Effects of doxorubicin and ruthenium red on intracellular Ca2+ stores in skinned rabbit mesenteric smooth-muscle fibres

Several agents are known to influence the contraction of skeletal and cardiac muscle via a modification of the Ca2+ release mechanism of the sarcoplasmic reticulum, e.g. caffeine, ryanodine, ruthenium red and doxorubicin. Of these substances, only the effects of caffeine and ryanodine have been described in smooth muscle. In this paper we describe the action of ruthenium red and doxorubicin on saponin-skinned mesenteric arteries of the rabbit. A high concentration (20 microM) of ruthenium red inhibited the Ca2+ release induced by low concentrations of caffeine, but had little effect on Ca2+ release induced by high concentrations (20 mM) of caffeine. This result indicates that the Ca2+ release channel of the internal Ca2+ store of smooth muscle cells is less sensitive to inhibition by ruthenium red than that of striated muscle. Doxorubicin in the micromolar range elicited a Ca2+ release and a concomitant contraction, essentially similar to its effect on skinned skeletal muscle cells. This work reveals further similarities between the Ca2+ release mechanisms of smooth and striated muscle, but the results also indicate that important differences between both systems may exist.     

Ca2+ sensitization of smooth muscle contractility induced by ruthenium red

The effects ofruthenium red (RuR) on contractility were examined in skinned fibers ofguinea pig smooth muscles, where sarcoplasmic reticulum function wasdestroyed by treatment with A-23187. Contractions of skinned fibers ofthe urinary bladder were enhanced by RuR in a concentration-dependentmanner (EC₅₀ = 60 µM at pCa6.0). The magnitude of contraction at pCa 6.0 was increased to 320% ofcontrol by 100 µM RuR. Qualitatively, the same results were obtainedin skinned fibers prepared from the ileal longitudinal smooth musclelayer and mesenteric artery. The maximal contraction induced by pCa 4.5 was not affected significantly by RuR. The enhanced contraction by RuRwas not reversed by the addition of guanosine5'-O-(2-thiodiphosphate) or a peptideinhibitor of protein kinase C [PKC-(1931)]. Theapplication of microcystin, a potent protein phosphatase inhibitor,induced a tonic contraction of skinned smooth muscle at lowCa²⁺ concentration([Ca²⁺]; pCa > 8.0).RuR had a dual effect on the microcystin-induced contraction-to-enhancement ratio at low concentrations and suppression at highconcentrations. The relaxation following the decrease in[Ca²⁺] from pCa 5.0 to>8.0 was significantly slowed down by an addition of RuR.Phosphorylation of the myosin light chain at pCa 6.3 was significantlyincreased by RuR in skinned fibers of the guinea pig ileum. Theseresults indicate that RuR markedly increases theCa²⁺ sensitivity of thecontractile system, at least in part via inhibition of myosin lightchain phosphatase.     

Effects of caffeine on crayfish muscle fibers. I. Activation of contraction and induction of Ca spike electrogenesis

Contractions are evoked in single muscle fibers of crayfish by intracellular as well as extracellular applications of caffeine. Responses to external applications in concentrations above 2 mM could be induced indefinitely. With concentrations above 5 mM the caffeine-induced responses were highly repeatable. Tensions were transient even when the caffeine remained in the bath. There was no change in resting potential, but during the contraction the effective resistance decreased about 10%. A number of factors (change in pH, Ca, K, and Cl) modified the responses. The time course of the tension was greatly prolonged when the transverse tubular system (TTS) was s swollen and was again shortened when the TTS was caused to shrink. An increased permeability to Ca induced by caffeine was evidenced by the transformation of the normally graded electrical responses to Ca spikes, which are insensitive to tetrodotoxin. The overshoot is a function of both external Ca and caffeine. A 10-fold change in Ca changed the overshoot by 19 mv in the presence of 10 mM caffeine and by 29 mv in 80 mM caffeine. The role of the increased permeability to Ca for caffeine-induced contractions will be analyzed in the accompanying paper.     

Ca2+-independent contraction of uterine smooth muscle

Rat uterine smooth muscle shows sustained contraction to oxytocin in Ca2+-free medium with EGTA, that is called "Ca-free contraction"(1). Participation of the rise in cytosolic free Ca2+ in this Ca-free contraction was tested. In Ca-free contraction, the cytosolic free Ca2+ level was not changed at all as measured with fura-2. Further, the chelation of cytosolic free Ca2+ with quin-2 did not at all affect Ca-free contraction. These results strongly suggest that Ca-free contraction is not triggered by Ca2+.     

Tentacle contraction inDiscophrya collini: The effects of ionophore A23187 and ruthenium red on Ca2+-induced contraction and uptake of extracellular calcium

Summary The tentacles of the suctorian protozoonDiscophrya collini are stimulated to contract by externally applied Ca²⁺. The role of extracellular Ca²⁺ in tentacle contraction was studied by monitoring⁴⁵Ca²⁺ uptake, using ionophore A23187 to facilitate membrane transport of calcium and ruthenium red (RR) as an inhibitor of transport. The degree of tentacle retraction was dependent upon external Ca²⁺ concentration and studies with⁴⁵Ca²⁺ using scintillation counting indicated a linear relationship between external Ca²⁺ concentration and Ca²⁺ uptake. Uptake of Ca²⁺ was enhanced in the presence of the ionophore while RR caused little inhibition.⁴⁵Ca²⁺ uptake was only partially inhibited by RR when cells were subjected to a Ca²⁺, ionophore and RR mixture. Grain counts from light microscope autoradiographs after treatment of cells with⁴⁵Ca²⁺/ionophore,⁴⁵Ca²⁺/RR or⁴⁵Ca²⁺ alone showed heavy, light and intermediate labelling respectively. In all instances the grains were evenly distributed within the cell.These observations are interpreted as supporting the suggestion that the ionophore enhances both the uptake of extracellular Ca²⁺ and release of Ca²⁺from an internal source, while the RR could only partially prevent movement of Ca²⁺ through the plasma mebrane. A model is presented suggesting that tentacle retraction is mediated by cytosolic Ca²⁺ levels which are determined by the fluxing of Ca²⁺ across the plasma membrane and the membrane of elongate dense bodies which act as internal Ca²⁺ reservoirs.     

Regulation of the Ca2+ dependence of smooth muscle contraction.

Cellular mechanisms for the regulation of Ca(2+)-dependent myosin light chain phosphorylation were investigated in bovine tracheal smooth muscle. Increases in the free intracellular Ca2+ concentration ([Ca2+]i), light chain phosphorylation, and force were proportional to carbachol concentration. KCaM, the concentration of Ca2+/calmodulin required for half-maximal activation of myosin light chain kinase, also increased proportionally, presumably due to Ca(2+)-dependent phosphorylation of the kinase. Isoproterenol treatment inhibited agonist-induced contraction by decreasing [Ca2+]i and thereby light chain phosphorylation. Depolarization by increasing concentrations of KCl also resulted in proportional increases in [Ca2+]i, KCaM, light chain phosphorylation, and force. However, the [Ca2+]i required to obtain a given value of either light chain phosphorylation or KCaM was greater in KCl-depolarized tissues compared to carbachol-treated tissues. In muscles contracted with KCl, isoproterenol treatment resulted in diminished light chain phosphorylation and force without alterations in [Ca2+]i or KCaM. Thus, isoproterenol inhibition of KCl-induced contraction results from a cellular mechanism different from that found in agonist-induced contraction. In neither case does isoproterenol produce relaxation by altering the calmodulin activation properties of myosin light chain kinase.     

Interaction of ruthenium red with Ca2(+)-binding proteins.

The interaction of ruthenium red, [(NH3)5Ru-O-Ru(NH3)4-O-Ru(NH3)5]Cl6.4H2O, with various Ca2(+)-binding proteins was studied. Ruthenium red inhibited Ca2+ binding to the sarcoplasmic reticulum protein, calsequestrin, immobilized on Sepharose 4B. Furthermore, ruthenium red bound to calsequestrin with high affinity (Kd = 0.7 microM; Bmax = 218 nmol/mg protein). The dye stained calsequestrin in sodium dodecyl sulfate-polyacrylamide gels or on nitrocellulose paper and was displaced by Ca2+ (Ki = 1.4 mM). The specificity of ruthenium red staining of several Ca2(+)-binding proteins was investigated by comparison with two other detection methods, 45Ca2+ autoradiography and the Stains-all reaction. Ruthenium red bound to the same proteins detected by the 45Ca2+ overlay technique. Ruthenium red stained both the erythrocyte Band 3 anion transporter and the Ca2(+)-ATPase of skeletal muscle sarcoplasmic reticulum. Ruthenium red also stained the EF hand conformation Ca2(+)-binding proteins, calmodulin, troponin C, and S-100. This inorganic dye provides a simple, rapid method for detecting various types of Ca2(+)-binding proteins following electrophoresis.     

Effects of impaired Ca2+ homeostasis on contraction in postinfarction myocytes.

The significance of altered Ca2+ influx and efflux pathways on contractile abnormalities of myocytes isolated from rat hearts 3 wk after myocardial infarction (MI) was investigated by varying extracellular Ca2+ concentration ([Ca2+]o, 0.6-5.0 mM) and pacing frequency (0.1-5.0 Hz). Myocytes isolated from 3-wk MI hearts were significantly longer than those from sham-treated (Sham) hearts (125 +/- 1 vs. 114 +/- 1 micrometer, P < 0.0001). At high [Ca2+]o and low pacing frequency, conditions that preferentially favored Ca2+ influx over efflux, Sham myocytes shortened to a greater extent than 3-wk MI myocytes. Conversely, under conditions that favored Ca2+ efflux (low [Ca2+]o and high pacing frequency), MI myocytes shortened more than Sham myocytes. At intermediate [Ca2+]o and pacing frequencies, differences in steady-state contraction amplitudes between Sham and MI myocytes were no longer significant. Collectively, the interpretation of these data was that Ca2+ influx and efflux pathways were subnormal in MI myocytes and that they contributed to abnormal cellular contractile behavior. Because Na+/Ca2+ exchange activity, but not whole cell Ca2+ current, was depressed in 3-wk MI rat myocytes, our results on steady-state contraction are consistent with, but not proof of, the hypothesis that depressed Na+/Ca2+ exchange accounted for abnormal contractility in MI myocytes. The effects of depressed Na+/Ca2+ exchange on MI myocyte mechanical activity were further evaluated in relaxation from caffeine-induced contractures. Because Ca2+ uptake by sarcoplasmic reticulum was inhibited by caffeine and with the assumption that intracellular Na+ and membrane potential were similar between Sham and MI myocytes, myocyte relaxation from caffeine-induced contracture can be taken as an estimate of Ca2+ extrusion by Na+/Ca2+ exchange. In MI myocytes, in which Na+/Ca2+ exchange activity was depressed, the half time of relaxation (1.54 +/- 0.14 s) was significantly (P < 0.02) prolonged compared with that measured in Sham myocytes (1.10 +/- 0.10 s).     

Effects of ruthenium red on activation of Ca2(+)-dependent cyclic nucleotide phosphodiesterase.

Ruthenium red inhibited Ca2(+)-dependent phosphodiesterase (Ca2(+)-PDE) selectively with an IC50 value of 15 microM. Increasing calmodulin concentration in the presence of both 100 microM and 4000 microM Ca2+ completely reversed the inhibition of Ca2(+)-PDE activity by ruthenium red. Ruthenium red-induced inhibition of Ca2(+)-PDE activity was also overcome by increasing the concentration of Ca2+ in the presence of both 200 ng and 2000 ng calmodulin, in sharp contrast to fluphenazine-induced inhibition of Ca2(+)-PDE. These results indicate that ruthenium red has distinct inhibitory mechanism which differs from that of calmodulin antagonists previously reported.     

Ca2+-induced Ca2+ desensitization of myosin light chain phosphorylation and contraction in phasic smooth muscle

The temporal relationship between Ca2+-induced contraction and phosphorylation of 20 kDa myosin light chain (MLC) during a step increase in Ca2+ was investigated using permeabilized phasic smooth muscle from rabbit portal vein and guinea-pig ileum at 25°C. We describe here a Ca2+-induced Ca2+ desensitization phenomenon in which a transient rise in MLC phosphorylation is followed by a transient rise in contractile force. During and after the peak contraction, the force to phosphorylation ratio remained constant. Further treatment with cytochalasin D, an actin fragmenting agent, did not affect the transient increase in phosphorylation, but blocked force development. Together, these results indicate that the transient phosphorylation causes the transient contraction and that neither inhomogeneous contractility nor reduced thin filament integrity effects the transient phosphorylation. Lastly, we show that known inhibitors to MLC kinase kinases and to a Ca2+-dependent protein phosphatase did not eliminate the desensitized contractile force. This study suggests that the Ca2+-induced Ca2+ desensitization phenomenon in phasic smooth muscle does not result from any of the known intrinsic mechanisms involved with other aspects of smooth muscle contractility.     

Effects of BAPTA on force and Ca2+ transient during isometric contraction of frog muscle fibers

The effects of1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraaceticacid (BAPTA) on force and intracellularCa²⁺ transient were studied duringisometric twitches and tetanuses in single frog muscle fibers. BAPTAwas added to the bathing solution in its permeant AM form (50 and 100 µM). There was no clear correlation between the changes in force andthe changes in Ca²⁺ transient.Thus during twitch stimulation BAPTA did not suppress theCa²⁺ transient until the force hadbeen reduced to <50% of its control value. At the same time, thepeak myoplasmic free Ca²⁺concentration reached during tetanic stimulation was markedly increased, whereas the force was slightlyreduced by BAPTA. The effects of BAPTA were not duplicated by usinganother Ca²⁺ chelator, EGTA,indicating that BAPTA may act differently as aCa²⁺ chelator. Stiffnessmeasurements suggest that the decrease in mechanical performance in thepresence of BAPTA is attributable to a reduced number of active crossbridges. The results could mean that BAPTA, under the conditions used,inhibits the binding of Ca²⁺ totroponin C resulting in a reduced state of activation of the contractile system.

     

Effect of ruthenium red upon Ca2+ and Mn2+ uptake in Saccharomyces cerevisiae. Comparison with the effect of La3+

The initial rate of both Ca2+ and Mn2+ uptake is inhibited by ruthenium red to about the same extent as by equivalent concentrations of La3+. The inhibition of Ca2+ uptake, however, is relieved during further incubation with ruthenium red. On preincubating the cells with ruthenium red even a stimulation of divalent cation uptake can be found. Relieve of the inhibition of divalent cation uptake is accompanied by K+ efflux. Both ruthenium red and La3+ displace Ca2+ very effectively from binding sites at the cell surface. The inhibition of initial Ca2+ uptake is accompanied by a reduction in the binding of Ca2+.     

Tianeptine's effects on spontaneous and Ca2+-induced uterine smooth muscle contraction

Tianeptine is a novel anti-depressant with an efficacy equivalent to that of classical anti-depressants. Additional beneficial effects include neuroprotection, anti-stress and anti-ulcer properties whose molecular mechanisms are still not completely understood but may involve changes in the anti-oxidant defence system. Herein, we have studied the effects of tianeptine on both contractile activity of isolated rat uteri and components of the endogenous anti-oxidative defence system. Tianeptine-induced dose-dependent inhibition of both spontaneous and Ca2+-induced contraction of uterine smooth muscle. The effect was more pronounced in the latter. Tianeptine treatment increased glutathione peroxidase (GSH-Px) and catalase (CAT) activities in spontaneous and Ca2+-stimulated uteri. A significant decrease in glutathione-reductase (GR) activity in both spontaneous and Ca2+-induced uterine contractions after tianeptine treatment indicated a reduction in reduced glutathione and consequently a shift toward a more oxidised state in the treated uteri. In spontaneously contracting uteri, tianeptine caused a decrease in copper-zinc SOD (CuZnSOD) activity. Tianeptine's anti-depressant effects may be accomplished by triggering a cascade of cellular adaptations including inhibition of smooth muscle contractility and an adequate anti-oxidative protection response.     

Ca2+-dependent facilitated shortening in isotonic contraction of trachealis muscle

We compared isotonic shortening with isometric force generation as a function of external Ca2+ in 166 tracheal smooth muscle (TSM) strips from 27 mongrel dogs in vitro. Concentration-response curves were generated with muscarinic stimulation (acetylcholine, ACh), alpha-adrenergic receptor activation (norepinephrine after beta-adrenoceptor blockade, NE), serotonin (5-HT), and KCl-substituted Krebs-Henseleit solution. The concentrations of 5-HT causing half-maximal shortening (ECS50, 1.54 +/- 0.14 X 10(-7) M) and half-maximal active isometric tension (ECT50, 1.72 +/- 0.30 X 10(-7) M) were similar (P = NS). Likewise, ECS50 (21.9 +/- 0.7 mM) and ECT50, (22.0 +/- 0.9 mM) were similar for KCl. In contrast, facilitated isotonic shortening (i.e., greater isotonic shortening for comparable degrees of force generation) was elicited with ACh and NE for all levels of force generation between 15 and 85% of maximum and for all concentrations of ACh from 3 X 10(-8) to 3 X 10(-5) M (P less than 0.05 for all points). Facilitated isotonic shortening also was elicited for all concentrations of NE from 10(-8) to 10(-6) M (P less than 0.05 for all points). Removal of Ca2+ from the perfusate substantially reduced the potency of ACh (P less than 0.001) and abolished differences between ECS50 (2.23 +/- 0.28 X 10(-5) M) and ECT50 (2.50 +/- 0.46 X 10(-5) M, P = NS). We demonstrate that for comparable degrees of force generation, muscarinic and alpha-adrenergic receptor activation cause greater isotonic shortening than KCl or 5-HT and that this facilitated shortening is associated with the concentration of external Ca2+.