Ontogeny of apelin and its receptor in the rodent gastrointestinal tract

doi:10.1016/j.regpep.2009.07.016

Regulatory Peptides

Volume 158, Issues 1–3, 27 November 2009, Pages 32-39

https://doi.org/10.1016/j.regpep.2009.07.016 Get rights and content

Abstract

Apelin is the endogenous ligand for the APJ receptor and both apelin and APJ are expressed in the gastrointestinal (GI) tract. The aim of this study was to define ontogeny of apelin and APJ in the developing rodent GI tract by measuring expression levels and characterizing abundance and cellular localization at an embryonic stage (E18.5 or E21), two postnatal stages (P4, P16) and in the adult. Apelin and APJ mRNA levels were measured by real time RT-PCR, apelin and APJ-containing cells were identified by immunohistochemical (IHC) staining. Gastric, duodenal and colonic apelin and APJ mRNA levels were highest at birth and declined postnatally. In the postnatal rat stomach, few apelin peptide-containing cells were identified, the density of gastric apelin-containing cells increased progressively after weaning and into adulthood. A robust APJ immunostaining was observed postnatally in the epithelium, intestinal goblet cells and in smooth muscle cells. In the adult rat, APJ immunostaining in the surface epithelium and goblet cells decreased markedly. During the early postnatal period, in an apelin-deficient mouse, APJ expression and immunostaining in the gut were reduced suggesting that apelin regulates APJ. Together, our data support a role for the apelin–APJ system in the regulation of smooth muscle, epithelial and goblet cell function in the GI tract.

Introduction

Apelin is the endogenous ligand for the APJ receptor [1]. The APJ receptor is a member of the G-protein-coupled receptor (GPCR) family [2] and is structurally related to the angiotensin and CXC chemokine receptors [3], [4]. Apelin was discovered by screening tissue extracts for their effects on extracellular acidification and inhibition of cAMP formation in a Chinese hamster ovary cell line transfected with the APJ cDNA [1]. Rat, mouse, cow and human apelin cDNAs have been characterized [1], [5] and encode a 77-amino-acid precursor peptide. A 36-amino-acid variant of apelin is the apparent parent peptide.

Apelin and APJ have a widespread distribution in the body [6], [7], [8]. Apelin and APJ are expressed in the brain, kidney, adipose tissue, heart, lung, retina, mammary gland and gastrointestinal tract (GI) [5], [9], [10], [11], [12], [13], [14], [15], [16], [17]. Apelin exerts a broad range of physiological actions including effects on heart contractility, blood pressure, blood vessel growth, appetite and drinking behavior, pituitary hormone secretion and the hypothalamic–pituitary–adrenal axis [17], [18], [19], [20], [21], [22], [23], [24]. In the GI tract and pancreas, apelin has been shown to influence gastric acid secretion as well as intestinal and pancreatic hormone secretions [17], [25], [26].

During pregnancy and lactation, breast apelin expression increases ~ 7- to 20-fold [5], [27] and significant amounts of apelin are ingested by neonates. A putative target of ingested apelin is the GI tract, however, the extent to which APJ is expressed postnatally in the GI tract, and more importantly, where APJ is localized in the GI tract is not known.

The purpose of the present study, therefore, was to characterize APJ and apelin expression (mRNA levels) profiles as well as localization and abundance of APJ protein and apelin peptide in the developing mouse and rat GI tract. Additionally, the influence of apelin gene knockout on APJ mRNA and immunostaining intensity was investigated.

Section snippets

Animals

All animal experiments were done in accordance with mandated standards of humane care and were approved by the Institutional Animal Care and Use Committees at the University of Texas Medical Branch and Stanford University. C57/BL6 mice (Fig. 1, Fig. 3), 129SV mice (Fig. 6, Fig. 7) and Sprague–Dawley rats (Fig. 1, Fig. 2, Fig. 3, Fig. 4, Fig. 5) were maintained in air-conditioned and light-regulated rooms (lights on, 0600–1800 h) and given access to food and water ad libitum. All tissues were

Ontogeny of apelin and APJ expression in the rat GI tract

The aim of this study was to characterize apelin and APJ expression (mRNA levels) in the developing rat stomach, duodenum and colon. We measured apelin and APJ expression at a single embryonic stage (E21), two postnatal stages (P4, P16), and in the adult rat (~ 1–3 months old).

In the rat stomach, apelin expression levels were maximal at the E21 and P4 stages, and then declined progressively into adulthood. Apelin expression levels in the rat stomach were significantly lower at the P16 and the

Discussion

The aim of the present study was to characterize the ontogeny of apelin and APJ expression as well as ontogeny, localization and abundance of apelin peptide and APJ protein in the developing rodent GI tract. Furthermore, the influence of apelin gene knockout on APJ expression and immunostaining intensity in the developing mouse GI tract was examined. In this study, we demonstrate that expression levels of apelin and APJ are regulated developmentally and in a parallel fashion. In the rat and

Acknowledgments

This work is supported by grants from the National Institutes of Health (P01 DK35608), Broad Medical Research Program (IBD-0118), and Crohn's and Colitis Foundation of America (Ref. #1821).

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The apelin/APJ signaling system and cytoprotection: Role of its cross-talk with kappa opioid receptor
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Apelin, a regulatory peptide, is an endogenous ligand of the apelin receptor (APJ), which belongs to the G protein-coupled receptor family. The peptide and its receptor are distributed in animal and human tissues, including the cardiovascular and central nervous systems, and studies indicate that apelin signaling could play a role in cytoprotection of cells where it is found. Apelin activity may be modulated by interactions of the APJ receptor with other receptors that result in heteromerization. The interaction of the APJ with other receptor systems increases the signaling repertoire of apelin, thereby allowing it to exert a widened degree of control over cellular physiological functions. This article reviews studies which provide evidence for the physiological importance of APJ/Kappa opioid receptor (KOR) heterodimers in several critical cellular processes, including cell proliferation, and results are discussed that support a role of this peptide and the APJ receptor in functioning of the cardiovascular, gastrointestinal and central nervous systems under normal conditions and pathology, as well as novel signal transduction characteristics resulting following the interaction of APJ and KOR. A better understanding of the cellular protective actions of apelin, and the physiological outcomes from interaction of its receptor with other receptor types could lead to new pharmaceutical targets for various diseases affecting among other organs, the heart, the gastrointestinal system and the brain.
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The apelin/APJ system, which has a widespread distribution in the body, is involved in the regulation of physiological and pathophysiological mechanisms such as regulation of blood pressure, stress response, immunological activities, obesity, diabetes, inflammation, and neurodegenerative diseases. Apelin also undertakes various tasks in the digestive system such as cell proliferation, secretion of hormones (eg. cholecystokinin and histamine), modulation of gastric and pancreatic secretions, and motility response. Recent researchs have reported that apelin exerts gastroprotective effects by regulating the components of gastric mucosal barrier. Mucosal disorders that can develop in the stomach due to different reasons such as microbial infections, drug use, smoking habits, alcohol consumption, exposure to stress, pepsin, and acid affect a significant proportion of the human population in the world. This review discusses the role of apelin in the protective mechanisms of gastric mucosa against harmful effects.
Peripheral apelin mediates visceral hypersensitivity and impaired gut barrier in a rat irritable bowel syndrome model
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Citation Excerpt :
APJ is expressed in various organs, such as brain, heart, lung, kidney, spleen, ovary and gut, and plays physiological roles in regulating cardiovascular function, angiogenesis, fluid homeostasis and energy metabolism (O'Carroll et al., 2013). In GI tract, APJ and apelin are detected in epithelial cells (Han et al., 2007), myenteric plexus neurons (Bülbül et al., 2020), smooth muscle (Wang et al., 2009) and enteroendocrine cells (Susaki et al., 2005), suggesting that apelinergic system may modulate GI function. Actually, peripheral injection of apelin was demonstrated to inhibit gastric emptying and colonic transit in rodents (Bülbül et al., 2017).
Growing evidence indicates that visceral hypersensitivity and impaired gut barrier play an important role in the pathophysiology of irritable bowel syndrome (IBS). In animal models, these changes are known to be mediated via corticotropin-releasing factor (CRF)-Toll like receptor 4 (TLR4)-proinflammatory cytokine signaling. Apelin, an endogenous ligand of APJ, was reported to modulate CRF-induced enhanced colonic motility. In this context, we hypothesized that apelin also modulates visceral sensation and gut barrier, and tested this hypothesis. We measured visceral pain threshold in response to colonic balloon distention by abdominal muscle contractions assessed by electromyogram in rats. Colonic permeability was estimated by quantifying the absorbed Evans blue in colonic tissue. Intraperitoneal (ip) administration of [Ala13]-apelin-13, an APJ antagonist, blocked lipopolysaccharide (LPS)- or CRF-induced visceral hypersensitivity and colonic hyperpermeability (IBS model) in a dose-response manner. These inhibitory effects were blocked by compound C, an AMPK inhibitor, N^G-nitro-L-arginine methyl ester, a nitric oxide (NO) synthesis inhibitor or naloxone in the LPS model. On the other hand, ip [Pyr1]-apelin-13, an APJ agonist, caused visceral hypersensitivity and colonic hyperpermeability, and these effects were reversed by astressin, a CRF receptor antagonist, TAK-242, a TLR4 antagonist or anakinra, an interleukin-1 receptor antagonist. APJ system modulated CRF-TLR4-proinflammatory cytokine signaling to cause visceral hypersensitivity and colonic hyperpermeability. APJ antagonist blocked these GI changes in IBS models, which were mediated via AMPK, NO and opioid signaling. Apelin may contribute to the IBS pathophysiology, and the inhibition of apelinergic signaling may be a promising therapeutic option for IBS.
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Citation Excerpt :
Mota et al., 2004; Medhurst et al., 2003). Both apelin and apelin receptor are widely distributed in mucosal enteroendocrine cells, myenteric plexus neurons and smooth muscle cells in the gastrointestinal (GI) tract (Susaki et al., 2005; Han et al., 2008; Wang et al., 2009). Previous studies have found that apelin plays important roles in regulating GI functions, including increasing gastric acid secretion (Ohno et al., 2012), regulating food intake and appetite (Sunter et al., 2003; O'Shea et al., 2003), inhibiting tissue maturation in the GI tract(Antushevich et al., 2015), stimulating cholecystokinin (CCK) secretion and affecting the activity of some gastric and pancreatic enzymes(Wang et al., 2004; Antuschevich et al., 2016).
Apelin is a novel neuropeptide identified as the endogenous ligand for the apelin receptor. Apelin and its receptor are widely distributed in the gastrointestinal tract. Studies have reported that apelin-13 is involved in modulating gastrointestinal motility; however, the evidence is insufficient and the relevant mechanism is still not fully clear. Consequently, our study designed to explore the effect induced by exogenous apelin-13, to analyze the mechanism of action in isolated rat colons and colonic smooth muscle cells. The spontaneous contractions of colonic smooth muscle strips from rat were measured in an organ bath system. L-type calcium currents and large conductance Ca²⁺-activated K⁺ (BK_Ca) currents in rat colonic smooth muscle cells were investigated using the electrophysiological patch-clamp technique.
Apelin-13 decreased the spontaneous contractile activity of colonic smooth muscle strips in a dose-dependent manner, and the inhibitory effect was not abolished by tetrodotoxin. The electrophysiological recordings revealed that apelin-13 reduced the crest currents of L-type calcium in a concentration-dependent manner in colonic smooth muscle cells at the test potential of 0 mV. Moreover, apelin-13 moved the current-voltage (I–V) curves of L-type calcium channels upward, but did not change their contour. Furthermore, the characteristics of L-type calcium channels with steady-state activation and steady-state inactivation were not significantly changed. Similarly, application of apelin-13 also significantly decreased BK_Ca currents in a concentration-dependent manner. In conclusion, apelin-13 inhibited the spontaneous contractile activity of isolated rat colons via the suppression of L-type calcium channels and BK_Ca channels in colonic smooth muscle cells.
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Among the numerous biological active products of preproapelin, apelin-13 has been shown to be the most abundant form with a greater affinity for APJ in rodents (De Mota et al., 2000; Medhurst et al., 2003). Within the GI tract, wide distribution of apelin/APJ receptor system suggests that the released apelin from gut may exhibit a regulatory role in GI functions (Han et al., 2008; Han et al., 2007; Susaki et al., 2005; Wang et al., 2004; Wang et al., 2009). Recently, acute stress-induced increase in gastric apelin content was demonstrated in rats (Izgut-Uysal et al., 2014), furthermore, peripheral administration of apelin has been shown to induce the release of cholecystokinin (CCK), the well-defined alimentary peptide hormone which is released in the postprandial stage in response to the meal ingestion (Bulbul et al., 2017a; Flemstrom et al., 2011; Wattez et al., 2013).
Stress increases the apelin content in gut, while exogenous peripheral apelin has been shown to induce cholecystokinin (CCK) release. The present study was designed to elucidate (i) the effect of acute stress on enteric production of apelin and CCK, (ii) the role of APJ receptors in apelin-induced CCK release depending on the nutritional status. CCK levels were assayed in portal vein blood samples obtained from stressed (ARS) and non-stressed (NS) rats previously injected with APJ receptor antagonist F13A or vehicle. Duodenal expressions of apelin, CCK and APJ receptor were detected by immunohistochemistry. ARS increased the CCK release which was abolished by selective APJ receptor antagonist F13A. The stimulatory effect of ARS on CCK production was only observed in rats fed ad-libitum. Apelin and CCK expressions were upregulated by ARS. In addition to the duodenal I cells, APJ receptor was also detected in CCK-producing myenteric neurons. Enteric apelin appears to regulate the stress-induced changes in GI functions through CCK. Therefore, apelin/APJ receptor systems seem to be a therapeutic target for the treatment of stress-related gastrointestinal disorders.

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Ontogeny of apelin and its receptor in the rodent gastrointestinal tract

Abstract

Introduction

Section snippets

Animals

Ontogeny of apelin and APJ expression in the rat GI tract

Discussion

Acknowledgments

Biochem Biophys Res Commun

Gene

Mech Dev

Mech Dev

Biochim Biophys Acta

Pharmacol Ther

Trends Pharmacol Sci

Cell Signal

J Biol Chem

Biochem Biophys Res Commun

Biochim Biophys Acta

Regul Pept

Regul Pept

Biochim Biophys Acta

J Mol Cell Cardiol

Dev Biol

Neurosci Lett

Biochem Biophys Res Commun

Regul Pept