Adenosine relaxes vagina smooth muscle through the cyclic guanosine monophosphate– and cyclic guanosine monophosphate–dependent pathways
Authors: Cellai Ilaria , Filippi Sandra , Comeglio Paolo , Guarnieri Giulia , Acciai Gabriele , Cancedda Chiara , Cipriani Sarah , Maseroli Elisa , Rastrelli Giulia , Morelli Annamaria , Maggi Mario , Vignozzi Linda 
Published: November 2024

Abstract

Background
In males, adenosine (ADO) is known to relax penile smooth muscles, although its role in the vagina is not yet fully elucidated.

Aim
This study investigated the effect of ADO on vagina smooth muscle activity, using a validated female Sprague–Dawley rat model.

Methods
Contractility studies, using noradrenaline-precontracted vaginal strips, tested the effects of ADORA1/3 antagonists and ADORA2A/2B antagonists and agonists. Increasing doses of ADO were tested after in vivo or in vitro treatment with Nω-nitro-L-arginine-methyl-ester hydrochloride (L-NAME) or with guanylate or adenylate cyclase inhibitors. Immunopositivity for ADORA2A and ADORA2B was assessed, and messenger RNA (mRNA) analysis was performed. Cyclic ADO monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP) were quantified both in rat vagina smooth muscle cells (rvSMCs) and in vaginal tissues with increasing doses of ADO.

Outcomes
Demonstrating ADO’s role in the relaxing/contractile mechanism in distal vagina smooth muscle.

Results
All ADO receptors mRNAs were expressed in vaginal tissue, with a prevalent content of ADORA2B. A high expression of genes regulating ADO catabolism (ADK) and de novo synthesis (NT5E) was found. In vaginal strips, ADO induced relaxation with IC50 = 144.7 μM and a flat pseudo-Hill coefficient value = -0.42, indicating an activity on heterogeneous receptors. Blocking ADORA1/3 shifted ADO response to the left and with a steeper slope. ADORA2A/2B agonists showed a higher potency than ADO in inducing relaxation. Immunolocalization confirmed the presence of ADORA2A/2B in vaginal musculature, in the blood vessels endothelium, and in the epithelium. ADO stimulation of vagina tissues induced a significant increase in cAMP and cGMP contents. Experiments on rvSMCs confirmed that ADO time- and dose-dependently stimulated cAMP production in these cells. However, ADORA2A/2B antagonists, although reducing the ADO-induced relaxation, did not completely block it. A similar inhibition was obtained by blocking adenylate cyclase. Overall, these findings suggest that ADO relaxation involves other pathways, eg, nitric oxide (NO)/cGMP. Accordingly, blocking NO formation through L-NAME substantially blunted ADO responsiveness, as it does the block of cGMP formation through 1H-[1,2,4]oxadiazolo-[4,3-a]quinoxalin−1-one. Simultaneous incubation with cGMP and cAMP blockers completely inhibited ADO responsiveness.

Clinical Translation
The study highlights ADO’s role in regulating vaginal smooth muscle activity, suggesting its potential effect on the vagina.

Strengths and Limitations
This is the first study on ADO in the vagina, although the results are preliminary and limited to the rat model.

Conclusion
These results show that ADO acts as a vaginal relaxing modulator through selective activation of receptors involving not only cAMP but also cGMP.