RESUMEN
Popular psychotropic drugs, like the antidepressant selective serotonin reuptake inhibitors (SSRIs) and tricyclic antidepressants (TCAs), and the mood stabilizer lithium, may have skeletal effects. In particular, preclinical observations suggest a direct negative effect of SSRIs on the skeleton. A potential caveat in studies of the skeletal effects of psychotropic drugs is the hypoactive (skeletal unloading) phenotype they induce. The aim of this study was to investigate the contribution of physical inactivity to the skeletal effects of psychotropic drugs by studying bone changes in cage control and tail suspended mice treated with either vehicle, SSRI, TCA or lithium. Tail suspension was used to control for drug differences on physical activity levels by normalizing skeletal loading between groups. The psychotropic drugs were found to have contrasting skeletal effects which were independent of drug effects on animal physical activity levels. The latter was evident by an absence of statistical interactions between the activity and drug groups. Pharmacological inhibition of the 5-hydroxytryptamine (5-HT) transporter (5-HTT) using a SSRI reduced in vivo gains in lower extremity BMD, and negatively altered ex vivo measures of femoral and spinal bone density, architecture and mechanical properties. These effects were mediated by a decrease in bone formation without a change in bone resorption suggesting that the SSRI had anti-anabolic skeletal effects. In contrast, glycogen synthase kinase-3[beta] (GSK-3[beta]) inhibition using lithium had anabolic effects improving in vivo gains in BMD via an increase in bone formation, while TCA-mediated inhibition of the norepinephrine transporter had minimal skeletal effect. The observed negative skeletal effect of 5-HTT inhibition, combined with recent findings of direct and indirect effects of 5-HT on bone formation, are of interest given the frequent prescription of SSRIs for the treatment of depression and other affective disorders. Likewise, the anabolic effect of GSK-3[beta] inhibition using lithium reconfirms the importance of Wnt/beta-catenin signaling in the skeleton and it's targeting by recent drug discovery efforts. In conclusion, the current study demonstrates that different psychotropic drugs with differing underlying mechanisms of action have contrasting skeletal effects and that these effects do not result indirectly via the generation of animal physical inactivity.
Asunto(s)
Densidad Ósea/efectos de los fármacos , Remodelación Ósea/efectos de los fármacos , Desipramina/farmacología , Fémur/efectos de los fármacos , Fluoxetina/farmacología , Cloruro de Litio/farmacología , Actividad Motora/efectos de los fármacos , Columna Vertebral/efectos de los fármacos , Absorciometría de Fotón , Análisis de Varianza , Animales , Antidepresivos de Segunda Generación/farmacología , Antidepresivos Tricíclicos/farmacología , Antimaníacos/farmacología , Fuerza Compresiva/fisiología , Ensayo de Inmunoadsorción Enzimática , Femenino , Fémur/diagnóstico por imagen , Suspensión Trasera , Ratones , Distribución Aleatoria , Serotonina/sangre , Columna Vertebral/diagnóstico por imagenRESUMEN
Evidence regarding a functional serotonin (5-hydroxytryptamine) signaling system in bone has generated considerable recent interest. The specific biochemical nature of serotoninergic pathways and their direct and/or indirect effects on bone metabolism are still unclear. Clinical evidence supports an effect of serotonin and altered serotonin signaling on bone metabolism. Serotonin is involved in the pathophysiology of depression, and therefore studies of depression and antidepressant treatments (as modulators of the serotonin system) are relevant with regard to bone outcomes. Studies on the effect of depression on bone mineral density (BMD) and fractures have been mixed. Studies on the associations between antidepressant use and BMD and/or fractures are more consistent. SSRIs have been associated with lower BMD and increased rates of bone loss, as well as increased rates of fracture after accounting for falls. These studies are limited by confounding because depression is potentially associated with both the outcome of interest (BMD and fracture) and the exposure (SSRIs). With mounting evidence for an effect on bone, this review considers the question of causality and whether selective serotonin reuptake inhibitors should be considered among those medications that contribute to bone loss, and therefore prompt clinicians to evaluate BMD proactively. Future research will be required to confirm the serotoninergic effects on bone and the biochemical pathways involved, and to identify clinical implications for treatment based on this novel pathway.
Asunto(s)
Antidepresivos/farmacología , Huesos/efectos de los fármacos , Inhibidores Selectivos de la Recaptación de Serotonina/farmacología , Adulto , Antidepresivos/efectos adversos , Antidepresivos/uso terapéutico , Densidad Ósea/efectos de los fármacos , Depresión/tratamiento farmacológico , Humanos , Inhibidores Selectivos de la Recaptación de Serotonina/efectos adversos , Inhibidores Selectivos de la Recaptación de Serotonina/uso terapéuticoRESUMEN
PURPOSE OF REVIEW: Osteoporosis in men is increasingly recognized as an important health problem. New research contributes to our knowledge of gender differences in osteoporosis risk, diagnosis and management. We undertook this review to summarize recent developments in the field of male osteoporosis. RECENT FINDINGS: The paper reviews recently published studies that reveal new insights into male osteoporosis. It addresses epidemiology, risk factors, use of clinical risk assessment tools, diagnosis and treatment. New data continue to suggest that men have higher mortality rates than women after hip fracture, and that men may experience fractures at higher bone mineral density values than women. Treatments for osteoporosis have been studied mostly in women, but trials including both men and women are now being conducted. Likewise, there are several newer cohorts with bone and fracture outcomes that include men and women. The Osteoporotic Fractures in Men (MrOS) study is the first United States-based cohort to include only men; this study is contributing importantly to our understanding of epidemiology and risk factors for osteoporosis in men. SUMMARY: Men and their physicians should be aware of the risk for osteoporosis and the gender differences that exist within this disease. Further research is needed to continue to understand differences in pathophysiology, epidemiology and risk factors, and to promote appropriate therapies among men.
Asunto(s)
Osteoporosis , Adulto , Anciano , Humanos , Masculino , Persona de Mediana Edad , Osteoporosis/diagnóstico , Osteoporosis/epidemiología , Osteoporosis/terapia , Medición de Riesgo , Factores de Riesgo , Factores SexualesRESUMEN
BACKGROUND: Selective serotonin reuptake inhibitors (SSRIs) are a widely used class of antidepressants that block the serotonin transporter. Osteoblasts and osteocytes express functional serotonin transporters; serotonin transporter gene disruption in mice results in osteopenia; and SSRI use has been associated with increased risk of hip fracture. METHODS: To determine whether SSRI use is associated with lower bone mineral density (BMD) in older men and to compare the results for SSRIs with those of other antidepressants, we performed a cross-sectional analysis of data from 5995 men 65 years and older participating in the prospective cohort Osteoporotic Fractures in Men Study. Main outcome measures included medication use; BMD at the femoral neck, greater trochanter, and lumbar spine measured by dual-energy x-ray absorptiometry; and potential covariates. RESULTS: In adjusted analyses, mean BMD among SSRI users (n=160) was 3.9% lower at the total hip and 5.9% lower at the lumbar spine compared with BMD in men reporting no antidepressant use (n=5708 [P=.002 for total hip; P<.001 for lumbar spine]). There was no significant difference among users of trazodone hydrochloride (n=52) or tricyclic antidepressants (n=99) compared with nonusers. Adjusting for variables that could be associated with BMD and/or SSRI use did not significantly alter these results. The observed difference in BMD for SSRIs is similar to that seen with glucocorticoids. CONCLUSIONS: In this population of men, BMD was lower among those reporting current SSRI use, but not among users of other antidepressants. Further research is needed to confirm this finding in light of widespread SSRI use and potentially important clinical implications.
Asunto(s)
Densidad Ósea/efectos de los fármacos , Fracturas Óseas/etiología , Osteoporosis/inducido químicamente , Inhibidores Selectivos de la Recaptación de Serotonina/efectos adversos , Absorciometría de Fotón , Anciano , Estudios Transversales , Depresión/tratamiento farmacológico , Cuello Femoral/diagnóstico por imagen , Estudios de Seguimiento , Fracturas Óseas/diagnóstico por imagen , Fracturas Óseas/epidemiología , Cadera/diagnóstico por imagen , Humanos , Incidencia , Vértebras Lumbares/diagnóstico por imagen , Masculino , Persona de Mediana Edad , Osteoporosis/complicaciones , Osteoporosis/metabolismo , Estudios Prospectivos , Factores de Riesgo , Inhibidores Selectivos de la Recaptación de Serotonina/uso terapéutico , Estados Unidos/epidemiologíaRESUMEN
Neurotransmitter regulation of bone metabolism has been a subject of increasing interest and investigation. We reported previously that osteoblastic cells express a functional serotonin (5-HT) signal transduction system, with mechanisms for responding to and regulating uptake of 5-HT. The clonal murine osteocytic cell line, MLO-Y4, demonstrates expression of the serotonin transporter (5-HTT), and the 5-HT1A, and 5-HT2A receptors by real-time RT-PCR and immunoblot analysis. Immunohistochemistry using antibodies for the 5-HTT, and the 5-HT1A and 5-HT2A receptors reveals expression of all three proteins in both osteoblasts and osteocytes in rat tibia. 5-HTT binding sites were demonstrated in the MLO-Y4 cells with nanomolar affinity for the stable cocaine analog [125I]RTI-55. Imipramine and fluoxetine, antagonists with specificity for 5-HTT, show the highest potency to antagonize [125I]RTI-55 binding in the MLO-Y4 cells. GBR-12935, a relatively selective dopamine transporter antagonist, had a much lower potency, as did desipramine, a selective norepinephrine transporter antagonist. The maximal [3H]5-HT uptake rate in MLO-Y4 cells was 2.85 pmol/15 min/well, with a Km value of 290 nM. Imipramine and fluoxetine inhibited specific [3H]5-HT uptake with IC50 values in the nanomolar range. 5-HT rapidly stimulated PGE2 release from MLO-Y4 cells; the EC50 for 5-HT was 0.1 microM, with a 3-fold increase seen at 60 min. The rate-limiting enzyme for serotonin synthesis, tryptophan hydroxylase, is expressed in MLO-Y4 cells as well as osteoblastic MC3T3-E1 cells. Thus, osteocytes, as well as osteoblasts, are capable of 5-HT synthesis, and express functional receptor and transporter components of the 5-HT signal transduction system.
Asunto(s)
Osteocitos/metabolismo , Receptor de Serotonina 5-HT1A/genética , Receptor de Serotonina 5-HT1A/metabolismo , Receptor de Serotonina 5-HT2A/genética , Receptor de Serotonina 5-HT2A/metabolismo , Proteínas de Transporte de Serotonina en la Membrana Plasmática/genética , Proteínas de Transporte de Serotonina en la Membrana Plasmática/metabolismo , Animales , Western Blotting , Línea Celular , Expresión Génica , Inmunohistoquímica , Cinética , Ratones , Osteoblastos/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ratas , Ratas Wistar , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Tibia/metabolismoRESUMEN
Medical residents may be vulnerable to low vitamin D status because of long work hours and lack of sun exposure. We conducted a prospective cohort study to measure serum 25-hydroxyvitamin D concentrations among internal medicine residents, document seasonal variation in vitamin D status, and assess risk factors for inadequate vitamin D stores. Dietary intake of calcium and vitamin D, lifestyle characteristics, and serum concentrations of 25(OH)-vitamin D and intact parathyroid hormone (iPTH) were measured in 35 resident volunteers before and after the winter season. A total of 63-69% of medical residents consumed <400 IU/day of vitamin D; 61-67% consumed <1000 mg/day of calcium. Twenty-five (74%) had lower serum 25(OH)-vitamin D concentrations and 23 (68%) had higher serum iPTH in the spring than in the fall. Nine (26%) residents had serum concentrations of 25(OH)-vitamin D of <20 ng/mL in the fall; and sixteen (47%) in the spring. Seven residents (20%) had serum concentrations of 25(OH)-vitamin D of <20 ng/mL at both time-periods; Eighteen residents (51.4%) had 25(OH)-vitamin D levels of <20 ng/mL for at least one of the time-periods. Medical residents are at risk for hypovitaminosis D, particularly during the winter months and should be aware of the need to supplement their vitamin D stores. Insufficient vitamin D status and inadequate vitamin D intake may have long-term implications for bone health in these individuals. Increased educational efforts to promote healthy dietary and lifestyle choices that allow attainment and maintenance of skeletal health are appropriate in this population.
Asunto(s)
Medicina Interna , Internado y Residencia , Deficiencia de Vitamina D/sangre , Vitamina D/análogos & derivados , Adulto , Calcio/sangre , Calcio de la Dieta , Femenino , Humanos , Estilo de Vida , Masculino , Persona de Mediana Edad , Oregon/epidemiología , Hormona Paratiroidea/sangre , Estudios Prospectivos , Estaciones del Año , Vitamina D/sangre , Deficiencia de Vitamina D/epidemiologíaRESUMEN
Neurotransmitter regulation of bone metabolism has been a subject of increasing interest and investigation. Dopamine (DA) has been reported to have effects on calcium and phosphorus metabolism. The dopamine transporter (DAT) is believed to control the temporal and spatial activity of released DA by rapid uptake of the neurotransmitter into presynaptic terminals. We have evaluated the histologic and biomechanical properties of the skeleton in mice homozygous for deletion of the DA transporter gene (DAT (-/-)) to help delineate the role of DA in bone biology. We have demonstrated that DAT (-/-) mice have reduced bone mass and strength. DAT (-/-) animals have shorter femur length and dry weight, and lower ash calcium content. Cancellous bone volume in the DAT (-/-) proximal tibial metaphysis is significantly decreased with reduced trabecular thickness. DAT (-/-) vertebrae have lower cancellous bone volume as a consequence of increased trabecular spacing and reduced trabecular number, and cortical thickness and bone area in the femoral diaphysis are reduced. The ultimate bending load (femoral strength) for the DAT (-/-) mice is 30% lower than the wild-type mice. Thus, deletion of the DAT gene results in deficiencies in skeletal structure and integrity. Since serotonin (5-HT) plays a role as a regulator of craniofacial morphogenesis, we explored the expression and function of 5-HT receptors and the 5-HT transporter (5-HTT) in bone. Primary cultures of rat osteoblasts (rOB) and a variety of clonal osteoblastic cell lines including ROS 17/2.8, UMR 106-H5 and Py1a show mRNA expression for the 5-HTT, and the 5-HT(1A), 5-HT(1D), 5-HT(2A) and 5-HT(2B) receptors by RT-PCR analysis and immunoblot. A relatively high density of nanomolar affinity 5-HTT binding sites is present in ROS 17/2.8 and UMR 106-H5 cells. The maximal [(3)H]5-HT uptake rate in ROS cells was 110 pmol/10 min/well, with a K(m) value of 1.13 microM. In normal differentiating rOB cultures, 5-HTT functional activity was observed initially at day 25, and activity increased by almost eight-fold at day 31. In mature rOB cultures, the estimated density of [(125)I]RTI-55 binding sites was 600 fmol/mg protein. PMA treatment caused a significant 40% reduction in the maximal uptake rate of [(3)H]5-HT, an effect prevented by pretreatment with staurosporine. 5-HT potentiates the PTH-induced increase in AP-1 activity in UMR 106-H5 cells. In 5-HTT (-/-) animals, cancellous bone volume (BV/TV) in the lumbar vertebrae is reduced, with a trend toward decreased trabecular thickness and trabecular number. These results demonstrate that osteoblastic cells express a functional serotonin system, with mechanisms for responding to and regulating uptake of 5-HT, and disruption of the 5-HTT gene may cause osteopenia.
RESUMEN
Neurotransmitter regulation of bone metabolism has been the subject of increasing interest and investigation. Because serotonin (5-HT) plays a role as a regulator of craniofacial morphogenesis, we investigated the expression and function of 5-HT receptors and the 5-HT transporter (5-HTT) in bone. Primary cultures of rat osteoblasts (rOB) and a variety of clonal osteoblastic cell lines, including ROS 17/2.8, UMR 106-H5, and Py1a, showed mRNA expression for 5-HTT as well as the 5-HT(1A), 5-HT(1D), 5-HT(2A), and 5-HT(2B) receptors by reverse transcription-polymerase chain reaction (RT-PCR) analysis. Protein expression of the 5-HT(1A), 5-HT(2A), and 5-HT(2B) receptors was confirmed by immunoblot. 5-HTT binding sites were assessed in ROS 17/2.8 and UMR 106-H5 cells by binding of the stable cocaine analog [125I]RTI-55, which showed a relatively high density of nanomolar affinity binding sites. Imipramine and fluoxetine, antagonists with specificity for 5-HTT, showed the highest potency to antagonize [125I]RTI-55 binding in ROS and UMR cells. GBR-12935, a relatively selective dopamine transporter antagonist, had a much lower potency, as did desipramine, a selective norepinephrine transporter antagonist. The maximal [3H]5-HT uptake rate in ROS cells was 110 pmol/10 min per well, with a K(m) value of 1.13 micromol/L. Imipramine and fluoxetine inhibited specific [3H]5-HT uptake with IC(50) values in the nanomolar range. In normal differentiating rOB cultures, 5-HTT functional activity was observed initially at day 25, and activity increased almost eightfold by day 31. In mature rOB cultures, the estimated density of [125I]RTI-55 binding sites was 600 fmol/mg protein. Functional downregulation of transporter activity was assessed after PMA treatment, which caused a significant 40% reduction in the maximal uptake rate of [3H]5-HT, an effect that was prevented by pretreatment with staurosporine. The affinity of 5-HT for the transporter was significantly increased following PMA treatment. We assessed the functional significance of expression of the 5-HT receptors by investigating the interaction between 5-HT and parathyroid hormone (PTH) signaling. 5-HT potentiates the PTH-induced increase in AP-1 activity in UMR cells. These results demonstrate that osteoblastic cells express a functional serotonin system, with mechanisms for responding to and regulating uptake of 5-HT.
Asunto(s)
Proteínas Portadoras/genética , Glicoproteínas de Membrana/genética , Proteínas de Transporte de Membrana , Proteínas del Tejido Nervioso , Osteoblastos/metabolismo , Receptores de Serotonina/genética , Serotonina/farmacocinética , Animales , Carcinógenos/farmacología , Proteínas Portadoras/metabolismo , Regulación hacia Abajo/efectos de los fármacos , Expresión Génica/fisiología , Radioisótopos de Yodo , Glicoproteínas de Membrana/metabolismo , Osteoblastos/citología , Osteosarcoma , Hormona Paratiroidea/fisiología , ARN Mensajero/análisis , Ensayo de Unión Radioligante , Ratas , Receptor de Serotonina 5-HT1D , Receptor de Serotonina 5-HT2A , Receptor de Serotonina 5-HT2B , Receptores de Serotonina/metabolismo , Receptores de Serotonina 5-HT1 , Proteínas de Transporte de Serotonina en la Membrana Plasmática , Acetato de Tetradecanoilforbol/farmacología , Tritio , Células Tumorales CultivadasRESUMEN
The age-related decline in beta-adrenergic receptor (beta-AR)-mediated vasorelaxation is associated with desensitization of beta-ARs without significant downregulation. The primary mode of this homologous beta-AR desensitization, in general, is via G protein receptor kinases (GRK). Therefore, we hypothesize that age-related changes in GRKs are causative to this etiology in rat aorta. Herein, we investigate the activity and cellular distribution (cytoplasmic vs. membrane) of several GRK isoforms and beta-arrestin proteins. GRK activity was assessed in extracts from aortic tissue of 6-wk, 6-mo, 12-mo, and 24-mo-old male Fischer-344 rats using a rhodopsin phosphorylation assay. We also performed immunoblots on lysates from aorta with specific antibodies to GRK-2, -3, -5, and beta-arrestin-1. Results show an age-related increase in GRK activity. Furthermore, expression of GRK-2 (cytoplasmic and membrane), GRK-3 (cytoplasmic and membrane), and beta-arrestin (soluble) increased with advancing age, whereas GRK-5 (membrane) expression remained unchanged. These results suggest that age is associated with increased activity and expression of specific GRKs. This increase likely results in enhanced phosphorylation and desensitization of beta-ARs. These biochemical changes are consistent with observed aging physiology.
Asunto(s)
Envejecimiento , Aorta/enzimología , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Animales , Aorta/ultraestructura , Arrestinas/análisis , Arrestinas/metabolismo , Membrana Celular/enzimología , Proteínas Quinasas Dependientes de AMP Cíclico/análisis , Citoplasma/enzimología , Quinasa 3 del Receptor Acoplado a Proteína-G , Quinasa 5 del Receptor Acoplado a Proteína-G , Immunoblotting , Isoenzimas/análisis , Isoenzimas/metabolismo , Masculino , Fosforilación , Proteínas Serina-Treonina Quinasas/análisis , Ratas , Ratas Endogámicas F344 , Rodopsina/metabolismo , Vasodilatación , Quinasas de Receptores Adrenérgicos beta , beta-Arrestina 1 , beta-ArrestinasRESUMEN
Rapid phosphorylation of many G-protein-coupled receptors (GPCRs) by G-protein-coupled receptor kinases (GRKs) accompanies stimulus-driven desensitization. Recent evidence suggests that GRKs and their associated arresting proteins, beta-arrestins, function as essential elements in the GPCR-mediated mitogen-activated protein (MAP) kinase signaling cascade. We investigated the interaction between GRKs and MAP kinase activation by growth factors in UMR 106-H5 osteoblastic cells stably expressing a dominant negative mutant of GRK2 (K220R). Expression of K220R in osteoblastic cells results in reduced cellular proliferation, both basally and in response to insulin-like growth factor-1 (IGF-1), and blunting of IGF-1- and EGF-induced MAP kinase activation. Reduced MAP kinase activation is not associated with alterations in IGF-1-receptor autophosphorylation. Both a constitutively active Ras mutant and PMA fully activate MAP kinase in K220R cells. We found that disruption of the GRK2 gene results in: (1) reduced osteoblast proliferation in response to growth factors, and (2) impaired receptor tyrosine kinase activation of mitogenic signaling pathways. Thus, GRK2 may regulate growth factor responsiveness in osteoblasts by modulating multiprotein complex formation following receptor tyrosine kinase activation.
Asunto(s)
Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Osteoblastos/enzimología , Animales , División Celular/efectos de los fármacos , Línea Celular , Proteínas Quinasas Dependientes de AMP Cíclico/genética , Activación Enzimática/efectos de los fármacos , Factor de Crecimiento Epidérmico/farmacología , Proteínas de Unión al GTP/metabolismo , Factor I del Crecimiento Similar a la Insulina/farmacología , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Mutación , Osteoblastos/efectos de los fármacos , Osteoblastos/metabolismo , Fosforilación , Ratas , Receptores de Superficie Celular/metabolismo , Acetato de Tetradecanoilforbol/farmacología , Quinasas de Receptores Adrenérgicos betaRESUMEN
Dopamine (DA) has been reported to have effects on calcium and phosphorus metabolism. The dopamine transporter (DAT) is believed to control the temporal and spatial activity of released DA by rapid uptake of the neurotransmitter into presynaptic terminals. We have evaluated the histologic and biomechanical properties of the skeleton in mice homozygous for deletion of the DA transporter gene (DAT) to help delineate the role of DA in bone biology. We have demonstrated that DAT-/-mice have reduced bone mass and strength. DAT-/- animals had shorter femur length and dry weight. Ash calcium content of the femur was 32% lower in the DAT-/- mice than in the wild-type animals. Cancellous bone volume in the proximal tibial metaphysis was significantly lower in the DAT-/- animals (p < 0.04). There was a 32% reduction in trabecular thickness (p = NS). For the vertebrae, cancellous bone volume was again lower in the DAT-/- animals compared with wild-type as a consequence of increased trabecular spacing (p < 0.05) and reduced trabecular number (p < 0.05). Cortical thickness and bone area in the femoral diaphysis were reduced in the DAT-/-animals. The ultimate bending load (femoral strength) for the DAT-/- mice was 30% lower than the wild-type mice (p = 0.004). Thus, deletion of the DAT gene results in deficiencies in skeletal structure and integrity.
Asunto(s)
Proteínas Portadoras/genética , Dopamina/metabolismo , Homocigoto , Glicoproteínas de Membrana , Proteínas de Transporte de Membrana , Proteínas del Tejido Nervioso , Animales , Proteínas Portadoras/metabolismo , Proteínas de Transporte de Dopamina a través de la Membrana Plasmática , Ratones , Ratones Noqueados , FenotipoRESUMEN
Biologic responses to peptide calciotropic hormones, such as parathyroid hormone (PTH) and calcitonin, exhibit desensitization. As with most hormones, however, the mechanisms of desensitization are not completely understood. For the beta 2-adrenergic receptor (beta 2AR) system, which is coupled to adenylyl cyclase via the stimulatory guanine nucleotide-binding regulatory (G5) protein, homologous desensitization is mediated in part by a receptor-specific kinase (beta ARK) and a soluble cofactor (beta-arrestin). Recently, this system has been reported to be involved in rapid homologous desensitization of the PTH/parathyroid hormone receptor protein (PTHrP) receptor. We have identified the presence of this system in bone using reverse-transcriptase PCR. Nucleotide sequence of PCR fragments from ROS 17/2.8 cells revealed 100% identity with rat brain beta ARK1 and beta-arrestin 1 sequences. Northern analyses with RNA from ROS 17/2.8, UMR 106-H5 cells, and primary cultures of nontransformed neonatal rat calvariae demonstrated two mRNA species of 4 and 2.6 kilobases (kb) for beta ARK and 7.5 kb for beta-arrestin, comparable to those found in bovine brain. beta ARK-like activity was demonstrated in cytosolic extracts of the UMR 106-H5 cells by assessing phosphorylation of the retinal photoreceptor, rhodopsin, by the extracts. Phosphorylation was enhanced with light-activated rhodopsin and by bovine brain G beta gamma subunits; heparin inhibited phosphorylation. These findings are characteristic of beta ARK. Expression of beta-arrestin in the UMR 106-H5 cells was confirmed by immunoblot. Thus, osteoblastic cells express proteins, beta ARK, and beta-arrestin, which may regulate desensitization of calciotropic hormone receptors.
Asunto(s)
Arrestinas/biosíntesis , Proteínas Quinasas Dependientes de AMP Cíclico/biosíntesis , Osteoblastos/metabolismo , Animales , Arrestinas/genética , Secuencia de Bases , Northern Blotting , Southern Blotting , Western Blotting , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Reacción en Cadena de la Polimerasa , Ratas , Células Tumorales Cultivadas , Quinasas de Receptores Adrenérgicos beta , beta-Arrestina 1 , beta-ArrestinasRESUMEN
Activation of the G protein-coupled receptor for parathyroid hormone (PTH)/PTH-related protein (PTHrP) produces homologous desensitization of receptor signaling. We have shown recently that the opossum PTH/PTHrP receptor stably expressed in human embryonic kidney (HEK) 293 cells is phosphorylated upon agonist binding and upon activation of serine/threonine protein kinases (PKA and PKC), an event which for some G protein-coupled receptors has been linked to desensitization. To locate the sites of phosphorylation, mutated forms of the opossum PTH/PTHrP receptor were stably expressed in HEK 293 cells, and ligand-stimulated receptor phosphorylation was evaluated. The five serine and threonine residues of the third cytoplasmic loop of the receptor were not required for receptor phosphorylation. Basal and ligand-induced phosphorylation were, however, completely abolished upon deletion of all but the 16 juxtamembrane residues of the cytoplasmic C-terminal tail of the receptor, even though this truncated receptor resembled the wild-type receptor in its level of expression based on Western blotting and radioligand binding. To identify further the phosphorylation sites, the 129 amino acid C-terminal tail of the rat PTH/PTHrP receptor was expressed in E. coli as a recombinant glutathione S-transferase fusion protein. Elimination of a single PKA consensus site in the tail (serine 491) resulted in > or = 90% loss of PKA-mediated phosphorylation, identifying this as the preferential site for PKA, with two other sites (serine 473 and/or 475) being minor sites. Phosphorylation by PKC occurred largely in the proximal portion of the tail, whereas beta-adrenergic receptor kinase 1 (beta ARK1) phosphorylated more distally in the tail. The ability of these kinases to phosphorylate the PTH/PTHrP receptor at distinct sites on the cytoplasmic tail may allow differential regulation of receptor signaling and trafficking.
Asunto(s)
Proteínas/metabolismo , Receptores de Hormona Paratiroidea/metabolismo , Transducción de Señal , Secuencia de Aminoácidos , Animales , Línea Celular , Humanos , Datos de Secuencia Molecular , Proteína Relacionada con la Hormona Paratiroidea , Fosforilación , Ratas , Receptor de Hormona Paratiroídea Tipo 1 , Receptores de Hormona Paratiroidea/genética , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismoRESUMEN
Monoclonal antibody, LAS-2, directed against the alpha subunit of transducin (Gt alpha), inhibited Gt beta gamma-dependent, pertussis toxin-catalyzed ADP ribosylation of Gt alpha and was specific for Gt alpha. Immunoblotting studies on proteolytic fragments of Gt alpha were consistent with an amino-terminal epitope. To define the antibody recognition site, recombinant Gt alpha was synthesized in Escherichia coli cotransfected with or without yeast N-myristoyl-transferase. Amino-terminal fatty acylation of Gt alpha, verified by use of radiolabeled fatty acid, was required for immunoreactivity. LAS-2 did not react with a chimeric protein consisting of residues 1-9 of Gt alpha and the remainder Go alpha, regardless of its myristoylation. Immunoreactivity was observed when amino acids 1-17 of Gt alpha were present in a Go alpha chimera and the protein was amino-terminally myristoylated; there was no reactivity without myristoylation. It appears that the LAS-2 epitope requires both Gt alpha-specific sequence in amino acids 10-17 and a fatty acyl group in proximity to these residues. These results are consistent with the hypothesis that the myristoyl group is essential for protein structure; conceivably it "folds back" on and stabilizes the amino-terminal structure of Gt alpha as opposed to protruding from an amino-terminal alpha-helix and serving as an amino-terminal membrane anchor.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Proteínas de Unión al GTP/inmunología , Proteínas Recombinantes de Fusión/inmunología , Animales , Secuencia de Bases , Sitios de Unión de Anticuerpos , Bovinos , Epítopos , Ratones , Datos de Secuencia Molecular , Ácido Mirístico , Ácidos Mirísticos/metabolismo , Relación Estructura-ActividadRESUMEN
Cushing's syndrome in infancy is uncommon. In this report, we describe an infant with ACTH-independent Cushing's syndrome in which an activating mutation in the stimulatory G-protein (Gs alpha) was detected. The patient presented at 3 months of age with Cushingoid features, poor linear growth, and elevated liver enzymes. Plasma ACTH and dexamethasone suppression test results were consistent with ACTH-independent Cushing's syndrome, and a subsequent adrenalectomy revealed bilateral adrenocorticonodular hyperplasia. Asymptomatic lesions consistent with fibrous dysplasia were later detected on bone scan. Genomic DNA was extracted from adrenal, liver, and blood and amplified by polymerase chain reaction with Gs alpha exon 8 primers. Using allele-specific oligonucleotide hybridization, the DNA was probed for known Gs alpha-activating mutations. A point mutation coding for an arginine to cysteine substitution at codon 201 of exon 8 was detected in genomic DNA from this infant's adrenal, liver, and leukocytes. The mutation was detected in nodular adrenal tissue, but was essentially absent in normal adrenal tissue. Activating mutations in the Gs alpha gene have previously been described in GH-secreting tumors, thyroid adenomas, and the McCune-Albright syndrome and are probably involved in the pathogenesis of adrenocorticonodular hyperplasia in this infant with Cushing's syndrome.
Asunto(s)
Hiperplasia Suprarrenal Congénita/complicaciones , Síndrome de Cushing/complicaciones , Proteínas de Unión al GTP/genética , Mutación , Hiperplasia Suprarrenal Congénita/genética , Secuencia de Bases , Síndrome de Cushing/genética , ADN/análisis , Humanos , Lactante , Masculino , Hibridación de Ácido Nucleico , Sondas de Oligonucleótidos , Reacción en Cadena de la Polimerasa , Análisis de Secuencia de ADNRESUMEN
Although osteoporotic fractures have long been considered a special problem for postmenopausal women, osteoporosis is also an important clinical issue in men. The causes and clinical spectrum of osteoporosis is similar in men and women, but there are definite gender differences that reflect sexual differences in skeletal development, age-related bone loss, and secondary causes of metabolic bone disease.
Asunto(s)
Osteoporosis/fisiopatología , Adulto , Anciano , Envejecimiento/fisiología , Densidad Ósea/fisiología , Femenino , Fracturas Óseas/epidemiología , Humanos , Hipogonadismo/complicaciones , Incidencia , Masculino , Persona de Mediana Edad , Osteoporosis/complicaciones , Osteoporosis/tratamiento farmacológico , Osteoporosis/epidemiología , Factores SexualesRESUMEN
Activation of adenylyl cyclase by cholera toxin A subunit (CT-A) results from the ADP-ribosylation of the stimulatory guanine nucleotide binding protein (GS alpha). This process requires GTP and an endogenous guanine nucleotide binding protein known as ADP-ribosylation factor (ARF). One membrane (mARF) and two soluble forms (sARF I and sARF II) of ARF have been purified from bovine brain. Because the conditions reported to enhance the binding of guanine nucleotides by ARF differ from those observed to promote optimal activity, we sought to characterize the determinants influencing the functional interaction of guanine nucleotides with ARF. High-affinity GTP binding by sARF II (apparent KD of approximately 70 nM) required Mg2+, DMPC, and sodium cholate. sARF II, in DMPC/cholate, also enhanced CT-A ADP-ribosyltransferase activity (apparent EC50 for GTP of approximately 50 nM), although there was a delay before achievement of a maximal rate of sARF II stimulated toxin activity. The delay was abolished by incubation of sARF II with GTP at 30 degrees C before initiation of the assay. In contrast, a maximal rate of activation of toxin by sARF II, in 0.003% SDS, occurred without delay (apparent EC50 for GTP of approximately 5 microM). High-affinity GTP binding by sARF II was not detectable in SDS. Enhancement of CT-A ADP-ribosyltransferase activity by sARF II, therefore, can occur under conditions in which sARF II exhibits either a relatively low affinity or a relatively high affinity for GTP. The interaction of GTP with ARF under these conditions may reflect ways in which intracellular membrane and cytosolic environments modulate GTP-mediated activation of ARF.
Asunto(s)
Toxina del Cólera/metabolismo , Proteínas de la Membrana/metabolismo , Factores de Ribosilacion-ADP , Animales , Bovinos , Ácidos Cólicos/metabolismo , Citosol/metabolismo , Detergentes/farmacología , Dimiristoilfosfatidilcolina/metabolismo , Nucleótidos de Guanina/metabolismo , Guanosina Trifosfato/metabolismo , Membranas Intracelulares/metabolismo , Cinética , Fosfolípidos/farmacología , Poli(ADP-Ribosa) Polimerasas/metabolismo , Unión ProteicaRESUMEN
The blood magnesium status--plasma and red blood cell (RBC) concentration and mononuclear blood cell (MBC) content of magnesium and parathyroid hormone (PTH) were determined in 6 patients with chronic hypoparathyroidism and 9 patients with primary hyperparathyroidism before and after surgical cure of the disease. The magnesium content of MBCs and concentration of RBCs increased significantly (p less than 0.01) after the surgical correction of primary hyperparathyroidism. Patients with chronic hypoparathyroidism had significant increase (p less than 0.05) of magnesium in RBCs and MBCs compared with the control group. No significant difference was observed for the plasma magnesium concentration for hypoparathyroid patients compared with the control group and hyperparathyroid patients before and after surgical care. These results suggest that endogenous PTH affects the concentration of magnesium in RBCs and magnesium content in MBCs but does not alter significantly the concentration of magnesium in plasma.
Asunto(s)
Hiperparatiroidismo/sangre , Hipoparatiroidismo/sangre , Magnesio/sangre , Eritrocitos/metabolismo , Humanos , Hiperparatiroidismo/cirugía , Leucocitos Mononucleares/metabolismoRESUMEN
We describe a patient with an absent intestinal response to 1,25-dihydroxyvitamin D [1,25-(OH)2D] and the beneficial effects of treatment with high dose iv calcium infusion. The patient presented with severe rickets despite therapy with extraordinarily high doses of 1 alpha-hydroxyvitamin D3 or 1,25-(OH)2D3. Unidirectional intestinal fractional calcium absorption when he was not treated with any calciferol was 14% (normal, 20-70%), as measured with stable calcium isotopes; no increase in calcium absorption occurred when serum 1,25-(OH)2D levels were more than 50-fold elevated. Cultured skin fibroblasts contained no detectable 25-hydroxyvitamin D3-24-hydroxylase activity in response to 1,25-(OH)2D3 (10(-9)-10(-6) mol/L). High dose iv calcium infusions and oral phosphorus supplementation for 135 days improved or normalized biochemical parameters and resulted in radiographic healing of the rachitic lesions. We conclude that 1) this patient had no response to 1,25-(OH)2D3 in vivo and in vitro; 2) long term parenteral calcium infusions were effective therapy in managing the patient's severe resistance to 1,25-(OH)2D; and 3) stable calcium isotopes are useful for measuring low levels of fractional calcium absorption.
Asunto(s)
Calcitriol/fisiología , Calcio/uso terapéutico , Hipofosfatemia Familiar/tratamiento farmacológico , Absorción Intestinal , Fosfatasa Alcalina/sangre , Calcio/administración & dosificación , Calcio/farmacocinética , Preescolar , Fibroblastos/efectos de los fármacos , Humanos , Hipofosfatemia Familiar/genética , Masculino , Fósforo/administración & dosificación , Fósforo/uso terapéutico , Piel/efectos de los fármacosRESUMEN
Alopecia is a frequent feature in hereditary resistance to (1,25(OH)2D). We sought insight into this feature by analysing data from affected members of 30 kindreds. We assessed indices of mineral metabolism in one group with normal hair compared with a group with alopecia. Hereditary resistance to 1,25(OH)2D was diagnosed at an earlier age in alopecic patients (0.9 vs 3.3 years, P less than 0.05); this reflected late presentation of metabolic bone disease in some cases with normal hair and could not be attributed to early diagnosis resulting from the striking feature of alopecia. For untreated subjects, serum concentrations of calcium and 1,25(OH)2D were similar in both groups of patients. During calciferol therapy, however, the cases with alopecia showed lower serum calcium (1.9 vs 2.4 mmol/l, P less than 0.005), but higher serum 1,25(OH)2D (2900 v 340 pg/ml, P less than 0.005). Hair status did not predict the type of defect identified with cultured skin fibroblasts but did correlate with responsiveness of 25(OH)D 24-hydroxylase to 1,25(OH)2D3 in those cells. Cells from seven of eight kindreds with alopecia showed no 24-hydroxylase response to high doses of 1,25(OH)2D3 while cells from five of six kindreds with normal hair showed a 24-hydroxylase response to high doses of 1,25(OH)2D3. We conclude that in cases with hereditary resistance to 1,25(OH)2D alopecia reflects the more severe grades of this resistance based upon earlier age at time of diagnosis, lower potential for calcaemic response to calciferols, and lower potential for 24-hydroxylase response to 1,25(OH)2D3 by cultured skin fibroblasts.