Cyclic AMP response to recombinant human relaxin by cultured human endometrial cells—A specific and high throughput in vitro bioassay

doi:10.1016/0006-291X(90)91262-Q

Biochemical and Biophysical Research Communications

Volume 170, Issue 1, 16 July 1990, Pages 214-222

https://doi.org/10.1016/0006-291X(90)91262-Q Get rights and content

Abstract

A specific and high throughput 96-well format bioassay for recombinant human relaxin (rhRLX) has been developed using human endometrial cells (NHE cells). rhRLX caused a time- and dose-dependent stimulation of cyclic AMP (cAMP) with 1/2 maximal activity of 3.56±0.65 ng/ml (n=30). The range of the standard curve was 0.39 to 25 ng/ml with interplate precision of 17 and 22% CV for high and low controls respectively. The cAMP response requires forskolin and 3-isobutyl-1-methylxanthine, and is enhanced by prostaglandin E₂ and F_2α. The NHE cells do not respond to A or B chains of rhRLX, or a whole array of hormones. Preincubation of rhRLX with specific monoclonal antibody completely abolished the cAMP response. This bioassay has been used to determine the biological activity of several manufactured lots of recombinant human relaxin.

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Understanding relaxin signalling at the cellular level
2019, Molecular and Cellular Endocrinology
Citation Excerpt :
cAMP signals by activating protein kinase A (PKA), Epac, and cyclic nucleotide-gated ion channels. Relaxin administration acutely increases cAMP activity across a range of cell types, including normal human endometrial (NHE) cells (Fei et al., 1990), endometrial stromal cells (Bartsch et al., 2004), human myometrial cells (Heng et al., 2008), human decidual cells from fetal membranes (Kern and Bryant-Greenwood, 2009), and human decidual macrophages (Horton et al., 2011). Relaxin also increases cAMP activity in rat anterior pituitary cells (Cronin et al., 1987) and various human cancer cell types including MCF-7 breast cancer cells (Bigazzi et al., 1992), monocytic leukaemia THP-1 cells (Parsell et al., 1996), prostate cancer cells (Silvertown et al., 2007; Vinall et al., 2011), and some glioblastoma cells (Glogowska et al., 2013).
The peptide hormone relaxin mediates many biological actions including anti-fibrotic, vasodilatory, angiogenic, anti-inflammatory, anti-apoptotic, and organ protective effects across a range of tissues. At the cellular level, relaxin binds to the G protein-coupled receptor relaxin family peptide receptor 1 (RXFP1) to activate a variety of downstream signal transduction pathways. This signalling cascade is complex and also varies in diverse cellular backgrounds. Moreover, RXFP1 signalling shows crosstalk with other receptors to mediate some of its physiological functions. This review summarises known signalling pathways induced by acute versus chronic treatment with relaxin across a range of cell types, it describes RXFP1 crosstalk with other receptors, signalling pathways activated by other ligands targeting RXFP1, and it also outlines physiological relevance of RXFP1 signalling outputs. Comprehensive understanding of the mechanism of relaxin actions in fibrosis, vasodilation, as well as organ protection, will further support relaxin's clinical potential.
Membrane receptors: Structure and function of the relaxin family peptide receptors
2010, Molecular and Cellular Endocrinology
The receptors for members of the relaxin peptide family have only recently been discovered and are G-protein-coupled receptors (GPCRs). Relaxin and insulin-like peptide 3 (INSL3) interact with the leucine-rich-repeat-containing GPCRs (LGRs) LGR7 and LGR8, respectively. These receptors show closest similarity to the glycoprotein hormone receptors and contain large ectodomains with 10 leucine-rich repeats (LRRs) but are unique members of the LGR family (class C) as they have an LDL class A (LDLa) module at their N-terminus. In contrast, relaxin-3 and INSL5 interact with another class of type I GPCRs which lack a large ectodomain, the peptide receptors GPCR135 and GPCR142, respectively. These receptors are now classified as relaxin family peptide (RXFP) receptors, RXFP1 (LGR7), RXFP2 (LGR8), RXFP3 (GPCR135) and RXFP4 (GPCR142). This review outlines the identification of the peptides and receptors, their expression profiles and physiological roles and the functional interactions of the peptides with their unique receptors.
Relaxin receptor-LGR7 (RXFP1)
2008, xPharm: The Comprehensive Pharmacology Reference
A novel, simple bioactivity assay for relaxin based on inhibition of platelet aggregation
2007, Regulatory Peptides
In humans, the relaxin hormone family includes H1, H2 and H3 isoforms and insulin-like peptides 3 to 6. The ever-increasing interest in relaxin as potential new drug requires reliable methods to compare bioactivity of different relaxins. The existing bioassays include in vivo or ex vivo methods evaluating the organ-specific responses to relaxin and in vitro methods based on measurement of cAMP increase in relaxin receptor-bearing cells. We previously demonstrated that relaxin dose-dependently inhibits platelet aggregation. On this basis, we have developed a simple, reliable bioassay for relaxin used to compare purified porcine relaxin, assumed as reference standard, with two recombinant human H2 relaxins, H3 relaxin, insulin-like peptides 3 and 5.
Pre-incubation of platelets with relaxins (3, 10, 30,100, 300 ng/ml; 10 min.) caused the inhibition of ADP-induced platelet aggregation. Within the 10–100 ng/ml range, porcine relaxin showed the highest effects and a nearly linear dose–response correlation. Lower peptide concentrations were ineffective, as were insulin-like peptides 3 and 5 at any concentration assayed. Platelet inhibition was mediated by specific RXFP1 relaxin receptor and cGMP, whose intracellular levels dose-dependently increased upon relaxin. For comparison, we stimulated THP-1 cells, a relaxin receptor-bearing cell line, with porcine relaxin, human H2 and H3 relaxins at the above concentrations (15 min.). We observed a dose-related increase of intracellular cAMP similar to the trend of platelet inhibition. Insulin like peptide 5 was ineffective. In conclusion, this study shows that inhibition of platelet aggregation may be used to assess bioactivity of relaxin preparations for experimental and clinical purposes.
Relaxin signalling in THP-1 cells uses a novel phosphotyrosine-dependent pathway
2007, Molecular and Cellular Endocrinology
The heterodimeric peptide hormone relaxin acts through the novel G-protein coupled receptor LGR7 to elicit the production of cAMP in the human monocyte cell line THP-1. The very small number of receptors on the cell surface, and the lack of response in cell membranes imply the involvement of a cytoplasmic signal amplification process. Here we show that this process comprises a novel and specific tyrosine kinase activity close to the receptor, and involves neither protein kinase A, mitogen-activated protein kinase, nor phosphoinositide-3 kinase activities as major upstream components. Furthermore, this novel involvement of a tyrosine kinase activity is cell-type dependent, being largely absent from LGR7-transfected HEK293T cells, and receptor-dependent; vasoactive intestinal peptide or isoproterenol signalling in the same cells does not require this tyrosine kinase activity.
'Relaxin' the stiffened heart and arteries: The therapeutic potential for relaxin in the treatment of cardiovascular disease
2006, Pharmacology and Therapeutics
Although originally characterised as a reproductive hormone, relaxin has emerged as a multi-functional endocrine and paracrine factor that plays a number of important roles in several organs, including the normal and diseased cardiovascular system. The recent discovery of the H3/relaxin-3 gene, and the elusive receptors for relaxin (Relaxin family peptide receptor; RXFP1) and relaxin-3 (RXFP3/RXFP4) have led to the re-classification of a distinct relaxin peptide/receptor family. Additionally, the identification of relaxin and RXFP1 mRNA and/or relaxin binding sites in the heart and blood vessels has confirmed that the cardiovascular system is a target for relaxin peptides. While evidence for the production of relaxins within the cardiovascular system is limited, several studies have established that the relaxin genes are upregulated in the diseased human and rodent heart where they likely act as cardioprotective agents. The ability of relaxin to protect the heart is most likely mediated via its antifibrotic, anti-hypertrophic, anti-inflammatory and vasodilatory actions, but it may also directly stimulate myocardial regeneration and repair. This review describes relaxin and its primary receptor (RXFP1) in relation to the roles and effects of relaxin in the normal and pathological cardiovascular system. It is becoming increasingly clear that relaxin has a number of diverse physiological and pathological roles in the cardiovascular system that may have important therapeutic and clinical implications.

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Cyclic AMP response to recombinant human relaxin by cultured human endometrial cells—A specific and high throughput in vitro bioassay

Abstract

Biochem. Biophys. Res. Commun.

Ann. N. Y. Acad. Sci.

Endocrinology

Endocrinology

Endocrinology

J. Endocrinol.

J. Clin. Endocrinol. Metab.

Endocrinology