K+ supplementation increases muscle [Na+-K+-ATPase] and improves extrarenal K+ homeostasis in rats

Bundgaard, Henning, Thomas A. Schmidt, Jim S. Larsen, andKeld Kjeldsen. K⁺supplementation increases muscle[Na⁺-K⁺-ATPase]and improves extrarenal K⁺homeostasis in rats. J. Appl. Physiol.82(4): 1136-1144, 1997.Effects ofK⁺ supplementation (~200 mmolKCl/100 g chow) on plasma K⁺,K⁺ content, andNa⁺-K⁺-adeonsinetriphosphatase(ATPase) concentration([Na⁺-K⁺-ATPase])in skeletal muscles as well as on extrarenalK⁺ clearance were evaluated inrats. After 2 days of K⁺supplementation, hyperkalemia prevailed(K⁺-supplemented vs.weight-matched control animals) [5.1 ± 0.2 (SE) vs. 3.2 ± 0.1 mmol/l, P < 0.05, n = 5-6], and after 4 daysa significant increase in K⁺content was observed in gastrocnemius muscle (104 ± 2 vs. 97 ± 1 µmol/g wet wt, P < 0.05, n = 5-6). After 7 days ofK⁺ supplementation, a significantincrease in[³H]ouabain bindingsite concentration (344 ± 5 vs. 239 ± 8 pmol/g wet wt,P < 0.05, n = 4) was observed in gastrocnemiusmuscle. After 2 wk, increases in plasmaK⁺,K⁺ content, and[³H]ouabain bindingsite concentration in gastrocnemius muscle amounted to 40, 8, and 68%(P < 0.05) above values observed inweight-matched control animals, respectively. The latter change wasconfirmed by K⁺-dependentp-nitrophenyl phosphatase activitymeasurements. Fasting for 1 day reduced plasmaK⁺ andK⁺ content in gastrocnemius musclein rats that had been K⁺supplemented for 2 wk by 3.1 ± 0.3 mmol/l(P < 0.05, n = 5) and 15 ± 2 µmol/g wet wt(P < 0.05, n = 5), respectively. After induction of anesthesia, arterial plasma K⁺was measured during intravenous KCl infusion (0.75 mmolKCl · 100 g bodywt¹ · h¹).The K⁺-supplemented fasted groupdemonstrated a 42% (P < 0.05) lower plasma K⁺ rise, associated with asignificantly higher increase inK⁺ content in gastrocnemius muscleof 7 µmol/g wet wt (P < 0.05, n = 5) compared with their controlanimals. In conclusion, K⁺supplementation increases plasmaK⁺,K⁺ content, and[Na⁺-K⁺-ATPase]in skeletal muscles and improves extrarenalK⁺ clearance capacity.

     

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.

     

Age- and tissue-specific induction of NHE3 by glucocorticoids in the rat small intestine

Of the two known apical isoforms of theNa⁺/H⁺ exchanger (NHE) family, only the NHE3gene is regulated by glucocorticoids. The aim of these studies was toinvestigate the mechanisms underlying the effects of methylprednisolone(MP) on expression of NHE3 in the proximal and distal small intestineof suckling and adult rats. Immunoblots showed that the glucocorticoidresponsiveness in the proximal small intestine was greatest in sucklinganimals (NHE3/-actin: 0.43 ± 0.09 control vs. 1.57 ± 0.15 MP;P < 0.001), and responsiveness decreased with age with noeffect in adults (0.56 ± 0.14 vs. 0.64 ± 0.17). Distal smallintestine was responsive only in adult rats (0.49 ± 0.13 vs. 1.65 ± 0.09; P < 0.001). This pattern was confirmed at the mRNAlevel and by ²²Na⁺ uptake. Western blot and[³H]dexamethasone mesylate binding showed thatthe responsiveness of NHE3 to glucocorticoids is directly related tothe expression of glucocorticoid receptor (GR) in the small intestine.These studies suggest that loss and gain of glucocorticoidresponsiveness in the proximal and distal small intestine,respectively, are related to age- and segment-dependent expression of GR.

     

Dexamethasone treatment causes resistance to insulin-stimulated cellular potassium uptake in the rat

Patients treated with glucocorticoids have elevated skeletal muscle ouabain binding sites. The major Na⁺-K⁺-ATPase (NKA) isoform proteins found in muscle, ₂ and ₁, are increased by 50% in rats treated for 14 days with the synthetic glucocorticoid dexamethasone (DEX). This study addressed whether the DEX-induced increase in the muscle NKA pool leads to increased insulin-stimulated cellular K⁺ uptake that could precipitate hypokalemia. Rats were treated with DEX or vehicle via osmotic minipumps at one of two doses: 0.02 mg·kg^–1·day^–1 for 14 days (low DEX; n = 5 pairs) or 0.1 mg·kg^–1·day^–1 for 7 days (high DEX; n = 6 pairs). Insulin was infused at a rate of 5 mU·kg^–1·min^–1 over 2.5 h in conscious rats. Insulin-stimulated cellular K⁺ and glucose uptake rates were assessed in vivo by measuring the exogenous K⁺ infusion () and glucose infusion (G_inf) rates needed to maintain constant plasma K⁺ and glucose concentrations during insulin infusion. DEX at both doses decreased insulin-stimulated glucose uptake as previously reported. G_inf (in mmol·kg^–1·h^–1) was 10.2 ± 0.6 in vehicle-treated rats, 5.8 ± 0.8 in low-DEX-treated rats, and 5.2 ± 0.6 in high-DEX-treated rats. High DEX treatment also reduced insulin-stimulated K⁺ uptake. (in mmol·kg^–1·h^–1) was 0.53 ± 0.08 in vehicle-treated rats, 0.49 ± 0.14 in low-DEX-treated rats, and 0.27 ± 0.08 in high-DEX-treated rats. DEX treatment did not alter urinary K⁺ excretion. NKA ₂-isoform levels in the low-DEX-treated group, measured by immunoblotting, were unchanged, but they increased by 38 ± 15% (soleus) and by 67 ± 3% (gastrocnemius) in the high-DEX treatment group. The NKA ₁-isoform level was unchanged. These results provide novel evidence for the insulin resistance of K⁺ clearance during chronic DEX treatment. Insulin-stimulated cellular K⁺ uptake was significantly depressed despite increased muscle sodium pump pool size. skeletal muscle; sodium pump; Na⁺-K⁺-ATPase     

Purification of active Na+-K+-ATPase using a new ouabain-affinity column

Ouabain, aspecific inhibitor ofNa⁺-K⁺-ATPase,was coupled to epoxy agarose via a 13-atom spacer to make an affinitycolumn that specifically bindsNa⁺-K⁺-ATPase.Na⁺-K⁺-ATPasefrom rat and dog kidney was bound to the column and was eluted as afunction of enzyme conformation, altered by adding specificcombinations of ligands.Na⁺-K⁺-ATPasefrom both sources bound to the column in the presence of Na + ATP + Mgand in solutions containing 30 mM K. No binding was observed in thepresence of Na or Na + ATP. These experiments suggest thatNa⁺-K⁺-ATPasebinds to the column under the same conditions that it binds tountethered ouabain.Na⁺-K⁺-ATPasealready bound to the column was competitively eluted with excess freeNa + ouabain or with Na + ATP. The latter eluted active enzyme. Forcomparable amounts of boundNa⁺-K⁺-ATPase,Na + ouabain and Na + ATP eluted more rat than dogNa⁺-K⁺-ATPase,consistent with the lower affinity of the ratNa⁺-K⁺-ATPasefor ouabain. The ouabain-affinity column was used to purify activeNa⁺-K⁺-ATPasefrom rat kidney microsomes and rat adrenal glomerulosa cells. Thespecific activity of the kidney enzyme was increased from ~2 to 15 µmolP_i · mg¹ · min¹.Na⁺-K⁺-ATPasepurified from glomerulosa cells that were prelabeled with [³²P]orthophosphatewas phosphorylated on the -subunit, suggesting that these cellscontain a kinase that phosphorylatesNa⁺-K⁺-ATPase.

     

Quantitation of intracellular [Na+] in vivo by using TmDOTP5-asanNMRshiftreagentand extracellularmarker

A method ispresented to measure the absolute concentration of intracellularNa⁺([Na⁺]_i)in vivo by using interleaved ²³Na-and ³¹P-nuclear magnetic resonance(NMR) spectroscopy andTmDOTP⁵ as shift reagentand chemical marker of tissue extracellular space (ECS). The techniquewas used to determine[Na⁺]_iand relative ECS in livers of control rats (21 ± 3 and0.11 ± 0.02 mM, respectively) and in rats exposed to carbontetrachloride (103 ± 29 and 0.23 ± 0.03 mM, respectively). TheNMR measurements were confirmed independently on excised tissue samplesby using atomic absorption spectroscopy. The results confirm thatTmDOTP⁵ can be used as acombined cation shift reagent and ECS marker, thereby allowingquantitation of[Na⁺]_iin vivo by NMR.

     

In vivo fluorescence measurement of Na+ concentration in the pericryptal space of mouse descending colon

A methodinvolving surgical exposure of the colonic mucosa, fluorescent dyeaddition, and confocal microscopy has been developed for monitoringcolonic crypt function in vivo in mice. Na⁺ concentrationin the extracellular pericryptal space of descending colon was measuredusing a low-affinity Na⁺-sensitive fluorescent indicatorconsisting of an Na⁺-sensitive chromophore (sodium red) andan Na⁺-insensitive chromophore (Bodipy-fl) immobilized on200-nm-diameter polystyrene beads. The Na⁺ indicator beadsaccumulated in the pericryptal spaces surrounding the colonic cryptsafter a 1-h exposure of the colonic luminal surface to the beadsuspension. Na⁺ concentration ([Na⁺]) in thepericryptal space was 491 ± 62 mM (n = 4). Aftera 70-min exposure to amiloride (0.25 mM), pericryptal[Na⁺] was reduced to 152 ± 21 mM. Blockage of thecrypt lumen with mineral oil droplets reduced pericryptal[Na⁺] to 204 ± 44 mM. Exposure of the colonicmucosa to FITC-dextran (4.5 kDa) led to rapid accumulation of the dyeinto the crypt lumen with a half time of 19.8 ± 1.0 s, whichwas increased to 77.9 ± 6.0 s after amiloride treatment.These results establish an in vivo fluorescence method to measurecolonic crypt function and provide direct evidence for accumulation ofa hypertonic absorbate in the pericryptal space of descending colon.The pericryptal space represents the first example of a hypertonicextracellular compartment in mammals that is not created by acountercurrent amplification mechanism.

     

Acute effects of 17beta-estradiol on myocardial pH, Na+, and Ca2+ and ischemia-reperfusion injury

Evidence suggests that 1) ischemia-reperfusion injury is due largely to cytosolic Ca²⁺ accumulation resulting from functional coupling of Na⁺/Ca²⁺ exchange (NCE) with stimulated Na⁺/H⁺ exchange (NHE1) and 2) 17-estradiol (E2) stimulates release of NO, which inhibits NHE1. Thus we tested the hypothesis that acute E2 limits myocardial Na⁺ and therefore Ca²⁺ accumulation, thereby limiting ischemia-reperfusion injury. NMR was used to measure cytosolic pH (pH_i), Na⁺ (Na), and calcium concentration ([Ca²⁺]_i) in Krebs-Henseleit (KH)-perfused hearts from ovariectomized rats (OVX). Left ventricular developed pressure (LVDP) and lactate dehydrogenase (LDH) release were also measured. Control ischemia-reperfusion was 20 min of baseline perfusion, 40 min of global ischemia, and 40 min of reperfusion. The E2 protocol was identical, except that 1 nM E2 was included in the perfusate before ischemia and during reperfusion. E2 significantly limited the changes in pH_i, Na and [Ca²⁺]_i during ischemia (P < 0.05). In control OVX vs. OVX+E2, pH_i fell from 6.93 ± 0.03 to 5.98 ± 0.04 vs. 6.96 ± 0.04 to 6.68 ± 0.07; Na rose from 25 ± 6 to 109 ± 14 meq/kg dry wt vs. 25 ± 1 to 76 ± 3; [Ca²⁺]_i changed from 365 ± 69 to 1,248 ± 180 nM vs. 293 ± 66 to 202 ± 64 nM. E2 also improved recovery of LVDP and diminished release of LDH during reperfusion. Effects of E2 were diminished by 1 µM N-nitro-L-arginine methyl ester. Thus the data are consistent with the hypothesis. However, E2 limitation of increases in [Ca²⁺]_i is greater than can be accounted for by the thermodynamic effect of reduced Na accumulation on NCE. myocardial ischemia; Na⁺/H⁺ exchange; Na⁺/Ca²⁺ exchange; nuclear magnetic resonance; ischemic biology; ion channels/membrane transport; transplantation     

Sprint training attenuates myocyte hypertrophy and improves Ca2+ homeostasis in postinfarction myocytes

Zhang, Xue-Qian, Yuk-Chow Ng, Timothy I. Musch, Russell L. Moore, R. Zelis, and Joseph Y. Cheung. Sprint training attenuates myocyte hypertrophy and improvesCa²⁺ homeostasis in postinfarctionmyocytes. J. Appl. Physiol. 84(2): 544-552, 1998.Myocytes isolated from rat hearts 3 wk aftermyocardial infarction (MI) had decreasedNa⁺/Ca²⁺exchange currents(I_Na/Ca; 3 Na⁺ out:1Ca²⁺ in) and sarcoplasmicreticulum (SR)-releasable Ca²⁺contents. These defects in Ca²⁺regulation may contribute to abnormal contractility in MI myocytes. Because exercise training elicits positive adaptations in cardiac contractile function and myocardialCa²⁺ regulation, thepresent study examined whether 6-8 wk ofhigh-intensity sprint training (HIST) would ameliorate some of thecellular maladaptations observed in post-MI rats with limited exerciseactivity (Sed). In MI rats, HIST did not affect citrate synthaseactivities of plantaris muscles but significantly increased thepercentage of cardiac -myosin heavy chain (MHC) isoforms (57.2 ± 1.9 vs. 49.3 ± 3.5 in MI-HIST vs. MI-Sed, respectively;P  0.05). At the single myocytelevel, HIST attenuated cellular hypertrophy observed post-MI, asevidenced by reductions in cell lengths (112 ± 4 vs. 130 ± 5 µm in MI-HIST vs. MI-Sed, respectively;P  0.005) and cell capacitances (212 ± 8 vs. 242 ± 9 pF in MI-HIST vs. MI-Sed, respectively; P  0.015). ReverseI_Na/Ca wassignificantly lower (P  0.0001) inmyocytes from MI-Sed rats compared with those from rats that were shamoperated and sedentary. HIST significantly increased reverseI_Na/Ca(P  0.05) without affecting theamount ofNa⁺/Ca²⁺exchangers (detected by immunoblotting) in MI myocytes. SR-releasable Ca²⁺ content, as estimated byintegrating forwardI_Na/Ca duringcaffeine-induced SR Ca²⁺ release,was also significantly increased (P  0.02) by HIST in MI myocytes. We conclude that the enhanced cardiacoutput and stroke volume in post-MI rats subjected to HIST aremediated, at least in part, by reversal of cellular maladaptationspost-MI.

     

Respective oxidation of 13C-labeled lactate and glucose ingested simultaneously during exercise

Péronnet, F., Y. Burelle, D. Massicotte, C. Lavoie,and C. Hillaire-Marcel. Respective oxidation of¹³C-labeled lactate and glucoseingested simultaneously during exercise. J. Appl.Physiol. 82(2): 440-446, 1997.The purpose ofthis experiment was to measure, by using¹³C labeling, the oxidation rateof exogenous lactate (25 g, as Na⁺,K⁺,Ca²⁺, andMg²⁺ salts) and glucose (75 g)ingested simultaneously (in 1,000 ml of water) during prolongedexercise (120 min, 65 ± 3% maximum oxygen uptake in 6 male subjects). The percentage of exogenous glucose and lactateoxidized were similar (48 ± 3 vs. 45 ± 5%, respectively). However, because of the small amount of oral lactate that could be tolerated without gastrointestinal discomfort, the amountof exogenous lactate oxidized was much smaller than that of exogenousglucose (11.1 ± 0.5 vs. 36.3 ± 1.3 g, respectively) andcontributed to only 2.6 ± 0.4% of the energy yield(vs. 8.4 ± 1.9% for exogenous glucose). The cumulative amount ofexogenous glucose and lactate oxidized was similar to that observedwhen 100 g of[¹³C]glucose wereingested (47.3 ± 1.8 vs. 50.9 ± 1.2 g, respectively). When[¹³C]glucose wasingested, changes in the plasma glucose¹³C/¹²Cratio indicated that between 39 and 61% of plasma glucose derived fromexogenous glucose. On the other hand, the plasma glucose ¹³C/¹²Cratio remained unchanged when[¹³C]lactate wasingested, suggesting no prior conversion into glucose before oxidation.

     

Na+-K+-ATPase in frog esophagus mucociliary cell membranes: inhibition by protein kinase C activation

We examined protein kinase C (PKC)-dependentregulation ofNa⁺-K⁺-ATPasein frog mucociliary cells. Activation of PKC by12-O-tetradecanoylphorbol-13-acetate (TPA) or 1,2-dioctanoyl-sn-glycerol(diC8) either in intact cells or isolated membranes resulted in aspecific inhibition ofNa⁺-K⁺-ATPaseactivity by ~25-45%. The inhibitory effects in membranes exhibited time dependence and dose dependence [half-maximalinhibition concentration (IC₅₀) = 0.5 ± 0.1 nM and 2.4 ± 0.2 µM, respectively, for TPA anddiC8] and were not influenced byCa²⁺. Analysis of the ouabaininhibition pattern revealed the presence of twoNa⁺-K⁺-ATPaseisoforms with IC₅₀ values forcardiac glycoside of 2.6 ± 0.8 nM and 409 ± 65 nM,respectively. Most importantly, the isoform possessing a higheraffinity for ouabain was almost completely inhibited by TPA, whereasits counterpart was hardly sensitive to the PKC activator. The resultssuggest that, in frog mucociliary cells, PKC regulatesNa⁺-K⁺-ATPaseand that this action is related to the specificNa⁺-K⁺-ATPaseisoform.

     

Cytoskeletal modulation of sodium current in human jejunal circular smooth muscle cells

ANa⁺ current is present in human jejunal circular smoothmuscle cells. The aim of the present study was to determine the role ofthe cytoskeleton in the regulation of the Na⁺ current.Whole cell currents were recorded by using standard patch-clamptechniques with Cs⁺ in the pipette to block K⁺currents. Cytochalasin D and gelsolin were used to disrupt the actincytoskeleton and phalloidin to stabilize it. Colchicine was used todisassemble the microtubule cytoskeleton (and intermediate filaments)and paclitaxel to stabilize it. Acrylamide was used to disrupt theintermediate filament cytoskeleton. Perfusion of the recording chamberat 10 ml/min increased peak Na⁺ current recorded fromjejunal smooth muscle cells by 27 ± 3%. Cytochalasin D andgelsolin abolished the perfusion-induced increase in Na⁺current, whereas incubation with phalloidin, colchicine, paclitaxel, oracrylamide had no effect. In conclusion, the Na⁺ currentexpressed in human jejunal circular smooth muscle cells appears to beregulated by the cytoskeleton. An intact actin cytoskeleton is requiredfor perfusion-induced activation of the Na⁺ current.

     

Transepithelial resistance can be regulated by the intestinal brush-border Na(+)/H(+) exchanger NHE3

Initiation of intestinal Na⁺-glucose cotransport results intransient cell swelling and sustained increases in tight junction permeability. Since Na⁺/H⁺ exchange has beenimplicated in volume regulation after physiological cell swelling, wehypothesized that Na⁺/H⁺ exchange might also berequired for Na⁺-glucose cotransport-dependent tightjunction regulation. In Caco-2 monolayers with activeNa⁺-glucose cotransport, inhibition ofNa⁺/H⁺ exchange with 200 µM5-(N,N-dimethyl)- amiloride induced 36 ± 2% increases in transepithelial resistance (TER). Evaluation using multiple Na⁺/H⁺ exchange inhibitors showed thatinhibition of the Na⁺/H⁺ exchanger 3 (NHE3)isoform was most closely related to TER increases. TER increases due toNHE3 inhibition were related to cytoplasmic acidification becausecytoplasmic alkalinization with 5 mM NH₄Cl prevented bothcytoplasmic acidification and TER increases. However, NHE3 inhibitiondid not affect TER when Na⁺-glucose cotransport wasinhibited. Myosin II regulatory light chain (MLC) phosphorylationdecreased up to 43 ± 5% after inhibition ofNa⁺/H⁺ exchange, similar to previous studiesthat associate decreased MLC phosphorylation with increased TER afterinhibition of Na⁺-glucose cotransport. However, NHE3inhibitors did not diminish Na⁺-glucose cotransport. Thesedata demonstrate that inhibition of NHE3 results in decreased MLCphosphorylation and increased TER and suggest that NHE3 may participatein the signaling pathway of Na⁺-glucosecotransport-dependent tight junction regulation.

     

NHE3-dependent cytoplasmic alkalinization is triggered by Na(+)-glucose cotransport in intestinal epithelia

Cytoplasmic pH (pH_i) was evaluated duringNa⁺-glucose cotransport in Caco-2 intestinal epithelialcell monolayers. The pH_i increased by 0.069 ± 0.002 within 150 s after initiation of Na⁺-glucosecotransport. This increase occurred in parallel with glucose uptake andrequired expression of the intestinal Na⁺-glucosecotransporter SGLT1. S-3226, a preferential inhibitor ofNa⁺/H⁺ exchanger (NHE) isoform 3 (NHE3),prevented cytoplasmic alkalinization after initiation ofNa⁺-glucose cotransport with an ED₅₀ of 0.35 µM, consistent with inhibition of NHE3, but not NHE1 or NHE2. Incontrast, HOE-694, a poor NHE3 inhibitor, failed to significantlyinhibit pH_i increases at <500 µM.Na⁺-glucose cotransport was also associated with activationof p38 mitogen-activated protein (MAP) kinase, and the p38 MAP kinase inhibitors PD-169316 and SB-202190 prevented pH_i increasesby 100 ± 0.1 and 86 ± 0.1%, respectively. Conversely,activation of p38 MAP kinase with anisomycin induced NHE3-dependentcytoplasmic alkalinization in the absence of Na⁺-glucosecotransport. These data show that NHE3-dependent cytoplasmic alkalinization occurs after initiation of SGLT1-mediatedNa⁺-glucose cotransport and that the mechanism of this NHE3activation requires p38 MAP kinase activity. This coordinatedregulation of glucose (SGLT1) and Na⁺ (NHE3) absorptiveprocesses may represent a functional activation of absorptiveenterocytes by luminal nutrients.

     

Effects of NaHCO3 loading on acid-base balance, lactate concentration, and performance in racing greyhounds

This investigation examined the effects ofNaHCO₃ loading on lactateconcentration ([La]), acid-base balance, and performance for a 603.5-m sprint task. Ten greyhounds completed aNaHCO₃ (300 mg/kg body weight) andcontrol trial in a crossover design. Results are expressed as means ± SE. Presprint differences (P < 0.05) were found for NaHCO₃ vs.control, respectively, for blood pH (7.47 ± 0.01 vs. 7.42 ± 0.01), HCO₃ (28.4 ± 0.4 vs. 23.5 ± 0.3 meq/l), and base excess (5.0 ± 0.3 vs. 0.2 ± 0.3 meq/l). Peak blood [La] increased(P < 0.05) inNaHCO₃ vs. control (20.4 ± 1.6 vs. 16.9 ± 1.3 mM, respectively). Relative to control,NaHCO₃ produced a greater(P < 0.05) reduction in blood baseexcess (18.5 ± 1.4 vs. 14.1 ± 0.8 meq/l) andHCO₃ (17.4 ± 1.2 vs.12.8 ± 0.7 meq/l) from presprint to postexercise. Postexercise peak muscle H⁺concentration ([H⁺])was higher (P < 0.05) inNaHCO₃ vs. control (158.8 ± 8.8 vs. 137.0 ± 5.3 nM, respectively). Muscle[H⁺] recoveryhalf-time (7.2 ± 1.6 vs. 11.3 ± 1.6 min) and time to predosevalues (22.2 ± 2.4 vs. 32.9 ± 4.0 min) were reduced(P < 0.05) inNaHCO₃ vs. control, respectively.No differences were found in blood[H⁺] or blood[La] recovery curves or performance times.NaHCO₃ increased postexerciseblood [La] but did not reduce the muscle or blood acid-basedisturbance associated with a 603.5-m sprint or significantly affectperformance.

     

Structure, function, and genomic organization of human Na(+)-dependent high-affinity dicarboxylate transporter

We have clonedand functionally characterized the human Na⁺-dependenthigh-affinity dicarboxylate transporter (hNaDC3) from placenta. ThehNaDC3 cDNA codes for a protein of 602 amino acids with 12 transmembrane domains. When expressed in mammalian cells, the clonedtransporter mediates the transport of succinate in the presence ofNa⁺ [concentration of substrate necessary for half-maximaltransport (K_t) for succinate = 20 ± 1 µM]. Dimethylsuccinate also interacts with hNaDC3. TheNa⁺-to-succinate stoichiometry is 3:1 and concentration ofNa⁺ necessary for half-maximal transport(K^Na+_0.5) is 49 ± 1 mM as determined by uptake studies withradiolabeled succinate. When expressed in Xenopuslaevis oocytes, hNaDC3 induces Na⁺-dependent inwardcurrents in the presence of succinate and dimethylsuccinate. At amembrane potential of 50 mV,K^Suc_0.5 is 102 ± 20 µM andK^Na+_0.5 is 22 ± 4 mM as determined by the electrophysiological approach. Simultaneous measurements of succinate-evoked charge transfer andradiolabeled succinate uptake in hNaDC3-expressing oocytes indicate acharge-to-succinate ratio of 1:1 for the transport process, suggestinga Na⁺-to-succinate stoichiometry of 3:1. pH titration ofcitrate-induced currents shows that hNaDC3 accepts preferentially thedivalent anionic form of citrate as a substrate. Li⁺inhibits succinate-induced currents in the presence of Na⁺.Functional analysis of rat-human and human-rat NaDC3 chimeric transporters indicates that the catalytic domain of the transporter lies in the carboxy-terminal half of the protein. The humanNaDC3 gene is located on chromosome20q12-13.1, as evidenced by fluorescent in situ hybridization. Thegene is >80 kbp long and consists of 13 exons and 12 introns.

     

Sodium/calcium exchange in amphibian skeletal muscle fibers and isolated transverse tubules

TheNa⁺/Ca²⁺ exchanger participates inCa²⁺ homeostasis in a variety of cells and has a key rolein cardiac muscle physiology. We studied in this work the exchanger ofamphibian skeletal muscle, using both isolated inside-out transversetubule vesicles and single muscle fibers. In vesicles, increasingextravesicular (intracellular) Na⁺ concentrationcooperatively stimulated Ca²⁺ efflux (reverse mode), withthe Hill number equal to 2.8. In contrast to the stimulation of thecardiac exchanger, increasing extravesicular (cytoplasmic)Ca²⁺ concentration ([Ca²⁺]) inhibited thisreverse activity with an IC₅₀ of 91 nM. Exchanger-mediated currents were measured at 15°C in single fibers voltage clamped at90 mV. Photolysis of a cytoplasmic caged Ca²⁺ compoundactivated an inward current (forward mode) of 23 ± 10 nA(n = 3), with an average current density of 0.6 µA/µF. External Na⁺ withdrawal generated an outwardcurrent (reverse mode) with an average current density of 0.36 ± 0.17 µA/µF (n = 6) but produced a minimal increasein cytosolic [Ca²⁺]. These results suggest that, inskeletal muscle, the main function of the exchanger is to removeCa²⁺ from the cells after stimulation.

     

Skeletal muscle ouabain binding sites are reduced in rats with chronic heart failure.

Intrinsic skeletal muscle abnormalities decrease muscular endurance in chronic heart failure (CHF). In CHF patients, the number of skeletal muscle Na(+)-K(+) pumps that have a high affinity for ouabain (i.e., the concentration of [(3)H]ouabain binding sites) is reduced, and this reduction is correlated with peak oxygen uptake. The present investigation determined whether the concentration of skeletal muscle [(3)H]ouabain binding sites found during CHF is related to 1) severity of the disease state, 2) muscle fiber type composition, and/or 3) endurance capacity. Four muscles were chosen that represented slow-twitch oxidative (SO), fast-twitch oxidative glycolytic (FOG), fast-twitch glycolytic (FG), and mixed fiber types. Measurements were obtained 8-10 wk postsurgery in 23 myocardial infarcted (MI) and 18 sham-operated control (sham) rats. Eighteen rats had moderate left ventricular (LV) dysfunction [LV end-diastolic pressure (LVEDP) < 20 mmHg], and five had severe LV dysfunction (LVEDP > 20 mmHg). Rats with severe LV dysfunction had significant pulmonary congestion and were likely in a chronic state of compensated congestive failure as indicated by an approximately twofold increase in both lung and right ventricle weight. Run time to fatigue and maximal oxygen uptake (VO(2 max)) were significantly reduced ( downward arrow39 and downward arrow28%, respectively) in the rats with severe LV dysfunction and correlated with the magnitude of LV dysfunction as indicated by LVEDP (run time: r = 0.60, n = 21, P < 0.01 and VO(2 max): r = 0.93, n = 13, P < 0.01). In addition, run time to fatigue was significantly correlated with VO(2 max) (r = 0.87, n = 15, P < 0.01). The concentration of [(3)H]ouabain binding sites (B(max)) was significantly reduced (21-28%) in the three muscles comprised primarily of oxidative fibers [soleus: 259 +/- 14 vs. 188 +/- 17; plantaris: 295 +/- 17 vs. 229 +/- 18; red portion of gastrocnemius: 326 +/- 17 vs. 260 +/- 14 pmol/g wet tissue wt]. In addition, B(max) was significantly correlated with VO(2 max) (soleus: r = 0.54, n = 15, P < 0.05; plantaris: r = 0.59, n = 15, P < 0.05; red portion of gastrocnemius: r = 0.65, n = 15, P < 0.01). These results suggest that downregulation of Na(+)-K(+) pumps that possess a high affinity for ouabain in oxidative skeletal muscle may play an important role in the exercise intolerance that attends severe LV dysfunction in CHF.     

Aldosterone induces ras methylation in A6 epithelia

Aldosterone increases Na⁺ reabsorption by renalepithelial cells: the acute actions (<4 h) appear to be promoted byprotein methylation. This paper describes the relationship betweenprotein methylation and aldosterone's action and describesaldosterone-mediated targets for methylation in cultured renal cells(A6). Aldosterone increases protein methylation from 7.90 ± 0.60 to 20.1 ± 0.80 methyl ester cpm/µg protein. Aldosteronestimulates protein methylation by increasing methyltransferase activityfrom 14.0 ± 0.64 in aldosterone-depleted cells to 31.8 ± 2.60 methyl ester cpm/µg protein per hour in aldosterone-treated cells. Three known methyltransferase inhibitors reduce thealdosterone-induced increase in methyltransferase activity. One ofthese inhibitors, the isoprenyl-cysteine methyltransferase-specificinhibitor,S-trans,trans-farnesylthiosalicylic acid, completely blocks aldosterone-induced protein methylation and also aldosterone-induced short-circuit current. Aldosterone inducesprotein methylation in two molecular weight ranges: near 90 kDa andaround 20 kDa. The lower molecular weight range is the weight of smallG proteins, and aldosterone does increase both Ras protein 1.6-fold andRas methylation almost 12-fold. Also, Ras antisense oligonucleotidesreduce the activity of Na⁺ channels by about fivefold. Weconclude that 1) protein methylation is essential foraldosterone-induced increases in Na⁺ transport;2) one target for methylation is p21^ras; and3) inhibition of Ras expression or Ras methylation inhibits Na⁺ channel activity.

     

Contribution of Na(+)-K(+)-Cl(-) cotransporter to high-[K(+)](o)- induced swelling and EAA release in astrocytes

We hypothesized that highextracellular K⁺ concentration([K⁺]_o)-mediated stimulation ofNa⁺-K⁺-Cl cotransporter isoform 1 (NKCC1) may result in a net gain of K⁺ and Cland thus lead to high-[K⁺]_o-induced swellingand glutamate release. In the current study, relative cell volumechanges were determined in astrocytes. Under 75 mM[K⁺]_o, astrocytes swelled by 20.2 ± 4.9%. This high-[K⁺]_o-mediated swelling wasabolished by the NKCC1 inhibitor bumetanide (10 µM, 1.0 ± 3.1%; P < 0.05). Intracellular³⁶Cl accumulation was increased from acontrol value of 0.39 ± 0.06 to 0.68 ± 0.05 µmol/mgprotein in response to 75 mM [K⁺]_o. Thisincrease was significantly reduced by bumetanide (P < 0.05). Basal intracellular Na⁺ concentration([Na⁺]_i) was reduced from 19.1 ± 0.8 to16.8 ± 1.9 mM by bumetanide (P < 0.05).[Na⁺]_i decreased to 8.4 ± 1.0 mM under75 mM [K⁺]_o and was further reduced to5.2 ± 1.7 mM by bumetanide. In addition, the recovery rate of[Na⁺]_i on return to 5.8 mM[K⁺]_o was decreased by 40% in the presenceof bumetanide (P < 0.05). Bumetanide inhibitedhigh-[K⁺]_o-induced ¹⁴C-labeledD-aspartate release by ~50% (P < 0.05).These results suggest that NKCC1 contributes tohigh-[K⁺]_o-induced astrocyte swelling andglutamate release.