Effects of metabolic inhibition on cytoplasmic calcium and contraction in smooth muscle of rat portal vein

Contractions in the rat portal vein, evoked by spontaneous action potentials or depolarizing high-K⁺ solution, are rapidly and reversibly inhibited by hypoxia or respiratory blockade. Intracellular free calcium ([Ca²⁺]_i) was measured using Fura-2 to evaluate the effects of metabolic blockade on excitation–contraction coupling. Spontaneous contractions were associated with transient increases in [Ca²⁺]_i. During exposure to cyanide (0.2–0.4 mm ) or 2,4-dinitrophenol (30 μm ) the duration and amplitude of the Ca²⁺ transients were decreased, leading to a decreased mean time integral of the individual [Ca²⁺]_i transient, and corresponding decrease in the duration and amplitude of the contraction. Basal [Ca²⁺]_i was increased in the presence of the metabolic inhibitors. High-K⁺ (40 mM) contractions caused a sustained increase in [Ca²⁺]_i, which was not inhibited by exposure to cyanide, although the amplitude of the associated contraction was greatly reduced. Together with the earlier demonstration of decreased 20 kD myosin light chain phosphorylation under these conditions, this indicates that the activation of contraction is influenced by metabolism via the energy dependence of the light chain phosphorylation reaction. Thus at least three steps in the excitation–contraction sequence are influenced by inhibition of oxidative metabolism: membrane excitation, light chain phosphorylation, and the cross-bridge cycle. This provides mechanisms for a high degree of metabolic sensitivity of vascular tone, of importance for the adaptation of blood flow to tissue metabolic demands.     

苯丙氨酸对自发性高血压大鼠血管平滑肌细胞内钙的影响

目的 探索苯丙氨酸对自发性高血压大鼠血管平滑肌细胞内钙的影响，方法 将ＳＨＲ和ＷＫＹ的血管平滑肌细胞在不同浓度苯丙酸培养液中培养一定时间后，加入（＾４５ＣａＣｌ２）温育一定时间后，测定进入细胞内（＾４５Ｃａ＾２＋）的量；而后将在不同浓度苯丙氨酸培养液中培养一定时间后的ＳＨＲ和ＷＫＹ的血管平滑肌细胞同ＰＳＳ液温育，采用Ｆｕｒａ－２／ＡＭ荧光指示剂测定不同浓度苯丙氨酸对细胞内游离Ｃａ＾２＋的影响。结果     

Electrical and mechanical responses to inhibition of cell respiration in vascular smooth muscle of the rat portal vein

Metabolic regulation of contractility in vascular smooth muscle was studied in the spontaneously active rat portal vein using respiratory depression by cyanide (0.2-2.0 mM) as a model for tissue hypoxia. Intracellular recordings of electrical activity were done with concomitant registration of force development. Average membrane potential in the absence of cyanide was -61 +/- 1 mV (n = 27). Addition of cyanide to normal Krebs solution resulted in a reduction of force amplitude and the number of action potentials per burst, with a relatively more pronounced effect on the mechanical activity. At moderate levels of inhibition of force amplitude the frequency of spontaneous bursts of action potentials transiently increased concomitant with a slight depolarization, but after prolonged (15-20 min) exposure to cyanide the membrane repolarized to the level prior to cyanide addition and the burst frequency decreased to be equal to or lower than that in the absence of cyanide. Higher concentrations of cyanide totally inhibited spontaneous mechanical and electrical activity. In contrast to the results with glucose, it was found that when beta-hydroxybutyrate was used as substrate the addition of 2 mM cyanide led to a marked hyperpolarization (13 +/- 1 mV) after total inhibition of spontaneous activity. The hyperpolarization was not prevented by administration of 4-aminopyridine (2.5 mM) or tetraethylammonium (4-6 mM) prior to the addition of cyanide. To investigate the effects of increased metabolic demand on the relation between force and membrane potential in cyanide-treated muscle, high-K+ (40 mM) contractures were studied. Contractures were associated with depolarization of 34 +/- 3 mV (n = 5). 1 mM cyanide reduced the amplitude of the contractures to about 9% of control with a moderate reduction in the amount of depolarization (28 +/- 1 mV, n = 5). It is concluded that the decrease of mechanical activity during respiratory inhibition may partly reflect a reduction in the number of spikes per burst but that other mechanisms, independent of membrane activity, also contribute to the inhibition. The increase of glycolysis during respiratory inhibition seems to prevent more pronounced changes in membrane potential.     

Calcium-dependence of the calcium-activated chloride current in smooth muscle cells of rat portal vein

Ca²⁺-activated Cl^– current in freshly isolated smooth muscle cells from rat portal vein was studied using the whole-cell patch-clamp technique. Simultaneously, the free-cytosolic Ca²⁺ concentration (Ca_i) was estimated using emission from the dye Indo-1. Pretreatment of the cells with amytal and carbonyl-cyanide-m-chlorophenylhydrazone, which reduced the intracellular adenosine triphosphate concentration, was used to weaken the cellular Ca²⁺ homeostatic system. Ca_i of treated cells slowly increased during perfusion with an external Ca²⁺-containing solution. This rise in Ca_i gradually activated a Ca²⁺-dependent Cl^– current which allowed the study of the relationship between activation of this current and Ca_i levels. The threshold Ca_i for activation of Cl^– channels was around 180 nM and full activation occurred at 600 nM. The Ca_i dependence of the Cl^– channels was not changed during application of noradrenaline and did not depend on the membrane potential. The gating of Ca²⁺-dependent Cl^– channels of rat portal vein myocytes seems to be mainly controlled by intracellular Ca²⁺     

Contractile and metabolic characteristics of creatine-depleted vascular smooth muscle of the rat portal vein

The functional consequences of phosphocreatine (PCr) depletion for mechanical properties, O2 consumption, and lactate production of the rat portal vein were investigated. After feeding rats for 8-9 weeks on a diet containing 2% beta-guanidino propionic acid (BGPA), PCr of the portal vein was reduced to 14% of control, whereas ATP was unchanged. No significant change was found in the level of spontaneous contractile activity or the force developed in a high-K+ contracture. Lactate production and the relationship between contractile force and O2 consumption were uninfluenced by BGPA treatment. The force-velocity relation of electrically stimulated portal veins showed no influence of BGPA treatment on Vmax. To investigate whether decrease in PCr influenced the response to metabolic stress, portal veins were exposed to graded concentrations (0.1-0.5 mM) of cyanide to depress cellular respiration. Veins from control and BGPA-treated rats showed the same relative decrease of contractile activity and O2 consumption, and the same increase in lactate production. Cyanide treatment resulting in a reduction of electrically stimulated force to 70-80% of the original gave a reduction of Vmax to 85-90%. The relative degree of reduction was uninfluenced by BGPA treatment. Reduction of PCr content thus does not affect the functional properties of metabolism or contractility under normoxic conditions. Furthermore, it can be inferred that the PCr reduction known to occur in smooth muscle exposed to hypoxia (L?vgren & Hellstrand 1985) is not in itself the major factor causing hypoxic inhibition of mechanical activity.     

Effects of calcium and sodium on contracture tension in the smooth muscle of the rat portal vein

Summary The purpose of the present study was to determine the quantitative relationship between membrane potential (or [K⁺]₀) and contracture tension in the smooth muscle of the rat portal vein, and to examine the influence of Ca⁺⁺ and Na⁺ on this relationship. However, electrical all-or-none responses were successfully abolished only in Na⁺-free sucrose-Krebs due to hyperpolarization and in K⁺-high Krebs due to depolarization. It did not seem possible to eliminate spike discharge at intermediate levels of membrane potential without the simultaneous loss of contractility. In the hyperpolarized state the muscle remained relaxed despite the very low levels of [Na⁺]₀ and despite increases in [Ca⁺⁺]₀ up to 20 mM. The depolarized portal vein developed a contracture which was intimately dependent on [Ca⁺⁺]₀, the threshold concentration being of the order of 0.1 to 0.3 mM. Spike-induced, phasic contractions showed a similar Ca⁺⁺-dependence. Variations in [Na⁺]₀ had only a slight and irregular influence on the Ca⁺⁺ dose-response curve of the depolarized muscles.Differences in the effects of Na⁺ on the rate of rise and the rate of fall of the contracture tension, respectively, suggested that Na⁺ is more important for the removal of Ca⁺⁺ from the contractile system than for the supply of Ca⁺⁺ to the system. With regard to the interaction of Ca⁺⁺ and Na⁺ in the excitation-contraction coupling the vascular smooth muscle seemed to differ from both heart muscle and skeletal muscle.The present study was supported by grants from the Swedish Medical Research Council (B 70-14x-28-06 A), from Air Force Office of Scientific Research through the European Office of Aerospace Research, OAR, United States Air Force under Contract F 1052-68-C-0044, from U.S. Public Health Service (HE-05678-08), from AB Hässle, Göteborg, and from the Deutsche Forschungsgemeinschaft (Bi 122/1).     

ATP对原代培养大鼠膀胱Cajal间质细胞及逼尿肌细胞兴奋性的影响

目的探讨外源性ATP对大鼠膀胱Cajal间质细胞（ICC）兴奋性的影响。方法采用胶原酶消化法急性分离SD大鼠膀胱ICC和逼尿肌细胞（DSMC）,通过激光共聚焦技术检测外源性ATP作用前后ICC及DSMC内Ca2＋信号的变化。结果胶原酶P消化后,在干细胞因子（SCF）的刺激下,经c-k it免疫荧光染色阳性的ICC呈长梭形或纺锤形,胞体较小,并且带有分枝状突起,而DSMC呈纤维形或短棒状,二者形态有明显区别。F luo-4 NW染色后,激光共聚焦显微镜观察显示,静止状态下ICC内[Ca2＋]i平均荧光强度（55.16±3.23）显著强于平滑肌细胞（45.59±2.08）,P〈0.05;20μmol/L和100μmol/L ATP可诱发体外培养的ICC及DSMC细胞内[Ca2＋]i一过性升高,随后缓慢下降。结论在SCF刺激条件下,SD大鼠膀胱ICC和DSMC可在体外稳定混合培养。外源性ATP可兴奋体外混合培养的ICC和DSMC。     

Comparison of VIP and β2--adrenoceptor-induced relaxations in the circular and longitudinal smooth muscle layers of the rat portal vein

Comparison of VIP and β₂-_-adrenoceptor-induced relaxations in the circular and longitudinal smooth muscle layers of the rat portal vein. Acta Physiol Scand 130, 601–607. Received I I November 1986, accepted 26 February 1987. ISSN 00014772. Department of Physiology, University of Bergen, Norwegian Defence Research Establishment, Kjeller, Norway. The relative importance of VIP in reduction of vascular tone was studied in circular and longitudinal preparations of the VIP-innervated rat portal vein. Exogenous VIP inhibited the methoxamine-evoked contractures in the atropine-blocked preparations with a lower potency in the inner, circular (pD₂= 6.4±0.5, n= 6) than in the outer, longitudinal layer (pD₂= 7.7 ± 0.1, n = 6). VIP was also a less efficient relaxant (intrinsic activity (a) = 0.60 ±0.16, n = 6) of the inner than of the outer layer (a= I. 00). The selective (salbutamol) and the non-selective (isoproterenol) β₂-_-agonists completely relaxed the methoxamine contractures in both layers and the potency (isoproterenol) was higher in the inner (pD₂= 6.39 ± 0.32, n = 6) than in the outer layer (pD₂= 5.67 ±0.34, n= 6). Plasma from the portal-mesenteric vein of anaesthetized, fasting rats contained 0.036 nM VIP (median, n= 17). that is, several orders of magnitude lower than the range of VIP concentrations relaxing the methoxamine contracted vein preparations via VIP receptors of the apamin-blockable category. The results support the hypothesis that a₁-_-adrenoceptor-induced contractions in the circular layer are predominately relaxed via β₂-_-adrenoceptors while relaxation of the outer layer may occur via VIP receptors, probably activated by local release of the neuropeptide.     

Contractile properties during development of hypertrophy of the smooth muscle in the rat portal vein

Structural and mechanical alterations during hypertrophy of the rat portal vein were investigated. Growth of the vessel was induced by a partial ligature of the vessel causing an increased transmural pressure. Vessel segments from animals kept with ligature for 1, 3, 5 and 7 days, were compared with vessels from sham-operated animals. Maximal active force and vessel cross-sectional area increased with time in the ligated group. On day 7, force and cross-sectional area at the optimal length, were markedly increased in the ligated group (21.1 +/- 1.0 mN, 0.55 +/- 0.04 mm2, n = 9) compared with the control vessels (11.7 +/- 1.0 mN, 0.30 +/- 0.02 mm2, n = 7). Light and electron microscopy of preparations fixed at optimal length showed that the amount of smooth muscle and the cross-sectional area of cell profiles were almost doubled in the ligated group on day 7, consistent with hypertrophy of the smooth muscle. The force per smooth muscle cell area was similar in the two groups (ligated: 132 +/- 15; control: 145 +/- 16 mN mm-2, n = 4-5). The maximal shortening velocity was significantly lower in the hypertrophied group (ligated: 0.28 +/- 0.02; control: 0.41 +/- 0.01 optimal length s-1, n = 6). In chemically skinned preparations, activated by maximal thiophosphorylation of the myosin light chains, force was higher in the ligated group compared to the controls but no difference in maximal shortening velocity was observed. In conclusion, the increased transmural pressure is associated with a rapid increase in the amount of smooth muscle in the portal vein. The mechanical data show that after 7 days the force generating ability of the contractile system has increased in proportion to the smooth muscle cell mass. The unaltered maximal shortening velocity in the skinned hypertrophied preparations suggests that the kinetic properties of the maximally activated contractile system are unaltered. The decreased maximal shortening velocity in the intact hypertrophied preparations may reflect alterations in the excitation-contraction coupling.     

Evidence for the involvement of the cyclooxygenase-metabolic pathway in diclofenac-induced inhibition of spontaneous contraction of rat portal vein smooth muscle cells.

The effects of diclofenac, a cyclooxygenase (COX) inhibitor, were investigated on spontaneous phasic contractions of longitudinal preparations of the rat portal vein. Diclofenac produced a concentration-dependent decrease in the amplitude of these spontaneous phasic contractions. Diclofenac (30 microM) decreased the amplitude of the spontaneous phasic increase in the F340/F380 ratio of Fura PE3, an indicator of intracellular Ca2+ concentration. It also reduced the number of action potentials in each burst discharge without changing the resting membrane potential of longitudinal smooth muscle cells. The extent of the distribution of Lucifer Yellow injected into a smooth muscle cell was decreased in the presence of diclofenac (30 microM). Both AH6809, a prostanoid EP receptor antagonist, and SQ22536, an adenylate cyclase inhibitor, decreased the amplitude of the spontaneous contractions. On the other hand, neither ozagrel, a thromboxane synthase inhibitor, nor SQ29548, a prostanoid TP receptor antagonist, significantly affected spontaneous contractions. These results indicate that diclofenac inhibits the amplitude of spontaneous contractions of the rat portal vein through inhibition of electrical activity, which may be related to an inhibition of the cyclooxygenase pathway.     

Differential actions of exogenous and intracellular spermine on contractile activity in smooth muscle of rat portal vein

Effects of the naturally occurring polyamine spermine on electrical and contractile properties of the rat portal vein were studied. 1 mM spermine nearly abolished spike activity and spontaneous contractions and decreased the intracellular Ca²⁺ concentration ([Ca²⁺],). The phasic force responses to 0.1 and 1 μM phenylephrine were partially inhibited, but not the sustain plateau contraction caused by 5 /IM phenylephrine. The Ca²⁺-force relation in high-K⁺ (128 mM)-depolarized veins was shifted to the right, EC₅₀ for Ca²⁺ increasing from 0.50 ± 0.03 mM (control, n= 8) to 0.65 ± 0.06 and to 0.94 ± 0.03 at 1 (n – 4) and 10 (n = 3) mM spermine, respectively. However, at a Ca²⁺ concentration of 2.5 mM, giving maximal force, there was no effect of spermine (1 mM) on either force or [Ca²⁺],. Whereas extracellular spermine thus reduced contractile activity at moderate levels of stimulation, increased intracellular concentration of spermine potentiated the force response to Ca²⁺. Intracellular loading of spermine by reversible permeabilization increased its concentration by 2–3 times. The spontaneous activity and response to phenylephrine were unchanged. However, the Ca²⁺-force relation of depolarized veins was shifted to the left, EC₅₀ decreasing from 0.51 ± 0.04 mM in controls (n= 7) to 0.36 ± 0.02 mM in the loaded veins (n= 9). Spermine increased Ca²⁺-activated force in portal veins permeabilized with β-escin. The degree of potentiation was consistent with observed effects in spermine-loaded intact veins. The results suggest that spermine at physiological intracellular concentration may contribute to the determination of Ca²⁺ sensitivity in vascular smooth muscle cells.     

Free cytosolic calcium during spontaneous contractions in smooth muscle of the guinea-pig mesotubarium

The free intracellular calcium ion concentration ([Ca²⁺]_i) was measured simultaneously with isometric force in strips of guinea-pig mesotubarium using the Fura-2 technique. During the relaxed period (5–15 min) between spontaneous contractions [Ca²⁺]_i continues to decrease after full mechanical relaxation to reach a minimal level of 86±8 nM (n=9) just before the start of the next contraction. During the spontaneous contractions (5–15 min) [Ca²⁺]_i reached a maximum of 211±19 nM and then oscillated between 155±16 nM and 194±9 nM. Increased extracellular Ca²⁺ concentration to 10 mM from the standard concentration of 1.5 mM caused a decreased frequency of spontaneous contractions and an increase in [Ca²⁺]_i both in the relaxed and contracted states. In 10 mM extracellular Ca²⁺, addition of AlF₄ ^–, as 1 mM NaF + 10 M AlCl₃, caused a sustained increase in [Ca²⁺]_i and maintained force. Addition of verapamil (10 M) in this situation decreased [Ca²⁺]_i to the resting level. The results suggest that the cyclic appearance of trains of action potentials is related to variation in [Ca²⁺]_i, possibly via inactivation of Ca²⁺-dependent K⁺ channels.     

Combined effects of hypoxia and endurance training on lipid metabolism in rat skeletal muscle

Aim: To determine whether endurance training can counterbalance the negative effects of hypoxia on mitochondrial phosphorylation and expression of the long chain mitochondrial fatty acid transporter muscle carnitine palmitoyl transferase 1 (mCPT‐1). Methods: Male Wistar rats were exposed either to hypobaric hypoxia (at a simulated altitude of ≈4000 m, PIO₂ ≈ 90 mmHg) or to normoxia (sea level) for 5 weeks. In each environment, rats were randomly assigned to two groups. The trained group went through a 5‐week endurance training programme. The control group remained sedentary for the same time period. Muscle fatty acid oxidation capacity was evaluated after the 5‐week period on isolated mitochondria prepared from quadriceps muscles with the use of palmitoylcarnitine or pamitoylCoA + carnitine. Results: Chronic hypoxia decreased basal (V₀, ?31% with pamitoylCoA + carnitine and ?21% with palmitoylcarnitine, P < 0.05) and maximal (V_max, ?31% with pamitoylCoA + carnitine, P < 0.05) respiration rates, hydroxyacylCoA dehydrogenase activity (?48%, P < 0.05), mCPT‐1 activity index (?34%, P < 0.05) and mCPT‐1 protein content (?34%, P < 0.05). Five weeks of endurance training in hypoxia brought V₀, mCPT‐1 activity index and mCPT‐1 protein content values back to sedentary normoxic levels. Moreover, in the group trained in hypoxia, V_max reached a higher level than in the group that maintained a sedentary lifestyle in normoxia (24.2 nmol O₂· min^?1 · mg^?1 for hypoxic training vs. 19.9 nmol O₂ · min^?1 · mg^?1 for normoxic sedentarity, P < 0.05). Conclusion: Endurance training can attenuate chronic hypoxia‐induced impairments in mitochondrial fatty acid oxidation. This training effect seems mostly mediated by mCPT‐1 activity rather than by mCPT‐1 content.     

Effects of Ca2+ on force-velocity characteristics of normal and hypertrophic smooth muscle of the rat portal vein

Portal hypertension was induced in rats by partial ligation of the hepatic branches of the portal vein. After 5 days of hypertension the portal veins were taken out and mounted for isometric and quick-release experiments. Portal veins from sham-operated normal rats served as controls. The ligated veins had an increased cross-sectional area, indicating smooth-muscle hypertrophy. Although the absolute magnitude of active force of these veins was increased, the active force per cross-sectional area was decreased, indicating an alteration in the properties of the contractile system. No difference in the Ca2+ concentration-response relations to K+-activated intact control and hypertrophic veins was found. In chemically skinned preparations, devoid of functional plasma membranes, the hypertrophic veins had similar Ca2+ sensitivity (in the presence of I microM calmodulin) but a lower force per cross-sectional area. Force-velocity relations were determined in K+-activated intact preparations. In control veins a reduction in extracellular Ca2+ was associated with a significant reduction in both isometric force and maximal shortening velocity (Vmax). In hypertrophic veins the decreased isometric force at maximal activation was associated with a low Vmax. A comparison between hypertrophic and submaximally stimulated control vessels showed corresponding Vmax and isometric force values. We conclude that the low isometric force of hypertrophic veins is associated with a lower rate of cross-bridge turnover. This could be an effect of alterations in the activation mechanisms or in the intrinsic properties of the contractile system itself.     

Quantitative aspects of electrical and mechanical responses to anisosmolar solutions in the smooth muscle of the rat portal vein

The electrical and mechanical activity of the rat portal vein were studied quantitatively under prolonged exposure to solutions in which osmolality was varied by changes in sucrose content. Reducing osmolality by 15 or 30 mosm/kg below the control value of 290 mosm/kg caused enhanced electrical and mechanical activity whereas hyperosmolality up to 390 mosm/kg led to inhibition as demonstrated earlier. These responses faded under prolonged exposure. In hypoosmolality integrated mechanical activity returned to control after about 10–15 min while spike activity remained somewhat increased. Prolonged hyperosmolality was associated with return of spike discharge towards control frequency whereas the integrated contractile force reverted from initial inhibition to levels above control after some 10–15 min. Therefore, the integrated force per spike was increased by prolonged hyperosmolality and decreased by hypoosmolality. Variations in osmolality had similar effects on the amplitude of K⁺ contractures. The time course of the osmotic responses are discussed in relation to the dynamic effects of passive stretch and shortening in the portal vein. The relation that may exist between “inotropic” effects of osmolality and the contractures obtained in strongly hypertonic media is considered.