RESUMEN
AIMS: Increasingly, patients with bilateral hip arthritis wish to undergo staged total hip arthroplasty (THA). With the rise in demand for arthroplasty, perioperative risk assessment and counselling is crucial for shared decision making. However, it is unknown if complications that occur after a unilateral hip arthroplasty predict complications following surgery of the contralateral hip. PATIENTS AND METHODS: We used nationwide linked discharge data from the Healthcare Cost and Utilization Project between 2005 and 2014 to analyze the incidence and recurrence of complications following the first- and second-stage operations in staged bilateral total hip arthroplasty (BTHAs). Complications included perioperative medical adverse events within 30 to 60 days, and infection and mechanical complications within one year. Conditional probabilities and odds ratios (ORs) were calculated to determine whether experiencing a complication after the first stage of surgery increased the risk of developing the same complication after the second stage. RESULTS: A total of 13 829 patients (5790 men and 8039 women) who underwent staged BTHAs were analyzed. The mean age at first operation was 62.9 years (14 to 95). For eight of the 12 outcomes evaluated, patients who experienced the outcome following the first arthroplasty had a significantly increased probability and odds of developing that same complication following the second arthroplasty, compared with those who did not experience the complication after the first surgery. This was true for digestive complications (OR 25.67, 95% confidence interval (CI) 13.86 to 46.08; p < 0.001), urinary complications (OR 6.48, 95% CI 1.7 to 20.73; p = 0.01), haematoma (OR 12.17, 95% CI 4.55 to 31.14; p < 0.001), deep vein thrombosis (OR 4.82, 95% CI 2.34 to 9.65; p < 0.001), pulmonary embolism (OR 12.03, 95% CI 2.02 to 46.77; p = 0.01), deep hip infection (OR 534.21, 95% CI 314.96 to 909.25; p < 0.001), superficial hip infection (OR 1574.99, 95% CI 269.83 to 9291.81; p < 0.001), and mechanical malfunction (OR 117.49, 95% CI 91.55 to 150.34; p < 0.001). CONCLUSION: The occurrence of certain complications after unilateral THA is associated with an increased risk of the same complication occurring after staged arthroplasty of the contralateral hip. Patients who experience these complications after unilateral hip arthroplasty should be appropriately counselled regarding their risk profile prior to undergoing staged contralateral hip arthroplasty. Cite this article: Bone Joint J 2019;101-B(6 Supple B):77-83.
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Artroplastia de Reemplazo de Cadera/efectos adversos , Osteoartritis de la Cadera/cirugía , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Femenino , Humanos , Masculino , Persona de Mediana Edad , Complicaciones Posoperatorias/etiología , Recurrencia , Estudios Retrospectivos , Factores de Riesgo , Adulto JovenRESUMEN
The extracellular calcium-sensing receptor CaSR is expressed in blood vessels where its role is not completely understood. In this study, we tested the hypothesis that the CaSR expressed in vascular smooth muscle cells (VSMC) is directly involved in regulation of blood pressure and blood vessel tone. Mice with targeted CaSR gene ablation from vascular smooth muscle cells (VSMC) were generated by breeding exon 7 LoxP-CaSR mice with animals in which Cre recombinase is driven by a SM22α promoter (SM22α-Cre). Wire myography performed on Cre-negative [wild-type (WT)] and Cre-positive (SM22α)CaSR(Δflox/Δflox) [knockout (KO)] mice showed an endothelium-independent reduction in aorta and mesenteric artery contractility of KO compared with WT mice in response to KCl and to phenylephrine. Increasing extracellular calcium ion (Ca(2+)) concentrations (1-5 mM) evoked contraction in WT but only relaxation in KO aortas. Accordingly, diastolic and mean arterial blood pressures of KO animals were significantly reduced compared with WT, as measured by both tail cuff and radiotelemetry. This hypotension was mostly pronounced during the animals' active phase and was not rescued by either nitric oxide-synthase inhibition with nitro-l-arginine methyl ester or by a high-salt-supplemented diet. KO animals also exhibited cardiac remodeling, bradycardia, and reduced spontaneous activity in isolated hearts and cardiomyocyte-like cells. Our findings demonstrate a role for CaSR in the cardiovascular system and suggest that physiologically relevant changes in extracellular Ca(2+) concentrations could contribute to setting blood vessel tone levels and heart rate by directly acting on the cardiovascular CaSR.
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Presión Sanguínea , Señalización del Calcio , Calcio/metabolismo , Hipotensión/metabolismo , Músculo Liso Vascular/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Vasoconstricción , Vasodilatación , Animales , Aorta/metabolismo , Presión Sanguínea/efectos de los fármacos , Presión Sanguínea/genética , Bradicardia/genética , Bradicardia/metabolismo , Bradicardia/fisiopatología , Señalización del Calcio/efectos de los fármacos , Señalización del Calcio/genética , Relación Dosis-Respuesta a Droga , Predisposición Genética a la Enfermedad , Frecuencia Cardíaca , Hipotensión/genética , Hipotensión/fisiopatología , Arterias Mesentéricas/metabolismo , Ratones de la Cepa 129 , Ratones Endogámicos C57BL , Ratones Noqueados , Músculo Liso Vascular/efectos de los fármacos , Músculo Liso Vascular/fisiopatología , Miocitos Cardíacos/metabolismo , Fenotipo , Receptores Sensibles al Calcio , Receptores Acoplados a Proteínas G/deficiencia , Receptores Acoplados a Proteínas G/genética , Vasoconstricción/efectos de los fármacos , Vasoconstricción/genética , Vasoconstrictores/farmacología , Vasodilatación/efectos de los fármacos , Vasodilatación/genética , Vasodilatadores/farmacología , Remodelación VentricularRESUMEN
OBJECTIVE: Loss-of-function calcium-sensing receptor (CAR) mutations cause elevated parathyroid hormone (PTH) secretion and hypercalcaemia. Although full Car deletion is possible in mice, most human CAR mutations result from a single amino acid substitution that maintains partial function. However, here, we report a case of neonatal severe hyperparathyroidism (NSHPT) in which the truncated CaR lacks any transmembrane domain (CaR(R392X)), in effect a full CAR 'knockout'. CASE REPORT: The infant (daughter of distant cousins) presented with hypercalcaemia (5.5-6 âmmol/l corrected calcium (2.15-2.65)) and elevated PTH concentrations (650-950â pmol/l (12-81)) together with skeletal demineralisation. NSHPT was confirmed by CAR gene sequencing (homozygous c.1174C-to-T mutation) requiring total parathyroidectomy during which only two glands were located and removed, resulting in normalisation of her serum PTH/calcium levels. DESIGN AND METHODS: The R392X stop codon was inserted into human CAR and the resulting mutant (CaR(R392X)) expressed transiently in HEK-293 cells. RESULTS: CaR(R392X) expressed as a 54â kDa dimeric glycoprotein that was undetectable in conditioned medium or in the patient's urine. The membrane localisation observed for wild-type CaR in parathyroid gland and transfected HEK-293 cells was absent from the proband's parathyroid gland and from CaR(R392X)-transfected cells. Expression of the mutant was localised to endoplasmic reticulum consistent with its lack of functional activity. CONCLUSIONS: Intriguingly, the patient remained normocalcaemic throughout childhood (2.5âmM corrected calcium, 11âpg/ml PTH (10-71), age 8 years) but exhibited mild asymptomatic hypocalcaemia at age 10 years, now treated with 1-hydroxycholecalciferol and Ca2+ supplementation. Despite representing a virtual CAR knockout, the patient displays no obvious pathologies beyond her calcium homeostatic dysfunction.
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Sustitución de Aminoácidos , Hipercalcemia/etiología , Hiperparatiroidismo/diagnóstico , Hiperparatiroidismo/genética , Mutagénesis Insercional , Paratiroidectomía , Receptores Sensibles al Calcio/genética , Arginina , Calcio/sangre , Niño , Retículo Endoplásmico/genética , Retículo Endoplásmico/metabolismo , Femenino , Técnica del Anticuerpo Fluorescente , Células HEK293 , Humanos , Hipercalcemia/sangre , Hiperparatiroidismo/sangre , Hiperparatiroidismo/congénito , Immunoblotting , Lactante , Recién Nacido , Hormona Paratiroidea/sangre , Hormona Paratiroidea/genética , Paratiroidectomía/métodos , Receptores Sensibles al Calcio/metabolismo , Análisis de Secuencia de ADN/métodos , Índice de Severidad de la Enfermedad , Transfección , Resultado del TratamientoRESUMEN
Ruminant nutrition relies upon the symbiotic relationship that exists with microbial populations in the rumen. Urea transported across the ruminal epithelia and secreted by the salivary glands is a key source of nitrogen for microbial growth in the rumen. As ruminal urea transport can be mediated by specific UT-B urea transporters, this study investigated whether UT-B urea transporters were also present in the bovine salivary gland. Western blotting experiments detected only small amounts of UT-B protein in whole-cell lysate from the bovine parotid gland. In contrast, strong 32 to 34 and 40 kDa UT-B proteins were detected in parotid plasma membrane-enriched protein, showing the importance of using enriched samples. These signals were also detected in rumen and correspond to bovine UT-B1 and UT-B2 urea transporters, respectively. Further immunolocalization studies identified that these proteins were located in the ductal system of the parotid gland. This study, therefore, confirmed the presence of UT-B urea transporter protein in the bovine parotid salivary gland.
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Proteínas de Transporte de Membrana/análisis , Glándula Parótida/química , Animales , Western Blotting/veterinaria , Bovinos , Membrana Celular/química , Membrana Celular/fisiología , Femenino , Proteínas de Transporte de Membrana/fisiología , Glándula Parótida/fisiología , Rumen/química , Rumen/fisiología , Transportadores de UreaRESUMEN
BACKGROUND AND PURPOSE: The extracellular calcium-sensing receptor (CaR) in vascular endothelial cells activates endothelial intermediate-conductance, calcium-sensitive K(+) channels (IK(Ca)) indirectly leading to myocyte hyperpolarization. We determined whether CaR expression and function was modified in a rat model of type II diabetes. EXPERIMENTAL APPROACH: Pressure myography, western blotting, sharp microelectrode and K(+)-selective electrode recordings were used to investigate the functional expression of the CaR and IK(Ca) in rat mesenteric arteries. KEY RESULTS: Myocyte hyperpolarization to the CaR activator calindol was inhibited by Calhex 231. U46619-induced vessel contraction elevated the extracellular [K(+)] around the myocytes, and inhibition of this 'K(+) cloud' by iberiotoxin was needed to reveal calindol-induced vasodilatations. These were antagonized by Calhex 231 and significantly smaller in Zucker diabetic fatty rat (ZDF) vessels than in Zucker lean (ZL) controls. Myocyte hyperpolarizations to calindol were also smaller in ZDF than in ZL arteries. In ZDF vessels, endothelial cell CaR protein expression was reduced; IK(Ca) expression was also diminished, but IK(Ca)-generated hyperpolarizations mediated by 1-EBIO were unaffected. CONCLUSIONS AND IMPLICATIONS: The reduced CaR-mediated hyperpolarizing and vasodilator responses in ZDF arteries result from a decrease in CaR expression, rather than from a modification of IK(Ca) channels. Detection of CaR-mediated vasodilatation required the presence of iberiotoxin, suggesting a CaR contribution to vascular diameter, that is, inversely related to the degree of vasoconstriction. Compromise of the CaR pathway would favour the long-term development of a higher basal vascular tone and could contribute to the vascular complications associated with type II diabetes.
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Diabetes Mellitus Tipo 2/fisiopatología , Arterias Mesentéricas/metabolismo , Canales de Potasio Calcio-Activados/metabolismo , Receptores Sensibles al Calcio/metabolismo , Animales , Western Blotting , Electrofisiología , Regulación de la Expresión Génica , Masculino , Arterias Mesentéricas/patología , Microelectrodos , Células Musculares/metabolismo , Miografía , Ratas , Ratas Wistar , Ratas Zucker , Vasodilatación/fisiologíaRESUMEN
Diabetes mellitus is associated with altered iron homeostasis in both human and animal diabetic models. Iron is a metal oxidant capable of generating reactive oxygen species (ROS) and has been postulated to contribute to diabetic nephropathy. Two proteins involved in iron metabolism that are expressed in the kidney are the divalent metal transporter, DMT1 (Slc11a2), and the Transferrin Receptor (TfR). Thus, we investigated whether renal DMT1 or TfR expression is altered in diabetes, as this could potentially affect ROS generation and contribute to diabetic nephropathy. Rats were rendered diabetic with streptozotocin (STZ-diabetes) and renal DMT1 and TfR expression studied using semi-quantitative immunoblotting and immunofluorescence. In STZ-diabetic Sprague-Dawley rats, renal DMT1 expression was significantly reduced and TfR expression increased after 2 weeks. DMT1 downregulation was observed in both proximal tubules and collecting ducts. Renal DMT1 expression was also decreased in Wistar rats following 12 weeks of STZ-diabetes, an effect that was fully corrected by insulin-replacement but not by cotreatment with the aldose reductase inhibitor, sorbinil. Increased renal TfR expression was also observed in STZ-diabetic Wistar rats together with elevated cellular iron accumulation. Together these data demonstrate renal DMT1 downregulation and TfR upregulation in STZ-diabetes. Whilst the consequence of altered DMT1 expression on renal iron handling and oxidant damage remains to be determined, the attenuation of the putative lysosomal iron exit pathway in proximal tubules could potentially explain lysosomal iron accumulation reported in human diabetes and STZ-diabetic animals.
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Proteínas de Transporte de Catión/metabolismo , Diabetes Mellitus Experimental/metabolismo , Nefropatías Diabéticas/metabolismo , Hierro/metabolismo , Receptores de Transferrina/metabolismo , Animales , Proteínas de Transporte de Catión/análisis , Diabetes Mellitus Experimental/inducido químicamente , Regulación hacia Abajo , Riñón/química , Riñón/metabolismo , Masculino , Ratas , Ratas Sprague-Dawley , Ratas Wistar , Regulación hacia ArribaRESUMEN
We have demonstrated that inner medullary collecting duct (IMCD) heavy endosomes purified from rat kidney IMCD contain the type II protein kinase A (PKA) regulatory subunit (RII), protein phosphatase (PP)2B, PKCzeta, and an RII-binding protein (relative molecular mass ~90 kDa) representing a putative A kinase anchoring protein (AKAP). Affinity chromatography of detergent-solubilized endosomes on cAMP-agarose permits recovery of a protein complex consisting of the 90-kDa AKAP, RII, PP2B, and PKCzeta. With the use of small-particle flow cytometry, RII and PKCzeta were localized to an identical population of endosomes, suggesting that these proteins are components of an endosomal multiprotein complex. (32)P-labeled aquaporin-2 (AQP2) present in these PKA-phosphorylated endosomes was dephosphorylated in vitro by either addition of exogenous PP2B or by an endogenous endosomal phosphatase that was inhibited by the PP2B inhibitors EDTA and the cyclophilin-cyclosporin A complex. We conclude that IMCD heavy endosomes possess an AKAP multiprotein-signaling complex similar to that described previously in hippocampal neurons. This signaling complex potentially mediates the phosphorylation of AQP2 to regulate its trafficking into the IMCD apical membrane. In addition, the PP2B component of the AKAP-signaling complex could also dephosphorylate AQP2 in vivo.
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Acuaporinas/metabolismo , Calcineurina/metabolismo , Proteínas Portadoras/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Médula Renal/enzimología , Transducción de Señal , Animales , Acuaporina 2 , Acuaporina 6 , Autorradiografía , Calcineurina/análisis , Proteínas Portadoras/análisis , Proteína Quinasa Tipo II Dependiente de AMP Cíclico , Electroforesis en Gel de Poliacrilamida , Endosomas/química , Endosomas/enzimología , Citometría de Flujo , Técnicas de Inmunoadsorción , Médula Renal/ultraestructura , Túbulos Renales Colectores/enzimología , Túbulos Renales Colectores/ultraestructura , Masculino , Fosforilación , Proteína Quinasa C/análisis , Proteína Quinasa C/metabolismo , Ratas , Ratas Sprague-Dawley , Especificidad por SustratoRESUMEN
We have demonstrated that the kidney plays an important role in iron balance and that metabolically significant reabsorption of this ion occurs in the loop of Henle and the collecting ducts [Wareing M, Ferguson CJ, Green R, Riccardi D, and Smith CP. J Physiol (Lond) 524: 581-586, 2000]. To test the possibility that the divalent metal transporter DMT1 (Gunshin H, Mackenzie B, Berger UV, Gunshin Y, Romero MF, Boron WF, Nussberger S, Gollan JL, and Hediger MA. Nature 388: 482-488, 1997) could represent the apical route for iron entry in the kidney, we raised and affinity-purified an anti-DMT-1 polyclonal antibody and determined DMT-1 distribution in rat kidney by Western analysis, immunofluorescence, and confocal microscopy. The strongest DMT1-specific (i.e., peptide-protectable) immunoreactivity was found in the collecting ducts, in both principal and intercalated cells. Thick ascending limbs of Henle's loop and, more intensely, distal convoluted tubules exhibited apical immunostaining. Considerable intracellular DMT-1 immunoreactivity was seen throughout the nephron, particularly in S3 segments. The described distribution of DMT-1 protein is in agreement with our previous identification of nephron sites of iron reabsorption, suggesting that DMT-1 provides the molecular mechanism for apical iron entry in the distal nephron but not in the proximal tubule. Basolateral iron exit may be facilitated by a different system.
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Proteínas Portadoras/metabolismo , Proteínas de Transporte de Catión , Proteínas de Unión a Hierro , Riñón/citología , Riñón/metabolismo , Animales , Northern Blotting , Western Blotting , Proteínas Portadoras/inmunología , Inmunoglobulina G/biosíntesis , Inmunoglobulina G/inmunología , Inmunohistoquímica , Hierro/metabolismo , Masculino , Microscopía Confocal , Microsomas/metabolismo , ARN/biosíntesis , ARN/aislamiento & purificación , Ratas , Ratas WistarRESUMEN
BACKGROUND: The role of nitric oxide in intestinal ischemia-reperfusion is unclear-some studies link it to the harmful effects of ischemia-reperfusion, while others report it to be protective. We propose that nitric oxide levels diminish in the reperfusion period in conjunction with the onset of increased capillary permeability. The aim of this study is to determine the effect of supplementing nitric oxide synthase with its substrate, l-arginine, on development of local mucosal injury and systemic capillary leak. MATERIALS AND METHODS: Rats underwent 30 min of superior mesenteric artery occlusion followed by 4 h of reperfusion. The vehicle groups received l-arginine either intravenously (4 mg/kg/min) or into the intestinal lumen. The intravenous groups received l-arginine either before the ischemic event or after 30 min of reperfusion. Capillary leak in the gut and lung were measured, as were degree of mucosal injury and number of infiltrating neutrophils. Appropriate controls were performed. RESULTS: Thirty minutes of mesenteric ischemia followed by 4 h of reperfusion significantly increased gut and lung leak, neutrophil infiltration, and the severity of mucosal injury. l-Arginine given iv prior to ischemia inhibited lung leak, mucosal injury, and neutrophil infiltration. When arginine was given during the reperfusion period, lung leak and neutrophil infiltration but not mucosal injury were reduced. Intraluminal l-arginine reduced mucosa injury, but had no effect on capillary leak. CONCLUSIONS: Supplementation with l-arginine enhances NO production, resulting in reduced systemic endothelial dysfunction. This may act as a useful clinical adjunct in the management of trauma patients in preventing the development of ARDS and multiple organ failure.
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Arginina/farmacología , Intestinos/irrigación sanguínea , Isquemia/fisiopatología , Óxido Nítrico/biosíntesis , Daño por Reperfusión/fisiopatología , Animales , Permeabilidad Capilar/efectos de los fármacos , Mucosa Intestinal/patología , Intestinos/inervación , Isquemia/patología , Masculino , Neuronas/enzimología , Óxido Nítrico Sintasa/metabolismo , Óxido Nítrico Sintasa de Tipo I , Circulación Pulmonar , Ratas , Ratas Sprague-Dawley , Daño por Reperfusión/patologíaRESUMEN
Dietary phosphate (Pi) intake and parathyroid hormone (PTH) are essential regulators of proximal tubular (PT) Pi reabsorption; both factors are associated with adaptive changes in PT apical brush border membrane (BBM) Na/Pi-cotransport activity and specific transporter protein (NaPi-2) content. Urinary Pi excretion is also inversely correlated with luminal Ca2+ concentration ([Ca2+]) both in a PTH-dependent and -independent fashion. A cell-surface, Ca2+(/polyvalent cation)-sensing receptor (CaR) has been localized to the PT BBM with unknown function. To investigate whether PTH and/or dietary Pi intake could affect the distribution or the expression of the CaR, we evaluated their effects on rat kidney CaR and the NaPi-2 expression by Western blot analysis and immunofluorescence microscopy. A chronic high-Pi (1.2%) versus low-Pi (0.1%) diet and acute PTH (1-34) infusion significantly reduced the PT BBM expression of both NaPi-2 and CaR proteins. CaR-specific immunoreactivity in nephron segments other than the PT was not affected by PTH or Pi intake. These results suggest that reduced renal PT CaR expression by a high-Pi diet and by increased circulating PTH levels could contribute to the local control of PT handling of Ca2+ and Pi.
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Proteínas Portadoras/análisis , Túbulos Renales Proximales/química , Hormona Paratiroidea/farmacología , Fosfatos/farmacología , Receptores de Superficie Celular/análisis , Sodio/farmacología , Animales , Western Blotting , Dieta , Técnica del Anticuerpo Fluorescente , Túbulos Renales Proximales/efectos de los fármacos , Túbulos Renales Proximales/metabolismo , Masculino , Microscopía Fluorescente , Hormona Paratiroidea/sangre , Proteínas de Unión a Fosfato , Fosfatos/administración & dosificación , Fosfatos/metabolismo , Fosfatos/orina , Ratas , Ratas Wistar , Receptores Sensibles al CalcioRESUMEN
Vasopressin or AVP regulates water reabsorption by the kidney inner medullary collecting duct (IMCD) through the insertion and removal of aquaporin (AQP) 2 water channels into the IMCD apical membrane. AVP-elicited trafficking of AQP2 with the apical membrane occurs via a specialized population of vesicles that resemble synaptic vesicles in neurons. AQP2 vesicles and the IMCD apical membrane contain homologs of vesicle-targeting and signal transduction proteins found in neurons. Expression studies of AQP2, including human AQP2 mutants, suggest that the carboxyl-terminal domain of AQP2 is important in AQP2 trafficking, particularly as a site for cAMP-dependent protein kinase phosphorylation. These present data reveal that IMCD cells possess a complex integrated-signaling and vesicle-trafficking machinery that provides integration of AVP-elicited water transport with many other parameters within the IMCD cell as well as kidney.
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Acuaporinas/metabolismo , Vasopresinas/fisiología , Agua/metabolismo , Animales , Acuaporina 2 , Acuaporina 6 , Arginina Vasopresina/farmacología , Transporte Biológico/fisiología , Humanos , Médula Renal , Túbulos Renales Colectores/citología , Túbulos Renales Colectores/efectos de los fármacos , Túbulos Renales Colectores/metabolismoRESUMEN
BACKGROUND/PURPOSE: It is generally believed that differentiated thyroid cancer (DTC) in young patients has an excellent prognosis. This calls into question the need for more extensive surgical ablation of the thyroid gland with attendant risks of surgical complications. The purpose of this report was to investigate both the incidence of surgical morbidity and the impact of surgery on locoregional recurrence of disease. METHODS: The authors reviewed the clinical course of patients under 22 years of age treated for DTC within Department of Defense hospitals since 1950. Data were available for determination of surgical morbidity in 126 and for outcome in 105. RESULTS: The incidence of postoperative hypocalcemia was 17% and of recurrent laryngeal nerve injury 3%. Factors predictive of morbidity were (1) more extensive thyroid surgery (P = .023), and (2) the presence of gross tumor invasion (P = .022). The incidence of neck recurrence was analyzed among a cohort of 90 patients. A total of 19 (21%) patients had a local recurrence. The median time to recurrence was 24 months. The factor predictive of recurrence was the presence of gross invasion (P = .0001). A strong trend toward locoregional recurrence was found among patients with metastatic disease to more than five cervical nodes (P < .08). The primary operations on the thyroid and regional nodes were not significant predictors of neck recurrence. Among these 19 patients there have been no deaths, but 25% had persistent disease at a mean follow-up of 12.6 years. CONCLUSIONS: The incidence of surgical morbidity does increase with more extensive surgery. Outcome is predicted primarily by the initial extent of disease.
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Recurrencia Local de Neoplasia/patología , Neoplasias de la Tiroides/cirugía , Adolescente , Adulto , Distribución de Chi-Cuadrado , Niño , Femenino , Humanos , Masculino , Invasividad Neoplásica , Metástasis de la Neoplasia , Complicaciones Posoperatorias , Análisis de Supervivencia , Neoplasias de la Tiroides/patología , Resultado del TratamientoRESUMEN
Extracellular calcium/polyvalent cation-sensing receptors (CaR) couple to G proteins and contain highly conserved extracellular cysteine residues. Immunoblotting of proteins from rat kidney inner medullary collecting duct endosomes with CaR-specific antibodies reveals alterations in the apparent molecular mass of CaR depending on protein denaturation conditions. When denatured by SDS under nonreducing conditions, CaR migrates as a putative dimeric species of 240-310 kDa. This is twice the predicted molecular mass of the CaR monomer observed after SDS denaturation in the presence of sulfhydryl-reducing agents. In sucrose density gradients, Triton X-100-solubilized CaR sediments as a 220-kDa complex, not explainable by binding of G proteins to CaR monomers. Treatment of Triton-soluble CaR with divalent (Ca2+, Mg2+) and trivalent (Gd3+) metal ion CaR agonists, but not monovalent ions (Na+), partially shifts the electrophoretic mobility of CaR under reducing conditions from a predominantly monomeric to this putative dimeric species on immunoblots in a manner similar to their rank order of functional potency for CaR activation (Gd3+ >> Ca2+ > Mg2+). This Ca2+ effect is blocked by pretreatment with N-ethylmaleimide. We conclude that disulfide bonds present in CaRs mediate formation of dimers that are preserved in Triton X-100 solution. In addition, CaR exposure to Ca2+ induces formation of additional disulfide bonds within the Triton-soluble CaR complex.
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Disulfuros/química , Receptores de Superficie Celular/química , Animales , Calcio/farmacología , Cationes Bivalentes/farmacología , Secuencia Conservada , Cisteína/química , Dimerización , Endosomas/química , Túbulos Renales/química , Masculino , Peso Molecular , Octoxinol/farmacología , Desnaturalización Proteica , Ratas , Ratas Sprague-Dawley , Receptores Sensibles al Calcio , TemperaturaRESUMEN
To investigate how hypercalcemia blunts renal concentrating ability, alterations in basal and arginine vasopressin (AVP)-elicited osmotic water (Pf) and urea (Purea) permeabilities were measured in isolated perfused terminal inner medullary collecting ducts (IMCD) from control and chronically hypercalcemic rats after dihydrotachysterol (DHT) (M. Levi, L. Peterson, and T. Berl. Kidney Int. 23: 489-497, 1983) treatment. The IMCD Pf of DHT-treated rats did not increase significantly after AVP and was accompanied by a significant 87 +/- 4% reduction in aquaporin-2 (AQP-2) protein but not mRNA. In contrast, both basal and AVP-elicited IMCD Purea from DHT rats were significantly increased and accompanied by a significant 41 +/- 11% increase in AVP-regulated urea transporter protein content. Immunoblotting with anti-calcium/polyvalent cation-sensing receptor protein (CaR) antiserum revealed specific alterations in CaR bands in endosomes purified from the apical membranes of inner medulla of DHT rats. These data are the first detailed analyses of hypercalcemia-induced alterations in AVP-regulated permeabilities and membrane transporters in IMCD. We conclude that selective alterations in IMCD transport occur in hypercalcemia, permitting the body to dispose of excess calcium without forming calcium-containing renal stones.
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Calcio/metabolismo , Hipercalcemia/metabolismo , Hipercalcemia/fisiopatología , Médula Renal/metabolismo , Médula Renal/fisiopatología , Receptores de Vasopresinas/fisiología , Urea/metabolismo , Vasopresinas/fisiología , Agua/metabolismo , Animales , Endosomas/metabolismo , Tasa de Filtración Glomerular , Masculino , Ratas , Ratas Sprague-DawleyRESUMEN
To investigate membrane lipid metabolism during smooth-muscle activation, the role of phospholipase D (PLD) in the production of phosphatidate (PA) was studied in rat small arteries stimulated with noradrenaline. Incubation with [3H]myristate preferentially labelled phosphatidylcholine (PtdCho), and in the presence of 0.5% ethanol [3H]phosphatidylethanol ([3H]PEt) was formed, demonstrating PLD activity. Noradrenaline (NA) stimulation resulted in an increase in PtdCho derived [3H]PA and [3H]PEt formation, indicating PLD activation. Stimulation of [14C]choline release confirmed PLD-mediated hydrolysis of PtdCho. Propranolol, an inhibitor of PA phosphohydrolase, increased [3H]PA levels in non-stimulated tissue and decreased the rate of degradation of both [3H]PA and [3H]PEt, implying that this is an active route for PA metabolism in small arteries. However, [3H]diacylglycerol levels were not increased during NA stimulation. Fluoroaluminate increased [3H]PEt formation and [14C]choline release, whereas high K+ in the presence of alpha 1-adrenoceptor blockade did not. Pervanadate increased phosphotyrosine levels in small arteries, and markedly stimulated [3H]PEt formation and [14C]choline release. The combination of pervanadate and NA stimulation resulted in a dramatic increase in [3H]PEt formation, which was greater than the sum of the individual responses to the two agonists. Pervanadate and fluoroaluminate in combination appeared to give an additive response, whereas high K+ did not alter the pervanadate-induced formation of [3H]PEt. Phosphotyrosine levels were increased by NA in the presence of tyrosine phosphatase inhibitors. This effect was blocked by genistein, a tyrosine kinase inhibitor. These data demonstrate that in NA-stimulated small arteries PLD-induced PtdCho hydrolysis contributes to accumulation of PA, but not of diacylglycerol. Furthermore, regulation of PLD activity appears to require G-protein and tyrosine-phosphorylation-linked pathways.
Asunto(s)
Arterias/metabolismo , Proteínas de Unión al GTP/metabolismo , Norepinefrina/farmacología , Ácidos Fosfatidicos/biosíntesis , Fosfolipasa D/metabolismo , Tirosina/metabolismo , Animales , Hidrólisis , Técnicas In Vitro , Fosfatidilcolinas/metabolismo , Fosforilación , Ratas , Ratas Sprague-Dawley , Transducción de SeñalRESUMEN
In order to determine if different physicochemical properties exist among antihormone-receptor complexes, we have compared the interaction of the antiprogestin RU486 with progesterone receptor (PR) versus the triphenylethylene antiestrogen H1285 (4-(N,N-diethyl-aminoethoxy)-4'-methoxy-alpha-(p-hydroxyphenyl-alp ha'- ethylstilbene] with estrogen receptor (ER) from rabbit uterine tissue. Contrary to other reports, we observed no difference in the sedimentation properties of transformed PR (4S) when bound by the antagonist RU486 versus the progesterone agonist R5020 in either cytosol or DEAE partially-purified receptor preparations analyzed on sucrose gradients containing 0.3 M KCl. In addition, we found no difference in the sedimentation properties of these receptor preparations in the presence of 10 mM sodium molybdate: the nontransformed RU486-PR and nontransformed R5020-PR both sedimented as a 6S species. These same results were obtained when the receptor preparation and gradient analysis were performed in the absence of monothioglycerol. Likewise, there was no change in the sedimentation properties of the transformed PR when the receptor, partially purified in the absence of molybdate, was analyzed on sucrose gradients containing 10 mM sodium molybdate to prevent receptor alteration during centrifugation. From DNA-cellulose assays performed with partially purified PR in the absence of molybdate we determined that the 4S form of R5020-PR and RU486-PR is transformed receptor; whereas in the presence of molybdate, the 6S species is nontransformed. In contrast, we found a different pattern of sedimentation when comparing transformed antiestrogen-receptor complexes with transformed estrogen-receptor complexes. In this case, transformed H1285-ER sedimented as 6S and estradiol-ER sedimented as 4S. We conclude from these experiments that these two antihormones, RU486 and H1285, may have different mechanisms of action in their antagonism of steroid hormone action. Antiestrogen stabilizes the salt-transformed ER as a dimer while antiprogestin appears to permit dissociation of the oligomeric form of the receptor to the monomeric form.