Functional Significance of Angiotensin Receptor Type 2 in the Neuroplasticity of Autonomic Ganglia in (mRen2)27 Transgenic Hypertensive Rats

Figures

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  FIGURE 1.. Blood pressure measurements. Tail-cuff plethysmography was used to measure systolic blood pressure for 5 consecutive days in 12–16-week-old rats. Systolic pressure was significantly higher in (mRen2)27 rats when compared with HnSD rats. Values are mean ± SEM (*P < 0.05; n = 12). There was no difference in the pulse rate between strains (data not shown).

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  FIGURE 2.. Activity-independent responses to bath-applied angiotensin II peptide. In vitro ganglionic responses to a basal stimulation rate of 0.5 msec square wave pulses at 0.2 Hz were assessed between HnSD and (mRen2)27 animals in the presence of AngII. AngII (16–256 nM) was bath applied to SCG and the amplitude of CAP was expressed as a percentage of baseline. AngII elicited a dose-dependent increase in CAP, but was greatest in (mRen2)27 rats that peaked at 128 nM. There was a significant increase in the magnitude of CAP in response to each concentration from control, with the peak observed at 128 nM AngII concentration. Values are mean ± SEM (*P < 0.005, **P < 0.0001).

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  FIGURE 3.. Protein expression of AT1, AT2 receptor subtypes, Ang-(1-7)-mediated MAS receptor, and NOX4-specific protein density in SCG. AngII receptor density was measured by western blot hybridization using specific antibodies in ganglia isolated from (mRen2)27 hypertensive and HnSD rats. Top: Densitometry analyses of protein level normalized to β-actin. Image analyses of the signals are normalized to β-actin. Values in each panel are mean ± SEM; *P < 0.05. Representative western blot. Bottom: Representative western blot, respectively.

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  FIGURE 4.. Expression of mRNAs was studied by RT–PCR. Relative gene expression of AT1, AT2, MAS, and NOX4 mRNA profile in the SCG normalized to β-actin expression by the 2ΔΔCT comparative method and expressed as fold change compared with HnSD. Values are mean ± SEM. mRNA for AT2 is significantly lower in SCG of (mRen2)27 transgenic hypertensive animals compared with control (1.013 ± 0.105 vs. 0.171 ± 0.013 a.u.; n = 6; *P < 0.05).

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  FIGURE 5 (A-C).. The fluorescent targets and their corresponding antibody dilutions are as follows; for AT1—extracellular-ATTO Fluor-550; AT2—extracellular-ATTO Fluor-488 and MAS receptor –Fluor-488 tags (1° antibody delusion 1:50) were visualized by confocal microscopy. The 4′, 6-diamidino-2-phenylindole and F-actin merged images show the green fluorescence is specific for angiotensin receptors. Images were adjusted for brightness/contrast using the Zeiss LSM 800 image browser similar for all 3 panels. Fluorescence intensity was quantified by ImageJ software and cumulative data are presented in a bar graph. Values in each panel are mean ± SEM; *P < 0.05.

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  FIGURE 6.. AT1, AT2, MAS receptor, and ROS in SCG of HnSD and (mRen2)27. In HnSD SCG, AngII–AT1 GPCR results in increased basal level of sympathetic nerve activity and ROS generation. AngII–AT2 counteracts the SNA increase, supports ROS and NO generation. NOX4 regulates production of H2O2 and Ang-(1-7)–MAS complex regulates the NO production for optimal SNA resulting in idyllic gLTP. In (mRen2)27 SCG, AT1R function remains the same, but because of the low AT2 receptor density, there is an impaired NO generation and increased ROS production leading to increased SNA. NOX-4 is a multifunctional nicotinamide adenine dinucleotide phosphate and auto regulates ROS balance. The low NOX4 activates singlet oxygen (O2−) generation. The super oxide dismutase converts O2− to H2O2 resulting in increased ROS buildup. The impaired shielding arm of RAS at the level of SCG results in abnormal aggregation of H2O2 and decrease NO, culminating in increased sympathetic outflow and ultimately resulting in heightened gLTP.