Intracellular Ca2+ dynamics in isolated Vascular Smooth Muscle Cells (VSMCs) from rat distal pulmonary artery through a non enzimatic method

Vascular smooth muscle cells (VSMCs) show a considerable plasticity and can undergo profound and reversible phenotypic changes (from contractile to proliferative) in response to vascular damage or disease, that involved the expression of many contractile proteins, including SERCA2a. The aim of the project was to developed a non-enzimatic method for isolating VSMCs from rat distal pulmonary artery and charactherized their Ca2+ dynamics. Preliminary results showed 1) an increase of SERCA2a activity in SR microsomes at different passages and 2) the presence of phospholamban (PLN), the physiological inhibitor of SERCA2a. Funtional analysis showed an increase of intracellular Ca2+ following a perfusion of 1) ATP 100 µM in absence of extracellular Ca2+; 2) a solution with [K+]out 40 mM and 3) Caffeine (20 mM). The increase of Ca2+ induced by ATP, High K+out and caffeine were present respectively in 95%, 40-60% and 5% of isolated cell. At the end, the effect of ATP was abolished by SERCA2a blocker thapsigargin (1 µM), while the decay of Ca2+ transient was slower in absence of extracellular Na+ and in the presence of mithocondrial calcium uniporter (MCU) blocker Ru360 (1 µM), suggesting a role of both SERCA2a, Na+/Ca2+ exchanger and MCU in the removal of intracellular Ca2+. At the end, our methods permits to obtain a primary VSMCs from rat pulmonary artery that expressed the main Ca2+ handling proteins, in particular SERCA2a and PLN, that could be considered a promising therapeutic target to prevent phenotypic change in this cells in pathological condition.