Bed rest suppresses bioassayable growth hormone release in response to muscle activity

McCall, G. E., C. Goulet, R. E. Grindeland, J. A. Hodgson,A. J. Bigbee, and V. R. Edgerton. Bed rest suppresses bioassayable growth hormone release in response to muscle activity.J. Appl. Physiol. 83(6):2086-2090, 1997.Hormonal responses to muscle activity werestudied in eight men before (13 or 12 and 8 or 7 days), during (2 or 3, 8 or 9, and 13 or 14 days) and after (+2 or +3 and +10 or +11 days) 17 days of bed rest. Muscle activity consisted of a series of unilateral isometric plantar flexions, including 4 maximal voluntary contractions (MVCs), 48 contractions at30% MVC, and 12 contractions at 80% MVC, all performed at a 4:1-swork-to-rest ratio. Blood was collected before and immediately aftermuscle activity to measure plasma growth hormone by radioimmunoassay (IGH) and by bioassay (BGH) of tibia epiphyseal cartilage growth inhypophysectomized rats. Plasma IGH was unchanged by muscle activitybefore, during, or after bed rest. Before bed rest, muscle activityincreased (P < 0.05) BGH by 66% at13 or 12 days (2,146 ± 192 to 3,565 ± 197 µg/l)and by 92% at 8 or 7 days (2,162 ± 159 to 4,161 ± 204 µg/l). After 2 or 3 days of bed rest, there was no responseof BGH to the muscle activity, a pattern that persisted through 8 or 9 days of bed rest. However, after 13 or 14 days of bed rest, plasmaconcentration of BGH was significantly lower after than before muscleactivity (2,594 ± 211 to 2,085 ± 109 µg/l). After completionof bed rest, muscle activity increased BGH by 31% at 2 or 3 days(1,807 ± 117 to 2,379 ± 473 µg/l;P < 0.05), and by 10 or 11 days theBGH response was similar to that before bed rest (1,881 ± 75 to4,160 ± 315 µg/l; P < 0.05). These data demonstrate that the ambulatory state of an individual canhave a major impact on the release of BGH, but not IGH, in response toa single bout of muscle activity.

     

Kinetics of creatine kinase in an experimental model of low phosphocreatine and ATP in the normoxic heart

To study the dependence of the forward flux of creatine kinase(CK) on its substrates and products we designed an acute normoxic modelof steady-state depletion of phosphocreatine (PCr) and adenylate in theisovolumic acetate-perfused rat heart. Various concentrations of PCrand ATP were induced by prior perfusion with 2 deoxy-D-glucose in the presenceof insulin. The apparent rate constant(k_f) and theforward CK flux were measured under metabolic and contractile steadystate by progressive saturation-transfer³¹P nuclear magnetic resonance(NMR). At high adenylate content CK flux was constant for a twofoldreduction in PCr concentration ([PCr]); CK flux was 6.3 ± 0.6 mM/s (vs. 6.5 ± 0.2 mM/s in control) because of adoubling of k_f.Although, at the lowest ATP concentration and [PCr], CKflux was reduced by 50%, it nevertheless always remained higher thanATP synthesis estimated by parallel oxygen consumption measurement.NMR-measured flux was compared with the flux computed under thehypothesis of CK equilibrium. CK flux could not be fully predicted bythe concentrations of CK metabolites. This is discussed in terms ofmetabolite and CK isozyme compartmentation.

     

Transgenic livers expressing mitochondrial and cytosolic CK: mitochondrial CK modulates free ADP levels

The function of creatinekinase (CK) and its effect on phosphorus metabolites was studied inlivers of transgenic mice expressing human ubiquitous mitochondrial CK(CK-Mit) and rat brain CK (CK-B) isoenzymes and their combination.³¹P NMR spectroscopy and saturation transfer were recordedin livers of anesthetized mice to measure high-energy phosphates andhepatic CK activity. CK reaction velocity was related to total enzyme activity irrespective of the isoenzyme expressed, and it increased with increasing concentrations of creatine (Cr). The fluxesmediated by both isoenzymes in both directions (phosphocreatine or ATP synthesis) were equal. Over a 20-fold increase in CK-Mit activity (28-560 µmol · g wetwt¹ · min¹), the fraction ofphosphorylated Cr increased 1.6-fold. Hepatic free ADP concentrationscalculated by assuming equilibrium of the CK-catalyzed reaction in vivodecreased from 84 ± 9 to 38 ± 4 nmol/g wet wt. Calculatedfree ADP levels in mice expressing high levels of CK-B (920-1,635µmol · g wet wt¹ · min¹)were 52 ± 6 nmol/g wet wt. Mice expressing both isoenzymes had calculated free ADP levels of 36 ± 4 nmol/g wet wt. Thesefindings indicate that CK-Mit catalyzes its reaction equally well inboth directions and can lower hepatic apparent free ADP concentrations.

     

Contraction-induced increase in Vmax of palmitate uptake and oxidation in perfused skeletal muscle

To evaluate the effects of contractions on thekinetics of uptake and oxidation of palmitate in a physiological musclepreparation, rat hindquarters were perfused with glucose (6 mmol/l),albumin-bound [1-¹⁴C]palmitate, andvarying amounts of albumin-bound palmitate (200-2,200 µmol/l) atrest and during muscle contractions. When plotted against the unboundpalmitate concentration, palmitate uptake and oxidation displayedsimple Michaelis-Menten kinetics with estimated maximal velocity(V_max)and Michaelis-Menten constant(K_m) values of42.8 ± 3.8 (SE)nmol · min¹ · g¹and 13.4 ± 3.4 nmol/l for palmitate uptake and 3.8 ± 0.4 nmol · min¹ · g¹and 8.1 ± 2.9 nmol/l for palmitate oxidation, respectively, at rest.Whereas muscle contractions increased theV_maxfor both palmitate uptake and oxidation to 91.6 ± 10.1 and 16.5 ± 2.3 nmol · min¹ · g¹,respectively, theK_m remainedunchanged.V_maxand K_m estimates obtained from Hanes-Woolf plots (substrate concentration/velocity vs.substrate concentration) were not significantly different. In theresting perfused hindquarter, an increase in palmitate delivery from31.9 ± 0.9 to 48.7 ± 1.2 µmol · g¹ · h¹by increasing perfusate flow was associated with a decrease in thefractional uptake of palmitate so that the rates of uptake andoxidation of palmitate remained unchanged. It is concluded that therates of uptake and oxidation of long-chain fatty acids (LCFA) saturatewith an increase in the concentration of unbound LCFA in perfusedskeletal muscle and that muscle contractions, but not an increase inplasma flow, increase theV_maxfor LCFA uptake and oxidation. The data are consistent with the notion that uptake of LCFA in muscle may be mediated in part by a transport system.

     

Nitric oxide relaxes human myometrium by a cGMP-independent mechanism

The role of intracellular guanosine 3',5'-cyclicmonophosphate concentration([cGMP]_i) in nitricoxide (NO)-mediated relaxations in the uterus has become controversial.We found the NO donor S-nitroso-L-cysteine(CysNO) to potently (IC₅₀ = 30 nM)inhibit spontaneous contractions in the nonpregnant human myometrium. CysNO treatment increased[cGMP]_i significantly(P < 0.001), and this increase wasblocked by the guanylyl cyclase inhibitors methylene blue (10 µM) orLY-83583 (1 µM); however, pretreatment with these guanylyl cyclaseinhibitors failed to block CysNO-mediated relaxations. IntracellularcAMP concentrations were not altered by treatment of tissues with 10 µM CysNO. Incubation with the cGMP analogs 8-bromo-cGMP or-phenyl-1,N²-etheno-cGMPdid not significantly affect spontaneous contractility. Pretreatment oftissues with charybdotoxin [a calcium-dependent potassium channel(BK) blocker] completely reversed CysNO-induced relaxations. Weconclude that NO is a potent inhibitor of spontaneous contractileactivity in the nonpregnant human uterus and that, although guanylylcyclase and BK activities are increased by NO, increases in[cGMP]_i are notrequired for NO-induced relaxations in this tissue.

     

Rat hindlimb muscle blood flow during level and downhill locomotion

Duringeccentrically biased exercise (e.g., downhill locomotion), whole bodyoxygen consumption and blood lactate concentrations are lower thanduring level locomotion. These general systemic measurements indicatethat muscle metabolism is lower during downhill exercise. This studywas designed to test the hypothesis that hindlimb muscle blood flow iscorrespondingly lower during downhill vs. level exercise. Muscle bloodflow (determined by using radioactive microspheres) was measured inrats after 15 min of treadmill exercise at 15 m/min on the level (L,0°) or downhill (D, 17°). Blood flow to ankle extensormuscles was either lower (e.g., white gastrocnemius muscle: D, 9 ± 2; L, 15 ± 1 ml · min¹ · 100 g¹) or not different(e.g., soleus muscle: D, 250 ± 35; L, 230 ± 21 ml · min¹ · 100 g¹) in downhill vs. levelexercise. In contrast, blood flow to ankle flexor muscles was higher(e.g., extensor digitorum longus muscle: D, 53 ± 5; L, 31 ± 6 ml · min¹ · 100 g¹) during downhill vs.level exercise. When individual extensor and flexor muscle flows weresummed, total flow to the leg was lower during downhill exercise (D,3.24 ± 0.08; L, 3.47 ± 0.05 ml/min). These data indicate thatmuscle blood flow and metabolism are lower during eccentrically biasedexercise but are not uniformly reduced in all active muscles; i.e.,flows are equivalent in several ankle extensor muscles and higher inankle flexor muscles.     

Macro CK type 1 as a major component of serum creatine kinase activity in pregnant sheep

Electrophoretic separation of creatine kinase (ATP: creatine phosphotransferase, E.C. 2.7.3.2) isoenzymes in agarose gel was made in the serum of 93 sheep of three Slovenian domestic breeds (Solcavska, Pramenka and Bovska breeds). 44 sheep were pregnant between 112 and 135 days at the time of the blood collection. The median value of serum total CK activity for all animals investigated was 82 U/l. After direct immunoinhibition of CK with anti-M-CK monoclonal antibodies the total CK activity remained the same (88 U/l, P = 0.354). There were significant differences among breeds in CK activity for the Solcavska (101 U/l), Bovska (89 U/l) and Pramenka breeds (73 U/l), respectively (Kruskal–Wallis one way analysis of variance, P < 0.01), and between pregnant (105 U/l) and non-pregnant animals (76 U/l), irrespective of the breed (Mann–Whitney rank sum test, P < 0.05). According to electrophoresis, all non-pregnant sheep had exclusively free CK-BB serum bands activity. In all pregnant sheep coupled dimeric BB variant appeared as macro-CK type 1 in the range between 80% and 100% of total CK activity. The present study confirms the existence of an elimination mechanism for CK from the plasma abundance free CK-BB enzyme.     

More tetanic contractions are required for activating glucose transport maximally in trained muscle

Kawanaka, Kentaro, Izumi Tabata, and MitsuruHiguchi. More tetanic contractions are requiredfor activating glucose transport maximally in trained muscle.J. Appl. Physiol. 83(2): 429-433, 1997.Exercise training increases contraction-stimulated maximalglucose transport and muscle glycogen level in skeletal muscle.However, there is a possibility that more muscle contractions arerequired to maximally activate glucose transport in trained than inuntrained muscle, because increased glycogen level after training mayinhibit glucose transport. Therefore, the purpose of this study was toinvestigate the relationship between the increase in glucose transportand the number of tetanic contractions in trained and untrained muscle.Male rats swam 2 h/day for 15 days. In untrained epitrochlearis muscle,resting glycogen was 26.6 µmol glucose/g muscle. Ten, 10-s-longtetani at a rate of 1 contraction/min decreased glycogen level to 15.4 µmol glucose/g muscle and maximally increased2-deoxy-D-glucose(2-DG) transport. Training increasedcontraction-stimulated maximal 2-DG transport (+71%;P < 0.01), GLUT-4 protein content(+78%; P < 0.01), and restingglycogen level (to 39.3 µmol glucose/g muscle;P < 0.01) on the next day after thetraining ended, although this training effect might be due, at least inpart, to last bout of exercise. In trained muscle, 20 tetani werenecessary to maximally activate glucose transport. Twenty tetanidecreased muscle glycogen to a lower level than 10 tetani (18.9 vs.24.0 µmol glucose/g muscle; P < 0.01). Contraction-stimulated 2-DG transport was negatively correlatedwith postcontraction muscle glycogen level in trained (r = 0.60;P < 0.01) and untrained muscle(r = 0.57;P < 0.01).

     

Ascorbate enhancement of H1 histamine receptor sensitivity coincides with ascorbate oxidation inhibition by histamine receptors

Ascorbate has previously been shown to enhance both ₁- and ₂-adrenergic activity. This activity is mediated by ascorbate binding to the extracellular domain of the adrenergic receptor, which also decreases the oxidation rate of ascorbate. H1 histamine receptors have extracellular agonist or ascorbate binding sites with strong similarities to _1- and ₂-adrenergic receptors. Physiological concentrations of ascorbate (50 µM) significantly enhanced histamine contractions of rabbit aorta on the lower half of the histamine dose-response curve, increasing contractions of 0.1, 0.2, and 0.3 µM histamine by two- to threefold. Increases in ascorbate concentration significantly enhanced 0.2 µM histamine (5–500 µM ascorbate) and 0.3 µM histamine (15–500 µM ascorbate) in a dose-dependent manner. Histamine does not measurably oxidize over 20 h in oxygenated PSS at 37°C. Thus the ascorbate enhancement is independent of ascorbate's antioxidant effects. Ascorbate in solution oxidizes rapidly. Transfected histamine receptor membrane suspension with protein concentration at >3.1 µg/ml (56 nM maximum histamine receptor) decreases the oxidation rate of 392 µM ascorbate, and virtually no ascorbate oxidation occurs at >0.0004 mol histamine receptor/mol ascorbate. Histamine receptor membrane had an initial ascorbate oxidation inhibition rate of 0.094 min·µg protein^–1·ml^–1, compared with rates for transfected ANG II membrane (0.055 min·µg protein^–1·ml^–1), untransfected membrane (0.052 min·µg protein^–1·ml^–1), creatine kinase (0.0082 min·µg protein^–1·ml^–1), keyhole limpet hemocyanin (0.00092 min·µg protein^–1·ml^–1), and osmotically lysed aortic rings (0.00057 min·µg wet weight^–1·ml^–1). Ascorbate enhancement of seven-transmembrane-spanning membrane receptor activity occurs in both adrenergic and histaminergic receptors. These receptors may play a significant role in maintaining extracellular ascorbate in a reduced state. molecular complementarity; vitamin C; seven-transmembrane-spanning membrane receptors     

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.

     

Effect of caffeinated drinks on substrate metabolism, caffeine excretion, and performance

The effect ofaddition of different dosages of caffeine (Caf) to acarbohydrate-electrolyte solution (CES) on metabolism, Caf excretion,and performance was examined. Subjects(n = 15) ingested 8 ml/kg of waterplacebo (Pla-W), 7% CES (Pla-CES), or 7% CES with 150, 225, and 320 mg/l Caf (CES-150, CES-225, and CES-320, respectively) during a warm-upprotocol (20 min) and 3 ml/kg at one-third and two-thirds of a 1-h timetrial. Performance was improved with Caf supplementation: 62.5 ± 1.3, 61.5 ± 1.1, 60.4 ± 1.0, 58.9 ± 1.0, and 58.9 ± 1.2 min for Pla-W, Pla-CES, CES-150, CES-225, and CES-320, respectively.The postexercise urinary Caf concentration (range 1.3-2.5 µg/ml)was dose dependent and always far below the doping level of theInternational Olympic Committee (12 µg/ml) in all subjects. Sweat Cafexcretion during exercise exceeded postexercise early-void urinary Cafexcretion. Caffeinated CES did not enhance free fatty acidavailability, ruling out the fact that performance improvement resultedfrom enhanced fat oxidation. It is concluded that addition ofrelatively low amounts of Caf to CES improves performance and thatpostexercise urinary Caf concentration remained low.

     

Paradoxical helium and sulfur hexafluoride single-breath washouts in short-term vs. sustained microgravity

Lauzon, Anne-Marie, G. Kim Prisk, Ann R. Elliott, SylviaVerbanck, Manuel Paiva, and John B. West. Paradoxical helium andsulfur hexafluoride single-breath washouts in short-term vs. sustainedmicrogravity. J. Appl. Physiol. 82(3):859-865, 1997.During single-breath washouts in normal gravity (1 G), the phase III slope of sulfur hexafluoride(SF₆) is steeper than that ofhelium (He). Two mechanisms can account for this:1) the higher diffusivity of Heenhances its homogeneous distribution; and2) the lower diffusivity ofSF₆ results in a more peripherallocation of the diffusion front, where airway asymmetry is larger.These mechanisms were thought to be gravity independent. However, weshowed during the Spacelab Life Sciences-2 spaceflight that insustained microgravity (µG) theSF₆-to-He slope difference isabolished. We repeated the protocol during short periods (27 s) of µG(parabolic flights). The subjects performed a vital-capacityinspiration and expiration of a gas containing 5% He-1.25%SF₆-balanceO₂. As in sustained µG, thephase III slopes of He and SF₆decreased. However, during short-term µG, theSF₆-to-He slope differenceincreased from 0.17 ± 0.03%/l in 1 G to 0.29 ± 0.06%/l inµG, respectively. This is contrary to sustained µG, in which theSF₆-to-He slope difference decreased from 0.25 ± 0.03%/l in 1 G to 0.01 ± 0.06%/lin µG. The increase in phase III slope difference in short-term µGwas caused by a larger decrease of He phase III slope compared with that in sustained µG. This suggests that changes in peripheral gasmixing seen in sustained µG are mainly due to alterations in thediffusive-convective inhomogeneity of He that require >27 s of µGto occur. Changes in pulmonary blood volume distribution or cardiogenicmixing may explain the differences between the results found inshort-term and sustained µG.

     

Stretch-induced Ca(2+) release via an IP(3)-insensitive Ca(2+) channel

Various mechanicalstimuli increase the intracellular Ca²⁺ concentration([Ca²⁺]_i) in vascular smooth muscle cells(VSMC). A part of the increase in [Ca²⁺]_i isdue to the release of Ca²⁺ from intracellular stores. Wehave investigated the effect of mechanical stimulation produced bycyclical stretch on the release of Ca²⁺ from theintracellular stores. Permeabilized VSMC loaded with ⁴⁵Ca²⁺ were subjected to 7.5% average (15%maximal) cyclical stretch. This resulted in an increase in⁴⁵Ca²⁺ rate constant by 0.126 ± 0.0035. Inhibition of inositol 1,4,5-trisphosphate (IP₃),ryanodine, and nicotinic acid adenine dinucleotide phosphate channels(NAADP) with 50 µg/ml heparin, 50 µM ruthenium red, and 25 µMthio-NADP, respectively, did not block the increase in⁴⁵Ca²⁺ efflux in response to cyclical stretch.However, 10 µM lanthanum, 10 µM gadolinium, and 10 µMcytochalasin D but not 10 µM nocodazole inhibited the increase in⁴⁵Ca²⁺ efflux. This supports the existence of anovel stretch-sensitive intracellular Ca²⁺ store in VSMCthat is distinct from the IP₃-, ryanodine-, and NAADP-sensitive stores.

     

Development of myoglobin concentration and acid buffering capacity in harp (<Emphasis Type="Italic">Pagophilus groenlandicus</Emphasis>) and hooded (<Emphasis Type="Italic">Cystophora cristata</Emphasis>) seals from birth to maturity

Pinnipeds rely on muscle oxygen stores to help support aerobic diving, therefore muscle maturation may influence the behavioral ecology of young pinnipeds. To investigate the pattern of muscle development, myoglobin concentration ([Mb]) and acid buffering ability (β) was measured in ten muscles from 23 harp and 40 hooded seals of various ages. Adult [Mb] ranged from 28–97 to 35–104 mg g tissue⁻¹ in harp and hooded seals, respectively, with values increasing from the cervical, non-swimming muscles to the main swimming muscles of the lumbar region. Neonatal and weaned pup muscles exhibited lower (~30% adult values) and less variable [Mb] across the body than adults. In contrast, adult β showed little regional variation (60–90 slykes), while high pup values (~75% adult values) indicate significant in utero development. These findings suggest that intra-uterine conditions are sufficiently hypoxic to stimulate prenatal β development, but that [Mb] development requires additional postnatal signal such as exercise, and/or growth factors. However, because of limited development in both β and [Mb] during the nursing period, pups are weaned with muscles with lower aerobic and anaerobic capacities than those of adults.     

Optimization of ethanol,citric acid,and α-amylase production from date wastes by strains of <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>, <Emphasis Type="Italic">Aspergillus niger</Emphasis>, and <Emphasis Type="Italic">Candida guilliermondii</Emphasis>

The present study deals with submerged ethanol, citric acid, and α-amylase fermentation by Saccharomyces cerevisiae SDB, Aspergillus niger ANSS-B5, and Candida guilliermondii CGL-A10, using date wastes as the basal fermentation medium. The physical and chemical parameters influencing the production of these metabolites were optimized. As for the ethanol production, the optimum yield obtained was 136.00 ± 0.66 g/l under optimum conditions of an incubation period of 72 h, inoculum content of 4% (w/v), sugars concentration of 180.0 g/l, and ammonium phosphate concentration of 1.0 g/l. Concerning citric acid production, the cumulative effect of temperature (30°C), sugars concentration of 150.0 g/l, methanol concentration of 3.0%, initial pH of 3.5, ammonium nitrate concentration of 2.5 g/l, and potassium phosphate concentration of 2.5 g/l during the fermentation process of date wastes syrup did increase the citric acid production to 98.42 ± 1.41 g/l. For the production of α-amylase, the obtained result shows that the presence of starch strongly induces the production of α-amylase with a maximum at 5.0 g/l. Among the various nitrogen sources tested, urea at 5.0 g/l gave the maximum biomass and α-amylase estimated at 5.76 ± 0.56 g/l and 2,304.19 ± 31.08 μmol/l/min, respectively after 72 h incubation at 30°C, with an initial pH of 6.0 and potassium phosphate concentration of 6.0 g/l.     

Extracellular ATP stimulates volume decrease in Necturus red blood cells

This study examined whether extracellular ATP stimulatesregulatory volume decrease (RVD) in Necturusmaculosus (mudpuppy) red blood cells (RBCs). Thehemolytic index (a measure of osmotic fragility) decreased withextracellular ATP (50 µM). In contrast, the ATP scavenger hexokinase(2.5 U/ml, 1 mM glucose) increased osmotic fragility. In addition, theATP-dependent K⁺ channelantagonist glibenclamide (100 µM) increased the hemolytic index, andthis inhibition was reversed with ATP (50 µM). We also measured cellvolume recovery in response to hypotonic shock electronically with aCoulter counter. Extracellular ATP (50 µM) enhanced cell volumedecrease in a hypotonic (0.5×) Ringer solution. In contrast, hexokinase (2.5 U/ml) and apyrase (an ATP diphosphohydrolase, 2.5 U/ml)inhibited cell volume recovery. The inhibitory effect of hexokinase wasreversed with the Ca²⁺ ionophoreA-23187 (1 µM); it also was reversed with the cationophore gramicidin(5 µM in a choline-Ringer solution), indicating that ATP was linkedto K⁺ efflux. In addition,glibenclamide (100 µM) and gadolinium (10 µM) inhibited cell volumedecrease, and the effect of these agents was reversed with ATP (50 µM) and A-23187 (1 µM). Using the whole cell patch-clamp technique,we found that ATP (50 µM) stimulated a whole cell current underisosmotic conditions. In addition, apyrase (2.5 U/ml), glibenclamide(100 µM), and gadolinium (10 µM) inhibited whole cell currents thatwere activated during hypotonic swelling. The inhibitory effect ofapyrase was reversed with the nonhydrolyzable analog adenosine5'-O-(3-thiotriphosphate) (50 µM), and the effect of glibenclamide or gadolinium was reversed withATP (50 µM). Finally, anionic whole cell currents were activated withhypotonic swelling when ATP was the only significant charge carrier,suggesting that increases in cell volume led to ATP efflux through aconductive pathway. Taken together, these results indicate thatextracellular ATP stimulated cell volume decrease via aCa²⁺-dependent step that led toK⁺ efflux.

     

Estimation of thickness of airwaysurface liquid in ferret trachea in vitro

Duneclift, S., U. Wells, and J. Widdicombe. Estimationof thickness of airway surface liquid in ferret trachea in vitro. J. Appl. Physiol. 83(3): 761-767, 1997.The tracheae of ferrets and rabbits were mounted in vitro inorgan baths. While the tracheae were liquid filled, the permeabilitycoefficient ( P) was determined, and then while thetracheae were air filled, the percent clearance for^99mTc-labeleddiethylenetriaminepentaacetic acid (DTPA) was determined. The thicknessof airway surface liquid (ASL) was estimated by three methods.1) The initial concentration of^99mTc-DTPA and the total amount of^99mTc-DTPA (the sum of thatentering the outside medium, that draining from the trachea, and thatwashed out at the end of 40 min) gave the initial volume of ASL andthus its thickness. Mean values were 45.7 µm for the ferret and 41.9 µm for the rabbit. 2) Estimates ofASL thickness at the end of the 40-min period, based on the final^99mTc-DTPA concentration and theamount in the washout, were 42.9 µm for ferret and 45.4 µm forrabbit. 3) The ratio of Pto percent clearance gave mean ASL thickness values of 49.2 µm forthe ferret and 40.3 µm for the rabbit. Thus three separate methodsfor determining ASL thickness give very similar results, with means inthe range 40-49 µm. Administration of methacholine or atropineto ferret tracheae did not significantly change ASL thickness.

     

Peroxide resistance of ER Ca2+ pump in endothelium: implications to coronary artery function

We examined the effects of peroxide on the sarco(endo)plasmicreticulum Ca²⁺ (SERCA) pump in pigcoronary artery endothelium and smooth muscle at three organizationallevels: Ca²⁺ transport inpermeabilized cells, cytosolicCa²⁺ concentration in intactcells, and contractile function of artery rings. We monitored theATP-dependent, azide-insensitive, oxalate-stimulated ⁴⁵Ca²⁺uptake by saponin-permeabilized cultured cells. Low concentrations ofperoxide inhibited the uptake less effectively in endothelium than insmooth muscle whether we added the peroxide directly to theCa²⁺ uptake solution or treatedintact cells with peroxide and washed them before the permeabilization.An acylphosphate formation assay confirmed the greater resistance ofthe SERCA pump in endothelial cells than in smooth muscle cells.Pretreating smooth muscle cells with 300 µM peroxide inhibited (by 77 ± 2%) the cyclopiazonic acid (CPA)-induced increase in cytosolicCa²⁺ concentration in aCa²⁺-free solution, but it did notaffect the endothelial cells. Peroxide pretreatment inhibited theCPA-induced contraction in deendothelialized arteries with a 50%inhibitory concentration of 97 ± 13 µM, but up to 500 µMperoxide did not affect the endothelium-dependent, CPA-inducedrelaxation. Similarly, 500 µM peroxide inhibited the angiotensin-induced contractions in deendothelialized arteries by 93 ± 2%, but it inhibited the bradykinin-induced,endothelium-dependent relaxation by only 40 ± 13%. The greaterresistance of the endothelium to reactive oxygen may be importantduring ischemia-reperfusion or in the postinfection immune response.

     

Sago starch as a low-cost carbon source for exopolysaccharide production by Lactobacillus kefiranofaciens

Low-cost sago starch was used as a carbon source for production of the exopolysaccharide kefiran by Lactobacillus kefiranofaciens. A simultaneous saccharification and fermentation process of sago starch for kefiran production was evaluated. Factors affecting the process such as an initial pH, temperature, starch concentration, including a mixture of α-amylase and glucoamylase were determined. The highest kefiran concentration of 0.85 g/l was obtained at the initial pH of 5.5, temperature of 30 °C, starch concentration of 4% and mixed-enzymes with activity of 100 U/g-starch. The use of a mixture of α-amylase and glucoamylase could enhance the productivity compared to the use of α-amylase alone. The optimal ratio of α-amylase to glucoamylase of 60:40 gave the highest kefiran production rate of 11.83 mg/l/h. This study showed that sago starch could serve as a low-cost substrate for kefiran production.     

Intra- and extra-cellular organophosphorus hydrolase production with recombinant <Emphasis Type="Italic">E. coli</Emphasis> using fed-batch fermentation

A fed-batch fermentation process for the production of organophosphorus hydrolase (OPH) (EC 3.1.8.1) by E. coli pET812 was developed in this research. With batch fermentation, the maximum OPH concentrations attained by batch fermentation were as low as 4 × 10⁵ U/l because cell growth and OPH production were inhibited by a high initial concentration of glucose. To develop a fed-batch fermentation process for obtaining higher concentrations of OPH, highly concentrated glucose solution (500 g/l) was added intermittently or continuously to increase the carbon source concentration. Eventually, 3.2 × 10⁶ U/l of OPH was produced with fed-batch fermentation in 24 h. This was eight times higher than the yield with conventional batch fermentation. A total concentration of 399–441 mg of OPH was produced/l, which was four times higher than that reported when using E. coli. Nearly half (44%) of the produced OPH was secreted into the culture solution.