Antioxidant-independent ascorbate enhancement of catecholamine-induced contractions of vascular smooth muscle

Ascorbate reduces the oxidation rate of catecholamines and, by an independent mechanism, enhances rabbit aortic ring contractions initiated by catecholamines. The largest significantly different fractional increases in force produced by ascorbate enhancement of norepinephrine (NE), epinephrine, phenylpropanolamine (PPA), and ephedrine (Eph) are 5.5, 1.8, 1.6, and 1.3 times, respectively. In physiological salt solutions bubbled with 95% O(2) at 37 degrees C, NE, PPA, and Eph have oxidation rate constants of 1.24, 247, and 643 h, respectively. Ascorbate significantly enhances 100 nM NE contractions by at least twofold at all ascorbate concentrations >15 microM, including the entire physiological range of 40-100 microM. Ascorbate preloading and washout followed by NE exposure produces significantly greater contractions than NE without ascorbate preloading but significantly lower than NE simultaneously with ascorbate. Ascorbate does not enhance K(+)- or angiotensin II-induced contractions. Ascorbate enhancement of catecholamine contractions occurs in addition to the reduction in oxidation rate, because the increases in force occur faster than oxidation can occur, the increases occur with compounds that have negligible oxidation rates, and the increases occur when ascorbate and NE are not physically present together. These results are consistent with ascorbate acting on the adrenergic receptor. Ascorbate may play a role in shock and asthma treatments and potentiate the cardiovascular health consequences of PPA and Eph (Ephedra).     

A mathematical model for the spontaneous contractions of the isolated uterine smooth muscle from patients receiving progestin treatment

The in vitro spontaneous contractions of human myometrium samples can be described using a phenomenological model involving different cell states and adjustable parameters. In patients not receiving hormone treatment, the dynamic behavior could be described using a three-state model similar to the one we have already used to explain the oscillations of intra-uterine pressure during parturition. However, the shape of the spontaneous contractions of myometrium from patients on progestin treatment was different, due to a two-step relaxation regime including a latched phase which cannot be simulated using the previous model without introducing an ad hoc mechanism to account for the extra energy involved in this sustained contraction. One way to do this is to introduce an anomalous rate of ATP consumption, the biochemical reasons for which have not yet been elucidated and which cannot be mathematically simulated using our experimental data. An alternative explanation is the reduced cycling rate of actin-myosin cross-bridges known to occur during the latch-phase. Our experimental findings suggest a third possibility, namely a sol-gel transition with a specific relaxation time constant, which would maintain a significant part of the cell population in the contracted-state until the intracellular-medium returns to its normal fluid behavior.     

A steady-state electrochemical model of vascular smooth muscle cells

A model of the steady-state electrochemical response of vascular smooth muscle cells to external stimuli is presented, which accounts for K, Na, and Ca fluxes. The results of the model are broadly in accordance with experimental data 1), at various transmural pressures; 2), with channel and pump blockade; and 3), under manipulation of external ionic concentrations. The model exhibits dual stable states which sometimes coexist, and abrupt transitions between these states may account for nongraded responses in arteries as external potassium or pressure is varied. The simulations suggest that changes in the intracellular sodium concentration ([Na]i) often accompany smooth muscle responses. For example, [Na]i values vary threefold over the range of pressures from 10 to 100 mmHg.     

Tolerance to ethanol-induced contractions of vascular smooth muscle: Role of endothelium

Ethanol, at high concentrations, produced a dose-dependent contraction of male rat aortic rings, $\text{in vitro}$. Mechanical removal of endothelial cells from aortic rings of control rats resulted in a small, but significant, shift of the ethanol dose-response curve to the right without a change in the maximal contraction. Removing the endothelial cells of aortic rings obtained from rats intoxicated with ethanol for two days significantly shifted the ethanol dose-response curve to the left and significantly increased the maximal contraction induced by ethanol. A comparison of the ethanol dose-response curves in aortic rings with endothelium obtained from control rats with those obtained from intoxicated rats indicated a significant shift to the right with no change in maximal response. No significant changes were observed when the responses of aortic rings without endothelium obtained from control and intoxicated rats were compared. These observations confirm that tolerance to ethanol can be demonstrated in vascular smooth muscle. In addition, they demonstrate that the endothelium is required for the development of tolerance to ethanol in the aorta.     

Airway smooth muscle tone modulates mechanically induced cytoskeletal stiffening and remodeling.

The application of mechanical stresses to the airway smooth muscle (ASM) cell causes time-dependent cytoskeletal stiffening and remodeling (Deng L, Fairbank NJ, Fabry B, Smith PG, and Maksym GN. Am J Physiol Cell Physiol 287: C440-C448, 2004). We investigated here the extent to which these behaviors are modulated by the state of cell activation (tone). Localized mechanical stress was applied to the ASM cell in culture via oscillating beads (4.5 mum) that were tightly bound to the actin cytoskeleton (CSK). Tone was reduced from baseline level using a panel of relaxant agonists (10(-3) M dibutyryl cAMP, 10(-4) M forskolin, or 10(-6) M formoterol). To assess functional changes, we measured cell stiffness (G') using optical magnetic twisting cytometry, and to assess structural changes of the CSK we measured actin accumulation in the neighborhood of the bead. Applied mechanical stress caused a twofold increase in G' at 120 min. After cessation of applied stress, G' diminished only 24 +/- 6% (mean +/- SE) at 1 h, leaving substantial residual effects that were largely irreversible. However, applied stress-induced stiffening could be prevented by ablation of tone. Ablation of tone also inhibited the amount of actin accumulation induced by applied mechanical stress (P < 0.05). Thus the greater the contractile tone, the greater was applied stress-induced CSK stiffening and remodeling. As regards pathobiology of asthma, this suggests a maladaptive positive feedback in which tone potentiates ASM remodeling and stiffening that further increases stress and possibly leads to worsening airway function.     

SK but not IK channels regulate human detrusor smooth muscle spontaneous and nerve-evoked contractions

Animal studies suggest that the small (SK) and intermediate (IK) conductance Ca(2+)-activated K(+) channels may contribute to detrusor smooth muscle (DSM) excitability and contractility. However, the ability of SK and IK channels to control DSM spontaneous phasic and nerve-evoked contractions in human DSM remains unclear. We first investigated SK and IK channels molecular expression in native human DSM and further assessed their functional role using isometric DSM tension recordings and SK/IK channel-selective inhibitors. Quantitative PCR experiments revealed that SK3 channel mRNA expression in isolated DSM single cells was ～12- to 44-fold higher than SK1, SK2, and IK channels. RT-PCR studies at the single-cell level detected mRNA messages for SK3 channels but not SK1, SK2, and IK channels. Western blot and immunohistochemistry analysis further confirmed protein expression for the SK3 channel and lack of detectable protein expression for IK channel in whole DSM tissue. Apamin (1 μM), a selective SK channel inhibitor, significantly increased the spontaneous phasic contraction amplitude, muscle force integral, phasic contraction duration, and muscle tone of human DSM isolated strips. Apamin (1 μM) also increased the amplitude of human DSM electrical field stimulation (EFS)-induced contractions. However, TRAM-34 (1 μM), a selective IK channel inhibitor, had no effect on the spontaneous phasic and EFS-induced DSM contractions suggesting a lack of IK channel functional role in human DSM. In summary, our molecular and functional studies revealed that the SK, particularly the SK3 subtype, but not IK channels are expressed and regulate the spontaneous and nerve-evoked contractions in human DSM.     

A comparative and regional study of potassium induced relaxation of vascular smooth muscle

Summary The current study was undertaken to assess species and regional variations in the relaxation of vascular smooth muscle in response to potassium and in the ouabain sensitivity of this relaxation. The effect of species variation was investigated through the use of tail arteries from rats, dogs, cats, monkeys, and pigs; the effect of regional variation was studied in tail, middle cerebral, femoral, and posterior coronary arteries from baboons. Helical strips from all of these vessels were made to contract with norepinephrine or serotonin in a potassium-free solution. The vessels relaxed when potassium was added back to the solution. Strips of tail artery from rats, dogs, and monkeys showed greater relaxation in response to potassium than did strips from pigs and cats. Helical strips from tail, cerebral, and coronary arteries of the baboon relaxed to a greater degree in response to potassium than did strips from the femoral artery. Ouabain produced a concentration-dependent decrease in the magnitude of potassium relaxation in all vessel types. Half-maximal inhibition occurred at approximately 10^–8 to 10^–7 M in all arterial strips except those obtained from rat tail artery (5×10^–5 M). The inhibition of potassium relaxation by ouabain was fully reversed by 30 min exposure to a ouabain-free solution in only the rat tail artery strips. A component of potassium-induced relaxation in tail artery strips from monkeys and baboons was not inhibited by ouabain. The results show that the magnitude of response, potassium and ouabain sensitivity, and recovery from ouabain treatment of potassium relaxation are species related. The regional bed from which the vascular smooth muscle is derived also determines the magnitude and potassium sensitivity of the relaxation. These parameters of potassium-dependent relaxation may reflect corresponding differences in the electrogenic pumping of sodium and potassium among various animal species and various regional vascular beds.Abbreviations ATPase adenosine triphosphatase - PSS physiological salt solution - C contractile magnitude from baseline in milligrams - R relaxation measured as residual force above baseline in milligrams - SEM standard error of the mean These studies were supported by NHLBI grant HL-18575Dr. Webb was a Post-doctoral Research Fellow of the Michigan Heart Association during this investigation     

Peroxide induced activation of glycogen phosphorylase A activity in vascular smooth muscle

1-Phenoxy-2-propanone, 1-chloro-3-phenoxy-2-propanone, and 1-fluoro-3-phenoxy-2-propanone are competitive acetylcholinesterase inhibitors with K_I values of 30, 0.85, and 2.2 μM, respectively, compared to 2 mM for 4-phenyl-2-butanone. The substituent effect on inhibition suggests that these compounds bind by formation of a tetrahedral adduct and are transition state analogs.Other evidence supports this conclusion: N-benzyl-2-chloroacetamide and 1-phenoxy-2-propanol are poor inhibitors (K_I = 11 and >10 mM); 1-phenoxy-2-propanone and 1-chloro-3-phenoxy-2-propanone have K_I values 330 and 140 times smaller than K_m for corresponding substrates; and 1-chloro-3-phenoxy-2-propanone protects the enzyme against irreversible inhibition by CH₃SO₂F.     

Extracellular matrix-specific focal adhesions in vascular smooth muscle produce mechanically active adhesion sites

Integrin-mediated mechanotransduction in vascular smooth muscle cells (VSMCs) plays an important role in the physiological control of tissue blood flow and vascular resistance. To test whether force applied to specific extracellular matrix (ECM)-integrin interactions could induce myogenic-like mechanical activity at focal adhesion sites, we used atomic force microscopy (AFM) to apply controlled forces to specific ECM adhesion sites on arteriolar VSMCs. The tip of AFM probes were fused with a borosilicate bead (2 ~ 5 microm) coated with fibronectin (FN), collagen type I (CNI), laminin (LN), or vitronectin (VN). ECM-coated beads induced clustering of alpha(5)- and beta(3)-integrins and actin filaments at sites of bead-cell contact indicative of focal adhesion formation. Step increases of an upward (z-axis) pulling force (800 ~ 1,600 pN) applied to the bead-cell contact site for FN-specific focal adhesions induced a myogenic-like, force-generating response from the VSMC, resulting in a counteracting downward pull by the cell. This micromechanical event was blocked by cytochalasin D but was enhanced by jasplakinolide. Function-blocking antibodies to alpha(5)beta(1)- and alpha(v)beta(3)-integrins also blocked the micromechanical cell event in a concentration-dependent manner. Similar pulling experiments with CNI, VN, or LN failed to induce myogenic-like micromechanical events. Collectively, these results demonstrate that mechanical force applied to integrin-FN adhesion sites induces an actin-dependent, myogenic-like, micromechanical event. Focal adhesions formed by different ECM proteins exhibit different mechanical characteristics, and FN appears of particular relevance in its ability to strongly attach to VSMCs and to induce myogenic-like, force-generating reactions from sites of focal adhesion in response to externally applied forces.     

Chloride channel activity of vascular smooth muscle in the spontaneous hypertensive rats

To characterize the activity of the Ca2+-activated Cl- channels in vascular smooth muscle (VSM) of the spontaneous hypertensive rats (SHR), the isolated mesenteric vascular beds and tail artery strips were preparated from SHR and Wistar rats aged 7-8 weeks. The changes in contractile response to norepinphrine (NE) were taken as an index of vascular mortion. Results showed that the contractile responses of mesenteric arteries and tail arteries to NE in SHR were significantly greater than that in Wistar rats. The inhibition magnitude of the contractile response by Ca2+-activated Cl- channel blocker, niflumic acid in SHR was significantly less than that in Wistar rats. Decreasing the extracellular Cl- concentration increased the contractile response to NE significantly, but the amplitude of enhanced contractile response in SHR was greater than that in Wistar rats. It can be concluded that NE-induced contraction was enhanced in SHR, which is partly due to an increase in Cl- efflux through the Ca2+-activated Cl- channels. The chloride channel activity may be increased in association with the elevation of blood pressure.     

A structural bio-chemo-mechanical model for vascular smooth muscle cell traction force microscopy

Biomechanics and Modeling in Mechanobiology - Altered vascular smooth muscle cell (VSMC) contractility is both a response to and a driver for impaired arterial function, and the leading...     

UV-C照射诱导体外血管平滑肌细胞凋亡模型的建立

应用常规细胞培养超净台紫外消毒灯（２２０Ｗ,２２０Ｖ,５０Ｈｚ）发射的ＵＶ－Ｃ波段的紫外光源（２５４ｎｍ）,垂直照射距离其１０ｃｍ处的大鼠主动脉平滑肌细胞,发现经照射后细胞出现典型的凋亡形态学改变,如细胞变圆,染色质浓缩,细胞膜出泡,出现凋亡小体等;细胞面积,核面积及核／胞面积比均显著降低;且提取细胞ＤＮＡ的琼脂糖凝胶电泳呈现梯状图谱。从形态学和生化指标方面证明了ＵＶ－Ｃ照射可诱导体外血管ＳＭＣｓ     

Lipopolysaccharide induced vascular smooth muscle cells proliferation: A new potential therapeutic target for proliferative vascular diseases

Vascular smooth muscle cells (VSMCs) proliferation is involved in vascular atherosclerosis and restenosis. Recent studies have demonstrated that lipopolysaccharide (LPS) promotes VSMCs proliferation, but the signalling pathways which are involved are not completely understood. The purpose of this review was to summarize the existing knowledge of the role and molecular mechanisms involved in controlling VSMCs proliferation stimulated by LPS and mediated by toll‐like receptor 4 (TLR4) signalling pathways. Moreover, the potential inhibitors of TLR4 signalling for VSMCs proliferation in proliferative vascular diseases are discussed.     

同型半胱氨酸/半胱氨酸诱导血管平滑肌细胞中的新cDNA

同型半胱氨酸／半胱氨酸是体内的含硫氨基酸,近年来提出它们可以诱导血管平滑肌细胞的增殖,是心血管病的一个新的独立的危险因子．但其机制尚不了解,本研究目的在于克隆同型半胱氨酸／半胱氨酸诱导的新基因．应用差异显示即ＲＮＡｍａｐｐｉｎｇ的方法,从半胱氨酸诱导的大鼠血管平滑肌细胞中,寻找有差异的条带,克隆新的ｃＤＮＡ．结果分离出７种上调和４种下调ｃＤＮＡ,并且发现其中有４个是至今尚未发现的新的ｃＤＮＡ．应用Ｎｏｒｔｈｅｒｎｂｌｏｔ分析,这４个新的ｍＲＮＡ其长度分别为５．０、２．０、１．８和２．２ｋｂ．它们可能在血管平滑肌细胞增殖和心血管病的发病中具有重要意义．     

Signaling pathways of LIGHT induced macrophage migration and vascular smooth muscle cell proliferation

The biological actions of LIGHT, a member of the tumor necrosis factor superfamily, are mediated by the interaction with lymphotoxin-beta receptor (LTbetaR) and/or herpes virus entry mediator (HVEM). Previous study demonstrated high-level expressions of LIGHT and HVEM receptors in atherosclerotic plaques. To investigate the role of LIGHT in the functioning of macrophages and vascular smooth muscle cells (VSMC) in relation to atherogenesis, we determined the effects of LIGHT on macrophage migration and VSMC proliferation. We found LIGHT through HVEM activation can induce both events. LIGHT-induced macrophage migration was associated with activation of signaling kinases, including MAPKs, PI3K/Akt, NF-kappaB, Src members, and FAK. Proliferation of VSMC was also shown relating to the activation of MAPKs, PI3K/Akt, and NF-kappaB, which consequently led to alter the expression of cell cycle regulatory molecules. Down-regulation of p21, p27, and p53, and inversely up-regulation of cyclin D and RB hyper-phosphorylation were demonstrated. In conclusion, LIGHT acts as a novel mediator for macrophage migration and VSMC proliferation, suggesting its involvement in the atherogenesis.