Role of M2 muscarinic receptor in the airway response to methacholine of mice selected for minimal or maximal acute inflammatory response

Airway smooth muscle constriction induced by cholinergic agonists such as methacholine (MCh), which is typically increased in asthmatic patients, is regulated mainly by muscle muscarinic M3 receptors and negatively by vagal muscarinic M2 receptors. Here we evaluated basal (intrinsic) and allergen-induced (extrinsic) airway responses to MCh. We used two mouse lines selected to respond maximally (AIRmax) or minimally (AIRmin) to innate inflammatory stimuli. We found that in basal condition AIRmin mice responded more vigorously to MCh than AIRmax. Treatment with a specific M2 antagonist increased airway response of AIRmax but not of AIRmin mice. The expression of M2 receptors in the lung was significantly lower in AIRmin compared to AIRmax animals. AIRmax mice developed a more intense allergic inflammation than AIRmin, and both allergic mouse lines increased airway responses to MCh. However, gallamine treatment of allergic groups did not affect the responses to MCh. Our results confirm that low or dysfunctional M2 receptor activity is associated with increased airway responsiveness to MCh and that this trait was inherited during the selective breeding of AIRmin mice and was acquired by AIRmax mice during allergic lung inflammation

Emerging role of angiotensin type 2 receptor (AT2R)/Akt/NO pathway in vascular smooth muscle cell in the hyperthyroidism

Hyperthyroidism is characterized by increased vascular relaxation and decreased vascular contraction and is associated with augmented levels of triiodothyronine (T3) that contribute to the diminished systemic vascular resistance found in this condition. T3 leads to augmented NO production via PI3K/Akt signaling pathway, which in turn causes vascular smooth muscle cell (VSMC) relaxation; however, the underlying mechanisms involved remain largely unknown. Evidence from human and animal studies demonstrates that the renin-angiotensin system (RAS) plays a crucial role in vascular function and also mediates some of cardiovascular effects found during hyperthyroidism. Thus, in this study, we hypothesized that type 2 angiotensin II receptor (AT2R), a key component of RAS vasodilatory actions, mediates T3 induced-decreased vascular contraction. Marked induction of AT2R expression was observed in aortas from T3-induced hyperthyroid rats (Hyper). These vessels showed decreased protein levels of the contractile apparatus: α-actin, calponin and phosphorylated myosin light chain (p-MLC). Vascular reactivity studies showed that denuded aortic rings from Hyper rats exhibited decreased maximal contractile response to angiotensin II (AngII), which was attenuated in aortic rings pre-incubated with an AT2R blocker. Further study showed that cultured VSMC stimulated with T3 (0.1 µmol/L) for 24 hours had increased AT2R gene and protein expression. Augmented NO levels and decreased p-MLC levels were found in VSMC stimulated with T3, both of which were reversed by a PI3K/Akt inhibitor and AT2R blocker. These findings indicate for the first time that the AT2R/Akt/NO pathway contributes to decreased contractile responses in rat aorta, promoted by T3, and this mechanism is independent from the endothelium.

Effects of ischemia and reperfusion on subpopulations of rat enteric neurons expressing the P2X7 receptor

BACKGROUND: Intestinal ischemia followed by reperfusion (I/R) may occur following intestinal obstruction. In rats, I/R in the small intestine leads to structural changes accompanied by neuronal death. AIM: To analyze the impact of I/R injury on different neuronal populations in the myenteric plexus of rat ileum. METHODS: The ileal artery was occluded for 35 min and animals were euthanized 6, 24, and 72 h, and 1 week later. Immunohistochemistry was performed with antibodies against the P2X7 receptor as well as nitric oxide synthase (NOS), calbindin, calretinin, choline acetyltransferase (ChAT), or the pan-neuronal marker anti-HuC/D. RESULTS: Double immunolabeling demonstrated that 100% of NOS-, calbindin-, calretinin-, and ChAT-immunoreactive neurons in all groups expressed the P2X7 receptor. Following I/R, neuronal density decreased by 22.6% in P2X7 receptor-immunoreactive neurons, and decreased by 46.7, 38, 39.8, 21.7, and 20% in NOS-, calbindin-, calretinin-, ChAT-, and HuC/D-immunoreactive neurons, respectively, at 6, 24, and 72 h and 1 week following injury compared to the control and sham groups. We also observed a 14% increase in the neuronal cell body profile area of the NOS-immunoreactive neurons at 6 and 24 h post-I/R and a 14% increase in ChAT-immunoreactive neurons at 1 week following I/R. However, the average size of the calretinin-immunoreactive neurons was reduced by 12% at 6 h post-I/R and increased by 8% at 24 h post-I/R. CONCLUSIONS: This work demonstrates that I/R is associated with a significant loss of different subpopulations of neurons in the myenteric plexus accompanied by morphological changes, all of which may underlie conditions related to intestinal motility disorder