Relationship of intracellular free Ca²⁺ concentration and calcium-activated chloride channels of pulmonary artery smooth muscle cells in rats under hypoxic conditions

Abstract

To investigate the relationship between intracellular free Ca²⁺ concentration ([Ca²⁺]_i) and calcium-activated chloride (Cl_CB) channels of pulmonary artery smooth muscle cells (PASMCs) in rats under acute and chronic hypoxic conditions, acute hypoxia-induced contraction was observed in rat pulmonary artery by using routine blood vascular perfusionin vitro. The fluorescence Ca²⁺ indicator Fura-2/AM was used to observe [Ca²⁺]_i of rat PASMCs under normal and chronic hypoxic condition. The effect of Cl_CB channels on PASMCs proliferation was assessed by MTT assay. The Cl_CB channel blockers niflumic acid (NFA) and indaryloxyacetic acid (IAA-94) exerted inhibitory effects on acute hypoxia-evoked contractions in the pulmonary artery. Under chronic hypoxic condition, [Ca²⁺]_i was increased. Under normoxic condition, [Ca²⁺]_i was (123.63±18.98) nmol/L, and in hypoxic condition, [Ca²⁺]_i was (281.75±16.48) nmol/L (P<0.01). Under normoxic condition, [Ca²⁺]_i showed no significant change and no effect on Cl_CB channels was observed (P>0.05 Chronic hypoxia increased [Ca²⁺]_i which opened Cl_CB channels. The NFA and IAA-94 blocked the channels and decreased [Ca²⁺]_i from (281.75±16.48) nmol/L to (117.66±15.36) nmol/L (P<0.01). MTT assay showed that under chronic hypoxic condition NFA and IAA-94 decreased the value of absorbency (A value) from 0.459±0.058 to 0.224±0.025 (P<0.01). Hypoxia increased [Ca²⁺]_i which opened Cl_CB channels and had a positive-feedback in [Ca²⁺]_i. This may play an important role in hypoxic pulmonary hypertension. Under chronic hypoxic condition, Cl_CB channel may play a part in the regulation of proliferation of PASMCs.

Keywords

Ca²⁺-activated Cl⁻ channels / intracellular free Ca²⁺ concentration / pulmonary artery smooth muscle / hypoxia

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