The Mechanism Of Angiotensin-(1-7) On The Regulation Of Blood Pressure In The Ventrolateral Medulla Of Rats

The fast rhythm of life style and social competition nowadays keep people in nervous emotion and cause psychological stress, which lead to an increase of hypertension incidence in our country year by year. According to reports the number of new patients is increasing by 3 million per year. There are also more cases of young people with hypertension. Chronic hypertension leads to the damage of heart, brain, kidney etc, so it is harmful to the health of people.The renin-angiotensin system (RAS) plays a crucial role in the regulation of blood pressure and cardiovascular activity. According to reports in recent years, besides angiotensin II (Ang II) with a strong vasoconstriction activity, angiotensin-( 1-7) [Ang-(1-7)], ignored for a long time, is also considered as a biologically active member of RAS, which participates in the regulation of cardiovascular homeostasis. There are three pathways in the endogenous synthesis of Ang-(1-7): first, Ang-(1-7) is generated from Angiotensin I (Ang I) by prolylendopeptidase (PEP) or neutral endopeptidase (NEP); secondly, it is formed from Ang II by PEP, prolylcarboxypeptidase (PCP) or angiotensin-converting-enzyme-related carboxypeptidase 2 (ACE2); finally, it is produced from Ang-(1-9) by NEP or angiotensin converting enzyme (ACE). The first and second pathways are the primary ones determining the content of Ang-(1-7). Ang-(1-7) plays a role of vasodilation, diuresis, antiproliferative of cardiomyocyte and so on by increasing the synthesis of nitric oxide (NO) and the release of prostaglandins or bradykinin. In the central nervous system, some effects of Ang-( 1 -7) are opposite to that of Ang II, for example, no effects of increased thirsty and a salty appetite are produced by Ang-(1-7); some are similar to Ang II, such as the increased release of vasopressin when microinjected into the hypothalamus paraventricular nucleus of rats and the change of the cardiovascular activity when microinjected to the ventrolateral medulla (VLM). But the mechanisms for these effects have remained obscure up to now. There are the complex cooperating or opposing interaction between Ang-(1-7) and Ang II in different positions or conditions. Ang-(1-7) possibly has more potential than Ang II in terms of physiological or pathological significance. Therefore, research on the the mechanisms forthese effects of Ang-(1-7), especially its effect on blood pressure regulation in the central nervous system, is very important.Reports show that the VLM is a key area in the regulation of cardiovascular activity and it consists of the rostral VLM (RVLM) and the caudal VLM (CVLM). The RVLM is a very important brain area where the sympathetic vasoconstriction neurons are located. Stimulation of the neurons in the RVLM increases the sympathetic outflow and blood pressure. The neurons in the RVLM are capable of integrating the information from peripheral and upper nervous system and transferring to the sympathetic preganglionic neurons in the intermedial lateral column (IML) of the spinal cord, and finally completing the regulation of cardiovascular activity. Current data show that the CVLM has no direct neuronal projections to the IML and its regulation to the cardiovascular activity is exerted by the inhibition of the RVLM neurons. Studies prove that there is an increase of sympathetic activity in the stress induced hypertension with many neurotransmitters and neuromodulators participating in. In the VLM, there are many kinds of neurotransmitters and modulators such as catecholamine, amino acid, nitric oxide, angiotensin, etc.The therapeutic effect of acupuncture has been proved by substantive clinical practice; meanwhile, great progress has been achieved in the research carried out on animals dealing with the regulation effect of acupuncture to the autonomic nervous system that controls cardiovascular activity. The profound research on the central effects of Ang-(1-7), in particular on its regulation effect on the hypertension in the VLM combined with acupuncture, will provide theoretical evidence to understand the mechanisms related to hypertension and suggest new treatments for hypertension.By using the methods of microinjection, microdialysis, high pressure liquid chromatogram (HPLC) and electroacupunture, the current study analyzed the artery blood pressure, heart rate and the releases of amino acid to investigate the mechanisms of Ang-(1-7) involved in the regulation of blood pressure in the VLM of normal and stress-induced hypertensive rats.Section 1 The mechanism of Ang-(1-7) on regulation of blood pressure in the RVLM of rats1. The mechanism of Ang-(1-7) on regulation of blood pressure in the RVLM of normal ratsThe unilateral (right side) microinjection of Ang-(1-7) in the RVLM of rats (n=6) in a dose of 10 pmol (in a volume of 0.1 μ1, which was the same below), 25 pmol or 50 pmol increased the blood pressure in a dose-dependent manner (p<0.05). However, the unilateral microinjection Ang779 (100 pmol), a selective antagonist of Ang-(1-7) receptor, in the RVLM decreased the blood pressure (n=7, P<0.05). The microinjection of artifical cerebrospinal fluid (ACSF) (n=6) at the same site caused no obvious effect.To investigate the hypertensive effect of Ang-(1-7) in the RVLM and the mechanisms involved in this effect, we microinjected Ang-(1-7) into the RVLM while the blood pressure was recorded and microdialysis performed in the RVLM and IML at the level of T8 simultaneously for analyzing amino acid release by HPLC with fluorescence detector. The results showed that microinjection of Ang-(1-7) (25 pmol, n=11) could cause an increase of blood pressure, and the analysis of microdialysis samples showed that the aspartic acid(Asp) and glutamate (Glu) in the RVLM and IML increased while the Tau in the IML decreased(P<0.05). By contrast, microinjection of Ang779 (100 pmol, n=11) in the RVLM caused a decrease of blood pressure, and the Glu in the RVLM and IML also decreased while glycine (Gly) and taurine (Tau) increased in the same areas (P<0.05). To further verify the correlation between the change of the amino acid release and the blood pressure caused by Ang-(1-7), after the microinjection of kynurenic acid (Kyna) (1.2nmol, no obvious response observed in this dose, which was the same below dealing with the application of antagonists), a non-selective ionotropic antagonist of Glu, into the RVLM and IML, Ang-(1-7) was microinjected into the RVLM and did not elevate the blood pressure anymore. This indicated that the antagonist of Glu could partially block the hypertensive effect of Ang-(1-7) in the RVLM. After the microinjection of Gly or Tau receptor antagonist Strychnine or TAG into the RVLM or IML, the microinjection of Ang779 into the RVLM did not decrease the blood pressure anymore. The results indicated that the antagonist of Gly and Tau can partially block the hypotensive effect of Ang779 in the RVLM. The above results showed that the hypertensive effect of Ang-(1-7) in the RVLM was correlated with the increased releaseof Asp and Glu and the decreased release of Tau in the RVLM and IML.To test whether the hypertensive effect of Ang-(1-7) was mediated through the Ang II type 1 (AT₁) receptor, Dup753, a selective antagonist of AT₁ receptor, was used. After the microinjection of Ang779 (50 pmol, n=9), the microinjection of Ang-(1-7) into the RVLM did not cause an increase of blood pressure and the changes of amino acid release in the RVLM and IML while the pre-injection of ACSF or AT₁ receptor antagonist Dup753 (100 pmol, n=9) had no the effect and the release of Asp and Glu in the RVLM and IML still increased. These data indicated that the regulation of Ang-(1-7) in the blood pressure in the RVLM may be mediated through the specific receptor of Ang-(l-7).Since there were many neurotransmitters in the RVLM, the nitric oxide synthase (NOS) inhibitors were used to test whether the hypertensive effect of Ang-(1-7) was mediated by neurotransmitters other than amino acids. The pre-injection of 7-nitroindazole (7-NI)(0.5 pmol, n=7), a neuronal NO synthase inhibitor, into the RVLM of rats could partially block the hypertensive effect of Ang-(1-7) (25 pmol) (P<0.05) while the aminoguanidine (AG) (75 pmol, n=7), an inducible NO synthase inhibitor, had no the effect. This indicated that the regulation of Ang-(1-7) in the blood pressure in the RVLM may be correlated with NO derived in neuronal NOS.2. The mechanism of Ang-(1-7) on regulation of blood pressure in the RVLM of stress-induced hypertensive ratsAfter 15 days of stress caused by electric foot shock combined with buzzer noise, the blood pressure and the release of Asp and Glu in the RVLM and IML of stressed rats increased significantly. The systolic blood pressure (SBP) increased to 144.7±6.1mmHg as compared with the SBP of the control rats(116.3±4.7 mmHg, n=10) (P<0.05). If the administration of electroacupuncture was conducted on "Zusanli" acupoint during the last 5 days of the stress period, the SBP of these rats decreased to 123.1±5.2 mmHg and the release of Asp and Glu in the RVLM was significantly lowered than the stressed rats (p<0.01). The microinjection of Ang-(1-7) (25 pmol, n=10) into the RVLM of stressed rats also led to the elevated blood pressure and the increased release of Asp and Glu in the RVLM and IML. The effects of Ang-(1-7) mentioned above were more enhanced in the stressed rats than in the control group rats (no stress) (p<0.01). The administration of electroacupuncture on stressed rats could inhibit the enhancement of the effect caused byAng-(1-7) (P<0.05). The microinjection of Ang779(100 pmol, n=10) decreased the blood pressure and increased the release of Gly and Tau in the RVLM and IML. What's more, this effect of Ang779 was enhanced in stressed rats and the electroacupuncture could inhibit the enhancement mentioned above. All these data indicated that the pressor effect and the increased release of Asp and Glu in the RVLM and IML caused by Ang-(1-7) were enhanced in stressed rats; the depressor effect and the increased release of Gly and Tau caused by Ang779 in the RVLM and IML were also enhanced; The administration of electroacupuncture could eliminate this enhancement in the change of blood pressure and the release of amino acids.Section 2 The mechanism of Ang-(l-7) on regulation of blood pressure in the CVLM of rats1. The mechanism of Ang-(1-7) on regulation of blood pressure in the CVLM of normal ratsThe microinjection of Ang-(1-7) in a dose of 10, 25 and 50 pmol (0.1 μl, n=6) in the CVLM unilaterally (right side) lead to the decrease of blood pressure in a dose-dependent manner. By contrast, the microinjection of Ang779, an Ang-(1-7) selective antagonist (100 pmol, n=11) (P<0.05), in the CVLM lead to an increase of blood pressure (P<0.05) and heart rate. The microinjection of ACSF in the same site had no significant effects on the blood pressure and heart rate.To investigate the mechanism of hypotensive effect of Ang-(1-7) in the CVLM, the microdialysis was performed in the same site or in the RVLM during the microinjection of Ang-(1-7) or Ang779. Results showed that the microinjection of Ang-(1-7) (25 pmol, n=l 1) in the CVLM increased the excitatory amino acid Glu and decreased the inhibitory amino acid Tau in the CVLM. At the same time, it decreased the release of Glu and increased the release of GABA in the RVLM. By contrast, the microinjection of Ang779 (100 pmol, n=11) elevated the blood pressure with the reduced release of Glu and increased release of Tau in the CVLM, but increased the release of Glu and decreased the release of GABA in the RVLM. To further confirm the correlation between the hypotensive and the hypertensive effect of Ang-(1-7) and Ang779 in the CVLM, respectively, and the change of release of amino acid neurotransmitters, the Glu receptor antagonist Kyna(1.2 nmol, n=6) or Tau receptor antagonist TAG (1.3 nmol, n=6) wasused. The microinjection of Kyna or TAG in the CVLM distinctly attenuated the hypotensive effect and hypertensive effect caused by Ang-(1-7) and Ang779 in the CVLM (P<0.05), respectively. After the microinjection of GABA_A receptor antagonist bicuculline (Bicu) (0.25 nmol, n=6), but not ACSF (n=6), in the RVLM, the micro injection of Ang-(1-7) in the CVLM not lead to the decrease of blood pressure. These results showed that the hypotensive effect of Ang-(1-7) in the CVLM was correlated with the change of amino acid releases, that is, the increase of Glu and decrease of Tau in the CVLM and the increase of GABA and the decrease of Glu in the RVLM.Was the hypotensive effect of Ang-(1-7) in the CVLM mediated by the AT₁ receptor? In order to explore this problem, Dup753 (100 pmol, n=9), an antagonist of AT1 receptor, was microinjected into the CVLM before the injection of Ang-(1-7). In this condition, the decreased blood pressure, the reduced release of Tau and the increased release of Glu in the CVLM were still observed (P<0.05). By contrast, the microinjection of Ang-(1-7) into the CVLM after the injection of Ang779 (50 pmol, n=9), an Ang-(1-7) specific receptor antagonist, caused no change in the blood pressure and the release of amino acid in the CVLM (P<0.05). These results suggested that the hypotensive effect of the Ang-(1-7) in the CVLM might be mediated by the specific receptor of Ang-(1-7) with the change of the release of Glu and Tau in this brain area.Since there were many neurotransmitters in the CVLM, was there any possibility that the hypertensive effect of Ang-(1-7) was mediated by neurotransmitters other than amino acids? In the CVLM of rats, the microinjection of Ang-(1-7) following the pre-injection of 7-NI (0.5 pmol, n=7) caused no obvious changes in the blood pressure, so it seemed that 7-NI could partially block the hypotensive effect of Ang-(1-7) in the CVLM, but the AG (75 pmol, n=7) could not block the hypotensive effect of Ang-(1-7). All this evidence indicated that the regulation of Ang-(1-7) in the blood pressure may be correlated with NO derived neuronal NOS.2. The mechanism of Ang-(l-7) on the regulation of blood pressure in the CVLM of stress-induced hypertensive ratsThe microinjection of Ang-(1-7) (25 pmol, n=10) in the CVLM of stressed rats induced the decrease of blood pressure and the increase of Glu release and decrease of Tau release in the CVLM, while the microinjection of Ang779 (100 pmol, n=10) in theCVLM of stressed rats led to the increase of blood pressure and decreased the release of Glu and increased the release of Tau in the CVLM. The effects mentioned above were more obvious in stressed rats than in control rats (no stresss) (P<0.05). The microinjection of Ang-(1-7) and Ang779 in the CVLM of electroacupuncture rats caused a similar but weaker effect compared with the stressed rats (P<0.05). These data indicated that the effects of Ang-(1-7) and Ang779 in the CVLM of stressed rats were enhanced and electroacupuncture could antagonize the enhancement of the effects in stressed rats.Taken together, our current study suggested that:l.An endogenous Ang-(1-7) is distributed in the VLM. In normal condition, Ang-(1-7) can exert tonic pressor effect in the RVLM and tonic depressor effect in the CVLM mediated through the specific receptor of Ang-(1-7).2. The pressor effect of Ang-(1-7) in the RVLM is correlated with the increased release of Glu and Asp and the decreased release of Tau in this area as well as in the IML. The depressor effect of Ang-(1-7) in the CVLM is correlated with the increased release of Glu and the decreased release of Tau in this area.3. The depressor effect of Ang-(1-7) in the CVLM is implemented by the inhibition of GABA to the RVLM.4. The pressor and depressor effect of Ang-(1-7) in the RVLM and CVLM, respectively may be mediated partially through NO derived neuronal NOS.5. The release of Asp and Glu increased significantly in the RVLM and IML of stress-induced hypertension rats. The changes of blood pressure and release of amino acid in the VLM caused by Ang-(1-7) in stressed rats are enhanced and the electroacupunture can antagonize this enhancement by the inhibition of the releases of corresponding amino acid.