RTN TRH causes prolonged respiratory stimulation

Cream, Carlos L., Aihua Li, and Eugene E. Nattie. RTNTRH causes prolonged respiratory stimulation. J. Appl.Physiol. 83(3): 792-799, 1997.We injectedthyrotropin-releasing hormone (TRH; 10 nl; 0.25, 0.5, 1.0, or 10 mM),its inactive free acid form (TRHOH; 1 mM), or a metabolite with lowTRH-receptor binding affinity, histidine-proline diketopiperazine (cHP;1 mM), into the retrotrapezoid nucleus of anesthetized rats. Injectionlocation was verified by anatomic analysis. Lower doses (0.25-0.5mM) significantly increased both the product of integrated phrenicamplitude and frequency(Phr · f) and f for 20-30min compared with artificial cerebrospinal fluid control injections. Higher doses (1.0-10 mM) produced greater and long-lastingstimulation of Phr · f,Phr, and f and of blood pressure. Thisstimulation reached values 150% of baseline and durations of 270 minafter a single injection. TRHOH (1 mM ) or cHP (1 mM) had no effect onPhr but increased f, as did 1 mM TRH. We concludethat TRH has a very powerful stimulatory effect in the retrotrapezoidnucleus region on Phr · f, withthe Phr response seemingly specific for TRHreceptors. Similar responses of f to TRHOH and cHP suggest it may benonspecific.

     

Neural-mechanical coupling of breathing in REM sleep

Smith, C. A., K. S. Henderson, L. Xi, C.-M. Chow, P. R. Eastwood, and J. A. Dempsey. Neural-mechanical coupling of breathing in REM sleep. J. Appl.Physiol. 83(6): 1923-1932, 1997.During rapid-eye-movement (REM) sleep theventilatory response to airway occlusion is reduced. Possiblemechanisms are reduced chemosensitivity, mechanical impairment of thechest wall secondary to the atonia of REM sleep, or phasic REM eventsthat interrupt or fractionate ongoing diaphragm electromyogram (EMG)activity. To differentiate between these possibilities, we studiedthree chronically instrumented dogs before, during, and after15-20 s of airway occlusion during non-REM (NREM) and phasic REMsleep. We found that 1) for a given inspiratory time the integrated diaphragm EMG(Di) was similar or reduced in REM sleep relativeto NREM sleep; 2) for a givenDi in response to airway occlusion and thehyperpnea following occlusion, the mechanical output (flow or pressure)was similar or reduced during REM sleep relative to NREM sleep;3) for comparable durations ofairway occlusion the Di and integratedinspiratory tracheal pressure tended to be smaller and more variable inREM than in NREM sleep, and 4)significant fractionations (caused visible changes in trachealpressure) of the diaphragm EMG during airway occlusion inREM sleep occurred in ~40% of breathing efforts. Thus reducedand/or erratic mechanical output during and after airwayocclusion in REM sleep in terms of flow rate, tidal volume, and/or pressure generation is attributable largely to reduced neural activity of the diaphragm, which in turn is likely attributable to REM effects, causing reduced chemosensitivity at the level of theperipheral chemoreceptors or, more likely, at the central integrator.Chest wall distortion secondary to the atonia of REM sleep maycontribute to the reduced mechanical output following airway occlusionwhen ventilatory drive is highest.

     

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.

     

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.

     

Blood-gas measurements adjusted for temperature at three sites during incremental exercise in the horse

Rectal temperature(T_re) is often used to adjustmeasurements of blood gases, but these adjusted measurements may notapproximate temperatures during intense exercise at main sites of gasexchange: muscle and lung. To evaluate differences in blood gasesbetween sites, temperatures (T) were measured with thermocouples in the rectum (re), in mixed venous blood (), ingluteal muscle (mu), and on the skin (sk) in seven Arabian horses asthey underwent an incremental exercise test on a treadmill. Bloodsamples were drawn from the carotid artery and pulmonary artery (mixedvenous) 30 s before each increase in speed and during recovery. Blood gases and pH were measured at 37°C, and all variables were adjusted to T_re,, andT_mu. Adjusted variables duringexercise and recovery were significantly different from each other atthe three sites. Linear and polynomial equations described the timecourse of venous temperature and fromT_re andT_sk during exercise and fromT_sk during recovery.Interpretation of changes in muscle metabolism and gas exchanges basedon blood-gas measurements is improved if they are adjustedappropriately to T_mu or, which may be predicted fromT_sk in addition toT_re during strenuous exercise andfrom T_sk during recovery.

     

Absence of myofibrillar creatine kinase and diaphragm isometric function during repetitive activation

Creatine kinase(CK) provides ATP buffering in skeletal muscle and is expressed as1) cytosolic myofibrillar CK (M-CK)and 2) sarcomeric mitochondrial CK(ScCKmit) isoforms that differ in their subcellular localization. Wecompared the isometric contractile and fatigue properties of1) control CK-sufficient (Ctl),2) M-CK-deficient (M-CK[/]), and3) combined M-CK/ScCKmit-deficientnull mutant (CK[/]) diaphragm (Dia) todetermine the effect of the absence of M-CK activity on Dia performancein vitro. Baseline contractile properties were comparable across groupsexcept for specific force, which was ~16% lower inCK[/] Dia compared withM-CK[/] and Ctl Dia. During repetitiveactivation (40 Hz, duty cycle), force declined in all threegroups. This decline was significantly greater inCK[/] Dia compared with Ctl and M-CK[/] Dia. The pattern of forcedecline did not differ between M-CK[/] andCtl Dia. We conclude that Dia isometric muscle function is notabsolutely dependent on the presence of M-CK, whereas the completeabsence of CK acutely impairs isometric force generation duringrepetitive activation.

     

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.

     

Nitric oxide inhibits superoxide production by inflammatory polymorphonuclear leukocytes

Nitric oxide(NO ·) has a complex role in the inflammatory response. Inthis study, we modified the levels of endogenous NO · in vivoin an acute model of inflammation and evaluated the interactionsbetween NO · and superoxide anion() produced bypolymorphonuclear leukocytes (PMNs) accumulated in the inflamed area.We injected phosphate-buffered saline (control group), 6 µmol ofL-N⁵-(1-iminoethyl)ornithine(L-NIO group), or 6 µmol ofL-arginine (L-arginine group) into thegranuloma pouch induced by carrageenan in rats. plus (indicative of NO · generation) was 188 nmol in the exudate of the control group, but itdecreased in the L-NIO group(P < 0.05) and increased in theL-arginine group(P < 0.05). When PMNsfrom treated rats were incubated in vitro, the productionof superoxide anion () decreased by ~46% in theL-arginine group. Furthermore, was inhibited in PMNs whenL-arginine was addedto the incubation medium before phorbol 12-myristate 13-acetatestimulation but not when added simultaneously. Our results suggest aprotective role for NO · in inflammation, through theinactivation of NADPH oxidase and the consequent impairment of production for cell-mediatedinjury.

     

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.

     

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).

     

Regulation of glutamate transport and transport proteins in a placental cell line

We utilized HRP.1 cells derived from midgestation ratplacental labyrinth to determine that the primary pathway for glutamate uptake is via system X, a Na⁺-dependenttransport system. Kinetic parameters of system X activity were similar to those previously determined in rat and humanplacental membrane vesicle preparations. Amino acid depletion caused asignificant upregulation of system X activity at 6, 24, and 48 h. This increase was reversed by the addition ofglutamate and aspartate but not by the addition of -(methylamino)isobutyric acid. Immunoblot analysis of the three transport proteins previously associated with systemX activity indicated a trend toward an increase inGLT1, EAAC1, and GLAST1 immunoreactive protein contents by 48 h;cell surface expression of the same was enhanced by 24 h.Inhibition analysis suggested key roles for EAAC1 and GLAST1 in basalanionic amino acid transfer, with an enhanced role for GLT1 underconditions of amino acid depletion. In summary, amino acid availabilityas well as intracellular metabolism regulate anionic amino acid uptake into this placental cell line.

     

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.

     

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.

     

Pulmonary disposition of lipophilic amine compounds in the isolated perfused rabbit lung

Audi, S. H., C. A. Dawson, J. H. Linehan, G. S. Krenz, S. B. Ahlf, and D. L. Roerig. Pulmonary disposition of lipophilic aminecompounds in the isolated perfused rabbit lung. J. Appl. Physiol. 84(2): 516-530, 1998.We measured the pulmonaryvenous concentration vs. time curves for [³H]alfentanil,[¹⁴C]lidocaine, and [³H]codeine after thebolus injection of each of these lipophilic amine compounds (LAC) and avascular-reference indicator (fluorescein isothiocyanate-dextran) intothe pulmonary artery of isolated perfused rabbit lungs. A range offlows and perfusate albumin concentrations was studied. To evaluate theinformation content of the data, we developed a kinetic modeldescribing the pulmonary disposition of these LAC that was based onindicator dilution theory, and we sought a robust approach forinterpreting the estimated model parameters. We found that thedistribution of the kinetic model rate constants of the lipophilicamine-tissue interactions can be described by ,, and ,where is a measure of the capacity of the rapidlyequilibrating interactions between the lipophilic amineand the tissue; is a measure of the equilibrium capacity of the slowly equilibrating interactions between the lipophilic amine and the tissue; and isthe mean sojourn time. The values of , , andwere 0.8 ± 0.1 (SE), 0.6 ± 0.1, and 1.6 ± 0.5 s; 1.9 ± 0.1, 5.3 ± 0.4, and 5.6 ± 0.5 s; and 1.1 ± 0.1, 9.8 ± 0.4, and 4.7 ± 0.2 s for alfentanil, lidocaine, and codeine, respectively.These values for , , andreveal the relative dominance of the slowly equilibrating interactions for lidocaine and codeine in comparison with alfentanil. This approachto data analysis may have utility for the potential use of LAC toreveal and to quantify changes in lung tissue composition associatedwith lung disease.

     

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.

     

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.

     

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.

     

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.

     

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.

     

Liposome encapsulation attenuates hemoglobin-induced vasoconstriction in rabbit arterial segments

Rudolph, Alan S., Anthony Sulpizio, Paul Hieble, VictorMacdonald, Mark Chavez, and Giora Feuerstein. Liposomeencapsulation attenuates hemoglobin-induced vasoconstriction in rabbitarterial segments. J. Appl. Physiol.82(6): 1826-1835, 1997.Free hemoglobin (Hb) induces a potentvasoconstrictor response that may limit its therapeutic application asa red blood cell replacement. We have investigated whetherencapsulation of stroma-free Hb (SFHb) or cross-linked Hb (-Hb)in liposomes modulates Hb vasoactivity in isolated blood vessels.Relaxation of rabbit thoracic vessels was measured before and afterexposure to acellular SFHb, -Hb, and liposome-encapsulated SFHbor -Hb. SFHb and -Hb caused significant inhibition ofcarbachol-induced relaxation at 0.5 mg/dl, whereas encapsulationinhibited vessel relaxation at 30- to 60-fold higher Hb concentrations.The contractile response of rabbit ear arterial segments to electricalstimulation in the presence of acellular -Hb resulted in a 150%increase (EC₁₅₀) in contractileamplitude at 0.23 mg/dl, whereas theEC₁₅₀ for encapsulated -Hbwas 13.7 mg/dl. Mechanistic studies of the vasoconstrictor activity ofHb demonstrated that acellular -Hb had no effect onnorepinephrine release in the rabbit ear artery. In addition, neitheracellular nor encapsulated -Hb preparations inhibited endothelialnitric oxide (NO) synthase activity isolated from bovine pulmonaryartery. However, inhibition of vessel relaxation by acellular orencapsulated -Hb was reversed by the NO donor S-nitrosylpenacillamine, implicatingHb-NO binding as a possible mechanism for the vasoconstrictor response.In vitro stopped-flow kinetic studies of Hb-NO binding showed similarrates of reaction for conversion of oxyhemoglobin to methemoglobin(metHb; <2 ms), followed by rapid conversion of metHb to NO-Hb (300 ms) for both acellular and encapsulated -Hb, demonstrating thatliposome encapsulation does not retard NO-Hb binding. The attenuatedvasoactivity of encapsulated Hb may, therefore, result from the limitedaccess of encapsulated Hb to NO imposed by the physical size of theliposome and reduced penetration of Hb across the vascular endothelium.