Bani, Daniele

Nistri, Silvia

English

PubMed

Silvia Nistri

Daniele Bani

Springer - Publisher Connector

BioMed Central
Reproductive Biology and 
Endocrinology
ssOpen AcceHypothesis
Relaxin receptors and nitric oxide synthases: search for the 
missing link
Silvia Nistri and Daniele Bani*
Address: Dept. Anatomy, Histology & Forensic Medicine, University of Florence. V.le G.Pieraccini, 6 I-50139 Florence, Italy
Email: Silvia Nistri - silvianistri@virgilio.it; Daniele Bani* - daniele.bani@unifi.it
* Corresponding author    
relaxinnitric oxide synthasesG proteinResearchers involved in the study of relaxin have wel-
comed the article by Hsu et al. [1], who eventually identi-
fied the receptors for this hormone. In the past, using
labeled relaxin, specific binding sites have been found in
several target organs and tissues for this hormone [2–4],
but the exact molecular and functional nature of the relax-
in receptor remained elusive. Relaxin is structurally simi-
lar to insulin and insulin-like growth factors (IGFs) [5],
hence it seemed logical to assume that its putative recep-
tor should belong to the insulin family of receptors, which
are membrane-associated tyrosine kinases. Nonetheless,
studies in this direction have been unfruitful and even
misleading, as experienced by Dr Ivell and his team. In a
recent study they were at a very short step from a crucial
discovery, because they found that the relaxin-induced
cAMP accumulation in target cells in in vitro culture was
prevented by a pharmacologic inhibitor of G protein acti-
vation [6]. However, their attention being focused on ty-
rosine phosphorylation, they did not go deep into this
finding. Using a completely different approach, Hsu and
coworkers noticed that knock-out mice for the relaxin-like
factor/Insl3 (also known as Leydig cell relaxin) had an ab-
normal testis descent phenotype [7,8] which was similar
to that of mice with a disruption of a G protein-coupled
receptor (GPCR) gene [9]. On these grounds, they
screened the human and mouse genome for orphan
GPCRs and they could smartly demonstrate that relaxin is
a cognate ligand for two leucine-rich repeat-containing
GPCRs, LGR7 and LGR8, acting through Gs proteins. Acti-
vation of adenylate cyclase by Gs proteins can explain why
relaxin induces an elevation of cAMP in target cells and
tissues [5]. In particular, in the same article cited above
[1], Hsu and coworkers demonstrated that LGR7 and
LGR8 are capable of mediating the action of relaxin
through a cAMP-dependent pathway distinct from that of
insulin and IGF family ligands.
In recent years, our own studies and those of other inves-
tigators have provided increasing evidence that relaxin can
also act on several of its targets by increasing the expres-
sion and/or activity of nitric oxide synthase (NOS) isoen-
zymes, thereby promoting the generation of nitric oxide
(NO) [10–16]. Time is ripe for investigating how the new-
ly discovered relaxin GPCRs may account for this. Based
on the current literature, there are multiple pathways by
Published: 5 February 2003
Reproductive Biology and Endocrinology 2003, 1:5
Received: 28 January 2003
Accepted: 5 February 2003
This article is available from: http://www.RBEj.com/content/1/1/5
© 2003 Nistri and Bani; licensee BioMed Central Ltd. This is an Open Access article: verbatim copying and redistribution of this article are permitted in all 
media for any purpose, provided this notice is preserved along with the article's original URL.
Figure 1
Putative interactions between relaxin receptor sign-
aling and intrinsic NO pathway. AC: adenylate cyclase 
Akt: protein kinase B Gs: Gs proteins IκB inhibitor subunit of 
nuclear factor kappa-B LGR7-8: leucine-rich repeat-contain-
ing G protein-coupled receptors 7 and 8 NF-κB nuclear fac-
tor kappa-B NO: nitric oxide NOS II: inducible NO synthase 
NOS III: constitutive NO synthase PI3-K: phosphoinositide 3-
kinase PKA: protein kinase A RLX: relaxinPage 1 of 2
(page number not for citation purposes)
Reproductive Biology and Endocrinology 2003, 1 http://www.RBEj.com/content/1/1/5Publish with BioMed Central   and  every 
scientist can read your work free of charge
"BioMed Central will be the most significant development for 
disseminating the results of biomedical research in our lifetime."
Sir Paul Nurse, Cancer Research UK
Your research papers will be:
available free of charge to the entire biomedical community
peer reviewed and published immediately upon acceptance
cited in PubMed and archived on PubMed Central 
yours — you keep the copyright
Submit your manuscript here:
http://www.biomedcentral.com/info/publishing_adv.asp
BioMedcentral
which GPCRs can stimulate NO biosynthesis. In endothe-
lial cells, the best-described agonists for the constitutive
NOS III isoform, acetylcholine and bradykinin, activate
specific membrane-associated GPCRs [17,18]. Similar ef-
fects are exerted by surface estrogen receptors [19]. It has
been reported that NOS III can be also activated by direct
stimulation of GPCRs with sphingosine 1-phosphate
[20]. The classical signaling pathway appears to involve G
protein βγ subunits that, by means of phosphoinositide 3-
kinase (PI3-K), switch on protein kinase B (Akt), which in
turn activates eNOS by phosphorylation at Ser-1179 [20].
Another mechanism, which may either coexist in the same
cell or be alternatively operating in different cell types,
could involve cAMP and the inducible NOS II isoform. In
rat vascular smooth muscle cells, GPCR-activated ade-
nylate cyclase and the consequent rise in cAMP upregu-
lates protein kinase A (PKA) activity [21]. In turn, PKA is
able to phosphorilate and inactivate IkB-α, the inhibitor
subunit of the transcription factor NF-κB, thus allowing
NF-κB to translocate into the nucleus and to promote the
expression of NOS II [22]. This latter mechanism may be
operating in some relaxin targets, in which induction of
NOS II expression and/or high-output, sustained NO gen-
eration have been observed [11,12,15,16], NOS II being
far more active than NOS III [23]. As summarized in Fig-
ure 1, at present there are some clues but no definitive
data about the molecular mechanisms by which relaxin,
by binding to its GPCRs, may induce the activation of the
NO pathway in its targets. Hopefully, future research
would clarify this point.
References
1. Hsu SY, Nakabayashi K, Nishi S, Kumagai J, Kudo M, Sherwood OD
and Hsueh AJW Activation of orphan receptors by the hor-
mone relaxin. Science 2002, 295:671-673
2. Osheroff PL and Ho W Expression of relaxin mRNA and relaxin
receptors in postnatal and adult rat brain and hearts. J Biol
Chem 1993, 268:15193-15199
3. Min G and Sherwood OD Identification of specific relaxin-bind-
ing cells in the cervix, mammary glands, nipples, small intes-
tine, and skin of pregnant pigs. Biol Reprod 1996, 55:1243-1252
4. Kohsaka T, Min G, Lukas G, Trupin S, Trupin Campbell E and Sher-
wood OD Identification of specific relaxin-binding cells in the
human female. Biol Reprod 1998, 59:991-999
5. Sherwood OD Relaxin. in The Physiology of Reproduction (Edited by:
Knobil E, Neill JD) New York, Raven 1994, 1:861-1009
6. Bartsch O, Bartlick B and Ivell R Relaxin signalling links tyrosine
phosphorylation to phosphodiesterase and adenylyl cyclase
activity. Mol Hum Reprod 2001, 7:799-809
7. Zimmermann S, Steding G, Emmen JM, Brinkmann AO, Nayernia K,
Holstein AF, Engel W and Adham IM Targeted disruption of the
Insl3 gene causes bilateral cryptorchidism. Mol Endocrinol 1999,
13:681-691
8. Nef S and Parada LF Cryptorchidism in mice mutant for Insl3.
Nat Genet 1999, 22:295-299
9. Overbeek PA, Gorlov IP, Sutherland RW, Houston JB, Harrison WR,
Boettger-Tong HL, Bishop CE and Agoulnik AI A transgenic inser-
tion causing cryptorchidism in mice. Genesis 2001, 30:26-35
10. Bani D Relaxin: a pleiotropic hormone. Gen Pharmacol 1997,
28:13-22
11. Masini E, Bani D, Bello MG, Bigazzi M, Mannaioni PF and Bani Sacchi
T Relaxin counteracts myocardial damage induced by
ischemia-reperfusion in isolated guinea pig hearts: evidence
for an involvement of nitric oxide. Endocrinology 1997, 138:4713-
4720
12. Bani D, Failli P, Bello MG, Thiemermann C, Bani Sacchi T, Bigazzi M
and Masini E Relaxin activates the L-arginine-nitric oxide path-
way in vascular smooth muscle cells in culture. Hypertension
1998, 31:1240-1247
13. Bani D, Baccari MC, Nistri S, Calamai F, Bigazzi M and Bani Sacchi T
Relaxin upregulates the nitric oxide biosynthetic pathway in
the mouse uterus. Involvement in the inhibition of myome-
trial contractility. Endocrinology 1999, 140:4434-4441
14. Danielson LA, Sherwood OD and Conrad KP Relaxin is a potent
renal vasodilator in conscious rats. J Clin Invest 1999, 103:525-
533
15. Failli P, Nistri S, Quattrone S, Mazzetti L, Bigazzi M, Bani Sacchi T and
Bani D Relaxin up regulates inducible nitric oxide synthase
expression and nitric oxide generation in rat coronary en-
dothelial cells. FASEB J Dec. 14, 2001, 10.1096/fj.01-0569fje.
Summary 2002, 16:252-254
16. Bani D, Baccari MC, Quattrone S, Nistri S, Calamai F, Bigazzi M and
Bani Sacchi T Relaxin depresses small bowel motility through
a nitric oxide-mediated mechanism. Studies in mice. Biol
Reprod 2002, 66:778-784
17. Liu J, Conklin BR, Blin N, Yun J and Weiss J Identification of a re-
ceptor/G-protein contact site critical for signaling specificity
and G-protein activation. Proc Natl Acad Sci USA 1995, 92:11642-
11646
18. Vanhoutte PM Endothelial dysfunction and atherosclerosis. Eur
Heart J 1997, 18:E19-E29
19. Wyckoff MH, Chambliss KL, Mineo C, Yuhanna IS, Mendelsohn ME,
Mumby SM and Shaul PW Plasma membrane estrogen recep-
tors are coupled to endothelial nitric oxide synthase through
Gαi. J Biol Chem 2001, 276:27071-27076
20. Igarashi J and Michel T Sphingosine 1-phosphate and isoform-
specific activation of phosphoinositide 3-kinase beta. Evi-
dence for divergence and convergence of receptor-regulated
endothelial nitric-oxide synthase signaling pathways. J Biol
Chem 2001, 276:36281-36288
21. Kim NY, Pae HO, Kim YC, Choi CK, Rim JS, Lee HS, Kim YM and
Chung HT Pentoxifylline potentiates nitric oxide production
in interleukin-1beta-stimulated vascular smooth muscle
cells through cyclic AMP-dependent protein kinase A
pathway. Gen Pharmacol 2000, 35:205-211
22. Grimm S and Baeuerle PA Review article: the inducible tran-
scription factor NF-κB: structure-function relationship of its
protein subunits. Biochem J 1993, 290:297-308
23. Moncada S, Palmer RMJ and Higgs EA Nitric oxide physiology,
pathophysiology, and pharmacology. Pharmacol Rev 1991,
43:109-142Page 2 of 2
(page number not for citation purposes)


Florence Research