Targeting of calsequestrin to sarcoplasmic reticulum after deletions of its acidic carboxy terminus

Calsequestrin (CS) is theCa²⁺ binding protein of thejunctional sarcoplasmic reticulum (jSR) lumen. Recently, a chimericCS-HA1, obtained by adding the nine-amino-acid viral epitopehemagglutinin (HA1) to the COOH terminus of CS, was shown to becorrectly segregated to the sarcoplasmic reticulum [A. Nori, K. A. Nadalini, A. Martini, R. Rizzuto, A. Villa, and P. Volpe.Am. J. Physiol. 272 (Cell Physiol. 41): C1420-C1428,1997]. A putative targeting mechanism of CS to jSR implieselectrostatic interactions between negative charges on CS and positivecharges on intraluminal domains of jSR integral proteins, such astriadin and junctin. To test this hypothesis, 2 deletion mutants ofchimeric CS were engineered: CS-HA1Glu-Asp, in which the 14 acidicresidues[-Glu-(Asp)₅-Glu-(Asp)₇-] of the COOH-terminal tail were removed, andCS-HA149_COOH, in which thelast, mostly acidic, 49 residues of the COOH terminus were removed.Both mutant cDNAs were transiently transfected in HeLa cells, myoblastsof rat skeletal muscle primary cultures, or regenerating soleus musclefibers of adult rats. The expression and intracellular localization ofCS-HA1 mutants were studied by epifluorescence microscopy with use ofantibodies against CS or HA1. CS-HA1 mutants were shown to beexpressed, sorted, and correctly segregated to jSR. Thus short or longdeletions of the COOH-terminal acidic tail do not influence thetargeting mechanism of CS.

     

Model of beta -cell mitochondrial calcium handling and electrical activity. I.Cytoplasmic variables

We continue our development of a kineticmodel of bursting electrical activity in the pancreatic -cell( J. Keizer and G. Magnus. Biophys. J. 56: 229-242,1989), including the influence of Ca²⁺ handling by themitochondria. Our minimal model of mitochondrial Ca²⁺handling [G. Magnus and J. Keizer. Am. J. Physiol. 273 (Cell Physiol. 42): C717-C733, 1997] is expanded toinclude the D-glucose dependence of the rate of productionof mitochondrial reducing equivalents. The Ca²⁺ dependenceof the mitochondrial dehydrogenases, which is also included in themodel, plays only a small role in the simulations, since thedehydrogenases appear to be maximally activated when D-glucose concentrations are sufficient to producebursting. A previous model of ionic currents in the plasma membrane isupdated using a recent experimental characterization of the dependence of the conductance of the ATP-sensitive K⁺(K_ATP) current on adenine nucleotides. The resultingwhole cell model is complex, involving 12 dynamic variables that coupleCa²⁺ handling in the cytoplasm and the mitochondria withelectrical activity in the plasma and inner mitochondrial membranes.Simulations with the whole cell model give rise to bursting electricalactivity similar to that seen in pancreatic islets and clusters ofpancreatic -cells. The full D-glucose dose response ofelectrical activity is obtained if the cytosolic rate of ATP hydrolysisis a sigmoidal function of glucose. The simulations give the correctshape, period, and phase of the associated oscillations in cytosolicCa²⁺, predict that the conductance of the K_ATPcurrent oscillates out of phase with electrical activity [as recentlyobserved in ob/ob mice (O. Larsson, H. Kindmark, R. Bränstrom, B. Fredholm, and P.-O. Berggren. Proc. Natl. Acad.Sci. USA 93: 5161-5165, 1996)], and make other novelpredictions. In this model, bursting results because Ca²⁺uptake into mitochondria during the active phase reduces the mitochondrial inner membrane potential, reducing the rate of production of ATP, which in turn activates the K_ATP current andrepolarizes the plasma membrane.

     

Properties of ATP-dependent K(+) channels in adrenocortical cells

Bovine adrenocortical zona fasciculata (AZF)cells express a novel ATP-dependent K⁺-permeable channel(I_AC). Whole cell and single-channel recordings were used to characterize I_AC channels withrespect to ionic selectivity, conductance, and modulation bynucleotides, inorganic phosphates, and angiotensin II (ANG II). Inoutside-out patch recordings, the activity of unitaryI_AC channels is enhanced by ATP in the patchpipette. These channels were K⁺ selective with nomeasurable Na⁺ or Ca²⁺ conductance. Insymmetrical K⁺ solutions with physiological concentrationsof divalent cations (M²⁺), I_ACchannels were outwardly rectifying with outward and inward chordconductances of 94.5 and 27.0 pS, respectively. In the absence ofM²⁺, conductance was nearly ohmic. Hydrolysis-resistantnucleotides including AMP-PNP and NaUTP were more potent than MgATP asactivators of whole cell I_AC currents. Inorganicpolytriphosphate (PPP_i) dramatically enhancedI_AC activity. In current-clamp recordings, nucleotides and PPP_i produced resting potentials in AZFcells that correlated with their effectiveness in activatingI_AC. ANG II (10 nM) inhibited whole cellI_AC currents when patch pipettes contained 5 mMMgATP but was ineffective in the presence of 5 mM NaUTP and 1 mM MgATP.Inhibition by ANG II was not reduced by selective kinase antagonists.These results demonstrate that I_AC is adistinctive K⁺-selective channel whose activity isincreased by nucleotide triphosphates and PPP_i.Furthermore, they suggest a model for I_AC gatingthat is controlled through a cycle of ATP binding and hydrolysis.

     

Role of endogenous female hormones in hypoxic chemosensitivity

Tatsumi, Koichiro, Cheryl K. Pickett, Christopher R. Jacoby,John V. Weil, and Lorna G. Moore. Role of endogenous female hormones in hypoxic chemosensitivity. J. Appl.Physiol. 83(5): 1706-1710, 1997.Effective alveolar ventilation and hypoxicventilatory response (HVR) are higher in females than in males andafter endogenous or exogenous elevation of progesterone and estrogen.The contribution of normal physiological levels of ovarian hormones toresting ventilation and ventilatory control and whether their site(s) of action is central and/or peripheral are unclear.Accordingly, we examined resting ventilation, HVR, and hypercapnicventilatory responses (HCVR) before and 3 wk after ovariectomy in fivefemale cats. We also compared carotid sinus nerve (CSN) and centralnervous system translation responses to hypoxia in 6 ovariectomized and 24 intact female animals. Ovariectomy decreased serum progesterone butdid not change resting ventilation, end-tidalPCO₂, or HCVR (allP = NS). Ovariectomy reduced theHVR shape parameter A in the awake(38.9 ± 5.5 and 21.2 ± 3.0 before and after ovariectomy, respectively, P < 0.05) andanesthetized conditions. The CSN response to hypoxia was lower inovariectomized than in intact animals (shape parameterA = 22.6 ± 2.5 and 54.3 ± 3.5 in ovariectomized and intact animals, respectively,P < 0.05), but central nervous system translation of CSN activity into ventilation was similar inovariectomized and intact animals. We concluded that ovariectomy decreased ventilatory and CSN responsiveness to hypoxia, suggesting that the presence of physiological levels of ovarian hormones influences hypoxic chemosensitivity by acting primarily at peripheral sites.

     

In vitro bronchial responsiveness in two highly inbred rat strains

Wang, C. G., J. J. Almirall, C. S. Dolman, R. J. Dandurand,and D. H. Eidelman. In vitro bronchial responsiveness in twohighly inbred rat strains. J. Appl.Physiol. 82(5): 1445-1452, 1997.We investigatedmethacholine (MCh)-induced bronchoconstriction in explanted airwaysfrom Fischer and Lewis rats. Lung explants, 0.5- to 1.0-mm thick, wereprepared from agarose-inflated lungs of anesthetized 8- to 12-wk-oldmale rats. After overnight culture, videomicroscopy was used to recordbaseline images of the individual airways. Dose-response curves to MChwere then constructed by repeated administration of MCh; airways werereimaged 10 min after each MCh administration. Airway internal luminalarea(A_i)was measured at successive MCh concentrations from10⁹ to10¹ M. Inaddition to the effective concentration leading to 50% of the achievedmaximal response, we also determined the effective concentrationleading to a 40% reduction inA_i.Both the effective concentration leading to 50% of the achievedmaximal response and the concentration leading to a 40% reduction inA_iwere significantly lower among Fischer rat airways(P < 0.05). Airway closure was morecommon among Fischer rat airways (17%) than among those of Lewis rats(7.5%). Responsiveness of Fischer rat airways was more heterogeneousthan among Lewis airways; a larger number of Fischer rat airwaysexhibited high sensitivity to MCh. There was no relationship betweenresponsiveness and baselineA_iin either strain. In a second experiment, we measured the rate ofcontraction of explanted airways from lungs inflated to 50, 75, and100% of total lung capacity. The average rate of contraction in thefirst 15 s was higher in Fischer rat airways at each inflation volume.These data indicate that the hyperresponsiveness of the Fischer rat reflects the responsiveness of individual airways throughout the airwaytree and are consistent with the notion that in this model hyperresponsiveness is an intrinsic property of airway smooth muscle.

     

Effects of inhaled CO2 and added dead space on idiopathic central sleep apnea

Xie, Ailiang, Fiona Rankin, Ruth Rutherford, and T. DouglasBradley. Effects of inhaledCO₂ and added dead space on idiopathic central sleep apnea. J. Appl.Physiol. 82(3): 918-926, 1997.We hypothesizedthat reductions in arterial PCO₂ (Pa_CO2) below the apnea threshold play akey role in the pathogenesis of idiopathic central sleep apnea syndrome(ICSAS). If so, we reasoned that raisingPa_CO2 would abolish apneas in thesepatients. Accordingly, patients with ICSAS were studied overnight onfour occasions during which the fraction of end-tidalCO₂ and transcutaneous PCO₂ were measured: during room airbreathing (N1), alternating room airand CO₂ breathing(N2),CO₂ breathing all night(N3), and addition of dead space viaa face mask all night (N4).Central apneas were invariably preceded by reductions infraction of end-tidal CO₂. Bothadministration of a CO₂-enrichedgas mixture and addition of dead space induced 1- to 3-Torr increasesin transcutaneous PCO₂, whichvirtually eliminated apneas and hypopneas; they decreased from43.7 ± 7.3 apneas and hypopneas/h onN1 to 5.8 ± 0.9 apneas andhypopneas/h during N3(P < 0.005), from 43.8 ± 6.9 apneas and hypopneas/h during room air breathing to 5.9 ± 2.5 apneas and hypopneas/h of sleep duringCO₂ inhalation during N2 (P < 0.01), and to 11.6% of the room air level while the patients werebreathing through added dead space duringN4 (P < 0.005). Because raisingPa_CO2 through two different meansvirtually eliminated central sleep apneas, we conclude that centralapneas during sleep in ICSA are due to reductions inPa_CO2 below the apnea threshold.

     

Letters to the Editor

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

Mitchell, Claire H., Jin Jun Zhang, Liwei Wang, andTim J. C. Jacob. Volume-sensitive chloride current in pigmented ciliary epithelial cells: role of phospholipases. Am. J. Physiol. 272 (Cell Physiol. 41): C212-C222, 1997.Thewhole cell recording technique was used to examine an outwardlyrectifying chloride current activated by hypotonic shock in bovinepigmented ciliary epithelial (PCE) cells. Removal of internal andexternal Ca²⁺ did not affect the activation of thesecurrents, but they were abolished by the phospholipase C inhibitorneomycin. The current was blocked by5-nitro-2-(3-phenylpropylamino)benzoic acid,4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid, and4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) in avoltage-dependent manner, but tamoxifen, dideoxyforskolin, andquinidine did not affect it. This blocking profile differs from that ofthe volume-sensitive chloride channel in neighboring nonpigmentedciliary epithelial cells (Wu, J., J. J. Zhang, H. Koppel, and T. J. C. Jacob. J. Physiol. Lond. 491: 743-755, 1996), and thisdifference implies that the volume responses of the two cell types aremediated by different chloride channels (Jacob, T. J. C., and J. J. Zhang. J. Physiol. Lond. In press). Intracellular administration of guanosine 5'-O-(3-thiotriphosphate) (GTPS) to PCE cells induced a transient, time-independent, outwardly rectifying chloride current that closely resembled the current activated by hypotonic shock. DIDS produced a voltage-dependent blockof the GTPS-activated current similar to the block of the hypotonically activated current. Intracellular neomycin completely prevented activation of this current as did incubation of the cells incalphostin C, an inhibitor of protein kinase C (PKC). Removal ofCa²⁺ did not affect activation of the current by GTPSbut extended the duration of the response. Inhibition of phospholipaseA₂ (PLA₂) with p-bromophenacyl bromideprevented the activation of the hypotonically induced current and alsoinhibited the current once activated by hypotonic solution. Thefindings imply that the hypotonic response in PCE cells is mediated byboth phospholipase C (PLC) and PLA₂. Both phospholipasesgenerate arachidonic acid, and, in addition, the PLC pathway regulatesthe PLA₂ pathway via a PKC-dependent phosphorylation ofPLA₂.

     

Effect of prior O2 breathing on ventilatory response to sustained isocapnic hypoxia in adult humans

Honda, Y., H. Tani, A. Masuda, T. Kobayashi, T. Nishino, H. Kimura, S. Masuyama, and T. Kuriyama. Effect of priorO₂ breathing on ventilatoryresponse to sustained isocapnic hypoxia in adult humans.J. Appl. Physiol. 81(4):1627-1632, 1996.Sixteen healthy volunteers breathed 100%O₂ or room air for 10 min in random order, then their ventilatory response to sustained normocapnic hypoxia (80% arterial O₂saturation, as measured with a pulse oximeter) was studied for 20 min.In addition, to detect agents possibly responsible for the respiratorychanges, blood plasma of 10 of the 16 subjects was chemically analyzed.1) Preliminary O₂ breathing uniformly andsubstantially augmented hypoxic ventilatory responses.2) However, the profile ofventilatory response in terms of relative magnitude, i.e., biphasichypoxic ventilatory depression, remained nearly unchanged.3) Augmented ventilatory incrementby prior O₂ breathing wassignificantly correlated with increment in the plasma glutamine level.We conclude that preliminary O₂administration enhances hypoxic ventilatory response without affectingthe biphasic response pattern and speculate that the excitatory aminoacid neurotransmitter glutamate, possibly derived from augmentedglutamine, may, at least in part, play a role in this ventilatoryenhancement.

     

Interaction between airway edema and lung inflation on responsiveness of individual airways in vivo

Brown, Robert H., Wayne Mitzner, and Elizabeth M. Wagner.Interaction between airway edema and lung inflation onresponsiveness of individual airways in vivo. J. Appl.Physiol. 83(2): 366-370, 1997.Inflammatorychanges and airway wall thickening are suggested to cause increasedairway responsiveness in patients with asthma. In fivesheep, the dose-response relationships of individual airways weremeasured at different lung volumes to methacholine (MCh) before andafter wall thickening caused by the inflammatory mediator bradykininvia the bronchial artery. At 4 cmH₂O transpulmonary pressure(Ptp), 5 µg/ml MCh constricted the airways to a maximum of 18 ± 3%. At 30 cmH₂O Ptp, MCh resultedin less constriction (to 31 ± 5%). Bradykinin increased airwaywall area at 4 and 30 cmH₂O Ptp(159 ± 6 and 152 ± 4%, respectively;P < 0.0001). At 4 cmH₂O Ptp, bradykinin decreasedairway luminal area (13 ± 2%; P < 0.01), and the dose-response curve was significantly lower (P = 0.02). At 30 cmH₂O, postbradykinin, the maximalairway narrowing was not significantly different (26 ± 5%;P = 0.76). Bradykinin produced substantial airway wall thickening and slight potentiation ofthe MCh-induced airway constriction at low lung volume. At high lung volume, bradykinin increased wall thickness but had no effecton the MCh-induced airway constriction. We conclude that inflammatoryfluid leakage in the airways cannot be a primary cause of airwayhyperresponsiveness.

     

Effects of training duration on substrate turnover and oxidation during exercise

Phillips, S. M., H. J. Green, M. A. Tarnopolsky, G. J. F. Heigenhauser, R. E. Hill, and S. M. Grant. Effects of training duration on substrate turnover and oxidation during exercise. J. Appl. Physiol. 81(5):2182-2191, 1996.Adaptations in fat and carbohydrate metabolismafter a prolonged endurance training program were examined using stableisotope tracers of glucose([6,6-²H₂]glucose),glycerol([²H₅]glycerol),and palmitate([²H₂]palmitate).Active, but untrained, males exercised on a cycle for 2 h/day[60% pretraining peak O₂consumption (O_{2 peak}) = 44.3 ± 2.4 ml ^· kg^{1 ·} min¹]for a total of 31 days. Three cycle tests (90 min at 60% pretraining O_{2 peak}) wereadministered before training (PRE) and after 5 (5D) and 31 (31D) daysof training. Exercise increased the rate of glucose production(R_a) and utilization(R_d) as well as the rate oflipolysis (glycerol R_a) and freefatty acid turnover (FFA R_a/R_d).At 5D, training induced a 10% (P < 0.05) increase in total fat oxidation because of an increase inintramuscular triglyceride oxidation (+63%,P < 0.05) and a decreased glycogenoxidation (16%, P < 0.05).At 31D, total fat oxidation during exercise increased a further 58%(P < 0.01). The pattern of fatutilization during exercise at 31D showed a reduced reliance on plasmaFFA oxidation (FFA R_d) and agreater dependence on oxidation of intramuscular triglyceride, whichincreased more than twofold (P < 0.001). In addition, glucose R_aand R_d were reduced at all timepoints during exercise at 31D compared with PRE and 5D. We concludethat long-term training induces a progressive increase in fatutilization mediated by a greater oxidation of fats from intramuscularsources and a reduction in glucose oxidation. Initial changes arepresent as early as 5D and occur before increases in muscle maximalmitochondrial enzyme activity [S. M. Phillips, H. J. Green, M. A. Tarnopolsky, G. J. F. Heigenhauser, and S. M. Grant.Am. J. Physiol. 270 (Endocrinol. Metab. 33):E265-E272, 1996].

     

A model of the spontaneously breathing patient: applications to intrinsic PEEP and work of breathing

Schuessler, Thomas F., Stewart B. Gottfried, and Jason H. T. Bates. A model of the spontaneously breathing patient: applications to intrinsic PEEP and work of breathing.J. Appl. Physiol. 82(5):1694-1703, 1997.Intrinsic positive end-expiratory pressure(PEEP_i) and inspiratory work ofbreathing (WI) are important factors in the management of severe obstructive respiratory disease. Weused a computer model of spontaneously breathing patients with chronicobstructive pulmonary disease to assess the sensitivity of measurementtechniques for dynamic PEEP_i(PEEP_{i dyn}) andWI to expiratory muscle activity(EMA) and cardiogenic oscillations (CGO) on esophageal pressure.Without EMA and CGO, bothPEEP_{i dyn} andWI were accurately estimated(r = 0.999 and 0.95, respectively). Addition of moderate EMA causedPEEP_{i dyn} andWI to be systematically overestimated by 141 and 52%, respectively. Furthermore, CGOintroduced large random errors, obliterating the correlation betweenthe true and estimated values for bothPEEP_{i dyn}(r = 0.29) andWI (r = 0.38). Thus the accurateestimation of PEEP_{i dyn} andWI requires steps to be taken toameliorate the adverse effects of both EMA and CGO. Taking advantage ofour simulations, we also investigated the relationship betweenPEEP_{i dyn} and staticPEEP_i(PEEP_{i stat}). ThePEEP_{i dyn}/PEEP_{i stat}ratio decreased as stress adaptation in the lung was increased,suggesting that heterogeneity of expiratory flow limitation isresponsible for the discrepancies betweenPEEP_{i dyn} andPEEP_{i stat} thathave been reported in patients with severe airwayobstruction.

     

Effects of hypoxic pulmonary vasoconstriction on pulmonary gas exchange

Brimioulle, Serge, Philippe Lejeune, and Robert Naeije.Effects of hypoxic pulmonary vasoconstriction on pulmonary gasexchange. J. Appl. Physiol. 81(4):1535-1543, 1996.Several reports have suggested that hypoxicpulmonary vasoconstriction (HPV) might result in deterioration ofpulmonary gas exchange in severe hypoxia. We therefore investigated theeffects of HPV on gas exchange in normal and diseased lungs. Weincorporated a biphasic HPV stimulus-response curve observed in intactdogs (S. Brimioulle, P. Lejeune, J. L. Vachièry, M. Delcroix, R. Hallemans, and R. Naeije, J. Appl.Physiol. 77: 476-480, 1994) into a 50-compartment lung model (J. B. West, Respir.Physiol. 7: 88-110, 1969) to control the amount ofblood flow directed to each lung compartment according to the localhypoxic stimulus. The resulting model accurately reproduced the bloodgas modifications caused by HPV changes in dogs with acute lung injury.In single lung units, HPV had a moderate protective effect on alveolaroxygenation, which was maximal at near-normal alveolarPO₂ (75-80 Torr), mixed venousPO₂ (35 Torr), andPO₂ at which hemoglobin is 50%saturated (24 Torr). In simulated diseased lungs associated with40-60 Torr arterial PO₂,however, HPV increased arterial PO₂ by 15-20 Torr. We conclude that HPV can improve arterialoxygenation substantially in respiratory failure.

     

Functional reconstitution of an eicosanoid-modulated Cl- channel from bovine tracheal smooth muscle

We describe thebiochemical properties of an eicosanoid-modulated Clchannel and assess the mechanisms by which the epoxyeicosatrienoic acids (EETs) alter both its unitary conductance and its openprobability (P_o). After a purification protocolinvolving wheat-germ agglutinin affinity and anion-exchangechromatography, the proteins were sequentially inserted into liposomes,which were then fused into PLBs. Functional and biochemicalcharacterization tests confirm that the Cl channel is a55-kDa glycosylated monomer with voltage- and Ca²⁺concentration-independent activity. 5,6- and 8,9-EET decreased theconductance of the native channel (control conductance: 70 ± 5 pSin asymmetrical 50 mM trans/250 mM cis CsCl) in aconcentration-dependent manner, with respective 50% inhibitoryconcentration values of 0.31 and 0.42 µM. These regioisomerssimilarly decreased the conductance of the purified channel (controlconductance value: 75 ± 5 pS in asymmetrical 50 mMtrans/250 mM cis CsCl), which had been stripped of its native proteic and lipidic environment. On the other hand, 5,6- and 8,9-EETs decreased the P_o of the nativechannel with respective 50% inhibitory concentration values of 0.27 and 0.30 µM but failed to alter the P_o of thepurified protein. Thus we suggest that the effects of these EETs onchannel conductance likely result from direct interactions ofEET anions with the channel pore, whereas the alterationof P_o requires a lipid environment of specificcomposition that is lost on solubilization and purification of the protein.

     

Hyperpnea-induced bronchoconstriction is dependent on tachykinin-induced cysteinyl leukotriene synthesis

Yang, X. X., W. S. Powell, M. Hojo, and J. G. Martin.Hyperpnea-induced bronchoconstriction is dependent ontachykinin-induced cysteinyl leukotriene synthesis. J. Appl. Physiol. 82(2): 538-544, 1997.The purposeof the study was to test the hypothesis that tachykinins mediatehyperpnea-induced bronchoconstriction indirectly by triggeringcysteinyl leukotriene (LT) synthesis in the airways. Guinea pigs(350-600 g) were anesthetized with xylazine and pentobarbital sodium and received hyperpnea challenge (tidal volume 3.5-4.0 ml,frequency 150 breaths/min) with either humidified isocapnic gas(n = 6) or dry gas(n = 7). Dry gas challenge wasperformed on animals that received MK-571(LTD₄ antagonist; 2 mg/kg iv; n = 5), capsaicin(n = 4), neurokinin (NK) antagonists[NK₁ (CP-99994) + NK₂ (SR-48968) (1 mg/kg iv);n = 6], or theH₁ antihistamine pyrilamine (2 mg/kg iv; n = 5). We measured thetracheal pressure and collected bile for 1 h before and 2 h afterhyperpnea challenge. We examined the biliary excretion of cysteinylLTs; the recovery of radioactivity in bile after instillation of 1 µCi [³H]LTC₄intratracheally averaged 24% within 4 h(n = 2). The major cysteinyl LTidentified was LTD₄ (32% recoveryof radioactivity). Cysteinyl LTs were purified from bile of animalsundergoing hyperpnea challenge by using reverse-phase high-pressureliquid chromatography and quantified by radioimmunoassay. There was asignificant increase in the peak value of tracheal pressure afterchallenge, indicating bronchoconstriction in dry gas-challenged animalsbut not after humidified gas challenge. MK-571, capsaicin, and NKantagonists prevented the bronchoconstriction; pyrilamine didnot. Cysteinyl LT levels in the bile after challenge weresignificantly increased from baseline in dry gas-challenged animals(P < 0.05) and were higher than inthe animals challenged with humidified gas or dry gas-challengedanimals treated with capsaicin or NK antagonists (P < 0.01). The results indicatethat isocapnic dry gas hyperpnea-induced bronchoconstriction is LTmediated and the role of tachykinins in the response is indirectthrough release of LTs. Endogenous histamine does not contribute to theresponse.

     

Effect of oxygen tension and rate of pressure reduction during decompression on central gas bubbles

Reinertsen, R. E., V. Flook, S. Koteng, and A. O. Brubakk.Effect of oxygen tension and rate of pressure reduction duringdecompression on central gas bubbles. J. Appl.Physiol. 84(1): 351-356, 1998.Reduction inascent speed and an increase in theO₂ tension in the inspired airhave been used to reduce the risk for decompression sickness. It haspreviously been reported that decompression speed andO₂ partial pressure are linearly related for human decompressions from saturation hyperbaric exposures. The constant of proportionality K(K = rate/partial pressure of inspiredO₂) indicates the incidence ofdecompression sickness. The present study investigated the relationshipamong decompression rate, partial pressure of inspiredO₂, and the number of central gasbubbles after a 3-h dive to 500 kPa while breathing nitrox with an O₂ content of 35 kPa. Weused transesophageal ultrasonic scanning to determine the number ofbubbles in the pulmonary artery of pigs. The results show that, for agiven level of decompression stress, decompression rate andO₂ tension in the inspired air canbe traded off against each other by using pulmonary artery bubbles asan end point. The results also seem to confirm that decompressions thathave a high K value are morestressful.

     

Regulation of the epithelial Na+ channel by intracellular Na+

The hypothesis that the intracellularNa⁺ concentration([Na⁺]_i)is a regulator of the epithelialNa⁺ channel (ENaC) was tested withthe Xenopus oocyte expression systemby utilizing a dual-electrode voltage clamp.[Na⁺]_iaveraged 48.1 ± 2.2 meq (n = 27)and was estimated from the amiloride-sensitive reversal potential.[Na⁺]_iwas increased by direct injection of 27.6 nl of 0.25 or 0.5 MNa₂SO₄.Within minutes of injection,[Na⁺]_istabilized and remained elevated at 97.8 ± 6.5 meq(n = 9) and 64.9 ± 4.4 (n = 5) meq 30 min after theinitial injection of 0.5 and 0.25 MNa₂SO₄,respectively. This increase of[Na⁺]_icaused a biphasic inhibition of ENaC currents. In oocytes injected with0.5 MNa₂SO₄(n = 9), a rapid decrease of inwardamiloride-sensitive slope conductance(g_Na) to 0.681 ± 0.030 of control within the first 3 min and a secondary, slowerdecrease to 0.304 ± 0.043 of control at 30 min were observed.Similar but smaller inhibitions were also observed with the injectionof 0.25 MNa₂SO₄.Injection of isotonicK₂SO₄(70 mM) or isotonicK₂SO₄made hypertonic with sucrose (70 mMK₂SO₄-1.2M sucrose) was without effect. Injection of a 0.5 M concentration ofeitherK₂SO₄,N-methyl-D-glucamine (NMDG) sulfate, or 0.75 M NMDG gluconate resulted in a much smaller initial inhibition (<14%) and little or no secondary decrease. Thusincreases of[Na⁺]_ihave multiple specific inhibitory effects on ENaC that can betemporally separated into a rapid phase that was complete within 2-3 min and a delayed slow phase that was observed between 5 and 30 min.

     

ATP-dependent regulation of inwardly rectifying K+ current in bovine retinal pigment epithelial cells

Inwardlyrectifying K⁺ current(I_Kir) infreshly isolated bovine retinal pigment epithelial (RPE) cells wasstudied in the whole cell recording configuration of the patch-clamptechnique. When cells were dialyzed with pipette solution containing noATP, I_Kir randown completely in <10 min [half time(t_1/2) = 1.9 min]. In contrast, dialysis with 2 mM ATP sustainedI_Kir for 10 min or more. Rundown was also prevented with 4 mM GTP or ADP. When 0.5 mMATP was used,I_Kir ran down by~71%. Mg²⁺ was a criticalcofactor because rundown occurred when the pipette solution contained 4 mM ATP but no Mg²⁺(t_1/2 = 1.8 min).I_Kir also randown when the pipette solution contained 4 mMMg²⁺ + 4 mM5'-adenylylimidodiphosphate(t_1/2 = 2.7 min)or 4 mM adenosine 5'-O-(3-thiotriphosphate)(t_1/2 = 1.9 min),nonhydrolyzable and poorly hydrolyzable ATP analogs, respectively. Weconclude that the sustained activity ofI_Kirin bovine RPE requires intracellular MgATP and that the underlyingmechanism may involve ATP hydrolysis.

     

Thermal and metabolic responses to cold-water immersion at knee, hip, and shoulder levels

Lee, Dae T., Michael M. Toner, William D. McArdle, IoannisS. Vrabas, and Kent B. Pandolf. Thermal and metabolic responses tocold-water immersion at knee, hip, and shoulder levels.J. Appl. Physiol. 82(5):1523-1530, 1997.To examine the effect of cold-water immersion atdifferent depths on thermal and metabolic responses, eight men (25 yrold, 16% body fat) attempted 12 tests: immersed to the knee (K), hip(H), and shoulder (Sh) in 15 and 25°C water during both rest (R) orleg cycling [35% peak oxygen uptake; (E)] for up to 135 min. At 15°C, rectal (T_re)and esophageal temperatures(T_es) between R and E were notdifferent in Sh and H groups (P > 0.05), whereas both in K group were higher during E than R(P < 0.05). At 25°C,T_re was higher(P < 0.05) during E than R at alldepths, whereas T_es during E washigher than during R in H and K groups.T_re remained at control levels inK-E at 15°C, K-E at 25°C, and in H-E groups at 25°C,whereas T_es remained unchanged inK-E at 15°C, in K-R at 15°C, and in all 25°C conditions (P > 0.05). During R and E, themagnitude of T_re change wasgreater (P < 0.05) than themagnitude of T_es change in Sh andH groups, whereas it was not different in the K group(P > 0.05). Total heat flow wasprogressive with water depth. During R at 15 and 25°C, heatproduction was not increased in K and H groups from control level(P > 0.05) but it did increase in Shgroup (P < 0.05). The increase inheat production during E compared with R was smaller(P < 0.05) in Sh (121 ± 7 W/m² at 15°C and 97 ± 6 W/m² at 25°C) than in H (156 ± 6 and 126 ± 5 W/m²,respectively) and K groups (155 ± 4 and 165 ± 6 W/m², respectively). These datasuggest that T_re andT_es respond differently duringpartial cold-water immersion. In addition, water levels above knee in15°C and above hip in 25°C cause depression of internal temperatures mainly due to insufficient heat production offsetting heatloss even during light exercise.

     

Amiloride-sensitive sodium current in everted Ambystoma initial collecting tubule: short-term insulin effects

Whole cellpatch-clamp techniques were used to investigate amiloride-sensitivesodium conductance (G_Na) in the everted initial collecting tubule of Ambystoma. Accessibility to both theapical and basolateral membranes made this preparation ideal forstudying the regulation of sodium transport by insulin.G_Na accounted for 20% of total cell conductance(G_T) under control conditions. A restingmembrane potential of 75 ± 2 mV (n = 7)together with the fact that G_T is stable withtime suggested that the cells studied were viable. Measurements ofcapacitance and use of a known uncoupling agent, heptanol, suggestedthat cells were not electrically coupled. Thus the values ofG_T and G_Na represented individual principal cells. Exposure of the basolateral membrane toinsulin (1 mU/ml) for 10-60 min significantly (P < 0.05) increased the normalized G_Na [1.2 ± 0.3 nS (n = 6) vs. 2.0 ± 0.4 nS(n = 6)]. Cell-attached patch-clamp techniques wereused to further elucidate the mechanism by which insulin increasesamiloride-sensitive epithelial sodium channel (ENaC) activity. In thepresence of insulin there was no apparent change in either the numberof active levels/patch or the conductance of ENaC. The openprobability increased significantly (P < 0.01) from0.21 ± 0.04 (n = 6) to 0.46 ± 0.07 (n = 6). Thus application of insulin enhanced sodium reabsorption by increasing the fraction of time the channel spent inthe open state.

     

Secretory modulation of basolateral membrane inwardly rectified K(+) channel in guinea pig distal colonic crypts

Cell-attached recordings revealedK⁺ channel activity in basolateral membranes ofguinea pig distal colonic crypts. Inwardly rectified currents wereapparent with a pipette solution containing 140 mM K⁺.Single-channel conductance () was 9 pS at the resting membrane potential. Another inward rectifier with  of 19 pS was observed occasionally. At a holding potential of 80 mV,  was 21 and 41 pS,respectively. Identity as K⁺ channels was confirmed afterpatch excision by changing the bath ion composition. From reversalpotentials, relative permeability of Na⁺ overK⁺ (P_Na/P_K)was 0.02 ± 0.02, withP_Rb/P_K = 1.1 andP_Cl/P_K < 0.03. Spontaneous open probability (P_o) of the 9-pSinward rectifier (^gpK_ir) was voltageindependent in cell-attached patches. Both a low(P_o = 0.09 ± 0.01) and a moderate(P_o = 0.41 ± 0.01) activity mode wereobserved. Excision moved ^gpK_ir to the mediumactivity mode; P_o of^gpK_ir was independent of bath Ca²⁺activity and bath acidification. Addition of Cl andK⁺ secretagogues altered P_o of^gpK_ir. Forskolin or carbachol (10 µM)activated the small-conductance ^gpK_ir inquiescent patches and increased P_o inlow-activity patches. K⁺ secretagogues, either epinephrine(5 µM) or prostaglandin E₂ (100 nM), decreasedP_o of ^gpK_ir in activepatches. This ^gpK_ir may be involved inelectrogenic secretion of Cl and K⁺ acrossthe colonic epithelium, which requires a large basolateral membraneK⁺ conductance during maximal Cl secretionand, presumably, a lower K⁺ conductance during primaryelectrogenic K⁺ secretion.