Acetylcholine inhibits hypoxia-induced tumor necrosis factor-α production via regulation of MAPKs phosphorylation in cardiomyocytes.

Recent findings have reported that up-regulation of tumor necrosis factor-alpha (TNF-α) induced by myocardial hypoxia aggravates cardiomyocyte injury. Acetylcholine (ACh), the principle vagal neurotransmitter, protects cardiomyocytes against hypoxia by inhibiting apoptosis. However, it is still unclear whether ACh regulates TNF-α production in cardiomyocytes after hypoxia. The concentration of extracellular TNF-α was increased in a time-dependent manner during hypoxia. Furthermore, ACh treatment also inhibited hypoxia-induced TNF-α mRNA and protein expression, caspase-3 activation, cell death and the production of reactive oxygen species (ROS) in cardiomyocytes. ACh treatment prevented the hypoxia-induced increase in p38 mitogen-activated protein kinase (MAPK) and c-Jun N-terminal kinase (JNK) phosphorylation, and increased extracellular signal-regulated kinase (ERK) phosphorylation. Co-treatment with atropine, a non-selective muscarinic acetylcholine receptor antagonist, or methoctramine, a selective type-2 muscarinic acetylcholine (M(2) ) receptor antagonist, abrogated the effects of ACh treatment in hypoxic cardiomyocytes. Co-treatment with hexamethonium, a non-selective nicotinic receptor antagonist, and methyllycaconitine, a selective alpha7-nicotinic acetylcholine receptor antagonist, had no effect on ACh-treated hypoxic cardiomyocytes. In conclusion, these results demonstrate that ACh activates the M(2) receptor, leading to regulation of MAPKs phosphorylation and, subsequently, down-regulation of TNF-α production. We have identified a novel pathway by which ACh mediates cardioprotection against hypoxic injury in cardiomyocytes.

Alterations of muscarinic acetylcholine receptors-2, 4 and α7-nicotinic acetylcholine receptor expression after ischaemia / reperfusion in the rat isolated heart.

1. Cardiac acetylcholine receptors are involved in the negative inotropic effect of the vagus and the protection of the stimulated vagal nerve against myocardial ischaemic injury. Acetylcholine receptors consist of five types of muscarinic acetylcholine receptors (M AChR) and several nicotinic acetylcholine receptors (nAChR). Notably, ischaemic heart disease is accompanied by substantial withdrawal of vagal activity. However, it is not entirely clear what the changes of M(2,4) AChR and α7-nAChR expression are after cardiac ischaemia/reperfusion (I/R) injury. 2. Cardiac functions were continuously recorded in Langendorff mode during 30 min of ischaemia and 60 min of reperfusion. Lactate dehydrogenase (LDH) leakage was measured. M(2,4) AChRs and α7-nAChR expression were measured by reverse transcription polymerase chain reaction and western blot. 3. In hearts exposed to I/R injury, left ventricular development pressure, heart rate and ± dP/dt decreased significantly compared with the controls. LDH leakage increased with respect to the controls during reperfusion. 4. In normal hearts, expression of M(2,4) AChR in the left ventricle were lower than in atria and the right ventricle, whereas expression of α7-nAChR was dramatically higher in the left ventricle and right ventricle than the atria. After reperfusion, the mRNA and protein expression of M(2) AChR increased notably in the left and right ventricle, and α7-nAChR was enhanced significantly in the left ventricle. M(4) AChR mRNA expression reduced notably after ischaemia and recovered to the control level after reperfusion in the atria, but the protein level did not change. 5. In conclusion, the increase in M(2) AChR and α7-nAChR after reperfusion might be the compensatory response to myocardial I/R injury, providing new information for treatment of myocardial I/R injury.

Exercise benefits cardiovascular health in hyperlipidemia rats correlating with changes of the cardiac vagus nerve.

The role of exercise training on hemodynamic parameters, blood lipid profiles, inflammatory cytokines, cholinesterase-positive nerves and muscarinic cholinergic (M(2)) receptors expression in the heart was investigated in Sprague-Dawley male rats with hyperlipidemia (HL). The rats were subjected to a high-fat diet and exercise training for 8 weeks, and then the hemodynamic parameters, the profiles of blood lipid and inflammatory cytokines, and the expression of cholinesterase-positive nerves and M(2) receptors were measured. HL rats displayed cardiac dysfunction, dysregulation of inflammatory cytokines, and decreased cholinesterase-positive nerves and M(2) receptors expression. The combination of hyperlipidemia with exercise training (AT) restored the profiles of blood lipids and the levels of inflammatory cytokines. In addition, AT and HL + AT improved cardiac function with increasing cholinesterase-positive nerves and M(2) receptors expression. Overall, these data show that the increased expression of cholinesterase-positive nerves and M(2) receptors in the heart is partially responsible for the benefits of exercise training on cardiac function in hyperlipidemia rats.

Involvement of volume-sensitive Cl- channels in the proliferation of human subcutaneous pre-adipocytes.

1. Obesity is a significant challenge in terms of public health and preventive medicine. Inhibition of pre-adipocyte proliferation is believed to be important in the proposed anti-obesity mechanism. The aim of the present study was to examine the interplay between Cl(-) channels and their possible involvement in the proliferation of undifferentiated human pre-adipocytes. 2. Pre-adipocytes were isolated from human abdominal subcutaneous adipose tissue. Membrane ion currents were recorded using the whole-cell patch-clamp technique. Expression of the Cl(-) channel ClC-3 gene and protein was determined by reverse transcription-polymerase chain reaction (RT-PCR) and western blot, respectively. Cell proliferation was evaluated using the [(3)H]-thymidine incorporation assay. 3. Electrophysiological recordings revealed a volume-sensitive Cl(-) current (I(Cl.vol)) expressed in pre-adipocytes that was activated under hyposmotic conditions (external osmolarity decreased by 80%) and inhibited by the Cl(-) channel blocker tamoxifen. 4. Expression of the ClC-3 channel gene and protein was confirmed by RT-PCR and western blot analysis. Blocking I(Cl.vol) with tamoxifen supressed the proliferation of pre-adipocytes in a concentration-dependent manner. 5. Collectively, the results of the present study indicate that the volume-sensitive Cl(-) channel participates in regulation of the proliferation of human subcutaneous pre-adipocytes.

[Vagal control of cardiac functions and vagal protection of ischemic myocardium].

The physiological activities of the cardiovascular system are under the control of autonomic nervous system (ANS). Recent researches have found that autonomic dysfunction, especially the withdrawal of vagal activity, was closely related to the etiology, course and prognosis of cardiovascular disease (CVD). Based on the current status and our achievements in this area, we discuss vagal regulation of different parts of the heart and the mechanism of vagal protection of myocardium. Using a force transducer and standard microelectrodes recording technology, we found that the vagus nerve transmitter--acetylcholine (ACh) had direct effects on ventricular myocytes in mammals: It inhibited the contractility and shortened the action potential duration of cardiac myocytes. We proved the existence of muscarinic receptors and vagal nerves innervation in ventricle with histochemical staining and molecular biological methods. Furthermore, ACh-activated potassium channel (KACh) was found in the ventricles of some animals by patch-clamp. Fade of the current (IK.ACh) to ACh in atrium was found in previous research, which was related to the muscarinic receptors and phosphorylation of G protein or potassium channel. However, the mechanism of the fade in ventricle needs to be further investigated. Combined with autonomic nervous evaluation methods (heart rate variability analysis) and relevant animal models, we studied the regulation of ANS during normal and morbid state, and proved the age-associated changes and compensatory effects of vagal control of hemodynamics after unilateral vagotomy. By increasing the vagal tension (ACh induced-preconditioning/postconditioning, aerobic exercise, beta receptor antagonist), we demonstrated protective effects of the vagus nerve on the ischemic myocardium and mechanism of the cholinergic anti-inflammatory effects on the inflammatory reaction induced by reperfusion injury. Evaluating cardiac autonomic nervous regulation and improving balance between sympathetic and vagal nerve will provide an important basis for the prevention and treatment of CVD.