Increase In Isometric Tension Research Articles

2297 The direct effect of trimethylamine N-oxide (TMAO) on cardiac muscle contractile mechanics

OBJECTIVES/SPECIFIC AIMS: The objective of this study was to determine if trimethylamine N-oxide (TMAO) alone could acutely alter cardiac contractile function on a beat-to-beat basis. METHODS/STUDY POPULATION: CD1 adult mouse hearts were extracted, attached to a force transducer, oxygenated, and paced within an organ bath. Changes in contractility were measured after pipetting or reverse perfusing TMAO through the aorta via a modified Langendorff apparatus to facilitate TMAO delivery into the myocardium. To determine if our findings translated to the human heart, we performed contractility experiments using human right atrial appendage biopsy tissue retrieved during cardiopulmonary bypass procedures. To investigate whether TMAO alters contractile rate, in a separate series of experiments, the atria and sinoatrial node of isolated hearts were kept intact to allow for spontaneous beating without artificial pacing and were treated with TMAO or vehicle. In addition, intracellular calcium measurements were performed on spontaneously beating embryonic rat cardiomyocytes after TMAO or vehicle treatment. RESULTS/ANTICIPATED RESULTS: We found acute exposure to TMAO, diluted into the organ bath, increased average contraction amplitude 20% and 41% at 300 µM and 3000 µM, respectively (p<0.05, n=6). Langendorff reverse perfusion of mouse hearts ex vivo with 300 µM TMAO generated an even greater response than nonperfusion peripheral exposure and increased isometric force 32% (p<0.05, n=3). Consistent with what we observed in mouse hearts, incubation of human atrial muscle tissue with TMAO at 3000 µM increased isometric tension 31% compared with vehicle (p<0.05, n=4). TMAO treatment (3000 µM) also increased average beating frequency of ex vivo mouse hearts by 27% compared with vehicle (p<0.05, n=3) and increased the spontaneous beating frequency of primary rat cardiomyocytes by 13% compared with vehicle treatment (p<0.05, n=4). DISCUSSION/SIGNIFICANCE OF IMPACT: TMAO, at pathological concentrations, directly increases the force and rate of cardiac contractility. Initially, these inotropic and chronotropic effects may be adaptive during CKD; however, chronic increases in isometric tension and beating frequency can promote cardiac remodeling and heart failure. Further translational studies are needed to understand the intricate relationship between the microbiome, kidneys, and heart and to examine if TMAO represents a therapeutic target for reducing cardiovascular mortality in CKD patients.

R21C Mutation in Cardiac Troponin I Imposes Differences in the Degree of Order and Kinetics of Myosin Cross-Bridges of Left and Right Ventricles

The effect of the TnI R21C mutation in the human cardiac troponin I, the mutation that is linked to hypertrophic cardiomyopathy, on muscles of the left (LV) and right (RV) ventricles was examined in the knock-in mice. Experiments were on a mesoscopic scale where the contribution of a single molecule makes difference to the overall signal. We examined 3-4 actin molecules in ex-vivo myofibrils prepared from LV and RV muscles. Experiments revealed that the mutation imposed significant differences in the distribution of actin angles: during contraction, actin from LV were much more tightly distributed compared to actin from RV. They also revealed that the mutation imposed significant difference on the kinetics cycle of cross-bridges (XBs) of the LV and RV: XBs from RV displayed a 3-fold decrease in the rate of power stroke and a 2-fold decrease in the rate of dissociation from thin filaments as compared to LV. We speculate that mutation operates differently in both ventricles because of the differences in external load between LV and RV. Kinetic results suggest that an increase in isometric tension observed in mutated RV results from an inability of XBs to dissociate promptly from thin filaments.

Electrophysiological properties of rat mesenteric lymphatic vessels and their regulation by stretch.

In mammals, lymph is propelled centrally primarily via the phasic contractions of collecting lymphatic vessels, known as lymphatic pumping. Electrophysiological studies conducted in guinea pig and sheep mesenteric lymphatic vessels indicate that contractions are initiated in the lymphatic muscle by nifedipine-sensitive action potentials (APs). Lymphatic pumping is highly sensitive to luminal fluid loading and the mechanical properties of this stretch-induced pumping have been consistently studied, in particular in rat mesenteric lymphatic vessels. However, membrane potential (Vm) and the electrophysiological events underlying stretch-induced lymphatic pumping have not been investigated in the rat. The aim of this study was thus to examine the properties of rat mesenteric lymphatic muscle Vm under resting conditions and to assess changes in Vm caused by distension. Lymphatic muscle Vm was measured with sharp intracellular microelectrodes either in unstretched conditions or under isometric tension provided by a wire-myograph. In unstretched vessels, Vm was -48 ± 2 mV (n=30). APs (amplitude ∼25 mV) were observed at a frequency of ∼8/min and were abolished by nifedipine. Under isometric tension, Vm was less polarized (-36 ± 1 mV, n=23), even at minimum tension. Increase in tension led to increase in contraction strength and contraction/AP frequency, while Vm was slightly hyperpolarized and AP amplitude not markedly altered. In our experimental conditions, rat lymphatic muscle has electrophysiological characteristics similar to that in other species. It responds to an increase in isometric tension with an increase in AP frequency, but resting Vm is not significantly affected.

Functional role of α 2-adrenoceptor subtypes in the cooling-enhanced vasoconstriction of isolated cutaneous digital veins of the horse

Cooling-enhanced contractile responses in cutaneous arteries have been reported to involve the activation of α 2C-adrenoceptors, but their role in cutaneous veins is not clearly understood. The aim was to pharmacologically characterize the subtype of postsynaptic α 2-adrenoceptors in the equine digital vein mediating contraction at two temperatures. The increase in isometric tension of endothelium-denuded equine digital vein in response to UK-14304 was studied in the absence and presence of relatively selective α 2-adrenoceptor antagonists at temperatures of either 30 °C (the peripheral digit temperature of horses maintained in a thermoneutral environment) or 22 °C. The response to UK-14304 was enhanced by cooling and antagonised by RX-821002 (α 2-adrenoceptor non-selective; 30 °C: apparent pK b = 8.5; 22 °C: pK b = 8.2) and yohimbine (α 2-adrenoceptor non-selective; 30 °C: apparent pK b = 7.2; 22 °C: apparent pK b = 7.4). The response at 30 °C was non-surmountably antagonised by BRL-44408 (α 2A-adrenoceptor-selective; apparent pK b = 8.9) and MK-912 (α 2C-adrenoceptor-selective: apparent pK b = 9.9). JP-1302 (α 2C-adrenoceptor-selective) surmountably antagonised the response with a low potency (apparent pK b = 5.6) at 30 °C. The response at 22 °C was surmountably antagonised by: BRL-44408 (α 2A-adrenoceptor-selective; apparent pK b = 6.5); MK-912 (α 2C-adrenoceptor-selective; pK b = 9.7) and JP-1302 (α 2C-adrenoceptor-selective; apparent pK b = 7.5). ARC-239 (α 2B-adrenoceptor-selective) did not affect the response at either temperature. The apparent potency of the different antagonists and their non-surmountable effect, suggests that the UK-14304 response at 30 °C could be mediated by α 2A-adrenoceptors and α 2C-adrenoceptors. By contrast, the α 2C-adrenoceptor appears to be the main α 2-adrenoceptor mediating the augmented response at 22 °C. It is unlikely that the α 2B-adrenoceptor subtype contributes at either temperature. These results support the participation of the α 2C-adrenoceptors in the cooling-enhanced response to UK-14304 in this cutaneous vein.

CD38 prevents morphine‐tolerance development in mouse distal colon

Although tolerance develops to the analgesic effects of morphine upon repeated administration, constipation continues to remain the major side effect. We recently reported that lack of tolerance to morphine‐induced effects in the colon contributes towards opioid‐induced bowel dysfunction. In the present study we examined the role of CD38, a membrane NAD(P)+ glycohydrolase in opiate tolerance. Increase in isometric tension of the circular muscle strips from the mouse distal colon were measured in response to morphine. Repeated administration of morphine did not induce tolerance in the colon (I: 100%, II: 103±10%, III: 114±16%). Incubation with nicotinamide, a negative feedback inhibitor of CD38 ADPribocyclase resulted in tolerance development to morphine (I: 100%, II: 85±14%, III: 38±9%; n=4). Similarly, tolerance also developed in the distal colon circular muscle of the CD38 knock out mice (I: 100%, II: 61±4%, III: 47±3%, IV: 46±9%; n=3). This study demonstrates the involvement of CD38 in the lack of tolerance development to morphine in distal colon. Inhibition of CD38 may be a potential therapeutic target to alleviate constipation effects of long‐term morphine therapy.

Rapid inhibitory effect of glucocorticoids on airway smooth muscle contractions in guinea pigs

The common disease asthma is characterized by the obstruction, inflammation and increased sensitivity of the airways. Glucocorticoids (GCs) are one of the most potent anti-inflammatory agents available for treating allergic disease. In this study, we report that the GC budesonide (BUD) can rapidly inhibit the histamine-induced contractions of airway smooth muscle in a process mediated by non-genomic mechanisms. The tracheas of albino Hartley guinea pigs were used. We measured the effects of BUD on the increased isometric tension of trachea segment rings and the shrinking of single airway smooth muscle cells (ASMCs) induced by histamine. With the application of each reagent, the changes in the isometric tension of the segment rings upon maximum contraction and at four time points were recorded. We found that BUD significantly suppressed the increase in isometric tension induced by histamine in guinea pigs within 15 min. We also observed that BUD can reduce the histamine-induced shrinking of single ASMCs in an even shorter time. Mifepristone (RU486) and actidione did not depress the inhibitory effect of BUD. The results preclude action via genomic-mediated responses that usually take several hours to occur. We conclude therefore that GCs have a rapid non-genomic inhibitory effect on guinea pig airway smooth muscle contractions, and provide a new way to investigate this non-genomic mechanism. Further study can provide theoretical evidence for the clinical application of GCs in asthma and other allergic diseases.

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.

Force enhancement by PEG during ramp stretches of skeletal muscle.

We have investigated the effects of a stronger actomyosin bond on force (Ps) during rapid stretch of active permeabilized rabbit psoas muscle fibers as a function of temperature from 5 to 30 degrees C. The strength of the actomyosin bond is enhanced by addition of polyethylene glycol (PEG), especially in pre-powerstroke states [Chinn et al. (2000) Biophys J 49: 437-451]. We have hypothesized that such states produce much of the force when activated muscles are stretched [Getz et al. (1998) Biophys J 75: 2971-2983]. Addition of PEG to activated fibers produced a small increase in isometric tension, Po (50-90 kN/m2), which was approximately independent of temperature. In contrast PEG produced a dramatic increase in Ps at low temperatures (200-300 kN/m2), but a modest increase at higher temperatures (70-90 kN/m2). We also measured Ps and Po in solutions containing the phosphate analog aluminum fluoride (AlF4) with and without PEG. In the absence of PEG, AlF4 reduced Po much more than Ps. Addition of PEG did not enhance Po, but enhanced Ps significantly. The contrasting effects of PEG on Ps and Po, and the effect of temperature can be explained by a model in which stretch force is produced by pre-powerstroke cross-bridges whose maximum distension is increased by PEG, and isometric force is produced by strongly bound cross-bridges whose bond strength is also enhanced by PEG, but to a lesser extent.

Regulation of Force Development Studied by Photolysis of Caged ADP in Rabbit Skinned Psoas Fibers

The present study examined the effects of Ca 2+ and strongly bound cross-bridges on tension development induced by changes in the concentration of MgADP. Addition of MgADP to the bath increased isometric tension over a wide range of [Ca 2+] in skinned fibers from rabbit psoas muscle. Tension-pCa (pCa is −log [Ca 2+]) relationships and stiffness measurements indicated that MgADP increased mean force per cross-bridge at maximal Ca 2+ and increased recruitment of cross-bridges at submaximal Ca 2+. Photolysis of caged ADP to cause a 0.5 mM MgADP jump initiated an increase in isometric tension under all conditions examined, even at pCa 6.4 where there was no active tension before ADP release. Tension increased monophasically with an observed rate constant, k ADP, which was similar in rate and Ca 2+ sensitivity to the rate constant of tension re-development, k tr, measured in the same fibers by a release-re-stretch protocol. The amplitude of the caged ADP tension transient had a bell-shaped dependence on Ca 2+, reaching a maximum at intermediate Ca 2+ (pCa 6). The role of strong binding cross-bridges in the ADP response was tested by treatment of fibers with a strong binding derivative of myosin subfragment 1 (NEM-S1). In the presence of NEM-S1, the rate and amplitude of the caged ADP response were no longer sensitive to variations in the level of activator Ca 2+. The results are consistent with a model in which ADP-bound cross-bridges cooperatively activate the thin filament regulatory system at submaximal Ca 2+. This cooperative interaction influences both the magnitude and kinetics of force generation in skeletal muscle.

Effect of temperature on elementary steps of the cross-bridge cycle in rabbit soleus slow-twitch muscle fibres.

1. Isometric tension, stiffness and the cross-bridge kinetics in rabbit soleus slow-twitch fibres (STFs) were studied in the temperature range 5-37 degrees C by sinusoidal analysis. 2. The effects of MgATP and phosphate (Pi) on the cross-bridge kinetics were studied, and the temperature dependence of the kinetic constants of elementary steps of the cross-bridge cycle was deduced in the range 20-37 degrees C. 3. The MgATP association constant (K1a) decreased when temperature was increased. The rate constants of the ATP-isomerization step (k1b and k-1b) and the cross-bridge detachment step (k2, and k(-2)) had Q10 values of 3-4, and hence their equilibrium constants (K1b and K2) changed little with temperature. 4. Q10 of the force generation step (k4) was the largest at 6.7; its reversal step (k(-4)) had a Q10 of 2.5, and hence its equilibrium constant (K4) increased significantly with temperature. The Pi association constant (K5) changed little with temperature. 5. The elementary steps of the cross-bridge cycle are more temperature sensitive in soleus STFs than in psoas, which are fast-twitch fibres. This is in accord with a higher temperature sensitivity of the apparent rate constants in STFs.T 6. The temperature dependence of the equilibrium constant of the force generation step (K4) was fitted to the modified Van't Hoff equation to deduce standard enthalpy change (DeltaH degrees; 70 +/- 20 kJ mol(-1)), standard entropy change (DeltaS degrees; 250 +/- 70 J mol(-1) K(-1)), and heat capacity change (DeltaCp; -12 +/- 5 kJ mol(-1) K(-1)). These results indicate that the force generation step is an entropy driven, endothermic reaction that accompanies a burial of large surface area. These observations are consistent with the hypothesis that hydrophobic interaction between residues of actin and myosin and between residues of the myosin head underlies the mechanism of force generation. 7. An increase of isometric tension with temperature is accounted for by the increased number of cross-bridges in tension generating states. Stiffness also increased with temperature, but to a lesser degree.

Histamine-Induced Increase in Isometric Tension of Smooth Muscle Is Mediated by Local Vagus Nerve in Human Bronchus

Background: The vagus nerve is reported to play an important role in the regulation of airway responsiveness. Objective: In the present study, we investigated the role of the local vagus nerve in the changes in isometric tension of smooth muscle induced by histamine in the human airway. Methods: Eight human lung tissue specimens were obtained at thoracic surgery, and 24 bronchial smooth muscle strips were used for isometric tension recording. The changes in isometric tension were induced by histamine in the presence or absence of physostigmine (10^–6 M), atropine (10^–6 M), and/or tetrodotoxin (10^–6 M). Results: We found that: (1) histamine induced a dose-dependent increase in the isometric tension in human bronchial smooth muscle; (2) physostigmine (10^–6 M) significantly potentiated the amplitude of the histamine-induced increase in isometric tension; (3) atropine (10^–6 M) significantly suppressed the histamine-induced increase in isometric tension; (4) tetrodotoxin (10^–6 M), did not affect the histamine-induced increase in isometric tension of smooth muscle, and (5) in the presence of tetrodotoxin, atropine significantly suppressed the histamine-induced increase in isometric tension. Conclusion: These findings suggest that the histamine-induced increase in isometric tension is mediated partly by acetylcholine, presumably released by the direct action of histamine on the vagus efferent nerve terminals without the central reflex via vagus nerve.

Heightened Vascular Reactivity Following Sympathetic Denervation Correlates with Increased Expression of C-Kit and Connexin43

P71 Short-term denervation produces supersensitivity to α-adrenergic agonists that resembles the heightened vascular reactivity observed in hypertensive subjects. Gap junctions, transmembrane channels responsible for cell-to-cell communication, may play a role in maintaining increased vascular reactivity. We examined the effects of denervation with intra-peritoneal reserpine 3 mg/kg/day (N=5) or topical 5% phenol-glycerol (N=7) on agonist-induced oscillatory activity. Wistar-Kyoto (WKY) rat tail arteries were exposed to step-wise addition of norepinephrine (NE) 10 -9 M-10 -5 M. The concentration for half-maximal response for NE-induced contraction was lower in reserpine- and phenol-treated vessels than controls (log EC 50 [mol]/L control -6.89 vs reserpine -7.16, p + -free solution produced an 8-fold increase in isometric tension in control vs denervated vessels (138.4±23.2 vs 17.2±4.5 % of contraction to phenylephrine 10 -6 M, p

Heightened Vascular Reactivity Following Sympathetic Denervation Correlates with Increased Expression of C-Kit and Connexin43

P71 Short-term denervation produces supersensitivity to α-adrenergic agonists that resembles the heightened vascular reactivity observed in hypertensive subjects. Gap junctions, transmembrane channels responsible for cell-to-cell communication, may play a role in maintaining increased vascular reactivity. We examined the effects of denervation with intra-peritoneal reserpine 3 mg/kg/day (N=5) or topical 5% phenol-glycerol (N=7) on agonist-induced oscillatory activity. Wistar-Kyoto (WKY) rat tail arteries were exposed to step-wise addition of norepinephrine (NE) 10 -9 M-10 -5 M. The concentration for half-maximal response for NE-induced contraction was lower in reserpine- and phenol-treated vessels than controls (log EC 50 [mol]/L control -6.89 vs reserpine -7.16, p<0.05); control -6.86 vs phenol -7.32, p<0.0001). Incubation in K + -free solution produced an 8-fold increase in isometric tension in control vs denervated vessels (138.4±23.2 vs 17.2±4.5 % of contraction to phenylephrine 10 -6 M, p<0.001). These findings are consistent with adrenergic denervation. Agonist-induced oscillatory activity was observed in 5/5 reserpine- and 4/7 phenol-treated vessels vs 0/12 controls (Fischer’s exact test p<0.05). The highly selective gap junction inhibitor, Gap27 (10 mM), nearly abolished NE-induced vascular smooth muscle oscillations. Reverse transcriptase polymerase chain reaction was carried out for connexin43 (Cx43) and c-Kit, a marker for non-neural interstitial cells that serve as pacemaker cells, using specific oligonucleotide primers. Following denervation, mRNA levels of Cx43 remained unchanged while levels of c-Kit increased markedly (0.16±0.03 vs 0.62±0.13 a.u., p<0.05). Western blot analysis revealed near doubling of Cx43 protein after denervation (80.0±13.7 vs 41.4±5.7 a.u., p<0.05). In conclusion, sympathetic denervation produced increased agonist-induced oscillatory contractile activity in vascular smooth muscle. The heightened vascular reactivity may result from upregulation of pacemaker cells and increased Cx43 expression.

The Role of Antioxidant Enzymes in the Control of Opossum Gallbladder Motility

Background. Superoxide rapidly oxidizes nitric oxide (NO) to form peroxynitrite, thus terminating the biological activity of NO. The aims of our study were to determine if superoxide alters the motor function of the gallbladder and to localize the antioxidant enzymes in the gallbladder.Materials and methods. Immunostaining and immunoblots were performed and enzyme activities were measured in the gallbladder. In physiologic experiments, force-displacement transducers recorded tension in gallbladder muscle strips. Superoxide was generated by the addition of xanthine with xanthine oxidase, while superoxide radicals were scavenged by the addition of superoxide dismutase (SOD) and catalase. SOD was inhibited by deithyldithiocarbamate.Results. Immunostaining demonstrated superoxide dismutase and catalase immunoreactivity in ganglia situated throughout the smooth muscle. Total superoxide dismutase activity was 115 ± 12 U/mg. Western blots detected expression of proteins of 19.4 kDa for copper/zinc SOD and 25.0 kDa for manganese SOD. Generation of superoxide increased isometric tension, while pretreatment with SOD prevented the increase in isometric tension induced by superoxide. Inhibition of SOD diminished the EFS-induced off response.Conclusions. We conclude that superoxide alters gallbladder motor function, and the presence of superoxide scavenging enzymes in enteric plexuses suggests that they may regulate gallbladder neuromuscular function by clearing endogenous superoxide.

Abnormalities in the contractile properties of colonic smooth muscle in idiopathic slow transit constipation

The underlying pathophysiology of idiopathic slow transit constipation (ISTC) remains unclear. At present, there is little evidence to implicate a smooth muscle myopathy in the aetiology of this condition. This study compared the effect of cisapride on the cholinergic response of colonic muscle strips from patients with this condition with that of control tissue. Isometric tension production was recorded from circular smooth muscle strips taken from five patients undergoing colectomy for ISTC in response to cumulative concentrations of carbachol (100 nmol/1-100 mumol/l) alone and in the presence of cisapride 400 nmol/l. Similar dose-response activity was obtained for a control group consisting of six patients undergoing resection for colorectal carcinoma. In the absence of cisapride, smooth muscle from patients with carcinoma exhibited a significantly lower sensitivity to cholinergic stimulation (agonist concentration required to produce half-maximal activation (EC50) 4.83 mumol/l) than that from patients with ISTC (EC50 1.63 mumol/l, P = 0.036), and also a greater maximal frequency of the oscillatory activity associated with the increase in isometric tension (0.070 versus 0.049 Hz, P = 0.035). Cisapride had no effect on the sensitivity to carbachol of the carcinoma tissue but brought about a significant reduction in the sensitivity of smooth muscle from patients with ISTC (EC50 3.24 mumol/l, P = 0.043). These findings indicate that colonic smooth muscle from patients with ISTC is hypersensitive to cholinergic stimulation and suggest the existence of a smooth muscle myopathy in this condition.

Smooth muscle energetic in the pig coronary artery during a calcium-dependent and a calcium-independent isometric contraction

Heat production by resting smooth muscle, was measured with a heat-flux micro calorimeter on cut-open segments of pig coronary artery superfused at 30 degrees C, was 0.93+/-0.06 (n=16) mW/g wet weight. Time courses of the increases in isometric tension and heat production with respect to basal during sustained supra maximal acetylcholine stimulation were qualitatively similar: initial peak tapering down to a supra basal plateau. Mean tension-associated heat production over 1 h was 0.16 J/g. During sustained exposure to phorbol 12,13-dibutyrate, supra basal tension and--with a 5-10 min initial delay--supra basal heat progressively increased to a plateau in about 40 min. Mean tension-associated heat production over 1 h was only 0.02 J/g with normal extracellular and intracellular mobilizable Ca2+ pools, and it was further reduced to 0.01 J/g with depleted Ca2+ pools. These results show that the maintenance--if not necessarily the building up--of tension under phorbol 12,13-dibutyrate does not entail any large dissipation of energy and is not dependent on the presence of normal Ca2+ pools.

Chloride-induced Ca2+ release from the sarcoplasmic reticulum of chemically skinned rabbit psoas fibers and isolated vesicles of terminal cisternae.

There is increasing evidence that Ca2+ release from sarcoplasmic reticulum (SR) of mammalian skeletal muscle is regulated or modified by several factors including ionic composition of the myoplasm. We have studied the effect of Cl- on the release of Ca2+ from the SR of rabbit skeletal muscle in both skinned psoas fibers and in isolated terminal cisternae vesicles. Ca2+ release from the SR in skinned fibers was inferred from increases in isometric tension and the amount of release was assessed by integrating the area under each tension transient. Ca2+ release from isolated SR was measured by rapid filtration of vesicles passively loaded with 45Ca2+. Ca2+ release from SR was stimulated in both preparations by exposure to a solution containing 191 mm choline-Cl, following pre-equilibration in Ca2+-loading solution that had propionate as the major anion. Controls using saponin (50 microg/ml), indicated that the release of Ca2+ was due to direct action of Cl- on the SR rather than via depolarization of T-tubules. Procaine (10 mM) totally blocked Cl-- and caffeine-elicited tension transients recorded using loading and release solutions having ([Na+] + [K+]) x [Cl-] product of 6487.69 mm2 and 12361.52 mm2, respectively, and blocked 60% of Ca2+ release in isolated SR vesicles. Surprisingly, procaine had only a minor effect on tension transients elicited by Cl- and caffeine together. The data from both preparations suggests that Cl- induces a relatively small amount of Ca2+ release from the SR by activating receptors other than RYR-1. In addition, Cl- may increase the Ca2+ sensitivity of RYR-1, which would then allow the small initial release of Ca2+ to facilitate further release of Ca2+ from the SR by Ca2+-induced Ca2+ release.

Mildly oxidized LDL evokes a sustained Ca(2+)-dependent retraction of vascular smooth muscle cells.

Oxidized low density lipoprotein (LDL) is thought to play a major role in atherogenesis. Atherosclerotic arteries exhibit structural changes associated with profound alterations in vascular tone that are potentially involved in arterial spasm and ischemic heart disease. We report here the role of oxidized LDL in the retraction of vascular smooth muscle cells. Mildly oxidized LDL elicited a broad and sustained peak in cytosolic calcium concentration ([Ca2+]i) in cultured arterial smooth muscle cells. Concomitant with the [Ca2+]i rise, oxidized LDL evoked a sustained and intense retraction of smooth muscle cells, as shown by the changes in cross-sectional area of single cells. Cell retraction was dependent on time, the concentration of oxidized LDL, and the level of LDL oxidation (native LDL induced neither a significant [Ca2+]i rise nor cell retraction). Oxidized LDL but not native LDL also elicited a delayed (12 +/- 2 hours) and sustained (14 +/- 2 hours) increase in isometric tension in deendothelialized arterial rings only, thus suggesting a protective role of intact endothelium. When triggered by nontoxic doses of oxidized LDL, retraction of cultured cells and the contractile response of aortic rings was reversible, whereas with higher (toxic) doses (> or = 200 micrograms apoB/mL), cell retraction was irreversible and led progressively to detachment and cell death. Cell retraction can be prevented in three ways: (1) by inhibiting LDL oxidation with supplements of antioxidants (indirect inhibition); (2) by blocking the pathogenic intracellular signaling elicited by oxidized LDL (direct inhibition), eg, by inhibiting calcium influx with EGTA or the calcium channel blocker nisoldipine or by blocking intracellular signaling (at a still-unknown step) by the lipophilic antioxidant alpha-tocopherol; and (3) by directly inhibiting myosin light chain kinase by 1-(5-chloronaphthalene-1-sulfonyl)-1H-hexahydro-1, 4-diazepine. In conclusion, oxidized LDL evoked a sustained and intense calcium-dependent retraction of cultured smooth muscle cell, which can be prevented by inhibiting LDL oxidation or by blocking the intracellular signaling induced by oxidized LDL.

Inotropic agent EMD-53998 weakens nucleotide and phosphate binding to cross bridges in porcine myocardium.

The effect of EMD-53998 (EMD) on elementary steps of the cross-bridge cycle in skinned porcine myocardium was studied to understand the positive inotropic mechanisms of EMD. The kinetic constants of the elementary steps were obtained by sinusoidal analysis and compared in the presence and absence of 50 microM EMD. In the presence of 50 microM EMD, the equilibrium constant of the cross-bridge detachment step decreases three times, and the equilibrium constant of the cross-bridge attachment (force generation) step increases two times. Our results further show that, in the presence of 50 microM EMD, the association constants of MgATP and MgADP decrease to one-ninth and one-third, respectively, and the association constant of phosphate decreases to one-third. These results indicate that EMD suppresses the nucleotide binding to cross bridges and increases cross-bridge resistance to phosphate accumulation in myocardium. These results predict that EMD decreases the number of detached cross bridges and increases the number of attached cross bridges. This prediction is consistent with the twofold increase of isometric tension with 50 microM EMD.

Distension reduces the vasoreactivity of the internal mammary artery.

Although the vasoreactivity of internal mammary artery grafts is beneficial in the long term, after mobilisation, vasospasm reduces flow through the vessel. This may be overcome and flow improved by distension. To assess the effects of distension on the vasoreactivity of the artery we have measured changes in isometric tension in rings of distended and undistended human internal mammary artery. For an equivalent wall tension, the diameter of the distended artery was 1922 microns (CL 1858 to 1986) and undistended 1684 microns (CL 1628 to 1739), (P < 0.001). In response to 25 mM potassium the increase in isometric tension, as a percent of resting tension, was 35% (CL 31 to 39) in undistended and 5% (CL 3 to 6) in distended segments (P < 0.001), with 10 microM noradrenaline increases were 57% (CL 47 to 66) and 3% (CL 2 to 5), respectively (P < 0.001). The reduction in developed tension in segments contracted with potassium and relaxed with 1 microM acetylcholine was 65% (CL 47 to 83) in undistended, and 15% (CL -1 to 32) in distended (P < 0.01). With 10 microM glyceryl trinitrate relaxation was 96% (CL 87 to 105) and 17% (CL -9 to 43), respectively (P < 0.01). Similarly, after contraction by noradrenaline, with acetylcholine, undistended segments relaxed by 50% (CL 44 to 55) and distended by 14% (CL 8 to 21) (P < 0.01), with glyceryl trinitrate relaxation was 90% (CL 80 to 99) and 7% (CL -4 to 19), respectively (P < 0.001). Distension produces a profound reduction in vasoreactivity of the internal mammary artery which is not due to endothelial damage alone.