Downregulation of thromboxane A2 and angiotensin II type 1 receptors associated with aging-related decrease in internal anal sphincter tone

Ipsita Mohanty,Jagmohan Singh,Satish Rattan

doi:10.1038/s41598-019-42894-4

Ipsita Mohanty, Jagmohan Singh + Show 1 more

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https://doi.org/10.1038/s41598-019-42894-4

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Journal: Scientific Reports	Publication Date: May 1, 2019
Citations: 7	License type: open-access

Affiliation: Thomas Jefferson University

Abstract

Aging-associated decrease in internal anal sphincter (IAS) tone (AADI) is a major contributor in the rectoanal incontinence (RI). To determine the pathogenesis of AADI, we investigated the effect of aging on GPCR activation and related downstream signaling. We particularly investigated two GPCRs that characterize IAS smooth muscle cells (SMCs): thromboxane A2 and angiotensin II type 1. Two groups of Fischer 344 rats (6-month-old [young group] and 26-month-old [old group]) were employed to determine the GPCR function by isometric contraction, the expressions of GPCRs, and their downstream regulatory signaling proteins (regulator of G-protein signaling 2, RGS2; GPCR Kinase 5, GRK5; and β-arrestin, Arrb2) using RT-PCR, qPCR, and western blot analyses. We used reversible biotinylation to monitor the GPCR trafficking using SMCs. Aging selectively attenuated thromboxane A2 and Ang II-induced IAS contraction. RT-PCR, qPCR, and WB data revealed a significant decrease in the expressions of the GPCRs and increase in the expression of RGS2, GRK5, and Arrb2. The increased GPCR internalization and decreased recycling under aging were validated by reversible biotinylation. We conclude that downregulation of GPCR, accompanied by upregulation of regulatory proteins, plays an important role in receptor desensitization and may be important underlying mechanisms of RI in certain aging patients.

Highlights

The myogenic basal tone in the internal anal sphincter (IAS) is crucial in rectoanal incontinence (RI)[1,2,3,4]
The studies reveal that aging leads to: 1) A decrease in the G-protein coupled receptors (GPCR) (TXA2-R and AT1-R)-mediated IAS tone, and in the fibroelastic properties (FEP) of the SM which correlate with decrease in the GPCR expression at gene and protein levels; 2) An increase in the expression of GPCR downstream signalling regulatory proteins, RGS2, GPCR kinase5 (GRK5), and Arrb[2]; and 3) Downregulation and compromise in the GPCR recycling leading to a decrease in the IAS tone
Our studies build on previously published studies[6,39] to show that AADI may be mediated by decrease in the response to GPCR (TXA2-R and AT1-R) activation

Summary

Introduction

The myogenic basal tone in the internal anal sphincter (IAS) is crucial in rectoanal incontinence (RI)[1,2,3,4]. The causes of RI are multifactorial, but a leading cause in humans is an aging-associated decrease in the IAS tone (AADI) and compromise in the fibroelastic properties (FEP) of the IAS5–7. Recent studies from different laboratories have shown that in animals and humans, unique myogenic properties of the IAS smooth muscle cells (SMCs) are characterized by the renin-angiotensin system (RAS) and arachidonic acid (AA) pathways, mediated through angiotensin II (Ang II) and thromboxane A2 (TXA2) by AT1-R and TXA2-R, respectively, via the G-protein coupled receptors (GPCR)[9,10,11,12,13,14]. Regulators of G-protein signaling (RGS; e.g., RGS2) play an important role in inhibiting GPCR signaling by accelerating Gα hydrolysis. C-terminal tail of the GPCR have been shown to play an important role in GPCR inactivation/internalization/ downregulation in different systems, by recruiting β-arrestin (Arrb), which binds to the GPCR on the internal surface[22,23,24,25]

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