不同前负荷条件下去神经平滑肌的应力松弛与膜电流特征

BACKGROUND: Muscle stretch test is a method for testing mechanical properties of denervated muscle under diverse loads. OBJECTIVE: To observe changes of spontaneous tension wave in myography and membrane electric current in aorta smooth muscle samples under several preload conditions. METHODS: Denervated intact smooth muscle samples were taken from the Kunming mouse aorta and urinary bladder wall. Smooth muscle samples were fixed on a micro positioning device and the first stretch was induced for a passive tension up to 1 g, this position was determined as the initial length (L0). Intermittently, with increasing sample length from L0 every 5 minutes, the 1st and 10th stretch were recorded as low- (L0+1) and high-preload (L0+10). 3% CaCl2 and 0.05% nitrendipine were dropped on the samples before L0+1 and L0+10 stretch were recorded. Membrane current changes were evaluated by glass microelectrodes and MultiClamp 700B Amplifier and pClamp 10 analyzer software. The membrane current changes after L0+1 and L0+10 were analyzed. RESULTS AND CONCLUSION: Prolonged stress relaxation phase was shortened with increasing preload in smooth muscle preparations. The stress relaxation phase in smooth muscle samples from urinary bladder wall was shorter than aorta samples. It was revealed that there were compliance differences between two kinds of smooth muscle samples. In aorta samples, myogenic spontaneous contraction amplitude during stress relaxation and membrane current were significantly increased with increasing preload. Increased amplitude and frequency of membrane current in aorta smooth muscle samples under high calcium concentration (3% CaCl2) were inhibited by L-type calcium channel blockade (0.05% nitrendipine). Collectively, increased preload in smooth muscle samples induced a directly decrease of compliance with an enlargement of myogenic spontaneous contraction amplitude. It appeared vital in particular in aorta samples. Membrane current significantly enhanced in spontaneous contraction period suggested the mechanical stretching ionic inward flow involved in this period. Membrane current amplitude was increased by high-preload under elevated calcium conditions; however, this change was suppressed by L-type calcium channel blockade. This study indicates quick stretching influences not only mechanically-gated channels in smooth muscle, but also the activation of L-type calcium channel. 中国组织工程研究杂志出版内容重点：组织构建;骨细胞;软骨细胞;细胞培养;成纤维细胞;血管内皮细胞;骨质疏松;组织工程     

天麻钩藤饮对SHR血清Ca2+浓度及血管平滑肌细胞钙通道的影响

目的：探讨天麻钩藤饮对自发性高血压大鼠(SHR)的血管平滑肌细胞钙通道电生理特征的影响，以期进一步阐明天麻钩藤饮对血压干预的作用机制。方法:选用12周龄自发性高血压雄性大鼠45只，随机分为5组，即天麻钩藤饮组（A组）、天麻钩藤饮去石决明组（B组）、硝苯地平组（C组）、石决明组（D组）、生理盐水对照组（E组）。治疗4周后，测定血清游离钙浓度；用全细胞模式膜片钳技术记录分析血管平滑肌细胞L型电压依赖性钙通道的特性。结果:用药前后天麻钩藤饮组和硝苯地平组血清游离钙浓度改变没有统计学意义(P>0.05)，天麻钩藤饮去石决明组、石决明组、生理盐水组血清游离钙浓度低于用药前 (P<0.05) ，以生理盐水组最明显(P<0.01)。天麻钩藤饮组、硝苯地平组有明显减少血管平滑肌细胞ICa-L内流的作用，石决明组作用较弱，天麻钩藤饮去石决明组、生理盐水组没有减少血管平滑肌细胞ICa-L内流的作用。结论:天麻钩藤饮可以提高血清游离钙离子浓度。天麻钩藤饮有明显的阻滞血管平滑肌细胞L型钙离子通道的作用，这可能是其降压作用机制之一。     

Interactions between membrane potential and intracellular calcium concentration in vascular smooth muscle

The intracellular calcium concentration is a major determinant of vascular tone. In the steady state it is regulated mainly by membrane potential. At the same time, several mechanisms regulating the calcium concentration, including the membrane potential, are influenced by the intracellular calcium concentration itself. There are thus multiple possible positive and negative feedback loops involved in calcium regulation. This review gives a brief overview of the different mechanisms involved, including calcium-dependent ion channels, exchangers, and ATPases, and discusses their role in agonist-mediated responses, in relation primarily to studies on the portal vein and mesenteric small arteries.     

Changes in resting membrane potential and contractility of innervated and denervated skeletal muscle free grafts in the rat

Summary In free orthotopic auto-grafts of the extensor digitorum muscle of rats a marked temporary decrease of resting membrane potential (RMP) of two superficial layers of muscle fibres is observed at 2 days with subsequent recovery 4 days after transplantation. Such a temporary decrease of the RMP is not observed in grafts of denervated muscle. This difference in change of RMP is apparently related to a temporary marked decrease or loss of contractility observed in innervated but not denervated graft and may explain in part the relatively more successful grafting of denervated muscles.     

Effects of motor nerve anesthesia and tenotomy on muscle membrane properties

Motor nerves to soleus muscles of rats were kept anesthetized for up to 7 days by applying solutions of lidocaine base or marcaine HCl. The anesthetic solutions were delivered from a subcutaneously located ALZA-minipump and reached the nerves through silastic cuffs. The ACh supersensitivity of muscles inactivated by nerve anesthesia for 3–7 days was comparable to that of muscles denervated for the same length of time.g _m/g_k (the ratio of total membrane conductance to the membrane K conductance) decreased from a normal value of 5–10 to less than two in 6–7 days, in anesthetic-inactivated and denervated muscles. The results were variable after 3 days of anesthesia.g _m/g_k of muscles which were tenotomized for 3 weeks was unchanged. The voltage-current curve for muscles kept in a solution containing 50 mM K propionate, which is steep at +50 mV, was less steep in denervated and anesthetic-inactivated, but not in tenotomized muscles, although atrophy was marked in all non-normal muscles.     

虎杖甙对正常人血管平滑肌细胞内钙和膜电位的调节作用

目的和方法：观察虎杖甙（ＰＤ）对人脐带动脉平滑肌细胞（ＶＳＭＣ）内游离钙、细胞膜电位的变化,以探讨ＰＤ对血管平滑肌的调节机制。用Ｆｌｕｏ－３－ＡＭ、ＤｉＢＡＣ４（３）标记培养的ＶＳＭＣ,在激光共聚焦显微镜上测定细胞内游离钙和膜电位变化。结果：给ＰＤ（０５ｍｍｏｌ／Ｌ）１０ｍｉｎ后,ＶＳＭＣ内游离钙浓度升高５６％±５６％。当ＰＤ加入前用维拉帕米和ＥＧＴＡ预处理后,则游离钙不再升高;ＥＧＴＡ和肝素预处理也抑制ＰＤ的升钙作用,而ＥＧＴＡ和普鲁卡因预处理则使细胞内钙显著升高。ＰＤ还可使ＶＳＭＣ膜电位去极化,加入钠通道阻断剂河豚毒素（２５μｍｏｌ／Ｌ）可完全阻断ＰＤ的去极化作用：加甲氰咪胍、维拉帕米、优降糖和利及丁预处理不能阻断ＰＤ去极化作用。结论：：ＰＤ可通过细胞外钙内流来增加细胞内游离钙浓度,并促进细胞外钠离子内流而导致细胞去极化     

Ryanodine modulation of45Ca efflux and tension in rabbit aortic smooth muscle

The effects of ryanodine on isometric tension development, net cellular Ca content and⁴⁵Ca efflux were measured in the isolated rabbit aorta. Pretreatment of the tissue with 10 M ryanodine for 30 min reduced the norepinephrine (NE)-induced tension of the aortic rings bathed in the absence of extracellular Ca²⁺ in parallel with a reduction in the NE-stimulated⁴⁵Ca efflux. Ryanodine alone caused a delayed moderate increase in⁴⁵Ca efflux, slowly increasing the fractional loss from 0.02/min to 0.03/min, without any increase in tension. The increased⁴⁵Ca efflux was accompanied by a decrease in the net cellular Ca content. When ryanodine and NE were administered in sequence during⁴⁵Ca efflux, we found a quantitative correlation between the ryanodine induced loss of⁴⁵Ca and the inhibition of the NE-induced⁴⁵Ca release. We conclude that the inhibition of the NE-induced contraction by 10 M ryanodine is the result of depletion of Ca from intracellular stores rather than inhibitions of Ca²⁺ release.Abbreviations used Ca total calcium - Ca²⁺ free calcium - NE norepinephrine - SR sarcoplasmic reticulum - PSS physiological salt solution     

Decrease of calcium binding by the red blood cell membrane in spontaneously hypertensive rats and in essential hypertension

Ca binding in the red blood cell (RBC) membrane of spontaneously hypertensive rats (SHR) and of patients with essential hypertension was studied. Under conditions of physiological concentration of free Ca in the incubation medium of RBC the outer part of the membrane binds 393±32 and 435±30 nmole of Ca per ml of RBC in rats and humans, respectively, without essential differences in the amount of Ca in hypertensive individuals as compared to the normotensive controls.The membrane of red blood cell ghosts (RBCgh) at concentrations of free Ca corresponding to its intracellular concentration binds 4.28±0.39 and 3.53±0.15 nmole of Ca per mg of protein of RBCgh in rats and humans, respectively. This part of membrane-bound Ca pool (most probably related to the inner part of the red blood cell membrane) is reduced by 48% in SHR and by 28% in patients with essential hypertension as compared to normotensive controls.It is suggested that the decrease of Ca binding ability of the RBC membrane in both types of hypertension studied may be a pattern of a more widespread cell membrane defect.     

Evidence of altered calcium accumulation and calcium binding by the membranes of adipocytes in spontaneously hypertensive rats

Calcium accumulation and calcium binding (ATP-dependent and ATP-independent calcium uptake) by the fragmented plasma membrane, the cytoplasmic reticulum and the mitochondria of isolated adipocytes obtained from spontaneously hypertensive (SHR, Kyoto-Wistar) and normotensive Wistar and Kyoto-Wistar rats were studied by means of isotopic (⁴⁵Ca) exchange in vitro.The value of Ca accumulation in the cytoplasmic reticulum fraction of adipocytes obtained from SHR was found decreased, while in the mitochondrial fraction it was considerably greater as compared to those in both normotensive control groups. Ca binding (ATP-independent) by the plasma membrane fraction of hypertensive rats was less than that of the normotensive rats. There was no difference between the groups studied in calcium binding ability for the cytoplasmic reticulum fraction of adipocytes.These results seem to indicate that the adipose cells of hypertensive rats have an alteration of the membrane mechanism maintaining intracellular calcium distribution.     

Silver ion-induced tension development and membrane depolarization in frog skeletal muscle fibres

Silver ions elicit dose-dependently a transient contracture in single fibres of bull-frog toe muscle placed in 0-Ca²⁺, Cl^–-free MOPS solution containing 3 mM Mg²⁺ and NO ₃ ^– . To elucidate the mechanisms involved, changes in membrane potential and in tension development were continuously measured following exposure to Ag⁺. The effect of Ag⁺ on contraction in fibres in which the membrane had been depolarized by elevating the external K⁺ concentration was also examined. The major findings of this investigation are as follows. (1) The mechanical threshold was shifted towards more negative potentials by 5 mV (–51 to –56 mV), when Ca²⁺ and Cl^– in the Ringer's solution were replaced with Mg²⁺ and NO ₃ ^– , respectively. (2) On the exposure of the fibres to 5 M Ag⁺, the membrane potential decreased by 1.6 mV from –87.8 mV and tension was developed. (3) In fibres soaked in a solution containing 10 mM K⁺ (corresponding to a membrane potential of –69.5 mV), 5 M Ag⁺ produced a large contracture similar to that seen in the control solution. (4) The Ag⁺-induced contracture was inactivated when more than 20 mM K⁺ was used. (5) The membrane depolarization evoked by either 20 or 50 M Hg²⁺ did not produce contraction. (6) Muscle fibres which had been exposed to 20 M Hg²⁺ for 5 min responded to 5 M Ag⁺ by a transient tension development. These findings strongly suggest that Ag⁺-induced tension development is not associated with depolarization of the surface membrane but rather is caused by specific actions of Ag⁺ on membrane proteins in the T-tubules.     

Single K+ channels in membrane evaginations of smooth muscle cells

Attempts have been made to apply the patch-clamp technique to enzymatically dispersed smooth muscle cells of frog and toad stomach. The rate of successful gigaseal formation has been extremely low, but better results can be obtained when patches are taken from membrane evaginations which develop on single cells after mechanical agitation and incubation in Ca²⁺-containing solutions at 25° C. Also ball-shaped single cells formed by the confluence of membrane evaginations were found to be equally useful for patch-clamp studies. Giga-seal formation was obtained in more than 80% of all attempts. Electron micrographs indicate that the myofilaments in membrane evaginations an in ball-shaped cells are separated from the cell membrane. Channel activity in membrane patches of such myoballs or evaginations is similar to the channel activity as found in intact cells. Two types of K⁺ channels (100 and 200 pS) have been observed that can be blocked by tetraethylammonium. Channels with the conductance of 200 pS are activated by intracellular Ca²⁺. The formation of evaginations has also been observed in other cells and may help to apply the patch-clamp technique to cells contaminated with surface coats.