Evaluation of the role of calcitonin deficiency in ovariectomy-induced osteopenia

Studies were carried out in rats to examine the role of calcitonin deficiency in the pathogenesis of ovariectomy-induced osteopenia. The parathyroid glands of 80 female Wistar rats were autotransplanted to their thigh muscle and the animals divided into 4 groups. Group 1 rats were sham ovariectomized, and thyroidectomized to make them calcitonin deficient; Group 2 rats were thyroidectomized, and ovariectomized to make them deficient in ovarian hormones as well; Group 3 rats were sham thyroidectomized and sham ovariectomized, and Group 4 rats were sham thyroidectomized and ovariectomized. A fifth group of rats were unoperated upon and served as controls. Thyroidectomized animals were maintained on thyroxine replacement and 11 months after ovariectomy all the animals were bled, killed and their femurs dissected out. In both the thyroid intact and thyroidectomized animals, ovariectomy decreased femur density significantly (P less than 0.01). Similarly, ovariectomy resulted in a decrease in femur calcium (P less than 0.01) in both groups of animals, and in a significant decrease in serum calcitonin (P less than 0.05) in the thyroid intact animals. We conclude from these findings that ovarian hormone deficiency can cause bone loss independently of lowering circulating calcitonin levels.     

Simvastatin did not prevent nor restore ovariectomy-induced bone loss in adult rats

Current published results on whether statins have beneficial effects on bone metabolism have been conflicting so far. In order to further investigate if statins were promising candidates for the treatment for osteoporosis, we conducted a study in which rats were ovariectomized (OVX) at 6 months of age, allowed to lose bone for 60 days and followed by oral administration of simvastatin at the dose levels of 0.3-10 mg/kg/d for 60 days. PGE2 (6 mg/kg) was used as a positive control. Study endpoints included bone histomorphometry on the proximal tibial metaphysis (PTM) and the tibial diaphysis (TX), dual-energy X-ray absorptiometry on the right femur and micro computed tomography (ICT) on the 5th lumbar vertebra (LV). After 120 days of OVX, cancellous bone lost by 80% in the PTM and 18% in the LV accompanied by increased bone formation and resorption. Simvastatin at all dose levels did not affect bone volume, bone formation rate and bone erosion surface when compared to 120 day ovariectomized animals at all bone sites studied. By contrast, PGE2 restored cancellous and cortical bone area to sham control levels. In conclusion, this study demonstrated that unlike PGE2, oral administration of simvastatin did not have effects on cancellous or cortical bone formation and resorption; and consequently was not able to prevent further bone loss or restore bone mass in the osteopenic, OVX rats.     

Asymmetric dimethylarginine, an endogenous NOS inhibitor, is actively metabolized in rat erythrocytes

N(G), N(G)-Dimethyl-L-arginine (asymmetric dimethylarginine: ADMA) is an endogenous competitive inhibitor of nitric oxide synthase (NOS). Plasma ADMA concentrations have been reported to increase in connection with diseases associated with an impaired endothelial L-arginine/NO pathway. In this study, we investigated the metabolism of ADMA in circulating blood cell populations to elucidate the regulatory mechanism of elevation of plasma ADMA, a novel risk factor for cardiovascular disease. We found by RT-PCR and Western blot analyses that protein arginine methyltransferase (PRMT)1 and dimethylarginine dimethylaminohydrolase (DDAH)-1, responsible for the biosynthesis and degradation of ADMA respectively, are expressed in erythrocytes (ECs), leukocytes, and platelets. We also identified a major ADMA-containing protein in ECs as catalase, confirmed by GST-pull down assay to bind to PRMT1 in vitro. This is the first report that the ADMA-metabolizing system, including the arginine methylation of proteins and the breakdown of free ADMA, occurs in circulating blood cell-populations, and that catalase in ECs might be a potential protein targeted by PRMT1.     

Prune suppresses ovariectomy-induced hypercholesterolemia in rats

Elevated cholesterol among women who have experienced natural or surgical menopause has been linked to ovarian hormone deficiency. The purpose of this study was to investigate the efficacy of prune, a good source of dietary fiber and phytochemicals, on lowering cholesterol in an ovariectomized (ovx) rat model. Forty-eight 90-day-old female Sprague-Dawley rats were randomly assigned to four groups: sham-operated (sham) + control diet, ovx + control diet, ovx + low-dose (LD; 5%) prune, and ovx + high-dose (HD; 25%) prune. After 45 days of treatment, rats were euthanized and tissues were collected for analyses. Ovariectomy elevated serum total cholesterol by 22% compared with sham, and HD prune diet prevented this increase without affecting high density lipoprotein cholesterol concentrations. Animals fed the HD prune diet had 13% lower liver total lipids compared with ovx animals. The findings of this study showed that prune exhibits hypocholesterolemic properties in ovarian hormone deficiency. Dose-response studies should be conducted to establish the effectiveness of prune in prevention of hypercholesterolemia in postmenopausal women who are not on estrogen replacement therapy and seek dietary alternatives. Mechanistic studies also are needed to establish its mode of action.     

Asymmetric dimethylarginine in children with homocystinuria or phenylketonuria

Plasma concentration of asymmetric dimethylarginine (ADMA), an endogenous inhibitor of nitric oxide (NO) synthesis from l-arginine and a cardiovascular risk factor, was found to be elevated in plasma of homocysteinemic adults. Enhanced cardiovascular risk due to homocystinuria and impaired renal function has been found in patients with phenylketonuria (PKU) on protein-restricted diet. However, it is still unknown whether ADMA synthesis is also elevated in children with homocystinuria due to cystathionine beta-synthase deficiency (classical homocystinuria), and whether ADMA may play a role in phenylketonuria in childhood. In the present study, we investigated the status of the l-arginine/NO pathway in six young patients with homocystinuria, in 52 young phenylketonuria patients on natural protein-restricted diet, and in age- and gender-matched healthy children serving as controls. ADMA in plasma and urine was determined by GC–MS/MS. The NO metabolites nitrate and nitrite in plasma and urine, and urinary dimethylamine (DMA), the dimethylarginine dimethylaminohydrolase (DDAH) metabolite of ADMA, were measured by GC–MS. Unlike urine ADMA excretion, plasma ADMA concentration in patients with homocystinuria was significantly higher than in controls (660 ± 158 vs. 475 ± 77 nM, P = 0.035). DMA excretion rate was considerably higher in children with homocystinuria as compared to controls (62.2 ± 24.5 vs. 6.5 ± 2.9 μmol/mmol creatinine, P = 0.068), indicating enhanced DDAH activity in this disease. In contrast and unexpectedly, phenylketonuria patients had significantly lower ADMA plasma concentrations compared to controls (512 ± 136 vs. 585 ± 125 nM, P = 0.009). Phenylketonuria patients and controls had similar l-arginine/ADMA molar ratios in plasma. Urinary nitrite excretion was significantly higher in phenylketonuria as compared to healthy controls (1.7 ± 1.7 vs. 0.7 ± 1.2 μmol/mmol creatinine, P = 0.003). Our study shows that the l-arginine/NO pathway is differently altered in children with phenylketonuria and homocystinuria. Analogous to hyperhomocysteinemic adults, elevated ADMA plasma concentrations could be a cardiovascular risk factor in children with homocystinuria. In phenylketonuria, the l-arginine/NO pathway seems not be altered. Delineation of the role of ADMA in childhood phenylketonuria and homocystinuria demands further investigation.     

Reduction of asymmetric dimethylarginine involved in the cardioprotective effect of losartan in spontaneously hypertensive rats

Previous studies have indicated that nitric oxide synthase (NOS) inhibitors can induce an increase of blood pressure and exacerbate myocardial injury induced by ischemia and reperfusion, whereas angiotensin II receptor antagonists protect the myocardium against injury induced by ischemia and reperfusion. Isolated hearts from male spontaneously hypertensive rats (SHR) or male Wistar-Kyoto rats (WKY) were subjected to 20 min global ischemia and 30 min reperfusion. Heart rate, coronary flow, left ventricular pressure, and its first derivatives (+/-dP/dt(max)) were recorded, and serum concentrations of asymmetric dimethylarginine (ADMA) and NO and the release of creatine kinase in coronary effluent were measured. The level of ADMA was significantly increased and the concentration of NO was decreased in SHR. Ischemia and reperfusion significantly inhibited the recovery of cardiac function and increased the release of creatine kinase, and ischemia and reperfusion-induced myocardial injury in SHR was aggravated compared with WKY. Vasodilation responses to acetylcholine of aortic rings were decreased in SHR. Treatment with losartan (30 mg/kg) for 14 days significantly lowered blood pressure, elevated the plasma level of NO, and decreased the plasma concentration of ADMA in SHR. Treatment with losartan significantly improved endothelium-dependent relaxation and cardiac function during ischemia and reperfusion in SHR. Exogenous ADMA also aggravated myocardial injury induced by ischemia and reperfusion in isolated perfused heart of WKY, as shown by increasing creatine kinase release and decreasing cardiac function. The present results suggest that the protective effect of losartan on myocardial injury induced by ischemia and reperfusion is related to the reduction of ADMA levels.     

Lithium suppresses ovariectomy-induced surges in plasma gonadotropins in rats

We have reported earlier that administration of lithium, the widely-used drug for the treatment of acute mania and prophylaxis of recurrent manic-depressive bipolar disorders, leads to a disruption of estrous cycle and a significant suppression of the proestrous surge of luteinizing hormone (LH) in a number of laboratory rodents. In this report we have examined the effects of this antimanic drug on plasma and pituitary levels of LH and follicle stimulating hormone (FSH) in rats following ovariectomy (OVX), an altered endocrine state in which the levels of serum LH and FSH are highly and chronically elevated. Adult OVX rats, maintained under standardized laboratory conditions (LD 12: 12; white lights on at 06.00 h, CST) were injected (ip) with lithium, 40 days post-operation, at a dosage of 3.0 and 2.0 mEq/Kg b. wt. for 3 and 7 days respectively (twice daily at 08.00 and 16.00 h). Control OVX rats received nothing or saline injections, whereas an intact control (C) received no surgical manipulation or drug injections of any kind. As expected, the levels of plasma LH and FSH in OVX (only) group showed nearly 6-fold and 75-fold increase respectively compared to those in C. Lithium injections in OVX rats for 3 and 7 days resulted in a significant reduction in plasma LH (P less than .005 and P less than .02 respectively) and FSH (P less than .001) levels when compared with those in the OVX control groups. Lithium also led to a significant reduction in the levels of pituitary LH after both 3 (P less than .02) and 7 days (P less than .02), but the levels of pituitary FSH remained unchanged. These results suggest that the pituitary gonadotropes constitute a definitive target for lithium's action, either directly or via the hypothalamus.     

Asymmetric dimethylarginine: metabolism, arginine paradox, pathophysiology

Pathophysiology of asymmetric dimethylarginine (ADMA), an endogenous competitive inhibitor of NO synthase, has been a subject of intensive research activity during last years. The ways of ADMA synthesis and degradation were studied. It was suggested that ADMA plays a considerable role in the realization of so called "Arginine paradox". This paradox refers to the dependence of cellular NO production on exogenous L-arginine despite the saturation of NOS enzymes with intracellular L-arginine. Close association was described between increase in blood ADMA level and endothelial dysfunction accompanied by related pathologies like atherosclerosis, renal insufficiency, hypertension and some others. Some studies have represented ADMA as a strong independent risk factor for cardiovascular complications. Possible reasons are discussed of some experimental data ambiguity as well as the limits of confidence in clinical ADMA analysis.     

SNARE function is not involved in early endosome docking

Docking and fusion of transport vesicles constitute elementary steps in intracellular membrane traffic. While docking is thought to be initiated by Rab-effector complexes, fusion is mediated by SNARE (N-ethylmaleimide-sensitive factor [NSF] attachment receptor) proteins. However, it has been recently debated whether SNAREs also play a role in the establishment or maintenance of a stably docked state. To address this question, we have investigated the SNARE dependence of docking and fusion of early endosomes, one of the central sorting compartments in the endocytic pathway. A new, fluorescence-based in vitro assay was developed, which allowed us to investigate fusion and docking in parallel. Similar to homotypic fusion, docking of early endosomes is dependent on the presence of ATP and requires physiological temperatures. Unlike fusion, docking is insensitive to the perturbation of SNARE function by means of soluble SNARE motifs, SNARE-specific F_ab fragments, or by a block of NSF activity. In contrast, as expected, docking is strongly reduced by interfering with the synthesis of phosphatidyl inositol (PI)-3 phosphate, with the function of Rab-GTPases, as well as with early endosomal autoantigen 1 (EEA1), an essential tethering factor. We conclude that docking of early endosomes is independent of SNARE function.     

Multidrug resistance P-glycoprotein is not involved in cholesterol esterification

The aim of the present paper is to reinvestigate the role of multidrug resistance P-glycoprotein MDR1 and MDR-associated protein (MRP1) in cholesterol esterification using well-characterized inhibitors. Using specific substrate efflux assay, we show that GF120918 (0.2 microM) and probenecid (5 mM) were specific inhibitors of MDR1 and MRP1, respectively. In HepG2 cells, neither of them affect the esterification of cholesterol derived from the uptake of cholesterol-rich lipoprotein, while both verapamil (100 microM) and progesterone (100 microM) were able to inhibit cholesterol esterification. Similar results were obtained with verapamil, progesterone, and GF120918 in the MDR1-overexpressing cells MCF7/ADR. The capacity of progesterone to reduce cholesterol esterification is not correlated with its ability to inhibit MDR1 but is rather due to direct inhibition of acyl-CoA:cholesterol acyltransferase (ACAT). We conclude that the esterification of cholesterol is not correlated with MDR1 or MRP1 activity, thus excluding their role in the intracellular transport of endocytosis-derived cholesterol.     

Nitric oxide is not involved in the control of vasopressin release during acute forced swimming in rats

Summary. Neurons of the hypothalamo-neurohypophyseal system (HNS) are known to contain high amounts of neuronal nitric oxide (NO) synthase (nNOS). NO produced by those neurons is commonly supposed to be involved as modulator in the release of the two nonapeptides vasopressin (AVP) and oxytocin into the blood stream. Previous studies showed that forced swimming fails to increase the release of AVP into the blood stream while its secretion into the hypothalamus is triggered. We investigated here whether hypothalamically acting NO contributes to the control of the AVP release into blood under forced swimming conditions. Intracerebral microdialysis and in situ hybridization were employed to analyze the activity of the nitrergic system within the supraoptic nucleus (SON), the hypothalamic origin of the HNS. A 10-min forced swimming session failed to significantly alter the local NO release as indicated both by nitrite and, the main by-product of NO synthesis, citrulline levels in microdialysis samples collected from the SON. Microdialysis administration of NO directly into the SON increased the concentration of AVP in plasma samples collected during simultaneous forced swimming. In an additional experiment the effect of the defined stressor exposure on the concentration of mRNA coding for nNOS within the SON was investigated by in situ hybridization. Forced swimming increased the expression of nNOS mRNA at two and four hours after onset of the stressor compared to untreated controls. Taken together, our results imply that NO within the SON does not contribute to the regulation of the secretory activity of HNS neurons during acute forced swimming. Increased nNOS mRNA in the SON after forced swimming and the increase in AVP release in the presence of exogenous NO under forced swimming points to a possible role of NO in the regulation of the HNS under repeated stressor exposure.Current address: Departments of Behavioral Neuroscience and Neurology, Oregon Health & Science University, Portland, OR 97239, U.S.A.     

Asymmetric dimethylarginine, endothelial nitric oxide bioavailability and mortality in sepsis

Background

Plasma concentrations of asymmetric dimethylarginine (ADMA), an endogenous inhibitor of nitric oxide synthase, are raised in patients with chronic vascular disease, causing increased cardiovascular risk and endothelial dysfunction, but the role of ADMA in acute inflammatory states is less well defined.

Methods and Results

In a prospective longitudinal study in 67 patients with acute sepsis and 31 controls, digital microvascular reactivity was measured by peripheral arterial tonometry and blood was collected at baseline and 2–4 days later. Plasma ADMA and L-arginine concentrations were determined by high performance liquid chromatography. Baseline plasma L-arginine: ADMA ratio was significantly lower in sepsis patients (median [IQR] 63 [45–103]) than in hospital controls (143 [123–166], p<0.0001) and correlated with microvascular reactivity (r = 0.34, R² = 0.12, p = 0.02). Baseline plasma ADMA was independently associated with 28-day mortality (Odds ratio [95% CI] for death in those in the highest quartile (≥0.66 µmol/L) = 20.8 [2.2–195.0], p = 0.008), and was independently correlated with severity of organ failure. Increase in ADMA over time correlated with increase in organ failure and decrease in microvascular reactivity.

Conclusions

Impaired endothelial and microvascular function due to decreased endothelial NO bioavailability is a potential mechanism linking increased plasma ADMA with organ failure and death in sepsis.     

Denitrification in lucerne nodules is not involved in nitrite detoxification

Dissimilatory reduction of ionic nitrogen oxides to gaseous forms such as nitrous oxide or nitrogen can be carried out by free living or symbiotic forms of some strains of Rhizobium meliloti. In this paper we investigate whether bacteroid denitrification plays a role in the alleviation of the inhibitory effects of nitrate on nitrogen fixation both in bacteroid incubations as in whole nodules. The presence of a constitutive nitrate reductase (NR) activity in isolated bacteroids caused nitrite accumulation in the incubation medium, and acetylene reduction activity in these bacteroids was progressively inhibited, since nitrite reductase (NiR) activity was unable to reduce all the nitrite produced by NR and denitrification occurred slowly. Even nodules infiltrated with nitrate and nitrite failed to increase gaseous forms of nitrogen substantially, indicating that nitrite availability was not limiting denitrification by bacteroids. In spite of the low rates of bacteroidal denitrification, the effect of nodule denitrification on the inhibition of nitrogen fixation by nitrate in whole plants was tested. For that purpose, lucerne plants (Medicago sativa L. cv. Aragon) were inoculated with two Rhizobium meliloti strains: 102-F-65 (non denitrifying) and 102-F-51 (a highly denitrifying strain). After a seven days nitrate treatment, both strains showed the same pattern of inhibition, and it occurred before any nitrate or nitrite accumulation within the nodules could be detected. This observation, together with the lack of alleviation of the ARA inhibition in the denitrifying strain, and the limited activity of dissimilatory nitrogen reduction present in these bacteroids, indicate a role other than nitrite detoxification for denitrification in nodules under natural conditions.     

Caspase-1 is not involved in experimental hepatitis in mouse

Experimental hepatitis induced by tumor necrosis factor in D-(+)-galactosamine-sensitized mice or by an agonistic anti-Fas antibody in normal mice is accompanied by dramatic apoptosis of hepatocytes. Apoptosis is the final result of activation of a cascade of caspases. We used caspase-1-/- mice, generated by gene targeting, to study the role of this protease in TNF- and anti-Fas-induced lethal hepatitis. We found that mutant mice exhibited the typical caspase-1-/- phenotype, since they resisted to a lethal injection of LPS and released no interleukin-1beta in the circulation, in contrast to wild-type littermates. When caspase-1-/- mice were challenged with different doses of tumor necrosis factor/D-(+)-galactosamine or with anti-Fas, no increased survival was observed compared with control mice. Furthermore, apoptosis in the livers of these mice and serum levels of alanine aminotransferase were not reduced. These data indicate that caspase-1 deficiency does not lead to reduced apoptosis in these models, either because caspase-1 is irrelevant in this model or because of functional redundancy.     

Hepatic venoconstriction is involved in anaphylactic hypotension in rats

We determined the roles of liver and splanchnic vascular bed in anaphylactic hypotension in anesthetized rats and the effects of anaphylaxis on hepatic vascular resistances and liver weight in isolated perfused rat livers. In anesthetized rats sensitized with ovalbumin (1 mg), an intravenous injection of 0.6 mg ovalbumin caused not only a decrease in systemic arterial pressure from 120 +/- 9 to 43 +/- 10 mmHg but also an increase in portal venous pressure that persisted for 20 min after the antigen injection (the portal hypertension phase). The elimination of the splanchnic vascular beds, by the occlusions of the celiac and mesenteric arteries, combined with total hepatectomy attenuated anaphylactic hypotension during the portal hypertension phase. For the isolated perfused rat liver experiment, the livers derived from sensitized rats were hemoperfused via the portal vein at a constant flow. Using the double-occlusion technique to estimate the hepatic sinusoidal pressure, presinusoidal (R(pre)) and postsinusoidal (R(post)) resistances were calculated. An injection of antigen (0.015 mg) caused venoconstriction characterized by an almost selective increase in R(pre) rather than R(post) and liver weight loss. Taken together, these results suggest that liver and splanchnic vascular beds are involved in anaphylactic hypotension presumably because of anaphylactic presinusoidal contraction-induced portal hypertension, which induced splanchnic congestion resulting in a decrease in circulating blood volume and thus systemic arterial hypotension.     

Protective effect of apigenin on ovariectomy-induced bone loss in rats

Recent studies have shown that apigenin not only inhibits bone resorption by osteoclasts but also induces osteoclast apoptosis. However, the influence of apigenin on osteoporosis in animals is relatively unknown. The purpose of this study was to examine the bone-protective effects of apigenin in estrogen-deficient ovariectomized rats. Three-month-old female Sprague-Dawley rats were either sham-operated or ovariectomized and fed AIN-93G diet for 7 weeks to induce bone loss. To confirm bone loss, we used a newly developed non-invasive technique involving zoom-in micro-computed tomography. Apigenin was administered at a dose of 10 mg/kg three times a week for 15 weeks. Our results indicate that apigenin not only increased the mineral content and density of the trabecular bone at the neck of the left femur, but also decreased body weight and dietary consumption. Moreover, our biochemical results indicate that apigenin has a positive effect on bone turnover. The present data suggest that apigenin should be considered for use in the treatment of osteoporosis.