Functional properties of skeletal muscle from transgenic animals with upregulated heat shock protein 70.

The influence of inducible heat stress proteins on protecting contracting skeletal muscle against fatigue-induced injury was investigated. A line of transgenic mice overexpressing the inducible form of the 72-kDa heat shock protein (HSP72) in skeletal muscles was used. We examined the relationship between muscle contractility and levels of the constitutive (HSC73) and inducible (HSP72) forms of the 72-kDa heat shock protein in intact, mouse extensor digitorum longus (EDL), soleus (SOL), and the diaphragm (DPH). In all transgenic muscles, HSP72 was expressed at higher levels compared with transgene-negative controls, where HSP72 was below the level of detection. At the same time, HSC73 levels were downregulated in all transgenic muscle types. Shipment-related stress caused an elevation in the levels of HSP72 in all muscles for 1 wk after arrival of the animals. We also found that, although no statistical differences in response to intermittent fatiguing stimulation in the contractile properties of intact transgene-positive muscles compared with their transgene-negative counterparts were observed, the response of intact transgene-positive EDL muscles to caffeine was enhanced. These findings demonstrate that elevated HSP72 does not protect EDL, SOL, or DPH muscles from the effects of intermittent fatiguing stimulation. However, HSP72 may influence the excitation-contraction coupling (ECC) process, either directly or indirectly, in EDL muscle. If the effects on ECC were indirect, then these results would suggest that manipulation of a specific gene might cause functional effects that seem independent of the manipulated gene/protein.

Increased superoxide production during fatigue in the perfused rat diaphragm.

This study test the hypothesis that a temporal relationship exists between the production of superoxide anion (O2-) and the contractile activity of perfused rat diaphragm. O2- levels were determined minute to minute by measuring the reduction of cytochrome c in the perfusate as the diaphragms were subjected to various levels of contractile activity. After equilibrating at low contractile rates (one 500 ms 80 Hz train/min), diaphragms were fatigued by increasing their contractile activity for 5 min (one 500 ms 80 Hz train/s) and then allowed to recover for 30 min (one 500 ms 80 Hz train/min). During equilibration, diaphragms did not produce O2- above the background level measured in the presence of superoxide dismutase (SOD). Within the first minute of fatigue-inducing stimulation, however, the rate of O2- production increased to 0.70 +/- 0.17 nmol/min and remained elevated until the recovery period when production returned towards baseline. SOD blocked this stimulation-related increase of O2-. Tension (+/-SOD) fell to 12% of the control value during the fatigue-inducing stimulation. During recovery the contractile response returned to 51% of control, indicating long-lasting effects on the contractile machinery. SOD did not limit fatigue or improve recovery, probably because it is a large protein that cannot cross cell membranes and protect the cells by scavenging O2- at its site of production.

Hemodynamic and metabolic effects of intravenous formyl-methionyl-leucyl-phenylalanine (FMLP) in rabbits.

We have studied the role of neutrophils and the effects of the chemotactic factor FMLP, using normal, neutropenic (after cyclophosphamide treatment) and C5a desArg-treated unanesthetized rabbits. The intravenous administration of FMLP in normal animals induces transient and dose-dependent hypotension, neutropenia and thrombocytopenia, with a maximal response in 3 minutes. When a bolus of 5 x 10(-9) moles FMLP was administered, maximal arterial hypotension (40 +/- 1.1 v.s. 90 +/- 1.1 mmHg in the controls, P < 0.001) accompanied by a significant increase in central venous pressure (0.89 +/- 0.9 v.s. -2.2 +/- 0.7 mmHg in the controls, P < 0.01) and a decrease in systemic vascular resistance (90 +/- 15.6 v.s 187 +/- 16.4 mmHg/L/min, P < 0.005). Plasma pH and bicarbonate were significantly reduced, with a parallel increase in plasma lactate levels. A similar reduction of arterial blood pressure was also noted in neutropenic animals (31 +/- 3% of pretreatment levels respectively). C5a des Arg caused neutropenia similar to that seen after 5 x 10(-9) moles FMLP (120 +/- 60/mm3 and 170 +/- 25/mm3 respectively), but it did not induce hypotension. These data suggest that simple neutrophil activation is not the mechanism of the FMLP-induced hypotension and that this chemotactic factor may interact with other cell types to produce its effects.

Positive inotropic effect of thyrotropin-releasing hormone on isolated rat hearts.

The effects of thyrotropin releasing hormone (TRH) on the contractility of electrically stimulated and perfused isolated rat hearts were investigated. TRH in the range of 0.1-10 mumol/l was found to exert a positive inotropic effect on cardiac contractility, which however qualitatively differed at lower vs. higher concentrations of the hormone: at 1 mumol/l, TRH was found to significantly enhance the rate of contraction as well as that of relaxation (by 23.2 +/- 3.7 and 27.8 +/- 7.7%, respectively), which culminated in an increased peak contractile force. However, at 10 mumol/l, the positive inotropic effect of TRH (i.e. the increase in peak contractile force) was smaller than at 1 mumol/l, which apparently was due to both a reduced TRH-induced elevation in the rate of contraction (12.4 +/- 3.2%) and a TRH-induced decrease in relaxation rate (11.1 +/- 8.1%). Since TRH is expressed in the heart, the above findings suggest that, in addition to its CNS-mediated cardiovascular effects, TRH modulates cardiac contractility as an autocrine regulator in a concentration-dependent manner, which likely involves more than one TRH receptor and associated signaling pathway.

Fatigue of rapid and slow onset in isolated perfused rat and mouse diaphragms.

Muscle fatigue was studied in the isolated perfused rat (66% oxidative fibers) and mouse (99% oxidative fibers) diaphragms. Both muscles displayed two fatigue patterns when exposed to 333-ms trains of 20-Hz stimulation. A rapid fatigue pattern appeared within each contractile train as an immediate progressive twitch-by-twitch diminution in contractility [a decrease in maximal isometric twitch tension (T) and maximal rate of T development]. An intertrain slow fatigue pattern also appeared as a progressive train-by-train diminution in contractility and an increased maximal rate of relaxation normalized to T. A reduction in the stimulatory frequency from 20 to 2 Hz caused a considerable diminution in the rapid fatigue pattern. These data suggest that rapid fatigue results from the time course of mechanical restitution, the time necessary for the Ca2+ channels of the sarcoplasmic reticulum to recover from inactivation. The slow fatigue pattern, on the other hand, is thought to be due to changes in the intracellular milieu. The difference in sensitivity of the rat and mouse diaphragms to rapid and slow fatigue is apparently related to differences in their fiber type composition. Thus, as would be expected, the mouse diaphragm, composed of only oxidative fibers, is less susceptible to slow fatigue compared with the rat diaphragm. On the other hand, it is more susceptible to rapid fatigue.

Inotropic influence of macrocyclic polyethers on tracheal smooth muscle.

Incubated guinea pig tracheal smooth muscle exhibited both positive and negative inotropic responses to a variety of crown ether analogs that ranged in size from 12-crown-4 to 30-crown-10 and included molecules whose lipophilicity was modified by the addition of benzo- and cyclohexo-substituents on the basic molecular framework. The inotropic influence of crown ethers may not only be due to their ionophoretic capabilities but may result from their ability to affect alterations in membrane physiology.

Inhibition of uterine contractility by progesterone and progesterone metabolites: mediation by progesterone and gamma amino butyric acidA receptor systems.

Progesterone and several progesterone metabolites are capable of inhibiting uterine contractility. Some progesterone metabolites have shown little or no affinity for the progesterone receptor but have been found to be potent modulators of the GABAA receptor system. This study examined whether the inhibition of uterine contraction by progesterone and its metabolites was progesterone receptor-mediated or gamma amino butyric acidA (GABAA) receptor-mediated. Uterine contractions were measured in annular rings of uterine tissue, 5 mm in length, from diestrous II rats, under a fixed tension of 1 gram. The steroids tested were 3 beta-hydroxy-5 beta-pregnan-20-one (6 micrograms/ml), 5 beta-pregnane-3,20-dione (10 micrograms/ml), 3 alpha-hydroxy-5 alpha-pregnan- 20-one (3 alpha,5 alpha-THP, 27.5 micrograms/ml), and progesterone (40 micrograms/ml). All compounds significantly inhibited spontaneous uterine contractions when compared to controls. No effect was seen by either 16 micrograms/ml of the progesterone antagonist, RU486, or 32 micrograms/ml of the GABAA antagonist, pictrotoxin, when administered alone. However, when uterine tissues were exposed to a combination of the steroid and the antagonist, the effect of 3 beta-hydroxy-5 beta-pregnan-20-one and 3 alpha,5 alpha-THP was blocked by picrotoxin but not by RU486, indicating that the action of these steroids was mediated through the GABAA system. The effect of 5 beta-pregnane-3,20-dione and progesterone was effectively blocked by RU486 but not by picrotoxin, suggesting that their actions were mediated through the progesterone receptor system. These results indicate that multiple mechanisms exist in the uterus for inhibiting uterine contractility by progesterone and its metabolites.

Theophylline, fatigue, and diaphragm contractility: cellular levels of 45Ca and cAMP.

The relationship between variations in diaphragmatic contractility and corresponding changes in total tissue levels of 45Ca and adenosine 3',5'-cyclic monophosphate (cAMP) was examined. The contractile performance of perfused contracting rat diaphragms was manipulated with theophylline (10(-4) M), induced fatigue, or both. The increased contractility associated with theophylline was related to significant increases in 45Ca levels without changes in cAMP levels. Fatigue-diminished contractility was associated with increases in both 45Ca and cAMP levels. The increased 45Ca and cAMP levels associated with fatigue persisted, even in the presence of theophylline. Calcium channel blockade with 10(-4) M verapamil blocked the positive inotropic influence of theophylline as well as the theophylline-associated increase in 45Ca levels. Verapamil had no effect on either the fatigue-associated decreases in contractility or the fatigue-enhanced 45Ca uptake. The results of this study strongly suggest that the enhanced contractility associated with theophylline is related to its influence on cellular calcium metabolism. The elevated level of isotopic calcium measured in fatigued muscle probably represents calcium sequestered in the sarcoplasmic reticulum, the result of cAMP-enhanced Ca-adenosine triphosphatase activity.

Effect of glucocorticoids on the response of airway smooth muscle to catecholamine and resorcinol beta-agonists.

The influence of hydrocortisone (11 beta, 17 alpha, 21-trihydroxy-pregn-4-ene-3,20-dione) or of methylprednisolone (6 alpha-methyl-11 beta, 17 alpha-21-trihydroxy-1,4-pregnadiene-3,20-dione) on the response of airway smooth muscle to a variety of beta-adrenergic bronchodilators was evaluated using incubated guinea pig tracheal rings, preconstricted with histamine. The adrenergic agonists chosen for this study included the nonselective beta 1- and beta 2-catecholamine, isoproterenol, the selective beta 2-catecholamine, rimiterol, and the selective beta 2-resorcinols, fenoterol and terbutaline. When the incubated rings were pretreated with 10-50 micrograms/mL of the steroids, there was a significant enhancement in smooth muscle sensitivity and reactivity to rimiterol and isoproterenol. Tracheal response to fenoterol or terbutaline, on the other hand, was not altered by the glucocorticoids. When used alone, neither steroid exerted an inotropic influence on the tracheal smooth muscle. The results of our study indicate that glucocorticoid enhancement of adrenergic bronchodilators is selective for catecholamines, and not for resorcinols.

Diltiazem, verapamil, and nifedipine inhibit theophylline-enhanced diaphragmatic contractility.

The ability of theophylline to limit diaphragmatic fatigue and to improve contractility appears to be dependent upon alterations in calcium metabolism. The mechanism responsible for these actions, however, remains unclear. We used perfused, contracting, intact rat diaphragm to measure the influence of therapeutic theophylline levels (10(-4) M) on tension development, in the presence or absence of the three most commonly used calcium channel blocking agents, or in the absence of calcium. Concentrations of diltiazem (10(-4) M), verapamil (10(-5) M), or nifedipine (10(-6) M) sufficient to completely block transmembrane calcium channels, were used. During the experiment, each diaphragm preparation was subjected to one of two fatiguing procedures: one to mimic that encountered during tachypnea and one to mimic that encountered during increased respiratory resistance. Our findings showed that therapeutic levels of theophylline were related to statistically significant (p less than 0.0005) increases in diaphragmatic contractility under control conditions and to statistically significant reductions in the sensitivity of the preparation to fatigue. All three calcium channel blockers negated the positive influence of theophylline. Zero calcium also prevented theophylline from enhancing the contractile properties of the diaphragm. It is our conclusion that theophylline enhances the contractile properties of the diaphragm by altering the transmembrane movement of calcium. Calcium antagonists, in turn, inhibit the beneficial effects of theophylline on diaphragm function.

Sleep apnea, proteinuria, and nephrotic syndrome.

Renal abnormalities in patients with obstructive sleep apnea syndrome (OSAS) have not been previously described. Medical records of patients who had been evaluated for possible sleep apnea syndrome and had had complete polysomnograms and urinalyses were reviewed to determine the frequency of proteinuria. High-grade proteinuria (greater than or equal to 3+ on urinalysis) was found in 6 of the 34 patients with obstructive sleep apnea, but in none of 34 patients in a control group matched for sex, age, and weight. In three patients, proteinuria was in the nephrotic range (3.5 g/24 h). The weight (mean +/- SD) of the patients with obstructive sleep apnea (112.7 +/- 35.3 kg) was not significantly different from the control group (109.2 +/- 30.3 kg). Microscopic examination of renal tissue in one patient with OSAS showed minimal changes. In four patients who were followed for 3 years, proteinuria improved after therapy for sleep apnea syndrome. We suggest that proteinuria may not be uncommon in patients with severe obstructive sleep apnea syndrome and may be reversible with correction of the sleep apnea syndrome.

Studies of mediator involvement in cooling-induced alterations of guinea pig tracheal smooth muscle contractility.

Heat loss from airway smooth muscle is a potent stimulus which causes substantial, but poorly understood, alterations in muscle tension. This study considered the involvement of endogenous mediators in cooling-induced tension changes in incubated guinea pig trachea. Smooth muscle tension was monitored in tracheal cylinders which were carefully cooled from 37 to 30 degrees C in the presence or absence of various inotropic mediators. In our study, cooling alone, at a rate of 1 degree C/min, was associated with an average loss of smooth muscle tension of 88.2 mg. Cooling tracheal tissue that had been previously exposed to 3 X 10(-6) M histamine, however, caused an additional increase in tracheal tension of 133 mg, over and above that caused by histamine alone. In the presence of 10(-5) M prostaglandin F2 alpha, or 10(-5) M thromboxane B2, cooling was associated with respective losses of smooth muscle tension of 211.4 and 211.2 mg, as compared to the tension associated with these mediators when they were used alone under control conditions. When the speed of tracheal cooling was increased to 40 degrees C/min, there was a slight increase in tension for 20 sec followed by a pronounced and sustained relaxation. The mechanisms involved in the response of airway smooth muscle to cooling are complex. The results of our study, however, suggest that mediators may play a role in the cooling-induced alterations of airway smooth muscle tension.

Regional contractile responses in pulmonary artery to alpha- and beta-adrenoceptor agonists.

Contractile sensitivity and reactivity to alpha- and beta-adrenoceptor stimulation was studied in incubated rabbit pulmonary artery cylindrical segments of differing diameters. Distinct differences were noted between the responses of extra- and intra-pulmonary pulmonary arteries to norepinephrine and isoproterenol. The sensitivity to norepinephrine was largest in the intrapulmonary pulmonary arteries. Arterial reactivity to norepinephrine was greatest in the larger of the intrapulmonary vessel segments, diminishing considerably as the vessels became smaller. Cocaine did not cause substantial alterations in the response of any of the arterial segments to the alpha-agonist. Phentolamine, however, exerted its influence primarily in the smaller arterial segments. Vascular sensitivity to isoproterenol was least in the intrapulmonary pulmonary arteries. These smaller vessel segments, however, were more reactive to isoproterenol than were the extrapulmonary pulmonary arterial segments. Propranolol, at a concentration of 10(-8) M, was an effective antagonist of the beta-agonist; at a concentration of 10(-7) M, however, this antagonist was related to isoproterenol-induced arterial contraction, apparently by stimulation of alpha-receptor sites. The results of this study demonstrated a regional heterogeneity in the contractile response of the pulmonary artery to alpha- and beta-stimulation. The extrapulmonary arterial segments were found to be more sensitive to beta-stimulation than were the smaller, intrapulmonary, segments. The intrapulmonary arterial segments, on the other hand, were found to be more sensitive to alpha-stimulation than were the extrapulmonary segments.

Hemorrhage-induced alterations of rabbit basilar artery reactivity and sensitivity to serotonin.

Subarachnoid hemorrhage has a profound effect on cerebrovascular reactivity. The present study noted a progressive change in the sensitivity and reactivity of rabbit basilar artery to serotonin after experimentally induced hemorrhage. The basilar artery exhibited an initial diminished response to serotonin for periods up to 6 hours after hemorrhage, whereafter the vessel gradually became hyperresponsive. The hypersensitivity became maximal 36 hours after hemorrhage and then began to return to normal. Such early onset of serotonin hypersensitivity and reactivity after subarachnoid hemorrhage has not been previously reported. The level of tension developed, however, suggests that serotonin alone is unlikely to cause vasospasm. The strict differentiation of spasm into early and delayed components is questioned.

Reactivity of rabbit basilar artery to alterations in extracellular potassium and calcium after subarachnoid hemorrhage.

Hemolysis of periarterial clots after subarachnoid hemorrhage may liberate large quantities of K+ into the vicinity of cerebral blood vessels and possibly change their sensitivity to endogenous vasoactive agents. The current study examined the influence of subarachnoid hemorrhage on the sensitivity of rabbit basilar arterial segments to K+ and Ca++. An analysis of K+ and Ca++ dose-response curves demonstrated that incubated arterial segments isolated from animals with subarachnoid hemorrhage were substantially more sensitive to these cations than were corresponding controls. We speculate that chronically elevated K+ levels in areas of periarterial clot lysis or brain ischemia might initiate vascular smooth muscle depolarization and vasospasm. Our data provide additional rationale for the use of calcium channel blockers in preventing or treating vasospasm in cases of subarachnoid hemorrhage.

Therapeutic effect of posture in sleep apnea.

Four patients with mild to moderate obstructive sleep apnea were monitored first in the supine posture to establish a diagnosis of obstructive sleep apnea (apnea indices 33, 12, 22, 36). A second polysomnogram, obtained while the patients slept in the lateral posture, showed a dramatic decrease in apnea (apnea indices 5, 0, 0.2, and 0) and snoring. Each patient had an enlarged uvula, which moved to the side in the lateral posture. We conclude that sleeping in the lateral posture may be therapeutic in some patients with obstructive sleep apneas.

The influence of lanthanum on the subcellular distribution of calcium in the perfused dog heart.

A newly developed method was used to make a direct study of the influence of lanthanum on the subcellular distribution of isotopic and elemental calcium in the isolated perfused dog heart. The trivalent cation was introduced into the heart for the last 5 min of a 60 min perfusion period. Developed pressure and dP/dt were monitored continually from a fluid filled balloon positioned within the left ventricular chamber. At the end of the perfusion period, calcium was fixed within the myocardium by rapid freezing followed by vacuum desiccation at -60 degrees C. Enriched populations of sarcolemma and mitochondria were then obtained utilizing a newly developed nonpolar density gradient ultracentrifugation technique. Lanthanum was found to decrease dP/dt by 82.5% without significant changes in contractile rate. Atomic absorption spectrophotometric analysis revealed that lanthanum was associated with a 48.8% decrease in sarcolemmal calcium and a 159.6% increase in mitochondrial calcium. Lanthanum caused a 40.2% increase in the mitochondrial tissue/medium 45Ca ratio without significantly altering the isotropic activity of the sarcolemma. The results confirm that lanthanum-induced negative inotropy is associated with a displacement of sarcolemma-associated calcium. Surprisingly, the diminished contractility occurs with an increase in mitochondrial-associated calcium.

Nonpolar density gradient ultracentrifugation in the direct determination of myocardial subcellular calcium.

A new method is described for making direct measurements of compartmentalized subcellular stores of calcium in the isolated perfused dog heart. Cellular calcium was immobilized by freezing the myocardium in diastole with an extremely cold fluorocarbon fluid (-125 degrees C). All subsequent procedures were conducted under conditions which prevented ionic diffusion, either at temperatures well below the freezing point of water or in the absence of water. Sarcolemmal and mitochondrial enriched fractions were segregated from dessicated, homogenized, myocardial tissue by ultracentrifugation utilizing density-gradients composed of blends of silicone and halocarbon on fluids within which physiological salts are insoluble. The total calcium content of these isolated fractions were then determined by atomic absorption spectrophotometry. Ouabain and epinephrine were subsequently used to alter the contractility of the perfused hearts and such contractile alterations were then related to changes noted in the calcium activity of the isolated subcellular fractions. In this study the calcium levels of the enriched mitochondrial fractions were elevated by both ouabain and epinephrine, while the calcium levels of the enriched sarcolemmal fractions were elevated only by ouabain. The advantage of this segregative procedure is that it prevents artifactual intercompartmental calcium rearrangement and preserves calcium levels to those initially fixed in situ at the time of freezing.

Temporal relationship of cyclic nucleotide levels and calcium exchange to histamine-induced tension development.

The purpose of these experiments was to study the temporal relationship between tension development in incubated guinea pig tracheal smooth muscle and changes in tissue levels of cAMP and cGMP, and isotopic Ca. Dose-response studies were performed with increasing concentrations of histamine both in the absence and presence of H1 receptor blockade using 10(-5) M diphenhydramine. The time course of tension development was subsequently determined in the presence of three concentrations of histamine shown to cause 50% (3 X 10(-6) M), 85% (9 X 10(-6) M), and 100% (5 X 10(-5) M) of maximal contraction. Tissue cyclic nucleotide and 45Ca levels were measured 20 sec, 1 min, and 6 min after the onset of contraction. For comparison, the influence of carbachol was also studied. Our findings demonstrate that there were no detectable alterations in tissue cAMP or cGMP levels during the initial phases of contractile change. In contrast, tissue isotopic Ca uptake increased early in histamine-induced contraction and was blocked by the H1 antagonist.

Crown ethers which influence cardiac and respiratory muscle contractility.

Guinea-pig tracheal smooth muscle and heart muscle demonstrated a variety of in vitro positive and negative inotropic responses to concentrations of crown ethers in the nmole/1 to mumole/1 range. It is suggested that these ionophoretic compounds have potential as therapeutic agents.