Decrease In Isometric Tension Research Articles

Myosin Phosphatase and Cofilin Mediate cAMP/cAMP-dependent Protein Kinase-induced Decline in Endothelial Cell Isometric Tension and Myosin II Regulatory Light Chain Phosphorylation

This study determined the effects of increased intracellular cAMP and cAMP-dependent protein kinase activation on endothelial cell basal and thrombin-induced isometric tension development. Elevation of cAMP and maximal cAMP-dependent protein kinase activation induced by 10 microm forskolin, 40 microm 3-isobutyl-1-methylxanthine caused a 50% reduction in myosin II regulatory light chain (RLC) phosphorylation and a 35% drop in isometric tension, but it did not inhibit thrombin-stimulated increases in RLC phosphorylation and isometric tension. Elevation of cAMP did not alter myosin light chain kinase catalytic activity. However, direct inhibition of myosin light chain kinase with KT5926 resulted in a 90% decrease in RLC phosphorylation and only a minimal decrease in isometric tension, but it prevented thrombin-induced increases in RLC phosphorylation and isometric tension development. We showed that elevated cAMP increases phosphorylation of RhoA 10-fold, and this is accompanied by a 60% decrease in RhoA activity and a 78% increase in RLC phosphatase activity. Evidence is presented that it is this inactivation of RhoA that regulates the decrease in isometric tension through a pathway involving cofilin. Activated cofilin correlates with increased F-actin severing activity in cell extracts from monolayers treated with forskolin/3-isobutyl-1-methylxanthine. Pretreatment of cultures with tautomycin, a protein phosphatase type 1 inhibitor, blocked the effect of cAMP on 1) the dephosphorylation of cofilin, 2) the decrease in RLC phosphorylation, and 3) the decrease in isometric tension. Together, these data provide in vivo evidence that elevated intracellular cAMP regulates endothelial cell isometric tension and RLC phosphorylation through inhibition of RhoA signaling and its downstream pathways that regulate myosin II activity and actin reorganization.

Modulation of actomyosin motor function by 1-hexanol.

This study examines the effects of 1-hexanol as a perturbing agent on actomyosin ATPase and its related functions in the concentration range between 0 and 20 mM. In this range the denaturation of myosin subfragment 1 (S1), as measured by the inactivation rate of its K-EDTA-ATPase, and depolymerization of F-actin were insignificant. Major findings showed that hexanol had the following effects which were fully reversible, (a) a marked activation of S1 MgATPase (approximately 10-fold at 20 mM) without greatly affecting the enhancement of tryptophan fluorescence by formation of S1.ADP.Pi intermediate and the rate of ADP release from S1.ADP; (b) an inhibition of the maximum actin-activated ATPase activity; (c) an increase in the affinity of S1 for actin in the presence of ATP and a decrease in the presence of ADP or the absence of nucleotide; (d) a reduction in the sliding velocity of actin filaments in in vitro motility assays with myosin, and (e) a decrease in isometric tension of single skinned muscle fibers. Thus, the effects of hexanol on actomyosin interaction are distinct for the weak and strong binding states, consistent with a change in the hydrophobic interaction in the interface between myosin and actin accompanying the transition from the weak to the strong binding state. Hexanol also accelerates the Pi release from S1.ADP.Pi, which is the transition step from the weak to the strong binding state. The fact that hexanol accelerates Pi release suggests that this alcohol perturbs the S1.ADP.Pi conformation. We speculate that this intermediate-specific structural perturbation is related to the inhibition of the maximum actin-activated ATPase, in vitro motility, and isometric tension.

Nitric oxide and endothelin in oxygen-dependent regulation of vascular tone of human umbilical vein.

We investigated the possible contribution of nitric oxide (NO) and endothelin (ET) to oxygen-dependent regulation of human umbilical vein vascular tone by simultaneous registration of intracellular membrane potential and isometric tension of vessel strips with and without NO synthase inhibition [10-4 M N omega-nitro-L-arginine methyl ester (L-NAME)], ETA receptor blockade (10(-5) M BQ-123), or ETB receptor blockade (10(-7) M BQ-788) at Po2 values in the bath solution between 5 and 104 mmHg. Increasing PO2 above the physiological intrauterine range resulted in depolarization and an increase of isometric tension, whereas lowering PO2 resulted in hyperpolarization and a decrease in isometric tension. Removal of the endothelium reversed these effects. At PO2 values below 39 mmHg, intact preparations treated with either L-NAME, BQ-788, or BQ-123 were more depolarized than controls. In the case of treatment with L-NAME or BQ-123, this was accompanied by an increase in isometric tension. We conclude that it is NO that mediates the hypoxic hyperpolarization and vasodilatation of the human umbilical vein and that ET, via activation of ETB1 receptors on endothelial cells, contributes to this effect.

The mechanism by which aminoglycoside antibiotics cause vasodilation of canine cerebral arteries.

The effects of aminoglycoside antibiotics were examined in canine cerebral arteries and in cultured cerebrovascular smooth muscle cells stimulated with oxyhemoglobin (OxyHb), a blood constituent which has been implicated in the pathogenesis of cerebrovascular spasm. In cerebral arterial rings precontracted with OxyHb (10 microM), the aminoglycosides caused a concentration-dependent decrease in isometric tension. The EC50s for the relaxation were 0.46+/-0.1 mM (n=6), 0.53+/-0.08 mM (n=12), 1.6+/-0.3 mM (n=7) and 3.9+/-0.5 mM (n=5) for neomycin, gentamicin, streptomycin and kanamycin, respectively. This order of potency corresponds approximately to the number of positive charges in the molecules. The aminoglycosides also inhibited the contractions to prostaglandin F2alpha (1 microM) and depolarizing concentrations of potassium chloride (60 mM). The order of potency was neomycin > gentamicin > streptomycin > kanamycin. The relaxation was maintained in vascular preparations denuded of endothelium. Neomycin (5 mM) abolished the Ca2+-independent contraction to PGF2alpha. In Fura-2-loaded cerebrovascular smooth muscle cells, OxyHb (1 microM) significantly enhanced the concentration of intracellular calcium ([Ca2+]i) by 330%. The administration of neomycin, gentamicin, kanamycin and streptomycin in concentrations corresponding to the EC50 from contractility studies, reduced the effects of OxyHb on [Ca2+]i by about 50% to 221+/-35 nM (n=7), 270+/-31 nM (n=7), 229+/-33 nM (n = 6) and 240+/-6 nM (n = 5), respectively. These results suggests that the effects of the aminoglycosides on the OxyHb-induced contraction and the long-term increase in [Ca2+]i, may arise from several effects, including inhibition of PLC, protection of calcium extrusion mechanisms, and interference with the process of [Ca2+]i accumulation.

RELAXATION OF VASCULAR SMOOTH MUSCLE INDUCED BY LOW‐POWER LASER RADIATION

The relaxation of rabbit aorta rings induced by low-power laser radiation was investigated in vitro to determine the location of the chromophore(s) responsible for this response and evaluate possible mechanisms. An action spectrum for relaxation was measured on rabbit thoracic aorta rings precontracted with norepinephrine. The decrease in isometric tension was measured during exposure to laser light (351-625 nm) delivered via a fiber optic to a small spot on the adventitial surface. The shortest UV wavelength (351 nm) was 35-fold more effective than 390 nm and 1700-fold more effective than 460 nm. Ultraviolet wavelengths also produced greater maximum relaxation (0.40-0.45) than visible wavelengths (0.20-0.25), suggesting that photovasorelaxation involves more than one chromophore. The adventitial layer was not necessary for photovasorelaxation, indicating that the light is absorbed by a chromophore in the medial layer. The same degree of relaxation was obtained on rings without adventitia when either one-half of the ring, or a small spot was irradiated indicating that communication between smooth muscle cells spreads a signal from the area illuminated to the entire ring. The mechanism for photovasorelaxation was investigated using potential inhibitors. N-monomethyl-L-arginine and N-amino-L-arginine, inhibitors of nitric oxide synthase, did not alter photovasorelaxation nor did indomethacin, an inhibitor of cyclooxygenase, and zinc protoporphyrin, an inhibitor of heme oxygenase.

Does pentobarbital anesthesia depress left ventricular contractility in dogs?

Although pentobarbital sodium (NP) anesthesia has been shown to depress left ventricular (LV) contractility in dogs, measurements of LV contractility in previous studies have been made soon after a bolus of NP was given when serum concentrations would be extremely high. In this study, we compared indexes of LV contractility during awake and anesthetized conditions. During anesthesia, measurements were obtained 1 h after an intravenous bolus of NP was given when serum concentrations were approximately 25 mg/l and above that reported to abolish pain. In 13 dogs, subendocardial ultrasonic crystal transducers and a high-fidelity pressure transducer were implanted into the LV. Measurements were obtained with and without prior treatment with propranolol to produce beta-adrenergic blockade. LV contractility was assessed by ejection fraction and the end-systolic pressure-volume relationship. The effect of NP on ventricular myocardium was also examined in an in vitro canine right trabecular preparation to compare in vivo and in vitro effects. In the in vivo study, the results showed no decrease in LV contractility during anesthesia regardless of whether propranolol was administered. The in vitro preparation showed only a minimal decrease in isometric tension at the concentrations used in the in vivo study. We conclude that NP anesthesia does not depress LV contractility when concentrations are maintained at approximately 25 mg/l.

Fatigue of mouse diaphragm muscle in isometric and isotonic contractions.

Fatiguabilities of mouse diaphragm muscle in vitro in isometric and isotonic contractions were compared in this study. Isolated mouse diaphragm muscle was stimulated repetitively to induce fatigue during both isometric and isotonic contractions. The supramaximal electrical stimulation used was a train of 100-Hz, 0.5-ms pulses delivered to the muscle every 2 s for 0.5 s. The percentage decrease in isometric tension from beginning to end of the fatiguing process was used as the index of fatigue. The experiments were carried out at different PO2 levels in both normal and zero-glucose Ringer solutions. It was found that fatigue developed more rapidly in isotonic contractions than in isometric ones. Also, the extracellular glucose level demonstrated little effect on the muscle's short-term fatiguability, whereas reductions in the extracellular PO2 exerted a profound effect, especially in the case of isotonic fatigue.

Response of isolated renal artery rings to adenosine and inosine

AbstractThe responses to adenosine and its analog, inosine, were compared in isolated rings of canine small renal arteries (0.5–1.3 mm o.d.). The rings were not contracted prior to nucleoside administration in order to detect any contraction response due to the nucleoside itself. Relaxation responses were elicited by progressively increasing the nucleoside concentration in the bathing medium. The maximal response (Rmax) was expressed as a percentage of the decrease in isometric tension. The ED50 for adenosine and inosine was 0.18 ± .002 μM and 202 ± 44 μM, respectively (P < 0.05) The Rmax for inosine was significantly less (P < 0.05) than that for adenosine, with a mean ratio of adenosine Rmax inosine Rmax of 1.47 ± 0.2. The results of these experiments indicated that adenosine and inosine directly relax isolated small renal arterial rings, although the classic in situ renal response to adenosine is increased resistance. Also, the relaxing action of adenosine, but not inosine, is significantly attenuated by 10 μM aminophylline. Further, renal arterial rings exhibit a greater sensitivity to adenosine than that previously reported for canine coronary artery rings, while the response to inosine is virtually identical.

Effect of lsoproterenol and EGTA on the volume‐pressure relationship of the in vitro whole bladder preparation

AbstractIn a previous study comparing the pharmacological response of smooth muscle strips isolated from rabbit urinary bladder with the response of the whole bladder preparation, we observed that although isoproterenol stimulated a marked decrease in isometric tension in the muscle strips, isoproterenol only slightly decreased intravesical pressure (whole bladder preparation).We have recently modified our whole bladder system so that we can now quantitatively determine the effect of drugs on bladder volume at constant pressure as well as on intravesical pressure at constant volume. Using this system we have reinvestigated the effect of isoproterenol and have demonstrated that although isoproterenol has only a slight effect on intravesical pressure, this minor effect on pressure resulted in a marked increase in whole bladder volume, and a corresponding increase in cystometric compliance.

Length dependence of calcium activated isometric force and immediate stiffness in living and glycerol extracted vascular smooth muscle.

Vascular smooth muscle series elasticity was examined in living and glycerinated preparations as a function of the tissue length or calcium elicited force. Isometrically contracted smooth muscle strips were submitted to small quick stretches and releases (rise time 1.5 ms). The resulting immediate tension changes were proportion to the length changes for length steps ranging from -0.5% L0 to +2% L0. Plotting the immediate tension changes versus the length steps resulted in force-extension diagrams of the series elasticity (T1-curves). The linear parts of the T1-curves extrapolated to a common abscissa intercept of about -1% to -2% L0 irrespective of the tissue length or the degree of calcium activated force. The slopes of the T1-curves taken as the stiffness of the series elasticity increased in proportion to the isometric tension and depended on the tissue length or the degree of calcium activation in a similar way as tension. It is concluded that tension changes due to changes in the calcium concentration or the tissue length are caused by a change in the number of attached crossbridges. Results obtained in "skinned" fibres were similar to the one obtained in living fibres indicating that electromechanical coupling was not a major factor in determining the decrease in isometric tension and stiffness at short lengths.

Lack of effect of hypercapnic acidosis on elasticity of cat papillary muscles.

Chages in external pH from 7.40 to 6.95 obtained by changing the pCO2 of the medium at constant bicarbonate concentration produced in cat papillary muscles a significant decrease in isometric tension with no changes in time to peak tension. Active and resting stiffness, as determined by two different methods, did not change under conditions of hypercapnic acidosis.

The positive inotropic action of insulin in the canine heart

The effects of glucagon-low insulin were studied on canine isolated papillary muscles incubated in Chenoweth-Kolle solution and in the pentobarbital anesthetized dog. Insulin, 12.5 mU/ml, increased developed isometric tension of papillary muscles which had equilibrated for 17 hrs, but not in muscles incubated up to 60 min. The decrease in isometric tension produced by propranolol, 10−6 M, but not that due to pentobarbital sodium, 80 μg/ml, was reversed by insulin. The positive inotropic effect of insulin was obtained when pyruvate, fumarate and glutamate replaced glucose as substrate in the Chenoweth-Kolle solution. The intracoronary administration of insulin, 0.1 U/kg, increased left ventricular isometric tension and the rate of tension development in dog hearts depressed with propranolol, 2 mg/kg. Insulin exerts a direct positive inotropic effect upon canine heart muscle depressed by propranolol or due to prolonged incubation. The effect is not dependent upon catecholamines, glucagon or glucose. The positive inotropic effect is not observed in non-depressed hearts or in barbiturate depressed hearts.