Central Glutamatergic Mechanism Involved In Improvement Of Exercise Training On Hypertension

Background and ObjectiveHypertension is characterized by elevated levels of blood pressure (BP) and sympathetic tone, which are closely related to a poor prognosis in this disease. It is well known that the rostral ventrolateral medulla (RVLM) is a common pathway for central control of sympathetic outflow, and plays a crucial role in maintaining resting BP and sympathetic tone. Abnormalities in structure and function of the RVLM have been suggested to be a major contributor to primary (neurogenic) hypertension. It has been documented that the imbalance between tonically active excitatory (glutamatergic) and inhibitory (GABAergic) inputs to RVLM vasomotor neurons is responsible for cardiovascular dysregulation in hypertension. Microinjection of the excitatory glutamate receptor antagonist kynurenic acid (KYN) into the RVLM produces a significant fall in resting BP in hypertensive rats but not in normotensive rats, suggesting that the tonic glutamatergic input to the RVLM is upregulated in hypertension. Accordingly, it is proposed that the increased tonic glutamatergic input to the RVLM is an important contributor to elevated levels of BP and sympathetic tone in hypertension.Growing evidence has demonstrated that exercise training (ExT) is capable of improving cardiovascular dysfunction in cardiovascular disorders such as hypertension and chronic heart failure. It is reported that ExT effectively lowers BP, decreases cardiac output, and enhances the baroreflex sensitivity in patients and animals with hypertension. However, the exact mechanism(s) by which ExT improves cardiovascular dysfunction in hypertension have not been fully understood. It is suggested that neural plasticity in the central cardiovascular networks is an important mechanism responsible for the effects of ExT on cardiovascular activity. It has been demonstrated that ExT is beneficial to hypertension via reducing the sympathetic activity. Moreover, ExT significantly attenuates the increase in BP and sympathetic activity induced by stimulation of the RVLM. suggesting the importance of the RVLM in mediating the effects of ExT on cardiovascular regulation. However, it is not clear if ExT alters the elevated tonically active glutamatergic inputs to RVLM neurons in hypertension.Therefore, this study was designed to determine the effect of ExT on tonically active glutamatergic inputs to the RVLM of spontaneously hypertensive rats (SHR). MethodsEight-week-old male normotensive Wistar-Kyoto (WKY) rats and SHR were preselected for their ability to walk on a treadmill and only active rats (80%) were used in this study. These active rats were randomly assigned either to the WKY rats sedentary group (WKY-Sedentary, WKY-Sed), the WKY rats ExT group (WKY-Training, WKY-ExT), the SHR sedentary group (SHR-Sedentary, SHR-Sed) or to the SHR ExT group (SHR-Training, SHR-ExT). ExT groups were subjected to low-intensity ExT on a motor-driven treadmill continuously for a period of12weeks (5days per week;60minutes per day). Using a noninvasive computerized tail-cuff system, BP and heart rate (HR) were measured at baseline (8weeks of age) and then every4weeks until the end of the study period. After the last exercise session (at the age of20weeks), animals were allowed at least24hours to recover from exercise to minimize alterations in cardiovascular control and further subjected to the following in vivo or in vitro experiments.After twelve weeks of ExT or Sed protocol, baroreflex-induced decrease in HR evoked by intravenous injection of phenylephrine (10μg/kg) was measured. The cardiovascular parameters (BP, HR, and renal sympathetic nerve activity, RSNA) were recorded continuously at least60minutes after bilateral injection of KYN (2.7nmol) into the RVLM. Before and after this set of experiments, baseline inte-RSNA and its maximal RSNA were measured to determine the sympathetic tone. Norepinephrine (NE) in24-hour urine was measured by high-performance liquid chromatography (HPLC). Citrate synthase (CS) concentration in soleus muscle was measured by ELISA. After RVLM tissues were punched and weighted, concentrations of glutamate and GABA in the RVLM were measured by HPLC. The protein expressions of glutamate receptors in the RVLM were measured by Western blot analysis and immunofluorescence. The protein expressions of c-fos and vesicular glutamate transporter2(VGLUT2) in the RVLM were measured by Western blot analysis. The gene expressions of glutamic acid decarboxylase (GAD) in the caudal ventrolateral medulla (CVLM) and RVLM were detected by real-time PCR.Results1. Assessment of ExT EfficacyAt the beginning of ExT protocol (at the age of8weeks), BP and HR were significantly higher in SHR-Sed than in WKY-Sed, which were elevated for the duration of the study. After12-week period of ExT, BP was significantly lower in SHR-ExT than in SHR-Sed, and HR was significantly decreased in SHR-ExT than in SHR-Sed. However, ExT did not change BP or HR in WKY rats. Baroreflex-induced decrease in HR evoked by intravenous injection of phenylephrine was significantly lower in SHR-Sed than in WKY-Sed, whereas it was reversed after ExT. Several values were measured for efficacy of ExT at the end of Sed or ExT protocol (at the age of20weeks). Body weight in ExT groups was significantly reduced compared with Sed groups. Soleus muscle weight was significantly increased in ExT groups than in Sed groups.24-hour urine excretion of NE was higher in SHR-Sed than in WKY-Sed, whereas it was significantly reduced following ExT protocol. In addition, the concentration of citrate synthase in soleus muscle was significantly higher in ExT than in Sed rats.2. Effect of ExT on the changes of BP,HR and RSNA evoked by blockade of glutamate receptors in the RVLM of ratsIn WKY-Sed or WKY-ExT groups, bilateral injection of KYN (2.7nmol) into the RVLM had little effect on resting BP, HR, and RSNA. However, bilateral microinjection of KYN into the RVLM produced a significant decrease in resting BP, HR, and RSNA in SHR-Sed. The degree of reduction in resting BP, HR, and RSNA induced by bilateral microinjection of KYN into the RVLM was significantly attenuated in SHR-ExT compared with SHR-Sed. It suggests that ExT attenuates the decrease in BP,HR and RSNA evoked by blockade of glutamate receptors in the RVLM of SHR.3. Effect of ExT on the concentration of glutamate and the protein expression of glutamate receptors in the RVLM of ratsThe concentration of glutamate in the RVLM was significantly increased in SHR-Sed than in WKY-Sed. However, it was significantly decreased in SHR-ExT compared with SHR-Sed. There was no significant difference of glutamate concentration in the RVLM between WKY-Sed and WKY-ExT. In addition, ExT significantly decreased protein expression of VGLUT2in the RVLM of SHR. The protein expression of glutamate receptors in the RVLM was significantly upregulated in SHR-Sed. However, the protein expression of glutamate receptors in the RVLM was significantly downregulated in SHR-ExT compared with SHR-Sed. There was no significant difference of glutamate receptors expressions in the RVLM between WKY-Sed and WKY-ExT. In addition, the protein expression of c-fos in the RVLM was significantly increased in SHR-Sed compared with WKY-Sed. However, a significant decrease in c-fos expression in the RVLM was observed in SHR-ExT compared with SHR-Sed. It suggests that ExT decreases the concentration of glutamate and the protein expression of glutamate receptors in the RVLM of SHR, which reduces the activity of SHR RVLM neurons. In addition, ExT has little effect on the concentration of glutamate and the protein expression of glutamate receptors in the RVLM of WKY.4. Effect of ExT on the concentration of GABA and the expression of GAD67in the RVLM of ratsThe concentration of GABA in the RVLM was significantly lower in SHR-Sed than in WKY-Sed. However, ExT significantly increased GABA concentration in SHR but not in WKY rats. The gene expression of GAD67in the RVLM was significantly lower in SHR-Sed than in WKY-Sed. ExT significantly increased the gene expression of GAD67in SHR but not in WKY rats. In the CVLM, the gene expression of GAD67was significantly lower in SHR-Sed than in WKY-Sed, whereas it was upregulated after ExT. It suggests that ExT increases the concentration of GABA and the expression of GAD67in the RVLM of SHR. In addition, ExT upregulates GAD67expression in the CVLM of SHR.ConclusionsThe present study has confirmed that low-intensity ExT downregulates the increased tonically active glutamatergic inputs to the RVLM in SHR. It is indicated that normalization of imbalance between excitatory and inhibitory inputs to RVLM neurons is an important mechanism responsible for the beneficial effects of ExT on sympathetic overactivity in hypertension.