Pathophysiological role of local inflammation in nitric oxide deficit in the carotid body in intermittent hypoxia

Dr Fung, Man Lung   (Principal Investigator (PI))

42

2016-10-01

1102080

Pathophysiological role of local inflammation in nitric oxide deficit in the carotid body in intermittent hypoxia

carotid chemoreceptor, cytokine, hypoxia, inflammation, sleep apnea

Physiology,Cardiovascular Research

Biology and Medicine (M)

17102316

General Research Fund (GRF)

2016

Completed

1 • Specific aim 1 is to determine the effect of anti-inflammatory drugs on the NO production in the CB or cultured cells in intermittent hypoxia. • Specific hypothesis 1 is that anti-inflammatory drugs ameliorate the IH-induced NO deficit in the CB. 2 • In-vivo studies - arterial pressures, serum and tissue oxidative markers, the NO level and the expression of NO synthases (NOSs) and the local inflammatory markers and signaling mediators in the CB will be obtained in the IH rats (with inspired oxygen levels altering between 5-21% per minute for 8 hours per day for 1-10 days), concurrently treated with dexamethasone or ibuprofen. Results will be compared with the IH and normoxic groups. • The NO bioavailability will be evaluated by the expression and functional studies measuring the expression level of NOSs (eNOS, nNOS, p-eNOS), and also electrochemical measurements of the constitutive NO release (level reduced by NOS inhibitors) and the NO elevation in responding to acute hypoxia. • Local inflammation will be examined by the expression of inflammatory mediators, cyclooxygenases (COX) and chemokines, and macrophage infiltration. Superior cervical ganglion will also be used as a control tissue. In addition, results will be confirmed by invivo studies using the wild-type and COX-knockout mice. • The CB hyperexcitability will be assessed by patch-clamp study of potassium currents and spectrofluorimetric study of intracellular calcium ([Ca++]i) in responding to acute hypoxia in the chemosensitive glomus cells in the CB. Electrophysiological study of the afferent nerve activity of the chemoreceptor will be performed on the CB in anesthetized rats. Results will be compared with those of the IH group and normoxic controls. 3 • In-vitro studies - In cultured PC12 cells as an oxygen-sensitive model or primary cultured type-I glomus cell of the CB, studies will be performed to examine the NO level and NOS expression, activation of NF-kb and signaling mediators (NF-kb activity, IKK phosphorylation, MAPKs) in the presence of vehicles, cytokine receptor antagonists, ERK/p-38/NF-kb inhibitors, COX inhibitors under hypoxic or control conditions. • The drug effects on the potassium current and [Ca2+]i responses to acute hypoxia in the PC12 or glomus cells will also be studied. The DNA-binding activity of NF-kb will be evaluated by EMSA. Western-blot will be performed to measure the total and phosphorylated levels of IKK and MAPKs. Results will be compared with those of the IH group and normoxic controls. • It is speculated that the decreased NO bioavailability and augmented chemoreceptor responses to acute hypoxia; the activation of NF-kb activity and the signaling mediators in the IH group, could be antagonized by anti-inflammatory drugs or the receptor blockers. 4 • Specific aim 2 is to examine how cytokines down-regulate the NO production and lead to functional changes in the carotid body in IH. • Specific hypothesis 2 is that the cytokines up-regulate the expression of NADPH oxidase and renin-angiotensin system, leading to the NO deficit in intermittent hypoxia. • In-vivo studies – as aforementioned the in-vivo parameters, the NO level and the expression of NO synthases (eNOS, nNOS, p-eNOS), NADPH oxidase subunits (Nox2, Nox4), renin-angiotensin system (angiotensinogen, angiotensin-converting enzymen, AT1 receptors) and the local inflammatory markers and signaling mediators in the CB will be obtained in the IH rats concurrently treated with NADPH oxidase inhibitor (apocynin) or AT1 receptor antagonist (losartan). • As stated in specific aim 1, the CB hyperexcitability will be evaluated by measurements of the afferent activity, potassium current and [Ca++]i elevation in responding to acute hypoxia. Results will be compared with the IH and normoxic groups. • In-vitro studies - the NO level and the NOS expression profile could be confirmed by studies using cultured PC12 cells or primary cultured type-I glomus cell of the CB. Studies will be performed to measure the NO level and NOS expression, the expressions of Nox subunits and renin-angiotensin system, activation of NF-kb and signaling mediators in the presence of vehicles, apocynin or lorsatan under hypoxic or control conditions. The hyperexcitability will be evaluated by measuring the potassium currents, the catecholamine secretion, [Ca++]i elevation in responding to acute hypoxia. In addition, the pharmacological effects will be confirmed by gene silencing of NADPH oxidase subunits Nox2 or Nox4 with siRNA.