Effect of ryanodine on sinus node recovery time determined in vitro

Evidence has indicated that the sarcoplasmic reticulum (SR) might be involved in the generation of spontaneous electrical activity in atrial pacemaker cells. We report the effect of disabling the SR with ryanodine (0.1 µM) on the sinus node recovery time (SNRT) measured in isolated right atria from 4-6-month-old male Wistar rats. Electrogram and isometric force were recorded at 36.5oC. Two methods for sinus node resetting were used: a) pulse: a single stimulus pulse interpolated at coupling intervals of 50, 65 or 80% of the regular spontaneous cycle length (RCL), and b) train: a 2-min train of pulses at intervals of 50, 65 or 80% of RCL. Corrected SNRT (cSNRT) was calculated as the difference between SNRT (first spontaneous cycle length after stimulation interruption) and RCL. Ryanodine only slightly increased RCL (<10%), but decreased developed force by 90%. When the pulse method was used, cSNRT (~40 ms), which represents intranodal/atrial conduction time, was independent of the coupling interval and unaffected by ryanodine. However, cSNRT obtained by the train method was significantly higher for shorter intervals between pulses, indicating the occurrence of overdrive suppression. In this case, ryanodine prolonged cSNRT in a rate-dependent fashion, with a greater effect at shorter intervals. These results indicate that: a) a functional SR, albeit important for force development, does not seem to play a major role in atrial automaticity in the rat; b) disruption of cell Ca2+ homeostasis by inhibition of SR function does not appear to affect conduction; however, it enhances overdrive-induced depression of sinusal automaticity.

Inhibitory effect of high [Mg2+] on the vasopressin-stimulated hydroosmotic permeability of the isolated perfused cortical collecting duct

High magnesium concentration inhibits the effect of arginine vasopressin (AVP) on smooth muscle contraction and platelet aggregation and also influences hepatocyte AVP receptor binding. The aim of this study was to determine the role of magnesium concentration [Mg2+] in AVP-stimulated water transport in the kidney collecting duct. The effect of low and high peritubular [Mg2+] on the AVP-stimulated osmotic water permeability coefficient (Pf) was evaluated in the isolated perfused rabbit cortical collecting duct (CCD). Control tubules bathed and perfused with standard Ringer bicarbonate solution containing 1 mM Mg2+ presented a Pf of 223.9 ± 27.2 µm/s. When Mg2+ was not added to the bathing solution, an increase in the AVP-stimulated Pf to 363.1 ± 57.2 µm/s (P<0.05) was observed. An elevation of Mg2+ to 5 mM resulted in a decrease in Pf to 202.9 ± 12.6 µm/s (P<0.05). This decrease in the AVP-stimulated Pf at 5 mM Mg2+ persisted when the CCDs were returned to 1 mM Mg2+, Pf = 130.2 ± 20.3 µm/s, and was not normalized by the addition of 8-[4-chlorophenylthio]-adenosine 3',5'-cyclic monophosphate, a cAMP analogue, to the preparation. These data indicate that magnesium may play a modulatory role in the action of AVP on CCD osmotic water permeability, as observed in other tissues.

Anxiolytic effect of methylene blue microinjected into the dorsal periaqueductal gray matter

The dorsal periaqueductal gray (DPAG) has been implicated in the behavioral and autonomic expression of defensive reactions. Several results suggest that, along with GABA, glutamate and serotonin, nitric oxide (NO) may play a role in defense reactions mediated by this region. To further investigate this possibility we microinjected methylene blue (MB; 10, 30 or 100 nmol/0.5 µl) into the DPAG of rats submitted to the elevated plus-maze test, an animal model of anxiety. MB has been used as an inhibitor of soluble guanylate cyclase (sGC) to demonstrate cGMP-mediated processes, and there is evidence that NO may exert its biological effects by binding to the heme part of guanylate cyclase, causing an increase in cGMP levels. The results showed that MB (30 nmol) significantly increased the percent of time spent in the open arms (saline = 11.57 ± 1.54, MB = 18.5 ± 2.45, P<0.05) and tended to do the same with the percentage of open arm entries (saline = 25.8 ± 1.97, MB = 33.77 ± 3.07, P<0.10), but did not change the number of enclosed arm entries. The dose-response curve, however, had an inverted U shape. These results indicate that MB, within a limited dose range, has anxiolytic properties when microinjected into the DPAG.

Effect of fatty acids on leukocyte function

Fatty acids have various effects on immune and inflammatory responses, acting as intracellular and intercellular mediators. Polyunsaturated fatty acids (PUFAs) of the omega-3 family have overall suppressive effects, inhibiting lymphocyte proliferation, antibody and cytokine production, adhesion molecule expression, natural killer cell activity and triggering cell death. The omega-6 PUFAs have both inhibitory and stimulatory effects. The most studied of these is arachidonic acid that can be oxidized to eicosanoids, such as prostaglandins, leukotrienes and thromboxanes, all of which are potent mediators of inflammation. Nevertheless, it has been found that many of the effects of PUFA on immune and inflammatory responses are not dependent on eicosanoid generation. Fatty acids have also been found to modulate phagocytosis, reactive oxygen species production, cytokine production and leukocyte migration, also interfering with antigen presentation by macrophages. The importance of fatty acids in immune function has been corroborated by many clinical trials in which patients show improvement when submitted to fatty acid supplementation. Several mechanisms have been proposed to explain fatty acid modulation of immune response, such as changes in membrane fluidity and signal transduction pathways, regulation of gene transcription, protein acylation, and calcium release. In this review, evidence is presented to support the proposition that changes in cell metabolism also play an important role in the effect of fatty acids on leukocyte functioning, as fatty acids regulate glucose and glutamine metabolism and mitochondrial depolarization.

Blockade of NK-1 receptors in the lateral commissural nucleus tractus solitarii of awake rats had no effect on the cardiovascular responses to chemoreflex activation

The neurotransmission of the chemoreflex in the nucleus tractus solitarii (NTS), particularly of the sympatho-excitatory component, is not completely understood. There is evidence that substance P may play a role in the neurotransmission of the chemoreflex in the NTS. Microinjection of substance P (50 pmol/50 nl, N = 12, and 5 nmol/50 nl, N = 8) into the commissural NTS of unanesthetized rats produced a significant increase in mean arterial pressure (101 ± 1 vs 108 ± 2 and 107 ± 3 vs 115 ± 4 mmHg, respectively) and no significant changes in heart rate (328 ± 11 vs 347 ± 15 and 332 ± 7 vs 349 ± 13 bpm, respectively) 2 min after microinjection. Previous treatment with WIN, an NK-1 receptor antagonist (2.5 nmol/50 nl), microinjected into the NTS of a specific group of rats, blocked the pressor (11 ± 5 vs 1 ± 2 mmHg) and tachycardic (31 ± 6 vs 4 ± 3 bpm) responses to substance P (50 pmol/50 nl, N = 5) observed 10 min after microinjection. Bilateral microinjection of WIN into the lateral commissural NTS (N = 8) had no significant effect on the pressor (50 ± 4 vs 42 ± 6 mmHg) or bradycardic (-230 ± 16 vs -220 ± 36 bpm) responses to chemoreflex activation with potassium cyanide (iv). These data indicate that the activation of NK-1 receptors by substance P in the NTS produces an increase in baseline mean arterial pressure and heart rate. However, the data obtained with WIN suggest that substance P and NK-1 receptors do not play a major role in the neurotransmission of the chemoreflex in the lateral commissural NTS.

Effect of epithelial debridement on human cornea proteoglycans

Corneal transparency is attributed to the regular spacing and diameter of collagen fibrils, and proteoglycans may play a role in fibrillogenesis and matrix assembly. Corneal scar tissue is opaque and this opacity is explained by decreased ultrastructural order that may be related to proteoglycan composition. Thus, the objectives of the present study were to characterize the proteoglycans synthesized by human corneal explants and to investigate the effect of mechanical epithelial debridement. Human corneas unsuitable for transplants were immersed in F-12 culture medium and maintained under tissue culture conditions. The proteoglycans synthesized in 24 h were labeled metabolically by the addition of 35S-sulfate to the medium. These compounds were extracted by 4 M GuHCl and identified by a combination of agarose gel electrophoresis, enzymatic degradation with protease and mucopolysaccharidases, and immunoblotting. Decorin was identified as the main dermatan sulfate proteoglycan and keratan sulfate proteoglycans were also prominent components. When the glycosaminoglycan side chains were analyzed, only keratan sulfate and dermatan sulfate were detected (~50% each). Nevertheless, when these compounds were 35S-labeled metabolically, the label in dermatan sulfate was greater than in keratan sulfate, suggesting a lower synthesis rate for keratan sulfate. 35S-Heparan sulfate also appeared. The removal of the epithelial layer caused a decrease in heparan sulfate labeling and induced the synthesis of dermatan sulfate by the stroma. The increased deposit of dermatan sulfate proteoglycans in the stroma suggests a functional relationship between epithelium and stroma that could be related to the corneal opacity that may appear after epithelial cell debridement.

Nasopharyngeal colonization by pathogenic bacteria: effect of polymorphisms in innate immune genes of young children

Nasopharyngeal bacteria can asymptomatically colonize the nasopharynx of infants and young children but are also associated with the development of respiratory infections and diseases. Such nasopharyngeal bacteria include Streptococcus pneumoniae, Moraxella catarrhalis, Haemophilus influenzae and Staphylococcus aureus. The host defense against invading pathogens is largely relies germline-encoded pattern recognition receptors (PRR), which are expressed on the cells of innate immunity, and different cytokines. These include toll-like receptors (TLR), mannose-binding lectin (MBL) and different cytokines such as IL-17A. Single nucleotide polymorphisms (SNP) in these receptors and cytokines have been reported. The aim of this study was to investigate genetic polymorphisms in the genes for TLR2, 3 and 4, MBL as well as for IL-17A and their associations with nasopharyngeal pathogenic bacterial colonization during a two-year follow-up. The study revealed that polymorphisms in TLRs, MBL2 and IL17A are associated with the nasopharyngeal bacterial colonization in young children. Healthy young (2.6 months of age) children with variant types of MBL2, TLR2 R753Q or TLR4 D299G had an increased risk to be colonized by S. pneumonia, S. aureus or M. catarrhalis, respectively. Moreover, variant types of MBL2 in healthy children with might facilitate human rhinovirus (HRV)-induced S. pneumoniae colonization at 2.6 months of age. The polymorphism of TLR4 D299G was shown to be associated with M. catarrhalis colonization throughout the whole two-year follow-up (2.6, 13 and 24 months of age) and also with the bacterial load of this pathogen. Also, the polymorphism of IL17A G152A was shown to be associated with increased risk to be colonized by S. pneumoniae at 13 and 24 months of age. Furthermore, the results suggest that IL17A G152A has an effect on production of serum IL-17A already at young age. In conclusion, the results of this study indicate that polymorphisms in the key PRRs and IL17A seem to play an important role to colonization of S. pneumoniae, M. catarrhalis, and S. aureus in healthy young Finnish children. The nasopharyngeal colonization by these pathogenic bacteria may further promote the development of respiratory infections and may be related to development of asthma and allergy in the later life of children. These findings offer a possible explanation why some children have more respiratory infections than other children and provide a rational basis for future studies in this field.

The effect of an aerobic training program on the electrical remodeling of the heart: high-frequency components of the signal-averaged electrocardiogram are predictors of the maximal aerobic power

Increased heart rate variability (HRV) and high-frequency content of the terminal region of the ventricular activation of signal-averaged ECG (SAECG) have been reported in athletes. The present study investigates HRV and SAECG parameters as predictors of maximal aerobic power (VO2max) in athletes. HRV, SAECG and VO2max were determined in 18 high-performance long-distance (25 ± 6 years; 17 males) runners 24 h after a training session. Clinical visits, ECG and VO2max determination were scheduled for all athletes during thew training period. A group of 18 untrained healthy volunteers matched for age, gender, and body surface area was included as controls. SAECG was acquired in the resting supine position for 15 min and processed to extract average RR interval (Mean-RR) and root mean squared standard deviation (RMSSD) of the difference of two consecutive normal RR intervals. SAECG variables analyzed in the vector magnitude with 40-250 Hz band-pass bi-directional filtering were: total and 40-µV terminal (LAS40) duration of ventricular activation, RMS voltage of total (RMST) and of the 40-ms terminal region of ventricular activation. Linear and multivariate stepwise logistic regressions oriented by inter-group comparisons were adjusted in significant variables in order to predict VO2max, with a P < 0.05 considered to be significant. VO2max correlated significantly (P < 0.05) with RMST (r = 0.77), Mean-RR (r = 0.62), RMSSD (r = 0.47), and LAS40 (r = -0.39). RMST was the independent predictor of VO2max. In athletes, HRV and high-frequency components of the SAECG correlate with VO2max and the high-frequency content of SAECG is an independent predictor of VO2max.

Endothelin-1 receptors play a minor role in the protection against acute Trypanosoma cruzi infection in mice

Chagas' disease, caused by the protozoan Trypanosoma cruzi, is a major cause of cardiovascular disability in countries where it is endemic. Damage to the heart microvasculature has been proposed to be an important factor in the pathogenesis of heart dysfunction. Endothelin-1 (ET-1) is a potent vasoconstrictor and exerts its effects via specific ET A and ET B receptors. A few studies have suggested a role for ET-1 and its receptors in the pathogenesis of Chagas' disease. We investigated the effects of treatment with bosentan, an ET A/ET B receptor antagonist, on the course of T. cruzi infection (Y strain) in C57Bl/6 mice. Treatment with bosentan (100 mg kg-1 day-1) was given per os starting day 0 after infection until sacrifice. Bosentan significantly increased myocardial inflammation, with no effects on parasitemia. Although the total number of nests was similar, a lower number of intact amastigote nests was found in the heart of bosentan-treated animals. Bosentan failed to affect the infection-associated increase in the cardiac levels of the cytokines IFN-g and TNF-a and the chemokines CCL2/MCP-1, CCL3/MIP-1a and CCL5/RANTES. In vitro, pre-incubation with ET-1 (0.1 µM) 4 h before infection enhanced the uptake of the parasites by peritoneal macrophages, and this effect was abrogated when macrophages were pre-treated with bosentan (1 µM) 15 min before incubation with ET-1. However, ET-1 did not alter killing of intracellular parasites after 48 h of in vitro infection. Our data suggest that bosentan-treated mice have a delay in controlling parasitism which is compensated for exacerbated inflammation. Infection is eventually controlled in these animals and lethality is unchanged, demonstrating that ET-1 plays a minor role in the protection against acute murine T. cruzi infection.

TIMP-1 mediates the inhibitory effect of interleukin-6 on the proliferation of a hepatocarcinoma cell line in a STAT3-dependent manner

The tissue inhibitor of metalloproteinases (TIMP)-1 is a multifunctional protein which is not only an inhibitor of matrix metalloproteinases (MMPs) but also to have a possible "cytokine-like" action. Here, we first compared mRNA expression of TIMP-1 and MMP-9 in BEL-7402 (a hepatocellular carcinoma cell line), L-02 (a normal liver cell line) and QSG-7701 (a cell line derived from peripheral tissue of liver carcinoma) using real-time quantitative RT-PCR. By evaluating the variation of the MMP-9/TIMP-1 ratio as an index of reciprocal changes of the expression of the two genes, we observed that the MMP-9/TIMP-1 ratio was about 13- and 5-fold higher in BEL-7402 than in L-02 and QSG-7701, respectively. Significantly, overexpression of TIMP-1 decreased the MMP-9/TIMP-1 ratio in BEL-7402 and then inhibited the cell growth to 60% and reduced the migration to about 30%. Meanwhile, our data showed that interleukin-6 (IL-6) (100 ng/mL) could also inhibited the cell growth of BEL-7402. Further studies indicated that TIMP-1 mediated the inhibitory effect of IL-6 on BEL-7402 cell proliferation in a STAT3-dependent manner, which could further accelerate the expression of the cyclin-dependent kinase inhibitor p21. A dominant negative STAT3 mutant totally abolished IL-6-induced TIMP-1 expression and its biological functions. The present results demonstrate that TIMP-1 may be one of the mediators that regulate the inhibitory effect of IL-6 on BEL-7402 proliferation in which STAT3 signal transduction and p21 up-regulation also play important roles.

Effect of 5-HT1B receptor agonists injected into the prefrontal cortex on maternal aggression in rats

Serotonin (5-HT1B) receptors play an essential role in the inhibition of aggressive behavior in rodents. CP-94,253, a 5-HT1B receptor agonist, can reduce aggression in male mice when administered directly into the ventro-orbitofrontal (VO) prefrontal cortex (PFC). The objective of the current study was to assess the effects of two selective 5-HT1B receptor agonists (CP-94,253 and CP-93,129), microinjected into the VO PFC, on maternal aggressive behavior after social instigation in rats. CP-94,253 (0.56 µg/0.2 µL, N = 8, and 1.0 µg/0.2 µL, N = 8) or CP-93,129 (1.0 µg/0.2 µL, N = 9) was microinjected into the VO PFC of Wistar rats on the 9th day postpartum and 15 min thereafter the aggressive behavior by the resident female against a male intruder was recorded for 10 min. The frequency and duration of aggressive and non-aggressive behaviors were analyzed using ANOVA and post hoc tests. CP-93,129 significantly decreased maternal aggression. The frequency of lateral attacks, bites and pinnings was reduced compared to control, while the non-aggressive behaviors and maternal care were largely unaffected by this treatment. CP-94,253 had no significant effects on aggressive or non-aggressive behaviors when microinjected into the same area of female rats. CP-93,129, a specific 5-HT1B receptor agonist, administered into the VO PFC reduced maternal aggressive behavior, while the CP-94,253 agonist did not significantly affect this behavior after social instigation in female rats. We conclude that only the 5-HT1B receptor agonist CP-93,129 administered into the VO PFC decreased aggression in female rats postpartum after social instigation.

The effect of thyroid hormones on the white adipose tissue gene expression of PAI-1 and its serum concentration

Metabolic syndrome is associated with an increased risk of developing cardiovascular diseases and Plasminogen activator inhibitor 1 (PAI-1) overexpression may play a significant role in this process. A positive correlation between adipose tissue gene expression of PAI-1 and its serum concentration has been reported. Furthermore, high serum levels of thyroid hormones (T3 and T4) and PAI-1 have been observed in obese children. The present study evaluates the impact of thyroid hormone treatment on white adipose tissue PAI-1 gene expression and its serum concentration. Male Wistar rats (60 days old) were treated for three weeks with T4 (50 µg/day, Hyper) or with saline (control). Additionally, 3T3-L1 adipocytes were treated for 24 h with T4 (100 nM) or T3 (100 nM). PAI-1 gene expression was determined by real-time PCR, while the serum concentration of PAI-1 was measured by ELISA using a commercial kit (Innovative Research, USA). Both the serum concentration of PAI-1 and mRNA levels were similar between groups in retroperitoneal and epididymal white adipose tissue. Using 3T3-L1 adipocytes, in vitro treatment with T4 and T3 increased the gene expression of PAI-1, suggesting non-genomic and genomic effects, respectively. These results demonstrate that thyroid hormones have different effects in vitro and in vivo on PAI-1 gene expression in adipocytes.

Immunomodulatory effect of mesenchymal stem cells

Mesenchymal stem cells (MSC) are multipotential nonhematopoietic progenitor cells capable of differentiating into multiple mesenchymal tissues. MSC are able to reconstitute the functional human hematopoietic microenvironment and promote engraftment of hematopoietic stem cells. MSC constitutively express low levels of major histocompatibility complex-I molecules and do not express costimulatory molecules such as CD80, CD86 or CD40, thus lacking immunogenicity. Furthermore, they are able to suppress T- and B-lymphocyte activation and proliferation and may also affect dendritic cell maturation. Based on these properties, MSC are being used in regenerative medicine and also for the treatment of autoimmune diseases and graft-versus-host disease. On the other hand, MSC from patients diagnosed with myelodysplastic syndromes or multiple myeloma display abnormalities, which could play a role in the physiopathology of the disease. Finally, in patients with immune thrombocytopenic purpura, MSC have a reduced proliferative capacity and a lower inhibitory effect on T-cell proliferation compared with MSC from healthy donors.

Effect of activation of canonical Wnt signaling by the Wnt-3a protein on the susceptibility of PC12 cells to oxidative and apoptotic insults

Wnt proteins are involved in tissue development and their signaling pathways play an important role during embryogenesis. Wnt signaling can promote cell survival, which is beneficial for neurons, but could also lead to tumor development in different tissues. The present study investigated the effects of a Wnt protein on the susceptibility of a neural tumor cell line (PC12 cells) to the cytotoxic compounds ferrous sulfate (10 mM), staurosporine (100 and 500 nM), 3-nitropropionic acid (5 mM), and amyloid β-peptide (Aβ25-35; 50 µM). Cells (1 x 10(6) cells/mL) were treated with the Wnt-3a recombinant peptide (200 ng/mL) for 24 h before exposure to toxic insults. The Wnt-3a protein partially protected PC12 cells, with a 6-15% increase in cell viability in the presence of toxic agents, similar to the effect measured using the MTT and lactate dehydrogenase cell viability assays. The Wnt-3a protein increased protein expression of β-catenin by 52% compared to control. These findings suggest that Wnt signaling can protect neural cells against apoptosis induced by toxic agents, which are relevant to the pathogenesis of Alzheimer’s and Huntington’s diseases.

Effect of image resolution manipulation in rearfoot angle measurements obtained with photogrammetry

The aim of this study was to investigate the influence of image resolution manipulation on the photogrammetric measurement of the rearfoot static angle. The study design was that of a reliability study. We evaluated 19 healthy young adults (11 females and 8 males). The photographs were taken at 1536 pixels in the greatest dimension, resized into four different resolutions (1200, 768, 600, 384 pixels) and analyzed by three equally trained examiners on a 96-pixels per inch (ppi) screen. An experienced physiotherapist marked the anatomic landmarks of rearfoot static angles on two occasions within a 1-week interval. Three different examiners had marked angles on digital pictures. The systematic error and the smallest detectable difference were calculated from the angle values between the image resolutions and times of evaluation. Different resolutions were compared by analysis of variance. Inter- and intra-examiner reliability was calculated by intra-class correlation coefficients (ICC). The rearfoot static angles obtained by the examiners in each resolution were not different (P > 0.05); however, the higher the image resolution the better the inter-examiner reliability. The intra-examiner reliability (within a 1-week interval) was considered to be unacceptable for all image resolutions (ICC range: 0.08-0.52). The whole body image of an adult with a minimum size of 768 pixels analyzed on a 96-ppi screen can provide very good inter-examiner reliability for photogrammetric measurements of rearfoot static angles (ICC range: 0.85-0.92), although the intra-examiner reliability within each resolution was not acceptable. Therefore, this method is not a proper tool for follow-up evaluations of patients within a therapeutic protocol.