(V)2.3), for the first time associated with CgA secretion in BON cells. Influx of Ca(2+) by activation of R-type channels may lead to an increase of intracellular Ca(2+), which stimulates CgA secretion. Thus, R-type channels could play an important role in certain clinical characteristics of NETs, such as the hypersecretion syndrome.
Asunto(s)
Canales de Calcio Tipo R/fisiología , Señalización del Calcio/fisiología , Cromograninas/metabolismo , Tumores Neuroendocrinos/patología , Neoplasias Pancreáticas/patología , Bario/metabolismo , Calcio/metabolismo , Bloqueadores de los Canales de Calcio/farmacología , Señalización del Calcio/efectos de los fármacos , Cromogranina A , Humanos , Líquido Intracelular/metabolismo , Transporte Iónico , Proteínas de Neoplasias/metabolismo , Tumores Neuroendocrinos/metabolismo , Neoplasias Pancreáticas/metabolismo , Técnicas de Placa-Clamp , Venenos de Araña/farmacología , Células Tumorales Cultivadas/metabolismoRESUMEN
The effect of protein tyrosine kinases (PTKs) on L-type calcium channel currents was studied in cultured rat and human retinal pigment epithelial cells. Barium currents through L-type channels were measured in the perforated patch-clamp technique and identified by using the L-type calcium channel opener Bay K8644 (10(-6) M). Application of the PTK blockers genistein (5 x 10(-6) M) or lavendustin A (5 x 10(-6) M) led to a decrease of L-type currents. The inactive genistein analog daidzein (10(-5) M) showed no effect on calcium channels. Intracellular application of pp60(c-src) (30 U/ml) via the patch-pipette during the conventional whole-cell configuration led to an increase of L-type currents. The protein kinase A and protein kinase G blocker H9 (10(-6) M) showed no effect on L-type currents; genistein reduced the current in the presence of H9. The protein kinase C (PKC) blocker chelerythrine (10(-5) M) reduced the L-type current; additional inhibition of PTK by lavendustin showed an additional reduction of currents. Intracellular application of myristoylated PKC substrate (5 x 10(-5) M) for PKC inhibition led to a fast rundown of L-type current amplitudes. Intracellularly applied myristoylated PKC substrate (10(-4) M) together with pp60(c-src) showed no effect on L-type current. Up-regulation of PKC by 10(-6) M phorbol-12-myristate-13-acetate (PMA) had no effect on the L-type current amplitude. However, genistein in cells pretreated with PMA led to an increase of the L-type currents. Intracellular application of pp60(c-src) in PMA-treated cells led to a reduction of L-type currents. We conclude that in the resting cell, PTK and PKC regulate L-type calcium channels in an additive manner. L-type channels appeared as a site of integration of PTK activation and of PKC-dependent pathways. The activity of PKC determines whether PTK decreases or increases L-type channel activity.
Asunto(s)
Canales de Calcio/fisiología , Epitelio Pigmentado Ocular/metabolismo , Proteína Quinasa C/fisiología , Proteínas Tirosina Quinasas/fisiología , Animales , Canales de Calcio Tipo L , Células Cultivadas , Humanos , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas pp60(c-src)/fisiología , Ratas , Acetato de Tetradecanoilforbol/farmacologíaRESUMEN
The effect of protein tyrosine kinases (PTK) on L-type calcium channels in cultured retinal pigmented epithelium (RPE) from rats with retinal dystrophy was investigated. Barium currents through Bay K 8644 (10(-6) M) sensitive L-type channels were measured using the patch-clamp technique. The current density of L-type currents is twice as high and the inactivation time constants are much slower than in cells from nondystrophic control rats. Application of the PTK blockers genistein, lavendustin A, and herbimycin A (all 5 x 10(-6) M) led to an increase of L-type currents. Intracellular application of pp60c-src (30 U/ml) via the patch pipette led to a transient decrease of L-type currents. The protein kinase A (PKA) and PKG blocker H9 (10(-6) M) showed no effect on L-type currents. However, the protein kinase C blocker chelerythrine (10(-5) M) reduced these currents. Up-regulation of PKC by 10(-6) M 4beta-phorbol-12 myristate-13 acetate (PMA) led to a decrease of L-type currents. Additional application of genistein led to a further decrease of these currents. However, intracellular application of pp60(c-src) in PMA-treated cells led to a transient increase of L-type currents. Investigating the calcium response to bFGF application showed that RPE cells from RCS rats used different pathways than control RPE cells to increase cytosolic free calcium. This different pathway does not involve the activation of L-type channels. The present study with RPE cells from rats with retinal dystrophy shows a changed integration of PTK and PKC in channel regulation. Considering the altered response to bFGF in RCS-RPE cells, this disturbed regulation of L-type channels by tyrosine kinases is involved in the etiology of retinal degeneration in RCS rats.
Asunto(s)
Canales de Calcio/biosíntesis , Inhibidores Enzimáticos/farmacología , Epitelio Pigmentado Ocular/fisiopatología , Proteína Quinasa C/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Degeneración Retiniana/fisiopatología , Alcaloides , Animales , Animales Recién Nacidos , Benzofenantridinas , Benzoquinonas , Calcio/metabolismo , Canales de Calcio/efectos de los fármacos , Canales de Calcio/fisiología , Canales de Calcio Tipo L , Células Cultivadas , Factor 2 de Crecimiento de Fibroblastos/farmacología , Genisteína/farmacología , Lactamas Macrocíclicas , Potenciales de la Membrana/efectos de los fármacos , Nifedipino/farmacología , Técnicas de Placa-Clamp , Fenantridinas/farmacología , Fenoles/farmacología , Epitelio Pigmentado Ocular/efectos de los fármacos , Epitelio Pigmentado Ocular/fisiología , Proteína Quinasa C/antagonistas & inhibidores , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Quinonas/farmacología , Ratas , Valores de Referencia , Degeneración Retiniana/enzimología , Rifabutina/análogos & derivados , Acetato de Tetradecanoilforbol/farmacología , Regulación hacia Arriba/efectos de los fármacosRESUMEN
The Royal College of Surgeons (RCS) rat is the first known animal with inherited retinal degeneration. Despite the fact that the genetic defect is not known, the RCS rat is widely used for research in hereditary retinal dystrophies. This review tries to summarize observations which have been made in the RCS rat and to make an attempt to formulate candidate genes which may the cause for the retinal degeneration in this rat strain. The genetic defect in RCS rats causes the inability of the retinal pigment epithelium (RPE) to phagocytose shed photoreceptor outer segments. In normal rats or humans, this circadian process is regulated by both the cyclic adenosine monophosphate (cAMP) and the calcium/ inositol phosphate systems. The calcium/inositol phosphate system seems to be linked to the phagocytosis receptors which recognize photoreceptor outer membranes to initialize phagocytosis. The cAMP system appeared as modulator of the regulation of phagocytosis. An increase in the intracellular cAMP concentration is an 'off' signal for phagocytosis. In RPE cells from RCS rats many observations have been made which indicate a changed second messenger metabolism concerning both the cAMP and the calcium/inositol phosphate systems. The genetic defect seems to concern a protein which is involved in the initialization of a second messenger pathway. We conclude that the genes coding for the phagocytosis receptor or for proteins which are linked to receptors (for example G proteins) are good candidates for defective genes in RCS rats.
Asunto(s)
Modelos Animales de Enfermedad , Ratas Mutantes/genética , Degeneración Retiniana/genética , Animales , Humanos , Fagocitosis/fisiología , Células Fotorreceptoras de Vertebrados/fisiología , Epitelio Pigmentado Ocular/fisiopatología , Ratas , Degeneración Retiniana/fisiopatología , Sistemas de Mensajero Secundario/fisiologíaRESUMEN
This combined study of patch-clamp and intracellular Ca2+ ([Ca2+]i) measurement was undertaken in order to identify signaling pathways that lead to activation of Ca2+-dependent Cl- channels in cultured rat retinal pigment epithelial (RPE) cells. Intracellular application of InsP3 (10 microM) led to an increase in [Ca2+]i and activation of Cl- currents. In contrast, intracellular application of Ca2+ (10 microM) only induced transient activation of Cl- currents. After full activation by InsP3, currents were insensitive to removal of extracellular Ca2+ and to the blocker of ICRAC, La3+ (10 microM), despite the fact that both maneuvers led to a decline in [Ca2+]i. The InsP3-induced rise in Cl- conductance could be prevented either by thapsigargin-induced (1 microM) depletion of intracellular Ca2+ stores or by removal of Ca2+ prior to the experiment. The effect of InsP3 could be mimicked by intracellular application of the Ca2+-chelator BAPTA (10 mm). Block of PKC (chelerythrine, 1 microM) had no effect. Inhibition of Ca2+/calmodulin kinase (KN-63, KN-92; 5 microM) reduced Cl--conductance in 50% of the cells investigated without affecting [Ca2+]i. Inhibition of protein tyrosine kinase (50 microM tyrphostin 51, 5 microM genistein, 5 microM lavendustin) reduced an increase in [Ca2+]i and Cl- conductance. In summary, elevation of [Ca]i by InsP3 leads to activation of Cl- channels involving cytosolic Ca2+ stores and Ca2+ influx from extracellular space. Tyrosine kinases are essential for the Ca2+-independent maintenance of this conductance.
Asunto(s)
Calcio/fisiología , Canales de Cloruro/fisiología , Fosfatos de Inositol/fisiología , Activación del Canal Iónico/fisiología , Epitelio Pigmentado Ocular/fisiología , Proteínas Tirosina Quinasas/fisiología , Animales , Células Cultivadas , Técnicas de Placa-Clamp , Ratas , Transducción de SeñalRESUMEN
The aim of this study is to characterize the subtype of tyrosine kinase-regulated L-type Ca(2+) channels in retinal pigment epithelial (RPE) cells. Ca(2+) channel alpha1D-subunits were enriched by immunoprecipitation from membrane proteins isolated from rat RPE cells. Western blot analysis of the precipitates revealed coprecipitation of pp60(c-src). In addition, in precipitates obtained with antibodies against pp60(c-src), alpha1D-subunits were identified. The same was observed in immunoprecipitations from rat brain neurons. Tyrosine phosphorylation of alpha1D-subunits was confirmed using anti-phosphotyrosine antibodies. Ba(2+) currents through L-type channels in cultured rat RPE cells were increased by intracellular application of active pp60(c-src) (30 U/ml) (heat-inactivated pp60(c-src) had no effect). Thus, L-type channels of the neuroendocrine subtype can be expressed in epithelial cells and are activated by tyrosine kinase of the src subtype. This kind of regulation is also suggested for brain-derived neurons.