M. B. Teixeira

Double disruption of α2A- and α2C -adrenoceptors results in sympathetic hyperactivity and high-bone-mass phenotype

Evidence demonstrates that sympathetic nervous system (SNS) activation causes osteopenia via β2‐adrenoceptor (β2‐AR) signaling. Here we show that female mice with chronic sympathetic hyperactivity owing to double knockout of adrenoceptors that negatively regulate norepinephrine release, α2A‐AR and α2C‐AR (α2A/α2C‐ARKO), present an unexpected and generalized phenotype of high bone mass with decreased bone resorption and increased formation. In α2A/α2C‐ARKO versus wild‐type (WT) mice, micro–computed tomographic (µCT) analysis showed increased, better connected, and more plate‐shaped trabeculae in the femur and vertebra and increased cortical thickness in the vertebra, whereas biomechanical analysis showed increased tibial and femoral strength. Tibial mRNA expression of tartrate‐resistant acid phosphatase (TRACP) and receptor activator of NF‐κB (RANK), which are osteoclast‐related factors, was lower in knockout (KO) mice. Plasma leptin and brain mRNA levels of cocaine amphetamine–regulated transcript (CART), which are factors that centrally affect bone turnover, and serum levels of estradiol were similar between mice strains. Tibial β2‐AR mRNA expression also was similar in KO and WT littermates, whereas α2A‐, α2B‐ and α2C‐AR mRNAs were detected in the tibia of WT mice and in osteoblast‐like MC3T3‐E1 cells. By immunohistochemistry, we detected α2A‐, α2B‐, α2C‐ and β2‐ARs in osteoblasts, osteoclasts, and chondrocytes of 18.5‐day‐old mouse fetuses and 35‐day‐old mice. Finally, we showed that isolated osteoclasts in culture are responsive to the selective α2‐AR agonist clonidine and to the nonspecific α‐AR antagonist phentolamine. These findings suggest that β2‐AR is not the single adrenoceptor involved in bone turnover regulation and show that α2‐AR signaling also may mediate the SNS actions in the skeleton.

S-nitrosoglutathione reductase–dependent PPARγ denitrosylation participates in MSC-derived adipogenesis and osteogenesis

Bone marrow-derived mesenchymal stem cells (MSCs) are a common precursor of both adipocytes and osteoblasts. While it is appreciated that PPARγ regulates the balance between adipogenesis and osteogenesis, the roles of additional regulators of this process remain controversial. Here, we show that MSCs isolated from mice lacking S-nitrosoglutathione reductase, a denitrosylase that regulates protein S-nitrosylation, exhibited decreased adipogenesis and increased osteoblastogenesis compared with WT MSCs. Consistent with this cellular phenotype, S-nitrosoglutathione reductase-deficient mice were smaller, with reduced fat mass and increased bone formation that was accompanied by elevated bone resorption. WT and S-nitrosoglutathione reductase-deficient MSCs exhibited equivalent PPARγ expression; however, S-nitrosylation of PPARγ was elevated in S-nitrosoglutathione reductase-deficient MSCs, diminishing binding to its downstream target fatty acid-binding protein 4 (FABP4). We further identified Cys 139 of PPARγ as an S-nitrosylation site and demonstrated that S-nitrosylation of PPARγ inhibits its transcriptional activity, suggesting a feedback regulation of PPARγ transcriptional activity by NO-mediated S-nitrosylation. Together, these results reveal that S-nitrosoglutathione reductase-dependent modification of PPARγ alters the balance between adipocyte and osteoblast differentiation and provides checkpoint regulation of the lineage bifurcation of these 2 lineages. Moreover, these findings provide pathophysiological and therapeutic insights regarding MSC participation in adipogenesis and osteogenesis.

Lack of α2C-Adrenoceptor Results in Contrasting Phenotypes of Long Bones and Vertebra and Prevents the Thyrotoxicosis-Induced Osteopenia.

A series of studies have demonstrated that activation of the sympathetic nervous system (SNS) causes osteopenia via β2-adrenoceptor (β2-AR) signaling. However, in a recent study, we found an unexpected and generalized phenotype of high bone mass in female mice with chronic sympathetic hyperactivity, due to double gene inactivation of adrenoceptors that negatively regulate norepinephrine release, α2A-and α2C-AR (α2A/2C-AR-/-). These findings suggest that β2-AR is not the single adrenoceptor involved in bone turnover regulation and show that α2-AR signaling may also mediate the SNS actions in the skeleton. In addition, we found that α2A/2C-AR-/- animals are resistant to the thyrotoxicosis-induced osteopenia, suggesting that thyroid hormone (TH), when in supraphysiological levels, interacts with the SNS to control bone mass and structure, and that this interaction may also involve α2-AR signaling. In the present study, to further investigate these hypotheses and to discriminate the roles of α2-AR subtypes, we have evaluated the bone phenotype of mice with the single gene inactivation of α2C-AR subtype, which mRNA expression was previously shown to be down regulated by triiodothyronine (T3). A cohort of 30 day-old female α2CAR-/- mice and their wild-type (WT) controls were treated with a supraphysiological dose of T3 for 30 or 90 days, which induced a thyrotoxic state in both mouse lineages. The micro-computed tomographic (μCT) analysis showed that α2C-AR-/- mice present lower trabecular bone volume (BV/TV) and number (Tb.N), and increased trabecular separation (Tb.Sp) in the femur compared with WT mice; which was accompanied by decreased bone strength (determined by the three-point bending test) in the femur and tibia. The opposite was observed in the vertebra, where α2C-AR-/- mice show increased BV/TV, Tb.N and trabecular thickness (Tb.Th), and decreased Tb.Sp, compared with WT animals. In spite of the contrasting bone phenotypes of the femur and L5, thyrotoxicosis negatively regulated most of the micro architectural features of the trabecular bone in both skeletal sites of WT, but not of α2C-AR-/- mice. T3 treatment also decreased biomechanical properties (maximum load and ultimate load) in the femur and tibia of WT, but not of knockout mice. The mRNA expression of osteocalcin, a marker of mature osteoblasts, and tartrate-resistant acid phosphatase, which is expressed by osteoclasts and is involved in collagen degradation, was increased by T3 treatment only in WT, and not in α2C-AR-/- mice. Altogether, these findings suggest that α2C-AR subtype mediates the effects of the SNS in the bone in a skeletal site-dependent manner, and that thyrotoxicosis depends on α2C-AR signaling to promote bone loss, which sustains the hypothesis of a TH-SNS interaction to modulate bone remodeling and structure.

Thyroid hormone interacts with the sympathetic nervous system to modulate bone mass and structure in young adult mice

To investigate whether thyroid hormone (TH) interacts with the sympathetic nervous system (SNS) to modulate bone mass and structure, we studied the effects of daily T3 treatment in a supraphysiological dose for 12 wk on the bone of young adult mice with chronic sympathetic hyperactivity owing to double-gene disruption of adrenoceptors that negatively regulate norepinephrine release, α(2A)-AR, and α(2C)-AR (α(2A/2C)-AR(-/-) mice). As expected, T3 treatment caused a generalized decrease in the areal bone mineral density (aBMD) of WT mice (determined by DEXA), followed by deleterious effects on the trabecular and cortical bone microstructural parameters (determined by μCT) of the femur and vertebra and on the biomechanical properties (maximum load, ultimate load, and stiffness) of the femur. Surprisingly, α(2A/2C)-AR(-/-) mice were resistant to most of these T3-induced negative effects. Interestingly, the mRNA expression of osteoprotegerin, a protein that limits osteoclast activity, was upregulated and downregulated by T3 in the bone of α(2A/2C)-AR(-/-) and WT mice, respectively. β1-AR mRNA expression and IGF-I serum levels, which exert bone anabolic effects, were increased by T3 treatment only in α(2A/2C)-AR(-/-) mice. As expected, T3 inhibited the cell growth of calvaria-derived osteoblasts isolated from WT mice, but this effect was abolished or reverted in cells isolated from KO mice. Collectively, these findings support the hypothesis of a TH-SNS interaction to control bone mass and structure of young adult mice and suggests that this interaction may involve α2-AR signaling. Finally, the present findings offer new insights into the mechanisms through which TH regulates bone mass, structure, and physiology.

Potassium distribution applied through vinasse in oxisol and red nitosol.

No solo, o potassio e movel e, portanto, sujeito a perdas por lixiviacao. Rica em potassio, a vinhaca e geralmente usada como fertilizante, especialmente nos canaviais. Mas o uso excessivo desta substância pode contaminar rios e lencois freaticos pelo excesso de potassio, se a aplicacao nao for manejada adequadamente. Portanto, objetivou-se, com o presente trabalho, avaliar o deslocamento do potassio em colunas segmentadas com dois tipos de solo nao saturado sob aplicacao da vinhaca. Foram confeccionadas colunas de acrilico com dimensoes de 75 cm de altura por 5 cm de diâmetro, preenchidas com dois tipos de solo, arenoso (Latossolo Vermelho) e argiloso (Nitossolo). Os resultados mostraram que a percolacao do K + esta diretamente relacionada com o tipo de solo, interagindo-se mais com a fracao solida do solo argiloso quando comparado com o solo arenoso. No solo argiloso houve maior concentracao de potassio nas primeiras camadas de solo, ao contrario do solo arenoso onde a distribuicao foi mais homogenea entre as camadas.

Global Disruption of α2A Adrenoceptor Barely Affects Bone Tissue but Minimizes the Detrimental Effects of Thyrotoxicosis on Cortical Bone

Evidence shows that sympathetic nervous system (SNS) activation inhibits bone formation and activates bone resorption leading to bone loss. Because thyroid hormone (TH) interacts with the SNS to control several physiological processes, we raised the hypothesis that this interaction also controls bone remodeling. We have previously shown that mice with double-gene inactivation of α2A- and -adrenoceptors (α2A/2C-AR−/−) present high bone mass (HBM) phenotype and resistance to thyrotoxicosis-induced osteopenia, which supports a TH-SNS interaction to control bone mass and suggests that it involves α2-AR signaling. Accordingly, we detected expression of α2A-AR, α2B-AR and α2C-AR in the skeleton, and that triiodothyronine (T3) modulates α2C-AR mRNA expression in the bone. Later, we found that mice with single-gene inactivation of α2C-AR (α2C-AR−/−) present low bone mass in the femur and HBM in the vertebra, but that both skeletal sites are resistant to TH-induce osteopenia, showing that the SNS actions occur in a skeletal site-dependent manner, and that thyrotoxicosis depends on α2C-AR signaling to promote bone loss. To further dissect the specific roles of α2-AR subtypes, in this study, we evaluated the skeletal phenotype of mice with single-gene inactivation of α2A-AR (α2A-AR−/−), and the effect of daily treatment with a supraphysiological dose of T3, for 4 or 12 weeks, on bone microarchitecture and bone resistance to fracture. Micro-computed tomographic (μCT) analysis revealed normal trabecular and cortical bone structure in the femur and vertebra of euthyroid α2A-AR−/− mice. Thyrotoxicosis was more detrimental to femoral trabecular bone in α2A-AR−/− than in WT mice, whereas this bone compartment had been previously shown to present resistance to thyrotoxicosis in α2C-AR−/− mice. Altogether these findings reveal that TH excess depends on α2C-AR signaling to negatively affect femoral trabecular bone. In contrast, thyrotoxicosis was more deleterious to femoral and vertebral cortical bone in WT than in α2A-AR−/− mice, suggesting that α2A-AR signaling contributes to TH actions on cortical bone. These findings further support a TH-SNS interaction to control bone physiology, and suggest that α2A-AR and α2C-AR signaling pathways have key roles in the mechanisms through which thyrotoxicosis promotes its detrimental effects on bone remodeling, structure and resistance to fracture.

Drippers flow disturbances due to application of CO2 for irrigation water.

O presente trabalho avaliou a dinâmica do uso de CO 2 em gotejadores autocompensantes, com emissores posicionados tanto para baixo quanto para cima, observando a ocorrencia de histerese para curvas de vazao em funcao da pressao crescente e decrescente em diferentes modelos para diferentes concentracoes de CO 2 . O experimento foi conduzido em uma bancada de ensaios em estrutura metalica instalada no Laboratorio de Irrigacao do DER-ESALQ/USP, localizada no municipio de Piracicaba – SP. Utilizaram-se cinco modelos de gotejadores (M1 a M5) que foram expostos aos seguintes tratamentos: a) pressoes: 50, 100, 150, 200, 250, 300, 350 e 400 kPa; b) posicao dos emissores: para baixo e para cima; c) concentracao de CO 2 : zero, 50, 100, 200 e 400% da dissolucao maxima de CO 2 em agua. Foram observados altos valores de coeficiente de variacao de vazao quando se aplicou a concentracao 400% da dissolucao de CO 2 em agua.