Angiotensin peptide synthesis and cyclic nucleotide modulation in sympathetic stellate ganglia

Figures

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  Fig. 1. Angiotensin synthesizing enzymes and precursors are expressed in human stellate ganglia. In human stellate ganglia the presence of the mRNA transcripts encoding Agt (n = 4), Ren (n = 3), Ace (n = 4), Ace2 (n = 3), Agtr1 (n = 4), Agtr2 (n = 3) and Mas1 (n = 4) were confirmed by qRT-PCR. The qRT-PCR raw counts for the genes of interest were normalized to the control gene B2m using the ∆CT method and expressed as ∆CT mean ± SEM (a). ELISAs were used to demonstrate the protein expression of the relevant proteins of interest including Agt, Ren, AngII, ACE2 and Ang1–7 in human stellate ganglia. Agt was found to be highly expressed in human stellate ganglia (n = 2, ~53,694 pg/mg), as was Ren (n = 3, 2005 ± 388 pg/mg). AngII (n = 3, 188.7 ± 15.37 pg/mg), ACE2 (n = 2, 171.9 ± 2.60 pg/mg) and Ang1–7 (n = 3, 179.9 ± 6.13 pg/mg) were also identified and were found to have similar levels of expression (b). Data are displayed as mean ± SEM. A model diagram depicts AngII and Ang1–7 release from the stellate ganglia and the proposed pre-and post-synaptic effects.

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  Fig. 2. The principal component (PCA) plot illustrates the variation of the stellate ganglia transcriptome between strains (a). The MA plot depicts the relationship between the number of counts per gene, the size of the fold change and the significance of fold changes. Significance is indicated by red spheres on a gene-by-gene basis. Non-significant transcripts are shown in grey (b).

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  Fig. 3. Transcripts of angiotensin synthesizing genes were observed in the rat sympathetic stellate ganglia in the RNA-seq dataset. The transcriptome of the sympathetic stellate ganglia was sequenced using stellate ganglia extracted from four-week-old male Wistar rats (n = 5) and age-matched male prehypertensive SHR (preSHR, n = 5). A KEGG analysis was carried out using the differentially expressed transcripts where the gene input was selected using the Benjamini-Hochburg p.adj < 0.05. The KEGG group ‘Renin Secretion’ was found to be significantly altered in the preSHR ganglia, where the gene input was selected using the Benjamini-Hochburg p.adj < 0.05 (a). A full list of the genes, the fold changes and respective levels of significance are reported in Table 4. The AngII and Ang1–7 synthesis pathways are outlined (b). Transcripts encoding the enzymes and precursors classically involved in the synthesis of AngII and Ang1–7 were identified in young rat stellate ganglia (b), where the relevant transcripts included Angiotensinogen (Agt), Renin (Ren) and the Angiotensin Converting Enzymes (Ace, Ace2). The transcripts for AngII receptors type 1 and 2 (Agtr1a, Agtr1b, Agtr2) and for the Ang1–7…

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  Fig. 4. Angiotensinergic mRNA transcript validation by qRT-PCR in rat stellate ganglia. The presence of the RNA transcripts Agt, Ren, Ace, Ace2, Agtr1a, Agtr1b, Agtr2 and Mas1 was confirmed by qRT-PCR in sympathetic stellate ganglia from four-week Wistar and preSHR ganglia (a), and 16-week adult Wistar and SHR (b). The qRT-PCR raw counts were first normalized to a control gene B2m as per the comparative (∆CT) method [48]. Each data point corresponds to one stellate RNA sample from one rat. Data are displayed as ∆CT mean ± SEM. FRET microscopy was conducted on sympathetic stellate neurons obtained from Wistar (n = 11 rats, 3 cultures, 20 cells) and preSHR rats (n = 9 rats, 3 cultures, 19 cells). Cells were transduced with the cGi500 FRET sensor and randomly selected for imaging. Increases in cGMP generation was observed in sympathetic neurons in response to Ang1–7 and AngII (c, d). Maximal FRET changes were evoked following administration of a combination of the NO-donor Sin-1 (10 μM) and the PDE inhibitor IBMX (100 μM). There was significantly greater cGMP generation in response to AngII in Wistar vs. preSHR neurons (two-way ANOVA, p = .0403). Peak FRET changes were obtained in response…

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  Fig. 5. Model diagram depicts angiotensin synthesis and pre- and post-synaptic signaling pathways. In sympathetic stellate neurons, the classical pathway for Angiotensin II (AngII) synthesis occurs by sequential enzymatic cleavage of Angiotensinogen (Agt) by renin and Angiotensin Converting Enzymes (ACE). AngII is hydrolyzed by ACE2 producing the bioactive metabolite of Angiotensin 1–7 (Ang1–7). We identified the presence of precursors and transcripts encoding these enzymes and depict here a proposed model for angiotensin synthesis (a). We also identified the presence of AngII and Ang1–7 receptors on sympathetic stellate ganglia of human and rat. AngII has been shown to elevate intracellular Ca2+ and enhance noradrenaline release via actions at AT1R [59,60]. Conversely Ang1–7-dependent activation of its cognate receptor, Mas R, has been shown to couple to NO in the brain and several other receptor sites [61]. In this study, we show that administration of both AngII and Ang1–7 elevate cGMP in the rat stellate ganglia. We and others have previously demonstrated the importance of NO-cGMP signaling in reducing [Ca2+]i [47,63] and end-organ transmission in peripheral sympathetic stellate…