Proceedings of The Physiological Society

Europhysiology 2018 (London, UK) (2018) Proc Physiol Soc 41, PCA328

Poster Communications

Adenosine and L-arginine transport are modulated by an alkaline intracellular pH in human umbilical vein endothelial cells from gestational diabetes mellitus

L. Sobrevia1,2,3, G. Fuentes4, M. Ramirez4

1. Pontificia Universidad Catolica de Chile, Santiago, Chile. 2. Universidad de Sevilla, Seville, Spain. 3. UQ Centre for Clinical Research (UQCCR), University of Queensland, Brisbane, Queensland, Australia. 4. Universidad de Antofagasta, Antofagasta, Chile.

Human umbilical vein endothelial cells (HUVECs) from gestational diabetes mellitus (GDM) pregnancies show reduced transport of the endogenous vasodilator nucleoside adenosine via the human equilibrative nucleoside transporters 1 and 2 (hENT1/2) (1) but increased transport of the amino acid L-arginine via the human cationic amino acid transporter 1 (hCAT-1) and activation of the endothelial nitric oxide (NO) synthase (2). Increased NO generation is seen in an alkaline medium and elevated NO reduced the hENT1/2 expression and activity in HUVECs (3). Thus, we evaluated whether HUVECs from GDM show an altered intracellular pH (pHi) and its consequences on the hENT1/2 and hCAT-1 transport activity. HUVECs were isolated (collagenase digestion) from full term normal (n = 22) or GDM (n = 22) pregnancies collected at the Clinical Hospital CHRISTUS-UC (Chile). The investigation conforms to the principles outlined in the Declaration of Helsinki. HUVECs were cultured in medium 199 supplemented with sera (20%) and antibiotics up to passage 2 under standard conditions (37 °C, 5% O2, 5% CO2). The pHi was measured in cells loaded (10 min) with the fluorescent pH-sensitive probe 2,7-bicarboxyethyl-5,6-carboxyfluorescein acetoxymethyl ester (BCECF-AM, 12 mmol/L) and exposed to NH4Cl (0.1 - 20 mmol/L) (i.e., acid pulse). Basal and pHi recovery rate (dpHi/dt) were estimated (up to 360 s) in cells exposed to 5 μmol/L 5-N,N-hexamethylene amiloride (HMA, Na+/H+ exchangers general inhibitor), 0.1 μmol/L zoniporide (NHE1 inhibitor), 0.1 μmol/L concanamycin A (V-ATPases inhibitor), or 10 μmol/L Schering (H+/K+-ATPase inhibitor). HUVECs from GDM show higher basal pHi compared with cells from normal pregnancies (pHi = 7.6 ± 0.1 vs. 7.2 ± 0.1, respectively) (values are mean ± S.E.M., compared by unpaired ANOVA, P<0.04) in the absence of inhibitors. Zoniporide and HMA reduced the basal pHi (pHi = 7.3 ± 0.2 and 7.2 ± 0.3, respectively) in GDM to those in normal pregnancies. The dpHi/dt in GDM was higher (2.1 ± 0.3 fold) than in normal pregnancies. Zoniporide and HMA reversed this increase in dpHi/dt to values in normal pregnancies. The GDM-reduced hENT1/2-mediated adenosine maximal transport capacity at basal pHi was reversed by NH4Cl-induced intracellular acidification (EC50 = 1.3 ± 0.2 mmol/L NH4Cl). The GDM-increased hCAT-1-mediated L-arginine maximal transport capacity was reversed by zoniporide- and HMA-induced intracellular acidification to values normal pregnancies. In conclusion, HUVECs from GDM pregnancies show an alkaline pHi which results in inactivation of hENT1/2 but increased hCAT-1 activity.

Where applicable, experiments conform with Society ethical requirements