Genome and Hormones: Gender Differences in Physiology: Selected Contribution: Estrogen receptor-alpha gene transfer inhibits proliferation and NF-kappa B activation in VSM cells from female rats

Epidemiological studies have demonstrated that hormonereplacement therapy with estrogen (E₂) or E₂plus progesterone in postmenopausal women decreases the age-associatedrisk of cardiovascular disease by 30-50%. Treatment of vascularsmooth muscle (VSM) cells with physiological concentrations ofE₂ has been shown to inhibit growth factor-stimulated cellproliferation. In this study, we tested the hypothesis thatE₂ inhibits the age-associated increase in VSM cellproliferation by inhibiting nuclear factor (NF)-B pathway. Weinvestigated the effects of E₂ treatment andadenovirus-mediated estrogen receptor (ER)- gene transfer on cellproliferation and NF-B activation using VSM cells cultured from3-mo-old and 24-mo-old Fischer 344 female rats. Our results demonstratethat VSM cell proliferation was significantly increased(P < 0.05) in aged compared with young adult femalerats. Treatment of VSM cells with physiological concentrations ofE₂ inhibited VSM cell proliferation, and this inhibitionwas significantly greater (P < 0.05) in cells from aged female rats compared with young adults. The inhibitory effects ofE₂ on cell proliferation in aged female rats weresignificantly potentiated by overexpression of the human ER- geneinto VSM cells. Constitutive and interleukin (IL)-1-stimulatedNF-B activation was significantly greater (P < 0.05) in VSM cells from aged compared with young female rats.E₂ treatment of VSM cells from aged female rats inhibitedboth constitutive and IL-1-stimulated NF-B activation. ER-gene transfer into VSM cells from aged female rats further augmentedthe inhibitory effects of E₂. In conclusion, our data demonstrate that constitutive and IL-1-stimulated NF-B activation is increased in VSM cells from aged female rats due to loss of E₂ and this can be restored back to normal levels by ER-gene transfer and E₂ treatment. In addition, increasedNF-B signaling may be responsible for increased incidence ofcardiovascular disease in postmenopausal females.

     

Ouabain and substrate affinities of human Na(+)-K(+)-ATPase alpha(1)beta(1), alpha(2)beta(1), and alpha(3)beta(1) when expressed separately in yeast cells

HumanNa⁺-K⁺-ATPase₁₁,₂₁, and ₃₁heterodimers were expressed individually in yeast, and ouabainbinding and ATP hydrolysis were measured in membrane fractions. Theouabain equilibrium dissociation constant was 13-17 nM for₁₁ and ₃₁at 37°C and 32 nM for ₂₁, indicatingthat the human -subunit isoforms have a similar high affinity forcardiac glycosides. K_0.5 values for antagonism of ouabain binding by K⁺ were ranked in order as follows:₂ (6.3 ± 2.4 mM) > ₃(1.6 ± 0.5 mM)  ₁ (0.9 ± 0.6 mM),and K_0.5 values for Na⁺ antagonismof ouabain binding to all heterodimers were 9.5-13.8 mM. Themolecular turnover for ATP hydrolysis by₁₁ (6,652 min¹) was abouttwice as high as that by ₃₁ (3,145 min¹). These properties of the human heterodimersexpressed in yeast are in good agreement with properties of the humanNa⁺-K⁺-ATPase expressed in Xenopusoocytes (G Crambert, U Hasler, AT Beggah, C Yu, NN Modyanov, J-DHorisberger, L Lelievie, and K Geering. J Biol Chem275: 1976-1986, 2000). In contrast to Na⁺ pumpsexpressed in Xenopus oocytes, the₂₁ complex in yeast membranes wassignificantly less stable than ₁₁ or₃₁, resulting in a lower functionalexpression level. The ₂₁ complex was also more easily denatured by SDS than was the₁₁ or the₃₁ complex.

     

Quantitative analysis of transpleural flux in the isolated lung

Li, M. H., J. Hildebrandt, and M. P. Hlastala.Quantitative analysis of transpleural flux in the isolated lung.J. Appl. Physiol. 82(2): 545-551, 1997.In this study, the loss of inert gas through the pleura of anisolated ventilated and perfused rabbit lung was assessed theoreticallyand experimentally. A mathematical model was used to represent an idealhomogeneous lung placed within a box with gas flow(box) surrounding the lung. Thealveoli are assumed to be ventilated with room air(A) andperfused at constant flow () containinginert gases (x) with various perfusate-air partition coefficients(_p,x).The ratio of transpleural flux of gas(pl_x)to its total delivery to the lung via pulmonary artery( ),representing fractional losses across the pleura, can be shown todepend on four dimensionless ratios:1)_p,x,2) the ratio of alveolar ventilation to perfusion(A/), 3) the ratioof the pleural diffusing capacity(Dpl_x) to the conductance ofthe alveolar ventilation (Dpl_x /A_g,where _g is the capacitancecoefficient of gas), and 4) theratio of extrapleural (box) ventilation to alveolar ventilation(box/A).Experiments were performed in isolated perfused and ventilated rabbitlungs. The perfusate was a buffer solution containing six dissolvedinert gases covering the entire 10⁵-fold range of_p,x usedin the multiple inert gas elimination technique. Steady-state inert gasconcentrations were measured in the pulmonary arterial perfusate,pulmonary venous effluent, exhaled gas, and box effluent gas. Theexperimental data could be described satisfactorily by thesingle-compartment model. It is concluded that a simple theoreticalmodel is a useful tool for predicting transpleural flux from isolatedlung preparations, with known ventilation and perfusion, for inertgases within a wide range of .

     

Association of chest wall motion and tidal volume responses during CO2 rebreathing

Yan, Sheng, Pawel Sliwinski, and Peter T. Macklem.Association of chest wall motion and tidal volume responses during CO₂ rebreathing.J. Appl. Physiol. 81(4):1528-1534, 1996.The purpose of this study is to investigate theeffect of chest wall configuration at end expiration on tidal volume(VT) response duringCO₂ rebreathing. In a group of 11 healthy male subjects, the changes in end-expiratory andend-inspiratory volume of the rib cage (Vrc,E andVrc,I, respectively) and abdomen (Vab,E and Vab,I, respectively) measured by linearizedmagnetometers were expressed as a function of end-tidalPCO₂(PET_CO2). The changes inend-expiratory and end-inspiratory volumes of the chest wall(Vcw,E and Vcw,I,respectively) were calculated as the sum of the respectiverib cage and abdominal volumes. The magnetometer coils were placed atthe level of the nipples and 1-2 cm above the umbilicus andcalibrated during quiet breathing against theVT measured from apneumotachograph. TheVrc,E/PET_CO2 slope was quite variable among subjects. It was significantly positive (P < 0.05) in fivesubjects, significantly negative in four subjects(P < 0.05), and not different fromzero in the remaining two subjects. TheVab,E/PET_CO2slope was significantly negative in all subjects(P < 0.05) with a much smallerintersubject variation, probably suggesting a relatively more uniformrecruitment of abdominal expiratory muscles and a variable recruitmentof rib cage muscles during CO₂rebreathing in different subjects. As a group, the meanVrc,E/PET_CO2,Vab,E/PET_CO2, andVcw,E/PET_CO2slopes were 0.010 ± 0.034, 0.030 ± 0.007, and0.020 ± 0.032 l / Torr, respectively;only theVab,E/PET_CO2 slope was significantly different from zero. More interestingly, theindividualVT/PET_CO2slope was negatively associated with theVrc,E/PET_CO2(r = 0.68,P = 0.021) and Vcw,E/PET_CO2slopes (r = 0.63,P = 0.037) but was not associated withtheVab,E/PET_CO2slope (r = 0.40, P = 0.223). There was no correlation oftheVrc,E/PET_CO2 andVcw,E/PET_CO2slopes with age, body size, forced expiratory volume in 1 s, orexpiratory time. The groupVab,I/PET_CO2 slope (0.004 ± 0.014 l / Torr) was not significantlydifferent from zero despite theVT nearly being tripled at theend of CO₂ rebreathing. Inconclusion, the individual VTresponse to CO₂, althoughindependent of Vab,E, is a function ofVrc,E to the extent that as theVrc,E/PET_CO2slope increases (more positive) among subjects, theVT response toCO₂ decreases. These results maybe explained on the basis of the respiratory muscle actions andinteractions on the rib cage.

     

Mitochondrial redox state as a potential detector of liver dysoxia in vivo

Dysoxia canbe defined as ATP flux decreasing in proportion toO₂ availability with preserved ATPdemand. Hepatic venous -hydroxybutyrate-to-acetoacetate ratio(-OHB/AcAc) estimates liver mitochondrial NADH/NAD and may detectthe onset of dysoxia. During partial dysoxia (as opposed to anoxia),however, flow may be adequate in some liver regions, diluting effluentfrom dysoxic regions, thereby rendering venous -OHB/AcAc unreliable.To address this concern, we estimated tissue ATP whilegradually reducing liver blood flow of swine to zero in a nuclearmagnetic resonance spectrometer. ATP flux decreasing withO₂ availability was taken asO₂ uptake(O₂) decreasing inproportion to O₂ delivery(O₂);and preserved ATP demand was taken as increasingP_i/ATP.O₂, tissueP_i/ATP, and venous -OHB/AcAcwere plotted againstO₂to identify critical inflection points. Tissue dysoxia required meanO₂for the group to be critical for bothO₂ and forP_i/ATP. CriticalO₂values for O₂ andP_i/ATP of 4.07 ± 1.07 and 2.39 ± 1.18 (SE) ml · 100 g¹ · min¹,respectively, were not statistically significantly different but notclearly the same, suggesting the possibility that dysoxia might havecommenced after O₂ begandecreasing, i.e., that there could have been"O₂ conformity." CriticalO₂for venous -OHB/AcAc was 2.44 ± 0.46 ml · 100 g¹ · min¹(P = NS), nearly the same as that forP_i/ATP, supporting venous -OHB/AcAc as a detector of dysoxia. All issues considered, tissue mitochondrial redox state seems to be an appropriate detector ofdysoxia in liver.

     

Identification of mediators stimulating proliferation and matrix synthesis of rat pancreatic stellate cells

The aim of thisstudy was to identify fibrogenic mediators stimulatingactivation, proliferation, and/or matrix synthesis of rat pancreaticstellate cells (PSC). PSC were isolated from the pancreas of normalWistar rats and from rats with cerulein pancreatitis. Cell activationwas demonstrated by immunofluorescence microscopy of smooth muscle-actin (SMA) and real-time quantitative RT-PCR of SMA, fibronectin,and transforming growth factor (TGF)-₁. Proliferationwas measured by bromodeoxyuridine incorporation. Matrix synthesis wasdemonstrated on the protein and mRNA level. Within a few days inprimary culture, PSC changed their phenotype from fat-storing toSMA-positive myofibroblast-like cells expressing platelet-derivedgrowth factor (PDGF) - and PDGF -receptors. TGF-₁and tumor necrosis factor (TNF)- accelerated the change in thecells' phenotype. Addition of 50 ng/ml PDGF and 5 ng/ml basicfibroblast growth factor (bFGF) to cultured PSC significantly stimulated cell proliferation (4.37 ± 0.49- and 2.96 ± 0.39-fold of control). Fibronectin synthesis calculated on the basis of DNA was stimulated by 5 ng/ml bFGF (3.44 ± 1.13-fold), 5 ng/ml TGF-₁ (2.46 ± 0.89-fold), 20 ng/ml PDGF (2.27 ± 0.68-fold), and 50 ng/ml TGF- (1.87 ± 0.19-fold). As shownby RT-PCR, PSC express predominantly the splice variant EIII-A offibronectin. Immunofluorescence microscopy and Northern blot confirmedthat in particular bFGF and TGF-₁ stimulated thesynthesis of fibronectin and collagens type I and III. In conclusion,our data demonstrate that 1) TGF-₁ andTNF- accelerate the change in the cell phenotype, 2) PDGF represents the most effective mitogen, and 3) bFGF,TGF-₁, PDGF, and, to a lesser extent, TGF- stimulateextracellular matrix synthesis of cultured rat PSC.

     

Letters to the Editor

The following is the abstract of the article discussed in thesubsequent letter:

Koga, Shunsaku, Tomoyuki Shiojiri, Narihiko Kondo,and Thomas J. Barstow. Effect of increased muscle temperature on oxygen uptake kinetics during exercise. J. Appl. Physiol.83(4): 1333-1338, 1997.To test whether increased muscletemperature (T_m) would improve O₂ uptake(O₂) kinetics, seven menperformed transitions from rest to a moderate work rate [below theestimated lactate threshold (LT_est)] and a heavy workrate (O₂ = 50% of thedifference between LT_est and peakO₂) under conditions of normal T_m (N) and increased T_m (H), produced bywearing hot water-perfused pants before exercise. QuadricepsT_m was significantly higher in H, but rectal temperaturewas similar for the two conditions. There were no significantdifferences in the amplitudes of the fast component ofO₂ or in the time constantsof the on and off transients for moderate and heavy exercise betweenthe two conditions. The increment inO₂ between the 3rd and 6thmin of heavy exercise was slightly but significantly smaller for H thanfor N. These data suggest that elevated T_m before exercise onset, which would have been expected to increase O₂delivery and off-loading to the muscle, had no appreciable effect onthe fast exponential component ofO₂ kinetics (invariant timeconstant). These data further suggest that elevated T_m doesnot contribute to the slow component ofO₂ during heavy exercise.

     

Adrenergic beta 1- and beta 1+2-receptor blockade suppress the natural killer cell response to head-up tilt in humans

Klokker, M., N. H. Secher, P. Madsen, M. Pedersen, and B. K. Pedersen. Adrenergic ₁-and ₁₊₂-receptor blockade suppress the natural killer cell response to head-up tilt in humans. J. Appl. Physiol. 83(5):1492-1498, 1997.To evaluate stress-induced changes in bloodleukocytes with emphasis on the natural killer (NK) cells, eight malevolunteers were followed during three trials of head-up tilt withadrenergic ₁- (metoprolol) and₁₊₂- (propranolol) blockade andwith saline (control) infusions. The ₁- and₁₊₂-receptor blockade did notaffect the appearance of presyncopal symptoms, but the head-up tiltinduced a transient lymphocytosis that was abolished by₁₊₂-receptor blockade but notby ₁-receptor blockade. Head-uptilt also resulted in delayed neutrophilia, which was insensitive to-receptor blockade. Lymphocyte subset analysis revealed that thehead-up tilt resulted in a twofold increase in the percentage andabsolute number of CD3/CD16⁺andCD3/CD56⁺NK cells in peripheral blood and that this increase was partially blocked by metoprolol and abolished by propranolol. The NKcell activity on a per NK cell basis did not change during head-up tilt, indicating that the cytotoxic capability of NK cells recruited tocirculation is unchanged. The data suggest that the head-up tilt-induced lymphocytosis was due mainly toCD16⁺ andCD56⁺ NK cells and that theirrecruitment to the blood was inhibited by₁- and especially₁₊₂-receptor blockade. Thusstress-induced recruitment of lymphocytes, and of NK cells inparticular, is mediated by epinephrine through activation of-receptors on the lymphocytes.

     

Muscle capillarization, O2 diffusion distance, and VO2 kinetics in old and young individuals

Chilibeck, P. D., D. H. Paterson, D. A. Cunningham, A. W. Taylor, and E. G. Noble. Muscle capillarization,O₂ diffusion distance, andO₂ kinetics in old andyoung individuals. J. Appl. Physiol.82(1): 63-69, 1997.The relationships between muscle capillarization, estimated O₂diffusion distance from capillary to mitochondria, andO₂ uptake(O₂) kineticswere studied in 11 young (mean age, 25.9 yr) and 9 old (mean age, 66.0 yr) adults. O₂kinetics were determined by calculating the time constants () forthe phase 2 O₂ adjustment to andrecovery from the average of 12 repeats of a 6-min, moderate-intensityplantar flexion exercise. Muscle capillarization was determined fromcross sections of biopsy material taken from lateral gastrocnemius.Young and old groups had similarO₂ kinetics(O₂-on = 44 vs. 48 s;O₂-off = 33 vs. 44 s, for young and old, respectively), muscle capillarization, andestimated O₂ diffusion distances.Muscle capillarization, expressed as capillary density or averagenumber of capillary contacts per fiber/average fiber area, and theestimates of diffusion distance were significantly correlated toO₂-off kinetics in theyoung (r = 0.68 to 0.83;P < 0.05). We conclude that1) capillarization andO₂ kinetics during exerciseof a muscle group accustomed to everyday activity (e.g., walking) arewell maintained in old individuals, and2) in the young, recovery of O₂ after exercise isfaster, with a greater capillary supply over a given muscle fiber areaor shorter O₂ diffusion distances.

     

Chronic beta -blockade increases skeletal muscle beta -adrenergicreceptor density and enhances contractile force

Murphy, René J. L., Phillip F. Gardiner, Guy Rousseau,Michel Bouvier, and Louise Béliveau. Chronic -blockadeincreases skeletal muscle -adrenergic-receptor density and enhancescontractile force. J. Appl. Physiol.83(2): 459-465, 1997.The effects of a chronic 14-dayadministration of a selective₂-adrenergic-receptor antagonist (ICI-118551) on skeletal muscle were evaluated in female Sprague-Dawley rats. Chronic ICI-118551 treatment did not modify musclemass, oxidative potential, or protein concentration of the medialgastrocnemius muscle, suggesting that maintenance of these skeletalmuscle characteristics is not dependent on₂-adrenergic-receptor stimulation. However, the drug treatment increased-adrenergic-receptor density of the lateral gastrocnemius (42%) andcaused an increase in specific (g/g) isometric in situ contractileforces of the medial gastrocnemius [twitch, 56%; tetanic (200 Hz), 28%]. The elevated contractile forces observed after achronic treatment with ICI-118551 were completely abolished when the₂-adrenergic antagonist wasalso administered acutely before measurement of contractile forces,suggesting that this response is₂-adrenergic-receptor dependent. Possible mechanisms for the increased forces were studied. Caffeine administration potentiated twitch forces but had little effecton tetanic force in control animals. Administration of dibutyryladenosine 3,5-cyclic monophosphate in control animals also resulted in small increases of twitch force but did not modify tetanic forces. We conclude that increases in -adrenergic-receptor density and the stimulation of the receptors by endogenouscatecholamines appear to be responsible for increased contractileforces but that the mechanism remains to be demonstrated.

     

Ventilatory long-term facilitation in unanesthetized rats

Wetested the hypothesis that unanesthetized rats exhibit ventilatorylong-term facilitation (LTF) after intermittent, but not continuous,hypoxia. Minute ventilation (E) and carbon dioxide production (CO₂) were measured inunanesthetized, unrestrained male Sprague-Dawley rats via barometricplethysmography before, during, and after exposure to continuous orintermittent hypoxia. Hypoxia was either isocapnic [inspiredO₂ fraction (FI_O2) = 0.08-0.09 and inspired CO₂ fraction(FI_CO2) = 0.04] or poikilocapnic(FI_O2 = 0.11 andFI_CO2 = 0.00). Sixty minutes afterintermittent hypoxia, E orE/CO₂ wassignificantly greater than baseline in both isocapnic and poikilocapnicconditions. In contrast, 60 min after continuous hypoxia,E andE/CO₂ were notsignificantly different from baseline values. These data demonstrateventilatory LTF after intermittent hypoxia in unanesthetized rats.Ventilatory LTF appeared similar in its magnitude (after accounting forCO₂ feedback), time course, and dependence on intermittenthypoxia to phrenic LTF previously observed in anesthetized,vagotomized, paralyzed rats.

     

Vascular tree structure affects lung blood flow heterogeneity simulated in three dimensions

Parker, James C., Chris B. Cave, Jeffrey L. Ardell, CharlesR. Hamm, and Susan G. Williams. Vascular treestructure affects lung blood flow heterogeneity simulated in threedimensions. J. Appl. Physiol. 83(4):1370-1382, 1997.Pulmonary arterial tree structures related toblood flow heterogeneity were simulated by using a symmetrical,bifurcating model in three-dimensional space. The branch angle (),daughter-parent length ratio(r_L), branchrotation angle (), and branch fraction of parent flow () for asingle bifurcation were defined and repeated sequentially through 11 generations. With  fixed at 90°, tree structures were generatedwith  between 60 and 90°,r_L between 0.65 and 0.85, and an initial segment length of 5.6 cm and sectioned into1-cm³ samples for analysis. Bloodflow relative dispersions (RD%) between 52 and 42% and fractaldimensions (D_s)between 1.20 and 1.15 in 1-cm³samples were observed even with equal branch flows. When  0.5, RD% increased, butD_s eitherdecreased with gravity bias of higher branch flows or increased withrandom assignment of higher flows. Blood flow gradients along gravityand centripetal vectors increased with biased flow assignment of higherflows, and blood flows correlated negatively with distance only when   0.5. Thus a recursive branching vascular tree structuresimulated D_s andRD% values for blood flow heterogeneity similar to those observedexperimentally in the pulmonary circulation due to differences in thenumber of terminal arterioles per1-cm³ sample, but blood flowgradients and a negative correlation of flows with distance requiredunequal partitioning of blood flows at branchpoints.

     

Combined heart rate and activity improve estimates of oxygen consumption and carbon dioxide production rates

Moon, Jon K., and Nancy F. Butte. Combined heart rateand activity improve estimates of oxygen consumption and carbon dioxideproduction rates. J. Appl. Physiol.81(4): 1754-1761, 1996.Oxygen consumption(O₂) andcarbon dioxide production (CO₂) rates were measuredby electronically recording heart rate (HR) and physical activity (PA).Mean daily O₂ andCO₂ measurements by HR andPA were validated in adults (n = 10 women and 10 men) with room calorimeters. Thirteen linear and nonlinear functions of HR alone and HR combined with PA were tested as models of24-h O₂ andCO₂. Mean sleepO₂ andCO₂ were similar to basalmetabolic rates and were accurately estimated from HR alone[respective mean errors were 0.2 ± 0.8 (SD) and0.4 ± 0.6%]. The range of prediction errorsfor 24-h O₂ andCO₂ was smallestfor a model that used PA to assign HR for each minute to separateactive and inactive curves(O₂, 3.3 ± 3.5%; CO₂, 4.6 ± 3%). There were no significant correlations betweenO₂ orCO₂ errors and subject age,weight, fat mass, ratio of daily to basal energy expenditure rate, orfitness. O₂,CO₂, and energy expenditurerecorded for 3 free-living days were 5.6 ± 0.9 ml ^· min^{1 ·} kg¹,4.7 ± 0.8 ml ^· min^{1 ·} kg¹,and 7.8 ± 1.6 kJ/min, respectively. Combined HR and PA measured 24-h O₂ andCO₂ with a precisionsimilar to alternative methods.

     

Evidence for functional role of epsilonPKC isozyme in the regulation of cardiac Na(+) channels

Investigation of the role ofindividual protein kinase C (PKC) isozymes in the regulation ofNa⁺ channels has been largely limited by the lack ofisozyme-selective modulators. Here we used a novel peptide-specificactivator (V1-7) of PKC and other peptide isozyme-specificinhibitors in addition to the general PKC activator phorbol12-myristate 13-acetate (PMA) to dissect the role of individual PKCs inthe regulation of the human cardiac Na⁺ channel hH1,heterologously expressed in Xenopus oocytes. Peptides wereinjected individually or in combination into the oocyte. Whole cellNa⁺ current (I_Na) was recorded usingtwo-electrode voltage clamp. V1-7 (100 nM) and PMA (100 nM)inhibited I_Na by 31 ± 5% and 44 ± 8% (at 20 mV), respectively. These effects were not seen with thescrambled peptide for V1-7 (100 nM) or the PMA analog4-phorbol 12,13-didecanoate (100 nM). However, V1-7-and PMA-induced I_Na inhibition was abolished byV1-2, a peptide-specific antagonist of PKC. Furthermore,PMA-induced I_Na inhibition was not altered by100 nM peptide-specific inhibitors for -, -, -, or PKC. PMAand V1-7 induced translocation of PKC from soluble toparticulate fraction in Xenopus oocytes. This translocationwas antagonized by V1-2. In native rat ventricular myocytes,PMA and V1-7 also inhibited I_Na; thisinhibition was antagonized by V1-2. In conclusion, the resultsprovide evidence for selective regulation of cardiac Na⁺channels by PKC isozyme.

     

Role for anions in pulmonary endothelial permeability

Griffin, M. Pamela. Role for anions in pulmonaryendothelial permeability. J. Appl.Physiol. 83(2): 615-622, 1997.-Adrenergic stimulation reduces albumin permeation across pulmonary artery endothelial monolayers and induces changes in cell morphology that aremediated by Cl flux. Wetested the hypothesis that anion-mediated changes in endothelial cellsresult in changes in endothelial permeability. We measured permeationof radiolabeled albumin across bovine pulmonary arterial endothelialmonolayers when the extracellular anion was Cl,Br,I,F, acetate(Ac), gluconate(G), and propionate(Pr). Permeability toalbumin (P_albumin)was calculated before and after addition of 0.2 mM of thephosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX), whichreduces permeability. InCl, theP_albumin was 3.05 ± 0.86 × 10⁶ cm/s andfell by 70% with the addition of IBMX. The initialP_albumin was lowest forPr andAc. InitialP_albumin was higher inBr,I,G, andF than inCl. A permeability ratiowas calculated to examine the IBMX effect. The greatest IBMX effect wasseen when Cl was theextracellular anion, and the order among halide anions wasCl > Br > I > F. Although the level ofextracellular Ca²⁺ concentration([Ca²⁺]_o)varied over a wide range in the anion solutions,[Ca²⁺]_odid not systematically affect endothelial permeability in this system.When Cl was theextracellular anion, varying[Ca²⁺]_ofrom 0.2 to 2.8 mM caused a change in initialP_albumin but no changein the IBMX effect. The anion channel blockers4-acetamido-4-isothiocyanotostilbene-2,2-disulfonic acid(0.25 mM) and anthracene-9-carboxylic acid (0.5 mM) significantly altered initialP_albumin and the IBMXeffect. The anion transport blockers bumetanide (0.2 mM) and furosemide(1 mM) had no such effects. We conclude that extracellular anionsinfluence bovine pulmonary arterial endothelial permeability and thatthe pharmacological profile fits better with the activity of anionchannels than with other anion transport processes.

     

Improved fatigue resistance not associated with maximum oxygen consumption in creatine-depleted rats

Tanaka, T., Y. Ohira, M. Danda, H. Hatta, and I. Nishi.Improved fatigue resistance not associated with maximum oxygen consumption in creatine-depleted rats. J. Appl.Physiol. 82 (6): 1911-1917, 1997.Effects offeeding of either creatine or its analog -guanidinopropionic acid(-GPA) on endurance work capacity and oxygen consumption werestudied in rats. Resting high-energy phosphate contents inhindlimb muscles were lower in the -GPA group and higher in thecreatine group than in controls. The glycogen contents in restinghindlimb muscles of rats fed -GPA were significantly higher thanthose in controls. The endurance run and swimming times to exhaustionwere significantly greater (32-70%) in the -GPA group than inthe control and creatine groups. However, there were nobeneficial effects on the maximum oxygen consumption (O_{2 max}) and oxygentransport capacity of blood by the feeding of -GPA. None of theseparameters were significantly influenced by creatine supply. Bothmaximum exercise time andO_{2 max} in the -GPAgroup were not changed by normalization of glycogen levels. Theactivities of mitochondrial enzymes in skeletal muscles were higher inthe -GPA group than in the controls. Thus endurance capacity isimproved if the respiratory capacity of muscles is increased, even whenthe contents of high-energy phosphates in muscles are lower. Increasedendurance capacity was not directly associated with the elevated levelsof muscle glycogen, oxygen transport capacity of blood, orO_{2 max}.

     

TGF-beta effects on epithelial ion transport and barrier: reduced Cl- secretion blocked by a p38 MAPK inhibitor

Growthfactors affect a variety of epithelial functions. We examined theability of TGF- to modulate epithelial ion transport andpermeability. Filter-grown monolayers of human colonic epithelia, T84and HT-29 cells, were treated with TGF- (0.1-100 ng/ml,15 min-72 h) or infected with an adenoviral vector encodingTGF- (Ad-TGF) for 144 h. Ion transport (i.e., short-circuitcurrent, I_sc) and transepithelial resistance(TER) were assessed in Ussing chambers. Neither recombinant TGF- norAd-TGF infection affected baseline I_sc;however, exposure to 1 ng/ml TGF- led to a significant (30-50%) reduction in the I_sc responses toforskolin, vasoactive intestinal peptide, and cholera toxin (agentsthat evoke Cl secretion via cAMP mobilization) and to thecell-permeant dibutyryl cAMP. Pharmacological analysis of signalingpathways revealed that the inhibition of cAMP-driven epithelialCl secretion by TGF- was blocked by pretreatment withSB-203580, a specific inhibitor of p38 MAPK, but not by inhibitors ofJNK, ERK1/2 MAPK, or phosphatidylinositol 3'-kinase. TGF- enhanced the barrier function of the treated monolayers by up to threefold asassessed by TER; however, this event was temporally displaced from thealtered I_sc response, being statisticallysignificant only at 72 h posttreatment. Thus, in addition toTGF- promotion of epithelial barrier function, we show that thisgrowth factor also reduces responsiveness to cAMP-dependentsecretagogues in a chronic manner and speculate that this serves as abraking mechanism to limit secretory enteropathies.

     

Mechanical and metabolic determination of VO2 and fatigue during repetitive isometric contractions in situ

Repetitiveisometric tetanic contractions (1/s) of the caninegastrocnemius-plantaris muscle were studied either at optimal length(L_o) or shortlength (L_s;~0.9 · L_o),to determine the effects of initial length on mechanical and metabolicperformance in situ. Respective averages of mechanical and metabolicvariables were(L_o vs.L_s, allP < 0.05) passive tension (preload) = 55 vs. 6 g/g, maximal active tetanic tension(P_o) = 544 vs. 174 (0.38 · P_o)g/g, maximal blood flow () = 2.0 vs. 1.4 ml · min¹ · g¹,and maximal oxygen uptake(O₂) = 12 vs. 9 µmol · min¹ · g¹.Tension at L_odecreased to0.64 · P_o over20 min of repetitive contractions, demonstrating fatigue; there were nosignificant changes in tension atL_s. In separatemuscles contracting atL_o, was set to that measured atL_s (1.1 ml · min¹ · g¹),resulting in decreased O₂(7 µmol · min¹ · g¹),and rapid fatigue, to0.44 · P_o. Thesedata demonstrate that 1)muscles at L_ohave higher andO₂ values than those at L_s;2) fatigue occurs atL_o with highO₂, adjusting metabolic demand (tension output) to match supply; and3) the lack of fatigue atL_s with lowertension, , andO₂ suggestsadequate matching of metabolic demand, set low by shortmuscle length, with supply optimized by low preload. Thesedifferences in tension andO₂ betweenL_o andL_s groupsindicate that muscles contracting isometrically at initial lengthsshorter than L_oare working under submaximal conditions.

     

Respiratory muscle work compromises leg blood flow during maximal exercise

Harms, Craig A., Mark A. Babcock, Steven R. McClaran, DavidF. Pegelow, Glenn A. Nickele, William B. Nelson, and Jerome A. Dempsey.Respiratory muscle work compromises leg blood flow during maximalexercise. J. Appl. Physiol.82(5): 1573-1583, 1997.We hypothesized that duringexercise at maximal O₂ consumption (O_{2 max}),high demand for respiratory muscle blood flow() would elicit locomotor muscle vasoconstrictionand compromise limb . Seven male cyclists(O_{2 max} 64 ± 6 ml · kg¹ · min¹)each completed 14 exercise bouts of 2.5-min duration atO_{2 max} on a cycleergometer during two testing sessions. Inspiratory muscle work waseither 1) reduced via aproportional-assist ventilator, 2)increased via graded resistive loads, or3) was not manipulated (control).Arterial (brachial) and venous (femoral) blood samples, arterial bloodpressure, leg (legs;thermodilution), esophageal pressure, andO₂ consumption(O₂) weremeasured. Within each subject and across all subjects, at constantmaximal work rate, significant correlations existed(r = 0.74-0.90;P < 0.05) between work of breathing(Wb) and legs (inverse), leg vascular resistance (LVR), and leg O₂(O_{2 legs};inverse), and between LVR and norepinephrine spillover. Mean arterialpressure did not change with changes in Wb nor did tidal volume orminute ventilation. For a ±50% change from control in Wb,legs changed 2 l/min or 11% of control, LVRchanged 13% of control, and O₂extraction did not change; thusO_{2 legs}changed 0.4 l/min or 10% of control. TotalO_{2 max} was unchangedwith loading but fell 9.3% with unloading; thusO_{2 legs}as a percentage of totalO_{2 max} was 81% incontrol, increased to 89% with respiratory muscle unloading, anddecreased to 71% with respiratory muscle loading. We conclude that Wbnormally incurred during maximal exercise causes vasoconstriction inlocomotor muscles and compromises locomotor muscle perfusion andO₂.

     

Hypoxia- and normoxia-induced reversibility of autonomic control in Andean guinea pig heart

León-Velarde, Fabiola, Jean-Paul Richalet, Juan-CarlosChavez, Rachid Kacimi, Maria Rivera-Chira, José-Antonio Palacios, and Daniel Clark. Hypoxia- and normoxia-induced reversibility ofautonomic control in Andean guinea pig heart. J. Appl.Physiol. 81(5): 2229-2234, 1996.We hereindescribe the regulation of cardiac receptors in a typical high-altitudenative animal. Heart rate response to isoproterenol(HR_Iso)(beats ^· min^{1 ·} mgIso ^· kg¹)and atropine, the density of -adrenergic(_AR) and muscarinic (M₂) receptors, and theventricular content of norepinephrine (NE) and dopamine (DA) werestudied in guinea pigs (Caviaporcellus). Animals native to Lima, Peru (150 m) werestudied at sea level (SL) and after 5 wk at 4,300-m altitude (SL-HA).Animals native to Rancas [Pasco, Peru (4,300 m)] werestudied at high altitude (HA) and after 5 wk at SL (HA-SL). HA animalshad a lower HR_Iso, maximum numberof _AR binding sites(B_max),_AR dissociation constant (K_d), NE, andDA (P < 0.05) and a higherM₂B_max(P < 0.001) when compared with theSL group. HA-SL showed an increase of theHR_Iso, _ARK_d, and NE(P < 0.05) and a decrease of theM₂B_max andK_d (P < 0.0001) when compared with theHA group. The present study demonstrates the differential regulationand reversibility of the autonomic control in the guinea pig heart.