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BACKGROUND AND AIMS: Spontaneous subarachnoid hemorrhage (SAH) from a brain aneurysm, if untreated in the acute phase, leads to loss of functional independence in about 30% of patients and death in 27-44%. To evaluate for SAH, the American College of Emergency Physicians (ACEP) Clinical Policy recommends obtaining a non-contrast brain computed tomography (CT) scan followed by a lumbar puncture (LP) if the CT is negative. On the other hand, current evidence from prospectively collected data suggests that CT alone may be sufficient to rule out SAH in patients who present within 6 h of symptom onset while anecdotal evidence suggests that CT angiogram (CTA) may be used to detect aneurysms, which are the probable cause of SAH. Since many different options are available to emergency physicians, we examined their practice pattern variation by observing their diagnostic approaches and their adherence to the ACEP Clinical Policy. METHODS: We developed, validated, and distributed a survey to emergency physicians at three practice sites: (1) Stanford Healthcare, California, (2) Intermountain Healthcare (five emergency departments), Utah, and (3) Ottawa General Hospital, Toronto. The survey questions examined physician knowledge on CT and LP's test performance and used case-based scenarios to assess diagnostic approaches, variation in practice, and adherence to guidelines. Results were presented as proportions with 95% CIs. RESULTS: Of the 216 physicians surveyed, we received 168 responses (77.8%). The responses by site were: (1) (n = 38, 23.2%), (2) (n = 70, 42.7%), (3) (n = 56, 34.1%). To the CT and LP test performance question, most physicians indicated that CT alone detects > 90% of SAH in those with a confirmed SAH [n = 150 (89.3%, 95% CI 83.6-93.5]. To the case-based questions, most physicians indicated that they would perform a CTA along with a CT [n = 110 (65.5%, 95% CI 57.8-72.6)], some indicated a LP along with a CT [n = 57, 33.9% 95% CI 26.8-41.6)], and a few indicated both a CTA and a LP [n = 16, 9.5%, 95% CI 5.5-15.0]. We also observed practice site variation in the proportion of physicians who indicated that they would use CTA: (1) (n = 25, 65.8%), (2) (n = 54, 77.1%), and (3) (n = 28, 50.0%) (p = 0.006). CONCLUSIONS: Survey responses indicate that physicians use some or all of the imaging tests, with or without LP to diagnose SAH. We observed variation in the use of CTA by site and academic setting and divergence from ACEP Clinical Policy.
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Angiografía por Tomografía Computarizada/estadística & datos numéricos , Medicina de Emergencia , Médicos , Pautas de la Práctica en Medicina/estadística & datos numéricos , Hemorragia Subaracnoidea/diagnóstico , Adulto , California , Canadá , Angiografía Cerebral/estadística & datos numéricos , Femenino , Humanos , Masculino , Persona de Mediana Edad , Ontario , Punción Espinal/estadística & datos numéricos , Encuestas y Cuestionarios , Tomografía Computarizada por Rayos X/estadística & datos numéricos , Estados Unidos , Utah , Adulto JovenRESUMEN
The retinal degeneration model rd10 contains a missense mutation of the catalytic PDE6 ß subunit, which hydrolyzes cGMP in response to light. This model produces cell death more slowly than others caused by PDE6 loss of function, making it of particular interest for studying potential therapeutics. We used morphology, biochemistry, and single-cell physiology to examine the mechanism of rd10 degeneration. Our results show that the mutation produces no alteration of Pde6b RNA but does dramatically decrease maximal and basal PDE6 activity, apparently caused by a decrease in protein stability and transport. The enzymatic properties of the remaining mutant PDE6 appear to be nearly normal. We demonstrate that an increase in free cGMP, which would result from decreased PDE6 activity and serve to increase opening of the cGMP-gated channels and calcium influx, is an underlying cause of cell death: degeneration of rd10/Cngb1-/- double mutants is slower than the parent rd10 line. Paradoxically, degeneration in rd10/Cngb1-/- is also slower than in Cngb1-/- This rescue is correlated with a lowering of cGMP content in Cngb1-/- retinas and suggests that it may be caused by mislocalization of active PDE6. Single-cell recordings from rd10 rods show that the rates of rise and decay of the response are significantly slower; simulations indicate that these changes are primarily the result of the decrease in PDE6 concentration and rod collecting area. Together, these results provide insights into the complex mechanisms that underlie rd10-mediated retinal degeneration and a cautionary note for analysis of therapeutic interventions.
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Calcio/metabolismo , GMP Cíclico/metabolismo , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 6/genética , Canales Catiónicos Regulados por Nucleótidos Cíclicos/genética , Proteínas del Tejido Nervioso/genética , Degeneración Retiniana/genética , Células Fotorreceptoras Retinianas Bastones/metabolismo , Animales , Muerte Celular , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 6/deficiencia , Canales Catiónicos Regulados por Nucleótidos Cíclicos/deficiencia , Modelos Animales de Enfermedad , Regulación de la Expresión Génica , Transporte Iónico , Potenciales de la Membrana/fisiología , Ratones , Ratones Noqueados , Mutación Missense , Proteínas del Tejido Nervioso/deficiencia , Estabilidad Proteica , Transporte de Proteínas , Degeneración Retiniana/metabolismo , Degeneración Retiniana/patología , Células Fotorreceptoras Retinianas Bastones/patología , Transducción de Señal , Análisis de la Célula Individual , Factores de TiempoRESUMEN
KEY POINTS: Recoverin is a small molecular-weight, calcium-binding protein in rod outer segments that can modulate the rate of rhodopsin phosphorylation. We describe two additional and perhaps more important functions during photoreceptor light adaptation. Recoverin influences the rate of change of adaptation. In wild-type rods, sensitivity and response integration time adapt with similar time constants of 150-200 ms. In Rv-/- rods lacking recoverin, sensitivity declines faster and integration time is already shorter and not significantly altered. During steady light exposure, rod circulating current slowly increases during a time course of tens of seconds, gradually extending the operating range of the rod. In Rv-/- rods, this mechanism is deleted, steady-state currents are already larger and rods saturate at brighter intensities. We propose that recoverin modulates spontaneous and light-activated phophodiesterase-6, the phototransduction effector enzyme, to increase sensitivity in dim light but improve responsiveness to change in brighter illumination. ABSTRACT: Recoverin is a small molecular-weight, calcium-binding protein in rod outer segments that binds to G-protein receptor kinase 1 and can alter the rate of rhodopsin phosphorylation. A change in phosphorylation should change the lifetime of light-activated rhodopsin and the gain of phototransduction, but deletion of recoverin has little effect on the sensitivity of rods either in the dark or in dim-to-moderate background light. We describe two additional functions perhaps of greater physiological significance. (i) When the ambient intensity increases, sensitivity and integration time decrease in wild-type (WT) rods with similar time constants of 150-200 ms. Recoverin is part of the mechanism controlling this process because, in Rv-/- rods lacking recoverin, sensitivity declines more rapidly and integration time is already shorter and not further altered. (ii) During steady light exposure, WT rod circulating current slowly increases during a time course of tens of seconds, gradually extending the operating range of the rod. In Rv-/- rods, this mechanism is also deleted, steady-state currents are already larger and rods saturate at brighter intensities. We argue that neither (i) nor (ii) can be caused by modulation of rhodopsin phosphorylation but may instead be produced by direct modulation of phophodiesterase-6 (PDE6), the phototransduction effector enzyme. We propose that recoverin in dark-adapted rods keeps the integration time long and the spontaneous PDE6 rate relatively high to improve sensitivity. In background light, the integration time is decreased to facilitate detection of change and motion and the spontaneous PDE6 rate decreases to augment the rod working range.
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Adaptación Fisiológica , Recoverina/metabolismo , Células Fotorreceptoras Retinianas Bastones/metabolismo , Animales , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 6/metabolismo , Ratones , Ratones Endogámicos C57BL , Recoverina/genética , Células Fotorreceptoras Retinianas Bastones/fisiología , Visión OcularRESUMEN
BACKGROUND: A volar locking plate (VLP) is the most frequently used form of implant used for open reduction and internal fixation of distal radius fractures. They are known to have a complication rate of up to 27%. We hypothesized that plate design could influence complication rates. METHODS: We performed a review of patients undergoing VLP fixation for distal radius fracture. A total of 228 patients underwent fixation with the Distal Volar Radial Anatomical (DVR) plate; 388 patients underwent fixation with the VariAx plate. Independent observers performed blinded case note and radiographic review, to assess for the quality of reduction, and complications for the inserted VLP. RESULTS: Mean time to surgery was 6.0 days; mean follow-up was 17.5 weeks. Mean age was 56.5 years. The quality of reduction was classified as anatomical (46%), good (36.3%), moderate (13.0%), or poor (3.9%). Complications were identified in 109 patients (17%). Plate prominence was seen in 133 patients (21%). The DVR plate was less prominent ( P < .001) and had better overall radiographic appearances ( P = .025). Flexor tendon complications were related to plate prominence ( P = .005). Inferior reduction was associated with increased time to surgery ( P = .020). CONCLUSIONS: This study highlights the importance of prompt surgery, effective fracture reduction, and careful plate positioning to avoid volar prominence.
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Placas Óseas , Fijación Interna de Fracturas , Fracturas del Radio/diagnóstico por imagen , Fracturas del Radio/cirugía , Humanos , Persona de Mediana Edad , Complicaciones Posoperatorias , Fracturas del Radio/clasificación , Estudios Retrospectivos , Traumatismos de los Tendones/diagnóstico por imagen , Tiempo de TratamientoRESUMEN
INTRODUCTION: Little data exists to help urgent care (UC) clinicians predict morbidity and mortality risk. Age, systolic blood pressure (SBP), and heart rate (HR) are easily obtainable and have been used in other settings to predict short-term risk of deterioration. We hypothesized that there is a relationship between advancing age, SBP, HR, and short-term health outcomes in the UC setting. METHODS: We collected retrospective data from 28 UC clinics and 22 hospitals in the Intermountain Healthcare system between years 2008-2013. Adult patients (≥18 years) were included if they had a unique UC visit and HR or SBP data. Three endpoints following UC visit were assessed: emergency department (ED) visit within three days, hospitalization within three days, and death within seven days. We analyzed associations between age, SBP, HR and endpoints using local regression with a binomial likelihood. Five age groups were chosen from previously published national surveys. Vital sign (VS) distributions were determined for each age group, and the central tendency was compared against previously published norms (90-120mmHg for SBP and 60-100bpm for HR.). RESULTS: A total of 1,720,207 encounters (714,339 unique patients) met the inclusion criteria; 51,446 encounters (2.99%) had ED visit within three days; 12,397 (0.72%) experienced hospitalization within three days; 302 (0.02%) died within seven days of UC visit. Heart rate and SBP combined with advanced age predicted the probability of ED visit (p<0.0001) and hospitalization (p<0.0001) following UC visit. Significant associations between advancing age and death (p<0.0001), and VS and death (p<0.0001) were observed. Odds ratios of risk were highest for elderly patients with lower SBP or higher HR. Observed distributions of SBP were higher than published normal ranges for all age groups. CONCLUSION: Among adults seeking care in the UC, associations between HR and SBP and likelihood of ED visits and hospitalization were more pronounced with advancing age. Death following UC visit had a more limited association with advancing age or the VS evaluated. Rapidly increasing risk below SBP of 100-110 mmHg in older patients suggests that accepted normal ranges for SBP may need to be redefined for patients treated in the UC clinic.
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Atención Ambulatoria/estadística & datos numéricos , Presión Sanguínea , Servicio de Urgencia en Hospital/estadística & datos numéricos , Frecuencia Cardíaca , Monitoreo Fisiológico/estadística & datos numéricos , Mortalidad , Adulto , Anciano , Envejecimiento , Estudios Transversales , Femenino , Hospitalización/tendencias , Humanos , Masculino , Persona de Mediana Edad , Médicos , Estudios Retrospectivos , Factores de RiesgoRESUMEN
Sarcoidosis is an idiopathic inflammatory disorder characterized by the presence of non-caseating tissue granulomas most commonly affecting lungs, lymph nodes and skin. Sarcoid skeletal involvement is relatively uncommon and in particular tenosynovitis. We describe an unusual case of sarcoidosis presenting with granulomatous tenosynovitis as the only manifestation of the disease, illustrating the radiological findings on different modalities followed by a review of the literature. Radiologists and clinicians should be aware of tenosynovitis as a manifestation of sarcoidosis as early and therefore appropriate treatment significantly alters patient's outcome and prognosis.
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Sarcoidosis/complicaciones , Sarcoidosis/diagnóstico , Tenosinovitis/etiología , Adulto , Diagnóstico Diferencial , Quimioterapia Combinada , Dedos/diagnóstico por imagen , Dedos/patología , Glucocorticoides/uso terapéutico , Humanos , Masculino , Metotrexato/uso terapéutico , Prednisolona/uso terapéutico , Radiografía , Sarcoidosis/tratamiento farmacológico , Sarcoidosis/etiología , Resultado del Tratamiento , UltrasonografíaRESUMEN
Light stimulates rhodopsin in a retinal rod to activate the G protein transducin, which binds to phosphodiesterase (PDE), relieving PDE inhibition and decreasing guanosine 3',5'-cyclic monophosphate (cGMP) concentration. The decrease in cGMP closes outer segment channels, producing the rod electrical response. Prolonged exposure to light decreases sensitivity and accelerates response kinetics in a process known as light adaptation, mediated at least in part by a decrease in outer segment Ca(2+). Recent evidence indicates that one of the mechanisms of adaptation in mammalian rods is down-regulation of PDE. To investigate the effect of light and a possible role of rhodopsin kinase (G protein-coupled receptor kinase 1 [GRK1]) and the GRK1-regulating protein recoverin on PDE modulation, we used transgenic mice with decreased expression of GTPase-accelerating proteins (GAPs) and, consequently, a less rapid decay of the light response. This slowed decay made the effects of genetic manipulation of GRK1 and recoverin easier to observe and interpret. We monitored the decay of the light response and of light-activated PDE by measuring the exponential response decay time (τREC) and the limiting time constant (τD), the latter of which directly reflects light-activated PDE decay under the conditions of our experiments. We found that, in GAP-underexpressing rods, steady background light decreased both τREC and τD, and the decrease in τD was nearly linear with the decrease in amplitude of the outer segment current. Background light had little effect on τREC or τD if the gene for recoverin was deleted. Moreover, in GAP-underexpressing rods, increased GRK1 expression or deletion of recoverin produced large and highly significant accelerations of τREC and τD. The simplest explanation of our results is that Ca(2+)-dependent regulation of GRK1 by recoverin modulates the decay of light-activated PDE, and that this modulation is responsible for acceleration of response decay and the increase in temporal resolution of rods in background light.
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Adaptación Ocular , Quinasa 1 del Receptor Acoplado a Proteína-G/metabolismo , Hidrolasas Diéster Fosfóricas/metabolismo , Recoverina/metabolismo , Animales , Calcio/metabolismo , Regulación hacia Abajo , Quinasa 1 del Receptor Acoplado a Proteína-G/genética , Proteínas Activadoras de GTPasa/genética , Proteínas Activadoras de GTPasa/metabolismo , Ratones , Ratones Endogámicos C57BL , Hidrolasas Diéster Fosfóricas/genética , Células Fotorreceptoras Retinianas Bastones/metabolismo , Células Fotorreceptoras Retinianas Bastones/fisiologíaRESUMEN
Previous experiments have shown that the insulin receptor (IR) is expressed in mammalian rods and contributes to the protection of photoreceptors during bright-light exposure. The role of the insulin receptor in the production of the light response is however unknown. We have used suction-electrode recording to examine the responses of rods after conditionally knocking down the insulin receptor. Our results show that these IR knock-down rods have an accelerated decay of the light response and a small decrease in sensitivity by comparison to littermate WT rods. Our results indicate that the insulin receptor may have some role in controlling the rate of rod response decay, but they exclude a major role of the insulin receptor pathway in phototransduction.
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Células Fotorreceptoras/metabolismo , Receptor de Insulina/genética , Células Fotorreceptoras Retinianas Bastones/metabolismo , Animales , Luz , Ratones , Ratones Noqueados , Estimulación Luminosa , Células Fotorreceptoras/efectos de la radiación , Receptor de Insulina/antagonistas & inhibidores , Células Fotorreceptoras Retinianas Bastones/efectos de la radiaciónRESUMEN
Anomalous muscles usually do not cause symptoms but are of academic interest mainly discovered during cadaveric dissection. An aberrant muscle belly arising from the index finger flexor digitorum superficialis tendon causing carpal tunnel syndrome is rare. The management of such an anatomical variant is dependent on whether the median nerve compression is associated with a palpable mass. A brief case highlighting important management principles along with a complete literature review is reported.
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Previous experiments have indicated that growth factor receptor-bound protein 14 (Grb14) may modulate rod photoreceptor cGMP-gated channels by decreasing channel affinity for cGMP; however, the function of Grb14 in rod physiology is not known. In this study, we examined the role of Grb14 by recording electrical responses from rods in which the gene for the Grb14 protein had been deleted. Suction-electrode recordings from single mouse rods showed that responses of dark-adapted Grb14(-/-) mice to brief flashes decayed more rapidly than strain-controlled wild type (WT) rods, with decreased values of both integration time and the exponential time course of decay (τREC). This result is consistent with an increase in channel affinity for cGMP produced by deletion of Grb14. However, Grb14(-/-) mouse rods also showed little change in dark current and a large and significant decrease in the limiting time constant τD, which are not consistent with an effect on channel affinity but seem rather to indicate modulation of the rate of inactivation of cyclic nucleotide phosphodiesterase 6 (PDE6). Grb14 has been reported to translocate from the inner to the outer segment in bright light, but we saw effects on response time course even in dark-adapted rods, although the effects were somewhat greater after rods had been adapted by exposure to bleaching illumination. Our results indicate that the mechanism of Grb14 action may be more complex than previously realized.
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Adaptación a la Oscuridad/fisiología , Proteínas del Ojo/metabolismo , Proteínas/metabolismo , Células Fotorreceptoras Retinianas Bastones/metabolismo , Proteínas Adaptadoras Transductoras de Señales , Animales , GMP Cíclico/genética , GMP Cíclico/metabolismo , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 6/genética , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 6/metabolismo , Proteínas del Ojo/genética , Ratones , Ratones Endogámicos BALB C , Ratones Noqueados , Transporte de Proteínas/fisiología , Proteínas/genética , Células Fotorreceptoras Retinianas Bastones/citologíaRESUMEN
Amphibian and mammalian rods can both detect single photons of light even though they differ greatly in physical dimensions, mammalian rods being much smaller in diameter than amphibian rods. To understand the changes in physiology and biochemistry required by such large differences in outer segment geometry, we developed a computational approach, taking into account the spatial organization of the outer segment divided into compartments, together with molecular dynamics simulations of the signaling cascade. We generated simulations of the single-photon response together with intrinsic background fluctuations in toad and mouse rods. Combining this computational approach with electrophysiological data from mouse rods, we determined key biochemical parameters. On average around one phosphodiesterase (PDE) molecule is spontaneously active per mouse compartment, similar to the value for toad, which is unexpected due to the much smaller diameter in mouse. A larger number of spontaneously active PDEs decreases dark noise, thereby improving detection of single photons; it also increases cGMP turnover, which accelerates the decay of the light response. These constraints explain the higher PDE density in mammalian compared with amphibian rods that compensates for the much smaller diameter of mammalian disks. We further find that the rate of cGMP hydrolysis by light-activated PDE is diffusion limited, which is not the case for spontaneously activated PDE. As a consequence, in the small outer segment of a mouse rod only a few activated PDEs are sufficient to generate a signal that overcomes noise, which permits a shorter lifetime of activated rhodopsin and greater temporal resolution.
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Fototransducción/fisiología , Modelos Biológicos , Fotones , Células Fotorreceptoras Retinianas Bastones/citología , Células Fotorreceptoras Retinianas Bastones/fisiología , Animales , Anuros , Tamaño de la Célula , GMP Cíclico/metabolismo , Hidrólisis , Ratones , Simulación de Dinámica Molecular , Hidrolasas Diéster Fosfóricas/metabolismo , Especificidad de la EspecieRESUMEN
INTRODUCTION: The concept of the golden patient (GP) was introduced to our busy teaching hospital, in April 2009, with the aim of improving our trauma theatre start times. The GP is a pre-selected first patient on the following day trauma list who is medically fit with a clear surgical plan. METHODS: This prospective study compared the trauma theatre start times over a two month period following the introduction of the GP, with a similar two month period prior to the introduction of the GP. A two-sided t-test was used to evaluate statistical significance between groups. RESULTS: Of the 55 planned trauma lists analysed, 42 had a designated GP on it (76%), 37 of which remained first on the actual trauma list (88%). The mean operation start time for the pre-GP lists was 10:03 compared to 09:33 for the actual GP lists (P<0.001). The reception, anaesthetic and operation start times for pre-GP lists compared with lists where no GP was selected were not statistically significant suggesting that the GP was the cause of the significance. CONCLUSION: The introduction of the GP to our trauma lists has made a significant improvement to theatre start times and consequently surgical theatre efficiency.
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Hospitales de Enseñanza , Quirófanos/organización & administración , Heridas y Lesiones/cirugía , Eficiencia Organizacional , Femenino , Hospitales de Enseñanza/organización & administración , Humanos , Masculino , Selección de Paciente , Guías de Práctica Clínica como Asunto , Estudios Prospectivos , Factores de Tiempo , Revisión de Utilización de Recursos , Listas de Espera , Carga de TrabajoRESUMEN
Light isomerizes 11-cis-retinal in a retinal rod and produces an active form of rhodopsin (Rh*) that binds to the G-protein transducin and activates the phototransduction cascade. Rh* is turned off by phosphorylation by rhodopsin kinase [G-protein-coupled receptor kinase 1 (GRK1)] and subsequent binding of arrestin. To evaluate the role of GRK1 in rod light response decay, we have generated the transgenic mouse RKS561L in which GRK1, which is normally present at only 2-3% of rhodopsin, is overexpressed by â¼12-fold. Overexpression of GRK1 increases the rate of Rh* phosphorylation and reduces the exponential decay constant of the response (τ(REC)) and the limiting time constant (τ(D)) both by â¼30%; these decreases are highly significant. Similar decreases are produced in Rv(-/-) rods, in which the GRK1-binding protein recoverin has been genetically deleted. These changes in response decay are produced by acceleration of light-activated phosphodiesterase (PDE*) decay rather than Rh* decay, because light-activated PDE* decay remains rate limiting for response decay in both RKS561L and Rv(-/-) rods. A model incorporating an effect of GRK1 on light-activated PDE* decay rate can satisfactorily account for the changes in response amplitude and waveform. Modulation of response decay in background light is nearly eliminated by deletion of recoverin. Our experiments indicate that rhodopsin kinase and recoverin, in addition to their well-known role in regulating the turning off of Rh*, can also modulate the decay of light-activated PDE*, and the effects of these proteins on light-activated PDE* decay may be responsible for the quickening of response recovery in background light.
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Quinasa 1 del Receptor Acoplado a Proteína-G/genética , Hidrolasas Diéster Fosfóricas/metabolismo , Recoverina/metabolismo , Células Fotorreceptoras Retinianas Bastones/metabolismo , Rodopsina/metabolismo , Potenciales de Acción/fisiología , Animales , Quinasa 1 del Receptor Acoplado a Proteína-G/metabolismo , Ratones , Ratones Transgénicos , Fosforilación , Estimulación Luminosa , Recoverina/genética , Transducina/metabolismoRESUMEN
Caveolin-1 (Cav-1), an integral component of caveolar membrane domains, is expressed in several retinal cell types, including photoreceptors, retinal vascular endothelial cells, Müller glia, and retinal pigment epithelium (RPE) cells. Recent evidence links Cav-1 to ocular diseases, including autoimmune uveitis, diabetic retinopathy, and primary open angle glaucoma, but its role in normal vision is largely undetermined. In this report, we show that ablation of Cav-1 results in reduced inner and outer retinal function as measured, in vivo, by electroretinography and manganese-enhanced MRI. Somewhat surprisingly, dark current and light sensitivity were normal in individual rods (recorded with suction electrode methods) from Cav-1 knock-out (KO) mice. Although photoreceptor function was largely normal, in vitro, the apparent K(+) affinity of the RPE-expressed α1-Na(+)/K(+)-ATPase was decreased in Cav-1 KO mice. Cav-1 KO retinas also displayed unusually tight adhesion with the RPE, which could be resolved by brief treatment with hyperosmotic medium, suggesting alterations in outer retinal fluid homeostasis. Collectively, these findings demonstrate that reduced retinal function resulting from Cav-1 ablation is not photoreceptor-intrinsic but rather involves impaired subretinal and/or RPE ion/fluid homeostasis.
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Caveolina 1/metabolismo , Microambiente Celular/fisiología , Homeostasis/fisiología , Células Fotorreceptoras de Vertebrados/metabolismo , Epitelio Pigmentado de la Retina/metabolismo , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , Uniones Estrechas/metabolismo , Animales , Caveolina 1/genética , Ratones , Ratones Noqueados , Células Fotorreceptoras de Vertebrados/citología , Potasio/metabolismo , Enfermedades de la Retina/genética , Enfermedades de la Retina/metabolismo , Epitelio Pigmentado de la Retina/citología , ATPasa Intercambiadora de Sodio-Potasio/genética , Uniones Estrechas/genéticaRESUMEN
The light-dependent decrease in cyclic guanosine monophosphate (cGMP) in the rod outer segment is produced by a phosphodiesterase (PDE6), consisting of catalytic α and ß subunits and two inhibitory γ subunits. The molecular mechanism of PDE6γ regulation of the catalytic subunits is uncertain. To study this mechanism in vivo, we introduced a modified Pde6g gene for PDE6γ into a line of Pde6g(tm1)/Pde6g(tm1) mice that do not express PDE6γ. The resulting ILE86TER mice have a PDE6γ that lacks the two final carboxyl-terminal Ile(86) and Ile(87) residues, a mutation previously shown in vitro to reduce inhibition by PDE6γ. ILE86TER rods showed a decreased sensitivity and rate of activation, probably the result of a decreased level of expression of PDE6 in ILE86TER rods. More importantly, they showed a decreased rate of decay of the photoresponse, consistent with decreased inhibition of PDE6 α and ß by PDE6γ. Furthermore, ILE86TER rods had a higher rate of spontaneous activation of PDE6 than WT rods. Circulating current in ILE86TER rods that also lacked both guanylyl cyclase activating proteins (GCAPs) could be increased several fold by perfusion with 100µM of the PDE6 inhibitor 3-isobutyl-1-methylxanthine (IBMX), consistent with a higher rate of dark PDE6 activity in the mutant photoreceptors. In contrast, IBMX had little effect on the circulating current of WT rods, unlike previous results from amphibians. Our results show for the first time that the Ile(86) and Ile(87) residues are necessary for normal inhibition of PDE6 catalytic activity in vivo, and that increased basal activity of PDE can be partially compensated by GCAP-dependent regulation of guanylyl cyclase.
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Fosfodiesterasas de Nucleótidos Cíclicos Tipo 6/genética , Células Fotorreceptoras Retinianas Bastones/metabolismo , Eliminación de Secuencia , Transducción de Señal , 1-Metil-3-Isobutilxantina/farmacología , Algoritmos , Animales , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 6/antagonistas & inhibidores , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 6/metabolismo , Proteínas del Ojo/antagonistas & inhibidores , Proteínas del Ojo/metabolismo , Femenino , Cinética , Luz , Masculino , Potenciales de la Membrana/efectos de la radiación , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos DBA , Ratones Transgénicos , Retina/metabolismo , Retina/patología , Degeneración Retiniana/genética , Degeneración Retiniana/patología , Células Fotorreceptoras Retinianas Bastones/efectos de los fármacosRESUMEN
The inhibitory subunit of rod cyclic guanosine monophosphate (cGMP) phosphodiesterase, PDE6γ, is a major component of rod transduction and is required to support photoreceptor integrity. The N74A allele of PDE6γ has previously been shown in experiments carried out in vitro to reduce the regulatory inhibition on the PDE6 catalytic core subunits, PDE6αß. This should, in intact rods, lead to an increase in basal (dark) PDE6 activity producing a state equivalent to light adaptation in the rods and we have examined this possibility using ERG and suction-electrode measurements. The murine opsin promoter was used to drive the expression of a mutant N74A and a wild-type PDE6γ control transgene in the photoreceptors of +/Pde6g(tm1) mice. This transgenic line was crossed with Pde6g(tm1)/Pde6g(tm1) mice to generate animals able to synthesize only the transgenic mutant PDE6γ. We find that the N74A mutation did not produce a significant decrease in circulating current, a decrease in sensitivity or affect the kinetics of the light response, all hallmarks of the light-adapted state. In an in vitro assay of the PDE purified from the N74A transgenic mice and control mice we could find no increase in basal activity of the mutant PDE6. Both the results from the physiology and the biochemistry experiments are consistent with the interpretation that the mutation causes a much milder phenotype in vivo than was predicted from observations made using a cell-free assay system. The in vivo regulation of PDE6γ on PDE6αß may be more dynamic and context-dependent than was replicated in vitro.
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Asparagina/metabolismo , GMP Cíclico/metabolismo , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 6/metabolismo , Retina/metabolismo , Células Fotorreceptoras Retinianas Bastones/metabolismo , Alelos , Animales , Dominio Catalítico , Electrorretinografía , Femenino , Genotipo , Immunoblotting , Masculino , Ratones , Ratones Transgénicos , Microscopía Electrónica de Transmisión , Mutación , Fenotipo , Epitelio Pigmentado de la Retina/ultraestructura , Células Fotorreceptoras Retinianas Bastones/ultraestructura , TransgenesRESUMEN
To characterize the sites of synaptic vesicle fusion in photoreceptors, we evaluated the three-dimensional structure of rod spherules from mice exposed to steady bright light or dark-adapted for periods ranging from 3 to 180 minutes using conical electron tomography. Conical tilt series from mice retinas were reconstructed using the weighted back projection algorithm, refined by projection matching and analyzed using semiautomatic density segmentation. In the light, rod spherules contained â¼470 vesicles that were hemi-fused and â¼187 vesicles that were fully fused (omega figures) with the plasma membrane. Active zones, defined by the presence of fully fused vesicles, extended along the entire area of contact between the rod spherule and the horizontal cell ending, and included the base of the ribbon, the slope of the synaptic ridge and ribbon-free regions apposed to horizontal cell axonal endings. There were transient changes of the rod spherules during dark adaptation. At early periods in the dark (3-15 minutes), there was a) an increase in the number of fully fused synaptic vesicles, b) a decrease in rod spherule volume, and c) an increase in the surface area of the contact between the rod spherule and horizontal cell endings. These changes partially compensate for the increase in the rod spherule plasma membrane following vesicle fusion. After 30 minutes of dark-adaptation, the rod spherules returned to dimensions similar to those measured in the light. These findings show that vesicle fusion occurs at both ribbon-associated and ribbon-free regions, and that transient changes in rod spherules and horizontal cell endings occur shortly after dark onset.
Asunto(s)
Tomografía con Microscopio Electrónico/métodos , Fusión de Membrana , Sinapsis/ultraestructura , Animales , Adaptación a la Oscuridad/efectos de la radiación , Femenino , Procesamiento de Imagen Asistido por Computador , Luz , Masculino , Fusión de Membrana/efectos de la radiación , Ratones , Ratones Endogámicos C57BL , Células Fotorreceptoras Retinianas Bastones/metabolismo , Células Fotorreceptoras Retinianas Bastones/efectos de la radiación , Células Fotorreceptoras Retinianas Bastones/ultraestructura , Sinapsis/metabolismo , Sinapsis/efectos de la radiaciónRESUMEN
UNLABELLED: Irritable bowel syndrome (IBS) is a common gastrointestinal disorder in which the pathophysiological mechanisms of the pain and hypersensitivity are not well understood. IBS patients frequently complain of pain in body regions somatotopically distinct from the gut, suggesting that central hyperalgesic mechanisms may be involved. In the current study, during the wind-up testing session, a series of 6 heat pulses were presented with an interstimulus interval (ISI) of 3 seconds. Following the 1st, 3rd, and 6th thermal stimuli, subjects were asked to rate the late thermal sensation or second pain. IBS patients who demonstrated temporal summation of pain (TSSP) then received dextromethorphan and placebo in a randomized, double-blind, fashion to block wind-up. The results showed: 1) a subset of IBS patients, but not controls, showed TSSP in response to a series of noxious heat pulses; and 2) TSSP was blocked by administration of dextromethorphan, an NMDA receptor antagonist. In summary, these findings further elucidate mechanisms of somatic hypersensitivity in a subset of IBS patients. Our results also support an etiologic basis for abnormal NMDA receptor mechanisms in some IBS patients. Future studies are needed to determine if NMDA receptor antagonists may be used to treat IBS patients. PERSPECTIVE: This study evaluates temporal summation of second pain in a subset of IBS patients that is blocked by Dextromethorphan, an NMDA receptor antagonist. Theses results could lead to the use of an NMDA receptor antagonist in the treatment of pain in a subset of IBS patients.
Asunto(s)
Dextrometorfano/farmacología , Antagonistas de Aminoácidos Excitadores/farmacología , Hiperalgesia/metabolismo , Síndrome del Colon Irritable/metabolismo , Umbral del Dolor/fisiología , Receptores de N-Metil-D-Aspartato/fisiología , Adulto , Método Doble Ciego , Femenino , Ácido Glutámico/fisiología , Humanos , Hiperalgesia/tratamiento farmacológico , Síndrome del Colon Irritable/tratamiento farmacológico , Masculino , Nociceptores/efectos de los fármacos , Nociceptores/fisiología , Dimensión del Dolor/métodos , Umbral del Dolor/efectos de los fármacos , Receptores de N-Metil-D-Aspartato/antagonistas & inhibidores , Transmisión Sináptica/efectos de los fármacos , Transmisión Sináptica/fisiologíaRESUMEN
Vertebrate photoreceptors are thought to adapt to light by a change in Ca(2+), which is postulated to mediate modulation of (1) excited rhodopsin (Rh*) by Ca(2+)-dependent binding of recoverin, (2) guanylyl cyclase activity via Ca(2+)-dependent GCAP proteins, and (3) cyclic nucleotide-gated channels by binding of Ca(2+)-calmodulin. Previous experiments genetically deleted recoverin and the GCAPs and showed that significant regulation of sensitivity survives removal of (1) and (2). We genetically deleted the channel Ca(2+)-calmodulin binding site in the mouse Mus musculus and found that removal of (3) alters response waveform, but removal of (3) or of (2) and (3) together still leaves much of adaptation intact. These experiments demonstrate that an important additional mechanism is required, which other experiments indicate may be regulation of phosphodiesterase 6 (PDE6). We therefore constructed a kinetic model in which light produces a Ca(2+)-mediated decrease in PDE6 decay rate, with the novel feature that both spontaneously activated and light-activated PDE6 are modulated. This model, together with Ca(2+)-dependent acceleration of guanylyl cyclase, can successfully account for changes in sensitivity and response waveform in background light.
Asunto(s)
Adaptación Ocular/fisiología , Canales Catiónicos Regulados por Nucleótidos Cíclicos/fisiología , Proteínas del Tejido Nervioso/fisiología , Células Fotorreceptoras Retinianas Bastones/fisiología , Adaptación Ocular/genética , Animales , Canales Catiónicos Regulados por Nucleótidos Cíclicos/deficiencia , Canales Catiónicos Regulados por Nucleótidos Cíclicos/genética , Marcación de Gen , Ratones , Ratones de la Cepa 129 , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Proteínas del Tejido Nervioso/deficiencia , Proteínas del Tejido Nervioso/genética , Estimulación Luminosa/métodosRESUMEN
The Ca(2+)-binding protein recoverin is thought to regulate rhodopsin kinase and to modulate the lifetime of the photoexcited state of rhodopsin (Rh*), the visual pigment of vertebrate rods. Recoverin has been postulated to inhibit the kinase in darkness, when Ca(2+) is high, and to be released from the disk membrane in light when Ca(2+) is low, accelerating rhodopsin phosphorylation and shortening the lifetime of Rh*. This proposal has remained controversial, in part because the normally rapid turnoff of Rh* has made Rh* modulation difficult to study in an intact rod. To circumvent this problem, we have made mice that underexpress rhodopsin kinase so that Rh* turnoff is rate limiting for the decay of the rod light response. We show that background light speeds the decay of Rh* turnoff, and that this no longer occurs in mice that have had recoverin knocked out. This is the first demonstration in an intact rod that light accelerates Rh* inactivation and that the Ca(2+)-binding protein recoverin may be required for the light-dependent modulation of Rh* lifetime.