Evidence for Na(+)-Ca2+ exchange and Ca(2+)-induced Ca2+ release in a cultured vascular smooth muscle cell line from the rat.

Measurements of intracellular calcium (Cai2+) and sodium (Nai+) have been made in single smooth muscle cells from the rat aortic cell line (A10) using the Ca(2+)- and Na(+)-sensitive dyes Fura-2 and SBFI (sodium-binding benzofuran isophthalate). The effects of manipulation of intracellular and extracellular Na+ on Cai2+ have been investigated. Reversal of the Na+ gradient in control cells does not result in any measurable increase in Cai2+ or change in the rate of recovery of the cells from agonist stimulation, suggesting that there is little functional Na(+)-Ca2+ exchange. In ouabain-pre-treated cells however, the recovery from agonist stimulation is significantly slowed, suggesting that in the presence of an elevated intracellular Na+ concentration there is an alteration in the Ca(2+)-handling mechanisms. Reversal of the Na+ gradient in ouabain-pre-treated cells results in a transient increase in Cai2+ followed by a slow secondary rise. The transient component of this rise is absent on a second activation of the cell or by prior mobilization of the intracellular stores of Ca2+ by agonist. Data presented in this paper suggest the possibility that the transient component is due to a Ca(2+)-induced Ca(2+)-release mechanism triggered by an initial influx of Ca2+. The mechanism underlying this influx is not known but may involve the Na(+)-Ca2+ exchanger operating in reverse. The possible modulation of the Na(+)-Ca2+ exchanger and Ca(2+)-induced Ca2+ release by internal Na+ is discussed.     

Autophagy inhibits PDGF-BB-induced calcification in vascular smooth muscle cells

AIM: To investigate the relationship between autophagy and calcification in vascular smooth muscle cells( VSMCs) after plateletderived growth factor( PDGF)-BB stimulation. METHODS: Cultured VSMCs were stimulated with PDGF-BB for different time,the expression of vascular calcification-related proteins and autophagy-related proteins were detected by Western blot. The interaction between Beclin1 and PI3KC3 was detected by co-immunoprecipitation. RESULTS: The expression of BMP2 and ALP showed a trend from decline to rise. ALP slumped at 12 h,and BMP2 slumped at 6 h. Moreover,the expression of Beclin-1 showed a trend from rise to decline,and peaked at 12 h. The conversion of LC3-Ⅰto Ⅱ increased in a time-dependent manner,and peaked at 24 h. The expression of BMP2 and ALP was increased in VSMCs incubated with PDGF-BB and autophagy inhibitor 3-MA,compared with PDGFBB-stimulated VSMCs. Furthermore,the interaction between Beclin1 and PI3KC3 was enhanced at 6 h after PDGF-BB stimulated,peaked at 12 h,and kept in high level at 24 h. Moreover,the phosphorylation level of Beclin1 was enhanced by PDGF-BB stimulation,and peaked at 6 h. CONCLUSION: Our findings demonstrate that PDGF-BB-induced autophagy inhibits VSMC calcification by enhancing Beclin1 phosphorylation and interaction between Beclin1 and PI3KC3.     

Globular adiponectin reduces vascular calcification via inhibition of ER-stress-mediated smooth muscle cell apoptosis

Objective: This study aims to explore the mechanism of globular adiponectin inhibiting vascular calcification. Methods: We established drug-induced rat vascular calcification model, globular adiponectin was given to observe the effect of globular Adiponectin on the degree of calcification. The markers of vascular calcification and apoptosis were also investigated. Meanwhile, the in vitro effect of globular Adiponectin on vascular calcification was also evaluated using primary cultured rat vascular smooth muscle cells. Results: We found that globular adiponectin could inhibit drug-induced rat vascular calcification significantly in vivo. The apoptosis of vascular smooth muscle cells was also reduced. The possible mechanism could be the down-regulation of endoplasmic reticulum stress by globular adiponectin. Experiments in primary cultured vascular smooth muscle cells also confirmed that globular adiponectin could reduce cell apoptosis to suppress vascular calcification via inhibition of endoplasmic reticulum stress. Conclusions: This study confirmed that globular adiponectin could suppress vascular calcification; one of the mechanisms could be inhibition of endoplasmic reticulum stress to reduce cell apoptosis. It could provide an effective method in the therapy of vascular calcification-associated diseases.     

Ca(2+)-dependent negative control mechanism for Ca(2+)-induced Ca2+ release in crayfish muscle.

The mechanism of termination of Ca(2+)-induced Ca2+ release (CICR) from the sarcoplasmic reticulum has been investigated in voltage clamped cut crayfish muscle fibres loaded with rhod-2. During depolarizing steps evoking calcium current (ICa), Ca2+ release was first activated. Then the release rapidly (tau approximately 6 ms) declined, as evidenced by the rate of change of the intracellular fluorescence signal representing a Ca2+ transient. The rapid termination of release was not accounted for by inactivation of the trigger ICa or depletion of Ca2+ from the SR, since the rate at which release declined was constant under conditions where the rate of ICa inactivation and the amount of Ca2+ released varied widely. Pre-elevations of [Ca2+]i with prepulses or photolysis of caged Ca2+ caused depression of Ca2+ release during a subsequent test pulse. When the rate of ICa onset was varied by applying voltage ramps with different slopes, currents with fast onset elicited larger Ca2+ release than calcium currents with slower onset, even though the amplitude of the currents was the same. These results suggest that a Ca(2+)-dependent negative control mechanism exists which mediates the termination of CICR independently of the duration of the trigger ICa and before significant depletion of Ca2+ in the SR occurs.     

Enhanced myosin phosphatase and Ca(2+)-uptake mediate adrenergic relaxation of airway smooth muscle

We examined the mechanisms underlying relaxations evoked by isoproterenol (Iso) in isolated porcine, bovine, or human tracheal and bronchial tissues (TSM and BSM, respectively). Iso had little effect against contractions evoked by high KCl, indicating that it does not directly suppress voltage-dependent Ca(2+)-influx nor directly inhibit myosin light chain kinase. Furthermore, Iso was equally potent against carbachol (CCh) contractions in the presence versus absence of nifedipine (10(-6) M), establishing that the primary action of Iso is not through membrane hyperpolarization. However, Iso relaxations in porcine/bovine BSM were significantly suppressed by inhibitors of the internal Ca(2+) pump (cyclopiazonic acid; 10(-5) M) or of myosin light chain phosphatase (calyculin; 10(-6) M). Myosin light chain phosphatase activity was assayed directly (using (32)P-labeled myosin) and found to be enhanced in a time- and concentration-dependent fashion by Iso. Iso relaxations in human airway tissues, on the other hand, were not significantly affected by either calyculin or cyclopiazonic acid. Thus, we conclude that Iso acts largely in a voltage-independent fashion: in nonhuman airways, this involves enhanced Ca(2+) pump activity (to decrease [Ca(2+)](i)) and myosin light chain phosphatase activation (to decrease Ca(2+)-sensitivity of the contractile apparatus), whereas in human airways the underlying mechanisms are still unclear.     

The carboxyl terminus of the epithelial Ca(2+) channel ECaC1 is involved in Ca(2+)-dependent inactivation

The family of epithelial Ca(2+) channels (ECaC) is a unique group of highly Ca(2+)-selective channels consisting of two members, ECaC1 and ECaC2. We used carboxyl terminal truncations and mutants to delineate the molecular determinants of the Ca(2+)-dependent inhibition of ECaC. To this end, rabbit ECaC1 was expressed heterologously with green fluorescent protein (GFP) in human embryonic kidney 293 (HEK293) cells using a bicistronic vector. Deletion of the last 30 amino acids of the carboxyl terminus of ECaC1 (G701X) decreased the Ca(2+) sensitivity significantly. Another critical sequence for Ca(2+)-dependent inactivation of ECaC1 was found upstream in the carboxyl terminus. Analysis of truncations at amino acid 635, 639, 646, 649 and 653 disclosed a critical sequence involved in Ca(2+)-dependent inactivation at positions 650-653. C653X showed decreased Ca(2+) sensitivity, comparable to G701X, while E649X lacked Ca(2+)-dependent inactivation. Interestingly, the number of green fluorescent cells, which is an index of the number of transfected cells, was significantly smaller for cells transfected with truncations shorter than E649 than for cells transfected with wild-type ECaC. However, the expression level of GFP was restored in the presence of the ECaC blocker ruthenium red, suggesting that these truncations resulted in deleterious Ca(2+) influx. In conclusion, we have identified two domains in the carboxyl terminus of ECaC1 that control Ca(2+)-dependent inactivation.     

Role of lysophosphatidylcholine in the desensitization of beta-adrenergic receptors by Ca(2+) sensitization in tracheal smooth muscle

Lysophosphatidylcholine (Lyso-PC) is generally considered to promote tissue inflammation. To determine the involvement of exogenous Lyso-PC in the beta-adrenergic desensitization by phospholipase A2, we examined the inhibitory effects of isoproterenol (ISO) on tension and intracellular Ca(2+) concentration by methacholine (MCh) after continuous exposure to Lyso-PC in guinea-pig tracheal smooth muscle, using isometric tension recordings and fura-2 signal (F340/F380 ratio). Pre- exposure to 10 microM Lyso-PC markedly reduced subsequent inhibition by 0.3 microM ISO against 1 microM MCh-induced contraction in a time-dependent manner. In contrast, values of percent F340/F380 ratio for MCh with ISO were not affected after exposure to Lyso-PC. In the presence of Y-27632, a selective rho-kinase inhibitor, a reduction in subsequent relaxation by ISO after exposure to Lyso-PC was inhibited in a concentration-dependent manner. Preincubation with cholera toxin also inhibited reduced responsiveness to ISO by Lyso-PC. Pre-exposure to Lyso-PC did not attenuate subsequent relaxation by agents that bypass beta-adrenergic receptors. These results indicate that continuous exposure to Lyso-PC may cause homologous desensitization of beta-adrenergic receptors via an augmentation in sensitivity to Ca(2+) by rho, a small G protein, in airway smooth muscle, and that activation of the stimulatory G protein of adenylyl cyclase, G(s), may prevent this phenomenon.     

牛磺酸减轻β-甘油磷酸诱导的大鼠血管平滑肌细胞钙化氧化应激在反流物所致食管粘膜损伤中的作用

目的：观察牛磺酸对钙化的形成及逆转作用的影响，探讨牛磺酸在血管钙化发生中的作用。方法：利用β-甘油磷酸制备钙化血管平滑肌细胞（VSMCs），测定细胞钙含量、碱性磷酸酶活性、[⁴⁵Ca]沉积及[³H]-胸腺嘧啶。结果：与对照组相比，钙化细胞的钙含量、ALP活性和[⁴⁵Ca]沉积均明显升高（P<0.05），而牛磺酸与β-甘油磷酸同时孵育呈剂量依赖性地抑制钙化发生。在牛磺酸逆转实验中，钙化细胞的钙含量、ALP活性和[⁴⁵Ca]沉积均较换液前明显降低（P<0.01）；且牛磺酸呈剂量依赖性地逆转已钙化细胞。与对照组相比，钙化细胞的细胞数量和[³H]-TdR掺入量增加（P<0.01），牛磺酸早期干预组显著抑制钙化细胞的增殖，但牛磺酸对已钙化的细胞,增殖抑制效应不明显。结论：牛磺酸可抑制细胞钙化形成且能逆转已形成钙化。     

血管平滑肌细胞凋亡在血管再狭窄中作用的研究进展

血管平滑肌细胞构成新生内膜增生的重要部分,且在血管腔内治疗术后再狭窄的心血管疾病的发生和发展中具有重要作用。血管平滑肌细胞凋亡能有效抑制血管球囊损伤和血管旁路移植术后新生内膜增生,从而可为血管术后再狭窄提供治疗手段。     

Biochemical evidence that cytokeratins are present in smooth muscle

Recent immunocytochemical studies have revealed that cytokeratin intermediate filaments, previously thought to be restricted to epithelial tissues, are present in muscle. In view of the implications of these reports for diagnostic pathology it is important to investigate by biochemical means whether these findings represent the presence of true cytokeratin proteins or an unexpected antigenic cross-reaction. In the present study intermediate filament proteins have been extracted from samples of human myometrium and identified by immunoblotting techniques using well characterized monoclonal antibodies, two against cytokeratins and two against desmin. The results show that the proteins of the appropriate molecular weight for cytokeratin were labelled by anti-cytokeratin antibodies and were quite distinct from those recognized by anti-desmin antibodies. This study therefore confirms previous immunocytochemical findings and emphasizes the need for caution when using anti-intermediate filament antibodies for diagnostic purposes.     

Hypoxic pulmonary vasoconstriction: cyclic adenosine diphosphate-ribose,smooth muscle Ca(2+) stores and the endothelium

Hypoxic pulmonary vasoconstriction (HPV) is unique to pulmonary arteries, and supports ventilation/perfusion matching. However, in diseases such as emphysema, HPV can promote hypoxic pulmonary hypertension (HPH), which ultimately leads to right heart failure. Since it was first described, the mechanisms underpinning HPV have remained obscure, and current therapies for HPH are poor. Previous investigations have suggested that HPV may be mediated by processes intrinsic to the pulmonary artery smooth muscle, and by the release of a vasoconstrictor(s) from the endothelium. It was thought that oxygen-sensitive ion channels in the smooth muscle cell membrane triggered HPV, and it has been argued that the endothelium-derived vasoconstrictor is endothelin-1. However, these proposals remain controversial. This review discusses the regulation by hypoxia of cyclic adenosine diphosphate-ribose production and Ca(2+) release from the sarcoplasmic reticulum in pulmonary artery smooth muscle. The role of these processes in triggering maintained HPV is then related to its subsequent progression due to vasoconstrictor(s) release from the endothelium.     

Acceleration of Ca(2+) repletion in the junctional sarcoplasmic reticulum and alternation of the Ca(2+)-induced Ca(2+)-release mechanism in hypertensive rat (SHR) cardiac muscle

We estimated the time taken for a repletion of the junctional sarcoplasmic reticulum (JSR) Ca(2+) stores from a family of mechanical restitution curves after twitches of various magnitudes in the cardiac muscle of hypertensive rats (SHR), using a method described previously (Tameyasu et al. Jpn J Physiol. 2004;54:209-19), to evaluate abnormality in Ca(2+) handling by cardiac JSR in hypertension. We found no differences in contractility or in the time course of mechanical restitution between SHR and the controls (WKY) at 3 weeks of age. In comparison to WKY, 7- and 20-week-old SHR showed a greater rested state contraction (RST) and similar or smaller rapid cooling contracture, suggesting that their JSR contains a similar amount of Ca(2+) at saturation, but releases more Ca(2+) upon stimulation. The adult SHR and WKY showed similar mechanical restitution time courses, but the adults had longer pretwitch latencies. The function G(t) representing the time course of JSR Ca(2+) store repletion in adult SHR exceeded the WKY value at t < or = 0.5 s, but the function H(t) representing JSR [Ca(2+)] change corresponding to the mechanical restitution after RST was smaller in the adult SHR at t < or = 0.5 s, resulting in smaller H(t)/G(t) in adult SHR at t < or = 0.5 s. Deviations of G(t), H(t), and H(t)/G(t) from WKY were greater at 20 weeks than at 7. The results suggest an acceleration of JSR Ca(2+) store repletion and an alternation of the Ca(2+)-induced release of Ca(2+ )from the JSR in young adult SHR.     

Inhibition of Ca2+ mobilization by caffeine in a cultured vascular smooth muscle cell line (A7r5).

The effects of caffeine on the resting level and agonist-induced changes in intracellular calcium ([Ca2+]i) have been studied in the vascular smooth muscle cell line A7r5. Caffeine (1-30 mM) lowers the resting [Ca2+]i by reducing the entry of Ca2+ and inhibits completely the mobilization of Ca2+ by arginine vasopressin. Application of forskolin, to elevate cAMP, does not affect the resting level of Ca2+i but does abolish the agonist-induced rise. These data add to the complexity of caffeine-induced changes in [Ca2+]i and point to a possible interaction between cAMP and other second messenger systems mobilizing Ca2+i in this cell type.