Focal central chemoreceptor sensitivity in the RTN studied with a CO2 diffusion pipette in vivo

Li, Aihua, and Eugene E. Nattie. Focal centralchemoreceptor sensitivity in the RTN studied with aCO₂ diffusion pipette in vivo.J. Appl. Physiol. 83(2): 420-428, 1997.We describe and use a CO₂diffusion pipette to produce a quickly reversible focal acidosis in theretrotrapezoid nucleus region of the rat brain stem. No tissueinjection is made. Instead, artificial cerebrospinal fluid (aCSF)equilibrated with CO₂ circulateswithin the micropipette, providing a source for continuedCO₂ diffusion into the tissue fromthe pipette tip. Tissue pH electrodes show the acidosis is limited to500 µm from the tip. In controls (aCSF equilibrated with air), 1-minpipette perfusions increased tissue pH slightly and decreased phrenicnerve amplitude. In moderate- andhigh-CO₂ groups (aCSF equilibratedwith 50 or 100% CO₂), 1-minperfusions significantly decreased tissue pH and increased phrenicnerve amplitude in a dose-dependent manner. The responses developed andreversed within minutes. Compared with our prior use of medullary acetazolamide injections to produce a focal acidosis, in this approachthe acidosis 1) arises and reversesquickly and 2) its intensity can bevaried. This allows study of sensitivity and mechanism. We concludefrom this initial experiment that retrotrapezoid nucleus regionchemoreceptors operate within the normal physiological range ofCO₂-induced tissue pH changes.

     

Cellular mechanisms involved in CO(2) and acid signaling in chemosensitive neurons

An increase in CO₂/H⁺ is a major stimulus for increased ventilation and is sensed by specialized brain stem neurons called central chemosensitive neurons. These neurons appear to be spread among numerous brain stem regions, and neurons from different regions have different levels of chemosensitivity. Early studies implicated changes of pH as playing a role in chemosensitive signaling, most likely by inhibiting a K⁺ channel, depolarizing chemosensitive neurons, and thereby increasing their firing rate. Considerable progress has been made over the past decade in understanding the cellular mechanisms of chemosensitive signaling using reduced preparations. Recent evidence has pointed to an important role of changes of intracellular pH in the response of central chemosensitive neurons to increased CO₂/H⁺ levels. The signaling mechanisms for chemosensitivity may also involve changes of extracellular pH, intracellular Ca²⁺, gap junctions, oxidative stress, glial cells, bicarbonate, CO₂, and neurotransmitters. The normal target for these signals is generally believed to be a K⁺ channel, although it is likely that many K⁺ channels as well as Ca²⁺ channels are involved as targets of chemosensitive signals. The results of studies of cellular signaling in central chemosensitive neurons are compared with results in other CO₂- and/or H⁺-sensitive cells, including peripheral chemoreceptors (carotid body glomus cells), invertebrate central chemoreceptors, avian intrapulmonary chemoreceptors, acid-sensitive taste receptor cells on the tongue, and pain-sensitive nociceptors. A multiple factors model is proposed for central chemosensitive neurons in which multiple signals that affect multiple ion channel targets result in the final neuronal response to changes in CO₂/H⁺. hypercapnia; brain stem; ventilation; peripheral chemoreceptor; glia; gap junction; glomus; channel; calcium; potassium; carbonic anhydrase; taste receptor; nociception     

Effect of changes in respiratory blood parameters on equine red blood cell K-Cl cotransporter

K influx intoequine red blood cells (RBCs) was measured using⁸⁶Rb as a tracer for K underconditions designed to mimic the changes in respiratory bloodparameters that occur in vivo during strenuous exercise. The effects onK influx of physiological changes in pH, cell volume,O₂ tension(PO₂),CO₂ tension(PCO₂), and bicarbonate and lactateconcentrations were defined. Physiological PO₂ exerted a dominant controllinginfluence on the H⁺-stimulatedCl-dependent K influx, consistent with effects on the K-Clcotransporter; PO₂ required forhalf-maximal activity was 37 ± 3 mmHg (4.9 kPa). AlthoughRBCs were swollen at low pH, results showed explicitly that the volumechange per se had little effect on K influx. Lactate had no effect onvolume- or H⁺-stimulated Kinfluxes, nor did bicarbonate or PCO₂affect the magnitude of K influxes after these stimuli or aftertreatment with protein kinase/phosphatase inhibitors. These resultsrepresent the first detailed report ofO₂ dependence ofH⁺-stimulated K-Cl cotransport inRBCs from any mammalian species. They emphasize the importance ofPO₂ in control of RBC K-Clcotransport.

     

Brain stem lesion size determined by DEAD red or conjugation of neurotoxin to fluorescent beads

Neurotoxinmicroinjected into the retrotrapezoid nucleus of anesthetized ratsdecreases phrenic activity and eliminates the response toCO₂. In unanesthetized rats, suchtreatment has no effect on awake, resting breathing and decreasesCO₂ sensitivity by 40% (M. Akilesh, M. Kamper, A. Li, and E. E. Nattie. J. Appl. Physiol. 82: 469-479, 1997). One important factorin explaining these disparate results is the actual size of theanatomic lesion. In the present study, we injected ibotenic acid intothe retrotrapezoid nucleus of anesthetized rats and evaluated lesionsize by using two new approaches: 1)DEAD red, a fluorescent probe that enters impaired cells through leakymembranes and binds to nucleic acids, and2) conjugation of toxin tofluorescent beads. With the use of DEAD red, the region containinglabeled dying cells was 313 ± 104 nl(n = 4), six times larger than theinitial injected volume, and the physiological effects on phrenicamplitude, the CO₂ response, andblood pressure began within minutes and were substantial. Withconjugated toxin, in theory, neuronal damage would be limited to theregion of detectable fluorescence (49 ± 10 nl;n = 4). Effects on phrenicamplitude, CO₂ sensitivity, andblood pressure were absent until ~2 h postinjection. Controlexperiments, with 2 h of in vitro incubation of theneurotoxin-microbead conjugate and injection of the supernatant aftercentrifugation, showed similar results that suggest release ofconjugated neurotoxin. We conclude that DEAD red provides a usefulmeans to monitor neuronal impairment in acute studies in vivo.Conjugation of neurotoxin to microbeads may be less reliable in this regard.

     

Oxidative stress decreases pHi and Na(+)/H(+) exchange and increases excitability of solitary complex neurons from rat brain slices

Putative chemoreceptors in the solitary complex (SC) are sensitive to hypercapnia and oxidative stress. We tested the hypothesis that oxidative stress stimulates SC neurons by a mechanism independent of intracellular pH (pH_i). pH_i was measured by using ratiometric fluorescence imaging microscopy, utilizing either the pH-sensitive fluorescent dye BCECF or, during whole cell recordings, pyranine in SC neurons in brain stem slices from rat pups. Oxidative stress decreased pH_i in 270 of 436 (62%) SC neurons tested. Chloramine-T (CT), N-chlorosuccinimide (NCS), dihydroxyfumaric acid, and H₂O₂ decreased pH_i by 0.19 ± 0.007, 0.20 ± 0.015, 0.15 ± 0.013, and 0.08 ± 0.002 pH unit, respectively. Hypercapnia decreased pH_i by 0.26 ± 0.006 pH unit (n = 95). The combination of hypercapnia and CT or NCS had an additive effect on pH_i, causing a 0.42 ± 0.03 (n = 21) pH unit acidification. CT slowed pH_i recovery mediated by Na⁺/H⁺ exchange (NHE) from NH₄Cl-induced acidification by 53% (n = 20) in -buffered medium and by 58% (n = 10) in HEPES-buffered medium. CT increased firing rate in 14 of 16 SC neurons, and there was no difference in the firing rate response to CT with or without a corresponding change in pH_i. These results indicate that oxidative stress 1) decreases pH_i in some SC neurons, 2) together with hypercapnia has an additive effect on pH_i, 3) partially inhibits NHE, and 4) directly affects excitability of CO₂/H⁺-chemosensitive SC neurons independently of pH_i changes. These findings suggest that oxidative stress acidifies SC neurons in part by inhibiting NHE, and this acidification may contribute ultimately to respiratory control dysfunction. hyperoxic hyperventilation; O₂ toxicity; pH regulation; brain stem; reactive oxygen species     

Skeletal muscle ECF pH error signal for exercise ventilatory control

Evans, Allison B., Larry W. Tsai, David A. Oelberg, HomayounKazemi, and David M. Systrom. Skeletal muscle ECF pH error signalfor exercise ventilatory control. J. Appl.Physiol. 84(1): 90-96, 1998.An autonomic reflexlinking exercising skeletal muscle metabolism to central ventilatorycontrol is thought to be mediated by neural afferents having freeendings that terminate in the interstitial fluid of muscle. Todetermine whether changes in muscle extracellular fluid pH(pH_e) can provide an errorsignal for exercise ventilatory control,pH_e was measured duringelectrically induced contraction by³¹P-magnetic resonancespectroscopy and the chemical shift of a phosphorylated, pH-sensitivemarker that distributes to the extracellular fluid (phenylphosphonicacid). Seven lightly anesthetized rats underwentunilateral continuous 5-Hz sciatic nerve stimulation in an 8.45-Tnuclear magnetic resonance magnet, which resulted in a mixed lacticacidosis and respiratory alkalosis, with no net change in arterial pH.Skeletal muscle intracellular pH fell from 7.30 ± 0.03 units atrest to 6.72 ± 0.05 units at 2.4 min of stimulation and then roseto 7.05 ± 0.01 units (P < 0.05), despite ongoing stimulation and muscle contraction.Despite arterial hypocapnia, pH_eshowed an immediate drop from its resting baseline of 7.40 ± 0.01 to 7.16 ± 0.04 units (P < 0.05)and remained acidic throughout the stimulation protocol. During the on-and off-transients for 5-Hz stimulation, changes in the pH gradientbetween intracellular and extracellular compartments suggestedtime-dependent recruitment of sarcolemmal ion-transport mechanisms.pH_e of exercising skeletal musclemeets temporal and qualitative criteria necessary for a ventilatorymetaboreflex mediator in a setting where arterial pH doesnot.

     

Effects of unilateral lesions of retrotrapezoid nucleus on breathing in awake rats

Akilesh, Manjapra R., Matthew Kamper, Aihua Li, and EugeneE. Nattie. Effects of unilateral lesions of retrotrapezoid nucleuson breathing in awake rats. J. Appl.Physiol. 82(2): 469-479, 1997.In anesthetizedrats, unilateral retrotrapezoid nucleus (RTN) lesions markedlydecreased baseline phrenic activity and the response toCO₂ (E. E. Nattie and A. Li.Respir. Physiol. 97: 63-77,1994). Here we evaluate the effects of such lesions on restingbreathing and on the response to hypercapnia and hypoxia inunanesthetized awake rats. We made unilateral injections [24 ± 7 (SE) nl] of ibotenic acid (IA; 50 mM), an excitatoryamino acid neurotoxin, in the RTN region(n = 7) located by stereotaxic coordinates and by field potentials induced by facial nervestimulation. Controls (n = 6) receivedRTN injections (80 ± 30 nl) of mock cerebrospinal fluid. A secondcontrol consisted of four animals with IA injections (24 ± 12 nl)outside the RTN region. Injected fluorescent beads allowed anatomicidentification of lesion location. Using whole body plethysmography, wemeasured ventilation in the awake state during room air, 7%CO₂ in air, and 10%O₂ breathing before and for 3 wkafter the RTN injections. There was no statistically significant effectof the IA injections on resting room air breathing in the lesion groupcompared with the control groups. We observed no apnea. The response to7% CO₂ in the lesion groupcompared with the control groups was significantly decreased, by 39%on average, for the final portion of the 3-wk study period. There wasno lesion effect on the ventilatory response to 10%O₂. In this unanesthetized model,other areas suppressed by anesthesia, e.g., the reticular activatingsystem, hypothalamus, and perhaps the contralateral RTN, may providetonic input to the respiratory centers that counters the loss of RTNactivity.

     

Cellular basis for contractile dysfunction in the diaphragm from a rabbit infarct model of heart failure

Abnormal respiratory muscle function isthought to contribute to breathlessness and exercise intolerance inheart failure but little is known about possible alterations in thefunction of such muscle. We have measured tetanic force andintracellular Ca²⁺ concentration([Ca²⁺]_i) in isolated, arteriallyperfused hemidiaphragm preparations from a rabbit coronary arteryligation model of heart failure. Increasing stimulation frequency(10-100 Hz) caused a progressive increase of force and[Ca²⁺]_i in control preparations,whereas force and [Ca²⁺]_i onlyincreased between 10 and 25 Hz stimulation (decreasing at higherfrequencies) in preparations from ligated animals. Cyclopiazonic acidproduced a dose-dependent shift in the relationship between stimulationfrequency and [Ca²⁺]_i in controlpreparations that was similar to the shift observed in the diaphragm ofcoronary-ligated animals. These data indicate that the in vitrocontractile characteristics of the diaphragm are significantly alteredin our model and that altered[Ca²⁺]_i regulation contributes tothe reduced diaphragm strength observed in heart failure.

     

Hypoxia, temperature, and pH/CO2 effects on respiratory discharge from a turtle brain stem preparation

Johnson, Stephen M., Rebecca A. Johnson, and Gordon S. Mitchell. Hypoxia, temperature, andpH/CO₂ effects on respiratory discharge from a turtle brain stem preparation. J. Appl. Physiol. 84(2): 649-660, 1998.An in vitrobrain stem preparation from adult turtles (Chrysemyspicta) was used to examine the effects of anoxia andincreased temperature and pH/CO₂on respiration-related motor output. At pH ~7.45, hypoglossal (XII)nerve roots produced patterns of rhythmic bursts (peaks) of discharge(0.74 ± 0.07 peaks/min, 10.0 ± 0.6 s duration) that werequantitatively similar to literature reports of respiratory activity inconscious, vagotomized turtles. Respiratory discharge was stable for 6 h at 22°C; at 32°C, peak amplitude and frequency progressivelyand reversibly decreased with time. Two hours of hypoxia had no effecton respiratory discharge. Acutely increasing bath temperature from 22 to 32°C decreased episode and peak duration and increased peakfrequency. Changes in pH/CO₂increased peak frequency from zero at pH 8.00-8.10 to maxima of0.81 ± 0.01 and 1.44 ± 0.02 peaks/min at 22°C (pH 7.32) and32°C (pH 7.46), respectively;pH/CO₂ sensitivity was similar atboth temperatures. We conclude that1) insensitivity to hypoxiaindicates that rhythmic discharge does not reflect gasping behavior,2) increased temperature altersrespiratory discharge, and 3)central pH/CO₂ sensitivity isunaffected by temperature in this preparation (i.e.,Q₁₀ ~1.0).

     

Regulation of the epithelial Na(+) channel by extracellular acidification

The effect of extracellular acidification wastested on the native epithelial Na⁺ channel (ENaC) in A6epithelia and on the cloned ENaC expressed in Xenopusoocytes. Channel activity was determined utilizing blocker-inducedfluctuation analysis in A6 epithelia and dual electrode voltage clampin oocytes. In A6 cells, a decrease of extracellular pH(pH_o) from 7.4 to 6.4 caused a slow stimulation of theamiloride-sensitive short-circuit current (I_Na)by 68.4 ± 11% (n = 9) at 60 min. This increaseof I_Na was attributed to an increase of openchannel and total channel (N_T) densities. Similar changes were observed with pH_o 5.4. The effects ofpH_o were blocked by buffering intracellularCa²⁺ with 5 µM1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid. Inoocytes, pH_o 6.4 elicited a small transient increase of theslope conductance of the cloned ENaC (11.4 ± 2.2% at 2 min)followed by a decrease to 83.7 ± 11.7% of control at 60 min (n = 6). Thus small decreases of pH_ostimulate the native ENaC by increasing N_T butdo not appreciably affect ENaC expressed in Xenopus oocytes.These effects are distinct from those observed with decreasingintracellular pH with permeant buffers that are known to inhibit ENaC.

     

The Ca2+-sensing receptor: a target for polyamines

The Ca²⁺-sensing receptor(CaR) is activated at physiological levels of externalCa²⁺(Ca_o) but is expressed in anumber of tissues that do not have well-established roles in thecontrol of Ca_o, including several regions of the brain and the intestine. Polyamines are endogenous polyvalent cations that can act as agonists for the CaR, as shown byour current studies of human embryonic kidney (HEK-293) cells transfected with the human CaR. Cellular parameters altered by polyamines included cytosolic freeCa²⁺(Ca_i), inositol phosphateproduction, and the activity of a nonselective cation channel. Sperminestimulated Ca_i transients inCaR-transfected HEK cells, with a concentration producing ahalf-maximal response (EC₅₀) of ~500µM in the presence of 0.5 mMCa²⁺, whereas sustained increasesin Ca_i had anEC₅₀ of ~200 µM. The order ofpotency was spermine > spermidine >> putrescine. Elevation ofCa_o shifted theEC₅₀ for spermine sharply to theleft, with substantial stimulation below 100 µM. Addition ofsubthreshold concentrations of spermine increased the sensitivity ofCaR-expressing HEK cells to Ca_o.Parathyroid hormone secretion from bovine parathyroid cells wasinhibited by 50% in the presence of 200 µM spermine, a responsesimilar to that elicited by 2.0 mMCa_o. These data suggest thatpolyamines could be effective agonists for the CaR, and severaltissues, including the brain, may use the CaR as a target for theactions of spermine and other endogenous polycationic agonists.

     

The Inhibition of Phloem Translocation by Ammonia

Bean plants (Phaseolus vulgaris L. cv. Fardenlosa Shiny) werelabelled with carbon-11 via their first trifoliate leaves when3-weeks-old and the transient inhibitions of translocation causedby the application of ammonium chloride solutions (10 mol m^–3)to a peeled region of stem were studied. At pH 6·5 theammonium was without effect. At pH 11·0 even a briefapplication inhibited translocation for many minutes, whilelonger applications inhibited translocation for considerablylonger. Solutions of 10 mol m^–3 sodium chloride were withouteffect at either pH. At pH 6·5 ammonium chloride solution contains predominantlyammonium ions (NH₄⁺) and at pH 11·0 predominantly dissolvedammonia gas (NH₃). Hence we conclude that phloem transport withinbean stems is inhibited by dissolved ammonia gas but not ammoniumions. Key words: Phloem translocation, transient inhibition, ammonia, ammonium ion     

Proton leak and CFTR in regulation of Golgi pH in respiratory epithelial cells

Work addressing whether cystic fibrosistransmembrane conductance regulator (CFTR) plays a role in regulatingorganelle pH has remained inconclusive. We engineered a pH-sensitiveexcitation ratiometric green fluorescent protein (pHERP) and targetedit to the Golgi with sialyltransferase (ST). As determined byratiometric imaging of cells expressing ST-pHERP, Golgi pH(pH_G) of HeLa cells was 6.4, while pH_G ofmutant (F508) and wild-type CFTR-expressing (WT-CFTR) respiratoryepithelia were 6.7-7.0. Comparison of genetically matched F508and WT-CFTR cells showed that the absence of CFTR statisticallyincreased Golgi acidity by 0.2 pH units, though this small differencewas unlikely to be physiologically important. Golgi pH was maintainedby a H⁺ vacuolar (V)-ATPase countered by a H⁺leak, which was unaffected by CFTR. To estimate Golgi proton permeability (P_H⁺), we modeledtransient changes in pH_G induced by inhibiting the V-ATPaseand by acidifying the cytosol. This analysis required knowing Golgibuffer capacity, which was pH dependent. Our in vivo estimate is thatGolgi P_H⁺ = 7.5 × 10⁴ cm/s when pH_G = 6.5, andsurprisingly, P_H⁺ decreased aspH_G decreased.

     

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

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

     

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.

     

Probing the extracellular release site of the plasma membrane calcium pump

Theplasma membrane Ca²⁺ pump is known to mediateCa²⁺/H⁺ exchange. Extracellular protonsactivated ⁴⁵Ca²⁺ efflux from human red bloodcells with a half-maximal inhibition constant of 2 nM when theintracellular pH was fixed. An increase in pH from 7.2 to 8.2 decreasedthe IC₅₀ for extracellular Ca²⁺ from ~33 to~6 mM. Changing the membrane potential by >54 mV had no effect onthe IC₅₀ for extracellular Ca²⁺. This arguesagainst Ca²⁺ release through a high-field access channel.Extracellular Ni²⁺ inhibited Ca²⁺ efflux withan IC₅₀ of 11 mM. Extracellular Cd²⁺ inhibitedwith an IC₅₀ of 1.5 mM, >10 times better thanCa²⁺. The Cd²⁺ IC₅₀ also decreasedwhen the pH was raised from 7.1 to 8.2, consistent withCa²⁺, Cd²⁺, and H⁺ competing forthe same site. The higher affinity for inhibition by Ni²⁺and Cd²⁺ is consistent with a histidine or cysteine as partof the release site. The cysteine reagent 2-(trimethylammonium)ethylmethanethiosulfonate did not inhibit Ca²⁺ efflux. Ourresults are consistent with the notion that the release site contains a histidine.

     

Hypoxic vasodilation in porcine coronary artery is preferentially inhibited by organ culture

Hypoxia (95% N₂-5%CO₂) elicits an endothelium-independent relaxation(45-80%) in freshly dissected porcine coronary arteries. Pairedartery rings cultured at 37°C in sterile DMEM (pH ~7.4) for 24 h contracted normally to KCl or 1 µM U-46619. However, relaxation inresponse to hypoxia was sharply attenuated compared with control (fresharteries or those stored at 4°C for 24 h). Hypoxicvasorelaxation in organ cultured vessels was reduced at both high andlow stimulation, indicating that both Ca²⁺-independent andCa²⁺-dependent components are altered. In contrast,relaxation to G-kinase (sodium nitroprusside) or A-kinase (forskolinand isoproterenol) activation was not significantly affected by organculture. Additionally, there was no difference in relaxation afterwashout of the stimulus, indicating that the inhibition is specific toacute hypoxia-induced relaxation. Simultaneous force and intracellularcalcium concentration ([Ca²⁺]_i) measurementsindicate the reduction in [Ca²⁺]_i concomitantwith hypoxia at low stimulus levels in these tissue is abolished byculture. Our results indicate that organ culture at 37°C specificallyattenuates hypoxic relaxation in vascular smooth muscle by alteringdynamics of [Ca²⁺]_i handling and decreasing aCa²⁺-independent component of relaxation. Thus organculture can be a novel tool for investigating the mechanisms ofhypoxia-induced vasodilation.

     

Uptake of Methylamine by Ulva rigida: Transport of Cations and Diffusion of Free Base

Methylamine¹ is taken up rapidly by disks cut from fronds ofUlva rigida (‘Ulva lactuca’) and can be accumulatedat concentrations several hundred times greater than those inthe bathing medium. At pH 8.0 (the pH of sea-water) the relationshipbetween influx and concentration is normally linear up to 0.1–0.3mM, followed by a second, less steep linear phase, the slopeof which decreases or increases with decreasing or increasingexternal pH. When pH is greater than 9.0, however, net uptakesoon ceases. Methylamine influx is greatly reduced at low temperature,by low concentrations of ammonia and, depending on the lengthof storage of the material, by darkness. Influx is also greatlyreduced when disks are pretreated in solutions containing ammoniaand to a much lesser extent when they are pretreated in methylamine,imidazol or nitrate. Methylamine influx lowers intracellularK⁺, increases Cl^– and has no effect on Na⁺. We suggestthat the first linear phase of influx versus concentration reflectsthe operation of an amine cation porter that is rate-limitedby diffusion of CH₃NH₃⁺ through the external unstirred layer,and that the second phase is due to diffusion of CH₃NH₂ intothe tissue.     

Age-related differences in Na+-dependent Ca2+ accumulation in rabbit hearts exposed to hypoxia and acidification

In this study, we test the hypothesisthat in newborn hearts (as in adults) hypoxia and acidificationstimulate increased Na⁺ uptake, in part via pH-regulatoryNa⁺/H⁺ exchange. Resulting increases inintracellular Na⁺ (Na_i) alter the force drivingthe Na⁺/Ca²⁺ exchanger and lead to increasedintracellular Ca²⁺. NMR spectroscopy measuredNa_i and cytosolic Ca²⁺ concentration([Ca²⁺]_i) and pH (pH_i) inisolated, Langendorff-perfused 4- to 7-day-old rabbit hearts. AfterNa⁺/K⁺ ATPase inhibition, hypoxic hearts gainedNa⁺, whereas normoxic controls did not [19 ± 3.4 to139 ± 14.6 vs. 22 ± 1.9 to 22 ± 2.5 (SE) meq/kg drywt, respectively]. In normoxic hearts acidified using theNH₄Cl prepulse, pH_i fell rapidly and recovered,whereas Na_i rose from 31 ± 18.2 to 117.7 ± 20.5 meq/kg dry wt. Both protocols caused increases in [Ca]_i;however, [Ca]_i increased less in newborn hearts than inadults (P < 0.05). Increases in Na_i and[Ca]_i were inhibited by theNa⁺/H⁺ exchange inhibitormethylisobutylamiloride (MIA, 40 µM; P < 0.05), aswell as by increasing perfusate osmolarity (+30 mosM) immediately before and during hypoxia (P < 0.05). The data supportthe hypothesis that in newborn hearts, like adults, increases inNa_i and [Ca]_i during hypoxia and afternormoxic acidification are in large part the result of increased uptakevia Na⁺/H⁺ and Na⁺/Ca²⁺exchange, respectively. However, for similar hypoxia and acidification protocols, this increase in [Ca]_i is less in newborn thanadult hearts.

     

Human cytomegalovirus infection enhances osmotic stimulation of Na+/H+ exchange in human fibroblasts

Infection withhuman cytomegalovirus (HCMV) causes an enlargement (cytomegaly) ofhuman fibroblasts (MRC-5). As a first step toward determining whethersolute uptake, mediated in part by Na⁺/H⁺exchange, is responsible for the development of cytomegaly, we studiedthe effects of HCMV infection on intracellular pH(pH_i) regulation (nominalCO₂/concn = 0) by comparing cytomegalic cells with mock-infected cells.Seventy-two hours after HCMV infection of MRC-5 cells we observed thefollowing changes relative to mock-infected cells: restingpH_i is 0.1-0.2 pH unit morealkaline; the intrinsic buffering power of the cytoplasm was reduced by~40-50%; acid-loadingH⁺-equivalent fluxes were reduced;and there were alterations of Na⁺/H⁺exchanger (NHE) properties, including an alkaline shift of the pH_i dependence of activity, areduction of the apparent affinity for extracellularNa⁺, and an increase of theapparent maximum velocity and a large increase in stimulation by ahyperosmotic challenge. These results indicate that HCMV infectionexerts a profound effect on functional properties of the NHE, onacid-loading mechanisms, and on intrinsic cellular buffering power.These effects are consistent with a role for the NHE in the developmentof cytomegaly.