Interaction between NO and oxytocin: Influence on LHRH release

Nitric oxide synthase (NOS)-containing neurons have been localized in various parts of the CNS. These neurons occur in the hypothalamus, mostly in the paraventricular and supraoptic nuclei and their axons project to the neural lobe of the pituitary gland. We have found that nitric oxide (NO) controls luteinizing hormone-releasing hormone (LHRH) release from the hypothalamus acting as a signal transducer in norepinephrine (NE)-induced LHRH release. LHRH not only releases LH from the pituitary but also induces sexual behavior. On the other hand, it is known that oxytocin also stimulates mating behavior and there is some evidence that oxytocin can increase NE release. Therefore, it occurred to us that oxytocin may also stimulate LHRH release via NE and NO. To test this hypothesis, we incubated medial basal hypothalamic (MBH) explants from adult male rats in vitro. Following a preincubation period of 30 min, MBH fragments were incubated in Krebs-Ringer bicarbonate buffer in the presence of various concentrations of oxytocin. Oxytocin released LHRH at concentrations ranging from 0.1 nM to 1 µM with a maximal stimulatory effect (P<0.001) at 0.1 µM, but with no stimulatory effect at 10 µM. That these effects were mediated by NO was shown by the fact that incubation of the tissues with NG-monomethyl-L-arginine (NMMA), a competitive inhibitor of NOS, blocked the stimulatory effects. Furthermore, the release of LHRH by oxytocin was also blocked by prazocin, an a1-adrenergic receptor antagonist, indicating that NE mediated this effect. Oxytocin at the same concentrations also increased the activity of NOS (P<0.01) as measured by the conversion of [14C]arginine to citrulline, which is produced in equimolar amounts with NO by the action of NOS. The release of LHRH induced by oxytocin was also accompanied by a significant (P<0.02) increase in the release of prostaglandin E2 (PGE2), a mediator of LHRH release that is released by NO. On the other hand, incubation of neural lobes with various concentrations of sodium nitroprusside (NP) (300 or 600 µM), a releaser of NO, revealed that NO acts to suppress (P<0.01) the release of oxytocin. Therefore, our results indicate that oxytocin releases LHRH by stimulating NOS via NE, resulting in an increased release of NO, which increases PGE2 release that in turn induces LHRH release. Furthermore, the released NO can act back on oxytocinergic terminals to suppress the release of oxytocin in an ultrashort-loop negative feedback

Effect of dimethylsulfoxide on sphingomyelinase activity and cholesterol metabolism in Niemann-Pick type C fibroblasts

Niemann-Pick type C (NPC) fibroblasts present a large concentration of cholesterol in their cytoplasm due to a still unidentified deficiency in cholesterol metabolism. The influence of dimethylsulfoxide (DMSO) on the amount of intracellular cholesterol was measured in 8 cultures of normal fibroblasts and in 7 fibroblast cultures from NPC patients. DMSO was added to the fibroblast cultures at three different concentrations (1, 2 and 4%, v/v) and the cultures were incubated for 24 h. Sphingomyelinase activity was significantly increased in both groups of cells only when incubated with 2% DMSO (59.4 ± 9.1 and 77.0 ± 9.1 nmol h-1 mg protein-1, controls without and with 2% DMSO, respectively; 47.7 ± 5.2 and 55.8 ± 4.1 nmol h-1 mg protein-1, NPC without and with 2% DMSO, respectively). However, none of the DMSO concentrations used altered the amount of cholesterol in the cytoplasm of NPC cells (0.704 ± 0.049, 0.659 ± 0.041, 0.688 ± 0.063 and 0.733 ± 0.088 mg/mg protein, without DMSO, 1% DMSO, 2% DMSO and 4% DMSO, respectively). This finding suggests that sphingomyelinase deficiency is a secondary defect in NPC and shows that DMSO failed to remove the stored cholesterol. These data do not support the use of DMSO in the treatment of NPC patients.

Relative influence of age, resting heart rate and sedentary life style in short-term analysis of heart rate variability

In order to assess the relative influence of age, resting heart rate (HR) and sedentary life style, heart rate variability (HRV) was studied in two different groups. The young group (YG) consisted of 9 sedentary subjects aged 15 to 20 years (YG-S) and of 9 nonsedentary volunteers (YG-NS) also aged 15 to 20. The elderly sedentary group (ESG) consisted of 16 sedentary subjects aged 39 to 82 years. HRV was assessed using a short-term procedure (5 min). R-R variability was calculated in the time-domain by means of the root mean square successive differences. Frequency-domain HRV was evaluated by power spectrum analysis considering high frequency and low frequency bands. In the YG the effort tolerance was ranked in a bicycle stress test. HR was similar for both groups while ESG showed a reduced HRV compared with YG. Within each group, HRV displayed a negative correlation with HR. Although YG-NS had better effort tolerance than YG-S, their HR and HRV were not significantly different. We conclude that HRV is reduced with increasing HR or age, regardless of life style. The results obtained in our short-term study agree with others of longer duration by showing that age and HR are the main determinants of HRV. Our results do not support the idea that changes in HRV are related to regular physical activity.

Effect of the acupoints ST-36 (Zusanli) and SP-6 (Sanyinjiao) on intestinal myoelectric activity of Wistar rats

Despite its ancient use as a therapeutic tool to treat several ailments, acupuncture still faces the challenge of scrutiny by Western science both in terms of its efficacy and in terms of the characterization of its effects and mechanisms of actions underlying these effects. We investigated under well-controlled and carefully characterized conditions the influence of electrical stimulation of acupuncture points ST-36 (Zusanli) and SP-6 (Sanyinjiao) on the myoelectric activity of the small intestine of 38 adult male Wistar rats. Electrical recordings obtained by means of four electrodes chronically implanted in the small intestine were used to assess the effects of acupuncture (electroacupuncture stimulation set at 2 Hz, intermittent stimulation, 1 V, for 30 min). Immobilization of the animals was associated with a consistent decrease (-8 ± 7%) in the myoelectric activity of the small intestine as measured by means of the root mean square. Conversely, acupuncture was able to significantly increase (overshoot) this activity compared to baseline (+44 ± 7%). In contrast, immobilized animals subjected to sham acupuncture had only modest (nonsignificant) increases in myoelectric activity (+9 ± 6%). Using carefully controlled conditions we confirmed previous noncontrolled studies on the ability of acupuncture to alter intestinal motility. The characterization of the topographic and temporal profiles of the effects observed here represents a basis for future dissection of the physiological and pharmacological systems underlying these effects.

Effect of perimuscular injection of Bothrops jararacussu venom on plasma creatine kinase levels in mice: influence of dose and volume

The effect of dose and volume of a perimuscular injection of Bothrops jararacussu venom on myonecrosis of skeletal muscle was studied in mice. An increase of the venom dose (0.25 to 2.0 µg/g) at a given volume (50 µl) resulted in an increase in plasma creatine kinase (CK) levels 2 h after injection. Plasma CK activity increased from the basal level of 129.27 ± 11.83 (N = 20) to 2392.80 ± 709.43 IU/l (N = 4) for the 1.0 µg/g dose. Histological analysis of extensor digitorum longus muscle 4 h after injection showed lesion of peripheral muscle fibers, disorganization of the bundles or the complete degeneration of muscle fibers. These lesions were more extensive when higher doses were injected. Furthermore, an increase in volume (12.5 to 100 µl) by dilution of a given dose (0.5 µg/g) also increased plasma CK levels from 482.31 ± 122.79 to 919.07 ± 133.33 IU/l (N = 4), respectively. These results indicate that care should be taken to standardize volumes and sites of venom injections.

Cardiovascular responses to locomotor activity and feeding in unrestrained three-toed sloths, Bradypus variegatus

Heart rate (HR) and systolic (SBP), diastolic (DBP) and mean (MBP) blood pressure were recorded by biotelemetry in nine conscious unrestrained sloths for 1 min every 15 min over a 24-h period. The animals were allowed to freely move in an acoustically isolated and temperature-controlled (24 ± 1ºC) experimental room with light-dark cycle (12/12 h). Behavior was closely monitored through a unidirectional visor and classified as resting (sitting or suspended), feeding (chewing and swallowing embauba leaves, Cecropia adenops), or locomotor activity around the tree trunk or on the room floor. Locomotor activity caused statistically significant increases in SBP (+8%, from 121 ± 22 to 131 ± 18 mmHg), DBP (+7%, from 86 ± 17 to 92 ± 10 mmHg), MBP (+8%, from 97 ± 19 to 105 ± 12 mmHg), and HR (+14%, from 84 ± 15 to 96 ± 15 bpm) compared to resting values, indicating a possible major influence of the autonomic nervous system on the modulation of cardiac function during this behavior. During feeding, the increase in blood pressure was even higher (SBP +27%, from 119 ± 21 to 151 ± 21 mmHg; DBP +21%, from 85 ± 16 to 103 ± 15 mmHg; MBP +24%, from 96 ± 17 to 119 ± 17 mmHg), while HR remained at 14% (from 84 ± 15 to 96 ± 10 bpm) above resting values. The proportionally greater increase in blood pressure than in HR during feeding suggests an increase in peripheral vascular resistance as part of the overall response to this behavior.

Mineralocorticoid modulation of central angiotensin-induced neuronal activity, water intake and sodium appetite

Central angiotensin II (AngII) stimulates water and salt solution intake. Pretreatment with low-dose mineralocorticoid (DOCA) enhances this AngII-induced intake of salt solutions (the synergy theory) in Wistar and Sprague Dawley rats but not in Fischer rats. This response is mediated via the AT-1 receptor. Electrophysiological experiments using iontophoretic application of AngII and the AT-1 receptor-specific non-peptide antagonist losartan showed excitation of neurons in the preoptic/medial septum region of urethane-anesthetized male Wistar rats. DOCA pretreatment further enhances this neuronal excitation in response to AngII and reduces the responses to losartan. This generated the hypothesis that DOCA-enhanced AngII-induced neuronal excitation is the neural support for the synergy theory. AT-2 receptors modulate these intake responses depending on sodium in the diet, and diuretic-induced dehydration during pregnancy produces a higher salt intake in the offspring. AngII-induced salt and water intakes were tested in offspring from Sprague Dawley mothers with only 1.8% NaCl to drink in which half were treated with furosemide. The important observations were a) the AT-1 antagonist alone suppressed intakes in offspring from mothers not treated with furosemide, b) both AT-1 and AT-2 antagonists suppressed intakes in offspring from furosemide-treated mothers, and c) combined administration of AT-1 and AT-2 antagonists greatly suppressed water intake in offspring from mothers not treated with furosemide. These results suggest that AT-1 and AT-2 receptors have variable properties (receptor number and/or second messengers). Furthermore, the activity and function of these central AngII receptors depend on the background mineralocorticoid levels. The exact mechanism of this influence, however, remains to be determined.

Protein tyrosine kinase inhibitors modify kainic acid-induced epileptiform activity and mossy fiber sprouting but do not protect against limbic cell death

Intrahippocampal administration of kainic acid (KA) induces synaptic release of neurotrophins, mainly brain-derived neurotrophic factor, which contributes to the acute neuronal excitation produced by the toxin. Two protein tyrosine kinase inhibitors, herbimycin A and K252a, were administered intracerebroventricularly, in a single dose, to attenuate neurotrophin signaling during the acute effects of KA, and their role in epileptogenesis was evaluated in adult, male Wistar rats weighing 250-300 g. The latency for the first Racine stage V seizure was 90 ± 8 min in saline controls (N = 4) which increased to 369 ± 71 and 322 ± 63 min in animals receiving herbimycin A (1.74 nmol, N = 4) and K252a (10 pmol, N = 4), respectively. Behavioral alterations were accompanied by diminished duration of EEG paroxysms in herbimycin A- and K252a-treated animals. Notwithstanding the reduction in seizure severity, cell death (60-90% of cell loss in KA-treated animals) in limbic regions was unchanged by herbimycin A and K252a. However, aberrant mossy fiber sprouting was significantly reduced in the ipsilateral dorsal hippocampus of K252a-treated animals. In this model of temporal lobe epilepsy, both protein kinase inhibitors diminished the acute epileptic activity triggered by KA and the ensuing morphological alterations in the dentate gyrus without diminishing cell loss. Our current data indicating that K252a, but not herbimycin, has an influence over KA-induced mossy fiber sprouting further suggest that protein tyrosine kinase receptors are not the only factors which control this plasticity. Further experiments are necessary to elucidate the exact signaling systems associated with this K252a effect.

Influence of renal denervation on blood pressure, sodium and water excretion in acute total obstructive apnea in rats

Obstructive apnea (OA) can exert significant effects on renal sympathetic nerve activity (RSNA) and hemodynamic parameters. The present study focuses on the modulatory actions of RSNA on OA-induced sodium and water retention. The experiments were performed in renal-denervated rats (D; N = 9), which were compared to sham (S; N = 9) rats. Mean arterial pressure (MAP) and heart rate (HR) were assessed via an intrafemoral catheter. A catheter was inserted into the bladder for urinary measurements. OA episodes were induced via occlusion of the catheter inserted into the trachea. After an equilibration period, OA was induced for 20 s every 2 min and the changes in urine, MAP, HR and RSNA were recorded. Renal denervation did not alter resting MAP (S: 113 ± 4 vs D: 115 ± 4 mmHg) or HR (S: 340 ± 12 vs D: 368 ± 11 bpm). An OA episode resulted in decreased HR and MAP in both groups, but D rats showed exacerbated hypotension and attenuated bradycardia (S: -12 ± 1 mmHg and -16 ± 2 bpm vs D: -16 ± 1 mmHg and 9 ± 2 bpm; P < 0.01). The basal urinary parameters did not change during or after OA in S rats. However, D rats showed significant increases both during and after OA. Renal sympathetic nerve activity in S rats increased (34 ± 9%) during apnea episodes. These results indicate that renal denervation induces elevations of sodium content and urine volume and alters bradycardia and hypotension patterns during total OA in unconscious rats.

Effects of a methanolic fraction of soybean seeds on the transcriptional activity of peroxisome proliferator-activated receptors (PPAR)

Since the anti-inflammatory, antidiabetic and hypolipidemic effects of soy isoflavones may be mediated by activation of peroxisome proliferator-activated receptors (PPAR), the present study investigated whether the methanolic fractions obtained from soybean seeds (E1) and soybean seed coats with hypocotyls (E2) could influence PPARα, PPARγ and PPARβ/δ transcriptional activity. The isoflavones from E1 and E2 were quantified by HPLC analysis. E1 and E2 were rich in isoflavones (daidzin, glycitin, genistin, malonyldaidzin, malonylglycitin, malonylgenistin, daidzein, glycitein, and genistein). Moreover, E1 and E2 showed no evidence of genetically modified material containing the gene CP4 EPSPS. To investigate PPAR transcriptional activity, human promonocytic U-937 cells were treated with E1 and E2 (200, 400, 800, and 1600 µg/mL), positive controls or vehicle. Data are reported as fold-activation of the luciferase reporter driven by the PPAR-responsive element. Dose-response analysis revealed that E1 and E2 induced the transcriptional activity of PPARα (P < 0.001), with activation comparable to that obtained with 0.1 mM bezafibrate (positive control) at 1600 µg/mL (4-fold) and 800 µg/mL (9-fold), respectively. In addition, dose-response analysis revealed that E1 and E2 activated PPARβ/δ (P < 0.05), and the activation at 800 µg/mL (4- and 9-fold, respectively) was comparable to that of 0.1 mM bezafibrate (positive control). However, no effect on PPARγ was observed. Activation of PPARα is consistent with the lipid-lowering activity of soy isoflavones in vivo, but further studies are needed to determine the physiological significance of PPARβ/δ activation.

Influence of the polymorphisms of the α-major regulatory element HS-40 on in vitro gene expression

The α-MRE is the major regulatory element responsible for the expression of human α-like globin genes. It is genetically polymorphic, and six different haplotypes, named A to F, have been identified in some population groups from Europe, Africa and Asia and in native Indians from two Brazilian Indian tribes. Most of the mutations that constitute the α-MRE haplotypes are located in flanking sequences of binding sites for nuclear factors. To our knowledge, there are no experimental studies evaluating whether such variability may influence the α-MRE enhancer activity. We analyzed and compared the expression of luciferase of nine constructs containing different α-MRE elements as enhancers. Genomic DNA samples from controls with A (wild-type α-MRE) and B haplotypes were used to generate C-F haplotypes by site-directed mutagenesis. In addition, three other elements containing only the G→A polymorphism at positions +130, +199, and +209, separately, were also tested. The different α-MRE elements were amplified and cloned into a plasmid containing the luciferase reporter gene and the SV40 promoter and used to transiently transfect K562 cells. A noticeable reduction in luciferase expression was observed with all constructs compared with the A haplotype. The greatest reductions occurred with the F haplotype (+96, C→A) and the isolated polymorphism +209, both located near the SP1 protein-binding sites believed not to be active in vivo. These are the first analyses of α-MRE polymorphisms on gene expression and demonstrate that these single nucleotide polymorphisms, although outside the binding sites for nuclear factors, are able to influence in vitro gene expression.

Influence of eNOS gene polymorphism on cardiometabolic parameters in response to physical training in postmenopausal women

The health-promoting effects of exercise training (ET) are related to nitric oxide (NO) production and/or its bioavailability. The objective of this study was to determine whether single nucleotide polymorphism of the endothelial NO synthase (eNOS) gene at positions -786T>C, G894T (Glu298Asp) and at the variable number of tandem repeat (VNTR) Intron 4b/a would interfere with the cardiometabolic responses of postmenopausal women submitted to physical training. Forty-nine postmenopausal women were trained in sessions of 30-40 min, 3 days a week for 8 weeks. Genotypes, oxidative stress status and cardiometabolic parameters were then evaluated in a double-blind design. Both systolic and diastolic blood pressure values were significantly reduced after ET, which was genotype-independent. However, women without eNOS gene polymorphism at position -786T>C (TT genotype) and Intron 4b/a (bb genotype) presented a better reduction of total cholesterol levels (-786T>C: before = 213 ± 12.1, after = 159.8 ± 14.4, Δ = -24.9% and Intron 4b/a: before = 211.8 ± 7.4, after = 180.12 ± 6.4 mg/dL, Δ = -15%), and LDL cholesterol (-786T>C: before = 146.1 ± 13.3, after = 82.8 ± 9.2, Δ = -43.3% and Intron 4b/a: before = 143.2 ± 8, after = 102.7 ± 5.8 mg/dL, Δ = -28.3%) in response to ET compared to those who carried the mutant allele. Superoxide dismutase activity was significantly increased in trained women whereas no changes were observed in malondialdehyde levels. Women without eNOS gene polymorphism at position -786T>C and Intron 4b/a showed a greater reduction of plasma cholesterol levels in response to ET. Furthermore, no genotype influence was observed on arterial blood pressure or oxidative stress status in this population.

The influence of stress on fear memory processes

It is well recognized that stressful experiences promote robust emotional memories, which are well remembered. The amygdaloid complex, principally the basolateral complex (BLA), plays a pivotal role in fear memory and in the modulation of stress-induced emotional responses. A large number of reports have revealed that GABAergic interneurons provide a powerful inhibitory control of the activity of projecting glutamatergic neurons in the BLA. Indeed, a reduced GABAergic control in the BLA is essential for the stress-induced influence on the emergence of associative fear memory and on the generation of long-term potentiation (LTP) in BLA neurons. The extracellular signal-regulated kinase (ERK) subfamily of the mitogen-activated protein kinase (MAPK) signaling pathway in the BLA plays a central role in the consolidation process and synaptic plasticity. In support of the view that stress facilitates long-term fear memory, stressed animals exhibited a phospho-ERK2 (pERK2) increase in the BLA, suggesting the involvement of this mechanism in the promoting influence of threatening stimuli on the consolidation fear memory. Moreover, the occurrence of reactivation-induced lability is prevented when fear memory is encoded under intense stressful conditions since the memory trace remains immune to disruption after recall in previously stressed animals. Thus, the underlying mechanism in retrieval-induced instability seems not to be functional in memories formed under stress. All these findings are indicative that stress influences both the consolidation and reconsolidation fear memory processes. Thus, it seems reasonable to propose that the emotional state generated by an environmental challenge critically modulates the formation and maintenance of long-term fear memory.

Genetic and environmental influences on blood pressure and physical activity: a study of nuclear families from Muzambinho, Brazil

Blood pressure (BP) and physical activity (PA) levels are inversely associated. Since genetic factors account for the observed variation in each of these traits, it is possible that part of their association may be related to common genetic and/or environmental influences. Thus, this study was designed to estimate the genetic and environmental correlations of BP and PA phenotypes in nuclear families from Muzambinho, Brazil. Families including 236 offspring (6 to 24 years) and their 82 fathers and 122 mothers (24 to 65 years) were evaluated. BP was measured, and total PA (TPA) was assessed by an interview (commuting, occupational, leisure time, and school time PA). Quantitative genetic modeling was used to estimate maximal heritability (h²), and genetic and environmental correlations. Heritability was significant for all phenotypes (systolic BP: h² = 0.37 ± 0.10, P < 0.05; diastolic BP: h² = 0.39 ± 0.09, P < 0.05; TPA: h² = 0.24 ± 0.09, P < 0.05). Significant genetic (r g) and environmental (r e) correlations were detected between systolic and diastolic BP (r g = 0.67 ± 0.12 and r e = 0.48 ± 0.08, P < 0.05). Genetic correlations between BP and TPA were not significant, while a tendency to an environmental cross-trait correlation was found between diastolic BP and TPA (r e = -0.18 ± 0.09, P = 0.057). In conclusion, BP and PA are under genetic influences. Systolic and diastolic BP share common genes and environmental influences. Diastolic BP and TPA are probably under similar environmental influences.

Influence of the dopaminergic system, CREB, and transcription factor-κB on cocaine neurotoxicity

Cocaine is a widely used drug and its abuse is associated with physical, psychiatric and social problems. Abnormalities in newborns have been demonstrated to be due to the toxic effects of cocaine during fetal development. The mechanism by which cocaine causes neurological damage is complex and involves interactions of the drug with several neurotransmitter systems, such as the increase of extracellular levels of dopamine and free radicals, and modulation of transcription factors. The aim of this review was to evaluate the importance of the dopaminergic system and the participation of inflammatory signaling in cocaine neurotoxicity. Our study showed that cocaine activates the transcription factors NF-κB and CREB, which regulate genes involved in cellular death. GBR 12909 (an inhibitor of dopamine reuptake), lidocaine (a local anesthetic), and dopamine did not activate NF-κB in the same way as cocaine. However, the attenuation of NF-κB activity after the pretreatment of the cells with SCH 23390, a D1 receptor antagonist, suggests that the activation of NF-κB by cocaine is, at least partially, due to activation of D1 receptors. NF-κB seems to have a protective role in these cells because its inhibition increased cellular death caused by cocaine. The increase in BDNF (brain-derived neurotrophic factor) mRNA can also be related to the protective role of both CREB and NF-κB transcription factors. An understanding of the mechanisms by which cocaine induces cell death in the brain will contribute to the development of new therapies for drug abusers, which can help to slow down the progress of degenerative processes.