Effect Of Cyclic Intermittent Hypoxia On Inhibition By NO In The Rat Carotid Body

Patients with obstructive sleep apnea (OSA) are at risk of systemicarterial hypertension. There is debate about the mechanisms by which cyclicintermittent hypoxia (CIH) of OSA might contribute to elevated arterialpressure. Sympathetic activity of patients with OSA was enhanced, andeffective treatment of sleep apnea with nasal continuous positive airwaypressure (nasal CPAP) reduces sympathetic activity and arterial pressure. Inaddition, CIH mimicking sleep apnea induce significant elevations of arterialpressure of rodent animsal; and this increase in pressure requires sustainedsympathoexcitation, because section of the renal nerves prior to the exposureto CIH prevents the rise in arterial pressure. These results suggested that onefactor contributting to elevated arterial pressure might be sustainedsympathoexcitation. Sympathetic activity is modulated by peripheral reflexactivity and central sympathetic processing. A number of studies suggests thatperipheral chemoreflex sensitivity is augmented by CIH exposure and thatenhanced chemosensitivity contributes to the sympathoexcitation andhypertension. Although these data suggest peripheral chemosensitivity isenhanced by CIH in patients with sleep apnea, few studies have examined themechanisms underlying enhanced chemosensitivity after CIH.Studies have shown that nitric oxide (NO) is a modulator ofchemosensitivity after CIH exposure. Physiologic concentration of NOprimarily plays an inhibitory role in chemosensitivity. NO is primarilyproduced by neuronal nitric oxide synthase (nNOS)-positive neurons whosesoma located in carotid sinus nerve(CSN) and glossopharyngeal nerve, andthese neurons directly innervate the carotid body. There are studies suggestingthat nNOS protein expression is decreased in rabbits with pacing-inducedcongestive heart failure (CHF). Furthermore, this decrease in nNOS contributes to enhanced chemosensitivity in CHF. Recently, there is evidencesuggestting nNOS protein is significant decreased in carotid bodies of CIHrats, but no evidence demonstrates the decrease contributing tochemosensitivity.In present study, we investigated the effect on nNOS protein and nNOSmRNA expression in carotid body and glomus cell after CIH exposure, and weobserved the effect on inhibition of NO in the rat carotid body after CIHexposure. Meanwhile, we tried to discussed the mechanisms by which CIHenhanced chemosensitivity.Objective: We exposed rats to CIH to investigate the effect of CIH onnNOS and its mRNA expression in carotid body and glomus cell and oninhibition by NO in the rat carotid body.Methods:1. Male Sprague-Dawley (SD) rats, weight240-250g, were purchased fromthe Laboratory Animal Center (Hebei Medical University, China) and housedin cages at20°C~22°C temperature,45%~55%relative humidity on a12hlight–dark cycle. The rats were divided into three groups: control group, shamgroup and CIH group. Animals were exposed to hypoxia during their normalrest period (light). Each day, animals were placed daily in commercial hypoxicchambers that were flushed with100%N2to inspired O2fraction (FIO2) nadirof8.2-10%for1min. Then the FIO2was allowed to return to21%graduallyover the remainder of each cycle. The exposure cycle was repeated every4min for8h/day,7days/week for5weeks. Sham animals experienced identicalhandling and exposure but chambers were flushed with room air. Controlanimals were housed in standard animal housing and were exposed to thesame light: dark conditions. After exposure cycle, animals were selectedrandomly for either physiological recordings or for molecular studies of thecarotid body.The indices as follow:(1) Physiological studiesUnder a dissecting microscope, we isolates left carotid sinus nerve (CSN) carefully. We examined the effects of CIH on arterial pressure,CSN dischargeand chemosensitivity. The effect of NO inhibition was then tested withadministering L-NNA, SNAP or L-arginine into the carotid artery.(2) Western blotting and RT-PCRCarotid bodies from sham and CIH rats after exposure to CIH wereharvested, and the expression of nNOS protein and nNOS mRNA wereexamined by Western blotting or RT-PCR.2. Glomus cells were obtained from SD rats (19~21day). We removed bothcarotid bodies from the carotid bifurcation and cleaned of connective tissueunder a dissecting microscope. Then the CBs were placed in enzymaticsolution. Obtained carotid body cells were placed in a hypoxia chamber whichwas placed in a incubator. The chamber was fiushed with100%N2makingFIO2gradually to0.1%. This process needed35min. Then100%O2wasflushed into the chamber leaveing the FIO2return to21%gradually, and thisprocess needed25min. Leaveing the exposure cycle was repeatedcontinuously. Carbon dioxide (CO2) concentration always remain at5%andincubator temperature is always maintained at37°C. Control cells weremaintained in an atmosphere containing5%CO2in room air at37°C. Cellsexposed to CIH or room air were used for immunocytochemistry.Then weobserved the change of nNOS expression on single cell under microscope.Results:1Effect of CIH on femoral arterial pressureFemoral arterial pressure of rats exposed to CIH was increased relative toSham animals or to Control animals (n=6for each group; CIH115.8±2.9mmHg; Sham91.5±2.8mmHg; Control90.0±1.0mmHg; P <0.01).2Effect of CIH on basal carotid sinus nerve (CSN) dischargeBasal CSN discharge was significantly augmented in CIH rats relative toSham or Control animals (n=6for each group; CIH35.9±2.2; Sham24.2±1.1; Control22.2±0.89; P <0.01).3Effect of CIH on CSN discharge in response to10%oxygenCIH animals had a greater increase in CSN discharge in response to10% O2than did Sham animals (CIH623.1±13.5%of baseline; Sham485.6±18.0%; P <0.01).4Effect of nNOS inhibitor L-NNA on CSN discharge in response to10%oxygen in the ratAdministration of the nNOS inhibitor L-NNA increased the CSNdischarge response to10%O2significantly more in Sham animals than inCIH animals (CIH699.1±15.6%of baseline; Sham786.4±50.4%ofbaseline).5Effect of NO donor SNAP and the NO precursor L-arginine on CSNdischarge in response to10%oxygen in the ratIn contrast, the NO donor SNAP (CIH264.0±10.8%of baseline; sham340.0±9.2%of baseline) and the NO precursor L-arginine (CIH390.2±15.6%of baseline; sham238.2±7.7%of baseline) decreased the CSNdischarge response to10%O2significantly more in CIH rats than in Shamrats.6Effect of CIH on expression of nNOS protein and nNOS mRNA in carotidbodynNOS mRNA and protein was significantly reduced in carotid bodies ofCIH animals relative to Sham animals.7Effect of CIH on expression of nNOS protein in glomus cellsnNOS protein was significantly reduced in CIH glomus cells relative toSham glomus cells.Conclusions: CIH decreased the expression of nNOS protein and nNOSmRNA in carotid body and its glomus cells. The decreased NO productionmay contribute to increased carotid body chemosensitivity after exposure toCIH.