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     

Tension response and45Ca release in vascular smooth muscle incubated in Ca-free solution

The contractile response of arterial smooth muscles induced by agonists as noradrenaline or histamine in Ca-free solution consists of two phases: an initial phasic component, which is transient and accompanied by a release of cellular Ca, and a small tonic component, which persists as long as the agonist is present. A second admission of the agonist without reexposure to Ca elicits only the tonic component. This tonic contraction differs in several respects from the phasic response obtained in Ca-free solution: it is independent of the duration of exposure to the Ca-free solution, it can be elicited many times without reexposure to Ca, and it is not accompanied by a measurable release of Ca from the cells.During superfusion with Ca-free solution, a tonic contraction is also induced by fluoride ions at concentrations exceeding 4 mM. The amplitude of this contraction is maximal at about 12 mM. Increasing the fluoride concentration shortens the delay between the addition of the F^– and the onset of the contraction. As is the case for the tonic noradrenaline-response, the F^–-induced contractions can be clicited many times without reexposure to Ca. The tonic contractions evoked by noradrenaline or histamine and by fluoride ions are additive. Both contractions are reversibly inhibited by caffeine, theophylline, Na-nitroprusside, papaverine and by nitroglycerine.The possiblity that these tonic contractions are not accompanied by an increase of the cytoplasmic Ca²⁺ concentration is discussed.This work was supported by the grant of the Fonds voor Wetenschappelijk Geneeskundig Onderzoek, Belgium, nr. 3.0087.74     

缺氧对兔胸主动脉平滑肌细胞增殖的影响

目的: 观察缺氧对培养的兔胸主动脉平滑肌细胞增殖的影响及其可能的机制。方法: 采用析因实验设计, 测定在正常或缺氧环境下, 不同浓度的胎牛血清对培养的血管平滑肌细胞数量、[³H]-TdR掺入量、MTT比色以及细胞cGMP水平的影响; 透射电镜观察细胞超微结构。结果: 缺氧组平滑肌细胞的数量、[³H]-TdR掺入量以及MTT比色显著多于常氧组(P<0.01); 电镜下可见缺氧组较正常组细胞器发达, 可见大量分泌胞, 少量髓样变线粒体; 缺氧组平滑肌细胞内的cGMP浓度明显高于常氧组。结论: 缺氧可促进血管平滑肌细胞DNA合成, 促进细胞增殖, 可能与提高细胞内的cGMP水平有关。     

Temperature-dependence of45Ca fluxes and contraction in vascular smooth muscle cells of rabbit ear artery

The effect of cooling from 35 to 20° C on the⁴⁵Ca-exchange and on the contractile response of rabbit ear artery has been investigated.The amplitude of the contraction induced by K-depolarization at 20° C is reduced to about 60% of its value at 35° C, whereas the response to noradrenaline is not significantly affected.Cooling induces a 2 to 4-fold reduction of the⁴⁵Ca-efflux rate. This effect also occurs in Ca-free medium and in solutions containing 1 mM La. It also occurs in Na-free medium and in tissues in which the transmembrane Na-gradient has been reduced.At 20° C, the⁴⁵Ca-influx in unstimulated tissues and in K-depolarized preparations is significantly lower than at 35° C. in Ca-depleted tissues, i.e. tissues in which the noradrenaline-sensitive Ca-store has been emptied by a stimulation with the agonist in Ca-free solution, the⁴⁵Ca-influx is not significantly affected by cooling.The gradual depletion of the noradrenaline-sensitive Ca-store in Ca-free solutions is at 20° C much slower than at 35° C. The amount of Ca released by noradrenaline is not affected by cooling, whereas for the same amount of Ca released the contractile response is higher at 20° C.These findings indicate that temperature affects the transmembrane Ca-extrusion and the Ca-influx through voltage-dependent channels. The properties of the noradrenaline-sensitive Ca-store are less sensitive to temperature.     

Effects of halothane on functionally skinned rabbit soleus muscle fibers: A correlation between tension transient and45Ca release

The mechanism(s) involved in the halothane-induced increase in skeletal muscle contraction was studied using functionally skinned soleus muscle fibers from rabbits: For the tension study, single functionally skinned fibers were individually mounted on two pairs of forceps, with one end attached to a photodiode tension transducer. Ca²⁺-activated tension development of the contractile proteins, and Ca²⁺ uptake and release from the sarcoplasmic reticulum (SR) using caffeine-induced tension transients were studied. To measure the amount of calcium, skinned fibers at 0.1 g/ml were used and 0.075 Ci⁴⁵Ca/ml was spiked in the solution 3 (pCa 6.5 and 1 mM [EGTA]) which promoted rapid loading of Ca²⁺. Halothane (1–3%) did not change the [Ca²⁺]-tension relationship; 2 and 3% halothane reduced the maximum Ca²⁺-activated tension by 6–7%. Halothane (1–3%) added to the solution 3, reduced⁴⁵Ca uptake by 3, 22 and 23%; however, the subsequent caffeine-induced tension transient and⁴⁵Ca release were increased by 10–40%. During the release phase only halothane increased both caffeine-induced tension transient and⁴⁵Ca release by 20–60%. The effects of halothane on the tension transient and on the⁴⁵Ca release were comparable. There was no dose-response relationship to the effects of halothane on the above parameters. It is concluded that halothane affects the SR by increasing its membrane permeability to Ca²⁺, resulting in an increase in myoplasmic [Ca²⁺] and thus in the twitch tension in skeletal muscle.     

Effect of isoprenaline on intracellular Ca uptake and on Ca influx in arterial smooth muscle

We have investigated the effect of isoprenaline on the amplitude of the transient contraction of isolated arteries induced by releasing the intracellular Ca with an agonist during incubation in a Ca-free solution. Under some conditions this force development is an indication of the amount of Ca in the intracellular store. Loading the store in high K⁺ solution in the presence of isoprenaline increases the amplitude of the subsequent contraction in Ca-free solution by 6 to 16% as compared to the control. The presence of isoprenaline during the loading procedure in the control solution with 5.9 mM K⁺ does not affect the amplitude of the subsequent contraction of the rabbit ear artery. In the rabbit coronary artery such pretreatment with isoprenaline inhibits the contraction to 40% of the control. These results indicate that -agonists may stimulate the Ca uptake in the intracellular store and, depending on the tissue, inhibit the influx of Ca.     

Alterations in45Ca distribution and movements in ileal longitudinal smooth muscle

The distribution and movements of⁴⁵Ca were examined in longitudinal smooth muscle isolated from guinea-pig ileum. Alterations in⁴⁵Ca distribution were induced by lack of equilibration prior to⁴⁵Ca incubation, decreased temperature, iodoacetic acid (IAA), decreased nonradioactive Ca⁺⁺ (increased specific activity of⁴⁵Ca), and increased K⁺, but not by acetylcholine, histamine or carbachol. Efflux of⁴⁵Ca was observed as two components with half-times of about 1 and 9.5 minutes. Either substitution of K⁺ for Na⁺ or addition of 80 mM K⁺ to O–Ca bathing solutions decreased the washout of⁴⁵Ca in a sustained manner, whereas addition of nonradioactive Ca⁺⁺ elicited both transient and maintained increases. Transient effects indicate a displacement of bound⁴⁵Ca; maintained increases could be attributed to a decreased reuptake of⁴⁵Ca. The K⁺-induced decrease in⁴⁵Ca efflux may result from a decreased cellular pool of available⁴⁵Ca after an inward shift of bound Ca⁺⁺. Absence of similar changes in⁴⁵Ca fluxes after exposure to stimulatory agents indicates that intracellular release and rebinding of Ca⁺⁺ may oscur without detectable changes in cellular Ca⁺⁺ or⁴⁵Ca fluxes.     

Lack of soluble carbonic anhydrase in aortic smooth muscle of the rabbit

Rabbit aorta was investigated for the occurrence of soluble, methazolamide-sensitive carbonic anhydrase (CA) by measuring electrometrically the rate of acidification of a weakly alkaline CO₂ solution buffered with 12.5 mM veronal/HCl at 0°C. For this purpose, aortae of 10 rabbits with the endothelium carefully preserved, were homogenized, centrifuged, and the supernatants pooled. The proteins were fractionated by FPLC, and tested for their CA activity. In control experiments, the pH change resulting from the spontaneous CO₂ hydration was found to be –0.92±0.01 pH units/min (mean±SE,n=10). Aliquots of the 30,000 dalton protein fraction corresponding to 100 mg of aortic tissue wet weight did not alter the hydration rate significantly (–0.94±0.01 pH units/min,n=10). Also, in the presence of 10^–4 M of the CA inhibitor, methazolamide, these rates were not altered significantly (–0.94±0.01 and –0.93±0.01 pH units/min, respectively,n=10). No CA activity was found in the other FPLC fractions, either. — These results suggest that soluble CA is absent from the myocytes and the endothelium of the rabbit aorta.Supported by the Deutsche Forschungsgemeinschaft within the SFB 90 Cardiovasculäres System     

Effects of Ca2+ channel agonist-antagonist enantiomers of dihydropyridine 202791 on insulin release, 45Ca uptake and electrical activity in isolated pancreatic islets

This is the first study using the selective agonist/antagonist stereoisomers of dihydropyridine 202791 to investigate stimulus-secretion coupling in pancreatic islet cells. We studied effects of the (+)(Ca2+ channel agonist) and (-)(Ca2+ channel antagonist) forms of the dihydropyridine, on 45calcium net uptake, insulin secretion, and membrane potential measured in rodent islets. The antagonist partially inhibited glucose-induced insulin secretion and Ca2+ uptake; however, the potassium-induced Ca2+ uptake was completely inhibited. The antagonist did not completely block glucose-evoked spike activity. Addition of the agonist enhanced insulin release and Ca2+ uptake in the presence of 5.6 mM-glucose, but did not increase insulin release or Ca2+ uptake in 16.7 mM-glucose. In the presence of tetraethylammonium (TEA), (+)202791 increased and (-)202791 decreased the duration of glucose-induced action potentials. The results again confirm the presence of a dihydropyridine-sensitive Ca2+ channel in pancreatic B-cells. In addition these data suggest that in these cells there is activation of a dihydropyridine-insensitive Ca2+ entry in the presence of glucose.     

K-channel blocking drugs induce histamine release and 45Ca uptake in isolated mast cells.

Histamine secretion and 45Ca uptake processes were studied in mast cells treated with four K+ channel blocking drugs in physiological saline and in media containing different ionic concentrations. Quinine, 4-aminopyridine and sparteine were effective as histamine-releasing agents when mast cells were incubated in physiologic saline solution. The dose-response profile obtained was in the range of 0.1-0.5 mM for quinine, 1-10 for 4-aminopyridine and 0.5-5 mM for sparteine and did not show significant differences between purified and unpurified mast cells. By contrast, tetraethylammonium (1-100 mM) did not induce histamine release. The presence of high K+ or Rb+ concentrations in the medium (Tris-K+ or Tris-Rb+, both at 150 mM) displaced the profile obtained to the right in cells stimulated with 4-aminopyridine or sparteine, but abolished histamine release induced by quinine. Additionally, all three K+ channel blockers increased 45Ca uptake in mast cells. The exact mechanism of the action of K+ channel blockers on mast cells is unknown. However, the fact that the drugs used were effective as histamine-releasing and 45Ca uptake promoters suggests both that mast cells might be endowed with a K+ channel activity and that the blockade of this should open certain calcium channels, leading to elevated intracellular Ca2+ levels which in turn activate mast cell secretion.     

Effects of acetaldehyde on responses of rabbit corpus cavernosal smooth muscle

Ethanol has various effects on male sexual activity under the influence of direct and indirect, in acute and chronic alcohol ingestion. However, whether acetaldehyde, a principal metabolite of ethanol, may affect penile erection directly has still not been elucidated. This present study was, therefore, designed to clarify the pharmacologic effects of the acetaldehyde on corpus cavernosal smooth muscle. Corpus cavernosal strips were prepared from rabbit penises. Isometric tension changes of rabbit corpus cavernosal strips to various drugs and electrical field stimulation (EFS) in an organ chamber were recorded with a pressure transducer after active muscle tone had been induced by phenylephrine (10(-5) mol/L). At the concentrations employed, acetaldehyde had no effect on the pH of the bathing medium. Acetaldehyde in each concentration did not significantly affect resting tone of the smooth muscle during 30 min incubation. Acetaldehyde suppressed contractility induced by phenylephrine and KCI at 10(-4) mol/L, and relaxation induced by EFS and bethanechol at 10(-3) mol/L and 10(-4) mol/L respectively, but acetaldehyde enhanced relaxation induced by ATP at high acetaldehyde level. Sodium nitroprusside-induced relaxation was not affected at any employed acetaldehyde concentration. This suggests that increasing the acetaldehyde level may contribute to male erectile dysfunction mainly by the inhibition of nitric oxide formation.     

Agonist-dependent modulation of Ca2+ sensitivity in rabbit pulmonary artery smooth muscle

The effects of the stable thromboxane analogue U46619, the ₁-adrenergic agent phenylephrine and depolarization with high K⁺ on cytoplasmic Ca²⁺ ([Ca²⁺]_i) and force development were determined in rabbit pulmonary artery smooth muscle. Following stimulation with each of the excitatory agents, the time course of the [Ca²⁺]_i/force relationship described counter-clockwise hysteresis loops with the rise and fall in [Ca²⁺]_i leading, respectively, contraction and relaxation. The rank order of the force/[Ca²⁺]_i ratios evoked by the different methods of stimulation was: U46619 > phenylephrine high K⁺. The difference between the actions of U46619 and phenylephrine was due to the lesser Ca²⁺-releasing and greater Ca²⁺-sensitizing action of U46619. Both U46619 and phenylephrine also released intracellular Ca²⁺ in intact (non-permeabilized) preparations. The effects of the two agonists on force, at constant free cytoplasmic [Ca²⁺] maintained with EGTA, were also determined in preparations permeabilized with staphylococcal -toxin, in which intracellularly stored Ca²⁺ was eliminated with A23187. Sensitization of the contractile response to Ca²⁺ by agonists was indicated by the contractile responses of permeabilized muscles to U46619 and to phenylephrine, in the presence of constant, highly buffered [Ca²⁺]_i. These contractions were inhibited by GDP[S] and could also be elicited by GTP. We conclude that, in addition to changing [Ca²⁺]_i, pharmacomechanical coupling can also modulate contraction by altering the sensitivity of the regulatory/contractile apparatus of smooth muscle to [Ca²⁺]_i, through a G-protein-coupled mechanism.     

Effects of exogenously applied calponin on Ca2+-regulated force in skinned smooth muscle of the rabbit mesenteric artery

To help elucidate the physiological role of calponin (a thin-filament-linked regulatory protein) in smooth muscle contraction, the effects of its exogenous application were investigated on actin-activated MgATPase activity in crude actomyosin from chicken gizzard, and on contraction induced by Ca²⁺-dependent and -independent means in arterial smooth muscle strips skinned by saponin or β-escin. Calponin concentration dependently inhibited actin-activated MgATPase activity with a proportional increase in its binding to actomyosin and also attenuated Ca²⁺-induced contractions, in the presence or absence of calmodulin, in skinned arterial strips. Calponin, when phosphorylated by protein kinase C, reduced both its ability to bind to actomyosin and its inhibitory action on actomyosin MgATPase. The phosphorylated calponin also had no effect on the maximum Ca²⁺-induced contraction in skinned smooth muscle, suggesting that these actions of calponin are not non-specific. Calponin attenuated the Ca²⁺-independent contraction observed in myosin light chain thio-phosphorylated strips, or on application of trypsin-treated myosin light chain kinase. However, calponin had no effect on maintained rigor contraction. These results suggest that in vascular smooth muscle, calponin may play a physiological role in the inhibition of Ca²⁺-regulated force, possibly through a direct action on active actin-myosin interactions.