<b><i>Uterine Artery Bulk RNA sequencing and transcriptomics</i></b> from non-pregnant wild type, <i>Rgs</i>2 KO, <i>Rgs5 </i>KO, and <i>Rgs2/5 </i>double knockout mice
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G protein regulation by regulators of G protein signaling (RGS) proteins of the R4/B family plays a key role in vascular tone maintenance; However, the regulatory mechanisms are poorly understood. Previous studies showed that the loss of G<sub>i/o</sub> and G<sub>q/11</sub> regulation by RGS2 and RGS5 is involved in augmented vascular tone and decreased uterine blood flow in mice. RGS2 and 5 are structurally and functionally closely related and are co-expressed in the resistance vasculature, including the uterine vascular bed. However, whether and how RGS2 and 5 coordinate their regulatory activities to finetune G protein signaling and regulate vascular tone are unclear. Here, we tested the hypothesis that the integrated activity of RGS2 and 5 modulates vascular tone by negatively regulating G<sub>i/o</sub> signaling to promote cAMP-dependent attenuation of uterine artery (UA) myogenic tone (MT). Using pressure myography, we examined MT of UA segments isolated from non-pregnant wild type (WT), <i>Rgs2</i><sup><em>-/-</em></sup>, <i>Rgs5</i><sup><em>-/-</em></sup>, and <i>Rgs2/5</i> dbKO mice in the absence and presence of exogenous cAMP or chemical inhibition of G<sub>i/o</sub> signaling, while nitric oxide tone was continuously suppressed with the eNOS inhibitor, L-NAME. We found that MT was reduced in <i>Rgs5</i><sup><em>-/-</em></sup> relative to WT or <i>Rgs2</i><sup><em>-/-</em></sup> UA in the absence or presence of L-NAME. Activating G<sub>i/o</sub> with dopamine increased, whereas exogenous cAMP decreased MT in <i>Rgs5</i><sup><em>-/-</em></sup> UA to similar levels in WT UA. Dual deletion of <i>Rgs2</i> and <i>5</i> abolished the reduction of MT due to the absence of only <i>Rgs5</i> and enhanced dopamine-induced G<sub>i/o</sub> effects in <i>Rgs2/5</i> dbKO UA. Conversely, and as in WT UA, G<sub>i/o</sub> inhibition with pertussis toxin or the application of exogenous cAMP decreased MT in <i>Rgs2/5</i>dbKO to similar levels in <i>Rgs5</i><sup><em>-/-</em></sup> UA. Application of the pan-phosphodiesterase (PDE) inhibitor, IBMX, concentration dependently decreased and normalized MT in all genotypes, and blocked dopamine-mediated MT augmentation in <i>Rgs2</i><sup><em>-/-</em></sup>, <i>Rgs5</i><sup><em>-/-</em></sup>, and <i>Rgs2/5</i> dbKO UA. These results indicate that activated G<sub>i/o</sub> augments MT by promoting PDE-mediated inhibition of cAMP-dependent vasodilatation; and while both RGS2 and 5 negatively regulate this novel G<sub>i/o</sub>-PDE-cAMP signaling pathway, RGS5 dampens the inhibitory efficacy of RGS2 towards G<sub>i/o</sub> in uterine arteries.
