Loss of resting bradycardia with detraining is associated with intrinsic heart rate changes

The mechanisms underlying the loss of resting bradycardia with detraining were studied in rats. The relative contribution of autonomic and non-autonomic mechanisms was studied in 26 male Wistar rats (180-220 g) randomly assigned to four groups: sedentary (S, N = 6), trained (T, N = 8), detrained for 1 week (D1, N = 6), and detrained for 2 weeks (D2, N = 6). T, D1 and D2 were treadmill trained 5 days/week for 60 min with a gradual increase towards 50% peak VO2. After the last training session, D1 and D2 were detrained for 1 and 2 weeks, respectively. The effect of the autonomic nervous system in causing training-induced resting bradycardia and in restoring heart rate (HR) to pre-exercise training level (PET) with detraining was examined indirectly after cardiac muscarinic and adrenergic receptor blockade. T rats significantly increased peak VO2 by 15 or 23.5% when compared to PET and S rats, respectively. Detraining reduced peak VO2 in both D1 and D2 rats by 22% compared to T rats, indicating loss of aerobic capacity. Resting HR was significantly lower in T and D1 rats than in S rats (313 ± 6.67 and 321 ± 6.01 vs 342 ± 12.2 bpm) and was associated with a significantly decreased intrinsic HR (368 ± 6.1 and 362 ± 7.3 vs 390 ± 8 bpm). Two weeks of detraining reversed the resting HR near PET (335 ± 6.01 bpm) due to an increased intrinsic HR in D2 rats compared with T and D1 rats (376 ± 8.8 bpm). The present study provides the first evidence of intrinsic HR-mediated loss of resting bradycardia with detraining in rats.

Dexamethasone blocks the migration of the human neuroblastoma cell line SK-N-SH

Glucocorticoids (Gc) influence the differentiation of neural crest-derived cells such as those composing sympathoadrenal tumors like pheochromocytomas, as well as neuroblastomas and gangliomas. In order to obtain further information on the effects of Gc on cells evolving from the neural crest, we have used the human neuroblastoma cell line SK-N-SH to analyze: 1) the presence and the binding characteristics of Gc receptors in these cells, 2) the effect of dexamethasone (Dex) on the migration of SK-N-SH cells, and 3) the effect of Dex on the organization of the cytoskeleton of SK-N-SH cells. We show that: 1) receptors that bind [³H]-Dex with high affinity and high capacity (Kd of 9.6 nM, Bmax of 47 fmol/mg cytosolic protein, corresponding to 28,303 sites/cell) are present in cytosolic preparations of SK-N-SH cells, and 2) treatment with Dex (in the range of 10 nM to 1 µM) has an inhibitory effect (from 100% to 74 and 43%, respectively) on the chemotaxis of SK-N-SH cells elicited by fetal bovine serum. This inhibition is completely reversed by the Gc receptor antagonist RU486 (1 µM), and 3) as demonstrated by fluorescent phalloidin-actin detection, the effect of Dex (100 nM) on SK-N-SH cell migration is accompanied by modifications of the cytoskeleton organization that appear with stress fibers. These modifications did not take place in the presence of 1 µM RU486. The present data demonstrate for the first time that Dex affects the migration of neuroblastoma cells as well as their cytoskeleton organization by interacting with specific receptors. These findings provide new insights on the mechanism(s) of action of Gc on cells originating in the neural crest.

Inducible nitric oxide synthase and tumor necrosis factor-alpha in delayed gastric emptying and gastrointestinal transit induced by lipopolysaccharide in mice

Gastrointestinal motility disturbances during endotoxemia are probably caused by lipopolysaccharide (LPS)-induced factors: candidates include nitric oxide (NO), tumor necrosis factor-alpha (TNF-alpha), interleukin-1ß, and interleukin-6. Flow cytometry was used to determine the effects of LPS and these factors on gastric emptying (evaluated indirectly by determining percent gastric retention; %GR) and gastrointestinal transit (GIT) in male BALB/c mice (23-28 g). NO (300 µg/mouse, N = 8) and TNF-alpha (2 µg/mouse, N = 7) increased (P < 0.01) GR and delayed GIT, mimicking the effect of LPS (50 µg/mouse). During early endotoxemia (1.5 h after LPS), inhibition of inducible NO synthase (iNOS) by a selective inhibitor, 1400 W (150 µg/mouse, N = 11), but not antibody neutralization of TNF-alpha (200 µg/mouse, N = 11), reversed the increase of GR (%GR 78.8 ± 3.3 vs 47.2 ± 7.5%) and the delay of GIT (geometric center 3.7 ± 0.4 vs 5.6 ± 0.2). During late endotoxemia (8 h after LPS), both iNOS inhibition (N = 9) and TNF-alpha neutralization (N = 9) reversed the increase of GR (%GR 33.7 ± 2.0 vs 19.1 ± 2.6% (1400 W) and 20.1 ± 2.0% (anti-TNF-alpha)), but only TNF-alpha neutralization reversed the delay of GIT (geometric center 3.9 ± 0.4 vs 5.9 ± 0.2). These findings suggest that iNOS, but not TNF-alpha, is associated with delayed gastric emptying and GIT during early endotoxemia and that during late endotoxemia, both factors are associated with delayed gastric emptying, but only TNF-alpha is associated with delayed GIT.

Matching salt intake to physiological need in rats using foraging protocols

Several studies of the quantitative relationship between sodium need and sodium intake in rats are reviewed. Using acute diuretic treatment 24 h beforehand, intake matches need fairly accurately when intake is spread out in time by using a hypotonic solution of NaCl. In contrast, using a hypertonic solution, intake is typically double the need. Using the same diuretic treatment, although the natriuresis occurs within ~1 h, the appetite appears only slowly over 24 h. Increased plasma levels of aldosterone parallel the increased intake; however, treatment with metyrapone blocks the rise in aldosterone but has no effect on appetite. Satiation of sodium appetite was studied in rats using sodium loss induced by chronic diuretic treatment and daily salt consumption sessions. When a simulated foraging cost was imposed on NaCl access in the form of a progressive ratio lever press task, rats showed satiation for NaCl (break point) after consuming an amount close to their estimated deficit. The chronic diuretic regimen produced hypovolemia and large increases in plasma aldosterone concentration and renin activity. These parameters were reversed to or toward non-depleted control values at the time of behavioral satiation in the progressive ratio protocol. Satiation mechanisms for sodium appetite thus do appear to exist. However, they do not operate quantitatively when concentrated salt is available at no effort, but instead allow overconsumption. There are reasons to believe that such a bias toward overconsumption may have been beneficial over evolutionary time, but such biasing for salt and other commodities is maladaptive in a resource-rich environment.

Cl- and regulation of pH by MDCK-C11 cells

The interaction between H+ extrusion via H+-ATPase and Cl- conductance was studied in the C11 clone of MDCK cells, akin to the intercalated cells of the collecting duct. Cell pH (pHi) was measured by fluorescence microscopy using the fluorescein-derived probe BCECF-AM. Control recovery rate measured after a 20 mM NH4Cl acid pulse was 0.136 ± 0.008 pH units/min (dpHi/dt) in Na+ Ringer and 0.032 ± 0.003 in the absence of Na+ (0 Na+). With 0 Na+ plus the Cl- channel inhibitor NPPB (10 µM), recovery was reduced to 0.014 ± 0.001 dpHi/dt. 8-Br-cAMP, known to activate CFTR Cl- channels, increased dpHi/dt in 0 Na+ to 0.061 ± 0.009 and also in the presence of 46 nM concanamycin and 50 µM Schering 28080. Since it is thought that the Cl- dependence of H+-ATPase might be due to its electrogenic nature and the establishment of a +PD (potential difference) across the cell membrane, the effect of 10 µM valinomycin at high (100 mM) K+ was tested in our cells. In Na+ Ringer, dpHi/dt was increased, but no effect was detected in 0 Na+ Ringer in the presence of NPPB, indicating that in intact C11 cells the effect of blocking Cl- channels on dpHi/dt was not due to an adverse electrical gradient. The effect of 100 µM ATP was studied in 0 Na+ Ringer solution; this treatment caused a significant inhibition of dpHi/dt, reversed by 50 µM Bapta. We have shown that H+-ATPase present in MDCK C11 cells depends on Cl- ions and their channels, being regulated by cAMP and ATP, but not by the electrical gradient established by electrogenic H+ transport.

Do early life factors influence body mass index in adolescents?

The association between early life factors and body mass index (BMI) in adulthood has been demonstrated in developed countries. The aim of the present study was to assess the influence of early life factors (birth weight, gestational age, maternal smoking, and social class) on BMI in young adulthood with adjustment for adult socioeconomic position. A cohort study was carried out in 1978/79 with 6827 mother-child pairs from Ribeirão Preto city, located in the most developed economic area of the country. Biological, economic and social variables and newborn anthropometric measurements were obtained shortly after delivery. In 1996, 1189 males from this cohort, 34.3% of the original male population, were submitted to anthropometric measurements and were asked about their current schooling on the occasion of army recruitment. A multiple linear regression model was applied to determine variables associated with BMI. Mean BMI was 22.7 (95%CI = 22.5-23.0). After adjustment, BMI was 1.22 kg/m² higher among infants born with high birth weight (³4000 g), 1.21 kg/m² higher among individuals of low social class at birth and 0.69 kg/m² higher among individuals whose mothers smoked during pregnancy (P < 0.05). The association between social class at birth and BMI remained statistically significant (P < 0.05) even after adjustment for adult schooling. These findings suggest that early life social influences on BMI were more important and were not reversed by late socioeconomic position. Therefore, prevention of overweight and obesity should focus not only on changes in adult life styles but also on factors such as high birth weight.

Antinociceptive properties in mice of a lectin isolated from the marine alga Amansia multifida Lamouroux

The antinociceptive effects of a lectin (LEC) isolated from the marine alga Amansia multifida were determined in Swiss mice. The LEC (1, 5, and 10 mg/kg) inhibited acetic acid-induced abdominal writhings in a dose-dependent manner after intraperitoneal or oral administration. A partial but significant inhibition of writhings was observed after the combination of LEC (10 mg/kg) with avidin (1 mg/kg), a potent inhibitor of the hemmaglutinant activity of the lectin. However, total writhing inhibition was demonstrable in the group of mice treated with LEC plus mannose (1 mg/kg), as compared to LEC alone or to control groups. Furthermore, avidin and mainly mannose also play a role in antinociception, somehow facilitating the interaction of LEC with its active cell sites. In the formalin test, although both phases of the response were significantly inhibited, the effect of LEC was predominant during phase 2, causing inhibition of licking time that ranged from 48 to 88% after oral (5 and 10 mg/kg) and intraperitoneal (1 to 5 mg/kg) administration. As is the case with morphine, the effect of LEC (2 mg/kg) was reversed by naloxone (2 mg/kg), indicating the involvement of the opioid system. LEC was also effective in the hot-plate test, producing inhibitory responses to the thermal stimulus, and its effects were blocked by naloxone. In the pentobarbital-induced sleeping time, although LEC did not alter the onset of sleep significantly, it increased the time of sleep within the same dose range compared to control. These results show that LEC presents antinociceptive effects of both central and peripheral origin, possibly involving the participation of the opioid system.

L-histidine reduces inhibitory avoidance in Carassius auratus submitted to cerebellar ablation

The effect of post-training treatment with L-histidine (LH) on the memory consolidation of inhibitory avoidance was investigated in Carassius auratus submitted to cerebellar ablation. The inhibitory avoidance procedure included 3 days: one habituation day, one training day (5 trials, T1-T5) and one test day. On the training day, each fish was placed individually in a white compartment separated from a black compartment by a sliding door. When the fish crossed into the black compartment, a weight was dropped in front of it (aversive stimulus) and the time to cross was recorded. Saline or LH (100 mg/kg) was injected intraperitoneally 10 min after the trials. Data were log10 transformed and analyzed by ANOVA and the Student-Newman-Keuls test (P < 0.05). In T5, all groups [ablation/LH (N = 15; 189.60 ± 32.52), ablation/saline (N = 14; 204.29 ± 28.95), sham/LH (N = 14; 232.36 ± 28.15), and sham/saline (N = 15; 249.07 ± 25.82)] had similar latencies that were significantly higher than T1 latencies [ablation/LH (89.33 ± 20.41), ablation/saline (97.00 ± 25.16), sham/LH (73.86 ± 18.42), and sham/saline (56.71 ± 17.59)], suggesting acquisition of inhibitory avoidance. For the test, there was a significant reduction in latencies of ablation/LH (61.53 ± 17.70) and sham/saline (52.79 ± 25.37) groups compared to the ablation/saline (213.64 ± 29.57) and sham/LH (199.43 ± 24.48) groups, showing that cerebellum ablation facilitated retention of inhibitory avoidance and LH reversed the effect of ablation. The results support other evidence that LH impairs memory consolidation and/or reduces the interpretation of aversion value.

Biomechanics and structural adaptations of the rat femur after hindlimb suspension and treadmill running

We microscopically and mechanically evaluated the femurs of rats subjected to hindlimb unloading (tail suspension) followed by treadmill training. Female Wistar rats were randomly divided into five groups containing 12-14 rats: control I (118 days old), control II (139 days old), suspended (tail suspension for 28 days), suspended-released (released for 21 days after 28 days of suspension), and suspended-trained (trained for 21 days after 28 days of suspension). We measured bone resistance by bending-compression mechanical tests of the entire proximal half of the femur and three-point bending tests of diaphyseal cortical bone. We determined bone microstructure by tetracycline labeling of trabecular and cortical bone. We found that tail suspension weakened bone (ultimate load = 86.3 ± 13.5 N, tenacity modulus = 0.027 ± 0.011 MPa·m vs ultimate load = 101.5 ± 10.5 N, tenacity modulus = 0.019 ± 0.006 MPa·m in control I animals). The tenacity modulus for suspended and released animals was 0.023 ± 0.010 MPa·m vs 0.046 ± 0.018 MPa·m for trained animals and 0.035 ± 0.010 MPa·m for control animals. These data indicate that normal activity and training resulted in recovered bone resistance, but suspended-released rats presented femoral head flattening and earlier closure of the growth plate. Microscopically, we found that suspension inhibited new bone subperiosteal and endosteal formation. The bone disuse atrophy secondary to hypoactivity in rats can be reversed by an early regime of exercising, which is more advantageous than ordinary cage activities alone.

Antinociceptive effects of the essential oil of Mentha x villosa leaf and its major constituent piperitenone oxide in mice

Mentha x villosa Huds (Labiatae) is an aromatic herb widely used in folk medicine. Since the essential oil of the herb has many pharmacological activities, including antispasmodic effects, we determined whether the oil and its major constituent, piperitenone oxide (PO), have antinociceptive activity. The essential oil of M. x villosa (EOMV) and PO administered orally at 200 mg/kg (vehicle: 0.1% Tween 80 in water) significantly reduced the writhings induced by acetic acid from control values of 59.5 ± 3.1 s (N = 10) to 31.9 ± 2.8 s (N = 10) and 23.8 ± 3.4 s (N = 10), respectively. When administered at 100 and 200 mg/kg, EOMV reduced the paw licking time for the second phase of the formalin test from the control value of 20.6 ± 2.1 s (N = 13) to 5.3 ± 2.2 s (N = 12) and 2.7 ± 1.2 s (N = 18), respectively. At 100 and 200 mg/kg, PO reduced this second phase to 8.3 ± 2.7 s (N = 12) and 3.0 ± 1.2 s (N = 10), respectively. This effect of EOMV and PO was not reversed by naloxone. EOMV and PO had no significant effect on the first phase of the formalin test. As evaluated by the hot-plate and tail immersion test, EOMV and PO, at doses up to 200 mg/kg, showed no analgesic activity. These results show that EOMV and PO have antinociceptive activity and suggest that this effect is probably an indirect anti-inflammatory effect, which does not involve the central nervous system.

Influence of progesterone on GAD65 and GAD67 mRNA expression in the dorsolateral striatum and prefrontal cortex of female rats repeatedly treated with cocaine

Female rats are intensely affected by cocaine, with estrogen probably playing an important role in this effect. Progesterone modulates the GABA system and attenuates the effects of cocaine; however, there is no information about its relevance in changing GABA synthesis pathways after cocaine administration to female rats. Our objective was to investigate the influence of progesterone on the effects of repeated cocaine administration on the isoenzymes of glutamic acid decarboxylase (GAD65 and GAD67) mRNA in brain areas involved in the addiction circuitry. Ovariectomized, intact and progesterone replacement-treated female rats received saline or cocaine (30 mg/kg, ip) acutely or repeatedly. GAD isoenzyme mRNA levels were determined in the dorsolateral striatum (dSTR) and prefrontal cortex (PFC) by RT-PCR, showing that repeated, but not acute, cocaine decreased GADs/β-actin mRNA ratio in the dSTR irrespective of the hormonal condition (GAD65: P < 0.001; and GAD67: P = 0.004). In the PFC, repeated cocaine decreased GAD65 and increased GAD67 mRNA ratio (P < 0.05). Progesterone replacement decreased both GAD isoenzymes mRNA ratio after acute cocaine in the PFC (P < 0.001) and repeated cocaine treatment reversed this decrease (P < 0.001). These results suggest that cocaine does not immediately affect GAD mRNA expression, while repeated cocaine decreases both GAD65 and GAD67 mRNA in the dSTR of female rats, independently of their hormonal conditions. In the PFC, repeated cocaine increases the expression of GAD isoenzymes, which were decreased due to progesterone replacement.

Participation of endogenous opioids in the antinociception induced by resistance exercise in rats

Exercise is a low-cost intervention that promotes health and contributes to the maintenance of the quality of life. The present study was designed to investigate the influence of different resistance exercise protocols on the nociceptive threshold of rats. Female Wistar rats were used to perform exercises in a weight-lifting exercise model. The following groups were examined (N = 6 per group): untrained rats (control group); an acute protocol group consisting of rats submitted to 15 sets of 15 repetitions of resistance exercise (acute group); rats exercised with 3 sets of 10 repetitions, three times per week for 12 weeks (trained group), and a group consisting of trained rats that were further submitted to the acute protocol (trained-acute group). The nociceptive threshold was measured by the paw-withdrawal test, in which the withdrawal threshold (escape reaction) was measured by an apparatus applying force to the plantar surface of the animal paw. The opioid antagonist naloxone (2 mg/kg) was administered subcutaneously 10 min before the exercise protocols. The trained group demonstrated antinociception only up to day 45 of the 12-week training period. A significant increase (37%, P < 0.05) in the nociceptive threshold was produced immediately after exercise, decreasing to 15% after 15 min, when the acute exercise protocol was used. Naloxone reversed this effect. These data show that the acute resistance exercise protocol was effective in producing antinociception for 15 min. This antinociceptive effect is mediated by the activation of opioid receptors.

A safety and feasibility study of cell therapy in dilated cardiomyopathy

The aim of this study was to determine if bone marrow mononuclear cell (BMMC) transplantation is safe for moderate to severe idiopathic dilated cardiomyopathy (IDC). Clinical trials have shown that this procedure is safe and effective for ischemic patients, but little information is available regarding non-ischemic patients. Twenty-four patients with IDC, optimized therapy, age 46 ± 11.6 years, 17 males, NYHA classes II-IV, and left ventricular ejection fraction <35% were enrolled in the study. Clinical evaluation at baseline and 6 months after stem cell therapy to assess heart function included echocardiogram, magnetic resonance imaging, cardiopulmonary test, Minnesota Quality of Life Questionnaire, and NYHA classification. After cell transplantation 1 patient showed a transient increase in enzyme levels and 2 patients presented arrhythmias that were reversed within 72 h. Four patients died during follow-up, between 6 and 12 weeks after therapy. Clinical evaluation showed improvement in most patients as reflected by statistically significant decreases in Minnesota Quality of Life Questionnaire (63 ± 17.9 baseline vs 28.8 ± 16.75 at 6 months) and in class III-IV NYHA patients (18/24 baseline vs 2/20 at 6 months). Cardiopulmonary exercise tests demonstrated increased peak oxygen consumption (12.2 ± 2.4 at baseline vs 15.8 ± 7.1 mL·kg-1·min-1 at 6 months) and walked distance (377.2 ± 85.4 vs 444.1 ± 77.9 m at 6 months) in the 6-min walk test, which was not accompanied by increased left ventricular ejection fraction. Our findings indicate that BMMC therapy in IDC patients with severe ventricular dysfunction is feasible and that larger, randomized and placebo-controlled trials are warranted.

Exercise training and detraining modify the morphological and mechanical properties of single cardiac myocytes obtained from spontaneously hypertensive rats

We determined the effects of exercise training and detraining on the morphological and mechanical properties of left ventricular myocytes in 4-month-old spontaneously hypertensive rats (SHR) randomly divided into the following groups: sedentary for 8 weeks (SED-8), sedentary for 12 weeks (SED-12), treadmill-running trained for 8 weeks (TRA, 16 m/min, 60 min/day, 5 days/week), and treadmill-running trained for 8 weeks followed by 4 weeks of detraining (DET). At sacrifice, left ventricular myocytes were isolated enzymatically, and resting cell length, width, and cell shortening after stimulation at a frequency of 1 Hz (~25°C) were measured. Cell length was greater in TRA than in SED-8 (161.30 ± 1.01 vs 156.10 ± 1.02 μm, P < 0.05, 667 vs 618 cells, respectively) and remained larger after detraining. Cell width and volume were unaffected by either exercise training or detraining. Cell length to width ratio was higher in TRA than in SED-8 (8.50 ± 0.08 vs 8.22 ± 0.10, P < 0.05) and was maintained after detraining. Exercise training did not affect cell shortening, which was unchanged with detraining. TRA cells exhibited higher maximum velocity of shortening than SED-8 (102.01 ± 4.50 vs 82.01 ± 5.30 μm/s, P < 0.05, 70 cells per group), with almost complete regression after detraining. The maximum velocity of relengthening was higher in TRA cells than in SED-8 (88.20 ± 4.01 vs70.01 ± 4.80 μm/s, P < 0.05), returning to sedentary values with detraining. Therefore, exercise training affected left ventricle remodeling in SHR towards eccentric hypertrophy, which remained after detraining. It also improved single left ventricular myocyte contractile function, which was reversed by detraining.

E3 ubiquitin ligase Cbl-b potentiates the apoptotic action of arsenic trioxide by inhibiting the PI3K/Akt pathway

Arsenic trioxide (ATO) is a strong inducer of apoptosis in malignant hematological cells. Inducible phosphatidyl inositol 3 kinase (PI3K)-Akt activation promotes resistance to ATO. In the present study, we evaluated whether E3 ubiquitin ligase Cbl-b, a negative regulator of PI3K activation, is involved in the action of ATO. The effect of ATO on cell viability was measured by the Trypan blue exclusion assay or by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Apoptosis was determined by flow cytometry and protein expression was assayed by Western blotting. ATO decreased the viability of HL60 cells and induced cellular apoptosis, which was accompanied by transient activation of Akt. The PI3K/Akt inhibitor, LY294002, significantly increased ATO-induced apoptosis (P < 0.05). In addition, ATO up-regulated the expression of Cbl-b proteins. Furthermore, ATO inhibited cell viability with an IC50 of 18.54 μM at 24 h in rat basophilic leukemia-2H3 cells. ATO induced cellular apoptosis with transient activation of Akt and Cbl-b was also up-regulated. Rat basophilic leukemia-2H3 cells transfected with a dominant negative (DN) Cbl-b mutation showed overexpression of Cbl-b (DN) and enhanced Akt activation. Compared with cells transfected with vector, ATO-induced apoptosis was decreased and G2/M phase cells were increased at the same concentration (P < 0.05). The PI3K/Akt inhibitor, LY294002, re-sensitized Cbl-b (DN) overexpressing cells to ATO and reversed G2/M arrest (P < 0.05). Taken together, these results suggest that Cbl-b potentiates the apoptotic action of ATO by inhibition of the PI3K/Akt pathway.