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.

     

Force relaxes before the fall of cytosolic calcium in the photomechanical response of rat sphincter pupillae

In the ratsphincter pupillae, as in other smooth muscles, the primary signaltransduction cascade for agonist activation is receptor  G protein phospholipase C  inositol trisphosphate  intracellularCa²⁺ concentration ([Ca²⁺]_i)  calmodulin  myosin light chain kinase  phosphorylated myosin  force development. Light stimulation of isolated sphincters pupillaecan be very precisely controlled, and precise reproducible photomechanical responses (PMRs) result. This precision makes the PMRideal for testing models of regulation of smooth muscle myosinphosphorylation. We measured force and[Ca²⁺]_i concurrently in sphincter pupillaefollowing stimulation by light flashes of varying duration andintensity. We sampled at unusually short (0.01-0.02 s) intervalsto adequately test a PMR model based on the myosin phosphorylationcascade. We found, surprisingly, contrary to the behavior of intestinalmuscle and predictions of the phosphorylation model, that during PMRsforce begins to decay while [Ca²⁺]_i is stillrising. We conclude that control of contraction in the sphincterpupillae probably involves an inhibitory process as well as activationby [Ca²⁺]_i.

     

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.

     

Arachidonic acid mediates dual effect of TNF-alpha on Ca2+ transients and contraction of adult rat cardiomyocytes

Tumor necrosisfactor (TNF)- has a biphasic effect on heart contractility andstimulates phospholipase A₂ (PLA₂) incardiomyocytes. Because arachidonic acid (AA) exerts a dual effect onintracellular Ca²⁺ concentration([Ca²⁺]_i) transients, we investigated thepossible role of AA as a mediator of TNF- on[Ca²⁺]_i transients and contraction withelectrically stimulated adult rat cardiac myocytes. At a lowconcentration (10 ng/ml) TNF- produced a 40% increase in theamplitude of both [Ca²⁺]_i transients andcontraction within 40 min. At a high concentration (50 ng/ml) TNF-evoked a biphasic effect comprising an initial positive effect peakingat 5 min, followed by a sustained negative effect leading to50-40% decreases in [Ca²⁺]_i transientsand contraction after 30 min. Both the positive and negative effects ofTNF- were reproduced by AA and blocked by arachidonyltrifluoromethylketone (AACOCF3), an inhibitor of cytosolic PLA₂.Lipoxygenase and cyclooxygenase inhibitors reproduced the high-doseeffects of TNF- and AA. The negative effects of TNF- and AA werealso reproduced by sphingosine and were abrogated by the ceramidaseinhibitor n-oleoylethanolamine. These results point out thekey role of the cytosolic PLA₂/AA pathway in mediating thecontractile effects of TNF-.

     

Na+ pump alpha 2-subunit expression modulates Ca2+ signaling

The role of the Na⁺ pump₂-subunit in Ca²⁺ signaling was examined inprimary cultured astrocytes from wild-type(₂^+/+ = WT) mouse fetuses and thosewith a null mutation in one [₂^+/ = heterozygote (Het)] or both [₂^/ = knockout (KO)] ₂ genes. Na⁺ pump catalytic() subunit expression was measured by immunoblot; cytosol[Na⁺] ([Na⁺]_cyt) and[Ca²⁺] ([Ca²⁺]_cyt) weremeasured with sodium-binding benzofuran isophthalate and fura 2 byusing digital imaging. Astrocytes express Na⁺ pumpswith both ₁- (80% of total ) and₂- (20% of total ) subunits. Het astrocytesexpress 50% of normal ₂; those from KO express none.Expression of ₁ is normal in both Het and KO cells.Resting [Na⁺]_cyt = 6.5 mM in WT, 6.8 mMin Het (P > 0.05 vs. WT), and 8.0 mM in KO cells(P < 0.001); 500 nM ouabain (inhibits only₂) equalized [Na⁺]_cyt at 8 mMin all three cell types. Resting[Ca²⁺]_cyt = 132 nM in WT, 162 nM in Het,and 196 nM in KO cells (both P < 0.001 vs. WT).Cyclopiazonic acid (CPA), which inhibits endoplasmic reticulum (ER)Ca²⁺ pumps and unloads the ER, induces transient (inCa²⁺-free media) or sustained (in Ca²⁺-repletemedia) elevation of [Ca²⁺]_cyt. TheseCa²⁺ responses to 10 µM CPA were augmented in Het as wellas KO cells. When CPA was applied in Ca²⁺-free media, thereintroduction of Ca²⁺ induced significantly largertransient rises in [Ca²⁺]_cyt (due toCa²⁺ entry through store-operated channels) in Het and KOcells than in WT cells. These results correlate with published evidencethat ₂ Na⁺ pumps andNa⁺/Ca²⁺ exchangers are confined to plasmamembrane microdomains that overlie the ER. The data suggest thatselective reduction of ₂ Na⁺ pump activitycan elevate local [Na⁺] and, viaNa⁺/Ca²⁺ exchange, [Ca²⁺] in thetiny volume of cytosol between the plasma membrane and ER. This, inturn, augments adjacent ER Ca²⁺ stores and therebyamplifies Ca²⁺ signaling without elevating bulk[Na⁺]_cyt.

     

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.

     

Synergism between toxin-gamma from Brazilian scorpion Tityus serrulatus and veratridine in chromaffin cells

Toxin- (T)from the Brazilian scorpion Tityusserrulatus venom caused a concentration- andtime-dependent increase in the release of norepinephrine andepinephrine from bovine adrenal medullary chromaffin cells. T was~200-fold more potent than veratridine judged fromEC₅₀ values, although the maximalsecretory efficacy of veratridine was 10-fold greater than that of T(1.2 vs. 12 µg/ml of catecholamine release). The combination of both toxins produced a synergistic effect that was particularly drastic at 5 mM extracellular Ca²⁺concentration([Ca²⁺]_o),when 30 µM veratridine plus 0.45 µM T were used. T (0.45 µM) doubled the basal uptake of⁴⁵Ca²⁺,whereas veratridine (100 µM) tripled it. Again, a drastic synergism in enhancing Ca²⁺ entry was seenwhen T and veratridine were combined; this was particularlypronounced at 5 mM[Ca²⁺]_o.Veratridine induced oscillations of cytosolicCa²⁺ concentration([Ca²⁺]_i)in single fura 2-loaded cells without elevation of basal levels. Incontrast, T elevated basal[Ca²⁺]_ilevels, causing only small oscillations. When added together, T andveratridine elevated the basal levels of[Ca²⁺]_iwithout causing large oscillations. T shifted the current-voltage (I-V) curve forNa⁺ channel current to the left.The combination of T with veratridine increased the shift of theI-V curve to the left, resulting in agreater recruitment of Na⁺channels at more hyperpolarizing potentials. This led to enhanced andmore rapid accumulation of Na⁺ inthe cell, causing cell depolarization, the opening of voltage-dependent Ca²⁺ channels, andCa²⁺ entry and secretion.

     

Acidosis antagonizes intracellular calcium response to kappa -opioid receptor stimulation in the rat heart

To study the effects of -opioid receptor stimulation onintracellular Ca²⁺ concentration([Ca²⁺]_i)homeostasis during extracellular acidosis, we determined the effects of-opioid receptor stimulation on[Ca²⁺]_iresponses during extracellular acidosis in isolated single ratventricular myocytes, by a spectrofluorometric method. U-50488H (10-30 µM), a selective -opioid receptor agonist, dosedependently decreased the electrically induced[Ca²⁺]_itransient, which results from the influx ofCa²⁺ and the subsequentmobilization of Ca²⁺ from thesarcoplasmic reticulum (SR). U-50488H (30 µM) also increased theresting[Ca²⁺]_iand inhibited the[Ca²⁺]_itransient induced by caffeine, which mobilizesCa²⁺ from the SR, indicating thatthe effects of the -opioid receptor agonist involved mobilization ofCa²⁺ from its intracellular poolinto the cytoplasm. The Ca²⁺responses to 30 µM U-50488H were abolished by 5 µMnor-binaltorphimine, a selective -opioid receptorantagonist, indicating that the event was mediated by the -opioidreceptor. The effects of the agonist on[Ca²⁺]_iand the electrically induced[Ca²⁺]_itransient were significantly attenuated when the extracellular pH(pH_e) was loweredto 6.8, which itself reduced intracellular pH(pH_i) and increased[Ca²⁺]_i.The inhibitory effects of U-50488H were restored during extracellular acidosis in the presence of 10 µM ethylisopropyl amiloride, a potentNa⁺/H⁺exchange blocker, or 0.2 mM Ni²⁺,a putativeNa⁺/Ca²⁺exchange blocker. The observations indicate that acidosismay antagonize the effects of -opioid receptor stimulation viaNa⁺/H⁺andNa⁺/Ca²⁺exchanges. When glucose at 50 mM, known to activate theNa⁺/H⁺exchange, was added, both the resting[Ca²⁺]_iand pH_i increased. Interestingly,the effects of U-50488H on [Ca²⁺]_iand the electrically induced[Ca²⁺]_itransient during superfusion with glucose were significantly attenuated; this mimicked the responses during extracellular acidosis. When a high-Ca²⁺ (3 mM) solutionwas superfused, the resting[Ca²⁺]_iincreased; the increase was abolished by 0.2 mMNi²⁺, but thepH_i remained unchanged. Like theresponses to superfusion with high-concentration glucose andextracellular acidosis, the responses of the[Ca²⁺]_iand electrically induced[Ca²⁺]_itransients to 30 µM U-50488H were also significantly attenuated. Results from the present study demonstrated for the first time thatextracellular acidosis antagonizes the effects of -opioid receptorstimulation on the mobilization ofCa²⁺ from SR. Activation of bothNa⁺/H⁺andNa⁺/Ca²⁺exchanges, leading to an elevation of[Ca²⁺]_i,may be responsible for the antagonistic action of extracellular acidosis against -opioid receptor stimulation.

     

Impaired G(s)alpha and adenylyl cyclase cause beta-adrenoceptor desensitization in chronically hypoxic rat hearts

The effectsof -adrenoceptor stimulation with isoproterenol on electricallyinduced contraction and intracellular calcium ([Ca²⁺]_i) transient, and cAMP inmyocytes from both hypertrophied right and nonhypertrophied leftventricles of rats exposed to 10% oxygen for 4 wk, were significantlyattenuated. The increased [Ca²⁺]_i transientin response to cholera toxin was abolished, whereas increased cAMPafter NaF significantly attenuated. The biologically activeisoform, G_s-small (45 kDa), was reduced while thebiologically inactive isoform, G_s-large (52 kDa),increased. The increased electrically induced[Ca²⁺]_i transient and cAMP with 10-100µM forskolin were significantly attenuated in chronically hypoxicrats. The content of G_i2, the predominantisoform of G_i protein in the heart, was unchanged. Resultsindicate that impaired functions of G_s protein and adenylyl cyclase cause -adrenoceptor desensitization. The impaired function of the G_s protein may be due to reducedG_s-small and/or increased G_s-large, whichdoes not result from changes in G_i protein. Responses toall treatments were the same for right and left ventricles, indicatingthat the impaired cardiac functions are not secondary to cardiac hypertrophy.

     

[Ca2+]i-reducing action of cAMP in rat pancreatic beta -cells: involvement of thapsigargin-sensitive stores

In the presentstudy, we examined the ability of adenosine 3',5'-cyclicmonophosphate (cAMP) to reduce elevated levels of cytosolicCa²⁺ concentration([Ca²⁺]_i)in pancreatic -cells.[Ca²⁺]_iand reduced pyridine nucleotide, NAD(P)H, were measured in rat single-cells by fura 2 and autofluorescence microfluorometry. Sustained[Ca²⁺]_ielevation, induced by high KCl (25 mM) at a basal glucose concentration (2.8 mM), was substantially reduced by cAMP-increasing agents, dibutyryl cAMP (DBcAMP, 5 mM), an adenylyl cyclase activatorforskolin (10 µM), and an incretin glucagon-likepeptide-1-(7-36) amide (10⁹ M), as well as byglucose (16.7 mM). The[Ca²⁺]_i-reducingeffects of cAMP were greater at elevated glucose (8.3-16.7 mM)than at basal glucose (2.8 mM). An inhibitor of protein kinase A (PKA),H-89, counteracted[Ca²⁺]_i-reducingeffects of cAMP but not those of glucose. Okadaic acid, a phosphataseinhibitor, at 10-100 nM also reduced sustained [Ca²⁺]_ielevation in a concentration-dependent manner. Glucose, but not DBcAMP,increased NAD(P)H in -cells.[Ca²⁺]_i-reducingeffects of cAMP were inhibited by 0.3 µM thapsigargin, an inhibitorof the endoplasmic reticulum (ER)Ca²⁺ pump. In contrast,[Ca²⁺]_i-reducingeffects of cAMP were not altered by ryanodine, an ERCa²⁺-release inhibitor,Na⁺-free conditions, or diazoxide,an ATP-sensitive K⁺ channelopener. In conclusion, the cAMP-PKA pathway reduces[Ca²⁺]_ielevation by sequestering Ca²⁺ inthapsigargin-sensitive stores. This process does not involve, but ispotentiated by, activation of -cell metabolism. Together with theknown[Ca²⁺]_i-increasingaction of cAMP, our results reveal dual regulation of -cell[Ca²⁺]_iby the cAMP-signaling pathway and by a physiological incretin.

     

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.

     

Importance of the lactate anion in control of breathing

Hardarson, Thorir, Jon O. Skarphedinsson, and TorarinnSveinsson. Importance of the lactate anion in control ofbreathing. J. Appl. Physiol. 84(2):411-416, 1998.The purpose of this study was to examine theeffects of raising the arterialLa andK⁺ levels on minute ventilation(E) in rats. EitherLa or KCl solutions wereinfused in anesthetized spontaneously breathing Wistar rats to raisethe respective ion arterial concentration ([La] and[K⁺]) gradually tolevels similar to those observed during strenuous exercise.E, blood pressure, and heart rate wererecorded continuously, and arterial[La],[K⁺], pH, and bloodgases were repeatedly measured from blood samples. To prevent changesin pH during the Lainfusions, a solution of sodium lactate and lactic acid was used. Raising [La] to13.2 ± 0.6 (SE) mM induced a 47.0 ± 4.0% increase inE without any concomitant changes ineither pH or PCO₂. Raising[K⁺] to 7.8 ± 0.11 mM resulted in a 20.3 ± 5.28% increase inE without changes in pH. Thus ourresults show that Laitself, apart from lactic acidosis, may be important in increasing E during strenuous exercise, and weconfirm earlier results regarding the role of arterial[K⁺] in the control ofE during exercise.

     

Contributions of pulmonary perfusion and ventilation to heterogeneity in VA/Q measured by PET

Treppo, Steven, Srboljub M. Mijailovich, and José G. Venegas. Contributions of pulmonary perfusion and ventilation toheterogeneity in A/measured by PET. J. Appl. Physiol. 82(4): 1163-1176, 1997. To estimate the contributions of the heterogeneity in regionalperfusion () and alveolar ventilation(A) to that of ventilation-perfusionratio (A/), we haverefined positron emission tomography (PET) techniques to image localdistributions of andA per unit of gas volume content(s and sA,respectively) and VA/ indogs. sA was assessed in two ways:1) the washout of ¹³NN tracer after equilibrationby rebreathing (sA_i), and2) the ratio of an apneic image after a bolus intravenousinfusion of ¹³NN-saline solution to an image collectedduring a steady-state intravenous infusion of the same solution(sA_p).sA_p was systematically higher than sA_i in allanimals, and there was a high spatial correlation betweens andsA_p in both body positions(mean correlation was 0.69 prone and 0.81 supine) suggesting thatventilation to well-perfused units was higher than to those poorlyperfused. In the prone position, the spatial distributions ofs, sA_p, and A/ were fairlyuniform with no significant gravitational gradients; however, in thesupine position, these variables were significantly more heterogeneous,mostly because of significant gravitational gradients (15, 5.5, and10%/cm, respectively) accounting for 73, 33, and 66% of thecorresponding coefficient of variation (CV)² values. Weconclude that, in the prone position, gravitational forces in blood andlung tissues are largely balanced out by dorsoventral differences inlung structure. In the supine position, effects of gravity andstructure become additive, resulting in substantial gravitationalgradients in s andsA_p, with the higherheterogeneity inA/ caused by agravitational gradient in s, only partially compensated by that in sA.

     

Regulation of arterial tone by smooth muscle myosin type II

Theinitiation of contractile force in arterial smooth muscle (SM) isbelieved to be regulated by the intracellular Ca²⁺concentration and SM myosin type II phosphorylation. We tested thehypothesis that SM myosin type II operates as a molecular motor proteinin electromechanical, but not in protein kinase C (PKC)-induced,contraction of small resistance-sized cerebral arteries. We utilized aSM type II myosin heavy chain (MHC) knockout mouse model and measuredarterial wall Ca²⁺ concentration([Ca²⁺]_i) and the diameter of pressurizedcerebral arteries (30-100 µm) by means of digital fluorescencevideo imaging. Intravasal pressure elevation caused a graded[Ca²⁺]_i increase and constricted cerebralarteries of neonatal wild-type mice by 20-30%. In contrast,intravasal pressure elevation caused a graded increase of[Ca²⁺]_i without constriction in (/)MHC-deficient arteries. KCl (60 mM) induced a further[Ca²⁺]_i increase but failed to inducevasoconstriction of (/) MHC-deficient cerebral arteries. Activationof PKC by phorbol ester (phorbol 12-myristate 13-acetate, 100 nM)induced a strong, sustained constriction of (/) MHC-deficientcerebral arteries without changing [Ca²⁺]_i.These results demonstrate a major role for SM type II myosin in thedevelopment of myogenic tone and Ca²⁺-dependentconstriction of resistance-sized cerebral arteries. In contrast, thesustained contractile response did not depend on myosin andintracellular Ca²⁺ but instead depended on PKC. We suggestthat SM myosin type II operates as a molecular motor protein in thedevelopment of myogenic tone but not in pharmacomechanical coupling byPKC in cerebral arteries. Thus PKC-dependent phosphorylation ofcytoskeletal proteins may be responsible for sustained contraction invascular SM.

     

O2 uptake kinetics after acetazolamide administration during moderate- and heavy-intensity exercise

Inhibition of carbonic anhydrase (CA) isassociated with a lower plasma lactate concentration([La]_pl)during fatiguing exercise. We hypothesized that a lower[La]_plmay be associated with faster O₂uptake (O₂) kinetics during constant-load exercise. Seven men performed cycle ergometer exercise during control (Con) and acute CA inhibition with acetazolamide (Acz,10 mg/kg body wt iv). On 6 separate days, each subject performed 6-minstep transitions in work rate from 0 to 100 W (below ventilatory threshold,<ET)or to a O₂ corresponding to~50% of the difference between the work rate atET and peakO₂(>ET).Gas exchange was measured breath by breath. Trials were interpolated at1-s intervals and ensemble averaged to yield a single response. The mean response time (MRT, i.e., time to 63% of total exponential increase) for on- and off-transients was determined using a two- (<ET) or athree-component exponential model(>ET).Arterialized venous blood was sampled from a dorsal hand vein andanalyzed for[La]_pl.MRT was similar during Con (31.2 ± 2.6 and 32.7 ± 1.2 s for onand off, respectively) and Acz (30.9 ± 3.0 and 31.4 ± 1.5 s for on and off, respectively) for work rates<ET. Atwork rates >ET, MRTwas similar between Con (69.1 ± 6.1 and 50.4 ± 3.5 s for on andoff, respectively) and Acz (69.7 ± 5.9 and 53.8 ± 3.8 s for on and off, respectively). On- and off-MRTs were slower for>ET thanfor <ETexercise.[La]_plincreased above 0-W cycling values during<ET and>ET exercise but was lower at the end of the transition during Acz (1.4 ± 0.2 and 7.1 ± 0.5 mmol/l for<ET and>ET,respectively) than during Con (2.0 ± 0.2 and 9.8 ± 0.9 mmol/lfor <ETand >ET,respectively). CA inhibition does not affectO₂ utilization at the onset of<ET or>ETexercise, suggesting that the contribution of oxidative phosphorylationto the energy demand is not affected by acute CA inhibition with Acz.

     

Hypoproteinemia, strong-ion difference, and acid-base status in critically ill patients

Thepresent study was a prospective, nonrandomized, observationalexamination of the relationship among hypoproteinemia and electrolyteand acid-base status in a critical care population of patients. A totalof 219 arterial blood samples reviewed from 91 patients was analyzedfor arterial blood gas, electrolytes, lactate, and total protein.Plasma strong-ion difference ([SID]) was calculated from[Na⁺] + [K⁺]  [Cl]  [La].Total protein concentration was used to derive the total concentration of weak acid([A]_tot).[A]_tot encompassed arange of 18.7 to 9.0 meq/l, whereas [SID] varied from 48.1 to 26.6 meq/l and was directly correlated with[A]_tot. The decline in[SID] was primarily attributable to an increase in[Cl]. A directcorrelation was also noted betweenPCO₂ and [SID], but notbetween PCO₂ and[A]_tot. The decrease in [SID] and PCO₂ wassuch that neither [H⁺]nor [HCO₃] changedsignificantly with[A]_tot.

     

Kinetics of oxygen uptake at the onset of exercise in boys and men

The objective of this study was to compare theO₂ uptake(O₂) kinetics at the onsetof heavy exercise in boys and men. Nine boys, aged 9-12 yr, and 8 men, aged 19-27 yr, performed a continuous incremental cyclingtask to determine peak O₂(O_{2 peak}).On 2 other days, subjects performed each day four cycling tasks at 80 rpm, each consisting of 2 min of unloaded cycling followed twice bycycling at 50%O_{2 peak} for 3.5 min,once by cycling at 100%O_{2 peak} for 2 min,and once by cycling at 130%O_{2 peak} for 75 s.O₂ deficit was not significantlydifferent between boys and men (respectively, 50%O_{2 peak} task: 6.6 ± 11.1 vs. 5.5 ± 7.3 ml · min¹ · kg¹;100% O_{2 peak} task:28.5 ± 8.1 vs. 31.8 ± 6.3 ml · min¹ · kg¹;and 130%O_{2 peak}task: 30.1 ± 5.7 vs. 35.8 ± 5.3 ml · min¹ · kg¹).To assess the kinetics, phase I was excluded from analysis. Phase IIO₂ kinetics could bedescribed in all cases by a monoexponential function. ANOVA revealed nodifferences in time constants between boys and men (respectively, 50%O_{2 peak}task: 22.8 ± 5.1 vs. 26.4 ± 4.1 s; 100%O_{2 peak} task: 28.0 ± 6.0 vs. 28.1 ± 4.4 s; and 130%O_{2 peak} task: 19.8 ± 4.1 vs. 20.7 ± 5.7 s). In conclusion, O₂ deficit and fast-componentO₂ on-transientsare similar in boys and men, even at high exercise intensities, whichis in contrast to the findings of other studies employing simplermethods of analysis. The previous interpretation that children relyless on nonoxidative energy pathways at the onset of heavy exercise isnot supported by our findings.

     

Changes in maximum oxygen uptake during prolonged training, overtraining, and detraining in horses

Tyler, Catherine M., Lorraine C. Golland, David L. Evans,David R. Hodgson, and Reuben J. Rose. Changes in maximum oxygenuptake during prolonged training, overtraining, and detraining inhorses. J. Appl. Physiol. 81(5):2244-2249, 1996.Thirteen standardbred horses were trained asfollows: phase 1 (endurance training, 7 wk),phase 2 (high-intensity training, 9 wk),phase 3 (overload training, 18 wk), andphase 4 (detraining, 12 wk). Inphase 3, the horses were divided intotwo groups: overload training (OLT) and control (C). The OLT groupexercised at greater intensities, frequencies, and durations than groupC. Overtraining occurred after 31 wk of training and was defined as asignificant decrease in treadmill run time in response to astandardized exercise test. In the OLT group, there was a significantdecrease in body weight (P < 0.05).From pretraining values of 117 ± 2 (SE)ml ^· kg^{1 ·} min¹,maximal O₂ uptake(O_{2 max}) increased by15% at the end of phase 1, and when signs of overtraining werefirst seen in the OLT group,O_{2 max} was 29%higher (151 ± 2 ml ^· kg^{1 ·} min¹in both C and OLT groups) than pretraining values. There was nosignificant reduction inO_{2 max} until after 6 wk detraining whenO_{2 max} was 137 ± 2 ml ^· kg^{1 ·} min¹.By 12 wk detraining, meanO_{2 max} was134 ± 2 ml ^· kg^{1 ·} min¹,still 15% above pretraining values. When overtraining developed, O_{2 max} was notdifferent between C and OLT groups, but maximal values forCO₂ production (147 vs. 159 ml ^· kg^{1 ·} min¹)and respiratory exchange ratio (1.04 vs. 1.11) were lower in the OLTgroup. Overtraining was not associated with a decrease inO_{2 max} and, afterprolonged training, decreases inO_{2 max} occurredslowly during detraining.

     

Impaired [Ca(2+)](i) and pH(i) responses to kappa-opioid receptor stimulation in the heart of chronically hypoxic rats

-Opioid receptor (-OR)stimulation with U50,488H, a selective -OR agonist, or activation ofprotein kinase C (PKC) with 4-phorbol 12-myristate 13-acetate (PMA), anactivator of PKC, decreased the electrically induced intracellularCa²⁺ ([Ca²⁺]_i) transient andincreased the intracellular pH (pH_i) in single ventricularmyocytes of rats subjected to 10% oxygen for 4 wk. The effects ofU50,488H were abolished by nor-binaltorphimine, a selective -ORantagonist, and calphostin C, a specific inhibitor of PKC, while theeffects of PMA were abolished by calphostin C andethylisopropylamiloride (EIPA), a potent Na⁺/H⁺exchange blocker. In both right hypertrophied and leftnonhypertrophied ventricles of chronically hypoxic rats, the effects ofU50,488H or PMA on [Ca²⁺]_i transient andpH_i were significantly attenuated and completely abolished,respectively. Results are first evidence that the[Ca²⁺]_i and pH_i responses to-OR stimulation are attenuated in the chronically hypoxic rat heart,which may be due to reduced responses to PKC activation. Responses toall treatments were the same for right and left ventricles, indicatingthat the functional impairment is independent of hypertrophy. -ORmRNA expression was the same in right and left ventricles of bothnormoxic and hypoxic rats, indicating no regional specificity.

     

Excitation wavelengths for fura 2 provide a linear relationship between [Ca(2+)] and fluorescence ratio

We proposed andtested the use of nontraditional excitation wavelengths(₁ and ₂) and an emission wavelength(_em) to define conditions under which free calciumconcentration and a fluorescence ratio are linearly related.Fluorescence spectra were determined for aqueous solutions thatcontained 25 µM fura 2, 125 mM K⁺, and either 0 mM or 0.1 mM Ca²⁺. Effectively linear relationships between[Ca²⁺] and a fluorescence ratio, i.e., <5% bias when[Ca²⁺]  5 × dissociation constant, were apparentwhen ₁  400 nm, ₂  370 nm, and_em  510 nm. Combinations with longer ₁and _em and/or with shorter ₂ reduced thisbias further. Although the method described does not obviate thecomplications that surround the correction for fluorescence background,choosing a nontraditional combination of excitation and emissionwavelengths offers several practical advantages over more traditionalfura 2 fluorescence methodologies in a variety of experimental settings.