Dynamic Changes Of Mitochondrial Ca²⁺ In Pulmonary Artery Smooth Muscle Cells Under Hypoxia And Its Mechanism

It is well known that elevated cytosolic free Ca²⁺ concentration ([Ca²⁺]i) in pulnary artery smooth muscle cells (PASMCs) plays critical roles in hypoxic pulmonary vasoconstriction. Mitochondria are one of the main intracellular calcium stores for maitaining cytosolic calcium homeostasis. A lot of evidences indicated that mitochondria are important in the development of HPV, but the contribution of mitochondrial calcium handling in HPV is poorly understood. In this study, we observed the dynamic changes of mitochondrial Ca²⁺ concentration ([Ca²⁺]mit) in PASMCs under hypoxia and explored the relationship among dynamic changes of [Ca²⁺]mit, [Ca²⁺]i and cellular membrane potential.Methods:Studies include 3 parts.Part 1: the PASMCs from adult Wistar mouse were isolated and cultured, then divided into 3 groups based on the loaded fluorescent indicators: Rhod-5F (5μmol/l), Di-8-ANEPPS (2μmmol/l), and Fluo-4/AM (4μmol/l). Hypoxia of the PASMCs was induced by sodium dithionite (Na₂S₂O₄, 2 mmol/L), which can make solution anoxia whithin 1 min by its deoxidized characteristic. The dynamic changes of [Ca²⁺]mit and the cell membrane potential, as well as [Ca²⁺]i were observed under confocal microscopy.Part 2: the cell culturing and grouping was done as Part 1, but 18mmol/L KCl was added to the three groups to change the cellular membrane potential. The dynamic changes of [Ca²⁺]mit and the cell membrane potential, as well as [Ca²⁺]i were observed under confocal microscopy.Part 3: the cell culturing and grouping was done as Part 1, but angiotensinâ…¡(10^-6 mol/L), nitric oxide donator-SIN-1 (3-morpholino-sydnonimine, 100μmol/L) was added to the three groups to explore the role of vasodilative and vasoconstrictive reagents on the dynamic changes of [Ca²⁺]mit and the cell membrane potential, as well as [Ca²⁺]i. Results:1. The dynamic changes of the cell membrane potential, [Ca²⁺]i and [Ca²⁺]mit under hypoxia1.1 After Na₂S₂O₄ stimulation, the fluorescent intensity of PASMCs membrane potentials decreased dramatically, followed by a very quickly recover and got to the summit at about 100 s The membrane potential at summit was higher than that at the initial stage. After that, the membrane potential was decrease gradually and to the level lower than the initial stage. This data indicates that hypoxia induces dynamic changes in PASMCs memebrane potential, that is, hyperpolarization at first, then depolarization and hyperpolarization at last.1.2 The fluorescent intensity of [Ca²⁺]i increased gradually after hypoxia stimulation. Similar pattern of dynamic changes was found on the fluorescent intensity of [Ca²⁺]mit. This result suggested, as evidences supported, that [Ca²⁺]mit may be dependant on [Ca²⁺]i.2. The dynamic changes of [Ca²⁺]i and [Ca²⁺]mit after depolarization with KClThe fluorescent intensity of Di-8-ANEPPS increased rapidly to the maximum at 60 s after adding KC1 to PASMCs and then gradually decreased, but the cells were kept in depolarized state within the whole period of observation.The [Ca(2+)]i and [Ca²⁺]mit increased rapidly to the maximum at 60 s after adding KCl to PASMCs and then gradually decreased to the initial. Same patterns were existed in the dynamic changes of fluorescent intensity of [Ca²⁺]mit and [Ca²⁺]I, indicating that [Ca²⁺]mit may be dependent on [Ca²⁺]I, as considerable evidences supported.3. The effects of the vasoconstrictive and vasodilative reagents on the dynamic change of cell membrane potential, [Ca²⁺]i and [Ca²⁺]mit.3.1 The effect of Angâ…¡, a vasoconstrictive reagent, on the dynamic changes of cell membrane potential, [Ca²⁺]I, and [Ca²⁺]mit.3.1.1 Basically, there was no changes of PASMCs membrane potential after adding Angâ…¡within the observation period.3.1.2 The fluorescent intensity of [Ca²⁺]i was increased rapidly and got to the maximum intensity at 50 s after adding Angâ…¡, then, decreased to the initial.3.1.3 Different dynamic changes in the fluorescent intensity of [Ca²⁺]mit compared with that of [Ca²⁺]i after adding Angâ…¡, the fluorescent intensity increased before 20 s, then dramtically decreased to the lowest at 100 s, maintaining at this intensity for about 300 s, and then recovered to the initial at 600 s.3.2 The effect of SIN-1, a vasodilative reagent- nitric oxide donator, on the dynamic changes of cell membrane potential, [Ca²⁺]i, and [Ca²⁺]mit3.2.1 After adding the SIN-1, the fluorescent intensity of cell membrane potential decreased rapidly from 20 s and got to the bottom at 200 s, then kept on this level until the final of observation.3.2.2 A similar pattern of dynamic changes of [Ca²⁺]i fluorescent intensity was found after adding the SIN-1 to PASMCs, comparing with the dynamic changes of membrane potential.3.2.3. After adding the SIN-1, the fluorescent intensity of [Ca²⁺]mit decreased rapidly after 20 s and got to the bottom at 160 s, then gradually recovered and kept on this level lower than the initial until the final observation.Conclusions:1. Hypoxia elevates the level of [Ca²⁺]mit and [Ca²⁺]I, the [Ca²⁺]mit may be mainly dependant on the level of [Ca²⁺]i.2. The elevated [Ca²⁺]mit and [Ca²⁺]i under hypoxia can't be simply explained by the decrease of PASMCs membrane potential.3. The Angâ…¡and NO could involve in HPV through affecting the level of [Ca²⁺]mit.