The temperature dependence of post-vibration tension recovery in intact and skinned rat tracheal smooth muscle

Summary The contraction kinetics of rat tracheal smooth muscle were studied by analysing the time course of tension recovery after the cessation of a 2 s length vibration (100 Hz, sinusoidal) in activated preparations. An initial fast component of tension recovery reflects the rearrangement of those crossbridges previously detached by vibration. The subsequent slow component could be related to the kinetics of the regular actin-myosin interaction. Both components still occur independently of the duration and type of activation (electrical field stimulation and 0.1 mM1^–1 acetylcholine), the bath temperature (16, 28 and 37° C), and of the functional behaviour of the cell membrane (intact or Triton X-100 skinned preparations). The time constants of post-vibration tension recovery were increased distinctly during prolonged activation, low temperature, and after skinning (lack of calmodulin). The activation enthalpy was calculated according to Arrhenius by using the time constant of the slow component of post-vibration tension recovery. It amounted to 94.7 ±0.6 kJ mol^–1 for the intact preparation and 97.2 ±1.0 kj mol^–1 for the skinned one (temperature range 16–28° C). These results provide further evidence that vibration affects the contractile system directly and that the kinetics of post-vibration tension recovery reflect the kinetics of actin-myosin interaction.     

Tension development and calcium sensitivity in skinned muscle fibres of the frog

Calcium activated isometric tension development was measured in single skinned muscle fibres of the ileofibularis muscle of the frog. The experiments were carried out at 5°C, pH=6.9, 1 mM free Mg²⁺ and an ionic strength of 160 mM. A Hill curve was fitted to the isometrically developed tension at different Ca²⁺ concentrations by means of a non-linear least mean square approximation. At a sarcomere length of 2.15 m, the Ca²⁺ concentration for half maximum tension (K) was 1.6 M. This Ca²⁺ concentration decreased with increasing sarcomere length; at 2.7 m, K was 1.1 M and at 3.1 m, K was 0.9 M. Therefore, Ca sensitivity is increased at larger sarcomere lengths. Consequently, the optimal sarcomere length for tension development shifted to larger values when the Ca²⁺ concentration was lowered. Osmotic compression of the fibre at 2.15 m by means of 5% Dextran also caused an increase in Ca sensitivity (K was 1.0 M). At 2.7 m, addition of 5% Dextran hardly affected the Ca sensitivity. The possible role of the interfilament spacing in the explanation of these results discussed.     

雌激素对血管平滑肌的作用

血管平滑肌细胞是保持血管功能完整性的重要结构和功能单位,在动脉粥样硬化、高血压、冠心病及损伤后再狭窄的发病机制中具有关键性作用。本文对雌激素通过功能性的雌激素受体调节血管平滑肌细胞的结构和功能进行综述。     

参与失血性休克血管钙敏感性调节的信号分子

目的：观察Rho-激酶、PKC、PKG对失血性休克大鼠血管钙敏感性的调控作用。 方法： 取失血性休克大鼠肠系膜上动脉，利用离体血管环张力测定技术，用去极化状态下(120 mmol/L K+)血管环对梯度浓度Ca2+的收缩力反映钙敏感性，观察Rho-激酶激动剂血管紧张素Ⅱ（Ang-Ⅱ）、Rho-激酶抑制剂fasudil、PKC激动剂PMA、PKC拮抗剂staurosporine、PKG激动剂8Br-cGMP和PKG拮抗剂KT-5823对失血性休克血管钙敏感性的影响。 结果： Ang-Ⅱ、PMA、KT-5823可增高失血性休克血管的钙敏感性，表现为Ca2+的量效曲线明显左移，在Ca2+（3×10-2 mol/L）水平，Emax分别为0.630 g/mg、0.595 g/mg、0.624 g/mg，均明显高于休克组的0.377 g/mg(P<0.05，P<0.01)；fasudil、staurosporine、8Br-cGMP可降低失血性休克血管的钙敏感性，表现为Ca2+的量效曲线明显右移，在Ca2+（3×10-2 mol/L）水平，Emax分别为0.242 g/mg、0.230 g/mg、0.256 g/mg，均显著低于休克组(P<0.05，P<0.01)。 结论： Rho-激酶、PKC、PKG对失血性休克大鼠血管钙敏感性有调节作用，Rho-激酶、PKC可上调钙敏感性，PKG可下调钙敏感性。     

Cytosolic calcium levels in vascular smooth muscle

Increase in cytosolic Ca2+ level ([Ca2+] cyt) is prerequisite for smooth muscle contraction. Simultaneous measurements of [Ca2+] cyt and muscle tension give direct information for the Ca2(+)-regulation of smooth muscle. A fluorescent Ca2+ indicator, fura-2, is used for this purpose. Comparison between [Ca2+] cyt and muscle tension in vascular smooth muscle indicates that, although high K+ and receptor-agonists such as norepinephrine and prostaglandin F2 alpha induce sustained contraction by the sustained increase in [Ca2+] cyt, greater contraction is produced by receptor-agonists than high K+ at a given [Ca2+]cyt. Phorbol ester show similar effects as receptor-agonists, and it potentiates a high K(+)-induced contraction with little effect on [Ca2+]cyt. These results suggest that the contraction of smooth muscle is due to the increase in [Ca2+]cyt. Furthermore, receptor-agonists stimulate phosphatidylinositol turnover and generates diacyl glycerol which activates protein kinase C and may consequently increase the Ca2+ sensitivity of contractile elements. The [Ca2+]cyt -dependent portion of these contractions is inhibited by Ca2+ channel blockers such as verapamil by the decrease in [Ca2+]cyt. By contrast, increase in cyclic AMP by isoproterenol and forskolin inhibits smooth muscle contraction by the decrease in [Ca2+]cyt also by the decrease in the Ca2+ sensitivity of contractile elements. Increase in the cyclic GMP level by sodium nitroprusside show effects quite similar to those of cyclic AMP. Thus, contractility of vascular smooth muscle seems to be regulated by [Ca2+]cyt and also by Ca2+ sensitivity of the contractile elements. Furthermore, at least part of the receptor-mediated changes may be due to activation of protein kinase C.     

Influence of pH on isometric force development and relaxation in skinned vascular smooth muscle

The effects of pH (from pH values 6.50–7.10) on isometric tension development and relaxation were investigated in Triton X-100 skinned rat caudal artery. Helically cut skinned strips contracted in 21 M Ca²⁺ were studied with respect to maximal isometric tension (P_o) and rate of contraction (T_0.5C), and following relaxation in 18 nm Ca²⁺, the rate of relaxation (T_0.5R). Acidic pH (pH 6.50) decreased P_o to 87% of isometric force obtained at pH 6.90, and increased the rate of contraction as shown by a decrease of T_0.5C to 80%. In contrast, T_0.5R increased 4.5-fold, indicating that with a change of only 0.40 pH units, relaxation rates were dramatically decreased. pCa-tension curves at pH values 6.50, 6.70, 6.90 and 7.10 indicated no significant shift in half maximal activation (pCa₅₀) between pH 6.50 and 6.70, but a significant (P<0.01) shift in pCa₅₀ between pH 6.70 ([Ca²⁺]=0.46 M) and pH 7.10 ([Ca²⁺]=0.87 M). Compared to contractions at pH 6.90, myosin light chain (LC₂₀) phosphorylation at pH 6.50 was significantly greater at 30 and 60 s into contraction but not significantly different at 3–10 min. At both pH 6.50 and 6.90, dephosphorylation was rapid and substantially preceded relaxation; LC₂₀ dephosphorylation and relaxation occurred more rapidly at pH 6.90 than at 6.50. At pH 6.50 and 6.90, relax solutions made with increased Ca²⁺ buffering capacity showed no effect in enhancing T_0.5R, suggesting the difference between relaxation rates was not due to Ca²⁺ diffusion limitations from the skinned strip. We suggest pH changes can after the contractile and relaxation responses in vascular smooth muscle and these effects may be related to LC₂₀ phosphorylation/dephosphorylation regulatory mechanisms.Abbreviations PIPES piperazine-N,N-bis-(2-ethanesulfonic acid) - EGTA 5-ethyleneglycol-bis-(-aminoethyl ether)-N,N,N,N-tetraacetic acid - DTT dithiothreitol - HDTA 1,6-diaminohexane-N,N,N,N-tetraacetic acid - SDS sodium dodecyl sulfate J. P. Gardner was supported in part by an AHA N.J. affiliate postdoctoral fellowship     

Temperature sensitivity of force and shortening velocity in maximally activated skinned smooth muscle

We have studied the temperature dependence of isometric force, rate of force development and maximal shortening velocity (V _max) in skinned guinea-pig taenia coli smooth muscle. To eliminate the influence of temperature on activation mechanisms, maximally thiophosphorylated preparations were used. Isometric force in the range 2–35°C was maximal at 22°C with a decrease of 25% at 2°C and 10% at 35°C. Rate of tension development from rigor after photolytic release of ATP increased four-fold between 5°C and 30°C. V _max increased with a Q₁₀ of about 2 (1.6, range 5–15°C, and 2.2, range 22–30°C). The temperature dependence of the rate of tension development indicates rate-limitation by transitions into force-generating states or by the hydrolysis reaction. The temperature dependence of V _max reflects effects of temperature on reactions (e.g. the ADP-release) associated with cross-bridge detachment. The small temperature dependence of steady-state force in smooth compared with skeletal muscle suggests differences in the cross-bridge reactions controlling the number of attached force-generating states in the two muscle types. This revised version was published online in August 2006 with corrections to the Cover Date.     

The effects of a calcium dependent protease on the ultrastructure and contractile mechanics of skinned uterine smooth muscle

Summary In situ substrates for a vascular smooth muscle calcium-dependent protease (CDP) were investigated using a chemically skinned uterine smooth muscle preparation. Treatment of skinned smooth muscles with CDP had no effect on the total content of actin and myosin. Electron microscopical observations demonstrated that membrane plaques, cytoplasmic dense bodies, and intermediate filaments were all degraded by CDP. In addition, CDP reduced both isometric force and isotonic shortening velocity of contracted muscles in a concentration and time-dependent manner. Treatment of contracting muscles with CDP resulted in a condensation of myofilaments away from the plasma membrane concurrent with the loss of contractility. The condensation of myofilaments was ATP-dependent and could be inhibited by removal of ATP prior to proteolysis. The effects of proteolysis on smooth muscle ultrastructure and contractility support previously proposed models which assign a role to cytoskeletal elements in coordinating the molecular interaction of actomyosin to produce muscle contraction. The loss of cytoskeletal structures following protease treatment suggests that one of the functions of CDP in smooth muscle may be the disassembly of the cell cytoskeleton.     

Changes of intracellular calcium concentrations by phenylephrine in renal arterial smooth muscle cells

In smooth muscle cells isolated from swine renal interlobar arteries, phenylephrine (PE) at concentrations of 1-10 microM produced biphasic increases of the intracellular calcium concentration. An early transient rise was followed by a maintained plateau. The maintained component was sensitive to extracellular calcium, in contrast to the early transient, which was still observed in nominally calcium-free solution. Nifedipine (1 microM) and NiCl2 (100 microM) only weakly affected the calcium signal, suggesting that voltage-sensitive calcium channels play only a minor role in the PE-induced changes in intracellular calcium. Thapsigargin (0.5 microM) elevated the intracellular calcium concentration and depressed both the early transient and the maintained component of the PE response. In calciumfree medium PE induced a transient rise of the intracellular calcium concentration with a depressed plateau. Readmission of calcium elevated the intracellular calcium concentration above the baseline. Both components of the PE-induced calcium signal were completely abolished when the cells were pretreated with the phospholipase C (PLC) inhibitor U73122 (2 microM). LaCl3 (100 microM, 1 mM), an inhibitor of calcium-release-activated current (ICRAC), had no effect on the PE-induced calcium signal. GdCl3 (50 microM), SKF 96365 (10 microM) and flufenamic acid (100 microM), reported to inhibit nonselective cation channels, blocked or transiently reduced the maintained calcium signal. Several protein kinase inhibitors such as genistein (10 microM), H7 (50 microM), H89 (1 microM) and bisindolylmaleimide (0.2 microM) reduced the maintained calcium signal. We conclude that the initial transient spike of the PE-induced calcium signal is due to release of calcium from inositol 1,4,5-trisphosphate-sensitive calcium stores evoked by alpha 1-adrenoceptor-coupled stimulation of PLC and that the maintained component is due to capacitative calcium entry, which is modulated by protein kinases.     

Electron microscopic localization of calcium in vascular smooth muscle

Potassium pyroantimonate has been employed in this study to localize calcium in the vascular smooth muscle of the thoracic aorta of the rabbit. The pyroantimonate ion precipitates sodium, magnesium and calcium. Incubation of the isolated thoracic aorta in a high potassium bathing medium which does not contain sodium, magnesium or calcium depletes the tissue of sodium. Addition of 10.8 mM CaCl₂ to the incubation medium results in welllocalized depositions of reaction product, presumably that of calcium pyroantimonate, in mitochondria, sarcoplasmic reticulum, and at the plasma membrane. Some or all of these organelles may, therefore, play a vital role in the contractionrelaxation cycle of vascular smooth muscle.     

The effect of calcium on the maximum velocity of shortening in skinned skeletal muscle fibres of the rabbit

Summary The relationship between maximum shortening velocity (V _max) and free calcium concentration has been studied in skinned single fibres from rabbit psoas and soleus muscles. At both 10 and 15° C,V _max measured in the psoas fibres was found to decrease by 40% when the pCa (-log[Ca²⁺]) was increased from 5.49 (maximally activating) to 6.21. Further decreases inV _max were observed when the pCa was increased to 6.32.V _max measured in soleus fibres at 15° C also decreased when the Ca²⁺ concentration was lowered, though the magnitude of this effect was slightly less than in the psoas fibres. Thus, a distinct effect of Ca²⁺ uponV _max has been shown to occur in mammalian skeletal muscle. The occurrence of this effect in both fast and slow muscle types may indicate that the underlying mechanism in the two cases is similar.     

Regulation of force and shortening velocity by calcium and myosin phosphorylation in chemically skinned smooth muscle

 The phosphatase inhibitor okadaic acid (OA) was used to study the relationship between [Ca²⁺], rates of phosphorylation/dephosphorylation and the mechanical properties of smooth muscle fibres. Force/velocity relationships were determined with the isotonic quick release technique in chemically skinned guinea-pig taenia coli muscles at 22° C. In the maximally thiophosphorylated muscle neither OA (10 μM) nor Ca²⁺ (increase from pCa 9.0 to pCa 4.5) influenced the force-velocity relationship. When the degree of activation was altered by varying [Ca²⁺] in the presence of 0.5 μM calmodulin, both force and the maximal shortening velocity (V _max) were altered. At pCa 5.75, at which force was about 35% of the maximal at pCa 4.5, V _max was 55% of the maximal value. When OA was introduced into fibres at pCa 6.0, force was increased from less than 5% to 100% of the maximal force obtained in pCa 4.5. The relationship between the degree of myosin light chain phosphorylation and force was similar in the two types of activation; varied [OA] at constant [Ca²⁺] and at varied [Ca²⁺]. The relation between force and V _max when the degree of activation was altered with OA was almost identical to that obtained with varied [Ca²⁺]. The results show that Ca²⁺ and OA do not influence force or V _max in the maximally phosphorylated state and suggest that the level of myosin light chain phosphorylation is the major factor determining V _max. The finding that the relationship between force and V _max was similar when activation was altered with OA and Ca²⁺ suggests, however, that alterations in the absolute rates of phosphorylation and dephosphorylation at a constant phosphorylation level do not influence the mechanical properties of the skinned smooth muscle fibres. Received: 1 December 1995 / Received after revision: 20 June 1996 / Accepted 12 July 1996     

The effect of sarcomere length on triad locationin intact feline caudofemoralis muscle fibres

The location of triads within a mammalian skeletal muscle sarcomere has traditionally been defined as ‘at the A-I junction’. We attempted to verify this statement by examining systematically the location of triads within the sarcomere over the physiological range of sarcomere lengths. This study was conducted using intact feline muscle fibres from caudofemoralis – an exclusively fast-twitch muscle from the hindlimb. Our results in intact fibres indicate that the distance between the Z-band and triad (ZT) is relatively constant over the range of sarcomere lengths (SLs) examined in this study (1.8–3.4μm). The slope between ZT and SL was measured to be 0.06 ± 0.01 (r=0.36, p < 0.001) while the slope between the M-line to triad distance (MT) and SL was measured to be 0.440.01 (r > 0.9, p < 0.001). The mean ZT was 0.52 ± 0.07μm, which corresponds to a triad location approximately halfway along the thin filaments. These results do not support the traditional statement regarding triad location. Nor do these results support a similar recent study conducted using chemically skinned muscle fibres from rat extensor digitorum longus (also a homogeneously fast-twitch muscle of the hindlimb), in which a slope of 0.25 was observed between ZT and SL (r > 0.9, p < 0.01). These results are, however, in qualitative agreement with results using intact fibres from fast-twitch rat semitendinosus. Based upon known morphology, we suggest that the only structure supporting triad position is the SR itself, and that a non-homogeneous distribution of the SR within the sarcomere might be responsible for maintaining triad location near the mid-region of the thin filaments. We also suggest that there might be optimal design reasons for locating the triads at the mid-region of the thin filaments. This revised version was published online in July 2006 with corrections to the Cover Date.     

Calcium sensitivity and energetics of contraction in skinned smooth muscle of the guinea pigtaenia coli at altered pH

Calcium-sensitivity of contraction, force-velocity relation and ATP hydrolysis rate at different pH (6.2–7.8) were investigated in skinned smooth muscle preparations from the guinea pigtaenia coli. Varied free-calcium levels were buffered by 4 mM BAPTA (1,2-bis(2-aminophenoxy)-ethane-N,N,N,N-tetraacetic acid) which has calcium binding properties little affected by pH. A small increase of calcium-sensitivity of contraction was seen at pH 6.2 compared to 6.9 and 7.8 (ED50 shift of about 0.15 pCa units). The isometric force andV _max in fibres activated either by calcium or by thiophosphorylation of the myosin light chains were each reduced by about 15% at pH 6.2 compared to 6.9 and 7.8. Following an isotonic quick release the shortening velocity decreases with time. This effect was more pronounced at pH 6.2 than at pH 6.9 or 7.8. The ATP hydrolysis rates in relaxed and thiophosphorylated fibres were essentially unaffected by alteration in pH between 6.2 and 7.8. Due to the lower force, energetic cost of force maintenance was thus somewhat increased at pH 6.2. These results suggest that pH alteration between 6.2 and 7.8 have effects on the properties of the contractile machinery of the smooth muscle in the skinned guinea pigtaenia coli. The effects are however small and therefore probably of little functional importance over a pH range which should cover most cases of intracellular pH alteration under physiological or pathophysiological conditions.     

Effects of calcium and ADP on tension responses to step length increases in glycerinated Mytilus smooth muscle

To study the mechanical properties of various crossbridge states in the anterior byssus retractor muscle (ABRM) of Mytilus, the tension response of the glycerinated ABRM to a step increase of the length was examined in rigor solutions with saturating Ca2+ (Rca) and without added Ca2+ (R), rigor ones with ADP (AD) and with both ADP and saturating Ca2+ (ADca), and a low ATP, activating one. The application of ADca to a rigor ABRM caused a slow tension development of less than 0.083 kg/cm2 (n = 6) probably because of contaminant ATP in the presence of 0.25 mM p,p-di(adenosine-5')pentaphosphate (Ap5A) and 10 U/ml hexokinase with 2 mM glucose. The instantaneous stiffness in ADca was slightly smaller than that in the low ATP solution and greater than those in R, Rca, and AD, giving evidence that the stretch response reflected the mechanical property of the crossbridges. The rate of the tension decay during the initial 5 ms after the length change was completed was slowest in the ADca among the solutions examined, while during the initial 30-90 ms it was faster in the low ATP solution than R, Rca, AD, and ADca with little difference of the rate in the latter four solutions. The difference in the time course of the tension decay in between the low ATP solution and ADca may be taken to indicate that the high stiffness in ADca was not due to the formation of the tension-generating crossbridges but to the crossbridges with both bound Ca and ADP (AMCaADP) made directly from the rigor crossbridge (AM). Consequently, it was thought that AMCaADP was stiffer than AM and the crossbridges with either bound Ca (AMCa) or ADP (AM.ADP), the latter three kinds of the crossbridges being formed directly from AM, not as a result of ATP hydrolysis.     

Digoxin-norepinephrine response and calcium blocker effects in vascular smooth muscle.

The mechanism of potentiation by digoxin of the response of vascular smooth muscle to norepinephrine was investigated in 5-cm intact segments of rabbit carotid artery. Segments were mounted in a chamber and perfused at constant pressure while flow and upstream and downstream pressures were recorded and resistance was calculated. Each vessel was perfused with a submaximal vasoconstricting concentration of norepinephrine (6 x 10(-6)M) alone, in the presence of digoxin (6 x 10(-5)M), and during exposure to both digoxin and one of the following calcium antagonists: lanthanum chloride (5 x 10(-4)M procaine hydrochloride (5 x 10(-3)M), or verapamil (5 x 10(-5)M). Digoxin potentiated the response to norepinephrine alone by 20% (P less than 0.01), to norepinephrine plus lanthanum chloride by 10% (P less than 0.001), and to norepinephrine plus procaine hydrochloride by 17% (P less than 0.001). Digoxin did not potentiate the norepinephrine response in the presence of verapamil. These data suggest that the mechanism of digoxin potentiation of the norepinephrine response in vascular smooth muscle may involve an alteration in a cellular calcium sequestration or release process. The potential cellular sites that may contribute to this phenomenon are discussed.