Abstract

--- - Aims/hypothesis Gestational diabetes mellitus (GDM) is associated with fetoplacental endothelial dysfunction, including impaired extracellular clearance of adenosine, a vasodilator, in HUVECs. This study investigated the regulation of intracellular pH (pHi) and its impact on adenosine membrane transport in HUVECs. The hypothesis of this study was that Na+/H+ exchanger (NHE) isoform 1 (NHE1)-dependent pHi regulation differs between normal and GDM pregnancies depending on maternal pre-pregnancy BMI, leading to differential human equilibrative nucleoside transporters-mediated adenosine transport. - Methods HUVECs were isolated from 43 women with normal pregnancies and 23 with type A1 GDM and further stratified by maternal pre-pregnancy BMI into subgroups with normal weight, overweight and obesity. Data were also analysed as pooled groups (BMI >= 20 kg/m(2)). pHi and dpHi/dt were assessed in cells preloaded with the pH-sensitive fluorescent probe 2,7-bicarboxyethyl-5,6-carboxyfluorescein acetoxymethyl ester (12 mu mol/l), in the absence or presence of 20 mmol/l NH4Cl (acid pulse), the general NHEs inhibitor 5-N,N-hexamethylene amiloride (5 mu mol/l) or the NHE1-selective inhibitor zoniporide (100 nmol/l). Intrinsic buffering capacity and H+ flux were calculated. NHE1 protein abundance was quantified by western blotting, and adenosine transport kinetics (0-500 mu mol/l, 10 s) were determined. - Results GDM was linked to intracellular alkalinisation (similar to 0.6 pHi units vs normal pregnancies), increased activity of NHE1 and NHE isoforms 2 and 3 and reduced buffering capacity, with these effects varying by pre-pregnancy maternal BMI. Increased pHi recovery (similar to 3.8-fold) and NHE1 activity (similar to 4.8-fold) were observed in cells from women with GDM and pre-pregnancy overweight, while those with obesity (i.e. gestational diabesity) showed unaltered NHE1-mediated pHi recovery. Buffering capacity was reduced across most GDM groups, except in the overweight group. The GDM-reduced adenosine transport maximal capacity via human equilibrative nucleoside transporter isoform 2 was restored by intracellular acidification in GDM. - Conclusions/interpretation Pre-pregnancy maternal metabolic status influences endothelial adaptation or maladaptation to GDM. Stratifying GDM cases by pre-pregnancy maternal BMI uncovers subgroup-specific physiological responses, highlighting the importance of tailored approaches in understanding GDM pathophysiology.