Ultrastructure, calcium accumulation, and contractile response in smooth muscle

After removing extracellular Ca2+ with [ethylene bis(oxyethylenenitrilo)]tetraacetic acid, we found that the guinea pig vas deferens (VD) was mechanically responsive to electrical stimulation for a significantly greater length of time than was guinea pig taenia coli (TC). An obvious explanation for these findings is that the VD has more intracellular calcium available for contraction than does the TC. To determine if this explanation is plausible, the volume of internal storage structures within the two muscles was compared. It was found that the volumes of potential sequestering structures in the VD and TC are not significantly different. Next, the affinities of the storage structures for calcium were compared. The VD was found to accumulate approximately twice as much 45Ca as did the TC, as determined by 45Ca autoradiography. Calcium-45 was present to a greater extent in association with surface vesicles, sarcoplasmic reticulum (SR), and mitochondria than in the unassociated state within the cytoplasmic matrix. Based on the results of these experiments, we suggest that the VD and the TC of the guinea pig differ in the affinity of their storage sites for calcium.     

Cytokines induce airway smooth muscle cell hyperresponsiveness to contractile agonists

Inflammatory cytokines have been shown to play an important role in the pathogenesis of various inflammatory processes. In throat infections, intracellular inflammatory cytokines have been detected from the sites of inflammation. The present study aimed to evaluate serum cytokine levels of patients with throat infections and correlate them to the inflammatory parameters and type of inflammation. Significantly higher inflammatory cytokine levels (interleukin [IL]-6 > 7 pg/mL, IL-1 > 1 beta pg/mL, tumor necrosis factor alpha > 1 pg/mL) were detected in most of the patients as opposed to healthy controls. Clinical parameters of infection (fever > 38 degrees C, leukocytosis > 11,000 white blood cells per cubic millimeter, polymorphonuclear neutrophils > 75%) were significantly correlated with high levels of inflammatory cytokines: mainly IL-6 and tumor necrosis factor alpha, and to a lesser degree with IL-1 beta. No correlation, however, was found between the type of inflammation and cytokine levels. The present study indicates a role of inflammatory cytokines in the pathogenesis of throat infections and the need for an anti-inflammatory and anticytokine therapeutic approach.     

Cytoplasmic redox potential affects energetics and contractile reactivity of vascular smooth muscle

Variations in the cytoplasmic redox potential (Eh) and NADH/NAD ratio as determined by the ratio of reduced to oxidized intracellular metabolite redox couples may affect mitochondrial energetics and alter the excitability and contractile reactivity of vascular smooth muscle. To test these hypotheses, the cytoplasmic redox state was experimentally manipulated by incubating porcine carotid artery strips in various substrates. The redox potentials of the metabolite couples [lactate]/[pyruvate]i and [glycerol 3-phosphate]/[dihydroxyacetone phosphate]i varied linearly (r=0.945), indicating equilibrium between the two cytoplasmic redox systems and with cytoplasmic NADH/NAD. Incubation in physiological salt solution (PSS) containing 10 mm pyruvate ([lact]/[pyr]=0.6) increased O2 consumption approximately 45% and produced anaplerosis of the tricarboxylic acid (TCA cycle), whereas incubation with 10 mm lactate-PSS ([lact]/[pyr]i=47) was without effect. A hyperpolarizing dose of external KCl (10 mM) produced a decrease in resting tone of muscles incubated in either glucose-PSS (-0.8+/-0.8 g) or pyruvate-PSS (-2.1+/-0.8 g), but increased contraction in lactate-PSS (1.5+/-0.7 g) (n=12-18, P<0.05). The rate and magnitude of contraction with 80 mm KCl (depolarizing) was decreased in lactate-PSS (P=0.001). Slopes of KCl concentration-response curves indicated pyruvate>glucose>lactate (P<0.0001); EC50 in lactate (29. 1+/-1.0 mM) was less than that in either glucose (32.1+/-0.9 mm) or pyruvate (32.2+/-1.0 mM), P<0.03. The results are consistent with an effect of the cytoplasmic redox potential to influence the excitability of the smooth muscle and to affect mitochondrial energetics.     

Airway smooth muscle, tidal stretches, and dynamically determined contractile states

In the classic theory of airway lumen narrowing in asthma, active force in airway smooth muscle is presumed to be in static mechanical equilibrium with the external load against which the muscle has shortened. This theory is useful because it identifies the static equilibrium length toward which activated airway smooth muscle would tend if given enough time. The corresponding state toward which myosin-actin interactions would tend is called the latch state. But are the concepts of a static mechanical equilibrium and the latch state applicable in the setting of tidal loading, as occurs during breathing? To address this question, we have studied isolated, maximally contracted bovine tracheal smooth muscle subjected to tidal stretches imposed at 0.33 Hz. We measured the active force (F) and stiffness (E), which reflect numbers of actin-myosin interactions, and hysteresivity (eta) which reflects the rate of turnover of those interactions. When the amplitude of imposed tidal stretch (epsilon) was very small, 0.25% of muscle optimal length, the steady-state value of F approximated the isometric force, E was large, and eta was small. When epsilon was increased beyond 1%, however, F and E promptly decreased and eta promptly increased. The muscle could be maintained in these steady, dynamically determined contractile states for as long as the tidal stretches were sustained; when epsilon subsequently decreased back to 0.25%, F, E, and eta returned slowly toward their previous values. The provocative stretch amplitude required to cause active force or muscle stiffness to fall by half, or hysteresivity to double, was slightly greater than 2%. These observations are consistent with a direct effect of stretch upon bridge dynamics in which, with increasing tidal stretch amplitude, the number of actin-myosin interactions decreases and their rate of turnover increases. We conclude that the interactions of myosin with actin are at every instant tending toward those that would prevail in the isometric steady state, but tidal changes of muscle length cause an excess in the rate of detachment. These stretch-induced detachment events can come so fast compared with the rate of attachment that static equilibrium conditions are never attained. If so, then airway lumenal narrowing and the underlying contractile state would be governed by a dynamic mechanical process rather than by a mechanical equilibrium of static forces.     

Myosin heavy chain isoforms and dynamic contractile properties: skeletal versus smooth muscle

Torsades de pointes is a potentially life-threatening form of polymorphic ventricular tachyarrhythmia typically seen in the presence of repolarization-prolonging agents. We investigated this particular form of tachyarrhythmia in the isolated, perfused rabbit heart. The experimental model was designed to reproduce conditions that are clinically known to be associated with an increased propensity to the development of torsades de pointes. The class III agent clofilium (1 microM) and d,l-sotalol (10 microM), as well as the antibiotic erythromycin (30-150 microM) were infused in the presence of either normal (5.88 mM) or low (1.5 mM) potassium concentration in sinus-driven or atrioventricular (AV)-blocked hearts. Ventricular tachyarrhythmias spontaneously emerged in the clofilium-, d,l-sotalol-, and erythromycin-treated AV-blocked hearts. The episodes showed typical features of torsades de pointes found in humans. They developed within 4-12 min after the onset of infusion, were normally nonsustained, and only rarely degenerated into ventricular fibrillation. Electrical stimulation at cycle lengths <600 ms and perfusion with MgSO4 suppressed arrhythmic activity. In the d,l-sotalol- and erythromycin-treated hearts, torsades de pointes occurred only in the presence of hypokalemia and bradycardia, whereas, in the presence of clofilium, bradycardia alone caused torsades de pointes. Monophasic action-potential recordings demonstrated early afterdepolarizations in endocardial and epicardial recordings. Thus the isolated AV-blocked rabbit heart represents a model for studying drug-related torsades de pointes and its mechanism.     

Effect of stretch on the contractile response of isolated portal vein smooth muscle

The character of spontaneous activity of the isolated preparation was shown to be affected only by the first stretching stimulus which was due to release of the intracellular Ca. Active contractile responses to stretching at the peak of the contracture due to perfusion of the preparations with the Krebs sodiumless solution, are directly correlated with the speed of the stretching. Stretching of the preparations in calciumless solution elicited no active response.     

The mechanism of stressor suppression of the contractile function and reactivity of aortic smooth muscle

Blockade of the NO synthesis did not change the stressor augmentation of the acetylcholine-induced endothelium-dependent relaxation. The immobilisation stress seems to suppress the contractile function and the reactivity of the aorta smooth muscle reactivity as the result of augmentation of the NO basal production in endotheliocytes.     

The influence of post-mortem conditions on contractile and relaxant responsiveness of guinea-pig isolated tracheal smooth muscle

Responsiveness to various contractile and relaxant agonists was assessed in tracheal preparations from guinea-pigs that had been incubated in situ at 4-37 degrees C for 0-168 h post-mortem. The potencies of histamine and acetylcholine were increased up to 168 h at 4 degrees C post-mortem and up to 24 h post-mortem at 22 degrees C. Histamine potency also increased with increasing post-mortem time at 37 degrees C. After 48 h at 22 degrees C and 8 h at 37 degrees C, responses to all spasmogens were abolished. Increases in histamine and acetylcholine potencies were similarly observed in tracheal tissue that had been removed at death and then incubated at 4 degrees C in oxygenated Krebs-bicarbonate solution for 0-168 h. The increased potency of these drugs may be explained by epithelial damage and/or loss of an epithelium-derived inhibitory factor (EpDIF). Both basal and spasmogen-stimulated increases in intracellular phosphoinositides fell with increasing time and ambient temperature post-mortem, despite the fact that contraction in response to these agonists could still be evoked. This suggests the selective failure of this signal transduction pathway and the maintenance of responsiveness via other mechanisms. The potencies and maximum effects of relaxant agonists remained unaltered in tracheal tissue with increasing time post-mortem, suggesting little change in the function of the appropriate receptor-signal transduction processes. This study has therefore demonstrated that at 4 degrees C. contractile and relaxant responses were preserved for up to 168 h post-mortem, although the modulatory influence of the epithelium on histamine and acetylcholine responses was rapidly lost.     

Glucose-induced oxidative stress in vascular contractile cells: comparison of aortic smooth muscle cells and retinal pericytes

Once onset of clinical rabies develops in an individual, death is inevitable. Thus, it is imperative that, for persons exposed or potentially exposed to rabies virus, prophylaxis must be instituted as soon as possible following the exposure. Local wound management is an essential part of postexposure rabies prophylaxis. Exposed persons should receive a recommended series of a tissue culture or cell culture origin vaccine. The number of doses and route of vaccination differ in various regions of the world and are discussed in the text. The administration of a rabies immune globulin is generally recommended in conjunction with the first dose of the rabies vaccine. Nerve tissue origin vaccines, although used extensively in some parts of the world, are not recommended if cell or tissue culture vaccines are available. Decision trees are presented in the text to aid in determining if rabies vaccine is necessary following a known or presumed exposure to the virus, along with a table outlining the various rabies vaccines available in the World. Rabies pre-exposure immunisation is recommended for those individuals at risk of exposure to the virus. Pre-exposure prophylaxis consists of 3 doses of an approved rabies vaccine administered either intramuscularly or intradermally on days 0, 7, and 21 or 28 with periodic booster doses or titre determination depending on the level of risk of potential exposure to the virus.     

Anti-implantation activity of antiestrogens and mifepristone

PURPOSE: Strong durable bonds between resin cements and metal alloys are critical to the success of resin-bonded, resin-veneered, or resin-retained prostheses. However, few comprehensive, comparative evaluations of materials or the fatigue effects of thermal cycling have been reported. The rate of strength loss may be a more important predictor of long-term success than bond strength. The purpose of this study was to investigate the effects of artificial aging by thermal cycling and resin cement type on the bond strengths to a base metal alloy. MATERIAL AND METHODS: This study investigated the effect of the number of thermal cycles (0, 1, 10, 100, 1,000, and 10,000) on the bond strengths of nine fixed prosthodontic resin cements. Specimens were assigned randomly to thermal cycle number/cement type test groups. Cylinders of a base metal alloy were bonded in an end-to-end configuration. One end of each bonded specimen was insulated, and the specimen was thermal cycled. Then, the bonds were tested in shear and bond strengths calculated. RESULTS: Two-way ANOVA revealed that the effects of cement type, the number of thermal cycles, and their interaction all significantly affected bond strength (p < .0001). Multiple range analysis showed that some cements had significant trends to lose bond strength with thermal cycling (p < .05), while others did not (p > .05). CONCLUSIONS: Both the type of resin cement and the amount of thermal cycling influenced bond strength to a base metal alloy. Some materials displayed more rapid loss of bond strength than others.     

Effects of chronic oral administration of levcromakalim on in vitro contractile responses of arterial smooth muscle

It has been shown that oral administration of 0.038-0.15 mg/kg levcromakalim elicits a dose-related antihypertensive response in spontaneously hypertensive rats (Clapham et al., Arzneim. Forsch. 41 (1991) 385). In the present study, we examined the effects of long term administration of a high dose of levcromakalim on in vitro vascular contractility. Levcromakalim (2.25 mg/kg/day) was administered to the rats for 2 weeks and the thoracic aorta was then isolated. The levcromakalim treatment markedly reduced the relaxant effect of levcromakalim itself on norepinephrine-induced contraction. Relaxant effects of sodium nitroprusside and 8-bromo-cGMP were also attenuated by the levcromakalim treatment, although the relaxant effects of verapamil and forskolin were unchanged. The levcromakalim treatment decreased the threshold concentration for KCl and norepinephrine to induce contraction. The chronic levcromakalim treatment did not affect the cGMP production due to 3-isobutyl-1-methylxanthine and/or sodium nitroprusside. The aorta isolated from spontaneous hypertensive rats did not exhibit spontaneous activity in normal solution. After treatment with levcromakalim, however, the aorta showed spontaneous rhythmic contractions. Verapamil (10 microM) completely suppressed the spontaneous activity and decreased the basal tension below the original level. Similar to the effects of chronic treatment with levcromakalim, high-K+ solution (15.4 mM) augmented the contractile response to norepinephrine in the aorta of normotensive rats and induced rhythmic contractions in the aorta of spontaneously hypertensive rats. These results suggest that chronic treatment with a high dose of levcromakalim attenuates not only the effects of levcromakalim itself but also the cGMP-mediated relaxation, possibly by desensitizing the K+ channel.     

Exteroceptive suppression of temporalis muscle activity during migraine attack and migraine interval before and after treatment with sumatriptan

Acute myocardial infarction is difficult enough to manage without the complications that commonly occur in the first days and weeks that follow. This article covers the most frequent complications, outlining when they are likely to occur, what the symptoms are, how they are best treated, and what sort of prognosis can be expected. In this way, the author provides a "map" of the days after infarction, showing the pitfalls and sand traps of clinical management.     

An hypothesis for the interpretation of the contractile response of vascular smooth muscle at the cellular level

The long preservation and recovery of functional (contractile) properties in cultured aortic smooth muscle cells, even after replating or deep-frozen storage and the measurement of their responses are now technically settled issues. We could thus study extensively the responses of single cultured cells from rat thoracic aorta. Responses were elicited by the addition of KCl 40 mmol/L without or with a calcium blocker PN 200-100 (10(-6) mol/L); angiotensin II (10(-11)-10(-6) mol/L) without or with antagonist (losartan 10(-5) mol/L); or serotonin (10(-9)-10(-4) mol/L) without or with antagonist (naftidrofuryl 10(-5) mol/L). Results thus obtained enabled us to propose a new hypothesis for the interpretation of the contractile responses of an elastic vascular smooth muscle. The different maximal effects of different agonists result mainly from the different proportions of cells they can mobilize; the agonist concentration-contraction relationship is mainly due to the increase of the proportion of cells involved up to a maximal value typical of the agonist used. An antagonist primarily reduce the proportion of cells an agonist can mobilize. Some of the consequences of this hypothesis are briefly outlined.     

Effect of preincubation on smooth muscle myosin light chain kinase activity

Phosphorylation of myosin regulatory light chain (RLC) catalysed by myosin light chain kinase (MLCK) is a key reaction in the regulation of actin-myosin interaction in smooth muscle. The activation of MLCK by calmodulin (CaM) and Ca2+ was investigated over a wide range of the enzyme concentrations using myosin or its RLC with Mw = 20 kDa as substrates. Kinase activation by CaM (at saturating Ca2+ concentrations) was characterized by positive cooperativity even though noncooperative activation would be expected from the established 1:1 binding stoichiometry between MLCK and CaM. The activation of the kinase by Ca2+ was also cooperative but only at relatively low CaM levels. This cooperativity was shown to result from time dependent changes in MLCK that take place during its incubation with Ca2+ and CaM before substrate addition in phosphorylation assays. As a result the kinase activity as a function of its concentration at constant CaM level was biphasic: there was the activity optimum at 1:1 ratio of CaM to MLCK and almost complete inhibition at 3 to 7 molar excess of kinase over CaM. Such changes that take place during 10 to 15 min preincubation with Ca2+ and CaM may involve the kinase supramolecular structure formation or/and its conformational rearrangements.     

Parasympathetic varicosity proliferation and synaptogenesis in rat eyelid smooth muscle after sympathectomy

A commonly used additive to tobacco products is cocoa. A sensitive an selective method was developed to measure caffeine, a marker for cocoa, in tobacco by using gas chromatography-mass spectrometry (GC-MS). Tobacco components usually produce high background signals in GC-MS analysis. Therefore, a series of extraction steps were designed to effectively purify the tobacco extracts. The analytical recovery of caffeine was 100 when [trimethyl-13C3] caffeine was used as an isotope-dilution reference. A linear calibration curve was generated with caffeine concentration ranging from 0.01 to 20 micrograms/ml. The detection limit of caffeine was 0.02 microgram/ml in the final solution. This method was applied to several commercial tobacco products, of which the corresponding caffeine levels varied from below the detection limit to 125 micrograms/g.     

Plasma progesterone concentrations in cows and steers after Repositol Progesterone treatment

Repositol progesterone was injected intramuscularly into 4 steers and 17 postpartum cows at a dose of 2.2 mg/kg body weight. In steers the plasma progesterone concentration rose to 6.8 ng/ml 1 day postinjection and declined to a mean concentration of 2.1 ng/ml on days 3 and 4 postinjection. Postpartum, presumed anestrus, cows reached a lesser progesterone concentration of 2.9 ng/ml 1 day postinjection and then declined to a lower mean of .7 ng/ml on days 3 and 4 postinjection. These data suggest different rates of metabolism and/or excretion of progesterone by cows and steers.     

Effects of long-term treatment with depot medroxy progesterone acetate for contraception on estrogenic activity

The aims of this study were to evaluate the serum estradiol level and the estrogenic activity in long-term DMPA users. From 1st January 1996 to 31st December 1996, fifty healthy women receiving DMPA for contraception for more than 36 months were recruited to the study. From the study, it was found that the mean duration of DMPA use was 59.1 +/- 30.7 months, the mean serum estradiol was 52.7 +/- 15.1 pg/ml and these women did not have problems of estrogen deficiency. This study revealed that long-term treatment with DMPA should not have any adverse effects on estrogenic activity.