Study On The Role And Modulation Mechanisms Of Calcium-activated Chloride Channels In Pulmonary Artery Smooth Muscle Cells In Rats

At present, the incidence of pulmonary hypertension and pulmonary heart disease is still high. Most of pulmonary hypertension is hypoxic pulmonary hypertension (HPH). The mechanism of hypoxic pulmonary hypertension has still not been fully elucidated. HPH has two pathophysiological characters: hypoxic pulmonary artery vasoconstriction (HPV) and hypoxic pulmonary vascular remodeling. HPV has been demonstrated not only in perfused lungs but also in pulmonary arterial rings denuded of endothelium and in single pulmonary arterial smooth muscle cell (PASMC). The role of ion channels function in HPH is the focus in various causes of HPH. Our laboratory has demonstrated that potassium channels have been played an important role in HPH. But the HPV has not been control completely by the opening agents of potassium channels. So the Cl^- channels were being focused by the researchers at present. It was been demonstrated that Cl^-channels had a closely relationship with essential hypertension. But the role of Cl^-channels in HPH has not been elucidated.Two major classes of Cl^- channels are expressed in pulmonary artery smooth muscle cells (PASMCs): Ca²⁺-activated Cl (Cl_Ca) channels and volume-sensitive Cl^-(Clswell) channes. Niflumic acid (NFA) and indaryloxyacetic acid (IAA-94) are special blockers of Cl_ca channels. They are dependent on a net increase in cytoplasmic free Ca²⁺ concentration ([Ca²⁺]_i). Studies have shown that Cl_Ca channels contribute tothe agonist-induced pulmonary artery contractions under physiological conditions. Acute and chronic hypoxic can increase [Ca²⁺]_i, but few studies if elevation of [Ca²⁺]_i can activate Cl_Ca channels. At present, the relationship of Cl_Ca channels and HPV or HPH has not been elucidated.It was disputed if increase in [Ca²⁺]_i generated by the influx of Ca²⁺ through either the voltage-dependent Ca²⁺ channels or by Ca²⁺ release from the sarcoplasmic reticulum or both. It was disputed if increase in [Ca²⁺]_i initiated coming from Ca²⁺ influx across the plasmalemma or sarcoplasmic reticulum under hypoxic condition. If the Ca²⁺ coming from sarcoplasmic reticulum, it was not yet known if Ca²⁺ came from the ryanodine receptor-seneitive or the IP3 receptor-seneitive stores or both? So far, there have been few reports about these questions. This study was aimed to investigate the roles of Cl_Ca channels in the HPV and hypoxic pulmonary artery remodeling in PASMCs in rat by utilizing immunohistochemistry staining technique, whole cell patch-clamp technique and fluorospectrophotometer.Part 1 Role and mechanisms of calcium-activated chloride channels in the regulation of pulmonary vascular tone in rats under physiological conditionsSubject 1 The signification and electrophysiological characteristics of calcium-activated chloride channels of pulmonary artery smooth muscle cells in rats under physiological conditionsObjective: To investigate the signification and the electrophysiological characteristics of calcium-activated chloride (Cl_Ca) channels of pulmonary artery smooth muscle cells (PASMCs) in rats under physiological conditions.Methods: Single PASMCs were obtained by the acute enzyme separation method(collagnase I plus papain). Ca²⁺-activated Cl^- currents (I_Cl(Ca)) were recorded by the conventional whole-cell patch clamp technique under bath solution with Ca²⁺or free Ca²⁺ conditions or put phenylephrine (PE) and Cyclopiazonic acid (CPA) into bath solution conditions.Results: (1) Cytoplasmic free Ca²⁺ concentration([Ca²⁺]_i was increased by PE and CPA ( P <0.01).(2) The Cl^- channel blockers Niflumic acid (NFA) and indaryloxyacetic acid (IAA-94) significantly and reversibly inhibited the Cl_Ca channels which can be opened by arised [Ca²⁺]_i.Conclusion: Cl_Ca channels are functionally expressed in PASMCs .An increased in [Ca²⁺]_i ,via Ca²⁺ influx or release, activates Cl_Ca channels and induces ICl(Ca). This Cl^- current contributes to the agonist-induced tension under physiological conditions.Subject 2 Role of calcium-activated chloride channels in the regulation of pulmonary vascular tone in rats under physiological conditionsObjective: To investigate the role of calcium-activated chloride channels and the Cl^-channel blockers niflumic acid (NFA) and indaryloxyacetic acid (IAA-94) in the regulation of vascularcontraction induced by phenylephrine (PE). Methods: The PE-induced contraction in rat pulmonary artery was observed by using routine blood vascular perfusion in vitro. The fluorescence Ca²⁺indicator Fura-2/AM was used to observe intracellular free Ca²⁺ concentration ([Ca²⁺]_i) of rat pulmonary artery smooth muscle cells (PASMCs) which were obtained by the acute enzyme separation method (collagnase I plus papain) on NFA and IAA-94 effects on PE-induced contraction. Changes of [Ca²⁺]_i in PASMCs were measured byspectrofluorometry.Results: The anion channel blockers NFA and IAA-94 produced inhibitory effects onPE-induced contractions in the pulmonary artery. NFA and IAA-94 negligibly affectedthe KCl-induced pulmonary artery contractions. PE can increase [Ca²⁺]_i and NFA andIAA-94 can decrease it.Conclusion: Calcium-activated chloride channels contribute to the agonist-inducedpulmonary artery contractions under physiological conditions, which may be a newclue to investigate the hypoxic pulmonary vasoconstriction.Part 2 Role and mechanisms of calcium-activated chloride channels in the regulation of pulmonary vasoconstriction in rats under acute hypoxia conditions Subject 1 Role of calcium-activated chloride channels in the regulation of pulmonary vasoconstriction in rats under acute hypoxia conditionsObjective: To investigate the role of calcium-activated chloride (Cl_Ca) channels in the regulation of hypoxic pulmonary vasoconstriction (HPV) induced by acute hypoxic conditions.Methods: Acute hypoxia-induced contraction was observed in rat pulmonary artery by using routine blood vascular perfusion in vitro; the fluorescence Ca²⁺ indicator Fura-2/AM was used to observe [Ca²⁺]_i; of rat PASMCs in normal (37℃,5%CO₂, 21%O₂, 74%N₂ ) and acute hypoxic(37℃ , 5%CO₂, 2%O₂, 93%N₂) conditions; Effects were observed on pulmonary artery tone and [Ca²⁺]_i by Cl_Cu channel blockers niflumic acid (NFA) and indaryloxyacetic acid (IAA-94) under acute hypoxicconditions.Results: (1) The Cl_Ca channel blockers NFA and IAA-94 produced no effects in the pulmonary artery tone in normoxic condition; The NFA and IAA-94 produced inhibitory effects on acute hypoxia-evoked contractions in the pulmonary artery. (2) under acute hypoxic condition, [Ca²⁺]_i was increased: In normoxic condition, [Ca²⁺]_i was ((95.73 ± 19.61))nmol/L, and in hypoxic condition, [Ca²⁺]_i was(247.50±22.15) nmol/L (P<0.01). (3) In normoxic condition, [Ca²⁺]i had no significant change and NFA and IAA-94 negligibly affected it (P>0.05); Acute hypoxic increased [Ca²⁺]_i which may open Cl_Ca channels. The NFA and IAA-94 blocked them and decreased [Ca²⁺]_i from (247.50 ± 22.15) nmol/L to (126.19±11.10)nmol/L(P<0.01). Conclusion: Acute hypoxic increased [Ca²⁺]_i which may open Cl_Ca channels and have a positive-feedback in [Ca²⁺]_i.This may play an important role in hypoxic pulmonary vasoconstriction.Subject 2 Effects and significance of acute hypoxia on calcium signal of sarcoplasmic reticulum in pulmonary artery smooth muscle in ratsObjective: To investigate the effects of acute hypoxia on calcium of sarcoplasmic reticulum in pulmonary artery smooth muscle in rats.Methods: Cell level: the fluorescence Ca²⁺ indicator Fura-2/AM was used to observe intracellular free Ca²⁺ concentration ([Ca²⁺]_i)of rat pulmonary artery smooth muscle cells ( PASMCs) through putting into ryanodine (RD) and cyclopiazonic acid (CPA) in normal (37℃, 5%CO₂, 21%O₂, 74%N₂ ) , acute hypoxic(37℃, 5%CO₂, 2%O₂, 93%N₂) under free Ca²⁺ conditions. Pulmonary artery ring level: Pulmonary artery tension was observed in rat by using routine blood vascular perfusion in vitro undersame conditions.Results: (1) Under acute hypoxic condition, [Ca²⁺]_i was increased: In normoxic condition, [Ca²⁺]_i was (96.99 ± 7.16) nmol/L, and in hypoxic condition, [Ca²⁺]_i was(257.06± 32.48)nmol/L (P<0.01). (2) Ryanodine or caffeine, an agent that depletes ryanodine receptor-seneitive Ca²⁺ stores, inhibited hypoxia-induced increases in [Ca²⁺]_i and [Ca²⁺]_i decreased to (108.71 ± 10.38)nmol/L (P<0.01); CPA or thapsigargin (TG) , an agent that depletes IP3 receptor-seneitive Ca²⁺ store, had no effects on [Ca²⁺]_i (P>0.05). (3) Acute hypoxia evoked pulmonary artery contractions: Pulmonary artery tension had no effects under normoxic condition and increased under acute hypoxia condition (49.28 ± 8.64) mg/mg(P<0.01). (4) Ryanodine or caffeine, an agent that depletes ryanodine receptor-seneitive Ca²⁺ stores, inhibited hypoxia-evoked contractions in the pulmonary artery. Diastolic rate was (91.75 ± 5.74) %(P<0.01); CPA or TG, an agent that depletes IP₃ receptor-seneitive Ca²⁺ store, had no effects on hypoxia-evoked contractions. Diastolic rate was (12.1±4.26) %(P>0.05) .Conclusion: The results indicate that release of Ca²⁺ from the ryanodine receptor-seneitive, but not the IP3 receptor-seneitive stores, contributes to the mechanism of hypoxic pulmonary vasoconstriction in rat. This is a mechanism intrinsic to pulmonary vascular smooth muscle without the need for Ca²⁺ influx across the plasmalemma or an endothelial factor.Part 3 Effects of calcium-activated chloride channels on proliferation and related-gene expression in pulmonary artery smooth muscle cells in rats under chronic hypoxic conditionsObjective: To investigate the effects of calcium-activated chloride (Cl_Ca) channels on the proliferation and related-gene expressions in pulmonary artery smooth muscle cells (PASMCs) in rats under chronic hypoxic conditions.Methods: The cultured PASMCs were divided into four groups under normoxic (37 ℃, 5%CO₂, 21%O₂. 74%N₂ ) and chronic hypoxic (37℃, 5%CO₂, 2%O₂, 93%N₂) conditions respectively: (1)control groups (N: normoxic, H: hypoxic, Nc, Hc): Cells were cultured in serum-free DMEM (SFM); (2)10 NFA groups (N_10nfa, H_10nfa) : Cells were cultured in SFM contain 10 μmol/L Niflumic acid (NFA); (3)50 NFA groups (N_50nfa, H_50nfa): Cells were cultured in SFM contain 50 μ mol/L NFA. (4) IAA-94 groups(N_IAA-94,H_IAA-94): Cells were cultured in SFM contain 10 μ mol/L indaryloxyacetic acid (IAA-94). The cells were observed by electron microscope; The cell cycles were observed by flow-cytometry; Cytoplasmic free Ca²⁺ concentration ([Ca²⁺]_i) in PASMCs were investigated by fluorescent quantitation using fluorospectrophotometer; The proliferation were detected by MTT assay; Immunocytochemistry staining was used to detect the effects of Cl_Ca channels blockers on the expressions of PCNA, c-fos and c-jun of PASMCs. Results: (1) The results showed that hypoxia initiated the change of PASMCs from contractile phenotype to synthetic phenotype. The synthetic phenotype smooth muscle cell was characterized by increase in endoplasmic reticulum and mitochondria, and by drease in SM-α-actin and muscle fiber under hypoxia condition. (2) Under chronic hypoxic condition, [Ca²⁺]_i was increased: In 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); In normoxic condition, [Ca²⁺]_i had no significant change and NFA and IAA-94 negligibly affected it (P>0.05), Chronic hypoxic increased [Ca²⁺]_i which may open Cl_Ca channels. The NFA and IAA-94 blocked them and decreased [Ca²⁺]i from (281.75 ± 16.48) nmol/L to (117.66±15.36)nmol/L (P<0.01).(3) MTT assay showed that in 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). (4) The expression of proliferating cell nucleus antigen were significantly increase in chronichypoxic condition (P<0.01); The NFA and IAA-94 were shown to significantly decrease them (P<0.01); But the NFA and IAA-94 did not show such effects in normoxic condition ( all P> 0.05) . (5) The numbers of S + G₂M PASMCs were significantly increase in chronic hypoxic condition (P<0.01); The NFA and IAA-94 were shown to significantly decrease them (P<0.01); But the NFA and IAA-94 did not show such effects in normoxic condition ( all P> 0.05) . (6) The expression of c-fos and c-jun were significantly increase in chronic hypoxic condition (P<0.01); The NFA and IAA-94 were shown to significantly decrease them ( P<0.01); But the NFA and IAA-94 did not show such effects in normoxic condition ( all P> 0.05) . Conclusion: Hypoxic increased [Ca²⁺]_i which may open Cl_Ca channels and had a positive-feedback in [Ca²⁺]_i This may play an important role in hypoxic pulmonary hypertension; In chronic hypoxic condition, Cl_Ca channel may have a role in the regulation of proliferation of PASMCs.