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Developmental changes in response to endothelins and receptor subtypes of isolated rat duodenum.

The response of isolated duodenum to endothelin-1, -3 and IRL 1620 (Suc-[Glu9,Ala11,15]endothelin-1 (8-21)), a selective endothelin ETB receptor agonist, was studied in both neonatal (1-week-old) and adult rats by recording mechanical activity isotonically. Endothelin-1, -3 and IRL 1620 (1-100 nM) elicited sustained contraction of neonatal duodenum, in a concentration-dependent manner, with a potency order of endothelin-1 = endothelin-3 > IRL 1620. The response to endothelin-1 and -3 (10-1000 nM) of adult duodenum was biphasic, i.e., transient relaxation followed by contraction, with a potency order of endothelin-1 > endothelin-3. The contractile response to endothelin-1 of adult but not neonatal duodenum was significantly antagonized by pretreatment with FR139317 (1 microM), an endothelin ETA receptor antagonist. An endothelin ETB receptor antagonist, RES-701-1 (3 microM), weakly antagonized the IRL 1620-induced contraction of neonatal duodenum. However, RES-701-1 (10 microM) did not affect the response to endothelin-1 of either adult or neonatal duodenum. These results indicate that the duodenal response to endothelins changes from a sustained contraction in neonates to a biphasic response in adults. The contractile response to endothelins of neonatal duodenum is suggested to be mediated through endothelin ETB receptors or possibly RES-701-1-resistant ETB receptor subtypes and contraction of adult duodenum through endothelin ETA receptors. The mechanism of the endothelin-induced response of duodenum was also studied.

Aging↗

Osmotic injury of PC-3 cells by hypertonic NaCl solutions at temperatures above 0 degrees C.

Cell injury due to osmotic dehydration, which is regarded as a major cause of injury during freeze-thaw processes, was examined closely using a perfusion microscope. Human prostatic adenocarcinoma cells (PC-3), which were put in a chamber, were subjected to hyperosmotic stresses by perfusing NaCl solutions of varying concentrations into the chamber. Cells were exposed to 2.5 and 4.5M NaCl solutions for 1-60 min by changing the concentrations at 0.2, 1, and 10 M/min. Decrease in cell viability was biphasic: the viability decreased first after the increase in NaCl concentration due to dehydration and then after return to isotonic conditions due to rehydration. Rehydration was substantially more responsible for cell injury than dehydration, which was marked at lower NaCl concentrations and lower temperatures. Injury resulting from contraction was negligible at the 2.5 M NaCl solution. While the hypertonic cell survival, which was determined without a return to isotonic conditions, was almost independent of time of exposure to hyperosmotic concentrations, the post-hypertonic survival after returning to isotonic conditions decreased with increasing exposure time, suggesting that the rehydration-induced injury was a consequence of time-dependent alteration of the plasma membrane. The post-hypertonic survival was lower for higher NaCl concentrations and higher temperatures, which was qualitatively consistent with previous studies. Effects of the rate of concentration change on the post-hypertonic cell survival were observed at 4.5 M; the highest rate of survival was obtained by slower increase and faster decrease in the NaCl concentration. However, the effect was negligible at 2.5 M.

Cell Line, Tumor↗

Characterization of PEG-mediated electrofusion of human erythrocytes.

Polyethylene glycol (PEG) and electrofusion were applied together in a simple and highly efficient cell fusion method. PEG (8000 M(r)) was used to bring human erythrocytes into contact, and a single 4.4 kV/cm, 80 microseconds duration pulse was applied to cell suspensions. The fusion yield (FY) is PEG concentration-dependent. A maximum FY (50%) was found at about 10% PEG. Higher PEG concentrations (> 10%) suppressed FY caused by colloid osmotic shrinkage. Morphological changes, such as colloidal osmotic swelling and shrinking, and the expanding and contraction of fusion lumen, when suspension media were changed from PBS to isotonic 15% dextran solutions, was examined by microscopy. FY was found to depend on both simple osmotic and colloidal-osmotic swelling. From the swelling behavior, we propose two types of electropores: the pre-fusion sites between cell pairs, and electropores on each individual cell connecting intracellular and extracellular space. The latter type is responsible for the colloidal osmotic swelling and shrinking of cell which, together with simple osmotic swelling, is responsible for expanding the pre-fusion sites into fusion lumens. Resealing of electropores resulted in reducing FY, but the FY can be restored by simple osmotic shock. Apparently, PEG plays two opposite roles in this fusion method; one is to promote pre-pulse and post-pulse cell-cell contact, protecting pre-fusion sites, and the other suppresses FY by colloid osmotic shrinkage of cells after pulsing, especially when high PEG concentration is used. 10% PEG 8000 represents the optimal combination of these properties.

Biophysical Phenomena↗

A possible effect of sulfhydryl reagents on the contractile activity of the rat detrusor muscle.

We aimed to investigate the effect of sulfhydryl (SH) inactivating agents, ethacrynic acid and N-ethylmaleimide, on the contractile activity of rat detrusor muscle. Wistar Kyoto rats weighing 150-250 g were anaesthetized with ketamine and bled to death. The urinary bladders were surgically removed and detrusor strips were mounted under 0.5 g tension in organ baths. The responses were recorded with isotonic transducers on polygraph paper. After an equilibrium period, the tissues were contracted by electrical field stimulation, acetylcholine, ethacrynic acid or N-ethylmaleimide and the effects of L-cysteine, glutathione, verapamil, Ca(2+)-free solution, sodium nitroprusside or atropine were then examined on these contractions. Verapamil, Ca(2+)-free solution or atropine significantly reduced the contractions elicited by electrical field stimulation and acetylcholine whereas L-cysteine, glutathione or sodium nitroprusside had no effect on the contractions in response to these stimuli. L-Cysteine, glutathione, verapamil or Ca(2+)-free solution significantly inhibited the contractions induced by ethacrynic acid or N-ethylmaleimide. Sodium nitroprusside slightly inhibited only the contraction induced by ethacrynic acid but not that with N-ethylmaleimide. Atropine has no action on the contractions in response to these SH reagents. These findings suggest that SH reagents may play a role in the contractile activity of rat detrusor muscle and this action seems to be related to the gating of Ca(2+) channels. Further experiments are needed to determine the cellular mechanism(s) of action by which these SH reagents act on the detrusor smooth muscle.

Acetylcholine↗

Oxygen modulation of superoxide radical injury in the krebs-perfused, isolated rabbit heart.

Myocardial malondialdehyde concentration (MDA) as an index of membrane lipoperoxidation was measured in previously ischemic isolated rabbit hearts reperfused with Krebs solution equilibrated with different oxygen concentrations. Hearts were subjected to a ischemic period of 30 min at 4 degrees C and then reperfused at 37 degrees C with a Krebs-Henseleit solution equilibrated with 95% oxygen (group 1), 65% oxygen (group 2), or 21% oxygen (group 3). MDA concentration in nanomoles per gram protein (mean +/- SEM) at the end of reperfusion in group 1 (n = 5) was 357 +/- 18; in group 2 (n = 5) 282 +/- 18; and in group 3 (n = 5) 246 +/- 16 (p =.0008, group 1 vs. group 3; p =.0109, group 1 vs. group 2). The results support that oxidative stress after ischemia and reperfusion is modulated by the oxygen concentration of the reperfusate in the crystalloid-perfused isolated rabbit heart such that higher oxygen concentrations are associated with greater oxidative stress.

Animals↗

Effects of rapid cooling on the mechanical and electrical activities of smooth muscles of guinea pig stomach and taenia coli.

The effects of rapid cooling on the mechanical and electrical activities of the guinea pig taenia coli and circular muscle of the stomach were investigated. Lowering the temperature from 32 degrees to 10 degrees C (cold shock) depolarized the membrane and increased the membrane resistance in both tissues. However, in the taenia coli, an initial reduction of membrane resistance was observed. In both tissues, contracture evoked by cold shock and rapid relaxation after rewarming, preceded the changes of membrane properties. Displacements of the membrane potential did not modify the amplitude of contracture under cold shock. Caffeine and thymol modified the membrane properties, but the effects of cold shock were still observed. The effects of cold shock were also observed on K-induced contracture. It was postulated that at least two different sites of sequestered bound Ca are located in these smooth muscles and are responsible for evoking the mechanical response. One component possesses a close relation to membrane and the other component is presumably sequestered within the muscle.

Animals↗

Control of isometric finger force in patients with cerebellar disease.

Control of isometric forces during grasping or handling of objects is an essential feature of all skilled manual performances. Previous studies of hand function in cerebellar patients were restricted to movements; force control was only investigated under isotonic conditions in combination with movements. Control of isometric forces during voluntary contraction of finger muscles was investigated in 31 patients with chronic cerebellar disease and in 20 normal controls. Eight patients with Friedreich's ataxia were considered to be typical for a disease affecting the afferents to the cerebellum; 4 patients with anterior lobe atrophy, which affects leg movements to a greater extent than finger movements, were compared with 3 patients with hemisphere lesions; 16 patients suffered from diffuse cerebellar atrophy. Using a recently developed microcomputer system for the continuous measurement of finger force, control of isometric force was studied in several tasks within the same subjects. The tasks included the maintenance of constant force output at different force levels with and without visual feedback, fast repetitive force changes, tracking of a sinusoidal target presented visually, and measurement of maximum grip force. The amount of voluntary contraction in terms of muscular power was normal in all patients; in all other tasks clear deficits were observed. The extent of the disturbances was highly variable between patients even within the same clinical subgroup. The detailed analysis of force traces revealed a great variety in types of disturbance. The frequently observed slowing of the speed in repetitive force changes can be attributed to different components in different patients and not only to difficulties at turning points as might be predicted from comparable movement studies. In addition, performance deficits across different tasks were not uniform, indicating dissociation of impairment. Deficits in maintaining force, which may be interpreted as dysmetria or due to tremor, do not necessarily imply an abnormality in diadochokinesis, as measured in fast repetitive force changes, nor does the converse apply.

Adolescent↗

Should normothermia be restored and maintained during resuscitation after trauma and hemorrhage?

BACKGROUND: Although hypothermia often occurs after trauma and has protective effects during ischemia and organ preservation, it remains unknown whether maintenance of hypothermia or restoring the body temperature to normothermia during resuscitation has any deleterious or beneficial effects on heart performance and organ blood flow after trauma-hemorrhage. METHODS: Male rats underwent laparotomy (i.e., induced trauma) and were exsanguinated to and maintained at a mean arterial pressure of 40 mm Hg until 40% of the maximum shed volume was returned in the form of Ringer's lactate. Body temperature decreased from approximately 36.5 degrees C to below 32 degrees C. The animals were then resuscitated with four times the volume of maximal bleedout with Ringer's lactate. In one group, body temperature was rewarmed to 37 degrees C during resuscitation. In another group, body temperature was maintained at hypothermia (32 degrees C) for 4 hours after resuscitation. In an additional group, the body temperature was kept at 37 degrees C during hemorrhage as well as during resuscitation. Left ventricle performance parameters such as maximal rate of left ventricular pressure increase and decrease (+/-dP/dt(max)) were measured up to 4 hours. Cardiac output and regional blood flow were determined by radioactive microspheres at 4 hours after the completion of resuscitation. RESULTS: The maintenance of normothermia during hemor. rhage or prolonged hypothermia after resuscitation depressed the left ventricular performance parameters, cardiac output, and regional blood flow in various organs. Rewarming the body to normothermia during resuscitation, however, significantly increased heart performance, cardiac output (from hypothermia 16.2 +/- 1.4 to 22.3 +/- 1.4 mL/min per 100 g body weight,p < 0.05) and total hepatic blood flow (from hypothermia 117.5 +/- 5.3 to 166.0 +/- 9.3 mL/min per 100 g tissue, p < .05). CONCLUSION: Our data indicate that restoration of normothermia during resuscitation improves cardiac function and hepatic blood flow compared with hypothermia.

Animals↗

Effects of local anaesthetics on the relationship between charge movements and contractile thresholds in frog skeletal muscle.

1. Charge movements to 10 mV steps at different potentials were studied in voltage-clamped frog skeletal muscle fibres in isotonic solutions that minimized ionic currents, under different pharmacological conditions. The earliest onset of detectable mechanical movement ('threshold') was assessed visually under magnification. 2. Charge movements in isotonic solutions were similar to those reported in hypertonic solutions, under identical pulse procedures. 3. In the absence of local anaesthetics, threshold occurred at a mean membrane potential of -55 mV, after the movement of 4.0 nC/muF of non-linear charge and when the membrane capacitance approximated values that corresponded to the onset of the 'Qgamma' charge movement component. 4. Lidocaine shifted the threshold in the hyperpolarizing direction to -62 mV, and reduced the amount of non-linear charge needed to reach threshold to 1.4 nC/muF. 5. The presence of tetracaine shifted threshold in the depolarizing direction to -39 mV, and more than doubled the amount of non-linear charge that had to move to reach threshold to 10.8 nC/muF. Much of this increase was attributed to the Qgamma component of charge movement. 6. It is concluded that more non-linear charge is required to initiate mechanical movement when calcium release by sarcoplasmic reticulum is inhibited by tetracaine, whereas less charge is so required when calcium re-uptake is being inhibited by lidocaine. Assuming earlier interpretations of the strength duration curve, this is consistent with charge movement preceding, rather than being a consequence of calcium release.

Anesthetics, Local↗

Ventilatory responses to muscular vibrations in healthy humans.

In healthy humans, we studied the effect of high-frequency mechanical vibrations applied unilaterally to the tendon of the biceps or triceps brachialis on ventilation and the breathing pattern. This stimulus preferentially activates the muscle spindle afferents. Increase of respiratory frequency and changes in the ventilatory timing started at the first or second inspiration during tendon stimulation, and no adaptation occurred as long as the vibrations continued. The tidal volume and mean inspiratory flow rate were only enhanced in individuals having high-frequency breathing during eupnea. The changes in ventilatory variables were observed when the motor response to vibrations was tested under isometric or isotonic conditions. Various experimental procedures enabled us to induce a tonic reflex contraction in either the vibrated muscle or the antagonist of no reflex contraction in either group of muscles. In all cases the increase in minute ventilation was identical. These changes in breathing pattern was not associated with a significant decrease in alveolar CO2 pressure and did not seem to be responsible for important variations in respiratory gas exchanges. The response to high-frequency vibrations was also studied after ventilation was increased with added dead space. The magnitude of hyperventilation an the pattern of ventilatory response produced by tendon stimulation did not change with increased ventilation. In conclusion, the stimulation of muscle spindles in human induces changes in ventilation and pattern of breathing , and the occurrence of a reflex muscular contraction does not seem necessary in order to obtain such effects.

Adult↗

Noninvasive measures of oxidative metabolism on working human muscles by near-infrared spectroscopy.

The purpose of this study was to determine whether the initial rate of hemoglobin and myoglobin deoxygenation during immediate postexercise ischemia, a reflection of muscle O2 consumption (VO2mus), can be a quantitative measure of muscle oxidative metabolism. The finger flexor muscles of five healthy men (aged 25-31 yr) were monitored by 31P-magnetic resonance spectroscopy for changes in phosphocreatine (PCr), Pi, and pH. Tests were conducted during 15 min of cuff ischemia and during 5 min of submaximal isotonic grip exercise at 10, 20, 30, and 40% of maximal voluntary contraction, one contraction every 4 s. The VO2mus changes were also monitored by near-infrared spectroscopy with continuous wave. The VO2mus during exercise was expressed relative to the resting value. The resting metabolic rate, calculated from the PCr breakdown rate after complete O2 depletion, was 0.0010 (SD) mM ATP/s. During submaximal exercise (pH > 6.9), the VO2mus increased with a rise in intensity (0.036 +/- 0.011, 0.054 +/- 0.016, 0.062 +/- 0.012, and 0.067 +/- 0.020 mM ATP/s during 10, 20, 30, and 40% maximal voluntary contraction, respectively) and showed significant correlation with changes in both calculated ADP and PCr values (r2 = 0.98 and r2 = 0.99, respectively). In conclusion, because of the significant correlation with regulatory metabolites (ADP and PCr) of oxidative phosphorylation, O2 decline rate in immediate postexercise ischemia determined by near-infrared spectroscopy with continuous wave can be utilized for the quantitative evaluation of localized muscle oxidative metabolism.

Adult↗

Limitation of myocardial reperfusion injury by intravenous perfluorochemicals. Role of neutrophil activation.

Neutrophil activation and infiltration into the ischemic myocardium after reperfusion may limit the amount of salvageable myocardium (reperfusion injury). The effects of intravenous perfluorochemicals (Fluosol-DA) on infarct size, ventricular contractility, and neutrophil function were assessed in an occlusion-reperfusion canine model. Closed-chest dogs were subjected to 90 minutes of left anterior descending artery occlusion followed by 24 hours of reperfusion. Animals were randomized to receive either Fluosol-DA (FDA, n = 8) or Ringer's lactate (CONT, n = 10) intravenously over 30 minutes just before left anterior descending artery reperfusion. Neutrophil demargination and infiltration into the myocardium were assessed in vivo with In111. Neutrophil chemotaxis, superoxide radical production, and lysozyme degranulation were evaluated ex vivo at baseline, 1 hour after occlusion, and 1 hour after reperfusion. Perfluorochemicals significantly reduced infarct size expressed as percent of area at risk (FDA, 7 +/- 4%; CONT, 24 +/- 6%; p less than 0.01). This was associated with positive wall motion in the jeopardized zone of Fluosol-DA animals compared with dyskinesis in control animals (FDA, +4.4 +/- 2.1%; CONT, -1.1 +/- 1.5%; p less than 0.05). Electron microscopy showed reduced neutrophil and erythrocyte plugging of capillaries with relative preservation of endothelial cells in the Fluosol-DA animals. Myocardial blood flow was greater in the ischemic endocardium of Fluosol-DA animals 1 hour after reperfusion (FDA, 1.23 +/- 0.21; CONT, 0.62 +/- 0.08 ml/g/min; p less than 0.01). Neutrophil demargination and infiltration into the ischemic myocardium was reduced in the animals treated with Fluosol-DA. (FDA, 2.5 +/- 0.7 x 10(3); CONT, 14.1 +/- 2.7 x 10(3) neutrophils/g; p less than 0.01). Neutrophil chemotaxis and lysozyme release were also markedly suppressed in the Fluosol-DA groups ex vivo. These results show that intravenous Fluosol-DA significantly reduces reperfusion injury with greater salvage of myocardium and improved left ventricular function. The chief mechanism of action of Fluosol-DA appears to be the suppression of neutrophil function.

Animals↗

Myocyte volume and function in response to osmotic stress: observations in the presence of an adenosine triphosphate-sensitive potassium channel opener.

BACKGROUND: Hypothermic hyperkalemic cardioplegia results in significant myocyte swelling and impaired contractility. These detrimental effects may be eliminated by the addition of an adenosine triphosphate-sensitive potassium (KATP) channel opener. This study evaluated the hypothesis that a KATP channel opener (diazoxide) would benefit volume homeostasis by limiting volume and subsequent contractility changes during osmotic stress. METHODS AND RESULTS: Isolated rabbit ventricular myocyte volume and contractility were evaluated using video microscopy and field stimulation after exposure to osmotic stress at 37 degrees C. Myocytes were exposed to Tyrode's physiological solution for 20 minutes and test solution for 20 minutes, and then reexposed to Tyrode's for 20 minutes. Test solutions included control Tyrode's (1T) and osmotically altered Tyrode's (2.6T, 0.9T, and 0.6T) solutions with or without the KATP channel opener diazoxide. Severe osmotic stress (2.6T and 0.6T) resulted in significant cell shrinkage and swelling, respectively. This was unchanged by the addition of diazoxide. Mild hyposmotic stress (0.9T) resulted in significant cell swelling that was eliminated by the addition of diazoxide. Cell swelling was associated with reduced contractility. CONCLUSIONS: Cell swelling, but not shrinkage, was detrimental to myocyte contractility. Diazoxide eliminated volume change due to mild hyposmotic stress, similar to that previously noted with hyperkalemic cardioplegia, but did not alter volume change secondary to severe osmotic stress.

Adenosine Triphosphate↗