(046) THE ROLE OF ADENOSINE IN THE RELAXANT MECHANISM OF RAT DISTAL VAGINA
Authors: Cipriani S , Cellai I , Filippi S , Comeglio P , Guarnieri G , Maseroli E , Rastrelli G , Morelli A , Maggi M , Vignozzi L 
Published: June 2024

Abstract

Introduction
Adenosine (ADO) is a purinergic neuromodulator that in the male exerts a positive effect on penile erection by controlling smooth muscle (SM) relaxation. However, its role in the relaxant/contractile pathways in the vagina is yet to be investigated.

Objective
The aim of this study was to characterize the effect of ADO stimulation on SM activity in distal vagina, evaluating its possible effect on the regulation of nitric oxide (NO) pathway using a validated animal model of female Sprague-Dawley rats.

Methods
The immunolocalization of ADORA2A and ADORA2B in distal vagina tissues was detected by immunohistological staining. In vitro contractility studies on noradrenaline (NA)-precontracted vaginal strips from intact Sprague-Dawley rats were performed; rat smooth muscle cells (rvSMCs) from distal vagina of intact animals were stimulated with ADO for cyclic adenosine monophosphate (cAMP) quantification. mRNA (messenger ribonucleic acid) isolated from vaginal tissue was analyzed by semi-quantitative Reverse Transcriptase-Polymerase Chain Reaction.

Results
All ADORAs mRNA were expressed in the vaginal tissue, with a significant prevalence of ADORA2B; furthermore, ADORA2A and ADORA2B immunolocalization highlighted that both receptors are highly expressed in the epithelium, in blood capillaries of the vascular bed and in the smooth muscle bundles of vagina wall. In vitro ADO stimulation induced a dose-dependent relaxation (IC50=31.9±2.38 μM), which was significantly reduced by the administration of NO-synthase inhibitor L-NAME; similar effects were observed after the mechanical ablation of endothelium and in vitro administration of the soluble guanylate cyclase inhibitor ODQ, highlighting the pivotal contribution of NO signaling to the ADO relaxant activity. In vitro stimulation with increasing doses of the selective ADO receptor ADORA2A agonist CGS2-1680 induced a dose-dependent relaxation (IC50=132.6±3.47 nM); a similar but less potent relaxant effect was observed with ADORA2B selective agonist BAY60-6583. The relaxing effect induced by ADO on NA-precontracted vaginal strips was completely counteracted by the stimulation with the re-spectively selective ADORA2A and ADORA2B antagonists SHC-442416 and PSB603; a similar effect was induced by ADORA1 selective antagonist SLV320, but not by ADORA3 selective antagonists VUF5574. The mRNA relative expression of enzymes regulating the ADO turnover [adenosine deami-nase (ADA), 5′-nucleotidase (5NTD), AMP deaminase type 1 and type 2 (AMPD1, AMPD2) and adenosine kinase (ADK)] showed a higher expression of genes regulating the de novo ADO/cAMP syn-thesis (ADK, 5NTD), compared to those acting on ADO degradation (ADA, AMPD1, AMPD2). Finally, stimulation of rvSMCs with increasing doses of ADO induced a significant production of intracellular cAMP after 3 minutes (1 and 10 μM).

Conclusions
These data show that ADO acts as an important relaxant modulator of the vagina through the activation of its receptors, specifically ADORA2A, and the consequent cAMP-dependent adenylate cyclase re-sponse. However, in vitro studies show that ADO also acts trough the NO-dependent pathway. These results therefore demonstrate for the first time a new relaxant pathway in distal vagina, ADO signaling, that results deeply intertwined with the well-known NO pathway. This finding unravels new biological mechanisms underlying vagina physiology that need to be further studied in future research.

Disclosure
No.