Aerobic exercise training engages cholinergic signaling to improve emphysema induced by cigarette smoke exposure in mice.

Lung inflammation is modulated by cholinergic signaling and exercise training protects mice against pulmonary emphysema development; however, whether exercise training engages cholinergic signaling is unknown. AIMS: As cholinergic signaling is directly linked to the vesicular acetylcholine transporter (VAChT) levels, we evaluated whether the effects of aerobic exercise training depend on the VAChT levels in mice with pulmonary emphysema. MAIN METHODS: Wild-type (WT) and mutant (KDHOM) mice (65-70% of reduction in VAChT levels) were exposed to cigarette smoke (30 min, 2×/day, 5×/week, 12 weeks) and submitted or not to aerobic exercise training on a treadmill (60 min/day, 5×/week, 12 weeks). Lung function and inflammation were evaluated. KEY FINDINGS: Cigarette smoke reduced body mass in mice (p < 0.001) and increased alveolar diameter (p < 0.001), inflammation (p < 0.001) and collagen deposition (p < 0.01) in lung tissue. Both trained groups improved their performance in the final physical test compared to the initial test (p < 0.001). In WT mice, exercise training protected against emphysema development (p < 0.05), reduced mononuclear cells infiltrate (p < 0.001) and increased MAC-2 positive cells in lung parenchyma (p < 0.05); however, these effects were not observed in KDHOM mice. The exercise training reduced iNOS-positive cells (p < 0.001) and collagen fibers deposition (p < 0.05) in lung parenchyma of WT and KDHOM mice, although KDHOM mice showed higher levels of iNOS-positive cells. SIGNIFICANCE: Our data suggest that the protective effects of aerobic exercise training on pulmonary emphysema are, at least in part, dependent on the integrity of the lung cholinergic signaling.

Neuronal cholinergic signaling constrains norepinephrine activity in the heart.

It is well known that cholinergic hypofunction contributes to cardiac pathology, yet, the mechanisms involved remain unclear. Our previous study has shown that genetically engineered model of cholinergic deficit, the vesicular acetylcholine transporter knockdown homozygous (VAChT KDHOM) mice, exhibit pathological cardiac remodeling and a gradual increase in cardiac mass with aging. Given that an increase in cardiac mass is often caused by adrenergic hyperactivity, we hypothesized that VAChT KDHOM mice might have an increase in cardiac norepinephrine (NE) levels. We thus investigated the temporal changes in NE content in the heart from 3-, 6-, and 12-mo-old VAChT mutants. Interestingly, mice with cholinergic hypofunction showed a gradual elevation in cardiac NE content, which was already increased at 6 mo of age. Consistent with this finding, 6-mo-old VAChT KDHOM mice showed enhanced sympathetic activity and a greater abundance of tyrosine hydroxylase positive sympathetic nerves in the heart. VAChT mutants exhibited an increase in peak calcium transient, and mitochondrial oxidative stress in cardiomyocytes along with enhanced G protein-coupled receptor kinase 5 (GRK5) and nuclear factor of activated T-cells (NFAT) staining in the heart. These are known targets of adrenergic signaling in the cell. Moreover, vagotomized-mice displayed an increase in cardiac NE content confirming the data obtained in VAChT KDHOM mice. Establishing a causal relationship between acetylcholine and NE, VAChT KDHOM mice treated with pyridostigmine, a cholinesterase inhibitor, showed reduced cardiac NE content, rescuing the phenotype. Our findings unveil a yet unrecognized role of cholinergic signaling as a modulator of cardiac NE, providing novel insights into the mechanisms that drive autonomic imbalance.

Increased cholinergic activity under conditions of low estrogen leads to adverse cardiac remodeling.

Cholinesterase inhibitors are used in postmenopausal women for the treatment of neurodegenerative diseases. Despite their widespread use in the clinical practice, little is known about the impact of augmented cholinergic signaling on cardiac function under reduced estrogen conditions. To address this gap, we subjected a genetically engineered murine model of systemic vesicular acetylcholine transporter overexpression (Chat-ChR2) to ovariectomy and evaluated cardiac parameters. Left-ventricular function was similar between Chat-ChR2 and wild-type (WT) mice. Following ovariectomy, WT mice showed signs of cardiac hypertrophy. Conversely, ovariectomized (OVX) Chat-ChR2 mice evolved to cardiac dilation and failure. Transcript levels for cardiac stress markers atrial natriuretic peptide (ANP) and B-type natriuretic peptide (BNP) were similarly upregulated in WT/OVX and Chat-ChR2/OVX mice. 17ß-Estradiol (E2) treatment normalized cardiac parameters in Chat-ChR2/OVX to the Chat-ChR2/SHAM levels, providing a link between E2 status and the aggravated cardiac response in this model. To investigate the cellular basis underlying the cardiac alterations, ventricular myocytes were isolated and their cellular area and contractility were assessed. Myocytes from WT/OVX mice were wider than WT/SHAM, an indicative of concentric hypertrophy, but their fractional shortening was similar. Conversely, Chat-ChR2/OVX myocytes were elongated and presented contractile dysfunction. E2 treatment again prevented the structural and functional changes in Chat-ChR2/OVX myocytes. We conclude that hypercholinergic mice under reduced estrogen conditions do not develop concentric hypertrophy, a critical compensatory adaptation, evolving toward cardiac dilation and failure. This study emphasizes the importance of understanding the consequences of cholinesterase inhibition, used clinically to treat dementia, for cardiac function in postmenopausal women.

Evaluation of the neuromuscular junction in a middle-aged mouse model of congenital myasthenic syndrome.

INTRODUCTION: Reduced expression of the vesicular acetylcholine transporter (VAChT) leads to changes in the distribution and shape of synaptic vesicles (SVs) at neuromuscular junctions (NMJs), suggesting vesicular acetylcholine (ACh) as a key component of synaptic structure and function. It is poorly understood how long-term changes in cholinergic transmission contribute to age- and disease-related degeneration in the motor system. METHODS: In this study we performed confocal imaging, electrophysiology, electron microscopy, and analyses of respiratory mechanics of the diaphragm NMJ components in 12-month-old wild-type (WT) and VAChTKDHOM mice. RESULTS: Diaphragms of NMJs of the VAChTKDHOM mice were similar to those in WT mice in number, colocalization, and fragmentation of pre-/postsynaptic components. However, they had increased spontaneous SV exocytosis, miniature endplate potential frequency, and diminished MEPP amplitude. No impairment in respiratory mechanics at rest was observed, probably due to the large neurotransmission safety factor of the diaphragm. DISCUSSION: The present findings help us to understand the consequences of reduced ACh release at the NMJs during aging.

Reduced expression of VAChT increases renal fibrosis.

Chronic kidney disease (CKD) is associated with several other long-lasting conditions such as diabetes and cardiovascular diseases and it is a significant contributor to mortality worldwide. Obstructive kidney disease is one of the leading causes of CKD in children and may result from a wide variety of pathologic processes. Recent studies have shown that α7 nicotinic acetylcholine receptor (α7 nAChR) activation in the cholinergic anti-inflammatory pathway reduces production of inflammatory mediators and consequently prevents tissue injury and death. Here, we examined the role of endogenous release of acetylcholine on the development of fibrosis in renal tissue using a model of unilateral ureter obstruction (UUO)-induced CKD, in which obstruction promotes inflammation-mediated kidney damages. To interfere with acetylcholine secretion, we used mice in which the vesicular acetylcholine transporter is genetically reduced (VAChT KD(hom) mice). We observed a higher renal damage in VAChT mutant mice when compared to wild type controls, exemplified by higher proteinuria and increased amount of type 1 collagen in the kidney tissue, indicating accentuated fibrogenesis. These results were accompanied by enhanced localized kidney inflammation, with increased TH1/TH17 profile response. Administration of PNU-282987, a selective agonist of α7 nAChR, significantly attenuated kidney injury after UUO in VAChT KD(hom) mice, indicating that the lack of acetylcholine release decrease the action of the cholinergic anti-inflammatory pathway, promoting an up-regulation of pro-inflammatory and pro-fibrotic pathways. These results suggest that physiological activation of the cholinergic anti-inflammatory pathway regulates inflammatory responses in the kidney suggesting a new therapeutic approach for kidney disease.

Vesicular acetylcholine transporter knock down-mice are more susceptible to inflammation, c-Fos expression and sickness behavior induced by lipopolysaccharide.

In addition to the well-known functions as a neurotransmitter, acetylcholine (ACh) can modulate of the immune system. Nonetheless, how endogenous ACh release inflammatory responses is still not clear. To address this question, we took advantage of an animal model with a decreased ACh release due a reduction (knockdown) in vesicular acetylcholine transporter (VAChT) expression (VAChT-KD(HOM)). These animals were challenged with lipopolysaccharide (LPS). Afterwards, we evaluated sickness behavior and quantified systemic and cerebral inflammation as well as neuronal activation in the dorsal vagal complex (DVC). VAChT-KD(HOM) mice that were injected with LPS (10mg/kg) showed increased mortality rate as compared to control mice. In line with this result, a low dose of LPS (0.1mg/kg) increased the levels of pro-inflammatory (TNF-α, IL-1ß, and IL-6) and anti-inflammatory (IL-10) cytokines in the spleen and brain of VAChT-KD(HOM) mice in comparison with controls. Similarly, serum levels of TNF-α and IL-6 were increased in VAChT-KD(HOM) mice. This excessive cytokine production was completely prevented by administration of a nicotinic receptor agonist (0.4mg/kg) prior to the LPS injection. Three hours after the LPS injection, c-Fos expression increased in the DVC region of VAChT-KD(HOM) mice compared to controls. In addition, VAChT-KD(HOM) mice showed behavioral changes such as lowered locomotor and exploratory activity and reduced social interaction after the LPS challenge, when compared to control mice. Taken together, our results show that the decreased ability to release ACh exacerbates systemic and cerebral inflammation and promotes neural activation and behavioral changes induced by LPS. In conclusion, our findings support the notion that activity of cholinergic pathways, which can be modulated by VAChT expression, controls inflammatory and neural responses to LPS challenge.

Autonomic nervous system modulation affects the inflammatory immune response in mice with acute Chagas disease.

The aim of the present study was to evaluate the effects of changes to the autonomic nervous system in mice during the acute phase of Chagas disease, which is an infection caused by the parasite Trypanosoma cruzi. The following types of mice were inoculated with T. cruzi (CHG): wild-type (WT) and vesicular acetylcholine transporter knockdown (KDVAChT) C57BL/6j mice; wild-type non-treated (NT) FVB mice; FVB mice treated with pyridostigmine bromide (PYR) or salbutamol (SALB); and ß(2)-adrenergic receptor knockout (KOß2) FVB mice. During infection and at 18-21 days after infection (acute phase), the survival curves, parasitaemia, electrocardiograms, heart rate variability, autonomic tonus and histopathology of the animals were evaluated. Negative control groups were matched for age, genetic background and treatment. The KDVAChT-CHG mice exhibited a significant shift in the electrocardiographic, autonomic and histopathological profiles towards a greater inflammatory immune response that was associated with a reduction in blood and tissue parasitism. In contrast, the CHG-PYR mice manifested reduced myocardial inflammation and lower blood and tissue parasitism. Similar results were observed in CHG-SALB animals. Unexpectedly, the KOß2-CHG mice exhibited less myocardial inflammation and higher blood and tissue parasitism, which were associated with reduced mortality. These findings could have been due to the increase in vagal tone observed in the KOß2 mice, which rendered them more similar to the CHG-PYR animals. In conclusion, our results indicate a marked immunomodulatory role for the parasympathetic and sympathetic autonomic nervous systems, which inhibit both the inflammatory immune response and parasite clearance during the acute phase of experimental Chagas heart disease in mice.

Antinociceptive effect of Brazilian armed spider venom toxin Tx3-3 in animal models of neuropathic pain.

Venoms peptides have produced exceptional sources for drug development to treat pain. In this study we examined the antinociceptive and side effects of Tx3-3, a peptide toxin isolated from Phoneutria nigriventer venom, which inhibits high-voltage-dependent calcium channels (VDCC), preferentially P/Q and R-type VDCC. We tested the effects of Tx3-3 in animal models of nociceptive (tail-flick test), neuropathic (partial sciatic nerve ligation and streptozotocin-induced diabetic neuropathy), and inflammatory (intraplantar complete Freund's adjuvant) pain. In the tail-flick test, both intrathecal (i.t.) and intracerebroventricular (i.c.v.) injection of Tx3-3 in mice caused a short-lasting effect (ED(50) and 95% confidence intervals of 8.8 [4.1-18.8] and 3.7 [1.6-8.4] pmol/site for i.t. and i.c.v. injection, respectively), without impairing motor functions, at least at doses 10-30 times higher than the effective dose. By comparison, ω-conotoxin MVIIC, a P/Q and N-type VDCC blocker derived from Conus magus venom, caused significant motor impairment at doses close to efficacious dose in tail flick test. Tx3-3 showed a long-lasting antinociceptive effect in neuropathic pain models. Intrathecal injection of Tx3-3 (30 pmol/site) decreased both mechanical allodynia produced by sciatic nerve injury in mice and streptozotocin-induced allodynia in mice and rats. On the other hand, i.t. injection of Tx3-3 did not alter inflammatory pain. Taken together, our data show that Tx3-3 shows prevalent antinociceptive effects in the neuropathic pain models and does not cause adverse motor effects at antinociceptive efficacious doses, suggesting that this peptide toxin holds promise as a novel therapeutic agent for the control of neuropathic pain. The Brazilian armed spider Tx3-3, a new P/Q and R-type calcium channel blocker, effectively alleviates allodynia in animal neuropathic pain models.

Reduced expression of mir15a in the blood of patients with oral squamous cell carcinoma is associated with tumor staging.

MicroRNAs (miRNAs) mir15a and let7a are important regulators of bcl-2, ras and c-myc proteins. Considering that these miRNAs are commonly altered in many human cancers and that these proteins are reported to be altered in oral squamous cell carcinoma (OSCC), we investigated them in a set of OSCC cases. The miRNAs as well as the proteins were evaluated in the tumor and blood of 20 patients by real-time quantitative PCR and immunohistochemistry, respectively. The expression of mir15a and bcl-2 proteins in the tumors was not associated with each other or with tumor staging. On the other hand, we found reduced expression of this miRNA in the blood of patients with an advanced stage of OSCC and with lymph node metastasis. The expression of let7a in the tumor and blood was not associated with tumor size, lymph node metastasis, tumor staging and immunoexpression of ras and c-myc proteins. In conclusion, the present study shows that reduced expression of mir15a is associated with OSCC staging.

Protective effect of retinal ischemia by blockers of voltage-dependent calcium channels and intracellular calcium stores.

In the present study, the neuroprotective effect of blockers of voltage-dependent calcium channels (VDCC) and intracellular calcium stores on retinal ischemic damage induced by oxygen deprivation-low glucose insult (ODLG) was investigated. Retinal damage induced by ODLG was dependent on the calcium concentration in the perfusion medium. When incubated in medium containing 2.4 mM CaCl(2), cell death in ischemic retinal slices treated with blockers of VDCC, omega-conotoxin GVIA (1.0 microM), omega-conotoxin MVIIC (100 nM) and nifedipine (1.0 microM), was reduced to 62 +/- 2.3, 46 +/- 4.3 and 47 +/- 3.9%, respectively. In the presence of blockers of intracellular calcium stores, dantrolene (100 microM) and 2-APB (100 microM), the cell death was reduced to 46 +/- 3.2 and 55 +/- 2.9%, respectively. Tetrodotoxin (1.0 microM), reducing the extent of the membrane depolarization reduces the magnitude of calcium influx trough VDCC causing a reduction of the cell death to 55 +/- 4.3. Lactate dehydrogenase content of untreated ischemic retinal slices was reduced by 37% and treatment of ischemic slices with BAPTA-AM (100 microM) or 2-APB (100 microM) abolished the leakage of LDH. Dantrolene (100 microM) and nifedipine (1.0 microM) partially blocked the induced reduction on the LDH content of retinal ischemic slices. Histological analysis of retinal ischemic slices showed 40% reduction of ganglion cells that was prevented by BAPTA-AM or dantrolene. 2-APB partially blocked this reduction whilst nifedipine had no effect, p > 0.95. Conclusion Blockers of VDCC and intracellular calcium-sensitive receptors exert neuroprotective effect on retinal ischemia.

A rat homologue of CED-6 is expressed in neurons and interacts with clathrin.

We isolated from a brain library a cDNA encoding an isoform of rat CED-6 that has not been previously described. This transcript results from alternative splicing of the ced-6 gene present on chromosome 9. We expressed this isoform as his-tagged protein in E. coli and used the purified protein to raise antibodies to investigate the expression of CED-6 in rat brain. Immunoblot analysis showed the presence of CED-6 as a doublet of approximately 34 and 33 kDa in cortex, hippocampus and cerebellum, indicating that the protein was present in different regions of the brain. Subcellular fractionation experiments showed that CED-6 immunoreactivity did not concentrate in GFAP-containing glial vesicles, whereas it showed a distribution similar to the synaptotagmin in synaptosomes-enriched fractions, suggesting that CED-6 is present in neurons. CED-6 immunoreactivity was also investigated using immunohistochemistry analysis and it was found in several brain regions, being particularly strong in the cell body of some groups of neurons such as Purkinje cell layer of cerebellum, and pyramidal cells of the hippocampal formation and also in epithelial cells from the choroid plexus. Importantly, CED-6 immunoreactivity colocalized with a neuronal marker but not with a glial marker. Considering that several PTB-containing proteins bind clathrin, we investigated whether rat CED-6 would also have this property. Yeast two-hybrid and GST pull-down analysis indicated that ratCED-6 interacts with clathrin and in cultured cells we detected colocalization between CED-6 and clathrin-coated vesicles. The present findings suggest that CED-6 may have a role in endocytic trafficking or signaling in neurons.

The effect of sevoflurane on intracellular calcium concentration from cholinergic cells.

The mechanism of action of volatile anesthetics is not completely understood. Calcium release from internal stores may alter signaling pathways that influence neurotransmission. Abnormalities of the regulation of intracellular calcium concentration ([Ca2+]i) from patients with malignant hyperthermia is a hallmark of this syndrome indicating the potential of these agents to interact with proteins involved in Ca2+ signaling. In the present study, a cholinergic cell line (SN56) was used to examine whether the release of calcium from intracellular stores occurs in the presence of sevoflurane. Changes in [Ca2+]i were measured using fluo-4, a fluorescent calcium sensitive dye and laser scanning confocal microscopy. Sevoflurane induced an increase on [Ca2+]i from SN56 cells. The sevoflurane-induced increase on [Ca2+]i remained even when the cells were perfused with medium lacking extracellular calcium. However, this effect was abolished by BAPTA-AM, a chelator of intracellular calcium, suggesting the involvement of intracellular Ca2+ stores. Using cyclopiazonic acid, an inhibitor of sarcoplasmic/endoplasmic reticulum Ca(2+)-ATPase, we investigated whether the depletion of intracellular Ca2+ stores interfered with the effect of sevoflurane. In the presence of this agent, sevoflurane caused a small but not significant rise on [Ca2+]i of the SN56 cells. Dantrolene, an inhibitor of ryanodine-sensitive calcium stores did not modify the sevoflurane increase on [Ca2+]i. Carbachol, a drug that releases Ca2+ from the IP3 pool, abolished the effect of sevoflurane. In addition, xestospongin D, a cell-permeant IP3 receptor antagonist, decreased significantly the sevoflurane increase on [Ca2+]i. Our data suggest that the sevoflurane-induced increase on [Ca2+]i from SN56 cells occurs through the release of calcium from IP3-sensitive calcium stores.

Dopamine release evoked by beta scorpion toxin, tityus gamma, in prefrontal cortical slices is mediated by intracellular calcium stores.

1. We have investigated the effect of tityus gamma (TiTX gamma) scorpion toxin on the release of [3H]dopamine in rat brain prefrontal cortical slices. The stimulatory effect of TiTX gamma on the release of [3H]dopamine was dose/time-dependent with an EC50 of 0.01 microM. 2. Tetrodotoxin blocked the TiTX gamma-induced release of [3H]dopamine, indicating the dependency for Na+ channels. 3. EGTA had no effect on the TiTX gamma-induced release of [3H]dopamine, indicating the process is independent of extracellular calcium. Release of [3H]dopamine evoked by TiTX gamma was inhibited by 57% by BAPTA, a chelator of intracellular calcium. 4. Xestospongin and 2-APB, putative blockers of IP3-sensitive release of intracellular calcium stores, caused an equal and significant inhibition of 24% of the TiTX gamma-induced release of [3H]dopamine, while the slight inhibition evoked by dantrolene, a putative blocker of ryanodine-sensitive calcium store was not significant. 5. Nomifensine and ascorbic acid, blockers of dopamine transporter (DAT), caused an inhibition of 27 and 29%, respectively, on the toxin-induced release of [3H]dopamine suggesting that most of the TiTX gamma-induced release of dopamine is not due to the reversal of Na+ gradient. 6. In conclusion the majority of the TiTX gamma-induced release of [3H]dopamine is exocytotic and mobilizes calcium from the intracellular IP3-sensitive calcium stores.