K+ channel pulmonary vasodilation in fetal lambs: role of endothelium-derived nitric oxide.

To define the role and mechanism of action of K+ channels in regulating fetal pulmonary vascular tone, we studied the hemodynamic effects of pinacidil (a K+ channel activator) and glibenclamide (a K+ channel blocker). The effects of pinacidil were compared with those of acetylcholine [an endothelium-derived relaxing factor- (EDRF) dependent pulmonary vasodilator] and 8-bromoguanosine 3',5'-cyclic monophosphate (8-bromo-cGMP, an EDRF-independent pulmonary vasodilator) before and after treatment with N omega-nitro-L-arginine [a competitive inhibitor of an EDRF, endothelium-derived nitric oxide (EDNO), synthesis], or L-arginine (the substrate for the formation of EDNO). In 14 unanesthetized fetal lambs in utero, catheters were inserted into the fetal pulmonary artery, descending aorta, left atrium, and superior vena cava to measure pressures and administer drugs. An ultrasonic flow transducer was placed around the left pulmonary artery to measure flow (QP) continuously. In eight animals, pinacidil, acetylcholine, and 8-bromo-cGMP caused similar acute maximal increases in QP of 128, 137, and 155 ml/min, respectively. After a 60-min infusion of N omega-nitro-L-arginine (2.07 +/- 0.27 mg.kg-1.min-1), the increase in QP caused by acetylcholine and pinacidil was significantly attenuated, by 84 and 68%, respectively, with only a 10% attenuation of the increase in QP caused by 8-bromo-cGMP. In six additional N omega-nitro-L-arginine-pretreated fetal lambs, infusion of L-arginine (32.2 +/- 4.3 mg.kg-1.min-1) restored the vasodilatory effects of acetylcholine and pinacidil. A 20-min infusion of glibenclamide (n = 6; 0.64 +/- 0.07 mg.kg-1.min-1) blocked the vasodilation by pinacidil but not acetylcholine.(ABSTRACT TRUNCATED AT 250 WORDS)


Influence of lung volume and alveolar pressure on reverse pulmonary venous blood flow.

We have reported that left atrial blood refluxes through the pulmonary veins to gas-exchanging tissue after pulmonary artery ligation. This reverse pulmonary venous flow (Qrpv) was observed only when lung volume was changed by ventilation. This was believed to drive Qrpv by alternately distending and compressing the alveolar and extra-alveolar vessels. Because lung and pulmonary vascular compliances change with lung volume, we studied the effect of positive end-expiratory pressure (PEEP) on the magnitude of Qrpv during constant-volume ventilation. In prone anesthetized goats (n = 8), using the right lung to maintain normal blood gases, we ligated the pulmonary and bronchial arterial inflow to the left lung and ventilated each lung separately. A solution of SF6, an inert gas, was infused into the left atrium. SF6 clearance from the left lung was determined by the Fick principle at 0, 5, 10, and 15 and again at 0 cmH2O PEEP and was used to measure Qrpv. Left atrial pressure remained nearly constant at 20 cmH2O because the increasing levels of PEEP were applied to the left lung only. Qrpv was three- to fourfold greater at 10 and 15 than at 0 cmH2O PEEP. At these higher levels of PEEP, there were greater excursions in alveolar pressure for the same ventilatory volume. We believe that larger excursions in transpulmonary pressure during tidal ventilation at higher levels of PEEP, which compressed alveolar vessels, resulted in the reflux of greater volumes of left atrial blood, through relatively noncompliant extra-alveolar veins into alveolar corner vessels, and more compliant extra-alveolar arteries.


Peripheral chemoreceptor modulation of the pulmonary vasculature in the cat.

The present study was undertaken to determine whether stimulation of the carotid and aortic bodies (cb and ab) could affect the pulmonary vasculature. Our hypothesis was that each promoted vasodilation and thus could modulate the pulmonary vasoconstrictor response to hypoxia. The experimental design of the first set of experiments took advantage of the facts that 1) the ab, but not the cb, increases its neural output in response to CO, whereas both respond to a decreased arterial PO2 (hypoxic hypoxia, HH) and 2) the aortic nerves in cats are easily transected. Hence, both cb and ab sent neural activity to the brain stem when the intact cat was exposed to 10% O2 in N2. Only the ab sent information during CO hypoxia (COH intact). Only the cb did so during HH in the cat in which the aortic nerves had been transected, removing the aortic body (HH abr); neither ab nor cb did so during COH abr. Fifteen anesthetized paralyzed artificially ventilated cats were fit with catheters in the femoral artery and vein, right and left atria, left ventricle, and pulmonary artery and with an aortic flow probe. In the HH intact and HH abr conditions, there was a significant rise in cardiac output, whereas pulmonary arterial pressure (Ppa) rose initially but then leveled off while cardiac output continued to rise. During the 15-min exposure to HH, pulmonary vascular resistance [PVR = (Ppa - Pla)/cardiac output, where Pla is left atrial pressure] rose initially and then decreased significantly at 2-3 min. In response to COH, PVR showed only a significant decrease. In the second set of experiments, seven cats were instrumented as above and had loops placed in the common carotid arteries for selectively perfusing the cbs. In response to a brief infusion of venous blood mixed with 0.3-0.5 micrograms NaCN, which selectively stimulated only the cb, aortic flow remained relatively constant while heart rate and Ppa - alveolar pressure difference decreased significantly; so also did PVR. These data are consistent with the hypothesis that stimulation of the ab and cb singly or together can provoke a significant pulmonary vasodilation in the anesthetized paralyzed artificially ventilated cat.


Effects of age and physical activity status on the speed-aerobic demand relationship of walking.

Older adults tend to show lower preferred walking speeds and higher aerobic demands per distance walked than young adults. It has been suggested that a more sedentary life-style contributes to diminished musculoskeletal functioning, which in turn contributes to poorer economy of motion in the aged and sedentary adults. The purpose of this study was to quantify the speed-aerobic demand relationship during walking for old (greater than 65 yr of age) and young adults and to determine whether physical activity status affects this relationship. Aerobic demands for 30 young and 30 old individuals representing sedentary and physically active groups were measured as the subjects performed treadmill walking at seven speeds ranging from 0.67 to 2.01 m/s. All four age/physical activity groups displayed U-shaped speed-aerobic demand curves with minimum gross oxygen consumption per unit distance walked (ml.kg-1.km-1) at 1.34 m/s. A statistically significant age effect on walking aerobic demand was observed, with old subjects showing an 8% higher mean aerobic demand than the young subjects. This age-related effect was not associated with shifts in the speed at which aerobic demand was minimized or with the preferred walking speed of older individuals falling on a less economical portion of the speed-aerobic demand curve. Rather, it was speculated that declines in force-generating capacity of muscle in the aged may require recruitment of additional motor units and perhaps an additional proportion of less economical fast twitch muscle fibers to generate necessary forces. Physical activity status had no significant effect on walking aerobic demand.(ABSTRACT TRUNCATED AT 250 WORDS)


Lung tissue behavior during methacholine challenge in rabbits in vivo.

Previous studies have shown that lung challenge with smooth muscle agonists increases tissue viscance (Vti), which is the pressure drop between the alveolus and the pleura divided by the flow. Passive inflation also increases Vti. The purpose of the present study was to measure the changes in Vti during positive end-expiratory pressure- (PEEP) induced changes in lung volume and with a concentration-response curve to methacholine (MCh) in rabbits and to compare the effects of induced constriction vs. passive lung inflation on tissue mechanics. Measurements were made in 10 anesthetized open-chest mechanically ventilated New Zealand male rabbits exposed first to increasing levels of PEEP (3-12 cmH2O) and then to increasing concentrations of MCh aerosol (0.5-128 mg/ml). Lung elastance (EL), lung resistance (RL), and Vti were determined by adjusting the equation of motion to tracheal and alveolar pressures during tidal ventilation. Our results show that under baseline conditions, Vti accounted for a major proportion of RL; during both passive lung inflation and MCh challenge this proportion increased progressively. For the same level of change in EL, however, the increase in Vti was larger during MCh challenge than during passive inflation; i.e., the relationship between energy storage and energy dissipation or hysteresivity was dramatically altered. These results are consistent with a MCh-induced change in the intrinsic rheological properties of lung tissues unrelated to lung volume change per se. Lung tissue constriction is one possible explanation.


Pulmonary hemodynamics in fetal lambs during development at normal and increased oxygen tension.

During the latter third of gestation, the number of resistance vessels in the lungs of the fetal sheep increases by 10-fold even after correction for lung growth. We measured pulmonary arterial pressure and blood flow directly and calculated total pulmonary resistance (pressure divided by flow) in intrauterine fetal lambs at 93-95 days and at 136 days of gestation (term is 145-148 days). In addition, we used a hyperbaric chamber to increase oxygen tension in the fetuses and measured the effect on the pulmonary circulation. When corrected for wet weight of the lungs, pulmonary blood flow did not change with advancing gestation (139 +/- 42 to 103 +/- 45 ml.100 g-1.min-1). Pulmonary arterial pressure increased (42 +/- 5 to 49 +/- 3 mmHg); thus total pulmonary resistance increased with advancing gestation from 0.32 +/- 0.12 to 0.55 +/- 0.21 mmHg.100 g.min.ml-1. If the blood flow is corrected for dry weight of the lungs, neither pulmonary blood flow nor total pulmonary resistance changed with advancing gestation. Increasing oxygen tension increased pulmonary blood flow 10-fold in the more mature fetuses but only 0.2-fold in the less mature fetuses. At the normal low oxygen tension of the fetus, pulmonary blood flow does not increase between these two points of gestation in the fetal lamb despite the increase in vessel density in the lungs. However, during elevated oxygen tension, pulmonary blood flow does increase in proportion to the increase in vessel density.


Response of laryngeal mechanoreceptors to high-frequency pressure oscillation.

High-frequency pressure oscillations (HFPO) in the upper airway induce arousal, activation of genioglossus muscle, and bronchoconstriction. The present study was designed to determine the response of superior laryngeal nerve afferent fibers to HFPO. In 10 anesthetized dogs spontaneously breathing through a tracheal cannula, the upper airway was converted to a closed system. The activity of thin bundles separated from the peripheral cut end of the superior laryngeal nerve was monitored. Of 104 mechanoreceptors identified, 87 were classified as respiratory modulated and 17 as non-respiratory modulated on the basis of their response to transmural pressure change and muscle activity. The responses of these fibers to HFPO of +/- 2.5 cmH2O at 10, 20, and 30 Hz were determined. Among the respiratory-modulated receptors, 86 of 87 increased their activity in response to HFPO. Of the 17 non-respiratory-modulated receptors, 12 receptors showing a random or tonic activity did not respond to HFPO, whereas the 5 that were silent during control condition responded exclusively to HFPO. Our results show that HFPO of similar frequency but much less magnitude than snoring is capable of activating the vast majority of laryngeal mechanoreceptors. Pressure-sensitive respiratory-modulated endings appear to mediate the arousal and genioglossal response, whereas non-respiratory-modulated receptors responding to HFPO presumably mediate the bronchoconstrictive response.


Effects of capillary red cell density on gas conductance of frog skin.

We tested experimentally the hypothesis that decreasing capillary red blood cell (RBC) density (dRBC) reduces the tissue diffusing capacity of frog skin to CO (DtiCO) and O2 (DtiO2). The effects of dRBC on CO2 transport were also assessed. C18O, O2, and CO2 transport between the skin and a cutaneous sample chamber on the belly of anesthetized (halothane) frogs (Rana pipiens) was measured by mass spectrometry, and the cutaneous conductances to C18O (GCO), O2 (GO2), and CO2 (GCO2) were calculated. The dRBC of the planar cutaneous capillary bed was measured by intravital fluorescent video microscopy. DtiCO and DtiO2 were calculated from a modification of the Roughton-Foster equation: 1/G = 1/Dti + 1/(theta RBC.dRBC), where theta RBC values were estimated from literature values. In one group of animals (n = 6), measurements were made before hemodilution (dRBC = 630 +/- 56 cells/mm2), after one hemodilution (dRBC = 349 +/- 50 cells/mm2), and after a second hemodilution (dRBC = 150 +/- 31 cells/mm2). In controls, time had no effect on GCO, GO2, or GCO2 (P greater than 0.42). Before hemodilution, GCO, GO2, and GCO2 were 0.069 +/- 0.010, 0.088 +/- 0.0012, and 1.23 +/- 0.010 nmol.min-1.Torr-1.cm-2, respectively, and lowering dRBC by hemodilution decreased all these parameters (P less than 0.025). The mean slopes of GCO, GO2, and GCO2 vs. dRBC were 6.0 +/- 1.3 x 10(-7), 7.2 +/- 2.3 x 10(-7), and 7.8 +/- 3.0 x 10(-6) nmol.min-1.Torr-1.RBC-1, respectively. Lowering dRBC also decreased DtiCO and DtiO2 (P less than 0.034). DtiCO and DtiO2 were 0.080 +/- 0.012 and 0.096 +/- 0.013 nmol.min-1.Torr-1.cm-2, respectively, before hemodilution. The mean slopes of DtiCO and DtiO2 vs. dRBC were 4.9 +/- 2.1 x 10(-7) and 6.5 +/- 2.8 x 10(-7) nmol.min-1.Torr-1.RBC-1, respectively. Hemodilution had no effect on perfused capillary density (P = 0.38). These results indicate that tissue diffusive conductance is proportional to dRBC. Regulation of dRBC may be an important mechanism modulating diffusive gas transport in tissue.


Determination of maximal oxygen consumption in exercising pregnant sheep.

Previous work with pregnant ewes has shown that acute bouts of exercise may cause changes in plasma hormone concentrations, blood flow distribution, and maternal and fetal temperatures. However, most of these studies do not quantify the chosen exercise intensity through measurement of oxygen consumption (VO2). Therefore the purpose of this study was to statistically model the VO2 response of pregnant sheep to treadmill (TM) exercise to determine the exercise intensities (% maximal VO2) of previous studies. Ewes with either single (n = 9) or twin (n = 5) fetuses were studied from 100 to 130 days of gestation. After 1-2 wk of TM habituation, maximal VO2 (VO2max) was determined by measurements of VO2 (open flow-through method) and blood lactate concentration. VO2 was measured as a function of TM incline (0, 3, 5, and 7 degree) and speed (0.8-3.4 m/s). VO2max averaged 57 +/- 7 (SD) ml.min-1.kg-1, and peak lactate concentration during exercise averaged 22 +/- 2 mmol/l. The relationship between VO2 (ml.min-1.kg-1) and incline (INC) and speed (SP) [VO2 = 0.70(INC) + 13.95(SP) + 1.07(INC x SP) - 1.18] was linear (r2 = 0.94). Our findings suggest that most previous research used exercise intensities less than 60% VO2max and indicate the need for further research that examines the effect of exercise during pregnancy at levels greater than 60% VO2max.


Respiratory muscle blood flow in exercising dogs after pneumonectomy.

In three foxhounds after left pneumonectomy, the relationships of ventilatory work and respiratory muscle (RM) blood flow to ventilation (VE) during steady-state exercise were examined. VE was measured using a specially constructed respiratory mask and a pneumotach; work of breathing was measured by the esophageal balloon technique. Blood flow to RM was measured by the radionuclide-labeled microsphere technique. Lung compliance after pneumonectomy was 55% of that before pneumonectomy; compliance of the thorax was unchanged. O2 uptake (VO2) of RM comprised only 5% of total body VO2 at exercise. At rest, inspiratory muscles received 62% and expiratory muscles 38% of the total O2 delivered to the RM (QO2RM). During exercise, inspiratory muscles received 59% and expiratory muscles 41% of total QO2RM. Blood flow per gram of muscle to the costal diaphragm was significantly higher than that to the crural diaphragm. The diaphragm, parasternals, and posterior cricoarytenoids were the most important inspiratory muscles, and internal intercostals and external obliques were the most important expiratory muscles for exercise. Up to a VE of 120 l/min through one lung, QO2RM constituted only a small fraction of total body VO2 during exercise and maximal vasodilation in the diaphragm was never approached.


Immediate diaphragmatic electromyogram responses to imperceptible mechanical loads in conscious humans.

We used an esophageal electrode to measure the amplitude and neural inspiratory and expiratory (N TE) timing responses of crural diaphragmatic electrical activity in response to flow-resistive (R) and elastic (E) loads at or below the threshold for conscious detection, applied pseudorandomly to the oral airway of eight normal subjects. We observed a rapid first-breath neural reflex that modified respiratory timing such that N TE lengthened significantly in response to R loads in six of eight subjects and shortened in response to E loading in six of seven subjects. The prolongation of N TE with R loading resulted primarily from lengthening the portion of N TE during which phasic activity in the diaphragm is absent (TE NDIA), whereas E loading shortened N TE mainly by reducing TE NDIA. Most subjects responded to both types of loading by decreasing mean tonic diaphragmatic activity, the average level of muscle activity that exists when no phasic changes are occurring, as well as its variability. The observed timing responses are consistent in direction with optimally adaptive pattern regulation, whereas the modulation of tonic activity may be useful in neural regulation of end-expiratory lung volume.


Cardiovascular responses of heart transplant recipients to graded exercise testing.

A group of orthotopic heart transplant (OHT, n = 28) and heart surgery (n = 19) patients, with similar ejection fractions and left ventricular end-diastolic pressures, were exercised to symptom-limited maximum to describe differences in cardiovascular and gas exchange responses. Testing was performed at a mean of 3 and 6 mo after surgery, respectively (P less than 0.05). OHT patients have a greater resting systolic and diastolic blood pressure (P less than 0.01) and a significantly greater (P less than 0.01) heart rate (HR) at rest in the supine and standing positions and during minutes 2 through 7 of supine recovery. Peak treadmill time was significantly less (P less than 0.01) in OHT patients. No significant differences were found for systolic blood pressure (SBP) during recovery, peak HR, ventilation, relative O2 uptake (VO2), body weight, ventilatory equivalents for O2 and CO2, O2 pulse, and HR-SBP product (peak HR x peak SBP). Peak pulse pressure, heart rate reserve, total VO2, and absolute VO2 at ventilatory threshold were significantly lower (P less than 0.01) in the OHT patients. We concluded that 1) complete cardiac decentralization is evident, 2) the significantly reduced VO2 at ventilatory threshold should be considered when activities of daily living are prescribed, and 3) SBP response is more appropriate than HR for assessing recovery of the decentralized heart after maximal exercise.


Metabolic effects of treadmill exercise training on the diabetic heart.

This study determined whether exercise training in rats would prevent the accumulation of lipids and depressed glucose utilization found in hearts from diabetic rats. Diabetes was induced by intravenous streptozotocin (60 mg/kg). Trained diabetic rats were run on a treadmill for 60 min, 27 m/min, 10% grade, 6 days/wk for 10 wk. Training of diabetic rats had no effect on glycemic control but decreased plasma lipids. In vivo myocardial long-chain acylcarnitine, acyl-CoA, and high-energy phosphate levels were similar in sedentary control, sedentary diabetic, and trained diabetic groups. The levels of myocardial triacylglycerol were similar in sedentary control and diabetic rats but decreased in trained diabetic rats. Hearts were perfused with buffer containing diabetic concentrations of glucose (22 mM) and palmitate (1.2 mM). D-[U-14C] glucose oxidation rates (14CO2 production) were depressed in hearts from sedentary diabetic rats relative to sedentary control rats. Hearts from trained diabetic rats exhibited increased glucose oxidation relative to those of sedentary diabetic rats, but this improvement was below that of the sedentary control rats. [9,10(-3)H]palmitate oxidation rates (3H2O production) were identical in all three groups. These findings suggest that exercise training resulted in a partial normalization of myocardial glucose utilization in diabetic rats.


Pulmonary artery occlusion is sufficient to increase pulmonary vascular permeability in rabbits.

Unilateral pulmonary artery obstruction (PAO) for 24-48 h, followed by reperfusion, results in pulmonary edema and lung inflammation. We hypothesized that lung injury actually occurred during the period of PAO but, because of low microvascular pressures during the period of occlusion, was not detected until perfusion was reestablished. To test this hypothesis, we studied 14 rabbits divided into three groups: group I rabbits underwent sham occlusion of the left pulmonary artery for 24 h; group II rabbits underwent PAO but were not reperfused; and group III rabbits were subjected to PAO and then reperfused for 4 h. The fluid filtration coefficient measured during a zone 3 no-flow hydrostatic stress (pulmonary arterial pressure = pulmonary venous pressure, both greater than alveolar pressure) in group I lungs was less than that of lungs in either group II or III [0.52 +/- 0.02 (SE) ml.min-1.cmH2O.100 g wet wt-1 vs. 0.94 +/- 0.11 and 0.86 +/- 0.13 for groups II and III, respectively, P less than 0.05]. The wet-to-dry weight ratio of the left lung measured after the zone 3 stress was applied for 20 min was 6.90 +/- 0.09 in group I rabbits and 9.21 +/- 0.75 and 11.75 +/- 0.44 in groups II and III, respectively (P less than 0.05). Radiolabeled microspheres demonstrated that flow to the left lung was diminished after the period of PAO (38 +/- 4, 9 +/- 5, and 2 +/- 1% of cardiac output in groups I, II, and III, respectively; P less than 0.05 for group I vs. groups II and III).(ABSTRACT TRUNCATED AT 250 WORDS)


Functional residual capacity and ventilation homogeneity in mechanically ventilated small neonates.

A modification of a computerized tracer gas (SF6) washout method was designed for serial measurements of functional residual capacity (FRC) and ventilation homogeneity in mechanically ventilated very-low-birth-weight infants with tidal volumes down to 4 ml. The method, which can be used regardless of the inspired O2 concentration, gave accurate and reproducible results in a lung model and good agreement compared with He dilution in rabbits. FRC was measured during 2-4 cmH2O of positive end-expiratory pressure (PEEP) in 15 neonates (700-1,950 g), most of them with mild-to-moderate respiratory distress syndrome. FRC increased with body weight and decreased (P less than 0.05) with increasing O2 requirement. Change to zero end-expiratory pressure caused an immediate decrease in FRC by 29% (P less than 0.01) and gave FRC (ml) = -1.4 + 17 x weight (kg) (r = 0.83). Five minutes after PEEP was discontinued (n = 12), FRC had decreased by a further 16% (P less than 0.01). The washout curves indicated a near-normal ventilation homogeneity not related to changes in PEEP. This was interpreted as evidence against the presence of large volumes of trapped alveolar gas.


Instant of vascular occlusion defined with laser-Doppler flowmetry.

Derivation of capillary pressure from tracings postarterial (AO) or -venous (VO) occlusion requires back extrapolation to an instant near the time of occlusion. This instant is difficult to identify because of pressure artifacts created by the occlusion maneuver. Theoretically, when the flow in the main artery (or veins) is stopped instantaneously, the flow in the arterioles (or venule) will stop after a short time delay (perhaps less than 100 ms). When flow had stopped in the main artery and in the arteriole, the pressure in the main artery at that instant would equal the pressure in the arterioles. We sought to identify the instant when flow stops in the arterioles and venules after AO and VO, respectively. In an isolated perfused dog left lower lobe preparation flow in the main vessels were monitored with inline flow probes, whereas flow in the microcirculation was monitored with laser-Doppler flow (LDF) probe placed on the lung surface. A sudden decline in arterial flow was detected by the LDF probe after 54 ms, while a sudden decline in venous flow was detected in the venules after 35 ms. These time delays were used as wave transmission time across the arterial and venous trees. Consequently, it was concluded that after AO, flow in the arterioles would stop 54 ms after it had become zero in the main artery, while after VO flow in the venules would stop 35 ms after it had become zero in the main vein. The pressure post-AO and post-VO was read at these instants (54 and 35 ms after flow in the main vessel reached zero).(ABSTRACT TRUNCATED AT 250 WORDS)


Low-frequency pulmonary impedance in rabbits and its response to inhaled methacholine.

We assessed pulmonary mechanics in six open-chest rabbits (3 young and 3 adult) by the forced oscillation technique between 0.16 and 10.64 Hz. Under control conditions, pulmonary resistance (RL) decreased markedly between 0.16 and 4 Hz, after which it became reasonably constant. Measurements of alveolar pressure from two alveolar capsules in each rabbit showed that the large decrease of RL with increasing frequency below 4 Hz was due to lung tissue rheology and that tissue resistance was close to zero above 4 Hz. Estimates of resistance and elastance, also obtained by fitting tidal ventilation data at 1 Hz to the equation of the linear single-compartment model, gave values for RL motion that were slightly higher than those obtained by forced oscillations at the same frequency, presumably because of the flow dependence of airways resistance. After treatment with increasing doses of aerosolized methacholine, RL and pulmonary elastance between 0.16 and 1.34 Hz progressively increased, as did the point at which the pulmonary reactance crossed zero (the resonant frequency). The alveolar pressure measurements showed the lung to become increasingly inhomogeneously ventilated in all six animals, whereas in the three younger rabbits lobar atelectasis developed at high methacholine concentrations and the alveolar capsules ceased to communicate with the central airways. We conclude that the low-frequency pulmonary impedance of rabbits exhibits the same qualitative features observed in other species and that it is a sensitive indicator of the changes in pulmonary mechanics occurring during bronchoconstriction.


A multipurpose instrument for quantitative intravital microscopy.

An in vivo microscope system has been developed that can measure fluorescence emission and/or light absorption at up to five wavelengths in a tissue area of 18-30 microns diam while imaging adjacent microcirculatory vessels with a video system. The system also incorporates a computer-controlled stage and data acquisition system for rapid and repeated measurements from a number of tissue sites. The tissue area monitored for fluorescence or absorption can be defined further by a confocal arrangement of the microscope optics. Tests of the system for NADH fluorescence measurements show good agreement between the fluorescence at 450 nm and NADH concentration in vitro and in skeletal muscle. The instrument can also be used simultaneously for spectrophotometric determination of O2 saturation and hematocrit in microcirculatory vessels. In vitro tests indicate suitable accuracy for such measurements. The open architecture and modular arrangement of the instrument facilitates its use for a variety of simultaneous measurements of parenchymal cell and microcirculatory function.


Role of extracellular Ca2+ in diaphragmatic contraction: effects of ouabain, monensin, and ryanodine.

The effects of zero extracellular Ca2+ on the contractility of rat diaphragmatic strips in vitro were studied in conjunction with various pharmacological agents known to influence the intracellular Ca2+ concentration: the Na+ ionophore, monensin, and the Na(+)-K+ pump inhibitor, ouabain, which enhance [Ca2+]i, caffeine, which induces Ca2+ release from the sarcoplasmic reticulum (SR), and ryanodine, which prevents Ca2+ retention by the SR. The effect of increasing [Ca2+]i on diaphragmatic contraction was assessed by comparing contractions induced by 120 mM K+ in the small muscle strips before and after the addition of ouabain or monensin. Monensin (20 microM) and ouabain (1-100 microM) augmented contractions up to threefold. Treatment of diaphragm strips with 3 nM ryanodine increased baseline tension 360% above the original resting tension but only if the diaphragm was electrically stimulated concurrently; 100 microM ryanodine induced contracture in quiescent tissue. High K+ contractures were of greater magnitude in the presence of ryanodine compared with control, and relaxation time was prolonged by greater than 200%. Ca(2+)-free conditions ameliorated these actions of ryanodine. Ryanodine reduced contractions induced by 10 mM caffeine and nearly abolished them in Ca(2+)-free solution. The data demonstrate that extracellular Ca2+ is important in certain types of contractile responses of the diaphragm and suggest that the processes necessary to utilize extracellular Ca2+ are present in the diaphragm.


Airway narrowing in excised canine lungs measured by high-resolution computed tomography.

The exact site of airway narrowing in asthma and chronic obstructive pulmonary disease is unknown. High-resolution computed tomography (HRCT) is a sensitive noninvasive imaging technique that can be used to measure airway dimensions. After determining the optimal computed tomographic parameters using a phantom, we measured lobe volume and airway dimensions of isolated canine lung lobes at a transpulmonary pressure of 25 cmH2O. These measurements were repeated after deflation and administration of aerosolized saline and carbachol (256 mg/ml). Lobe volume decreased with all treatments. The maximal lobar volume change was 26% at 6 cmH2O after carbachol. Average airway lumen area decreased with all treatments. After carbachol, at transpulmonary pressures of 25, 15, 10, 8, and 6 cmH2O, lumen area decreased by 7.3 +/- 4.1, 62.0 +/- 4.9, 77.5 +/- 3.0, 31.9 +/- 9.0, and 95.2 +/- 1.0% (SE), respectively. When the airways were divided into four categories on the basis of initial lumen diameter (less than 2, 2-4, 4-6, and greater than 6 mm), the greatest decreases in luminal area after carbachol were seen in intermediate-sized airways (2-4 mm, 56 +/- 4%; 4-6 mm, 59 +/- 3%). HRCT can be used to make accurate measurements of airway dimensions and airway narrowing in excised lungs. HRCT may allow measurement of airway wall thickness and determination of the site of airway narrowing in asthma.


Reversal of hyperdynamic response to continuous endotoxin administration by inhibition of NO synthesis.

Septic shock is characterized by an increase in cardiac output and a fall in systemic vascular resistance index and mean arterial pressure. Endotoxin alters the smooth muscle function of blood vessels, probably by means of an increased production of the potent vasodilator nitric oxide (NO). The present study was accomplished to determine how the inhibition of NO synthesis influences cardiovascular performance in an ovine model of hyperdynamic endotoxemia. Endotoxemia was induced in five range ewes (41 +/- 2 kg) by continuous infusion of Escherichia coli endotoxin (LPS, 10 ng.kg-1.min-1) over the entire study period. After 24 h of LPS infusion, cardiac output increased from 5.2 +/- 0.3 to 7.9 +/- 0.6 (SE) 1/min (P less than 0.05) and mean arterial pressure and systemic vascular resistance index fell from 92 +/- 5 to 79 +/- 6 mmHg (P = 0.08) and from 1,473 +/- 173 to 824 +/- 108 dyn.s.cm-5.m2 (P less than 0.05), respectively. The pulmonary shunt fraction increased from 0.23 +/- 0.03 to 0.32 +/- 0.03 (P less than 0.05). The intravenous administration of the NO synthase inhibitor N omega-nitro-L-arginine methyl ester (25 mg/kg) 24 h after the start of the LPS infusion changed these values to approximately baseline levels over the subsequent 4 h. Although N omega-nitro-L-arginine methyl ester increased pulmonary arterial pressure and pulmonary vascular resistance (P less than 0.05), right and left ventricular stroke volume index showed no significant changes. It is concluded that NO has a major function in cardiovascular performance in endotoxemia.(ABSTRACT TRUNCATED AT 250 WORDS)


A mathematical model of life-threatening hyperthermia during infancy.

A mathematical model was created to test the hypothesis that a partially covered febrile infant may develop potentially lethal temperature elevation. Infants may be at special risk to develop hyperthermia because, unlike older children, infants may not be able to remove blankets in response to temperature elevation. The model compared heat production (MTsk) with heat loss (Qtot). The difference between these terms is the excess energy (E): MTsk - Qtot = E. In most situations the simulated infant transfers heat to the environment as rapidly as it is produced (E less than 0), so hyperthermia does not result. In some situations, heat production exceeds heat loss (E greater than 0), causing progressive warming. The time was calculated for the simulated infant to progress from 41 to 43.4 degrees C (defined as a lethal end point). In certain circumstances, this may occur in less than 90 min. An infant at high risk of hyperthermia may not appear to be covered by a conspicuous excess of insulation (less than or equal to 3.5 cm may be sufficient). In many situations, heat loss is more closely determined by exposed body surface area than by blanket thickness. These findings have important implications for understanding the antecedents of hyperthermia in infants and may help in understanding the role of hyperthermia in certain pediatric illnesses.


An animal model of life-threatening hyperthermia during infancy.

A mathematical model of heat balance in human infants suggests that it may be possible for severe hyperthermia to develop if an infant is unable to remove his blankets in response to overheating (thermal entrapment). This hypothesis was tested in an animal model of weanling piglets. Ten piglets were warmed in a radiant heater to rectal temperature of 41 degrees C to simulate a fever. Animals in the experimental and control groups were removed from the heater and covered with ordinary infant blankets (to a thickness of approximately 3 cm). Endogenously produced heat caused the animals to warm to 42 degrees C. At this point, the control animals were uncovered. They rapidly cooled to normal body temperature. Animals in the experimental group remained covered until they expired from hyperthermia at 43.9 +/- 0.7 degrees C (SD) after 96 +/- 43 (SD) min. These data show that lethal hyperthermia may result from thermal entrapment. This finding may help clarify the role that hyperthermia may play in illnesses such as hemorrhagic shock and encephalopathy syndrome and some cases of sudden infant death syndrome.


Skeletal muscle carnitine metabolism in patients with unilateral peripheral arterial disease.

Patients with peripheral arterial disease (PAD) have abnormalities of carnitine metabolism that may contribute to their functional impairment. To test the hypothesis that muscle acylcarnitine generation (intermediates in oxidative metabolism) in patients with PAD provides a marker of the muscle dysfunction, 10 patients with unilateral PAD and 6 age-matched control subjects were studied at rest, and the patients were studied during exercise. At rest, biopsies of the gastrocnemius muscle in the patients' nonsymptomatic leg revealed a normal carnitine pool and lactate content compared with control subjects. In contrast, the patients' diseased leg had higher contents of lactate and long-chain acylcarnitines than controls. The muscle short-chain acylcarnitine content in the patients' diseased leg at rest was inversely correlated with peak exercise performance (r = -0.75, P less than 0.05). With graded treadmill exercise, only patients who exceeded their individual lactate threshold had an increase in muscle short-chain acylcarnitine content in the nonsymptomatic leg, which was identical to the muscle carnitine response in normal subjects. In the patients' diseased leg, muscle short-chain acylcarnitine content increased with exercise from 440 +/- 130 to 900 +/- 200 (SE) nmol/g (P less than 0.05). In contrast to the nonsymptomatic leg, there was no increase in muscle lactate content in the diseased leg with exercise, and the change in muscle carnitine metabolism was correlated with exercise duration (r = 0.82, P less than 0.01) and not with the lactate threshold. We conclude that energy metabolism in ischemic muscle of patients with PAD is characterized by the accumulation of acylcarnitines.(ABSTRACT TRUNCATED AT 250 WORDS)


Effect of fatigue on maximal inspiratory pressure-flow capacity.

The inspiratory muscles can be fatigued by repetitive contractions characterized by high force (inspiratory resistive loads) or high velocities of shortening (hyperpnea). The effects of fatigue induced by inspiratory resistive loaded breathing (pressure tasks) or by eucapnic hyperpnea (flow tasks) on maximal inspiratory pressure-flow capacity and rib cage and diaphragm strength were examined in five healthy adult subjects. Tasks consisted of sustaining an assigned breathing frequency, duty cycle, and either a "pressure-time product" of esophageal pressure (for the pressure tasks) or peak inspiratory flow rate (for the flow tasks). Esophageal pressure was measured during maximal inspiratory efforts against a closed glottis (Pesmax), maximal transdiaphragmatic pressure was measured during open-glottis expulsive maneuvers (Pdimax), and maximal inspiratory flow (VImax) was measured during maximal inspiratory efforts with no added external resistance before and after fatiguing pressure and flow tasks. The reduction in Pesmax) with pressure fatigue (-25 +/- 7%) was significantly greater than the change in Pesmax with flow fatigue (-8 +/- 8%, P less than 0.01). In contrast, the reductions in Pdimax (-11 +/- 8%) and VImax (-16 +/- 3%) with flow fatigue were greater than the changes in Pdimax (-0.6 +/- 4%, P less than 0.05) or VImax (-3 +/- 4%, P less than 0.05) with pressure fatigue. We conclude that respiratory muscle performance is dependent not only on the presence of fatigue but whether fatigue was induced by pressure tasks or flow tasks. The specific impairment of Pesmax and not of Pdimax or flow with pressure fatigue may reflect selective fatigue of the rib cage muscles.(ABSTRACT TRUNCATED AT 250 WORDS)


Cardiopulmonary adaptations to pneumonectomy in dogs. I. Maximal exercise performance.

Maximal exercise performance was evaluated in four adult foxhounds after right pneumonectomy (removal of 58% of lung) and compared with that in seven sham-operated control dogs 6 mo after surgery. Maximal O2 uptake (ml O2.min-1.kg-1) was 142.9 +/- 1.9 in the sham group and 123.0 +/- 3.8 in the pneumonectomy group, a reduction of 14% (P less than 0.001). Maximal stroke volume (ml/kg) was 2.59 +/- 0.10 in the sham group and 1.99 +/- 0.05 in the pneumonectomy group, a reduction of 23% (P less than 0.005). Lung diffusing capacity (DL(CO)) (ml.min-1.Torr-1.kg-1) reached 2.27 +/- 0.08 in the combined lungs of the sham group and 1.67 +/- 0.07 in the remaining lung of the pneumonectomy group (P less than 0.001). In the pneumonectomy group, DL(CO) of the left lung was 76% greater than that in the left lung of controls. Blood lactate concentration and hematocrit were significantly higher at exercise in the pneumonectomy group. We conclude that, in dogs after resection of 58% of lung, O2 uptake, cardiac output, stroke volume, and DL(CO) at maximal exercise were restricted. However, the magnitude of overall impairment was surprisingly small, indicating a remarkable ability to compensate for the loss of one lung. This compensation was achieved through the recruitment of reserves in DL(CO) in the remaining lung, the development of exercise-induced polycythemia, and the maintenance of a relatively large stroke volume in the face of an increased pulmonary vascular resistance.


Human tolerance to heat strain during exercise: influence of hydration.

This study determined whether 1) exhaustion from heat strain occurs at the same body temperatures during exercise in the heat when subjects are euhydrated as when they are hypohydrated, 2) aerobic fitness influences the body temperature at which exhaustion from heat strain occurs, and 3) curves could be developed to estimate exhaustion rates at a given level of physiological strain. Seventeen heat-acclimated men [maximal oxygen uptake (VO2max) from 45 to 65 ml.kg-1.min-1] attempted two heat stress tests (HSTs): one when euhydrated and one when hypohydrated by 8% of total body water. The HSTs consisted of 180 min of rest and treadmill walking (45% VO2max) in a hot-dry (ambient temperature 49 degrees C, relative humidity 20%) environment. The required evaporative cooling (Ereq) exceeded the maximal evaporative cooling capacity of the environment (Emax); thus thermal equilibrium could not be achieved and 27 of 34 HSTs ended by exhaustion from heat strain. Our findings concerning exhaustion from heat strain are 1) hypohydration reduced the core temperature that could be tolerated; 2) aerobic fitness, per se, did not influence the magnitude of heat strain that could be tolerated; 3) curves can be developed to estimate exhaustion rates for a given level of physiological strain; and 4) exhaustion was rarely associated with a core temperature up to 38 degrees C, and it always occurred before a temperature of 40 degrees C was achieved. These findings are applicable to heat-acclimated individuals performing moderate-intensity exercise under conditions where Ereq approximates or exceeds Emax and who have high skin temperatures.


A signal-averaging technique for the analysis of human muscle sympathetic nerve activity.

We present a signal-averaging technique for analysis of human muscle sympathetic nerve activity (SNA). Nerve traffic was averaged by coupling signal acquisition to electrocardiographic R waves. The amplitude of the averaged waveform was multiplied by the number of R waves sampled to provide a measure of SNA in arbitrary units. This was compared with SNA measured by manual digitization of hard-copy records. In nine volunteers, SNA was increased or decreased with stepwise infusions of nitroprusside or phenylephrine: there were 10 5-min periods of data in each subject. Across all subjects, the correlation between manual and signal-averaged measures of SNA was excellent during both nitroprusside (r = 0.98) and phenylephrine infusions (r = 0.91) and the slopes of the regression lines were near unity. In three periods of data collection, electrical artifacts were added randomly at frequencies of 0.5 and 0.07 Hz during playback of the signal into the computer. Signal-averaged estimates of SNA were unaffected by artifacts. This technique provides reliable observer-independent measures of SNA.


Contraction-activated glucose uptake is normal in insulin-resistant muscle of the obese Zucker rat.

The rates of muscle glucose uptake of lean and obese Zucker rats were assessed via hindlimb perfusion under basal conditions (no insulin), in the presence of a maximal insulin concentration (10 mU/ml), and after electrically stimulated muscle contraction in the absence of insulin. The perfusate contained 28 mM glucose and 7.5 microCi/mmol of 2-deoxy-D-[3H-(G)]glucose. Glucose uptake rates in the soleus (slow-twitch oxidative fibers), red gastrocnemius (fast-twitch oxidative-glycolytic fibers), and white gastrocnemius (fast-twitch glycolytic fibers) under basal conditions and after electrically stimulated muscle contraction were not significantly different between the lean and obese rats. However, the rate of glucose uptake during insulin stimulation was significantly lower for obese than for lean rats in all three fiber types. Significant correlations were found for insulin-stimulated glucose uptake and glucose transporter protein isoform (GLUT-4) content of soleus, red gastrocnemius, and white gastrocnemius of lean (r = 0.79) and obese (r = 0.65) rats. In contrast, the relationships between contraction-stimulated glucose uptake and muscle GLUT-4 content of lean and obese rats were negligible because of inordinately low contraction-stimulated glucose uptakes by the solei. These results suggest that maximal skeletal muscle glucose uptake of obese Zucker rats is resistant to stimulation by insulin but not to contractile activity. In addition, the relationship between contraction-stimulated glucose uptake and GLUT-4 content appears to be fiber-type specific.


Plasma Met-enkephalin and catecholamine responses to intense exercise in humans.

Native and cryptic Met-enkephalin and catecholamines are coreleased in response to stress. However, it is not known whether Met-enkephalin and catecholamines exhibit concurrent temporal relationships in response to exercise. The purpose of this investigation was to examine the corelease of catecholamines and Met-enkephalin in endurance-trained (n = 6) and untrained (n = 6) male subjects during a 6-min bout of exercise: 4 min at 70% of maximal O2 uptake (VO2max) followed by 2 min at 120% VO2max. Peak catecholamine levels were found at 1 min of recovery. In trained subjects, native Met-enkephalin peaked during exercise at 70% VO2max, declined during exercise at 120% VO2max, and returned to basal levels by 1 min of recovery. In the untrained subjects, native Met-enkephalin peaked at 120% VO2max (6 min) and returned to baseline by 5 min of recovery. In both groups, cryptic Met-enkephalin peaked at 70% VO2max and returned to basal levels during exercise at 120% VO2max. These data demonstrate that during exercise there is a temporal dissociation in plasma levels of Met-enkephalin and catecholamines.


Effect of leukopenia on pulmonary hypertension after heparin-protamine in pigs.

Heparin neutralization by protamine after cardiac surgery and cardiopulmonary bypass may be associated with complement activation, transient leukopenia, thromboxane A2 release, and severe pulmonary hypertension. The role of leukocytes in the heparin-protamine reaction was studied in leukopenic pigs (n = 9) and a control group (n = 8). Leukopenia was induced by pretreatment with cyclophosphamide (30 mg.kg-1.day-1) for 6-7 days. During general anesthesia and after catheterization, baseline recordings of hemodynamics were performed and blood samples were withdrawn. Heparin (250 IU/kg) was injected and measurements were repeated after 10 min. Protamine sulfate (100 mg) was then infused over 2 min and measurements were performed after 2, 5, and 15 min. Prostanoid concentrations were measured by radioimmunoassays. In additional in vitro experiments, the release of thromboxane B2 from washed platelets and leukocytes after heparin-protamine stimulation was measured. Pretreatment with cyclophosphamide reduced leukocyte counts by 95.5% and the number of neutrophils by greater than 99.9%. Protamine infusion increased mean pulmonary arterial pressure by 74 and 46% and pulmonary vascular resistance by 185 and 384% in control and leukopenic animals, respectively. Thromboxane B2 concentrations increased in both groups. Stimulation by heparin, protamine, or heparin and protamine in sequence did not induce any thromboxane A2 release from washed blood cells. It is concluded that leukocytes do not contribute to pulmonary hypertension after heparin-protamine.


Pulmonary vascular protein sieving capability after exposure to high vascular pressures.

We evaluated the ability of the canine in situ left lower lobe (LLL) vasculature to sieve endogenous plasma proteins of various molecular radii (34-124 A) after LLL arterial pressure had been transiently elevated to 23.8 +/- 0.9 (control group, n = 5) or 92.3 +/- 1.4 (SE) Torr (high-pressure group, n = 9) by restricting LLL venous outflow under conditions of constant flow. After LLL flow was returned to natural perfusion, left atrial pressure was elevated in step increments, and LLL lymph and blood samples were collected until filtration-independent lymph-to-plasma protein concentration ratios (CL/CP) were obtained. The osmotic reflection coefficients (sigma d) for total proteins and seven protein fractions (separated by gradient gel electrophoresis) were calculated. The average total protein sigma d of the high-pressure group [0.51 +/- 0.06 (SE)] was significantly lower than that of the control group (0.68 +/- 0.03). Several LLLs of the high-pressure group, however, exhibited normal sigma d's. Protein fraction CL/CP's decreased with increasing molecular radius in both groups, but the CL/CP-molecular radius relationship was displaced upward in the high-pressure group. Pore analysis suggested that the decreases in sigma d could be explained by increases in the fractional flow through a large-pore system.


Reduction in myotendinous junction surface area of rats subjected to 4-day spaceflight.

The surface area of myotendinous junctions (MTJs), expressed relative to the cross-sectional area of myofibrils attached to them, was determined using established morphometric techniques in which the digitlike processes of the cell at MTJs are modeled as circular paraboloids. The relative area, called the folding factor, was measured for six rats after a 4-day spaceflight and six control rats maintained in a vivarium under otherwise identical conditions. Spaceflight resulted in a significant reduction in relative MTJ surface area, from 19.7 +/- 2.3 (SD) in control animals to 13.3 +/- 2.5 for animals after spaceflight. Furthermore, space animals displayed increased numbers of fibroblasts enriched in rough endoplasmic reticulum near the MTJ, a greater number of ribosomes and mitochondria within muscle at the MTJ, and increased occurrence of lesions in the connective tissue near the MTJ. The results indicate that spaceflight, possibly through the removal of gravity-associated loading from muscle, causes a modification in MTJ structure and may result in injuries at MTJs after return to normal loading.


Hypertrophic response to unilateral concentric isokinetic resistance training.

The purposes of this study were to 1) determine the effect of concentric isokinetic training on strength and cross-sectional area (CSA) of selected extensor and flexor muscles of the forearm and leg, 2) examine the potential for preferential hypertrophy of individual muscles within a muscle group, 3) identify the location (proximal, middle, or distal level) of hypertrophy within an individual muscle, and 4) determine the effect of unilateral concentric isokinetic training on strength and hypertrophy of the contralateral limbs. Thirteen untrained male college students [mean age 25.1 +/- 6.1 (SD) yr] volunteered to perform six sets of 10 repetitions of extension and flexion of the nondominant limbs three times per week for 8 wk, using a Cybex II isokinetic dynamometer. Pretraining and posttraining peak torque and muscle CSA measurements for both the dominant and nondominant limbs were determined utilizing a Cybex II isokinetic dynamometer and magnetic resonance imaging scanner, respectively. The results indicated significant (P less than 0.0008) hypertrophy in all trained muscle groups as well as preferential hypertrophy of individual muscles and at specific levels. None of the muscles of the contralateral limbs increased significantly in CSA. In addition, significant (P less than 0.0008) increases in peak torque occurred for trained forearm extension and flexion as well as trained leg flexion. There were no significant increases in peak torque, however, for trained leg extension or for any movement in the contralateral limbs. These data suggest that concentric isokinetic training results in significant strength and hypertrophic responses in the trained limbs.


Aminophylline increases submaximum power but not intrinsic velocity of shortening of frog muscle.

We hypothesized that methylxanthines, such as aminophylline, increase the power developed by submaximally activated frog skeletal muscles by increasing the force developed at any given velocity of shortening. Frog semitendinosus muscles were excised and tested at 20 degrees C in oxygenated control and aminophylline Ringer solutions. Force-velocity relationships were determined and power was calculated from muscles stimulated at frequencies of 80 and 300 Hz. The 300-Hz frequency of stimulation produced a maximum rate of force development. In 50 and 500 microM aminophylline, twitch force increased by 25 +/- 12 and 75 +/- 13%, respectively. Aminophylline did not affect maximum isometric force generation or the shortening velocity at any relative load. At 80-Hz stimulation and in the presence of 500 microM aminophylline, power increased by an average of 11% at 10 of 14 relative loads. At maximum frequencies of stimulation, aminophylline had no effect on any measured parameter. We conclude that aminophylline increases the power developed by submaximally activated frog muscles through an increase in the force generated particularly at the lower velocities of shortening.


Human muscle fatigue after glycogen depletion: a 31P magnetic resonance study.

To differentiate the effects of high energy phosphates, pH, and [H2PO4-] on skeletal muscle fatigue, intracellular acidosis during handgrip exercise was attenuated by prolonged submaximal exercise. Healthy human subjects (n = 6) performed 5-min bouts of maximal rhythmic handgrip (RHG) before (CONTROL) and after prolonged (60-min) handgrip exercise (ATTEN-EX) designed to attenuate lactic acidosis in active muscle by partially depleting muscle glycogen. Concentrations of free intracellular phosphocreatine ([PCr]), adenosine triphosphate ([ATP]), and orthophosphate ([P(i)]) and pH were measured by 31P nuclear magnetic resonance spectroscopy and used to calculate adenosine diphosphate [ADP], [H2PO4-], and [HPO4(2-)]. Handgrip force output was measured with a dynamometer, and fatigue was determined by loss of maximal contractile force. After ATTEN-EX, the normal exercise-induced muscle acidosis was reduced. At peak CONTROL RHG, pH fell to 6.3 +/- 0.1 (SE) and muscle fatigue was correlated with [PCr] (r = 0.83), [P(i)] (r = 0.82), and [H2PO4-] (r = 0.81); [ADP] was 22.0 +/- 5.7 mumol/kg. At peak RHG after ATTEN-EX, pH was 6.9 +/- 0.1 and [ADP] was 116.1 +/- 18.2 mumol/kg, although [PCr] and [P(i)] were not different from CONTROL RHG (P greater than 0.05). After ATTEN-EX, fatigue correlated most closely with [ADP] (r = 0.84). The data indicate that skeletal muscle fatigue 1) is multifactorial, 2) can occur without decreased pH or increased [H2PO4-], and 3) is correlated with [ADP] after exercise-induced glycogen depletion.


Effects of hyperphosphatemia on diaphragmatic strength and endurance.

Effects of an infusion of Na2HPO4 on diaphragm strength, endurance, and magnitude of recovery were evaluated in in situ canine diaphragm strips. Results showed no effect on maximal isometric tetanic tension. Twitch tension and tension in the low- (10-Hz) frequency range were significantly increased (P less than 0.01). Time to fatigue (endurance) increased by 38 +/- 4.5% in the group that received phosphorus compared with its control and decreased by 18.5 +/- 2.5% in the group that received dextrose compared with its control (P less than 0.005). Recovery from fatigue was also significantly improved after the phosphorus infusion. Serum ATP and 2,3-diphosphoglycerate levels were unchanged throughout the experiment. The results of this study support the notion that hyperphosphatemia improves diaphragmatic endurance and recovery from fatigue. The mechanisms involved may in part be due to the phosphate-buffering effects, which limit the extent of the muscle intracellular acidosis produced with fatigue.


Pores of Kohn are filled in normal lungs: low-temperature scanning electron microscopy.

Interalveolar pores of Kohn, small uniform-sized epithelium-lined openings in alveolar walls of normal lung, have historically been demonstrated with electron-microscopic techniques that remove water. We show these pores to be present but almost invariably filled with material when water and surfactant are preserved in frozen hydrated lung examined with low-temperature scanning electron microscopy. In the normal mouse, 16 open empty pores per alveolus were found in instillation-fixed dried lung vs. less than 1 per alveolus in frozen hydrated lungs (P less than 0.001). In the normal rat, 13 pores were seen per alveolus in instillation-fixed dried lung vs. less than 1 per alveolus in frozen hydrated lungs (P less than 0.001). We suggest that pores of Kohn 1) function primarily as conduits for interalveolar movement of alveolar liquid, surfactant components, and macrophages, 2) provide distributed sites for tubular myelin storage without increasing gas diffusion pathway thickness in the alveolar subphase itself, and 3) do not function as pathways for collateral ventilation during normal breathing in the absence of atelectasis or obstruction.


Characteristics of spontaneous and evoked motility in the isolated perfused porcine duodenum.

The aims of the study were to evaluate characteristics of spontaneous motility and of the ascending excitatory peristaltic reflex (AEPR) and intraluminal cross-sectional area in the isolated perfused porcine duodenum. The parameters were measured by an intraluminal catheter by use of the perfused side-hole technique and impedance planimetry. Respiratory parameters such as pH and oxygen consumption and the arterial perfusion pressure were monitored and did not vary significantly throughout the study time. Spontaneous motility was intense at the beginning but declined and disappeared within 45-90 min. It was abolished by atropine, epinephrine, and UK-14,304 (an alpha 2-adrenoceptor agonist). Secondary motility was evoked by intraluminal balloon distensions by raising the balloon pressure to 1.5 kPa for 1-min periods. Reproducible results regarding the AEPR, external balloon diameters to elicit the AEPR, and intraluminal cross-sectional area were obtained. The order of potency (pD2 values) for inhibition of the AEPR was the selective M3-receptor antagonist 4-DAMP greater than atropine greater than the selective M2-receptor antagonist AFDX-116 greater than the selective M1-receptor antagonist pirenzepine greater than hexamethonium. 4-DAMP was 16 and 29 times more potent than AFDX-116 (P less than 0.02) and pirenzepine (P less than 0.02). None of the drugs altered the intraluminal cross-sectional area during the balloon distensions. The model provides the opportunity for physiological and pharmacological studies of duodenal motility and duodenal cross-sectional area devoid of extrinsic neural and endocrine effects. The abolishment of the AEPR by atropine is caused by blockade of the M3-receptor in the porcine duodenum.


Medullary CO2 chemoreceptor neuron identification by c-fos immunocytochemistry.

In a search for CO2 chemoreceptor neurons in the brain stem, we used immunocytochemistry to monitor the expression of neuronal c-fos, a marker of increased activity, after 1 h of exposure to CO2 in five groups of Sprague-Dawley rats (294 +/- 20 g): five air breathing controls, three breathing 10% CO2, three breathing 13% CO2, three breathing 15% CO2, and three breathing 15% CO2 and treated with morphine (10 mg/kg sc). After exposure the rats were anesthetized with pentobarbital sodium and perfused intracardially with 4% paraformaldehyde. The brain stem was removed and cryoprotected, and then 50-microns frozen sections were cut and immunostained for the fos protein. Brain stem fos-immunoreactive neurons were plotted and counted in the superficial 0.5 mm of the ventral medullary surface. Thirteen to 15% CO2 evoked fos-like immunoreactivity (FLI) in 321 +/- 146 neurons/rat. Significant CO2-induced labeling was confined within the superficial 150 microns: 67% of identified cells were less than 50 microns below the surface, greater than 90% between 1.0 and 3.0 mm from the midline, and approximately 60% in the rostral half of the medulla. Thirteen to 15% CO2 also evoked FLI in the area of the nucleus tractus solitarius but not in other medullary regions. Morphine (10 mg/kg sc) did not suppress high CO2-evoked FLI in either the ventral medullary surface or the nucleus tractus solitarius, although it eliminated excitement and hyperventilation. We suggest that respiratory CO2 chemoreceptor neurons can be identified in rats by their expression of c-fos after 1 h of hypercapnia.(ABSTRACT TRUNCATED AT 250 WORDS)


Nigericin forms highly stable complexes with lithium and cesium.

Nigericin is a monocarboxylic polyether molecule described as a mobile K+ ionophore unable to transport Li+ and Cs+ across natural or artificial membranes. This paper shows that the ion carrier molecule forms complexes of equivalent energy demands with Li+, Cs+, Na+, Rb+, and K+. This is in accordance with the similar values of the complex stability constants obtained from nigericin with the five alkali metal cations assayed. On the other hand, nigericin-alkali metal cation binding isotherms show faster rates for Li+ and Cs+ than for Na+, K+, and Rb+, in conditions where the carboxylic proton does not dissociate. Furthermore, proton NMR spectra of nigericin-Li+ and nigericin-Cs+ complexes show wide broadenings, suggesting strong cation interaction with the ionophore; in contrast, the complexes with Na+, K+, and Rb+ show only clear-cut chemical shifts. These latter results support the view that nigericin forms highly stable complexes with Li+ and Cs+ and contribute to the explanation for the inability of this ionophore to transport the former cations in conditions where it catalyzes a fast transport of K+ greater than Rb+ greater than Na+.


Hexokinase receptors: preferential enzyme binding in normal cells to nonmitochondrial sites and in transformed cells to mitochondrial sites.

Hexokinase plays an important role in normal glucose-utilizing tissues like brain and kidney, and an even more important role in highly malignant cancer cells where it is markedly overexpressed. In both cell types, normal and transformed, a significant portion of the total hexokinase activity is bound to particulate material that sediments upon differential centrifugation with the crude "mitochondrial" fraction. In the case of brain, particulate binding may constitute most of the total hexokinase activity of the cell, and in highly malignant tumor cells as much as 80 percent of the total. When a variety of techniques are rigorously applied to better define the particulate location of hexokinase within the crude "mitochondrial fraction," a striking difference is observed between the distribution of hexokinase in normal and transformed cells. Significantly, particulate hexokinase found in rat brain, kidney, or liver consistently distributes with nonmitochondrial membrane markers whereas the particulate hexokinase of highly glycolytic hepatoma cells distributes with outer mitochondrial membrane markers. These studies indicate that within normal tissues hexokinase binds preferentially to nonmitochondrial receptor sites but upon transformation of such cells to yield poorly differentiated, highly malignant tumors, the overexpressed enzyme binds preferentially to outer mitochondrial membrane receptors. These studies, taken together with the well-known observation that, once solubilized, the particulate hexokinase from a normal tissue can bind to isolated mitochondria, are consistent with the presence in normal tissues of at least two different types of particulate receptors for hexokinase with different subcellular locations. A model which explains this unique transformation-dependent shift in the intracellular location of hexokinase is proposed.


High-conductance pathways in mitochondrial membranes.

The outer and inner membranes of mitochondria have recently been studied with the patch clamp technique. What has emerged is still an ill-defined picture for either membrane, primarily for the wide range of conductances found. Interestingly, however, a few conductances (in the range of 10-80 pS) seem to be ubiquitously distributed. Parallel studies in situ and in reconstituted systems have allowed the assignment to distinct membrane locations of some conductances, whose physiological role is, however, not yet elucidated.


Cytoskeletal elements are required for the formation and maturation of autophagic vacuoles.

We evaluated the role of cytoskeletal elements in the degradation of endogenous proteins via autophagy using biochemical and morphological techniques. In the absence of exogenous amino acids, degradation of endogenous proteins was enhanced in cultured normal rat kidney cells. This enhanced degradative state was accompanied by a 4-fold increase in the occurrence of autophagic vacuoles. In the presence of drugs that induce the depolymerization of microfilaments (cytochalasins B and D) or microtubules (nocodazole), protein degradation was not enhanced in nutrient-deprived cells. Although these drugs had similar inhibitory effects on the protein degradation, their effect on autophagy differed. Cytochalasins B and D interfered with the formation of the autophagosome. In cells treated with these drugs, the fractional volume represented by autophagic vacuoles was not substantially increased despite nutrient depletion. On the contrary, nocodazole appeared to have no effect on the formation of autophagosomes. Instead, this drug suppressed the delivery of hydrolytic enzymes, thereby resulting in an accumulation of acidic autophagic vacuoles containing undegraded cellular components.


Transforming growth factor-beta 1 mediated alterations in ribonucleotide reductase gene expression in BALB/c 3T3 fibroblasts.

Transforming growth factor-beta 1 (TGF-beta 1) stimulated DNA synthesis (3-fold) in BALBc/3T3 fibroblasts following 24 hours of growth factor exposure. Since ribonucleotide reductase is important for the coordination of DNA synthesis and cell proliferation, we investigated the hypothesis that cells like BALB/c 3T3, which are TGF-beta 1 responsive, would exhibit modifications in expression of the gene for ribonucleotide reductase following growth factor treatment. We observed 2.6, 4.1, and 4.8-fold increases in ribonucleotide reductase activity following TGF-beta 1 exposure for 6, 12, and 24 hours, respectively. Increased ribonucleotide reductase R2 gene expression (3, 3.7, and 4.5-fold) and R1 gene expression (2,2.5, and 2.6-fold) were observed following 6, 12, and 24 hours of TGF-beta 1 treatment, respectively. Western blots indicated 2.2, 3.1, and 4.1-fold increases in protein R2 levels at 6, 12, and 24 hours exposure to TGF-beta 1, whereas 2.6 and 3.3-fold elevations in R1 protein levels were observed at 12 and 24 hours post-TGF-beta 1 exposure. These TGF-beta 1 mediated modifications in ribonucleotide reductase gene expression occurred, in part, prior to any detectable changes in the rate of DNA synthesis, demonstrating alterations in the normal regulation of ribonucleotide reductase. Furthermore, these alterations could be markedly reduced by prolonged pretreatment with 12-O-tetradecanoylphorbol-13-acetate (R2 gene expression increased by only 1.3, 1.5 and 2.3-fold after 6, 12, and 24 hours of TGF-beta 1 treatment, respectively), suggesting a role for a protein kinase C pathway in the TGF-beta 1 regulated changes in ribonucleotide reductase gene expression. These results indicate for the first time that TGF-beta 1 can regulate the expression of the two genes for ribonucleotide reductase in BALB/c 3T3 fibroblasts, and suggest that regulation of these genes plays an important role in critical events involved in growth factor modulation of normal and transformed cell proliferation.


Protein kinase C remains functionally active during TPA induced neuronal differentiation of SH-SY5Y human neuroblastoma cells.

SH-SY5Y human neuroblastoma cells can be induced to differentiate into a neuronal phenotype by treatment with 12-O-tetradecanoylphorbol-13-acetate (TPA). In other cell systems, TPA treatment frequently leads to down-regulation of protein kinase C (PKC). However, we now report that TPA-treated and non-treated SH-SY5Y cells express PKC-alpha, but not PKC-beta and PKC-gamma, mRNA. Furthermore, only a slight down-regulation of the PKC-alpha protein could be seen during prolonged treatment with 16 nM TPA, the concentration giving optimal differentiation. In contrast, a higher concentration of TPA (1.6 microM) results in a poor neuronal differentiation and a complete down-regulation of PKC-alpha. PKC-alpha was rapidly translocated to the particulate fraction and remained membrane bound for at least 4 days during treatment with 16 nM TPA. In such cells a sustained increased level of the phosphorylated form of a 80,000 Dalton PKC-substrate was found. In addition to this sustained augmented phosphorylation, administration of fresh TPA at day 4 caused a small but reproducible further increased level of phosphorylated substrate. When the PKC activity was measured by the histone phosphorylation assay a substantial fraction of the initial enzyme activity could still be detected after 4 days of TPA treatment. Taken together, the data demonstrate that PKC remains functionally active during TPA induced differentiation of SH-SY5Y cells, which may suggest a continuous role for the enzyme during the differentiation process.


Brefeldin A, thapsigargin, and AIF4- stimulate the accumulation of GRP78 mRNA in a cycloheximide dependent manner, whilst induction by hypoxia is independent of protein synthesis.

The glucose regulated proteins (GRPs) are major structural components of the endoplasmic reticulum (ER) and are involved in the import, folding, and processing of ER proteins. Expression of the glucose regulated proteins (GRP78 and GRP94) is greatly increased after cells are exposed to stress agents (including A23187 and tunicamycin) which inhibit ER function. Here, we demonstrate that three novel inhibitors of ER function, thapsigargin (which inhibits the ER Ca(2+)-ATPase), brefeldin A (an inhibitor of vesicle transport between the ER and Golgi) and AIF4-, (which inhibits trimeric G-proteins), can increase the expression of both GRP78 and 94. The common characteristic shared by activators of GRP expression is that they disrupt some function of the ER. The increased levels of GRPs may be a response to the accumulation of aberrant proteins in the ER or they may be increased in response to structural/functional damage to the ER. The increased accumulation of GRP78 mRNA after exposure of cells to either thapsigargin, brefeldin A, AIF4-, A23187, or tunicamycin can be blocked by pre-incubation in cycloheximide. In contrast, accumulation of GRPs after exposure to hypoxia was independent of cycloheximide. In addition, the protein kinase inhibitor genistein blocked the thapsigargin induced accumulation of GRP78 mRNA, whereas the protein phosphatase inhibitor okadaic acid caused increased accumulation of GRP78 mRNA. The data indicates that there are at least 2 mechanisms for induced expression of GRPs, one of which involves a phosphorylation step and requires new protein synthesis (e.g., thapsigargin, A23187) and one which is independent of both these steps (hypoxia).


EGF receptor regulation in normal mouse mammary gland.

Estrogen (E), progesterone (P), and epidermal growth factor (EGF) are known to regulate growth and development of the normal mammary gland, and it is possible that EGF may interact with E and/or P. Estrogen (ER), progesterone (PR), and EGF receptors (EGF-R) have been detected in both mammary epithelial and stromal cells, and the relative roles of the various cells types in hormone-dependent growth regulation are not known. The present studies were undertaken to determine if E and/or P influence EGF action by exerting a regulatory effect on EGF-R levels and which cell types are affected. The comparative effects of ovariectomy and hormone treatments on EGF-R levels were examined in immature, pubertal 5-week-old and sexually mature 10-week-old female mice. EGF-R were characterized as a single class of high affinity sites and EGF-R concentration was 2-fold higher in glands of 5-week-old mice. Ovariectomy had no significant effect on EGF-R concentration in either age group, and treatment with E and/or P had no effect on EGF-R levels in either epithelial or stromal cells in 5-week-old mice. In contrast, E+P treatment caused a 2-fold increase in receptor concentration in 10-week-old mice in the mammary epithelium. Thus it appears that the developmental state of the gland may determine the nature and extent of the interaction of of EGF, E, and P.


Transforming growth factor-beta 1 rapidly induces Hsp70 and Hsp90 molecular chaperones in cultured chicken embryo cells.

In this report we show that: (1) molecular chaperones in the heat shock protein (hsp) family are a new class of cellular proteins induced by Transforming Growth Factor-beta 1 (TGF beta), a cytokine present in serum, (2) rapid induction of Hsc70 precedes a general increase in protein synthesis and may be a preparatory event, (3) TGF beta is a potent regulator of overall protein synthesis in chicken embryo cells (CEC), and (4) isoforms of Hsp90 with different biochemical properties exist, raising the possibility that they may have different functions. TGF beta can substitute for serum in stimulating synthesis of members of the Hsp90 and Hsp70 families of stress proteins, whereas other cytokines, including PDGF, FGF, and EGF, were not effective nor did they enhance the stimulatory effect of TGF beta on the hsp's. Analysis of the induction of hsp's using one- and two-dimensional polyacrylamide gel electrophoresis indicated that members of the Hsp70 family of molecular chaperones were induced rapidly by TGF beta, reaching maximum rates of accumulation by 5 hours of treatment. Total protein synthesis increased more slowly, undergoing an approximately twofold increase in 24 hours. Using a modified protocol for two-dimensional gel electrophoresis, the Hsp90 protein family was separated into four isoelectric forms, two of which were phosphorylated (Hsp90-2 and -4). These phosphorylated isoforms turned over faster than the unphosphorylated forms of Hsp90. All four isoforms were heat inducible, but only Hsp90-2 and -3 were induced rapidly by TGF beta, again within 5 hours of treatment. The effects of serum on these protein families were similar to those of TGF beta, suggesting that this cytokine may be the serum component primarily responsible for up-regulating members of the Hsp90 and Hsp70 families. We hypothesize that cells rapidly increase their chaperoning capacity for newly synthesized polypeptides in preparation for an increase in the rate of synthesis of proteins up-regulated by TGF beta.


C-fos expression is hypersensitive to serum-stimulation in cultured cystic kidney cells from the C57BL/6J-cpk mouse.

Cystic kidneys of the C57BL/6J-cpk murine model of polycystic kidney disease show a marked overexpression of the proto-oncogenes c-fos, c-myc, and c-Ki-ras, consistent with an increased rate of cell proliferation and an altered state of differentiation. To determine if cystic cells have increased responsiveness to stimulation with mitogenic agents, quiescent primary cultures from normal and cystic cpk kidneys were treated with fetal bovine serum (FBS), 8-bromo-cAMP (cAMP), or epidermal growth factor (EGF). The level of c-fos induction following stimulation by FBS was found to be dramatically higher in cystic cells than in normal cells; whereas induction by cAMP or EGF was essentially the same in both cell types and much less than that seen in FBS-stimulated cells. To determine if this serum hypersensitivity reflects an increased proliferative state in vivo, c-fos induction was examined in cultures derived from normal kidneys stimulated to regenerate by folic acid-induced acute renal injury. As with cystic kidneys, the folic acid-injured kidneys showed increased c-fos responsiveness to FBS in cell culture. These experiments suggest that cystic and regenerating kidneys have an altered phenotypic state in vivo that is manifested in cell culture by serum hypersensitivity. However, whereas the folic acid-injured kidneys ultimately reestablish normal kidney function, cystic kidneys further progress to renal failure, suggesting that cystic epithelial cells are locked in this altered state of differentiation.


Cytokines involved in monocyte mediated tumor cell death and growth inhibition in serum-free medium.

In a serum-free culture system, the release of TNF, lI-1, lI-6, IFN-alpha, and IFN-beta during interaction of elutriated human monocytes (MO) with human tumor cells (TC) was studied by ELISA-technique. Contributions of these cytokines to inhibition of TC-growth and to induction of TC-death by supernatants (SU) gained from such MO/TC-interaction cultures were investigated using affinity chromatography for removal of individual cytokines. Although the TC used are relatively insensitive to recombinant human TNF, withdrawal of TNF causes 50% to 75% reduction of SU-induced TC-death rates, suggesting that susceptibility to TNF is raised during MO/TC-interaction by the other cytokines. Individual removal of other cytokines does not cause reduction of SU-mediated TC-death. However, combined withdrawal of lI-1 and IFN-alpha/beta causes in 2 of 4 TC-lines significant reduction of TC-death. Combined removal of TNF, IFN-alpha/beta, lI-1, and lI-6 leads to complete prevention of SU-mediated growth inhibitory and lytic effects, suggesting that besides these cytokines other signals are not involved significantly. SU-effects can be mimicked by appropriate combinations of authentic cytokines. The response of TC to SU- or cytokine-exposure is strikingly dependent on TC-density, leading at subconfluent TC-density exclusively to inhibition of growth and at postconfluent TC-density to induction of cell death. The principal effect of SU or cytokine combinations in this context seems to be the activation of growth inhibitory signal transduction pathways leading to TC-death in postconfluent TC-populations exclusively if growth stimulatory pathways are activated at the same time. Mouse L cells do not follow this reaction pattern: Their death is exclusively dependent on the presence of TNF in SU and they die upon SU-exposure at postconfluent as well as at subconfluent cell density.


Independent regulation of prostaglandin production and the stress response in human fibroblasts.

The stress, or heat shock response of eukaryotic cells is characterized by dramatic changes in the metabolism of responding cells, most notably the increased synthesis of a group of proteins known as heat shock proteins. In this study, we examined the relationship of prostaglandin synthesis/release to the stress response. Stress protein synthesis was induced with sodium arsenite, and prostaglandin E2 and prostacyclin (measured as 6-keto PGF1 alpha) levels were determined by enzyme immunoassay. The stress response was monitored by the incorporation of [35S]methionine and separation of protein by one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Prostaglandin synthesis and the stress response were both induced by sodium arsenite. However, aspirin, a cyclooxygenase inhibitor, inhibited arsenite-induced prostaglandin synthesis but did not inhibit stress protein synthesis. Conversely, the calcium ionophore A23187 also stimulated prostaglandin synthesis, but did not induce the stress response. The results of this study indicate that sodium arsenite, a stress response inducer, stimulates prostaglandin production, but this appears to be a correlative rather than causative occurrence in the stress response. Determination of the cytotoxicity of arsenite indicated a high correlation of stimulation of prostaglandin release with cytotoxicity.


Relationship between culture conditions and the dependency on mitochondrial function of mammalian cell proliferation.

In cultured mammalian cells, the relationship was investigated between mitochondrial function and proliferation under various culture conditions. Continuous inhibition of the expression of the mitochondrial genome was used to reduce the activity of enzymes involved in oxidative phosphorylation by 50% at every cell division. Under these conditions, culturing in relatively poor media resulted in arrest of the proliferation of most cell lines after 1 cell division. This was preceded by decreasing levels of ATP and increasing levels of ADP, suggesting that the ATP-generating capacity of the cells was limiting. Culturing in richer media led to arrest of the proliferation after 5 to 6 divisions, but accumulation of ADP was not observed. Addition of pyruvate to rich culture media and, at least for 1 cell line, increasing the CO2 levels, completely prevented proliferation arrest. Inability to synthesise metabolic precursors via mitochondrial intermediary metabolism probably explains growth arrest of cells cultured in rich media. Pyruvate and CO2 were, however, without effect on the proliferation arrest of cells cultured in relatively poor media. Therefore, pyruvate dependency for growth of cells without functional mitochondria holds true only under culture conditions where the ATP-generating capacity of the cells is not limiting.


Reversible G1 arrest of a human lung epithelial cell line by staurosporine.

Staurosporine, a microbial-derived protein kinase inhibitor, reversibly blocked non-synchronized, replicating cultures of the human lung epithelial cell line EKVX in the G1 phase of cell cycle and inhibited DNA synthesis and cell replication. The mechanism of this cell-cycle arrest in EKVX cells by staurosporine was likely due to inhibition of protein kinase C (PKC) because: 1) dose-dependent inhibition of DNA synthesis occurred at levels of staurosporine that inhibit phosphorylation of PKC substrate, 2) inhibition of DNA synthesis was also seen after treatment with another PKC inhibitor H7, but not by the chemically similar HA1004, which has a relative inhibitory specificity for cAMP-dependent protein kinase, and 3) the DNA synthesis was not inhibited by specific tyrosine kinase inhibitors Genistein and Lavendustin A at concentrations that inhibit tyrosine kinase activity. Removal of staurosporine from cell culture media resulted in a rebound in PKC activity and synchronized DNA synthesis in EKVX cultures. The reversibility of the inhibition was noted even after 5 days of treatment with staurosporine, and DNA synthesis remained synchronized for at least two rounds of cell replication after removal of staurosporine. Flow cytometric analysis confirmed that more than 90% of the cell population was blocked in the G1 phase after cells were treated with staurosporine for 24 h. Agents such as staurosporine may be useful for synchronizing cell populations to study cell-cycle specific biochemical events important for the regulation of cell replication in the EKVX cell line.


DNA-binding domain of RCC1 protein is not essential for coupling mitosis with DNA replication.

The RCC1 protein that is required for coupling mitosis with the S phase has a DNA-binding domain in the N-terminal region outside the repeat. We found that RCC1 protein without any DNA-binding activity complemented the tsBN2 mutation with the same efficiency as that of intact RCC1 protein. In ts+ transformants of tsBN2 cells transfected with the RCC1 cDNA lacking the DNA-binding domain, an endogenous RCC1 disappeared at 39.5 degrees C, and the deleted RCC1 protein encoded by the transfected cDNA was found in the cytoplasm, but a significant amount of it was also found in the nuclei. This deleted RCC1 protein was eluted from the nuclei with the same concentration of NaCl and DNase I as was used for the intact RCC1 protein in BHK21 cells. Furthermore, the deleted RCC1 protein co-migrated with the nucleosome fraction on sucrose density gradient analysis. These results indicate that the RCC1 protein binds chromatin with the aid of other unknown protein(s). Thus, the DNA-binding domain of RCC1 protein is not essential for coupling between the S and M phases, but was shown instead to function as a nuclear translocation signal.


Effects of vinblastine, podophyllotoxin and nocodazole on mitotic spindles. Implications for the role of microtubule dynamics in mitosis.

Inhibition of mitosis by many drugs that bind to tubulin has been attributed to depolymerization of microtubules. However, we found previously that low concentrations of vinblastine and vincristine blocked mitosis in HeLa cells with little or no depolymerization of spindle microtubules, and spindles appeared morphologically normal or nearly normal. In the present study, we characterized the effects of vinblastine, podophyllotoxin and nocodazole over broad concentration ranges on mitotic spindle organization in HeLa cells. These three drugs are known to affect the dynamics of microtubule polymerization in vitro and to depolymerize microtubules in cells. We wanted to probe further whether mitotic inhibition by these drugs is brought about by a more subtle effect on the microtubules than net microtubule depolymerization. We compared the effects of vinblastine, podophyllotoxin and nocodazole on the organization of spindle microtubules, chromosomes and centrosomes, and on the total mass of microtubules. Spindle organization was examined by immunofluorescence microscopy, and microtubule polymer mass was assayed on isolated cytoskeletons by a quantitative enzyme-linked immunoadsorbence assay for tubulin. As the drug concentration was increased, the organization of mitotic spindles changed in the same way with all three drugs. The changes were associated with mitotic arrest, but were not necessarily accompanied by net microtubule depolymerization. With podophyllotoxin, mitotic arrest was accompanied by microtubule depolymerization. In contrast, with vinblastine and nocodazole, mitotic arrest occurred in the presence of a full complement of spindle microtubules. All three drugs induced a nearly identical rearrangement of spindle microtubules, an increasingly aberrant organization of metaphase chromosomes, and fragmentation of centrosomes. The data suggest that these anti-mitotic drugs block mitosis primarily by inhibiting the dynamics of spindle microtubules rather than by simply depolymerizing the microtubules.


Subcellular imaging of calcium exchange in cultured cells with ion microscopy.

Calcium to calcium exchange between intracellular and extracellular pools has been imaged directly in individual cells using stable 44Ca (98.78% enrichment) in the nutrient medium and an isotopic imaging technique, ion microscopy. Observations were made by imaging mass 40 to determine the native intracellular calcium (40Ca), and mass 44 to localize the 44Ca from the extracellular medium that exchanged with cellular calcium. LLC-PK1 porcine kidney epithelial cells were exposed to the nutrient medium with 1.87 mM stable 44Ca for 0, 1, 2, 5, 8, 20, 60 and 90 min, and cryogenically prepared prior to ion microscopic analysis. The cell nucleus, the Golgi region and the remaining cell cytoplasm could be spatially resolved to within about 0.5 microns using the ion microscope. On the basis of the kinetics of 40Ca to 44Ca exchange it was observed that all three compartments had a rapidly exchanging pool of calcium, which took about 2 min to exchange. A moderately rapidly exchanging pool of calcium was identified between 2 and 20 min of calcium exchange. The cells had exchanged about 50% of their total internal calcium with the external calcium in less than 20 min. The remaining 50% of the cellular calcium could be classified as a slowly exchanging pool. Isotopic images of 39K and 23Na were recorded along with 40Ca and 44Ca images to assess the health status of the cell. Isotopic imaging has the unique ability to distinguish intracellular calcium from the extracellular calcium that enters cells and has enormous potential for studies of calcium transport under physiological and pathological conditions.


Differential expression of the VLA family of integrins along the crypt-villus axis in the human small intestine.

Regulation of epithelial cell proliferation, migration and differentiation under physiological conditions remains poorly understood. Interaction of the cells with their underlying basement membrane through integrins, a specific subset of cell surface binding proteins, is one potential mechanism. In the present work, I examined this hypothesis by investigating the distribution of a variety of epithelial basement membrane proteins and the expression of the members of the VLA family of integrins in the adult intestinal epithelium. Indeed, this rapidly renewing simple epithelium contains within its functional unit, the crypt-villus axis, essentially two distinct cell populations: the proliferative and undifferentiated crypt cells and the mature enterocytes on the villus. Although immunolocalization of basement membrane molecules revealed that laminin, type IV collagen and heparan sulfate proteoglycan are distributed homogeneously all along the crypt-villus axis, other non-exclusive basement membrane components were found differentially expressed. Tenascin was concentrated at the base of both villus and lower crypt cells while cellular fibronectin was mostly detected in association with the crypt cells. Moreover, VLA beta 1 as well as 5 of the 6 VLA alpha subunits tested were expressed by intestinal epithelial cells under specific patterns of staining. The beta 1 and alpha 6 subunits were strongly detected at the base of all enterocytes while alpha 5, also detected all along the crypt-villus axis, was weaker and consistently appeared with a punctated/interrupted pattern. On the other hand, the VLA alpha 1, alpha 2 and alpha 3 were expressed at the basolateral domains of enterocytes under distinctive crypt-villus gradients. The alpha 1 subunit was detected at the base of all epithelial cells but lateral staining was only observed in differentiating cells (middle and upper crypt). Finally, in most specimens, alpha 2 and alpha 3 displayed strictly complementary staining patterns for the lower crypt region (alpha 2+, alpha 3-) and the upper crypt-to-villus region (alpha 2-, alpha 3+). Taken together, these data emphasize that proliferation, migration and differentiation in the normal state are susceptible to various influences including compositional changes in the basement membrane and differential expression of receptors for these components.


Collagen-induced rapid morphogenesis of human mammary epithelial cells: the role of the alpha 2 beta 1 integrin.

The cell line MTSV1-7, originally derived by immortalizing mammary epithelial cells cultured from human milk was able to form three-dimensional structures in collagen gel. We have now found that these cells, cultured as a monolayer, are able to undergo rapid morphogenesis forming ridges and balls around collagen fibres, when soluble collagen type I is added to the medium. Monoclonal antibodies to the alpha 2 (P1E6) and beta 1- (mAB13) subunits of VLA-2, but not to the alpha 3-subunit (P1B5) of VLA-3, could block this collagen-induced rapid morphogenesis (CIRM). The effect of the antibodies on cell attachment, spreading, and migration on collagen gels was analyzed to identify alpha 2 beta 1 dependent steps which might be involved in CIRM. The results suggest that while other proteins, besides alpha 2 beta 1, are also involved in cell attachment and migration, cell spreading was specifically blocked by antibodies to the VLA-2, but not to the VLA-3 integrin. The results demonstrate that the alpha 2 beta 1 integrin plays a crucial role in the collagen-induced morphogenesis of human mammary epithelial cells and implicate the process of VLA-2-dependent cell spreading as an important step in this morphogenesis.


Antagonistic effects of TGF-beta 1 and MSF on fibroblast migration and hyaluronic acid synthesis. Possible implications for dermal wound healing.

The migration of adult skin fibroblasts into three-dimensional collagen gel matrices is differentially affected by cell density, with subconfluent cells displaying a significantly elevated level of migration compared to confluent ones. Fetal fibroblasts differ from adult cells in that they display an elevated level of migration at both subconfluent and confluent cell densities. We have previously reported that this difference in behaviour results from the secretion by fetal fibroblasts of a 'migration stimulating factor' (MSF) which is not made by their normal adult counterparts, and that MSF appears to act by stimulating the synthesis of hyaluronic acid (HA). Data presented in this communication indicate that (a) MSF specifically stimulates the synthesis of high molecular weight species of HA, (b) TGF-beta 1 inhibits the elevated migration of adult fibroblasts plated at subconfluent cell density, (c) under these conditions, TGF-beta 1 induces a parallel decrease in the synthesis of high molecular weight HA and increase in the synthesis of low molecular weight HA, (d) TGF-beta 1 is a potent antagonist of MSF, effectively blocking its stimulation of cell migration and synthesis of high molecular weight HA, and (e) the inhibition of fibroblast migration by TGF-beta 1 does not appear to be a chemotactic response dependent upon the existence of a concentration gradient of the cytokine. Our observations regarding the inhibitory effects of TGF-beta 1 on fibroblast migration into 3D collagen gels stand in marked contrast to various published reports indicating that this cytokine stimulates the migration of human skin fibroblasts through the pores of polycarbonate filters as used in modified Boyden chamber assays; this discrepancy underscores the importance of the substratum in modulating cellular response to cytokines. Our results are discussed in terms of the possible combined contribution of MSF and TGF-beta 1 to wound healing.


Cell cycle modification during the transitions between meiotic M-phases in mouse oocytes.

When metaphase II-arrested mouse oocytes (M II) are activated very soon after ovulation, they respond abortively by second polar body extrusion followed by another metaphase arrest (metaphase III, M III; Kubiak, 1989). The M II/M III transition resembles the natural transition between the first and second meiotic metaphases (M I/M II). We observed that a similar sequence of events takes place during these two transitions: after anaphase, a polar body is extruded, the microtubules of the midbody disappear rapidly and a new metaphase spindle forms. The MPM-2 monoclonal antibody (which reacts with phosphorylated proteins associated with the centrosome during M-phase) stains discrete foci of peri-centriolar material only in metaphase arrested oocytes; during both transitional periods, a diffuse staining is observed, suggesting that these centrosomal proteins are dephosphorylated, as in a normal interphase. However, the chromosomes always remain condensed and an interphase network of microtubules is never observed during the transitional periods. Incorporation of 32P into proteins increases specifically during the transitional periods. Pulse-chase experiments, after labeling of the oocytes in M phase with 32P, showed that a 62 kDa phosphoprotein band disappears at the time of polar body extrusion. Histone H1 kinase activity (which reflects the activity of the maturation promoting factor) drops during both transitional periods to the level characteristic of interphase and then increases when the new spindle forms. Both the M I/M II and M II/M III transitions require protein synthesis as demonstrated by the effect of puromycin. These results suggest that the two M-phase/M-phase transitions are probably driven by the same molecular mechanism.


Expression and function of ras proto-oncogene proteins in human sperm cells.

The presence and role of c-ras proteins were investigated in mature human sperm cells. The v-H-ras monoclonal antibody (mAb) against the c-ras protein, p21, reacted specifically with the acrosomal region of methanol-fixed as well as unfixed-live capacitated and non-capacitated human sperm cell in the indirect immunofluorescence technique. The v-H-ras mAb predominantly recognized c-ras protein of 21 kDa on the Western blot of lithium diiodosalicylate (LIS)-solubilized human sperm preparation. The incubation of sperm cells with v-H-ras mAb affected the sperm cell function in the human sperm penetration assay. The antibody significantly reduced the acrosome reaction and release of acrosin activity from the sperm cells. There was no effect of the mAb on percentage motility, although the mAb significantly affected various motility characteristics such as linearity, amplitude of lateral head displacement (ALH) and beat frequency, the motility parameters involved in the hyperactivation phenomenon of sperm cells leading to capacitation and acrosome reaction. These results suggest that the c-ras or c-ras-like proteins are present in mature sperm cell and may have a role in capacitation and/or acrosome reaction of human sperm cell.