Increase in Ca++ sensitivity of the contractile system by MCI-154, a novel cardiotonic agent, in chemically skinned fibers from the guinea pig papillary muscles

The effects of MCI-154, a novel cardiotonic agent, on the contractile protein system and the sarcoplasmic reticulum (SR) were investigated by using thin bundles of chemically skinned fibers from the guinea pig papillary muscles. In the skinned muscle fibers treated with 50 micrograms/ml of saponin, MCI-154 shifted the -log[Ca++]M-tension relation curve to the left and upward in the concentration-dependent manner (10(-7) to 10(-4) M). This was confirmed also in the skinned muscle fibers treated with 250 micrograms/ml of saponin which destroyed not only the surface membrane but also the function of SR. Sulmazole (10(-4) M) shifted the -log[Ca++]M-tension relation curve to the left but the effect was about 100 times less potent than that of MCI-154. Unlike MCI-154, sulmazole had little effect on the maximum tension development induced by -log[Ca++]M 4.4. Milrinone did not affect the Ca++-induced tension development in the skinned cardiac fibers. Higher concentration of MCI-154 (10(-4) M) also increased amplitude of -log[Mg-ATP]M-tension-curve in the absence of free Ca++ ion (bell-shaped curve) to the upward. Initial rate and plateau phase of Ca++ uptake by the SR in the skinned fibers treated with 50 micrograms/ml of saponin was increased slightly by MCI-154 at the concentrations of 10(-6) and 10(-4) M. MCI-154 had no effect on the Ca++-induced Ca++ release mechanism in the SR. These results suggest that an increase in Ca++ sensitivity of the contractile protein system is responsible for, at least in part, the mechanism of the positive inotropic action of MCI-154.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of pimobendan, a new cardiotonic agent, on contractile responses in single skeletal muscle fibres of the frog

Effects of a new cardiotonic agent, pimobendan, on contraction were investigated in single intact skeletal muscle fibres of the frog. Pimobendan increased twitch tension in a concentration-dependent manner regardless of the presence or absence of Ca2+ without any effect on tetanic tension, the resting membrane potential and the shape of the action potential. Pimobendan caused a further increase in twitch tension potentiated by caffeine (1 mM). Adenine, an inhibitor of Ca2+-induced Ca2+ release from the sarcoplasmic reticulum, inhibited twitch tension potentiated by caffeine but not by pimobendan, suggesting that twitch potentiation by pimobendan is not attributed to increases in Ca2+-induced Ca2+ release. Pimobendan failed to increase cAMP levels in the skeletal muscle, though forskolin significantly increased it without any effect on twitch tension. Contractile responses to high concentrations of caffeine and K+ were also potentiated by pimobendan. These results suggest that the potentiating effect of pimobendan on skeletal muscle contraction is mainly due to the increase in Ca2+ sensitivity to the contractile apparatus.     

Powerful activation of skeletal muscle actomyosin ATPase by goniodomin A is highly sensitive to troponin/tropomyosin complex

Goniodomin A has been shown to cause the conformational change of actin to modify actomyosin ATPase activity. Goniodomin A induced a potent stimulation of the actomyosin ATPase activities of the actin-myosin reconstituted system and natural actomyosin in the range of 10(-8) to 10(-7) M. When the concentration was increased above 10(-7) M, actomyosin ATPase activity was decreased. Interestingly, the troponin/tropomyosin complex caused a concentration-dependent inhibition of the goniodomin A-induced stimulation of actomyosin ATPase activity. In the presence of a high concentration of the troponin/tropomyosin complex, goniodomin A decreased actomyosin ATPase activity in a concentration-dependent manner. The enhancement of the ATPase activity of troponin/tropomyosin-free natural actomyosin by goniodomin A was larger than that obtained with natural actomyosin. Goniodomin A at lower concentrations enhanced the superprecipitation of natural actomyosin but decreased it at higher concentrations. The ATPase activity of skeletal muscle myofibrils and the contractile response of skinned fibers to Ca(2+) were never activated and were decreased by this compound, suggesting an inhibition by the troponin/tropomyosin complex. In the far ultraviolet circular dichroism, goniodomin A above 10(-8) M increased the negative ellipticity at 220 nm, suggesting an increase in the alpha-helical content of actin. These results suggest that goniodomin A increases and decreases actomyosin ATPase activity, probably through the stimulatory and inhibitory sites on actin, respectively. It is also suggested that the troponin/tropomyosin complex binds to actin to inhibit the goniodomin A-induced enhancement of actomyosin ATPase activity, probably by affecting the stimulatory site on the molecule.     

Potent stimulation of myofilament force and adenosine triphosphatase activity of canine cardiac muscle through a direct enhancement of troponin C Ca++ binding by MCI-154, a novel cardiotonic agent

In the present study we have analyzed a likely biochemical mechanism underlying the Ca++-sensitizing action of MCI-154 (6-[4-(4'-pyridyl)aminophenyl)-4,5-dihydro-3(2H)-pyridazinone hydrochloride), a novel cardiotonic agent, on the contractile protein system. MCI-154 (10(-7) to 10(-4) M) enhanced the tension development induced by -log molar-free Ca++ concentration (pCa) 5.8 in chemically skinned fiber from the canine right ventricular muscle in a concentration-dependent manner. At pCa 7.0, MCI-154 (10(-7) to 10(-4) M) markedly increased adenosine triphosphatase (ATPase) activities of canine myofibrils and reconstituted actomyosin. In myofibrils and reconstituted actomyosin, MCI-154 (10(-7) to 10(-4) M) caused a parallel shift of the pCa-ATPase activity relation curve to the left without affecting the maximum activity, suggesting an increase in Ca++ sensitivity. MCI-154 (10(-8) to 10(-4) M) had little effect on actin-activated, Mg++, Ca++ and (K+, EDTA)-ATPase activities of myosin. Ca++ binding to cardiac myofibrils or purified cardiac troponin was increased by 10(-4) M MCI-154. These results suggest that MCI-154 enhances Ca++ binding to cardiac troponin C to elevate the Ca++ sensitivity of myofilaments and thus may cause a positive inotropic action in cardiac muscle. MCI-154 may provide a valuable tool for studying the molecular mechanism by which Ca++ regulates the contractile system.     

Mechanism of inotropic action of xestoquinone, a novel cardiotonic agent isolated from a sea sponge.

Xestoquinone (XQN) isolated from the sea sponge Xestospongia sapra produced dose-dependent cardiotonic effects on guinea pig left and right atria. A direct action of XQN (1-30 microM) on the contractile machinery of cardiac myofilaments was demonstrated in chemically skinned fiber preparations from guinea pig papillary muscles. In atrial preparations, the XQN-induced inotropic effect was markedly inhibited by verapamil or nifedipine, but was not affected by practolol, chlorpheniramine, cimetidine, tetrodotoxin or reserpine. The Ca++ dependence curve for the contractile response of the atria was substantially shifted to the left by XQN (10 microM), and this XQN-induced shift was reversed by verapamil. The time-to-peak tension and relaxation times of the atrial contractions were shortened by XQN, and the action potential duration was markedly prolonged. Whole-cell patch clamp recordings in left atrial strips confirmed that XQN (30 microM) increased the slow inward current. However, there was a temporal dissociation between altered tension development and prolongation of the action potential duration. Cyclic AMP phosphodiesterase activity was inhibited and tissue cyclic AMP content of guinea pig left atria was increased by XQN (0.3-10 microM) in a concentration-dependent manner, but increases in cyclic AMP content did not occur in parallel with increases in contractile response. These observations suggest that an enhancement of intracellular cyclic AMP content and Ca++ influx across the cell membrane contribute to the late phase of XQN-caused cardiotonic responses, whereas the early phase may largely be elicited through direct activation of contractile elements. XQN may provide a novel leading compound for valuable cardiotonic agents.     

MCI-154, a novel cardiotonic agent, reverses the acidic pH-induced decrease in responses of cardiac myofilaments to Ca++: comparison with sulmazole and pimobendan

In the present study, we have examined the effects of decreasing pH from 7.0 to 6.6 on the tension developed by direct activation of the myofilaments in chemically skinned fibers from guinea pig papillary muscles. We then compared the effects of the novel inotropic agents MCI-154, pimobendan and sulmazole, which have direct action on cardiac myofilaments, on the acidic pH-induced changes in responses of the contractile system to Ca++. The reduction of pH from 7.0 to 6.8 shifted the pCa (-log[Ca++] M)-tension relation curve to the right with no change in maximum tension. However, the reduction of pH from 7.0 to 6.6 shifted the pCa tension relation curve to the right and also depressed maximum force development. These effects were reversible by returning to neutral pH (pH 7.0), but were not overcome by increasing the free [Ca++] (decreasing pCa from 4.4 to 4.0). The amplitude of pMg-ATP (-log[MgATP]M)-tension curve in the absence of free Ca++ (Ca++ less than 1 nM, bell-shaped curve) was shifted downward by reducing pH from 7.0 to 6.6. MCI-154 (1-100 microM) reversed the acidic pH-induced decrease of tension development which was activated by pCa 5.8 in a concentration-dependent manner. Moreover, the acidosis induced reductions of maximum tension (pCa, 4.4) and pMgATP 6.0-activated tension (Ca++ less than 1 nM) were also reversed by MCI-154 (1-100 microM) in a concentration-dependent manner.(ABSTRACT TRUNCATED AT 250 WORDS)     

Administration of endotoxin, tumor necrosis factor, or interleukin 1 to rats activates skeletal muscle branched-chain alpha-keto acid dehydrogenase.

Protein catabolic states (i.e., sepsis and trauma) are thought to be associated with accelerated oxidation of branched-chain amino acids (BCAA). Branched-chain alpha-keto acid dehydrogenase (BCKAD), the rate-limiting enzyme for BCAA oxidation by muscle, is regulated by phosphorylation/dephosphorylation. Skeletal muscle BCKAD was only 2-4% active in control rats. Intravenous injection of Salmonella enteritidis endotoxin (0.25-10 mg/kg) did not change total BCKAD activity, but increased the percent active enzyme in muscle three- to four-fold in 4-6 h. Identical results were observed in adrenalectomized rats pretreated with one dose of alpha-methylprednisolone (2.5 mg/kg i.p.) 30-60 min before saline or endotoxin injection, indicating that endotoxin's effect was not mediated by hypersecretion of adrenal hormones. Cortisone pretreatment of normal rats (100 mg/kg per d) for 2 d prevented endotoxin-induced activation of muscle BCKAD, suggesting that endogenous secretion products mediated BCKAD activation by endotoxin. Human recombinant tumor necrosis factor-alpha and/or IL-1 beta or alpha (50 micrograms/kg) increased muscle BCKAD activation two- to fourfold in normal rats 4-6 h after intravenous injection. We conclude that cytokine-mediated activation of muscle BCKAD may contribute to accelerated BCAA oxidation in septicemia.     

Electrophysiological effects of ACC-9358, a novel class I antiarrhythmic agent, on isolated canine Purkinje fibers and ventricular muscle

Class I antiarrhythmic agents are heterogeneous with respect to their cardiac electrophysiological effects and have been subdivided into three categories: la, lb and lc. The purpose of the present study was to determine the classification and investigate the mechanism of action of ACC-9358 [4-hydroxy-N-phenyl-3,5-bis (1-pyrrolidinyl-methyl)benzamide], a novel class I antiarrhythmic agent currently under clinical investigation. The effects of ACC-9358 on action potentials from isolated canine Purkinje fibers and ventricular muscle were examined using standard microelectrode techniques. In Purkinje fibers, ACC-9358 (1-50 microM) exerted a dose-dependent reduction in maximum upstroke velocity (Vmax) and action potential duration at 50 and 90% repolarization (APD50 and APD90). The reduction of Vmax was voltage-dependent (greater at an extracellular potassium concentration of 6 mM than at 2.7 mM), frequency-dependent (greater at a basic cycle length of 500 than at 2000 msec) and very slow in onset (rate constant of 0.017 action potentials-1) and offset (recovery half-time of 66.9 sec). In Purkinje fibers, ACC-9358 attenuated the action potential shortening effects of lidocaine but not that of nicardipine or nicorandil and shortened APD50 to a greater extent at a basic cycle length of 2000 than at 500 msec. In ventricular muscle, ACC-9358 (1-50 microM) exerted a dose-dependent reduction in Vmax and prolongation of APD50 and APD90.(ABSTRACT TRUNCATED AT 250 WORDS)     

Does the positive inotropic action of a novel cardiotonic agent, MCI-154, involve mechanisms other than cyclic AMP?

MCI-154 is a new positive inotropic agent with vasodilating property. Experiments were carried out in the canine isolated right ventricular muscle in order to elucidate whether or not cyclic AMP is involved in the positive inotropic effect (PIE) of MCI-154. MCI-154 (10(-7) to 10(-4) M) produced a concentration-dependent PIE amounting to 75% of the maximal effect of isoproterenol. MCI-154 did not affect the time to peak tension and had a tendency to shorten the relaxation time and total duration of contraction. Pindolol, reserpine-pretreatment or tetrodotoxin did not modify the PIE of MCI-154. MCI-154 increased the cyclic AMP levels only at 3 X 10(-4) M, whereas CI-914, of which chemical structure is similar to that of MCI-154, elevated definitely the cyclic AMP at the lower concentrations (10(-5) to 10(-4) M). Carbachol at a concentration known to decrease markedly the PIE of amrinone, milrinone and papaverine, did not affect the PIE of MCI-154. MCI-154 inhibited the activity of a crude phosphodiesterase (PDE) from the canine ventricular muscle and it enhanced the PIE of isoproterenol, which implied the involvement of cyclic AMP. However, the maximal inhibition of PDE by MCI-154 remained less than 18%. Amrinone, milrinone and papaverine inhibited more potently the PDE activity than MCI-154. These results suggest that the elevation of cyclic AMP levels is only partially involved in the PIE of MCI-154 in the canine right ventricular muscle, and that MCI-154 may have novel mechanisms of action different from those of amrinone, milrinone and CI-914 that are largely cyclic AMP-dependent.     

Development of a novel smart scaffold for human skeletal muscle regeneration

Skeletal muscle defects are notoriously difficult to manage and the current methods used are associated with many limitations. Engineered skeletal muscle tissue has the potential to provide a solution that circumvents these disadvantages. Our previous work has identified a novel three‐dimensionally aligned degradable phosphate glass fibre scaffold that can support myoblast differentiation and maturation. This current study has further developed the scaffold by encasing the fibres within a collagen gel to produce a smart composite scaffold that provides key biomimetic cues and supports the formation of a tissue that may be implanted in vivo. The constructs formed were approximately 30 mm long and microscopic examination confirmed favourable unidirectional cell alignment. There was good cell survival, and gene expression studies demonstrated upregulation of the myogenic regulatory factors and developmental and adult myosin heavy chain isoforms indicating myofibre formation and maturation respectively. Compared with the three‐dimensional glass fibre scaffolds, the composite scaffolds had later gene upregulation, however, the use of collagen gels reinforced with degradable aligned glass fibres offers the opportunity to create a tissue analogue that can be easily manipulated. Furthermore, the glass fibre ends could support tendon/bone formation, and the channels formed as the fibres degrade could allow for vascular ingrowth. Copyright © 2013 John Wiley & Sons, Ltd.     

Effects of pimobendan, a novel inotropic agent, on intracellular calcium and tension in isolated ferret ventricular muscle

1. In this study we have investigated the effects of a novel inotropic agent, pimobendan (UDCG 115-BS), on skinned and intact ventricular muscle from ferrets. 2. Pimobendan (20 or 100 mumol/l) increased tension at a given free [Ca2+] when applied to skinned ventricular muscle, i.e. it increased the Ca2+ sensitivity of the myofibrils. 3. Tension and intracellular free Ca2+ [( Ca2+]i) were measured simultaneously in intact papillary muscles using the aequorin technique. When 25 mumol/l pimobendan was added to the superfusing solution, a slowly developing positive ionotropic effect was produced, which was accompanied by an increase in the size of the systolic rise in [Ca2+]i (Ca2+ transients) with a similar time course. 4. In order to determine whether pimobendan increased the Ca2+ sensitivity of myofibrils in an intact papillary muscle, we compared the increase in Ca2+ transients and tension observed in response to changes in extracellular [Ca2+] with those observed in response to pimobendan. The result of this comparison was that in intact muscle pimobendan caused no apparent increase in myofibrillar Ca2+ sensitivity. 5. Pimobendan caused an abbreviation of the time course of the Ca2+ transients, but the twitch was slightly prolonged. 6. When isoprenaline was added to the superfusing solution, a positive inotropic effect was produced, which was accompanied by a marked increase in the size of the Ca2+ transients. Isoprenaline caused an abbreviation of the time course of both the Ca2+ transients and the twitch. When the Ca2+ sensitivity of the intact myofibrils was determined as described above, isoprenaline caused a desensitization. Pimobendan produced a sensitization when compared with isoprenaline. 7. These results are consistent with the hypothesis that pimobendan produces an inotropic effect in isolated cardiac muscle which is mediated both by an increase in Ca2+ sensitivity and by an increase in adenosine 3':5'-cyclic monophosphate due to its phosphodiesterase-inhibiting activity. Such a combination of activities may be particularly advantageous for an inotropic agent.     

Comparison of serum cardiac specific troponin-I with creatine kinase,creatine kinase-MB isoenzyme,tropomyosin, myoglobin and C-reactive protein release in marathon runners: cardiac or skeletal muscle trauma?

Problems arise in distinguishing skeletal from cardiac muscle trauma on the basis of serum enzyme tests following severe muscle exercise. The contributions of cardiac and skeletal sources have been assessed in eleven marathon runners by measuring pre- and post-race serum levels of cardiac-specific myofibrillar troponin-I together with total creatine kinase, creatine kinase-MB isoenzyme, myoglobin, myofibrillar tropomyosin and C-reactive protein. Total creatine kinase, creatine kinase-MB isoenzyme, tropomyosin and myoglobin were significantly elevated above pre-race levels in all runners between 1 h and 128 h post-race. Neither mean cardiac troponin-I nor C-reactive protein was elevated post-race. Nine out of sixty-three samples fulfilled conventional positive criteria for cardiac muscle damage on the basis of combined creatine kinase and creatine kinase-MB isoenzyme levels. Six runners had one or more positive samples. No samples had levels above twice the upper normal limit for either cardiac troponin-I or C-reactive protein. Correlation analysis of levels in each sample indicated skeletal and not cardiac muscle as the source of raised serum protein.     

Deficiency of skeletal muscle succinate dehydrogenase and aconitase. Pathophysiology of exercise in a novel human muscle oxidative defect.

We evaluated a 22-yr-old Swedish man with lifelong exercise intolerance marked by premature exertional muscle fatigue, dyspnea, and cardiac palpitations with superimposed episodes lasting days to weeks of increased muscle fatigability and weakness associated with painful muscle swelling and pigmenturia. Cycle exercise testing revealed low maximal oxygen uptake (12 ml/min per kg; healthy sedentary men = 39 +/- 5) with exaggerated increases in venous lactate and pyruvate in relation to oxygen uptake (VO2) but low lactate/pyruvate ratios in maximal exercise. The severe oxidative limitation was characterized by impaired muscle oxygen extraction indicated by subnormal systemic arteriovenous oxygen difference (a-v O2 diff) in maximal exercise (patient = 4.0 ml/dl, normal men = 16.7 +/- 2.1) despite normal oxygen carrying capacity and Hgb-O2 P50. In contrast maximal oxygen delivery (cardiac output, Q) was high compared to sedentary healthy men (Qmax, patient = 303 ml/min per kg, normal men 238 +/- 36) and the slope of increase in Q relative to VO2 (i.e., delta Q/delta VO2) from rest to exercise was exaggerated (delta Q/delta VO2, patient = 29, normal men = 4.7 +/- 0.6) indicating uncoupling of the normal approximately 1:1 relationship between oxygen delivery and utilization in dynamic exercise. Studies of isolated skeletal muscle mitochondria in our patient revealed markedly impaired succinate oxidation with normal glutamate oxidation implying a metabolic defect at the level of complex II of the mitochondrial respiratory chain. A defect in Complex II in skeletal muscle was confirmed by the finding of deficiency of succinate dehydrogenase as determined histochemically and biochemically. Immunoblot analysis showed low amounts of the 30-kD (iron-sulfur) and 13.5-kD proteins with near normal levels of the 70-kD protein of complex II. Deficiency of succinate dehydrogenase was associated with decreased levels of mitochondrial aconitase assessed enzymatically and immunologically whereas activities of other tricarboxylic acid cycle enzymes were increased compared to normal subjects. The exercise findings are consistent with the hypothesis that this defect impairs muscle oxidative metabolism by limiting the rate of NADH production by the tricarboxylic acid cycle.     

Presynaptic inhibition of sympathetic fibers participating in vasodilatation in response to K+-induced contraction of frog skeletal muscle

Experiments were performed on an autoperfused transparent frog muscle (m. cutaneus pectoris) with the purpose of elucidating possible mechanisms of functional hyperaemia. The diameter of a primary arteriole was followed before and after a K+-induced contracture lasting 20 s. The vasodilatation following this period of activity was expressed as the diameter change relative to the maximal possible diameter increase. The results emphasize adenosine as an important substance participating in vasodilatation in active skeletal muscle, its effect possibly being presynaptic (prejunctional) inhibition of sympathetic vasoconstrictor fibers to arteriolar smooth muscle (sympathetic uncoupling), in addition to the well-known direct effect (relaxation) on vascular smooth muscle cells. The presence of a strong resting sympathetic tone was demonstrated by local application of phentolamine (7.1 X 10(-5) M) that led to a large diameter increase. The purinoceptor blocking agent, 8-phenyltheophylline (8-PTP) invariably attenuated post-contraction vasodilatation, the adenosine transport inhibitor dipyridamole (10(-5) M) increased the hyperaemia response. Lack of sympathetic arteriolar tone during hyperaemia was demonstrated by the absence of a dilatory effect of phentolamine applied in the post-contraction period. Conversely, blockage of purinoceptors by 8-PTP during and after the contracture allowed sympathetic discharge to continue as verified by application of phentolamine, now leading to further vasodilatation. We suggest that presynaptic inhibition of sympathetic nerve endings by adenosine participates in vasodilatation in frog skeletal muscle.     

Enhanced cytokine-induced mechanical hyperalgesia in skeletal muscle produced by a novel mechanism in rats exposed to unpredictable sound stress

Stress exacerbates both experimental and clinical pain, most well-characterized in irritable bowel and fibromyalgia syndromes. Since it has been hypothesized that cytokines play an etiopathogenic role in fibromyalgia and other chronic widespread pain conditions, we investigated the relationship between stress and cytokines in a model of stress-induced chronic somatic pain. A series of experiments were performed to evaluate the impact of stress on the hyperalgesia-induced by endotoxin (lipopolysaccharide, LPS) and the role of two pro-inflammatory cytokines, interleukin-6 (IL-6) and tumor necrosis α (TNFα). Fourteen days after exposure to a 4-day protocol of unpredictable sound stress, the ability of systemic LPS (100 μg/kg, i.p) to elicit cytokine-mediated mechanical hyperalgesia was measured in gastrocnemius muscle. LPS-induced hyperalgesia was significantly greater in stressed rats, but when rats were treated intrathecally with antisense oligodeoxynucleotide (ODN), to decrease either the gp130 subunit of the IL-6 receptor or the TNFα receptor, in nociceptors, skeletal muscle hyperalgesia in sound stressed, but not control, rats was prevented. These data suggest that chronic stress alters signaling in the primary afferent nociceptor for the hyperalgesia induced by endogenously produced pro-inflammatory cytokines.     

Effects of pimobendan, a cardiotonic and vasodilating agent with phosphodiesterase inhibiting properties, on isolated arteries and veins of rats.

Effects of pimobendan (PBD) were investigated on isolated rat blood vessels. PBD dose-dependently relaxed aortic, femoral arterial and mesenteric venous preparations precontracted with KCl and reduced the amplitude of spontaneous contractions of portal venous preparations; the sensitivity to PBD was femoral greater than portal greater than mesenteric greater than aorta. Relaxation response of the femoral artery to PBD was not changed by propranolol and aminophylline. Glyceryl trinitrate (GTN), isoproterenol (ISO), forskolin and adenosine also elicited dose-dependent relaxations of femoral arteries; the rank order of potency (mean negative log EC50 value) was GTN greater than ISO greater than PBD = forskolin greater than adenosine. The relaxation responses to PBD and isobutyl methylaxanthine (IBMX) were not attenuated with removal of endothelial cells. In the femoral artery, methylene blue diminished GTN-induced relaxation but not PBD-induced relaxation. PBD and IBMX increased the relaxation responses of the artery to cyclic AMP-forming drugs (ISO, forskolin and adenosine) but not a cyclic GMP-forming drug (GTN). PBD and IBMX increased the relaxation response of mesenteric veins to ISO. The drugs noncompetitively inhibited arterial contractions accompanied by voltage-dependent and alpha-adrenoceptor-operated Ca2+ influxes. In the absence of extracellular Ca2+, PBD and IBMX reduced contractile responses of arteries to norepinephrine but not caffeine. The present results suggested that PBD relaxed the blood vessels, at least in part, through an intracellular accumulation of cyclic AMP.     

Reliability of a new,hand-held device for assessing skeletal muscle stiffness

OBJECTIVE: The aim of the study was to examine the test-retest reliability of the Myoton-2 myometer for measuring skeletal muscle viscoelastic stiffness. METHODS: Ten healthy volunteers took part. On day 1, the viscoelastic stiffness of the rectus femoris, vastus lateralis, biceps femoris, and gastrocnemius muscle (lateral and medial heads) was measured at rest using the Myoton-2 myometer. On day 2, the tests were repeated, and the rectus femoris was also examined during the maintenance of submaximal contractions of the quadriceps, and at a different resting muscle length. RESULTS: The myometer showed good to excellent test-retest reliability for all muscles (ICCs 0.80-0.93), except for the vastus lateralis (ICC 0.40). Viscoelastic stiffness showed a linear increase with increasing quadriceps' force output, and was higher in stretched than in shortened resting muscle. CONCLUSION: The Myoton-2 myometer is a reliable device for measuring the viscoelastic stiffness of resting muscle. Furthermore, viscoelastic stiffness showed the expected changes in response to increases in force output and muscle length, suggesting that the measurements were also valid. RELEVANCE: The results of this pilot study show that the Myoton-2 myometer is a simple, precise instrument for measuring muscle viscoelastic stiffness. If the findings can be confirmed in larger studies, further research should be carried out to examine its potential applications in the field of musculoskeletal medicine.     

A novel thienylhydrazone, (2-thienylidene)3,4-methylenedioxybenzoylhydrazine, increases inotropism and decreases fatigue of skeletal muscle

We report here the preclinical profile of etoricoxib (MK-0663) [5-chloro-2-(6-methylpyridin-3-yl)-3-(4-methylsulfonylphenyl) pyridine], a novel orally active agent that selectively inhibits cyclooxygenase-2 (COX-2), that has been developed for high selectivity in vitro using whole blood assays and sensitive COX-1 enzyme assays at low substrate concentration. Etoricoxib selectively inhibited COX-2 in human whole blood assays in vitro, with an IC(50) value of 1.1 +/- 0.1 microM for COX-2 (LPS-induced prostaglandin E2 synthesis), compared with an IC(50) value of 116 +/- 8 microM for COX-1 (serum thromboxane B2 generation after clotting of the blood). Using the ratio of IC(50) values (COX-1/COX-2), the selectivity ratio for the inhibition of COX-2 by etoricoxib in the human whole blood assay was 106, compared with values of 35, 30, 7.6, 7.3, 2.4, and 2.0 for rofecoxib, valdecoxib, celecoxib, nimesulide, etodolac, and meloxicam, respectively. Etoricoxib did not inhibit platelet or human recombinant COX-1 under most assay conditions (IC(50) > 100 microM). In a highly sensitive assay for COX-1 with U937 microsomes where the arachidonic acid concentration was lowered to 0.1 microM, IC(50) values of 12, 2, 0.25, and 0.05 microM were obtained for etoricoxib, rofecoxib, valdecoxib, and celecoxib, respectively. These differences in potency were in agreement with the dissociation constants (K(i)) for binding to COX-1 as estimated from an assay based on the ability of the compounds to delay the time-dependent inhibition by indomethacin. Etoricoxib was a potent inhibitor in models of carrageenan-induced paw edema (ID(50) = 0.64 mg/kg), carrageenan-induced paw hyperalgesia (ID(50) = 0.34 mg/kg), LPS-induced pyresis (ID(50) = 0.88 mg/kg), and adjuvant-induced arthritis (ID(50) = 0.6 mg/kg/day) in rats, without effects on gastrointestinal permeability up to a dose of 200 mg/kg/day for 10 days. In squirrel monkeys, etoricoxib reversed LPS-induced pyresis by 81% within 2 h of administration at a dose of 3 mg/kg and showed no effect in a fecal 51Cr excretion model of gastropathy at 100 mg/kg/day for 5 days, in contrast to lower doses of diclofenac or naproxen. In summary, etoricoxib represents a novel agent that selectively inhibits COX-2 with 106-fold selectivity in human whole blood assays in vitro and with the lowest potency of inhibition of COX-1 compared with other reported selective agents.     

A novel quercetin/β-cyclodextrin transdermal gel,combined or not with therapeutic ultrasound,reduces oxidative stress after skeletal muscle injury

A gel containing the inclusion complex of quercetin and β-cyclodextrin was developed in order to verify its effects, isolated or using phonophoresis, on oxidative biomarkers after skeletal muscle injury. 30 male rats were divided into one of five groups: Control (CTRL), Muscle Injury (MI), Therapeutic Pulsed Ultrasound (TPU), Therapeutic Pulsed Ultrasound plus Quercetin (TPU plus gel-QUE) or Quercetin gel (QUE). Quercetin gel was complexed with β-Cyclodextrin (β-CD) using chromatography (HPLC). TPU and quercetin application occurred with 2, 12, 24, 48, 72, 96 hours intervals after injury. Gastrocnemius muscle was injured by mechanical trauma. Lipid peroxidation, superoxide dismutase activity, and catalase activity were assessed. The inclusion complex exhibited adequate entrapment efficiency, relative density and pH. The viscosity of the complex showed a non-Newtonian pseudoplastic behavior. Quercetin/β-cyclodextrin gel reduced lipid peroxidation, superoxide dismutase activity and catalase activity compared to muscle injury group. Similarly, phonophoresis and TPU also reduced the levels of these oxidative biomarkers. In conclusion, quercetin/β-cyclodextrin transdermal gel reduces oxidative stress biomarkers after skeletal muscle injury irrespective of using phonophoresis.

A gel containing the inclusion complex of quercetin and β-cyclodextrin was developed in order to verify its effects, isolated or using phonophoresis, on oxidative biomarkers after skeletal muscle injury.