Sympatholytic properties of several AT1-receptor antagonists in the isolated rabbit thoracic aorta

OBJECTIVE: To evaluate the facilitating effect of angiotensin II on sympathetic neurotransmission to quantitatively compare the sympatho-inhibitory potencies of the selective AT1 -receptor antagonists losartan, irbesartan and telmisartan in the isolated rabbit thoracic aorta. DESIGN: To investigate the influence of pharmacological compounds on pre-junctional sympathetic transmission, the quantification of sympathetic transmitter release is the most straightforward approach. METHODS: To investigate the sympatholytic properties of AT1 -blockers, we studied their effects on the enhancement by angiotensin II of electrical field stimulation (EFS)-evoked (2 Hz) sympathetic transmission in a modified spillover model. RESULTS: Angiotensin II (0.01 nmol/l-0.1 micromol/l) caused a concentration-dependent enhancement of EFS-evoked noradrenaline release (control versus concentrations 0.1 nmol/l-0.1 micromol/l, P<0.05). The maximal augmentation, by almost 100%, was observed at a concentration of 1 nmol/l (FR2/FR1, 2.03 +/- 0.11 versus control, 0.99 +/- 0.03). Higher concentrations (up to 0.1 micromol/l) produced less than maximal facilitation. The AT1 -receptor antagonists losartan (0.1 nmol/l-0.1 micromol/l), telmisartan (0.01-10 nmol/l) and irbesartan (0.1 nmol/l-0.1 micromol/l) concentration dependently attenuated the angiotensin II-mediated (1 nmol/l) enhancement of EFS-evoked sympathetic outflow. The concentrations that reduced the enhancement by 50% (IC50 values, expressed as -log mol/l +/- SEM) were 9.05 +/- 0.16 losartan, 10.28 +/- 0.20 telmisartan and 9.20 +/- 0.23 irbesartan. Accordingly, the order of potency with respect to sympatho-inhibition proved telmisartan> irbesartan = losartan (where > signifies P<0.05). CONCLUSIONS: The facilitating effect of angiotensin II on the sequelae of neuronal stimulation appears to be mediated by pre-synaptically located AT1 -receptors. Facilitation can be concentration dependently attenuated by AT1 -blockade. The order of potency with respect to sympatho-inhibition is telmisartan irbesartan = losartan. These differences may be explained by differences in affinity for the pre-synaptic AT1 -receptor.     

Neurogenic nitric oxide release increases in mesenteric arteries from ouabain hypertensive rats

OBJECTIVES: We investigated whether chronic ouabain treatment changes the vasoconstrictor responses induced by electrical field stimulation (EFS) in endothelium-denuded rat superior mesenteric arteries and a possible role of neuronal nitric oxide (NO). METHOD: Mesenteric arteries from untreated and ouabain-treated rats (approximately equal to 8.0 microg/kg per day, for 5 weeks) were used in this study. Vascular reactivity was analyzed by isometric tension recording. Expression of the neuronal NO synthase isoform was analyzed by Western blot. Noradrenaline release was evaluated in segments incubated with [H]noradrenaline. RESULTS: Systolic (SBP) and diastolic (DBP) blood pressure were higher in ouabain-treated rats than in untreated rats (SBP, untreated: 120 +/- 3.5 mmHg versus ouabain-treated: 150 +/- 4.7 mmHg, P < 0.01; DBP, untreated: 87 +/- 3.0 mmHg versus ouabain-treated: 114 +/- 2.6 mmHg, P < 0.001). EFS-induced vasoconstrictions were smaller in arteries from ouabain-treated rats than in those from untreated animals, while the EFS-induced [H]noradrenaline release and the vasoconstriction induced by exogenous noradrenaline (1 nmol/l-10 micromol/l) remained unmodified. The non-selective NO synthase (NOS) inhibitor, N-nitro-L-arginine methyl ester (100 micromol/l), increased the EFS-induced vasoconstriction in mesenteric arteries from both groups, although the effect was more pronounced in segments from ouabain-treated rats. The selective neuronal NOS inhibitor, 7-nitroindazole (7-NI; 100 micromol/l) increased EFS-induced contraction only in segments from ouabain-treated rats. Neuronal NOS expression was greater in the mesenteric arteries from ouabain-treated rats than in those from untreated animals. Sodium nitroprusside (0.1 nmol/l-10 micromol/l) induced a similar vasodilatation in segments from both groups. CONCLUSIONS: These results suggest that chronic ouabain treatment is accompanied by an increase in neuronal NO release that reduces EFS-induced vasoconstriction.     

Promotion of murine hepatocarcinogenesis by testosterone is androgen receptor-dependent but not cell autonomous.

Tfm (testicular feminization) mutant mice lack functional androgen receptors. By studying liver tumor development in Tfm mice, we have shown that the greater susceptibility of male mice relative to female mice for liver tumor induction by N,N-diethylnitrosamine is androgen receptor-dependent. C57BL/6J normal and Tfm mutant mice were injected at 12 days of age with N,N-diethylnitrosamine (0.2 mumol/g, i.p.), and liver tumors were enumerated in 50-week-old animals. Normal males averaged 20 liver tumors per animal; Tfm males, 0.7; normal females, 0.6; and Tfm/+ heterozygous females, 1.5. The androgen receptor gene and the Tfm mutation are X chromosome linked. Because of random X chromosome inactivation, hepatocytes from Tfm/+ heterozygous female mice are mosaic with respect to the expression of mutant or wild-type receptors. To determine if testosterone acts directly as a liver tumor promoter, through the androgen receptor in preneoplastic hepatocytes, or by an indirect mechanism, we chronically treated these mosaic female mice with testosterone and measured the androgen receptor content of the resulting tumors. B6C3F1 Tfm/+ mosaic and +/+ wild-type female mice were injected i.p. at 12 days of age with N,N-diethylnitrosamine (0.1 mumol/g) and ovariectomized at 8 weeks of age. Half of the mice of each group subsequently received biweekly s.c. injections of testosterone (0.15 mg per mouse) for 30 weeks. Tumor multiplicity was the same for wild-type and Tfm/+ mosaic females treated with testosterone (31-32 tumors per animal at 38 weeks of age) and was increased relative to females not treated with testosterone (13-17 tumors per animal at 50 weeks of age). Testosterone treatment did not significantly increase the percentage of androgen receptor-positive tumors in Tfm/+ mosaic females: 58% of the tumors from Tfm/+ mosaic females treated with testosterone were receptor positive compared to 48% in Tfm/+ females not treated with testosterone and 92% in wild-type females treated with testosterone. Finally, the number of androgen receptors in the majority of liver tumors examined was greatly decreased relative to the surrounding normal liver tissue. We conclude that liver tumor promotion by testosterone requires a functional androgen receptor in the intact animal. However, this promotion is not cell autonomous; that is, the response of the preneoplastic hepatocyte is not dependent on the expression of functional receptor in the target cell.     

Gonadotropin treatment increases homocysteine levels in idiopathic hypogonadotropic hypogonadism: an indirect effect mediated by changes in body composition

The main objective of the present study was to examine the alterations in plasma total homocysteine (tHcy) concentrations during a testosterone-deficient state and after gonadotropin treatment for 6 Months in patients with idiopathic hypogonadotropic hypogonadism (IHH). Thirty-five newly diagnosed male patients with IHH (mean age 21.34+/-1.53 years) and 29 age- and body mass index-matched healthy males (mean age 21.52+/-1.77 years) were recruited into the study. Pretreatment levels of free testosterone (1.51+/-0.66 pg/ml), estradiol (21.37+/- 4.37 pg/ml), FSH (0.91+/-0.24 IU/l) and LH (1.25+/- 0.53 IU/l) were lower than controls (25.17+/-3.06 pg/ml, 31.00+/-4.96 pg/ml, 3.14+/-1.62 IU/l and 4.83+/-1.65 IU/l respectively) (P<0.001). They increased significantly after treatment (18.18+/-1.59 pg/ml, 27.97+/- 4.25 pg/ml, 2.41+/-0.27 IU/l and 2.79+/-0.19 IU/l respectively) (P<0.001). Patients with IHH had lower tHcy levels than controls (10.14+/-1.34 and 12.58+/- 2.29 micro mol/l respectively) (P<0.001). Plasma tHcy concentrations increased significantly (12.63+/-1.44 micromol/l) after 6 months of treatment (P<0.001). As compared with the controls, pretreatment levels of serum creatinine (63.54+/-13.01 vs 82.84+/-16.69 micromol/l), hemoglobin (12.98+/-0.56 vs 13.83+/-0.71 g/dl) and hematocrit (39.29+/-2.01 vs 41.38+/-1.95%) were significantly lower (P<0.001), and they increased significantly following treatment (80.24+/-11.93 micromol/l, 13.75+/-0.49 g/dl and 41.26+/-1.78% respectively) (P<0.001). The pretreatment folic acid and vitamin B(12) levels were significantly higher in patients when compared with controls (14.87+/-5.68 vs 12.52+/-4.98 nmol/l, P=0.034 and 289.75+/-92.34 vs 237.59+/-108.17 pmol/l, P=0.002 respectively). They decreased significantly after treatment (11.29+/-3.31 nmol/l and 228.51+/-54.33 pmol/l respectively) (P<0.001). The univariate and multivariate regression analysis results showed that only changes in creatinine, creatinine clearance, vitamin B12 and folic acid were independently associated with changes in tHcy levels in patients with IHH. In conclusion, the increase in plasma tHcy concentrations following gonadotropin treatment seems to be largely independent of changes in androgen levels.     

Testicular steroidogenesis in the testicular feminized (Tfm) mouse: loss of 17 alpha-hydroxylase activity.

Testicular feminized (Tfm) mice are totally insensitive to androgen and may be used to study the role of the androgen receptor in normal development and function. We have examined testicular and Leydig cell steroidogenesis in Tfm mice. Serum bioactive LH was high in Tfm mice but serum testosterone was low and this was associated with a severe reduction in testicular testosterone production in vitro. Examination of [3H]pregnenolone metabolism by testes of Tfm mice indicated that progesterone, rather than testosterone, was the major steroid produced. Leydig cells were isolated from normal and Tfm mice and from normal mice in which testicular descent was surgically prevented before puberty. As in whole testes, androgen production in response to human chorionic gonadotrophin was severely reduced in Leydig cells from testes of Tfm mice compared with normal or cryptorchid groups. In contrast, progesterone production by Leydig cells from testes of Tfm mice was markedly increased in comparison with other groups. Total steroid production (progesterone plus androstenedione plus testosterone), however, was only 24% of normal in Leydig cells from Tfm mice. The pattern of steroid production by Leydig cells from cryptorchid testes was similar to control, although total steroid production was reduced to about 50% (this was significantly higher than the Tfm group, P less than 0.05). The high progesterone/androgen ratio in testes from Tfm mice suggested that 17 alpha-hydroxylase was depleted in these animals. To confirm this, activity of the four major steroidogenic enzymes associated with the smooth endoplasmic reticulum was measured.(ABSTRACT TRUNCATED AT 250 WORDS)     

Measures of oxidative stress in heterozygous familial hypercholesterolaemia

Familial hypercholesterolaemia (FH) may be associated with increased oxidative stress which may contribute to atherogenesis. Plasma lipid hydroperoxides (ROOHs), 8-epi PGF(2alpha) and alpha-tocopherol were measured in normal subjects and in newly referred heterozygous FH patients and used as indices of oxidative stress. ROOH levels were higher (+16%), albeit non-significantly, in FH patients than in controls subjects (4.4+/-0.3 vs. 3.8+/-0.3 micromol/l; n=51 and 40, respectively). 8-epi PGF(2alpha) levels were significantly greater (+56%) in the FH patients than in controls (0.43+/-0.06 vs. 0.27+/-0.05 nmol/l; P<0.05; n=14 and 16, respectively). FH patients with vascular disease had significantly higher (+32%) levels of ROOH compared with patients without vascular disease (4.9+/-0.40 vs. 3.7+/-0.33 micromol/l; P<0.05; n=27 and 24, respectively). Similarly, 8-epi PGF(2alpha) concentrations were higher (+100%) in the FH patients with vascular disease than in those without it (0.6+/-0.08 vs. 0.3+/-0.10 nmol/l; P<0.05; n=6 and 8, respectively). Absolute alpha-tocopherol levels in FH patients were similar to those in controls (21.0+/-0.70 vs. 23.8+/-1.30 micromol/l). When alpha-tocopherol levels were expressed relative to cholesterol, however, the concentrations were found to be significantly lower (-43%) in FH patients than in controls (2.9+/-0.10 vs. 5.1+/-0.40 micromol/mmol, P<0.0005). There were no differences in absolute or cholesterol standardised alpha-tocopherol levels in patients with and without vascular disease. These data suggest that oxidative stress is increased in FH-patients and is particularly pronounced in those patients with vascular disease. It is possible that increased oxidative stress may precede the development of vascular disease.     

Ouabain treatment increases nitric oxide bioavailability and decreases superoxide anion production in cerebral vessels

OBJECTIVE: Chronic administration of ouabain induces hypertension and increases the contribution of nitric oxide to vasoconstrictor responses in peripheral arteries. The aim of this study was to analyse whether ouabain treatment alters the nitric oxide bioavailability in cerebral arteries. METHODS: Basilar arteries from control and ouabain-treated rats ( approximately 8.0 microg/day, 5 weeks) were used. Vascular reactivity was analysed by isometric tension recording, protein expression by western blot, nitric oxide levels by diaminofluorescein-induced fluorescence, superoxide anion (O2) production by ethidium fluorescence and lucigenin chemiluminescence and plasma total antioxidant status by a commercial kit. RESULTS: The relaxations induced by bradykinin (1 nmol/l-10 micromol/l) and L-arginine (0.01-300 micromol/l) and the contractile responses induced by both N-nitro-L-arginine methyl ester (0.1-100 micromol/l) and oxyhaemoglobin (0.01-10 micromol/l) were greater in arteries from ouabain-treated than control rats. However, the relaxation to diethylamine NONOate-nitric oxide (0.1 nmol/l-10 micromol/l) and the contractions to KCl (7.5-120 mmol/l) and 5-hydroxytryptamine (0.01-10 micromol/l) were similar in arteries from both groups. Ouabain treatment increased basal nitric oxide levels but did not modify endothelial and neuronal nitric oxide synthase protein expression. O2 production was lower in cerebral arteries from ouabain-treated rats; however, plasma total antioxidant status and vascular protein expression of Cu/Zn-superoxide dismutase, Mn-superoxide dismutase and extracellular superoxide dismutase were similar in both groups. CONCLUSION: Chronic ouabain treatment increased nitric oxide basal levels in basilar arteries probably due to the decreased O2 levels. This might be an adaptive mechanism of the cerebral vasculature to the increase in blood pressure.     

A comparison between the effects of low dose (1 microg) and standard dose (250 microg) ACTH stimulation tests on adrenal P450c17alpha enzyme activity in women with polycystic ovary syndrome

OBJECTIVE: Numerous studies have found elevated androgen production by the adrenal glands in patients with polycystic ovary syndrome (PCOS). However, the role and the mechanisms responsible for the adrenal androgen excess in women with PCOS are not well understood. DESIGN: Our aim was to compare 17-hydroxyprogesterone (17-OHP), androstenedione, dehydroepiandrosterone sulfate (DHEAS) and cortisol responses to a low dose (1 microg) ACTH stimulation test (LDT) with the responses to a standard dose (250 microg) ACTH stimulation test (SDT) in patients with PCOS. METHODS: Fifty women with PCOS (mean age 25.4+/-0.7 years) and 20 healthy women (mean age 27.3+/-2.2 years) were included in the study. The patients and controls underwent ACTH stimulation tests with 1 microg and 250 microg synthetic ACTH in the follicular phase of their cycles. Venous blood was drawn at 0, 30 and 60 min for determination of serum cortisol, 17-OHP, androstenedione and DHEAS levels. RESULTS: In PCOS subjects, peak and area under the curve (AUC) 17-OHP (9.3+/-0.3 nmol/l, 378.4+/-61 nmol/lx60 min), androstenedione (15.6+/-0.6 nmol/l, 806.4+/-52 nmol/lx60 min) and DHEAS (7.5+/-0.4 micromol/l, 385.6+/-25.5 micromol/lx60 min) responses to SDT were significantly higher than the levels in healthy women (respectively 5.7+/-0.3 nmol/l and 249.4+/-52.2 nmol/lx60 min for 17-OHP; 9.1+/-0.3 nmol/l and 413.7+/-31.6 nmol/lx60 min for androstenedione; 4.3+/-0.4 micromol/l and 224.9+/-24.5 micromol/lx60 min for DHEAS) (P<0.05). Peak and AUC cortisol responses to SDT were similar in PCOS and control subjects. Peak and AUC cortisol and 17-OHP responses to LDT in women with PCOS were similar to the values obtained in healthy women. Peak androstenedione (12.5+/-0.6 nmol/l) and peak (6.5+/-0.5 nmol/l) and AUC (336.3+/-22.4 micromol/lx60 min) DHEAS responses to LDT were significantly higher in women with PCOS. CONCLUSIONS: These results show that LDT is capable of revealing the adrenal hyperactivity in women with PCOS. Adrenal P450c17alpha enzyme dysregulation in PCOS is revealed by ACTH stimulation at a pharmacological dose (250 microg) but not by a physiological dose (1 microg). LDT is able to demonstrate adrenal hyperactivity characterized by an increase in DHEAS levels.     

Discontinuation of estrogen replacement therapy in GH-treated hypopituitary women alters androgen status and IGF-I

OBJECTIVE AND DESIGN: Compared with their male counterparts, healthy females secrete more growth hormone (GH) and those with GH-deficiency have lower insulin-like growth factor I (IGF-I) levels and are less responsive to GH substitution. To test whether this gender difference is related to sex hormones we measured androgen status and IGF-I related parameters in 38 hypopituitary women (mean (range) age 41.5 (20-58) years) during continued GH substitution as compared with a control group of 38 healthy women matched for age and menopausal status. Twenty six patients were studied twice: with estrogen replacement and after 28 days of estrogen discontinuation in a randomised design. RESULTS: The patients were androgen deficient compared with controls (median, range), dehydroepiandrosterone sulphate (DHEAS): 185 (99-7800) nmol/l vs 4400 (820-13,000) nmol/l, P=or<0.001; androstenedione: 0.5 (0.1-7.1) nmol/l vs 4.3 (1.6-8.8) nmol/l, P=or<0.001; dihydrotestosterone (DHT): 0.13 (0.09-0.54) nmol/l vs 0.55 (0.09-0.89) nmol/l, P=or<0.001; testosterone: 0.28 (0.09-1.56) nmol/l vs 1.1 (0.71-2.24) nmol/l, (P=or<0.001); free testosterone: 0.004 (0.001-0.030) nmol/l vs 0.016 (0.001-0.030) nmol/l, P=or<0.001. The circulating levels of IGF-I, IGF-II, IGF-binding protein 1 (IGFBP-1), and IGFBP-3 did not differ between patients and controls. The subgroup of patients receiving hydrocortisone (HC) replacement (n=24) had significantly lower levels of androgens (suppressed by 80-100%) as well as IGF-I and IGFBP-3 as compared with the patients not receiving HC. IGF-I was correlated to free testosterone in patients (r=0.57, P=0.0005) as well as controls (r=0.43, P=0.008), and free testosterone was a significant positive predictor of IGF-I. Estrogen discontinuation induced an increase in IGF-I (167+/-15 vs 206+/-14 microg/l, P=0.005 and IGFBP-3 (3887+/-139 vs 4309+/-138 microg/l, P=0.0005). Estrogen discontinuation was associated with a significant increase in median (range) free testosterone (0.004 (0-0.02) vs 0.0065 (0-0.03) nmol/l, P=0.001) and a significant decrease in median (range) sex-hormone binding globulin (SHBG; 93 (11-278) vs 55.5 (20-142) nmol/l, P=0.001). DeltaIGF-I correlated with DeltaSHBG (r=-0.45 P=0.033) and DeltaIGFBP-3 (r=0.67 P=or<0.001). In a regression model DeltaE2, Deltatestosterone, DeltaSHBG and DeltaIGFBP-3 explained 93% of the variation in DeltaIGF-I. CONCLUSIONS: Androgen levels are low in hypopituitary women and free testosterone correlates with IGF-I. Discontinuation of estrogen replacement in these patients induces elevations in IGF-I as well as free testosterone, and DeltaIGF-I correlated positively with Deltafree testosterone. These effects may contribute to the gender differences observed in the GH-IGF axis in healthy adults as well as in the responsiveness of hypopituitary patients to GH substitution.     

Maternal diet composition alters serum steroid and free fatty acid concentrations and vaginal pH in mice

We examined the effects of three maternal diets (very high fat (VHF), low fat (LF), and control (Purina 5015)) on serum steroids, free fatty acids (FFA), and vaginal pH in National Institutes of Health Swiss mice. Females were fed (VHF, n = 33; LF, n = 33; 5015, n = 48) from 4 to 16 weeks of age. Following breeding, female serum was collected at 0.5 (pre-implantation, early diestrus) or 8.5 (post-implantation, mid-diestrus) days post-coitus (dpc). The serum concentrations of 17beta-estradiol, testosterone, progesterone, and FFA were analyzed at both collection points, and vaginal pH at 0.5 dpc. Striking differences in steroids and FFA were observed at 0.5 dpc among the groups. Estradiol was higher in the VHF (14.1 +/- 3.0 pg/ml), compared with LF mice (5.2 +/- 2.3 pg/ml; P< or = 0.05). In contrast, 0.5 dpc testosterone was lower in the VHF (10.5 +/- 3.0 pg/ml) versus the LF group (32.7 +/- 8.4 pg/ml; P< or = 0.05). At 8.5 dpc, progesterone was higher in the VHF (89.6 +/- 6.7 ng/ml) versus the 5015 group (60.1 +/- 4.9 ng/ml; P< or = 0.05). VHF mice had higher FFA concentrations at 0.5 dpc (1.0 +/- 0.2 mmol/l) than LF and control mice (0.5 +/- 0.1 and 0.6 +/- 0.1 mmol/l respectively; P< or = 0.05). At 8.5 dpc, VHF females had higher serum FFA (0.8 +/- 0.1 mmol/l) than LF and control females (0.4 +/- 0.1 and 0.6 +/- 0.1 mmol/l; P< or = 0.05). Mean vaginal pH of VHF females (6.41 +/- 0.09) was lower than 5015 females (6.76 +/- 0.10; P< or = 0.05). These diet-induced alterations in serum steroid and FFA concentrations might affect several reproductive processes, including preferential fertilization by one class of sperm over the other and sex bias in pre- and post-implantational embryonic development.     

Cyclooxygenase inhibition converts the effect of nitric oxide synthase inhibition from infarct size reduction to expansion in isolated rabbit hearts.

Nitric oxide (NO) and prostacyclin (PGI2) are putative cardioprotective agents. Evidence indicates that there may be a reciprocal relationship involved in the synthesis of NO and PGI2, so that inhibiting the release of one mediator may promote the synthesis of the other. Therefore, we investigated the effects of concomitantly inhibiting NO and PGI2 synthesis, using NG-nitro-L-arginine (L-NOARG) or indomethacin, respectively, on infarct size. Langendorff-perfused rabbit hearts were assigned randomly to one of five treatment groups of n=6: control L-NOARG 100 micromol/l; indomethacin 3 micromol/l L-NOARG 100 micromol/l + indomethacin 3 micromol/l; or L-NOARG 100 micromol/l + L-arginine 1 mmol/l. After 30 min regional ischaemia and 120 min reperfusion, infarct size was assessed by tetrazolium staining. Infarct size was reduced significantly in hearts treated with L-NOARG (20.8+/-1.3%) compared to control hearts (34.7+/-0.4%). This reduction in infarct size was abolished by co-perfusing with a 10-fold excess of L-arginine (30.7+/-1.7%). While indomethacin alone had no effect (33.4+/-2.3%), perfusion with both L-NOARG and indomethacin resulted in a significant increase in infarct size (44.0+/-1.9%) compared to controls. Treatment with L-NOARG alone increased 6-keto PGF1alpha in coronary effluent prior to ischaemia (30.5+/-1.2 vs 16.6+/-1.3 pg/min/g in controls, P<0.05). This effect was reversed by co-perfusion with either L-arginine or indomethacin. These results indicate that the reduction in infarct size by L-NOARG may be due to increased PGI2 release. Concomitant administration of indomethacin negated this effect and revealed an adverse effect of NO synthase inhibition on infarct size.     

Critical role for CuZn-superoxide dismutase in preventing angiotensin II-induced endothelial dysfunction

The goal of the present study was to test the hypothesis that the CuZn isoform of superoxide dismutase (CuZnSOD) protects against angiotensin II (Ang II)-induced endothelial dysfunction. Vascular responses of carotid arteries from control, CuZnSOD-deficient (CuZnSOD(+/-)), and CuZnSOD transgenic mice were examined in vitro after overnight incubation with either vehicle or Ang II (1 or 10 nmol/L). In control mice, acetylcholine produced concentration-dependent relaxation that was not affected by 1 nmol/L Ang II. In contrast, relaxation to acetylcholine in arteries from CuZnSOD+/- mice was markedly and selectively attenuated after incubation with 1 nmol/L Ang II (eg, 100 micromol/L acetylcholine produced 93+/-6% and 44+/-15% relaxation in vehicle- and Ang II-treated arteries, respectively). A higher concentration of Ang II (10 nmol/L) selectively impaired relaxation to acetylcholine in arteries from control mice (eg, 100 micromol/L acetylcholine produced 96+/-4% and 45+/-7% relaxation in vehicle- and Ang II-treated vessels, respectively). In contrast, 10 nmol/L Ang II had no effect on responses to acetylcholine in carotid arteries from CuZnSOD transgenic mice (or in control mice treated with the superoxide scavenger Tiron [1 mmol/L]). Superoxide levels in control mice were higher in aorta treated with Ang II than with vehicle and were markedly reduced in CuZnSOD transgenic mice. These findings provide the first direct evidence that CuZnSOD limits Ang II-mediated impairment of endothelial function and that loss of 1 copy of the CuZnSOD gene is sufficient to enhance Ang II-induced vascular dysfunction.     

Sex steroids and body composition in men with cystic fibrosis

OBJECTIVE: Delayed sexual maturation and low body weight is common in cystic fibrosis (CF). Concomitant data on sex hormones and concomitant body composition are lacking in men with CF. DESIGN: Cross-sectional study. SUBJECTS AND METHODS: Serum levels of testosterone, 17beta-oestradiol (E(2)), 25-hydroxyvitamin D (25(OH)D), sex hormone-binding globulin (SHBG) and LH were measured by RIA and total and regional lean body mass (LBM), fat body mass (FBM), bone mineral content and bone mineral density (BMD) were assessed by dual-energy X-ray absorptiometry, in men with CF (n=40; age 24.7+/-5.4 years) and age-matched healthy controls (n=28; age 25.7+/-3.7). Only men without acute disease exacerbation or systemic glucocorticoid treatment were included. RESULTS: Mean levels of hormonal serum parameters differed significantly between healthy controls (testosterone=20.2+/-5.5 nmol/l; E(2)=95.0+/-20.2 pmol/l; 25(OH)D=62.8+/-28.3 nmol/l) and patients (testosterone=15.9+/-4.1 nmol/l; E(2)=60.7+/-19.4 pmol/l; 25(OH)D=39.5+/-17.8 nmol/l; P<0.001) while no difference was found for SHBG or LH. Eleven (for E(2), 19 of 40, for 25(OH)D, 20 of 40) out of 40 patients had serum testosterone levels 2 s.d. below the mean of normal. Men with CF showed a relative shift from FBM to LBM and a different body fat distribution compared with healthy controls (P<0.01). Testosterone was not correlated with weight, total or regional LBM or FBM, but significantly with BMD (r=0.32; P<0.05) independently from body height and 25(OH)D levels. E(2) was correlated with regional and total FBM (r=0.48; P<0.05). In a multiple regression analysis of the joint effect of testosterone and body components on E(2), a testosterone-independent effect was found for FBM. CONCLUSIONS: CF patients with stable disease have moderately reduced serum testosterone levels. This might already imply detrimental effects on bone. The change in LBM of patients appears to have no direct association with sex hormone levels while low FBM might cause reduced net conversion of serum testosterone to E(2) with possible effects on FBM distribution.     

Effects of adrenal androgens on the transplantable human prostate tumor PC-82.

The potential of the adrenal androgens androstenedione (A) and dehydroepiandrosterone (DHEA) to stimulate prostate tumor growth was investigated in the hormone-dependent human prostate tumor model PC-82, propagated in nude mice. Substitution of castrated mice bearing growth-arrested tumors with DHEA for 28 days, resulting in peripheral levels of 9.2 +/- 1.7 nmol/liter (mean +/- SEM), led to a decline of tumor burden comparable to that observed in castrated controls. Intratumor testosterone (T) and 5 alpha-dihydrotestosterone were similar to those detected in the castrated group. In contrast, high-dose A supplementation (peripheral level of 13.5 +/- 1.3 nmol/liter) in androgen ablated tumor-bearing mice resulted in tumor growth, although less pronounced than in T-resubstituted mice (T level of 18.8 +/- 1.5 nmol/l). Intraprostatic levels of androgens were not different between both groups. Substitution of castrated PC-82 tumor-bearing mice with low-dose A (2.5 +/- 0.4 nmol/l) neither stimulated growth of tumors nor did it lead to regression of PC-82 tumors. Proliferative activity as estimated by BrdU incorporation (S-phase cells) was not induced in these tumors. In conclusion, DHEA does not have a stimulatory effect on growth of PC-82 tumor tissue, but A is capable of inducing PC-82 tumor growth, most likely through peripheral conversion of A into T and 5 alpha-dihydrotestosterone.     

Effect of omega-3 fatty acids on lipid peroxidation and antioxidant enzyme status in type 2 diabetic patients

BACKGROUND: This study was conducted to investigate the effect of omega-3 fatty acids on lipid peroxidation and antioxidant enzyme activities in non-insulin dependent diabetic patients. METHODS: Thirty-four non-insulin dependent diabetic patients were selected for this study and they were initially treated with antidiabetic drugs alone for one month. This was followed by supplementation with omega-3 fatty acids (1,080 mg of EPA and 720 mg of DHA per day) along with the antidiabetic drugs for a period of two months. RESULTS: No change in glycaemic control was observed in diabetic patients at the end of two months of omega-3 fatty acids therapy along with antidiabetic drugs. The combined treatment significantly reduced serum triglycerides (2.07 +/- 0.94 mmol/l, before combined therapy vs 1.54 +/- 0.49 mmol/l after combined therapy, P<0.05) and increased HDL-cholesterol levels (0.93 +/- 0.099 mmol/l, before combined therapy vs 1.04 +/- 0.098 mmol/l after therapy, P<0.01). The raised lipid peroxide levels (5.14 +/- 0.61 micromol MDA/l in controls vs 6.36 +/- 1.56 micromol MDA/l in diabetic patients, P<0.001) were significantly decreased in these patients after the combined therapy (6.36 +/- 1.56 micromol MDA/l, before combined therapy vs 5.16 +/- 0.7 micromol MDA/l, after combined therapy, P<0.01). Among the erythrocyte antioxidant enzymes, the Glutathione peroxidase activity was increased (32.5 +/- 9.9 U/g Hb/min, before combined therapy vs 42.25 +/- 4.6 U/g Hb/min, after combined therapy, P<0.01) while no change was observed in Catalase (99.7 +/- 30.4 KU/g Hb before combined therapy vs 85.35 +/- 23.41 KU/g Hb, after combined therapy) and Superoxide dismutase activities (2.6 +/- 1.04 U/mg Hb/min, before therapy vs 3.01 +/- 1.08 U/mg Hb/min, after combined therapy) after the 2 months of combined treatment with antidiabetic agents and omega-3 fatty acids. CONCLUSION: Supplementation with omega-3 fatty acids has beneficial effects on serum triglycerides, HDL-cholesterol, lipid peroxidation and antioxidant enzymes, which may lead to decreased rate of occurrence of vascular complications in diabetes.     

Reduced antioxidant status in obese children with multimetabolic syndrome

BACKGROUND: In our previous study, the negative correlation found between plasma insulin levels and plasma alpha-tocopherol concentrations suggested that decreased antioxidant vitamin levels and reduced antioxidant capacity might be a characteristic feature of obese children with multimetabolic syndrome (MMS). OBJECTIVE: To investigate lipid-soluble antioxidant vitamin levels and total antioxidant status (TAS) in obese children with and without MMS and in controls. SUBJECTS: In total, 16 control children (age: 16.2+/-1.1 y, BMI: 20.7+/-1.9 kg/m(2), body fat (BF): 25.6+/-5.7%; mean+/-s.d.), 15 obese children (age: 13.4+/-2.1 y, BMI: 34.2+/-3.1 kg/m(2), BF: 36.9+/-5.8%,) and 17 obese children without MMS (age: 14.4+/-2.3 y, BMI: 30.4+/-6.2 kg/m(2), BF: 36.3+/-5.8%) were included in the study. METHODS: Body composition was determined by anthropometric methods. Vitamin analysis was carried out by high-performance liquid chromatography and TAS of the plasma was measured with commercially available kits. Plasma glucose, lipids and insulin were measured by standard laboratory methods. RESULTS: Plasma alpha-tocopherol and beta-carotene levels corrected for plasma lipids (cholesterol + triglyceride) were significantly (P<0.05) lower in obese children with MMS (2.4 (3.1) micromol/mmol and 12.3 (24.0) pmol/mmol, respectively, median (range from the first to the third quartile)), than in the obese without MMS (3.7 (0.9) micromol/mmol and 48.2 (27.7) pmol/mmol) and in the control group (3.8 (0.7) micromol/mmol and 86.6 (44.5) pmol/mmol). Plasma TAS values of the MMS group (1.2 (0.4) mmol/l) were also significantly (P<0.05) reduced as compared to obese children without MMS (1.62 (0.14) mmol/l) and to controls (1.58 (0.21) mmol/l). CONCLUSION: Obese children with MMS are prone to oxidative stress. Further investigations are necessary to determine if these children may benefit from vitamin E and beta-carotene supplementation.     

Simvastatin enhanced sodium nitroprusside-induced apoptosis of smooth muscle cells. An involvement of geranylgeraniol

A decrease in serum cholesterol is one of the most beneficial effects in anti-atherogenesis. Nitric oxide is also an anti-atherogenic substance, inducing vasodilation and inhibits proliferation of smooth muscle cells (SMC). Therefore, we examined sodium nitroprusside (SNP)-induced apoptosis of vascular SMC with respect to cholesterol metabolism. Cultured vascular SMC from bovine carotid arteries and rat aorta were used. Apoptosis was determined by propidium iodide assay. Treatment of the SMC with SNP(100 micromol/l-1 mmol/l ) for 6 h induced a little nuclear fragmentation. SNP (1 mmol/l ) elicited apoptosis in 4.4+/-2.2% of cells. Pretreatment of SMC with simvastatin (1 microg/ml, 2 days), a hydroxymethylglutaryl Coenzyme A (HMG CoA) reductase inhibitor, synergistically enhanced SNP-induced apoptosis (% apoptosis =15. 9+/-3.3%). Either mevalonate (100 micromol/l) or geranylgeraniol (30 micromol/l) recovered the simvastatin (1 microg/ml)-enhanced SMC apoptosis induced by SNP. Neither squalene (10 mmol/l) nor farnesol (30 micromol/l) had a recovery effect on the simvastatin-enhanced SMC apoptosis induced by SNP. Pretreatment with simvastatin (1 microg/ml) reduced total cholesterol content in SMC. Mevalonate (100 micromol/l) restored a decrease in total cholesterol content. However, incubation with LDL deficient serum did not enhance SNP-induced apoptosis of SMC, although treatment with LDL deficient serum decreased the total cholesterol content in SMC. These data suggested that decrease in HMG CoA reductase metabolites, especially geranylgeraniol might enhance the SNP-induced apoptosis of SMC, and that, apoptosis was not involved in a decrease in cholesterol of SMC.     

In men older than 70 years, total testosterone remains stable while free testosterone declines with age. The Health in Men Study

OBJECTIVE: An age-related decline in serum total and free testosterone concentration may contribute to ill health in men, but limited data are available for men > 70 years of age. We sought to determine the distribution and associations of reduced testosterone concentrations in older men. DESIGN: The Health in Men Study is a community-representative prospective cohort investigation of 4263 men aged > or = 70 years. Cross-sectional hormone data from 3645 men were analysed. METHODS: Early morning sera were assayed for total testosterone, sex hormone binding globulin (SHBG) and LH. Free testosterone was calculated using the Vermeulen method. RESULTS: Mean (+/- s.d.) serum total testosterone was 15.4 +/- 5.6 nmol/l (444 +/- 162 ng/dl), SHBG 42.4 +/- 16.7 nmol/l and free testosterone 278 +/- 96 pmol/l (8.01 +/- 2.78 ng/dl). Total testosterone correlated with SHBG (Spearman's r = 0.6, P < 0.0001). LH and SHBG increased with age (r = 0.2, P < 0.0001 for both). Instead of declining, total testosterone increased marginally (r = 0.04, P = 0.007) whilst free testosterone declined with age (r = -0.1, P < 0.0001). Free testosterone was inversely correlated with LH (r = -0.1, P < 0.0001). In multivariate analyses, increasing age, body mass index (BMI) and LH were associated with lower free testosterone. CONCLUSIONS: In men aged 70-89 years, modulation of androgen action may occur via an age-related increase in SHBG and reduction in free testosterone without a decline in total testosterone concentration. Increasing age, BMI and LH are independently associated with lower free testosterone. Further investigation would be required to assess the clinical consequences of low serum free testosterone, particularly in older men in whom total testosterone may be preserved.     

Inhibition of smooth muscle cell calcium mobilization and aortic ring contraction by lactone vastatins

OBJECTIVE: To determine the effects of vastatins on the contraction of rat aortic rings and to assess their effects on calcium mobilization using cultured smooth muscle cells from rat aorta. METHODS: Aortic rings from Sprague-Dawley rats were mounted on stainless steel wires to determine the generation of tension using force-displacement transducers. The tension (g) developed by angiotensin II (100 nmol/l) was measured under basal conditions and after 45 min incubation with 20 micromol/l simvastatin. The effect of 20 mol/l simvastatin, lovastatin, mevastatin and pravastatin on noradrenaline concentration-response curves and the angiotensin II-induced calcium mobilization was also evaluated. RESULTS: Addition of angiotensin II to aortic rings incubated in Krebs' Ringer bicarbonate medium produced tension generation (0.9 +/- 0.12 g = 100%). Treatment of aortic rings with simvastatin inhibited the angiotensin II-induced contraction 58 +/- 0.06%. To evaluate this effect further, dose-response curves with noradrenaline were measured in the presence and absence of 20 micromol/l simvastatin, lovastatin, mevastatin and pravastatin. The results indicate that simvastatin, lovastatin and mevastatin inhibited the contraction induced by noradrenaline (10 micromol/l) by about 50%. Pravastatin did not inhibit aortic ring contraction. Furthermore, the concentration required for 50% of the maximal contraction (EC50) by noradrenaline (6.2 +/- 0.1 nmol/l) was significantly increased by simvastatin, lovastatin, mevastatin and pravastatin. The inhibition of vascular contraction by vastatins appears to involve inhibition of 3-hydroxy-3-methylglutaryl (HMG)-CoA reductase activity because the inhibitory effect of simvastatin was reduced 50% by 10 mmol/l mevalonic acid. To determine whether the depression of vascular contraction by these agents was correlated with cell calcium changes, the angiotensin II-induced calcium mobilization was determined in Fura-2 loaded cells, before and after treatment with these inhibitors. Simvastatin, lovastatin and mevastatin significantly reduced the angiotensin II-induced calcium mobilization. The concentration that induced 50% inhibition was 3.3 micromol/l for simvastatin, 17.4 micromol/l for mevastatin and 21.7 micromol/l for lovastatin. No effect of pravastatin on calcium mobilization was observed. CONCLUSIONS: These findings suggest that lactone vastatins depress vascular contraction by reducing cytosolic calcium release in vascular smooth muscle cells. These agents also appear to exert competitive and non-competitive type antagonisms on noradrenaline action.     

Modulation of iron uptake in heart by L-type Ca2+ channel modifiers: possible implications in iron overload.

Heart failure is the leading cause of mortality in patients with transfusional iron (Fe) overload in which myocardial iron uptake ensues via a transferrin-independent process. We examined the ability of L-type Ca2+ channel modifiers to alter Fe2+ uptake by isolated rat hearts and ventricular myocytes. Perfusion of rat hearts with 100 nmol/L 59Fe2+ and 5 mmol/L ascorbate resulted in specific 59Fe2+ uptake of 20.4+/-1.9 ng of Fe per gram dry wt. Abolishing myocardial electrical excitability with 20 mmol/L KCl reduced specific 59Fe2+ uptake by 60+/-7% (P<0.01), which suggested that a component of myocardial Fe2+ uptake depends on membrane voltage. Accordingly, 59Fe2+ uptake was inhibited by 10 micromol/L nifedipine (45+/-12%, P<0.02) and 100 micromol/L Cd2+ (86+/-3%; P<0. 001) while being augmented by 100 nmol/L Bay K 8644 (61+/-18%, P<0. 01) or 100 nmol/L isoproterenol (40+/-12%, P<0.05). By contrast, uptake of 100 nmol/L ferric iron (59Fe3+) was significantly lower (1. 4+/-0.3 ng Fe per gram dry wt; P<0.001) compared with divalent iron. These data suggest that a component of Fe2+ uptake into heart occurs via the L-type Ca2+ channel in myocytes. To investigate this further, the effects of Fe2+ on cardiac myocyte L-type Ca2+ currents were measured. In the absence of Ca2+, noninactivating nitrendipine-sensitive Fe2+ currents were recorded with 15 mmol/L [Fe2+]o. Low concentrations of Fe2+ enhanced Ca2+ current amplitude and slowed inactivation rates, which was consistent with Fe2+ entry into the cell, whereas higher Fe2+ levels caused dose-dependent decreases in peak current. Fe3+ had no effect on current amplitude or decay. Combined, our data suggest that myocardial Fe2+ uptake occurs via L-type Ca2+ channels and that blockade of these channels might be useful in the treatment of patients with excessive serum iron levels.