Thermoregulatory responses to cold transients: effects of menstrual cycle in resting women

Effects of themenstrual cycle on heat loss and heat production(M) and core and skin temperatureresponses to cold were studied in six unacclimatized female nonsmokers(18-29 yr of age). Each woman, resting supine, was exposed to acold transient (ambient temperature = mean radiant temperature = 20 to5°C at 0.32°C/min, relative humidity = 50 ± 2%, wind speed = 1 m/s) in the follicular (F) phase(days 2-6) and midluteal (L)phase (days 19-23) of her menstrual cycle. Clothed in each of two ensembles with different thermal resistances, women performed multiple experiments in the F andL phases. Thermal resistance was 0.2 and 0.4 m² · K · W¹for ensembles A andB, respectively. Esophagealtemperature (T_es), mean weightedskin temperature(_sk),finger temperature (T_fing), andarea-weighted heat flux were recorded continuously. Rate of heat debt(S) and integrated mean bodytemperature(_b,i)were calculated by partitional calorimetry throughout the cold ramp. Extensive peripheral vasoconstriction in the F phase during early periods of the ramp elevated T_esabove thermoneutral levels. Shivering thermogenesis(M = M  M_basal,W /m²) was highly correlated withdeclines in_sk andT_fing(P <0.0001). There was a reducedslope in M as a function of_b,i inthe L phase with ensembles A(P < 0.02) andB (P < 0.01). Heat flux was higher andS was less in the L phases withensemble A(P < 0.05). An analytic modelrevealed that_sk andT_es contribute as additive inputsand T_fing has a multiplicativeeffect on the total control of Mduring cold transients(R² = 0.9).Endogenous hormonal levels at each menstrual cycle phase, coretemperature and_skinputs, vascular responses, and variations in body heat balance must beconsidered in quantifying thermoregulatory responses in women duringcold stress.

     

Cardiac output and mixed venous oxygen content measurements by a tracer bolus method: theory

Clark, Justin S., Yuxiang J. Lin, Michael J. Criddle,Antonio G. Cutillo, Adelbert H. Bigler, Fred L. Farr, and Attilio D. Renzetti, Jr. Cardiac output and mixed venous oxygen content measurements by a tracer bolus method: theory. J. Appl.Physiol. 83(3): 884-896, 1997.We present a bolus method ofinert-gas delivery to the lungs that facilitates application ofmultiple inert gases and the multiple inert-gas-exchange technique(MIGET) model to noninvasive measurements of cardiac output (CO) andcentral mixed venous oxygen contentReduction in recirculation error is made possible by 1)replacement of sinusoidal input functions with impulse inputs and2) replacement of steady-state analyses with transientanalyses. Recirculation error reduction increases the inert-gasselection to include common gases without unusually high (and difficultto find) tissue-to-blood partition coefficients for maximizing thesystemic filtering efficiency. This paper also presents a practicalmethod for determining the recirculation contributions to inert expiredprofiles in animals and determining their specific contributions toerrors in the calculations of CO and from simulationsapplied to published ventilation-perfusion ratio(/) profiles.Recirculation errors from common gases were found to be reducible tothe order of 5% or less for both CO and whereassimulation studies indicate that measurement bias contributions fromrecirculation, / mismatch, andthe / extractionprocess can be limited to 15% for subjects with severe/ mismatch and high inspiredoxygen fraction levels. These studies demonstrate a decreasinginfluence of / mismatch onparameter extraction bias as the number of inert gases are increased.However, the influence of measurement uncertainty on parameterextraction error limits improvement to six gases.

     

Effects of CO2-HCO<SUP>&minus;</SUP><SUB>3</SUB> on catecholamine efflux from cat carotid body

Iturriaga, Rodrigo, and Julio Alcayaga. Effects ofCO₂-on catecholamine efflux from cat carotid body. J. Appl. Physiol. 84(1): 60-68, 1998.Using achronoamperometric technique with carbon-fiber microelectrodes andneural recordings, we simultaneously measured the effects of thefollowing procedures on catecholamine efflux (CA) andfrequency of chemosensory discharges (f_x) fromsuperfused cat carotid body: 1) theaddition ofCO₂- to Tyrode solution previously buffered withN-2-hydroxyethylpiperazine-N -2-ethanesulfonicacid, maintaining pH at 7.40; 2)hypercapnia (10% CO₂, pH 7.10);3) hypoxia(PO₂ h  40 Torr) with andwithoutCO₂-;and 4) the impact of several bolusesof dopamine (DA; 10-100 µg) on hypoxic and hypercapnic challenges. WithCO₂-,hypoxia increased f_x which preceded CAincreases, whereas hypercapnia raised f_x but didnot consistently increase CA. Repeated stimuli induced similarf_x increases, but attenuated CA. AfterDA, hypoxia produced larger CA, which preceded chemosensoryresponses. WithoutCO₂-, hypoxia produced a similar pattern of CA andf_x responses. Switching to Tyrode solution withCO₂-at pH 7.40 raised f_x but did not increase CA.WithCO₂- and after DA, hypoxic-induced CAs were larger than in its absence. Results suggest that DA release is not essential for chemosensory excitation.

     

Exertional fatigue, sleep loss, and negative energy balance increase susceptibility to hypothermia

The purpose ofthis study was to determine how chronic exertional fatigue and sleepdeprivation coupled with negative energy balance affectthermoregulation during cold exposure. Eight men wearing only shortsand socks sat quietly during 4-h cold air exposure (10°C)immediately after (<2 h, A) they completed 61 days of strenuousmilitary training (energy expenditure ~4,150 kcal/day, energy intake~3,300 kcal/day, sleep ~4 h/day) and again after short (48 h, SR)and long (109 days, LR) recovery. Body weight decreased 7.4 kg frombefore training to A, then increased 6.4 kg by SR, with an additional6.4 kg increase by LR. Body fat averaged 12% during A and SR andincreased to 21% during LR. Rectal temperature(T_re) was lower before andduring cold air exposure for A than for SR and LR.T_re declined during cold exposurein A and SR but not LR. Mean weighted skin temperature(_sk)during cold exposure was higher in A and SR than in LR. Metabolic rate increased during all cold exposures, but it was lower during A and LRthan SR. The mean body temperature (0.67 T_re + 0.33 _sk) threshold for increasing metabolism was lower during A than SR and LR.Thus chronic exertional fatigue and sleep loss, combined withunderfeeding, reduced tissue insulation and blunted metabolic heatproduction, which compromised maintenance of body temperature. A shortperiod of rest, sleep, and refeeding restored the thermogenic responseto cold, but thermal balance in the cold remained compromised untilafter several weeks of recovery when tissue insulation had beenrestored.

     

Fast and slow components of cerebral blood flow response to step decreases in end-tidal PCO2 in humans

This study examined the dynamics of the middlecerebral artery (MCA) blood flow response to hypocapnia in humans(n = 6) by using transcranial Dopplerultrasound. In a control protocol, end-tidalPCO₂(PET_CO2) was heldnear eucapnia (1.5 Torr above resting) for 40 min. In ahypocapnic protocol, PET_CO2was held near eucapnia for 10 min, then at 15 Torr below eucapnia for20 min, and then near eucapnia for 10 min. During both protocols,subjects hyperventilated throughout andPET_CO2 and end-tidalPO₂ were controlled by using thedynamic end-tidal forcing technique. Beat-by-beat values werecalculated for the intensity-weighted mean velocity (_IWM),signal power (), and theirinstantaneous product(_IWM).A simple model consisting of a delay, gain terms, time constants(_f,on, _f,off) and baseline levels offlow for the on- and off-transients, and a gain term(g_s) and time constant(_s) for a second slower component was fitted to the hypocapnic protocol. The cerebral bloodflow response to hypocapnia was characterized by a significant (P < 0.001) slowprogressive adaptation in_IWM, with g_s = 1.26 %/Torr and_s = 427 s, that persistedthroughout the hypocapnic period. Finally, the responses at the onsetand relief of hypocapnia were asymmetric(P < 0.001), with_f,on (6.8 s) faster than_f,off (14.3 s).

     

Role of SGK in hormonal regulation of epithelial sodium channel in A6 cells

The purpose of this study was to examinethe role of the serum- and glucocorticoid-induced kinase (SGK) in theactivation of the epithelial sodium channel (ENaC) by aldosterone,arginine vasopressin (AVP), and insulin. We used atetracycline-inducible system to control the expression of wild-type(SGK), constitutively active (S425Dmutation; SGK), or inactive (K130Mmutation; SGK) SGK in A6 cellsindependently of hormonal stimulation. The effect of SGK expression onENaC activity was monitored by measuring transepithelialamiloride-sensitive short-circuit current (I_sc) of transfected A6 cell lines. Expression ofSGK orSGK and aldosterone stimulation haveadditive effects on I_sc. Although SGK could playsome role in the aldosterone response, our results suggest that othermechanisms take place. SGK abrogatesthe responses to AVP and insulin; hence, in the signaling pathways ofthese hormones there is a shared step that is stimulated by SGK.Because AVP and insulin induce fusion of vesicles to the apicalmembrane, our results support the notion that SGK promotes incorporation of channels in the apical membrane.

     

Peripheral O2 diffusion does not affect VO2 on-kinetics in isolated in situ canine muscle

To test thehypothesis that muscle O₂ uptake(O₂) on-kinetics islimited, at least in part, by peripheralO₂ diffusion, we determined theO₂ on-kinetics in1) normoxia (Control);2) hyperoxic gas breathing(Hyperoxia); and 3) hyperoxia andthe administration of a drug (RSR-13, Allos Therapeutics), whichright-shifts the Hb-O₂dissociation curve (Hyperoxia+RSR-13). The study was conducted inisolated canine gastrocnemius muscles(n = 5) during transitions from restto 3 min of electrically stimulated isometric tetanic contractions(200-ms trains, 50 Hz; 1 contraction/2 s; 60-70% peakO₂). In all conditions,before and during contractions, muscle was pump perfused withconstantly elevated blood flow (), at a levelmeasured at steady state during contractions in preliminary trials withspontaneous . Adenosine was infusedintra-arterially to prevent inordinate pressure increases with theelevated . was measuredcontinuously, arterial and popliteal venousO₂ concentrations were determinedat rest and at 5- to 7-s intervals during contractions, andO₂ was calculated as · arteriovenous O₂ content difference.PO₂ at 50%HbO₂saturation (P₅₀) was calculated.Mean capillary PO₂(c_O2)was estimated by numerical integration.P₅₀ was higher in Hyperoxia+RSR-13[40 ± 1 (SE) Torr] than in Control and in Hyperoxia (31 ± 1 Torr). After 15 s of contractions,c_O2was higher in Hyperoxia (97 ± 9 Torr) vs. Control (53 ± 3 Torr) and in Hyperoxia+RSR-13 (197 ± 39 Torr) vs. Hyperoxia. Thetime to reach 63% of the difference between baseline and steady-stateO₂ during contractions was 24.7 ± 2.7 s in Control, 26.3 ± 0.8 s in Hyperoxia, and 24.7 ± 1.1 s in Hyperoxia+RSR-13 (not significant). Enhancement ofperipheral O₂ diffusion (obtainedby increasedc_O2at constant O₂ delivery) duringthe rest-to-contraction (60-70% of peakO₂) transition did notaffect muscle O₂on-kinetics.

     

Pulmonary gas exchange during exercise in pigs

Increased ventilation-perfusion(A/)inequality is observed in ~50% of humans during heavy exercise andcontributes to the widening of the alveolar-arterialO₂ difference(A-aD_O2). Despite extensive investigation, the cause remains unknown. As a firststep to more direct examination of this problem, we developed an animalmodel. Eight Yucatan miniswine were studied at rest and duringtreadmill exercise at ~30, 50, and 85% of maximalO₂ consumption (O_{2 max}). Multipleinert-gas, blood-gas, and metabolic data were obtained. TheA-aD_O2increased from 0 ± 3 (SE) Torr at rest to 14 ± 2 Torr duringthe heaviest exercise level, but arterialPO₂(Pa_O2) remained at resting levels during exercise. There was normalA/inequality [log SD of the perfusion distribution(log) = 0.42 ± 0.04] at rest, and moderate increases(log = 0.68 ± 0.04, P < 0.0001) wereobserved with exercise. This result was reproducible on a separate day.TheA/inequality changes are similar to those reported in highly trainedhumans. However, in swine, unlike in humans, there was no inert gasevidence for pulmonary end-capillary diffusion limitation during heavyexercise; there was no systematic difference in the measuredPa_O2 and the Pa_O2 as predicted from the inertgases. These data suggest that the pig animal model iswell suited for studying the mechanism of exercise-inducedA/ inequality.

     

Kinetics of CO2 excretion and intravascular pH disequilibria during carbonic anhydrase inhibition

Cardenas, Victor, Jr., Thomas A. Heming, and Akhil Bidani.Kinetics of CO₂ excretion andintravascular pH disequilibria during carbonic anhydrase inhibition.J. Appl. Physiol. 84(2): 683-694, 1998.Inhibition of carbonic anhydrase (CA) activity (activity in redblood cells and activity available on capillary endothelium) results indecrements in CO₂ excretion(CO₂) and plasma-erythrocyteCO₂--H⁺disequilibrium as blood travels around the circulation. To investigate the kinetics of changes in blood PCO₂and pH during progressive CA inhibition, we used our previouslydetailed mathematical model of capillary gas exchange to analyzeexperimental data of CO₂ and blood-gas/pH parameters obtained from anesthetized, paralyzed, andmechanically ventilated dogs after treatment with acetazolamide (Actz,0-100 mg/kg iv). Arterial and mixed venous blood samples werecollected via indwelling femoral and pulmonary arterial catheters, respectively. Cardiac output was measured by thermodilution. End-tidal PCO₂, as a measure of alveolarPCO₂, was obtained from continuousrecords of airway PCO₂ above thecarina. Experimental results were analyzed with the aid of amathematical model of lung and tissue-gas exchange. Progressive CAinhibition was associated with stepwise increments in the equilibratedmixed venous-alveolar PCO₂ gradient(9, 19, and 26 Torr at 5, 20, and 100 mg/kg Actz, respectively). Themaximum decrements in CO₂were 10, 24, and 26% with 5, 20, and 100 mg/kg Actz, respectively,without full recovery ofCO₂ at 1 h postinfusion. Equilibrated arterial PCO₂overestimated alveolar PCO₂, andtissue PCO₂ was underestimated by themeasured equilibrated mixed venous bloodPCO₂. Mathematical model computations predicted hysteresis loops of the instantaneousCO₂--H⁺relationship and in vivo bloodPCO₂-pH relationship due to thefinite reaction times forCO₂--H⁺reactions. The shape of the hysteresis loops was affected by the extentof Actz inhibition of CA in red blood cells and plasma.

     

Lyn- and ERK-mediated vs. Ca2+-mediated neutrophil O2- responses with thermal injury

We evaluated thedependency of neutrophil O production on PTK-Lyn andMAPK-ERK1/2 in rats after thermal injury. Activation of PTK-Lyn wasassessed by immunoprecipitation. Phosphorylation of ERK1/2 was assessedby Western blot analysis. O production was measuredby isoluminol-enhanced luminometry. Imaging technique was employed tomeasure neutrophil [Ca²⁺]_i in individualcells. Thermal injury caused marked upregulation of Lyn and ERK1/2accompanying enhanced neutrophil O production.Treatment of rats with PTK blocker (AG556) or MAPK blocker (AG1478)before burn injury caused complete inhibition of the respective kinaseactivation. Both AG556 and AG1478 produced an ~66% inhibition inO production. Treatment with diltiazem (DZ) producedan ~37% inhibition of O production withoutaffecting Lyn or ERK1/2 activation with burn injury. Ca²⁺mobilization was upregulated with burn injury but not affected bytreatment of burn rats with AG556. Unlike the partial inhibition ofburn-induced O production by AG556, AG1478, or DZ,platelet-activating factor antagonist (PAFa) treatment of burn ratsproduced near complete inhibition of O production.PAFa treatment also blocked activation of Lyn. The findings suggestthat the near complete inhibition of O production byPAFa was a result of blockade of PTK as well as Ca²⁺signaling. Overall, our studies show that enhanced neutrophil O production after thermal injury is a result ofpotentiation of Ca²⁺-linked and -independent signalingtriggered by inflammatory agents such as PAF.

     

Phosphate-induced chondrocyte apoptosis is linked to nitric oxide generation

An elevation in inorganic phosphate(P_i) concentration activates epiphyseal chondrocyteapoptosis. To determine the mechanism of apoptosis,tibial chondrocytes were treated with P_i, andnitrate/nitrite (NO/NO) levelswere determined. P_i induced a threefold increase in the NO/NO concentration; inhibitorsof nitric oxide (NO) synthase activity and P_i transportsignificantly reduced NO/NO levels and prevented cell death. Furthermore, a dose-dependent increasein cell death was observed after exposure of chondrocytes toS-nitrosoglutathione. P_i increased caspase 3 activity 2.7-fold. Both caspase 1 and caspase 3 inhibitors protectedchondrocytes from P_i-induced apoptosis.P_i caused a significant decrease in the mitochondrialmembrane potential, while NO synthase inhibitors maintainedmitochondrial function. While P_i caused thiol depletion, inhibition of P_i uptake or NO generation served tomaintain glutathione levels. The results suggest that NO serves tomediate key metabolic events linked to P_i-dependentchondrocyte apoptosis.

     

Timolol may inhibit aqueous humor secretion by cAMP-independent action on ciliary epithelial cells

The -adrenergic antagonisttimolol reduces ciliary epithelial secretion in glaucomatous patients.Whether inhibition is mediated by reducing cAMP is unknown. Elementalcomposition of rabbit ciliary epithelium was studied by electron probeX-ray microanalysis. Volume of cultured bovine pigmented ciliaryepithelial (PE) cells was measured by electronic cell sizing;Ca²⁺ activity and pH were monitored with fura 2 and2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein, respectively. Timolol (10 µM) produced similar K and Cl losses fromciliary epithelia in HCO/CO₂ solutionbut had no effect in HCO/CO₂-free solution or in HCO/CO₂ solutioncontaining the carbonic anhydrase inhibitor acetazolamide. Inhibitionof Na⁺/H⁺ exchange by dimethylamiloride inHCO/CO₂ solution reduced Cl and Kcomparably to timolol. cAMP did not reverse timolol's effects. Timolol(100 nM, 10 µM) and levobunolol (10 µM) produced cAMP-independentinhibition of the regulatory volume increase (RVI) in PE cells andincreased intracellular Ca²⁺ and pH. IncreasingCa²⁺ with ionomycin also blocked the RVI. The resultsdocument a previously unrecognized cAMP-independent transport effect oftimolol. Inhibition of Cl/HCO exchangemay mediate timolol's inhibition of aqueous humor formation.

     

Ca(2+) regulation of gap junctional coupling in lens epithelial cells

The quantitative effects of Ca²⁺signaling on gap junctional coupling in lens epithelial cells have beendetermined using either the spread of Mn²⁺ that is imagedby its ability to quench the fluorescence of fura 2 or the spread ofthe fluorescent dye Alexa Fluor 594. Gap junctional coupling wasunaffected by a mechanically stimulated cell-to-cell Ca²⁺wave. Furthermore, when cytosolic Ca²⁺ concentration(Ca) increased after the addition of the agonistATP, coupling was unaffected during the period thatCa was maximal. However, coupling decreasedtransiently ~5-10 min after agonist addition whenCa returned to resting levels, indicating that thistransient decrease in coupling was unlikely due to a direct action ofCa on gap junctions. An increase inCa mediated by the ionophore ionomycin that wassustained for several minutes resulted in a more rapid and sustaineddecrease in coupling (IC₅₀ ~300 nM Ca²⁺, Hillcoefficient of 4), indicating that an increase in Ca alone could regulate gap junctions. Thus Ca increases that occurred during agonist stimulation and cell-to-cell Ca²⁺ waves were too transient to mediate a sustaineduncoupling of lens epithelial cells.

     

Functional and molecular evidence for Na+-HCO3- cotransporter in porcine vas deferens epithelia

This study focused on the role ofsodium-bicarbonate cotransporter (NBC1) in cAMP-stimulated iontransport in porcine vas deferens epithelium. Ion substitutionexperiments in modified Ussing chambers revealed that cAMP-mediatedstimulation was dependent on the presence of Na⁺,HCO, and Cl for a full response.HCO-dependent current was unaffected byacetazolamide, bumetanide, or amiloride but was inhibited bybasolateral 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid.Na⁺-driven, HCO-dependent,stilbene-inhibitable anion flux was observed across the basolateralmembrane of selectively permeabilized monolayers. Results ofradiotracer flux studies suggest a4,4'-dinitrostilbene-2,2'-disulfonate-sensitive stoichiometry of 2 baseequivalents per Na⁺. Antibodies raised against rat kidneyNBC epitopes (rkNBC; amino acids 338-391 and 928-1035)identified a single band of ~145 kDa. RT-PCR detected NBC1 message inporcine vas deferens epithelia. These results demonstrate that vasdeferens epithelial cells possess the proteins necessary for thevectoral transport of HCO and that these mechanismsare maintained in primary culture. Taken together, the results indicatethat vas deferens epithelia play an active role in male fertility andhave implications for our understanding of the relationship betweencystic fibrosis and congenital bilateral absence of the vas deferens.

     

Signaling by eNOS through a superoxide-dependent p42/44 mitogen-activated protein kinase pathway

Expression ofendothelial nitric oxide synthase (eNOS) in transfected U-937 cellsupregulates phorbol 12-myristate 13-acetate (PMA)-induced tumornecrosis factor- (TNF-) production through a superoxide(O)-dependent mechanism. Because mitogen-activatedprotein kinases (MAPK) have been shown to participate in both reactiveoxygen species signaling and TNF- regulation, their possible role ineNOS-derived O signal transduction was examined. Aredox-cycling agent, phenazine methosulfate, was found to bothupregulate TNF- (5.8 ± 1.0 fold; P = 0.01) andincrease the phosphorylation state of p42/44 MAPK (3.1 ± 0.2 fold; P = 0.01) in PMA-differentiated U-937 cells. AlthoughS-nitroso-N-acetylpenicillamine, a nitric oxide(NO) donor, also increased TNF- production, NO exposure led tophosphorylation of p38 MAPK, not p42/44 MAPK. Upregulation of TNF-production by eNOS transfection was associated with increases inactivated p42/44 MAPK (P = 0.001), whereas levels ofphosphorylated p38 MAPK were unaffected. Furthermore, cotransfectionwith Cu/Zn superoxide dismutase, which blocks TNF- upregulation byeNOS, also abolished the effects on p42/44 MAPK. Expression ofGln³⁶¹eNOS, a mutant that produces O but not NO, still resulted in p42/44 MAPK phosphorylation. In contrast, twoNADPH binding site deletion mutants of eNOS that lack oxidase activityhad no effect on p42/44 MAPK. Finally, PD-98059, a p42/44 MAPK pathwayinhibitor, blocked TNF- upregulation by eNOS (P = 0.02).Thus O produced by eNOS increases TNF- productionvia a mechanism that involves p42/44 MAPK activation.

     

Airway surface liquid pH in well-differentiated airway epithelial cell cultures and mouse trachea

Airway surface liquid (ASL) pH hasbeen proposed to be important in the pathophysiology of cysticfibrosis, asthma, and cough. Ratio image analysis was used to measurepH in the ASL after staining with the fluorescent pH indicator2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF)-dextran. ASL pH in bovine airway cell cultures grown at anair-liquid interface was 6.98 ± 0.06 in the absence and 6.81 ± 0.04 in the presence of HCO/CO₂. Steady-state ASL pH changed in parallel to changes in bath pH and wasacidified by Na⁺ or Cl replacement but wasnot affected by the inhibitors amiloride, glibenclamide, or4,4'-dinitrostilbene-2,2'-disulfonic acid. In response to suddenacidification or alkalization of the ASL by ~0.4 pH units byHCl/NaOH, ASL pH recovered to its initial value at a rate of 0.035 pHunits/min (HCO) and 0.060 pH units/min(+HCO); the pH recovery rate was reduced byamiloride and H₂DIDS. In anesthetized mice in which thetrachea was surgically exposed for measurement of BCECF-dextranfluorescence through the translucent tracheal wall, ASL pH was7.14 ± 0.01. ASL pH was sensitive to changes in blood pH createdby metabolic (HCl or NaHCO₃ infusion) or respiratory (hyperventilation, hypoventilation) mechanisms. ASL pH is thus primarily determined by basolateral fluid pH, andH⁺/OH transport between the ASL andbasolateral fluid involves amiloride-sensitive Na⁺/H⁺ exchange and stilbene-sensitiveCl/HCO exchange. The rapid response ofASL pH to changes in systemic acid-base status may contribute to airwayhypersensitivity in asthma and other airway diseases.

     

Neurotrophin-3 inhibits HCO3- absorption via a cAMP-dependent pathway in renal thick ascending limb

Neurotrophins are expressed in the adult kidney, but theirsignificance is unclear. We showed previously that nerve growth factor(NGF) inhibits HCO absorption in the rat medullarythick ascending limb (MTAL) via an extracellular signal-regulatedkinase (ERK)-dependent pathway. Here we examined whether otherneurotrophic factors affect MTAL HCO absorption.Brain-derived neurotrophic factor and glial cell line-derived neurotrophic factor had no effect. In contrast, neurotrophin-3 (NT-3,0.7 nM) inhibited HCO absorption by 40%(half-maximal inhibition at ~0.4 nM). Inhibition by NT-3 was additiveto inhibition by NGF. Inhibitors of ERK activation that blockinhibition by NGF had no effect on inhibition by NT-3. In contrast,8-bromo-cAMP or forskolin pretreatment blocked inhibition by NT-3 butnot NGF. Inhibition by NT-3 was also blocked by the specific proteinkinase A (PKA) inhibitor myristoylated PKI(14-22) amide and by vasopressin, which inhibits HCO absorption via cAMP. Inhibitors of phosphatidylinositol 3-kinase orprotein kinase C did not affect NT-3-induced inhibition, but inhibitionby NT-3 was eliminated by genistein, consistent with involvement of areceptor tyrosine kinase. These results demonstrate that NT-3 inhibitsHCO absorption via a cAMP- and PKA-dependentpathway. NT-3 and NGF regulate MTAL ion transport through differentsignal transduction mechanisms. These studies establish a direct rolefor NT-3 in regulation of renal tubule transport and identify the MTALas an important target for neurotrophins, which may be involved in thecontrol of renal acid excretion.

     

Expression, localization, and functional evaluation of CFTR in bovine corneal endothelial cells

HCO-dependentfluid secretion by the corneal endothelium controls corneal hydrationand maintains corneal transparency. Recently, it has been shown thatmRNA for the cystic fibrosis transmembrane conductance regulator (CFTR) is expressed in the corneal endothelium; however, protein expression, functional localization, and a possible role in HCO transport have not been reported. Immunoblotting for CFTR showed asingle band at ~170 kDa for both freshly isolated and primary cultures of bovine corneal endothelial cells. Indirectimmunofluorescence confocal microscopy indicated that CFTR locates tothe apical membrane. Relative changes in apical and basolateralchloride permeability were estimated by measuring the rate offluorescence quenching of the halide-sensitive indicator6-methoxy-N-ethylquinolinium iodide during Clinflux in the absence and presence of forskolin (FSK). Apical andbasolateral Cl permeability increased 10- and 3-fold,respectively, in the presence of 50 µM FSK. FSK-activated apicalchloride permeability was unaffected by H₂DIDs (250 µM);however, 5-nitro-2-(3-phenylpropyl-amino)benzoic acid (NPPB; 50 µM) and glibenclamide (100 µM) inhibited activated Clfluxes by 45% and 30%, respectively. FSK-activated basolateral Cl permeability was insensitive to NPPB, glibenclamide,or furosemide but was inhibited 80% by H₂DIDS.HCO permeability was estimated by measuring changesin intracellular pH in response to quickly lowering bath[HCO]. FSK (50 µM) increased apicalHCO permeability by twofold, which was inhibited42% by NPPB and 65% by glibenclamide. BasolateralHCO permeability was unaffected by FSK. Genistein(50 µM) significantly increased apical HCO andCl permeability by 1.8- and 16-fold, respectively. When50 µM genistein was combined with 50 µM FSK, there was no furtherincrease in Cl permeability; however,HCO permeability was reduced to the control level.In summary, we conclude that CFTR is present in the apical membrane ofbovine corneal endothelium and could contribute to transendothelialCl and HCO transport. Furthermore,there is a cAMP-activated Cl pathway on the basolateralmembrane that is not CFTR.

     

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 .

     

Role of glutamate transporters in the regulation of glutathione levels in human macrophages

Cysteine is the limiting precursor forglutathione synthesis. Because of its low bioavailability, cysteine isgenerally produced from cystine, which may be taken up through twodifferent transporters. The cystine/glutamate antiporter(x system) transports extracellular cystine inexchange for intracellular glutamate. The X_AG transportsystem takes up extracellular cystine, glutamate, and aspartate. Bothare sensitive to competition between cystine and glutamate, and excessextracellular glutamate thus inhibits glutathione synthesis, anonexcitotoxic mechanism for glutamate toxicity. We demonstratedpreviously that human macrophages express the glutamate transportersexcitatory amino acid transporter (EAAT)1 and EAAT2 (which do nottransport cystine, X system) and overcomecompetition for the use of cystine transporters. We now showthat macrophages take up cystine through the x andnot the X_AG system. We also found that glutamate, although competing with cystine uptake, dose-dependently increases glutathione synthesis. We used inhibitors to demonstrate that this increase ismediated by EAATs. EAAT expression in macrophages thus leads toglutamate-dependent enhancement of glutathione synthesis by providingintracellular glutamate for direct insertion in glutathione and alsofor fueling the intracellular pool of glutamate andtrans-stimulating the cystine/glutamate antiporter.