Effect of long-term L-thyroxine treatment on the activity of NO-synthases in tissues of rats with obesity induced by high-fat diet

Obesity, a metabolic syndrome (MS) component, disturbs macro- and microcirculation largely due to the attenuation of NO-dependent cascades leading to pathology of the cardiovascular system. Among the activators of NO-synthases (NOS), the enzymes catalyzing NO synthesis, are thyroid hormones. Since obesity and MS are characterized by reduced functions of the thyroid gland, the replacement therapy with thyroid hormones exhibiting vasodilator properties is one of the approaches to functional recovery of the cardiovascular system. However, there is no information so far about the effect of thyroid hormones on NOS activity in obesity. The aim of this work was to study the effect of 4-week treatment of rats with high-fat diet induced obesity with L-thyroxine (20 μg/kg daily) on functional activity of total NOS as well as its endothelial (eNOS) and neuronal (nNOS) isoforms in the brain, myocardium and skeletal muscles. Obese rats were found to exhibit the reduced level of thyroid hormones, impaired glucose tolerance and dyslipidemia. In the myocardium and skeletal muscles of obese rats, total NOS and eNOS activities were considerably reduces, while in the brain they did not alter very much. Long-term treatment of obese rats with L-thyroxine led to a substantial increase in total NOS and eNOS activities in the myocardium and skeletal muscles as well as to an increase in total NOS and nNOS activities in the brain with enzyme activities exceeding those in control. In healthy rats treated with L-thyroxine, total NOS and eNOS activities in the myocardium and skeletal muscles as well as total NOS in the brain were also substantially increased. The inducible NOS isoform (iNOS) was found to contribute significantly to an increase in total NOS activity both in obese rats and healthy rats treated with L-thyroxine; its activity was determined by calculation. Thus, 4-week L-thyroxine treatment of obese rats deficient in thyroid hormones led to a recovery of total NOS and eNOS activities in the myocardium and skeletal muscles reduced in obesity. This suggests a promising future for thyroxine therapy of vascular pathology in obesity and MS.     

Mammary radioiodine accumulation due to functional sodium iodide symporter expression in a benign fibroadenoma

The sodium iodide symporter (NIS) has been characterized to mediate the active transport of iodide not only in the thyroid gland but also in various non-thyroidal tissues, including lactating mammary gland and the majority of breast cancers, thereby offering the possibility of diagnostic and therapeutic radioiodine application in breast cancer. In this report, we present a 57-year-old patient with multifocal papillary thyroid carcinoma, who showed focal radioiodine accumulation in a lesion in the right breast on a posttherapy (131)I scan following radioiodine therapy. CT and MR-mammography showed a focal solid lesion in the right breast suggestive of a fibroadenoma, which was confirmed by histological examination. Immunostaining of paraffin-embedded tumor tissue sections using a human NIS antibody demonstrated NIS-specific immunoreactivity confined to epithelial cells of mammary ducts. In conclusion, in a thyroid cancer patient we identified a benign fibroadenoma of the breast expressing high levels of functionally active NIS protein as underlying cause of focal mammary radioiodine accumulation on a posttherapy (131)I scan. These data show for the first time that functional NIS expression is not restricted to lactating mammary gland and malignant breast tissue, but can also be detected in benign breast lesions, such as fibroadenomata of the breast.     

Signaling cascade that mediates endothelial nitric oxide synthase activation induced by atrial natriuretic peptide

Atrial natriuretic peptide (ANP) induces activation of nitric oxide-synthase (NOS). Aims: to identify the isoform of NOS involved in ANP effects, to study whether ANP modifies NOS expression and to investigate the signaling pathways and receptors involved in NOS stimulation. NOS activation induced by ANP would be mediated by endothelial NOS (eNOS) since neuronal or inducible NOS inhibition did not alter ANP effect. The peptide induced no changes in eNOS protein expression. NOS activity stimulated by ANP, in the kidney, aorta and left ventricle, was partially abolished by the NPR-A/B antagonist, as well as PKG inhibition, but no difference in atria was observed. 8-Br-cGMP partially mimicked the effect of ANP on NOS in all tissues. NOS stimulation by ANP in atria disappeared when G protein was inhibited, but this effect was partial in the other tissues. Calmodulin antagonist abolished NOS stimulation via ANP. Inhibition of the PLC, PKC or PI3 kinase/Akt pathway failed to alter NOS activation induced by ANP. ANP would activate eNOS in the aorta, heart and kidney without modifying the expression of the enzyme. ANP would interact with NPR-C coupled via G proteins leading to the activation of Ca(2+)-calmodulin-dependent NOS in atria; while in ventricle, aorta and kidney, ANP could also interact with NPR-A/B, increasing cGMP, which in turns activates PKG to stimulate eNOS.     

Early and Late Toxicity of Radioiodine Therapy: Detection and Management

ObjectiveTo review the early and late toxicity associated with radioiodine (¹³¹I) therapy, highlighting the need for early detection and, where possible, preventive measures.MethodsWe performed a literature search on MEDLINE using the terms radioiodine, ¹³¹I toxicity, complications, Graves disease, and thyroid cancer and chose the most relevant studies for this review. Where appropriate, we refer to our own published series of patients as well.ResultsUptake of ¹³¹I into the salivary glands, lacrimal glands, fetal thyroid, and adult thyroid accounts for the early toxic effects of radioiodine therapy. Delayed radiation effects to the gonads, bone marrow, and cell nuclei give rise to late complications. Toxicity may also arise from uptake into metastatic tumors located at vulnerable sites, including the spinal cord, brain, and lungs.ConclusionAlthough radioiodine therapy for benign and malignant thyroid disorders is usually well tolerated, clinicians involved in the management of thyroid disorders need to be aware of the potential toxicity of radioiodine and take all measures to reduce these effects to a minimum. (Endocr Pract. 2010;16:1064-1070)     

Nitric oxide-dependent penile erection in mice lacking neuronal nitric oxide synthase.

BACKGROUND: Nitric oxide (NO) has been implicated as a mediator of penile erection, because the neuronal isoform of NO synthase (NOS) is localized to the penile innervation and NOS inhibitors selectively block erections. NO can also be formed by two other NOS isoforms derived from distinct genes, inducible NOS (iNOS) and endothelial NOS (eNOS). To clarify the source of NO in penile function, we have examined mice with targeted deletion of the nNOS gene (nNOS- mice). MATERIALS AND METHODS: Mating behavior, electrophysiologically induced penile erection, isolated erectile tissue isometric tension, and eNOS localization by immunohistochemistry and Western blot were performed on nNOS- mice and wild-type controls. RESULTS: Both intact animal penile erections and isolated erectile tissue function are maintained in nNOS mice, in agreement with demonstrated normal sexual behaviors, but is stereospecifically blocked by the NOS inhibitor, L-nitroarginine methyl ester (L-NAME). eNOS is abundantly present in endothelium of penile vasculature and sinusoidal endothelium within the corpora cavemosa, with levels that are significantly higher in nNOS- mice than in wild-type controls. CONCLUSIONS: eNOS mediates NO-dependent penile erection in nNOS- animals and normal penile erection. These data clarify the role of nitric oxide in penile erection and may have implications for therapeutic agents with selective effects on NOS isoforms.     

A new method to evaluate the residual activity in patients undergoing 131I thyroid therapy

The radioiodine administration is a standard therapeutic approach to both benign thyroid diseases, such as hyperthyroidism, and carcinomas. The high administered ¹³¹I activities are of radiation protection concern, due to relevant patient residual contamination. The aim of this work was to develop a new procedure based on external radiometric surveys and on a mathematical model in order to estimate the ¹³¹I activity in patients undergoing hyperthyroidism radioiodine therapy.In the first stage of this study, a suitable detector was chosen and its response vs. activity was characterized. The experimental verification was performed measuring the ambient dose equivalent rate from patients receiving radioiodine administration. The results confirm the reliability of the proposed method, as shown by the slight differences between the administered activities and the ones calculated from external measurements. Furthermore, the same procedure was applied to detect the percentage residual activity in patients at two preset time intervals: 4 hours and 4 days after the radioiodine administration. The obtained results clearly highlight that the method can ensure a level of reliability compatible with the radiation protection purposes.     

Optimization of 131I ablation in patients with differentiated thyroid carcinoma: comparison of early outcomes of treatment with 100 mCi versus 60 mCi

INTRODUCTION: The aim of this study was to compare the early outcomes between two groups of patients with differentiated thyroid carcinoma (DTC) who received 60 or 100 mCi of (131)I for remnant ablation. MATERIAL AND METHODS: 224 DTC patients with primary tumor > 1 cm of diameter or multifocal were randomised into prospective clinical trial. Patients with extrathyroideal extension of primary tumor and nodal metastases or M1 were not enrolled. 99 patients received 60 mCi, and 125--100 mCi of radioiodine as the first ablative dose. RESULTS: The effectiveness of thyroid ablation was evaluated after one year, during endogenous TSH (thyroid stimulating hormone) stimulation, and after two years during Lthyroxine therapy. Whole body scintigraphy (WBS) was performed under thyroxine withdrawal and thyroglobulin serum level was assessed. Distant micrometastases were detected in 9.8% of patients by post-therapy WBS, 11 patients in group A treated with 60 mCi and 11 in group B treated with 100 mCi. In other patients no symptoms of persistent disease were detected. At one year follow up full remission was diagnosed in 176 patients: 76 in group A and 100 in group B. The remaining ones, 13.3% and 11.2% respectively, received the second course of (131)I for remnant ablation. There were no statistically significant differences in Tg (thyroglobulin) serum level either 12 or 24 months after 131I treatment. CONCLUSIONS: Our evaluation of early efficacy of adjuvant radioiodine treatment in low risk DTC patients shows no differences between two radioiodine activities - 60 and 100 mCi in relation to thyroid ablation. Thus, the activity of 60 mCi is recommended.     

Roles of nitric oxide in brain hypoxia-ischemia.

A large body of evidence has appeared over the last 6 years suggesting that nitric oxide biosynthesis is a key factor in the pathophysiological response of the brain to hypoxia-ischemia. Whilst studies on the influence of nitric oxide in this phenomenon initially offered conflicting conclusions, the use of better biochemical tools, such as selective inhibition of nitric oxide synthase (NOS) isoforms or transgenic animals, is progressively clarifying the precise role of nitric oxide in brain ischemia. Brain ischemia triggers a cascade of events, possibly mediated by excitatory amino acids, yielding the activation of the Ca2+-dependent NOS isoforms, i.e. neuronal NOS (nNOS) and endothelial NOS (eNOS). However, whereas the selective inhibition of nNOS is neuroprotective, selective inhibition of eNOS is neurotoxic. Furthermore, mainly in glial cells, delayed ischemia or reperfusion after an ischemic episode induces the expression of Ca2+-independent inducible NOS (iNOS), and its selective inhibition is neuroprotective. In conclusion, it appears that activation of nNOS or induction of iNOS mediates ischemic brain damage, possibly by mitochondrial dysfunction and energy depletion. However, there is a simultaneous compensatory response through eNOS activation within the endothelium of blood vessels, which mediates vasodilation and hence increases blood flow to the damaged brain area.     

Trends in thyroid imaging

The master choice for thyroid imaging remains the radioiodine thyroid scan with 123I or 99mTc. 131I has to be reserved for the follow-up of thyroid cancer only. Ultrasound may remain useful in certain situations but it does not integrate as it is the case with the radioisotopic scan together with morphological and functional information.     

Influence of initial large dose on subsequent uptake of therapeutic radioiodine in thyroid cancer patients

Fifty-two patients with differentiated thyroid cancer, following thyroidectomy were studied by administering a quantity of up to 5 mCi of [¹³¹I]sodium iodide. In most of these patients, radioiodine uptake values obtained with the subsequent therapeutic dose were markedly lower than those observed with the initial doses. This observation was verified in seven of the patients with differentiated thyroid cancer, by measuring the radioiodine uptake with a second dose of 4.5 mCi of [¹³¹I]sodium iodide. Calculations showed that the major etiology was probably therapeutic irradiation of the thyroid by the first dose.     

NO and NOS isoforms in the development of apoptosis in renal ischemia/reperfusion

Nitric oxide (NO) and the expression of endothelial (eNOS) and inducible (iNOS) isoforms of nitric oxide synthase (NOS) are recognized as important mediators of physiological and pathological processes of renal ischemia/reperfusion (I/R) injury, but little is known about their role in apoptosis. The ability of the eNOS/NO system to regulate the iNOS/NO system and thus promote apoptosis was assessed during experimental renal I/R. Renal caspase-3 activity and the number of TUNEL-positive cells increased with I/R, but decreased when NOS/NO systems were blocked with L-NIO (eNOS), 1400W (iNOS), and N-nitro-l-arginine methyl ester (L-NAME; a nonselective NOS inhibitor). I/R increased renal eNOS and iNOS expression as well as NO production. The NO increase was eNOS- and iNOS-dependent. Blockage of NOS/NO systems with L-NIO or L-NAME also resulted in a lower renal expression of iNOS and iNOS mRNA; in contrast, eNOS expression was not affected by iNOS-specific blockage. In conclusion, two pathways define the role of NOS/NO systems in the development of apoptosis during experimental renal I/R: a direct route, through eNOS overexpression and NO production, and an indirect route, through expression/activation of the iNOS/NO system, induced by eNOS.     

The treatment of multinodular large non-toxic goiter using repeated doses of radioiodine (preliminary report)

INTRODUCTION: The aim of study was to establish the effectiveness of radioiodine therapy using 131I in the group of patients with multinodular large non-toxic goiter. MATERIAL AND METHODS: Therapy was undertaken in female patients disqualified from surgery due to high risk and these patients who didn't agree to surgery. Studies were performed in 7 women (age range: 62-82 yrs) with large goiters (2nd degree according to WHO classification and goiter volume assessed by USG over 100 cm(3)). Serum TSH, fT4, fT3, antithyroid antibodies (TPOAb, TgAb, TRAb) levels, urinary iodine concentration (UIE) were estimated in all patients parallel with radioiodine uptake test (after 5 and 24 hours), 131I thyroid scintigraphy and fine needle biopsy to exclude neoplasmatic transformation. These studies and therapy with 22 mCi 131I were repeated every 3 months. RESULTS: Before therapy median thyroid volume was approximately 145 cm(3) and during therapy gradually decreased to 76 cm(3) after 6 months and to 65 cm(3) after 12 months. Increase of TRAb can be a inhibiting factor of thyroid volume reduction. Other antithyroid antibodies showed marked tendency to rise but without significant correlation with radioiodine uptake and goiter reduction. After 12 months we found 2 patients with clinical and laboratory hypothyroidism. CONCLUSIONS: In some cases of multinodular large non-toxic goiter, the radioiodine therapy can be the best alternative way for L-thyroxine treatment or surgery therapy. The fractionated radioiodine therapy of multinodular large non-toxic goiter is safe and effective method but continuation of nodules observation is necessary.     

Malignant struma ovarii: report of a case and review of the literature

Malignant struma ovarii is a very rare disease and therefore there is neither common agreement on treatment regimens nor sufficient follow-up experience. The case of a 38-year-old woman with malignant struma ovarii is described. The patient presented with a clinically silent ovarian neoplasm discovered incidentally during investigations for metrorrhagia. The ovarian mass was resected and the tumor was found to be a teratoma. Within the teratoma a papillary thyroid carcinoma of the follicular variant was found. Subsequently, in order to make follow-up of the patient possible by thyroglobulin measurement and radioiodine whole body scintigraphy, near-total thyroidectomy was performed and the thyroid was found to be normal on histology. Whole body radioiodine scintigraphy with (131)I and ablation of the thyroid remnant by the administration of 80 mCi (131)I was performed. The patient recovered uneventfully and is now well.     

Endothelial and inducible nitric oxide synthases in oocytes of cattle

Nitric oxide (NO) is a chemical messenger generated by the activity of the nitric oxide synthases (NOS). The NOS/NO system appears to be involved in oocyte maturation, but there are few studies on gene expression and protein activity in oocytes of cattle. The present study aimed to investigate gene expression and protein activity of NOS in immature and in vitro matured oocytes of cattle. The influence of pre-maturation culture with butyrolactone I in NOS gene expression was also assessed. The following experiments were performed: (1) detection of the endothelial (eNOS) and inducible (iNOS) isoforms in the ovary by immunohistochemistry; (2) detection of eNOS and iNOS in the oocytes before and after in vitro maturation (IVM) by immunofluorescence; (3) eNOS and iNOS mRNA and protein in immature and in vitro matured oocytes, with or without pre-maturation, by real time PCR and Western blotting, respectively; and (4) NOS activity in immature and in vitro matured oocytes by NADPH-diaphorase. eNOS and iNOS were detected in oocytes within all follicle categories (primary, secondary and tertiary), and other compartments of the ovary and in the cytoplasm of immature and in vitro matured oocytes. Amount of mRNA for both isoforms decreased after IVM, but was maintained after pre-maturation culture. The NOS protein was detected in immature (pre-mature or not) and was still detected in similar amount after pre-maturation and maturation for both isoforms. NOS activity was detected only in part of the immature oocytes. In conclusion, isoforms of NOS (eNOS and iNOS) are present in oocytes of cattle from early folliculogenesis up to maturation; in vitro maturation influences amount of mRNA and NOS activity.     

Expression of nitric oxide synthase isoforms is reduced in late-gestation ovine fetal brainstem

Fetal baroreflex responsiveness increases in late gestation. An important modulator of baroreflex activity is the generation of nitric oxide in the brainstem nuclei that integrate afferent and efferent reflex activity. The present study was designed to test the hypothesis that nitric oxide synthase (NOS) isoforms are expressed in the fetal brainstem and that the expression of one or more of these enzymes is reduced in late gestation. Brainstem tissue was rapidly collected from fetal sheep of known gestational ages (80, 100, 120, 130, 145 days gestation and 1 day and 1 wk postnatal). Neuronal (nNOS), inducible (iNOS), and endothelial (eNOS) mRNA was measured using real-time PCR methodology specific for ovine NOS isoforms. The three enzymes were measured at the protein level using Western blot methodology. In tissue prepared for histology separately, the cellular pattern of immunostaining was identified in medullae from late-gestation fetal sheep. Fetal brainstem contained mRNA and protein of all three NOS isoforms, with nNOS the most abundant, followed by iNOS and eNOS, respectively. nNOS and iNOS mRNA abundances were highest at 80 days' gestation, with statistically significant decreases in abundance in more mature fetuses and postnatal animals. nNOS and eNOS protein abundance also decreased as a function of developmental age. nNOS and eNOS were expressed in neurons, iNOS was expressed in glia, and eNOS was expressed in vascular endothelial cells. We conclude that all three isoforms of NOS are constitutively expressed within the fetal brainstem, and the expression of all three forms is reduced with advancing gestation. We speculate that the reduced expression of NOS in this brain region plays a role in the increased fetal baroreflex activity in late gestation.     

Lack of nitric oxide synthase depresses ion transporting enzyme function in cardiac muscle

Nitric oxide (NO*) is produced endogenously from NOS isoforms bound to sarcolemmal (SL) and sarcoplasmic reticulum (SR) membranes. To investigate whether locally generated NO* directly affects the activity of enzymes mediating ion active transport, we studied whether knockout of selected NOS isoforms would affect the functions of cardiac SL (Na+ + K+)-ATPase and SR Ca2+-ATPase. Cardiac SL and SR vesicles containing either SL (Na+ + K+)-ATPase or SR Ca2+-ATPase were isolated from mice lacking either nNOS or eNOS, or both, and tested for enzyme activities. Western blot analysis revealed that absence of single or double NOS isoforms did not interrupt the protein expression of SL (Na+ + K+)-ATPase and SR Ca2+-ATPase in cardiac muscle cells. However, lack of NOS isoforms in cardiac muscle significantly altered both (Na+ + K+)-ATPase activity and SR Ca2+-ATPase function. Our experimental results suggest that disrupted endogenous NO* production may change local redox conditions and lead to an unbalanced free radical homeostasis in cardiac muscle cells which, in turn, may affect key enzyme activities and membrane ion active transport systems in the heart.     

Intermittent pneumatic compression regulates expression of nitric oxide synthases in skeletal muscles

This study investigated the effects of intermittent pneumatic compression (IPC) on expression of nitric oxide synthase (NOS) isoforms in compressed (anterior tibialis, AT) and uncompressed (cremaster muscles, CM) skeletal muscles. Following IPC application of 0.5, 1, and 5h on both legs of rats, the endothelial NOS (eNOS) mRNA expression was significantly up-regulated to 1.2-, 1.8, and 2.7-fold from normal, respectively, in both AT and CM, and protein expression increased more than 1.5-fold of normal at each time point. Similarly, neuronal NOS expression was up-regulated, but to a lesser degree. In contrast, inducible NOS expression was significantly and time-dependently down-regulated in both muscles. After IPC cessation, eNOS levels returned to normal in both AT and CM. The results confirm our hypothesis that IPC-induced vasodilation is mediated by regulating expression of NOS isoforms, in particular eNOS, in both compressed and uncompressed skeletal muscles. The results also suggest the importance of precisely characterizing expression of each NOS isoform in tissue pathophysiology.     

Mitochondrial nitric oxide synthase is not eNOS, nNOS or iNOS

Recent studies indicated that there is a distinct mitochondrial nitric oxide synthase (mtNOS) enzyme, which may be identical to the other known NOS isoforms. We investigated the possible involvement of the endothelial, the neuronal, and the inducible NOS isoforms (eNOS, nNOS, iNOS, respectively) in mitochondrial NO production. Mouse liver mitochondria were prepared by Percoll gradient purification from wild-type and NOS knockout animals. NOS activity was measured by the arginine conversion assay, NO production of live mitochondria was visualized by the fluorescent probe DAF-FM with confocal microscopy and measured with flow cytometry. Western blotting or immunoprecipitation was performed with 12 different anti-NOS antibodies. Mitochondrial NOS was purified by arginine, 2,5 ADP and calmodulin affinity columns. We observed NO production and NOS activity in mitochondria, which was not attenuated by classic NOS inhibitors. We also detected low amounts of eNOS protein in the mitochondria, however, NO production and NOS activity were intact in eNOS knockout animals. Neither nNOS nor iNOS were present in the mitochondria. Furthermore, we could not find mitochondrial targeting signals in the sequences of either NOS proteins. Taken together, the presented data do not support the hypothesis that any of the known NOS enzymes are present in the mitochondria in physiologically relevant levels.     

Autoinhibition of endothelial nitric-oxide synthase. Identification of an electron transfer control element.

The primary sequences of the three mammalian nitric- oxide synthase (NOS) isoforms differ by the insertion of a 52-55-amino acid loop into the reductase domains of the endothelial (eNOS) and neuronal (nNOS), but not inducible (iNOS). On the basis of studies of peptide derivatives as inhibitors of.NO formation and calmodulin (CaM) binding (Salerno, J. C., Harris, D. E., Irizarry, K., Patel, B., Morales, A. J., Smith, S. M., Martasek, P., Roman, L. J., Masters, B. S., Jones, C. L., Weissman, B. A., Lane, P., Liu, Q., and Gross, S. S. (1997) J. Biol. Chem. 272, 29769-29777), the insert has been proposed to be an autoinhibitory element. We have examined the role of the insert in its native protein context by deleting the insert from both wild-type eNOS and from chimeras obtained by swapping the reductase domains of the three NOS isoforms. The Ca2+ concentrations required to activate the enzymes decrease significantly when the insert is deleted, consistent with suppression of autoinhibition. Furthermore, removal of the insert greatly enhances the maximal activity of wild-type eNOS, the least active of the three isoforms. Despite the correlation between reductase and overall enzymatic activity for the wild-type and chimeric NOS proteins, the loop-free eNOS still requires CaM to synthesize.NO. However, the reductive activity of the CaM-free, loop-deleted eNOS is enhanced significantly over that of CaM-free wild-type eNOS and approaches the same level as that of CaM-bound wild-type eNOS. Thus, the inhibitory effect of the loop on both the eNOS reductase and. NO-synthesizing activities may have an origin distinct from the loop's inhibitory effects on the binding of CaM and the concomitant activation of the reductase and.NO-synthesizing activities. The eNOS insert not only inhibits activation of the enzyme by CaM but also contributes to the relatively low overall activity of this NOS isoform.