Aging modifies receptor expression but not muscular contractile response to angiotensin II in rat jejunum

Abstract

The involvement of renin-angiotensin system in the modulation of gut motility and age-related changes in mRNA expression of angiotensin (Ang II) receptors (ATR) are well accepted. We aimed to characterize, in vitro, the contractile responses induced by Ang II, in jejunum from young (3-6 weeks old) and old rats (≥ 1 year old), to evaluate possible functional differences associated to changes in receptor expression. Mechanical responses to Ang II were examined in vitro as changes in isometric tension. ATR expression was assessed by qRT-PCR. Ang II induced a contractile effect, antagonized by losartan, AT1R antagonist, and increased by PD123319, AT2R antagonist, as well by neural blocker ω-conotoxin and by nitric oxide (NO) synthase inhibitor. No difference in the response was observed between young and old groups. AT1 receptor-mediated contractile response was decreased by U-73122, phospholipase C (PLC) inhibitor; or 2-aminoethoxy-diphenylborate (2-APB), inositol triphosphate (IP₃) receptor inhibitor; or nifedipine, L-type calcium channel blocker. Age-related changes in the expression of both AT1 receptor subtypes, AT1a and AT1b, and of AT2 receptors were detected. In conclusion, Ang II modulates the spontaneous contractility of rat jejunum via postjunctional AT1 receptors, involving Ca²⁺ mobilization from intracellular stores, via PLC/IP₃ pathway, and Ca²⁺ influx from extracellular space, via L-type channels. Prejunctional AT2 receptors would counteract AT1 receptor effects, via NO synthesis. The observed age-related differences in the expression of all AT receptor subtypes are not reflected in the muscular contractile response to Ang II.

Keywords: Aging; Angiotensin II; Angiotensin II receptors; Intestinal motility.

Fig. 1

Ang II responses in young and old jejunum preparations. A Original recordings showing the mechanical responses evoked by 10 μM CCh and 100 nM Ang II in jejunum of young and old rats. B Concentration-response curve for the excitatory effects induced by Ang II (0.1–300 nM) in jejunum of young and old rats. Data are means ± S.E.M. and are expressed as percentage of the maximal effect induced by 10 μM CCh (n = 9 for each group)

Fig. 2

Characterization of receptors involved in excitatory effects induced by Ang II in young and old jejunum preparations. Concentration-response curves to Ang II (0.1–300 nM) before and after losartan, AT1 receptor antagonist (100 nM, n = 5 for each group), and PD123319 (100 nM, n = 5 for each group), AT2 receptor antagonist, in jejunum from young (A) and old (B) rats. Data are means ± S.E.M. and are expressed as percentage of the maximal effect induced by 10 μM CCh. The values for the control curves are the means of the control data obtained before each treatment. *P < 0.05 compared to the respective own control condition

Fig. 3

Effects of inhibitor of enteric nervous system pathways and AT2 receptor antagonist on Ang II response. Histogram showing the effects of Ang II (50 nM) in the presence or absence of ω-conotoxin, Ca₂₊ voltage-gated neural channel blocker (0.1 μM, n = 4 for each group); or atropine (1 μM, n = 4 for each group), muscarinic receptor antagonist; or l-NAME (100 μM, n = 4 for each group), inhibitor of NO synthase; or PD123319 (100 nM, n = 5 for each group) in jejunum from young (A) or old (B) rats. Data are means ± S.E.M. and are expressed as percentage of the maximal effect induced by 10 μM CCh. The graphed value for the control bar is the mean of the control data obtained before each treatment. *P < 0.05 compared to the respective own control condition

Fig. 4

Intracellular signaling involved in Ang II response. Histogram showing the effects of Ang II (50 nM) in the presence or absence of U-73122, PLC inhibitor (10 μM, n = 4 for each group); 2-APB, membrane-permeant IP3 receptor inhibitor (20 μM, n = 4 for each group); ryanodine (10 μM, n = 4); and nifedipine, voltage-gated calcium channel antagonist (5 nM, n = 4 for each group), in jejunum from young (A) or old (B) rats. Data are means ± S.E.M. and are expressed as percentage of the maximal effect induced by 10 μM CCh. The graphed value for the control bar is the mean of the control data obtained before each treatment. *P < 0.05 compared to the respective own control condition

Fig. 5

Angiotensin II receptor expression analysis. Histograms showing levels of AT1a (A), AT1b R (B), and AT2 (C) mRNA expression in jejunum from young or old rats. Gene expression was normalized by β-actin. Results are expressed as mean ± S.E.M., n = 3 each. *P < 0.05 compared to old group