The effects of ATP, inorganic phosphate, protons, and lactate on isolated myofibrillar ATPase activity WQSAPARKHOUSE School of Kinesiology , Simon h s a r University, Burnabj, B. C. , Cartahfa V5A 1S6
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Received April 24, 1991 PARKHOUSE, W" S. 1992. The effects s f ATP, inorganic phosphate, protons, and lactate on isolated myofibrillar ATBase activity. Can. J. Physiol. Pharmacol. 70: 1 175- H 181. The purpose of this study was to examine the effects sf lactate, protons, inorganic phosphate, and ATP on myofibrillar ATPase activity. Myofibrils were isolated from carp (Cyprinius carpks L.) fast-twitch white muscle, and myofibrillar ATPase activities were assessed under maximal activating calcium levels (pCa 4.0) at 10°C in reaction media containing metabolic profiles similar to those seen in fatiguing muscles. The Ca2'-activated ATPase activity was assessed by an ATP regenerating assay that csupled the myofibrillar ATPase to pymvate kinase and lactate dehydrogenase. This assay allowed the effects of ATP, inorganic phosphate, protons, and lactate on myofibrillar ATPase activity to be assessed. The coupled assay was found to give similar myofibrillar ATPase kinetics, with the exception of higher maximal activities, to those seen with a standard end-point assay. Myofibrillar ATPase activity was depressed by 35% when ATP concentrations were lowered to 2.5 mM. Lowering ATP levels to 0.5 mM reduced the myofibrillar ATPase activities by 85 %. Lactate had no effect on myofibrillar ATBase activities. Inorganic phosphate levels up to about 20 mM significantly decreased the myofibrillar ATPase activities, after which further increases in inorganic phosphate content had minimal effects. The changes in ATPase activities were related to total inorganic phosphate, not ts the content of diprotonated inorganic phosphate. Myofibrillar ATPase activity was highest at pH 7.5 and lowest at pH 6.0. The interactive effects of low ATP, decreased pH, and high inorganic phosphate levels were not additive, giving similar decreases in activity to those produced by increased inorganic phosphate levels alone. These results suggest that alterations in metabolites accompanying exercise may regulate myofibrillar ATPase activity and contribute to fatigue by inhibiting myofibrillar ATP hydrolysis rates. Key words: fatigue, muscle, ATPase, fish, inorganic phosphate. -
PARKHOUSE, W S . 1992. The effects of ATP, inorganic phosphate, protons, and lactate on isolated myofibrillar ATPase activity. Can. J. Physiol. Pharmacol. 78 : 1175- 1181. Le but de cette Ctude a CtC d'examiner les effets du lactate. des protons, du phosphate inorganique et de 1'ATP sur l'activiti de 1'ATPase myofibrillaire. On a is016 des myofibrilles du muscle blanc 2i contraction rapide de la c a p e (Cygrisaius carpis L.) et CvaluC les activitCs de l'ATPase dans des conditions de taux de calcium d'activation maximale (pCA 4,0), h BO°C, dans des milieux reproduisant des conditions mdtaboliques similaires B celles des muscles en fatigue. On a 6valuC l'activitd d'ATPase activCe par le Ca2+ en recourant h un dosage regCnCrateur d7ATP, lequel a coup16 l'activit6 d'ATPase myofibrillaire h la pyruvate kinase et h la 1acticodCshydrogCmse. Ce dosage a permis d'dvaluer les effets de 19ATP,du phosphate inorganique, des protons et du lactate sur l'activitC dqATPasemyofibrillaire. I1 a donnk des cinCtiques d' ATPase myofibrillaire similaires, sauf pour ce qui est des activitCs maximales plus ClevCes. LqactivitCd'ATPase myofibrillaire a diminuC de 35% lorsque les concentrations dvATPont CtC abaissdes 2i 2,5 mM et de 85 % lorsque les concentrations snt Ctk rduites h 0,5mM. Le lactate n'a pas eu d9effetsur les activitCs d' ATPase myofibrillaire. Des taux de phosphate inorganique pouvant aller jusqu'h 20 mM ont significativernent rCduit les activitCs d'ATPase myofibrillaire; d'autres augmentations de la teneur en phosphate inorganique n'ont eu que des effets minimes. Les variations des activitCs dvATPase ont CtC relikes h la teneur totale en phosphate inorganique et non pas h la teneur en phosphate inorganique diprotonk. L'activitC d'ATPase myofibrillaire a kt6 maximale au pH 7,5 et a diminuC B des activitks minimales au pH 6,O. Les effets interactifs d'une faible teneur en ATP, d'un pH rCduit et de bux ClevCs de phosphate inorganique n'ont pas CtC additifs, donnant des diminutions d'activite sirnilaires B celles provoqutes par les augmentations de phosphate inorganique uniquement. Ces rCsultats suggkrent que des altCrations au niveau des rnCtabolites lors de l'exercice pourraient rCguler l'activitt d'ATPase myofibrillaire et contribuer h la fatigue par l'inhibition des taux d9hydrolyse le 1'ATP myofibrillaire. Mots cI&s : fatigue, muscle, ATPase, poisson, phosphate inorganique.
Introduction Many investigators have examined the relationships between fatiguing muscle preparations and the accumulation of metabolic end products (Kentish 1986; Fitts and Holloszy 1974; Sahlin et al. 1981; Parkhouse et id. 1988). In these studies, decreases in ATP levels and the accumulation of lactate, prstons, and inorganic phosphate (Pi; both total and diprstonated) have been suggested to (i) inhibit the energy-yielding pathways (Spriet 199B), ( i i ) result in large ionic imbalances that would affect membrane potentids (Parkhouse et al. 1987), (iii) compete with calcium for the calcium binding sites on troponin-@ (Fabiato and Fabiato 1978; Blanchard et al. 1984), (iv) reduce the rate and (or) amount of calcium released and sequestered by the sareoplasmic reticulum (Fabiato and Fabiato 1978), and (v) alter crossbridge kinetics (Kawai et al. Printed in Canada i ImprirnC au Canada
1987; Metzer and Moss 1987; Chase and Kushmerick 1988; Brozovich et al. 1988). Most of these investigations have utilized whole muscle, skinned fibers, or whole organism models to examine the potential causitive relationships between these metabolites and skeletal muscle fatigue. The relationship between these metabolites and mysfibrillar kinetics can only be hypothesized, since these altered metabolite levels also have effects on other cellular functions. Examining the effects of these metabolites on the isolated myofibrillar complex may provide insight into their interrelationships in the fatigue process. However, it is recognized that upon addition sf ATP, isolated myofibrils form a structure that could loosely be termed a logjam s f filaments, and that the ATPase activity of this undefined structure lie somewhere between that of purified actin and myosin and that
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Time (min) PIG. 2. Typical Ca2+-stimulated (pCa 4.0) myofibrillar ATPase activities of the coupled (C) and end-point (E) assay systems calculated every 30 s for a single isolation of carp myofibrils.
Fro. 1. (A) Carp white muscle Ca2+-stimulated myofibrillar ATPase activities at 10°C, pH 7.0, and 5 mM ATP, over the pCa range 5-8 for the end-point (E) and coupled (C) assay systems. (B) Will plot of the Ca2+-stimulated rnyofibrillar ATPase activities of the E and C assay systems.
of more intact fiber systems. Acknowledging these discrepancies, these structures allow myofibrillar ATPase kinetics to be probed in an intact stable preparation containing the contractile regulatory proteins. Therefore, the purpose of this investigation was to examine the effects s f lactate, protons, Pi, and ATP on myofibrdlar ATPase activity.
Methods Animnks and muscle san~pllng Carp (Cytbas'nims cargio L.) were obtained from a local supplier (Sun Valley Trout F a m , Mission, B.C.) and were kept in fresh, aerated, dechlorinated tap water at 10- 12'C prior to the experiments. Fish were sacrificed by decapitation, and the white muscle was rapidly excised from a site posterior to the dorsal fin and freezeclamped with aluminum tongs cooled in liquid nitrogen. Samples were stored at -80°C until analysis. Myofibrkblar isolarion The fish white muscle was homogenized in 20 volumes of ice-cold relaxing buffer (39 mM sodium borate; 25 mM KC1; 5 mM EDTA; pH 7.1) for 2 X 10 s with a tissue homogenizer (Polytron PT-10) at 80% of maximal speed. This buffer was designed to relax the muscle by chelating any calcium and magnesium present in the preparation. The honmogenate suspension was then centrifuged at 1000 x g for 12 min at 4°C and the supernatant discarded. The myofibrillar pellet was then resuspended in the relaxing buffer and the procedure repeated. The resulting myofibrillar pellet was subjected to two further resuspensions and centrifugations under identical conditions, with the exception that EDTA was excluded from the ice-cold buffer (39 mM sodium borate; 25 mM KC1; pH 7.1). These procedures were found to be sufficient to remove any remining EDTA. The myofibrillar pellet was subjected to another two resuspensions and
centrifugations at 1000 x g for 12 min at 4'C in a medium consisting of 50 mM Tris, 5 mM sodium azide (NaN,), 100 mM KC1, and 0-5% Triton X-100, pH 7.4. Sodium azide and Triton X-100 were included to inhibit mitochondria1 ATPase and to remove the sarcoplasmic reticulum and ~rcolemmalATPase, respectively. This procedure was sufficient for removal of contaminating ATPases, since no differences were observed in ATPase activities when sodium azide or ouabain was included in the assay media. The myofibrillar pellet was resuspended in a final ice-cold suspension medium consisting of 150 mM KC1 and 50 mM Tris, pH 7.4, to remove any traces of sodium azide or Triton X-BOO. The suspension was then centrifuged as described previously. The myofibrillar pellet was then resuspended in 10 volumes of suspension medium and passed through glass wool to remove any large particles or clumps of myofibrils. Passing the suspension through the glass wool resulted in a loss of 20 -40% of the protein yield. Aliquots (58 p L ) of suspended myofibrillar protein were taken in duplicate for protein determination by the method of Lowry et al. (1951), and all protein concentrations are expressed relative to wet muscle. Myofibrillar ATPase activity The norm1 myofibrillar ATPase assay (Belcastro et al. 1984; Pierce and Dhalla 1985) uses a large amount of protein and measures the accumulation of P, over a given time period (usually 2 - 5 min) . This procedure will be referred to as the end-point assay and results in the lowering of ATP levels in the medium and accumulation of ADP, making it difficult to assess the effects of these metabolites on the enzyme activity itself. Furthermore, alterations in ionic strength and the addition of ATP are known to affect the ATPase activity and structure of the myofibrillar complex in the end-pont assay. The use of an ATP regenerating assay that couples myofibrillar ATPase to pyruvate b a s e (PK) and lactate dehydrogenase (EDH) maintains ATP levels constant while allowing the use of very low levels of myofibrillar protein, owing to the sensitivity of monitoring NADH production as opposed to P, production. This assay, which will be referred to as the coupled assay, has been used previously to measure sarcoplasrmic reticulum Ca2+ATPase activity (Tate et al. 1988). The validity of this assay for use in the present study was assessed by comparing the results obtained between it and the end-point assay in terms of Ca2+-activated ATPase activity, Ca2+ sensitivities, and Ca2+ cooperativity (Fig. I), and the accumulations of Pi and ADP and declines in ATP as a function of assay time (Figs. 2-4).
PARKHOUSE
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EBJD- P O I N T
COUPLED
c2
-5 8
to,
Time (rnin)
FIG.3 . Amount of ATP hydrolyzed per assay for the coupled (C) and end-pint (E) assay systems as a function of assay run time. TABLE1. Mean control (C) and exhaustive (E) exercise metabolite levels (para01 . g-') in fish white muscle -
Rainbow trouta
Rainbow troutb
Rainbow troutC
Carp"
ATP C E ADP
e
E pi
C E Lactate C E
PH C E "Data from Parbouse et al. (1988). bData from Dobssn et al. (1987). 'Data from Miliigan and Wood (1 986). dData from Driedzic and Hochachka (1976). 'P, values are corrected for nonspecific hydrolysis of phosphocreatine (Kr)during sampling and freezing, assuming that !XI% of the total creatine pool would be in the form of PCr in control muscle. f ~ a l u e sare calculated based on the decrease in PCr and ATP.
Myofibrillar ATPase activities were assessed every 30 s and the concentrations of ATB and ADP were determined as outlined previously (Parkhouse et al. 1988). Inorganic phosphate levels were not detectable in the regenerating system by colorimetric assay and were calcuHated based on a stoichiometric relationship with ATP declines. The csncentrations of the effectors used reflect exercising values for fish white muscle (Table I). Free Ca2+ concentrations were determined with an Orion calcium-sensitive electrode by the method of Bers (1982). End-point assay Briefly, myofibrillar ATPase activity was determined in a medium containing 50 mM KC1, 20 mM Tris, 1 mM MgC12, and 208500 pglmL of mysfibrillar protein. Following a 5-min preincubatisn period at pCa 4, the reaction was started with 5 mM Mg . ATP. The reaction was terminated at 5 min by adding 20% trichloroacetic acid. The liberated Pi was determined in the protein-free supernatant (Taussky and Shsrr 1953).
Time (min)
FIG.4. Absolute change in ATP, ADP, and P, s f the end-point (E) and coupled (C) assay systems as a function of assay mn time. Coupled assay Mysfibrillar ATPase activity was determined in an ATP regenerating medium containing 50 mM KCB, 20 mM Tris, 1 mM MgCl,, 2 mM phosphoenolpymvate (PEP), 0.08 NABH, 5 mM Mg - ATP, 10 U LDH, and 10 U PK, pH 7.0. This assay medium was incubated in a thermostated cell holder for 5 min and the reaction initiated with 20 pg of myofibrillar protein. The reaction was initiated with protein in place of Mg * ATP, for ease in establishing a stable baseline. However, initiating the assay with Mg ATP resulted in no difference in ATPase activity. The reaction was followed at 340 nm on a Pye Unicam SP8880 spectrophotometer. The initial linear portion of the curve was used to determine maximal ATPase activities at pCa 4. ATPase activity was assessed in the presence and absence of sodium azide and ouabain to test for contamination by mitochondria1 and (or) sarcolemmal ATPases. All ATBase activities were corrected for nonspecific ATP hydrolysis and checked for maximal activating Ca2+ concentrations. As ionic strength has been found to affect myofibrillar ATPase activities measured with the end-point assay, the effects of ionic strength were examined in the coupled assay medium by varying KC1 contents between 56 and 150 mM. Under these conditions, no significant differences were found in the ATPase activities.
-
Data analyses Data are presented as means f SD. A Student's P-testwas used to analyze the differences in myofibrillar ATPase activities between eorresponding values of H,PQ,-. p < 0.05 was considered significant.
Results The two assays resulted in similar Ca2* sensitivities and CaB cooperativities, but the maximal Ca2*-activated ATPase activity was about 25% higher with the coupled system
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Inorganic phosphate (mM) FIG.5. Myofibrillar ATPase activity in response to altered ATP concentrations at maximal activating Ca2+ levels (pCa 4.0).
-
2
-5
0
Condition 0
10
20
30
40
Lactate (mM)
FIG.6. Myofibrillar PhTFPase activity in response to altered lactate (A) and proton (B) concentrations at maximal activating @a2' levels (pCa 4.0).
(Fig. I). In contrast to the end-point assay, the coupled assay demonstrated a linear relationship between Ca2+-activated ATPase activity and time (Fig. 2). Figure 3 shows the large differences in the amounts of ATP hydrolyzed per assay under the two assay systems, with the end-point assay hydrolyzing 10- 15 times more ATP per assay at any given time interval. Figure 4 shows the changes in assay medium ATB, ADP, and
FIG.7. Myofibrillar ATPase activity in response to altered inorganic phosphate (Pi) levels (A) and vari-ioaas conditions of ATP, pH, and Pi (B) at maximal activating Ca2+ levels (pCa 4.0). Condition A, 5 mM ATP, pH 7.8, B mM Pi; condition B, 5 mM ATP, pH 6.5, 1 mM Pi; condition C, 5 mM ATP, pH 7.0, 18 mM Pi; condition B, 5 mM ATPp pH 6.5, BO mM Pi; condition E, 2.5 mM ATP, pH 6.5, 18 mM Pi; condition F, 5 mM ATP, pH 7.0, 20 mM Pi; condition G, 2.5 mM ATP, pH 6.5, 20 mM Pi; condition H, 0.5 mM ATP, pH 6.0, 20 mM Pi.
Pi contents under the two assay conditions as a fbnction s f incubation time. The effects of ATP, lactate, pH, and Pi on myofibrillar ATPase activities are demonstrated in Figs. 5 - 7. The major findings sf this study were that ATPase activities decreased in the face of modest ATP declines; that the declines in ATPase activity were associated with total Pi content, not &Po4-, and that protons also depress the ATPase activity but that this effect is not additive to the Pi effect. Specifically, a reduction in ATP to 2.5 mM resulted in a 35 % decline in maximal Ca2+-activated myofibrillar ATPase activity. A further reduction in the incubation medium's ATB concentration to 0.5 mM was found to reduce the ATBase activity a arther 50%. Therefore, when ATP levels were very low, ATPase activity had decreased by 85 % (Fig. 5). Maximal &la2+-activatedrnysfibrillar ATPase activity was unaffected by increases in lactate concentration to 40 mM (Fig. $A). In contrast, a reduction in pH from 7.0 to 6.5 resulted in about a 35 % loss of ATPase activity (Fig. QB).A further decline in pH to 6.0 was found to reduce the ATPase activity to less than 20% of its activity at pH 7.8 (Fig. 6B).
TABLE2. MyofibriBlar ATPase versus total and diprotonated forms of inorganic phosphate (mean 9 SD)
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hsH
Total Pi CmM)
H2P04(mM>
Activity (rams1 . mg-' protein . min- ')
NOTE:H,PO,- values are calculated based on a pK of 6.81 (Gadian et al. 1981). "Significantly different from a11 other conditions ( p < 0.05).
A 1 mM Pi concentration was found to have no effect on the n~aximal Ca2+-activated my~fibrillar ATPase activity, with the activities still k i n g in the range of 400 nmol - mg-I protein . rnin-l (Fig. 7A). However, increasing the Pi levels to 10 mM resulted in approximately a 55% decline in this ATPase activity (Fig. 7A). A further increase in Pi levels to 20 mM resulted in a final ATPase activity that was less than 28% of its original level (Fig. 7A). Increasing the Pi content from 20 to 40 mM had relatively little effect on the rnyofibrillar ATPase activity. Pi primarily exists in the physiological pH range as either the mono- (HP04") or di-protonated (H2P04-) form. The reduction in ATPase activity was unrelated to changes in the concentration of the dipr~tonatedform (Table 2). At identical concentrations of total Pi (10 mM) but vastly different concentrations of H2PO4- (3.9 and 6.7 mM), myofibrillar ATPase activity was not significantly different. In contrast, at the same concentration of H2PO4- (7.8 mM) and vastly different total Pi concentrations (1 1.6 and 20 mM), the 12 and 72 f 8 nmol . mg-I ATPase activities were 165 min-I (mean f SD). A number of combinations of Pi, ATP, and pH were used to assess the interactive effects of these metabolites on the myofibrillar ATPase activity (Fig. 7B). Condition A, which uses metabolite levels representative of control muscle (pH 7.0, 5 mM ATP, 1 mM Pi), results in an activity of about 420 nmol mg-I protein - min-I. Decreasing the pH to 6.5 results in a decrease of about 35 % of the ATPase activity (condition B). Increasing the Pi concentration to 10 mM while maintaining control levels of ATP and pH resulted in about a 55 % drop in ATPase activity (condition C) . However, decreasing pH to 6.5 in addition to increasing the Pi content to 10 mM (condition D) resulted in no further lowering of the ATPase activity. Similarly, an additional decrease of ATP to 2.5 mM in addition to the changes in pH and Pi (condition E) did not further reduce the ATPase activity. Similarly to conditions C, D, and E, a fiarther reduction in pH and ATB had no fierther effect on rnyofibrillar ATPase depression (conditions G and H) than merely increasing Pi content to 20 mM alone (condition F) .
+
Biseussion Vakdity of the mg)ofibri&&ar ATPase assay The coupled assay system gave similar CaD sensitivities, CaD cooperativities, and maximal hydrolysis rates (albeit higher) to those obtained by the end-point assay system (Fig. 1). It is apparent that the coupled assay system buffered any ATP declines and ADP accumulations while minimizing the Pi increases (Figs. 2 -4). In contrast, the end-point assay results in large changes in these three metabolites. Accumulations of
lactate and protons would be stoichismetrically matched to the increased Pi levels in the coupled assay system. Ionic strength greatly affected the myofibrillar ATPase activity in the endpoint assay but had no effect on the coupled assay system. This finding, plus that of a steady hydrolysis rate with time as opposed to the oscillatory pattern observed in the end-point assay (Fig. 2), suggests that the lower protein content in the coupled assay serves to reduce the problems associated with its lack of filament arrays. Myofihrillar ATPase activity The decreased hydrolysis rate at higher Pi and proton concentrations and lower ATP levels implies that either fewer crossbridges are mobilized or cycling, or that the crossbridges cycle more slowly. The decreased hydrolysis rate was not due to a partial activation by Ca2+ because a further increase in Ca2' to pCa 4.0 did not increase the hydrolysis rate. The decreased hydrolysis rates in the presence of increased Pi do not appear to be related to changes in ionic strength, as altering the assay medium from 58 to 150 mM KC1 and including 40 mM lactate had no effect on ATPase activities. The combination of increased proton and Pi levels plus decreased ATP were ineffective in suppressing the myofibrillar ATPase activity to a greater extent than that produced by increased Pi alone. The concentrations of metabolites used in this study, with the exception of reducing pH to 6.0 and ATP to 0.5 mM to illustrate an extreme situation, reflect those values found in exercised fish (Table 1). Maximal myofibrillar ATPase activity under optimal (control) conditions resulted in activity of about 400 nmol . mg-I protein = min-l, which is similar to the values reported for fish white muscle by Johnston (1982) but 3.5 -4 times that reported for rat plantaris muscle (Bellcastro et al. 1988) when corrected for temperature (assuming a Qlo of 2). Eflects of ATP &c&iaes on rnj~ofibri&&ar ATPnse activity The shortening velocity of skinned fiber preparations is generally unaffected until ATP levels are less than 1 mM whereas the myofibrillar ATPase activity of the isolated carp myofibrils was decreased by 35 % at 2.5 mM ATP. In contrast, rat plantaris myofibrillar ATPase was relatively unaffected by a reduction of ATP to 2.5 mM (W. % . Parkhouse, unpublished observations). This finding may reflect the observation that fish white muscle does not maintain ATP levels to the same degree as mammalian muscle during increased metabolic demands such as exercise (Parkhouse et al. 1988; Dobson et al. 1987; Dawson et al. 1978). It appears that ATP may be a novel effector of fish white muscle myofibrillar ATPase activity. Efects of Pi on ATP knydrolj~sisrates Pi in muscle cells has been shown to increase in concentration as a result of fatiguing contractions (Dawson et al. 1978; Wilkie 1981; Parkhouse et al. 1988). Although Pi has consistently been found to decrease isometric tension in skinned fibers (Herzig et al. 1981; Altringham and Johnston 1985; Cooke and Pate 1985; Godt et al. 1985; Dawson et al. 1986; Kawai and Guth 1986; Kentish 1986; Kawai et al. 1987; Nosek et al. 1987; Brozovich et al. 1988), conflicting data exist regarding its effect on ATP hydrolysis rates. In the present study, myofibrillar ATPase activity was inhibited at a relatively low level of Pi (10 mM), with no hrther suppression of activity occurring beyond 20 mM Pi (Fig. 7A). Similarly to this study, Pi suppression of ATPase rate did not
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saturate over the 6 - 14 rnM range (Kawai et al. 1987). In that study, the Pi-mediated decrease in ATPase rate was less than the decrease in tension. These levels are frequently seen in active skeletal muscle and would therefore impart some regulation on the rate of ATP turnover (Dawson et al. 1978; Wilkie 1981). The inhibitory action of Pi on isolated myofibrillar ATPase activities supports some previous findings that phosphate inhibits the ATPase activity of isometric skinned fibers (Kawai et al. 1987). However, others (Altringham and Johnston 1985; Cooke and Pate 1985; Chase and Kushmerick 1988; Luney and Godt 1986) observed no effect, while Herzig et al. (1981) found an increased effect of Pi on the maximal velocity of shortening at neutral pH.
Eficts of lactate and pH on rnyofibriilar ATPase activity During high-intensity exercise requiring anaerobic glycolytic energy production, lactate accumulation and proton production are stoichiometrically matched. This accumulation of lactate anions and protons could potentially reduce the rate of ATP hydrolysis by the myofibrillar ATPase. The present study found no effect of lactate on myofibrdlar ATPase activity. This finding is in agreement with Chase and Kushmerick (1988) who similarly found no effect of 58 mM lactate on the velocity of unloaded shortening in either rabbit psoas or soleus muscle. In contrast, proton-induced altered Ca2+ kinetics and decreased crossbridge cycling rates are proposed to contribute to the reduced tension and decreased shortening velocities, respectively (Fabiato and Fabiato 1978; Blanchard et a%.1984; Metzger and Moss 1987; Chase and Kushmerick 1988). In the present investigation, despite maximal activating Ca2+ levels, myofibrillar ATPase activity was reduced in the presence of increased proton levels. This finding is in agreement with the findings of Chase and Kushmerick (1988), Metzger and Moss (1987), and Pate et ale (1987), who found shortening velocity of skinned fibers to increase as pH was increased over the physiological pH range. BBanchard et al. (1984) also found maximal ATPase activity of isolated rabbit psoas muscle myofibrils to be depressed by 16- 15 % at pH 6.5 whereas the present investigation found ATPase activity to be lowered by 35% under similar pH conditions. Furthermore, Blanchard et al. (1984) found no further suppression of activity at pH 6.2. These differences may be attributed to the higher glycoliytic capacity and ultimately proton-producing capacity of the carp white muscle as opposed to rabbit psoas muscle. Chase and Kushmerick (1988) and Metzger and Moss (1987) similarly found shortening velocities of fast muscles to be more affected by acidic pH than slow fibers. Eflects of pH and Pion myofibrillar ATPase activiv Of further interest are the interactive effects of both increasing proton and increasing Pi levels in terms of myofibrillar ATPase activity. Complicating the interpretation of these data is the fact that decreasing pH results in an increased content of the diprotonated form of Pi. The pK of Pi is a b u t 6.81 (Gadian et al. 1981), and at a physiological pH, Pi is relatively equally distributed between the di- and mono-protonated forms. However, as the pH drops with the onset of fatigue, a greater amount of the Pi within the muscle cell would be converted to the diprotonated form (Dawson et al. 1978). In the present study the reduction in ATPase activity was unrelated to the increasing content of H2PO4-. and the effects of pH and Pi on ATPase activity did not appear to be additive. This latter finding contrasts with the findings of Chase and Kushmerick (1988) in terms of the additive effects of pH and Pi on
shortening velocities at pH 6.0, However, in that study the additive effect was only about 10%, resulting in a find suppression of shortening velocity by about 50%. The additive effect may have been dampened in this study because of the relatively greater effect of Pi alone on myofibrillar ATPase activity. Of metabolic importance is the suggestion that Pi and protons, in addition to calcium, may serve to regulate myofibrillar ATPase hydrolysis rates. In conclusion, it appears that mysfibrillar ATPase activity is inhibited by increasing proton and Pi levels and reduced substrate (ATP) availability. This reduction in myofibrillar ATPase activity may decrease shortening velocity in exercising muscle and may serve to balance ATP hydrolysis rates to ATP production rates.
This work was supported by Naturd Sciences and Engineering Research Council of Canada operating grant. W.S.P. is gratefbl to Ian MacLean for his fine technical assistance. Altringham, J. D., and Johnston, I. A. 1985. Effects of phosphate on the contractile properties of fast and slow muscle fibers from antarctic fish. J. Physiol. (Lond.), 368: 491 -500. Belcastro, A. N., Turcotte, W., Rossiter, M., et a/. 1984. Myofibril ATPase activity of cardiac and skeletal muscle of exhaustively exercised rats. Ent. J. Biochem. 16: 297 - 303. Belcastro, A. N., Parkhouse, W. S., Dobson, G . P., and Gilchrist, J. S. 1988. Influence of exercise on cardiac and skeletal muscle myofibrillar proteins. Mol. Cell. Biochem. 83: 27 - 36. Bers, D. M . 1982. A simple method for accurate determination of free calcium in CaD-EGTA solutions. Am. J. PhysioI. 242: C404 - C408. Blanchard, E. M., Ban, B. S . , and Solaro, W. J. 1984. The effect of acidic pH on the ATPase activity and troponin Ca" 'binding of rabbit skeletal myofilaments. J. Biol. Chem. 259: 3 181- 3 186. Brozovich, F. V . , Yates, L. D., and Gordon, A. M . 1988. Muscle force and stiffness during activation and relaxation: implications for the actomyosin ATPase. J. Gen. Physiol. 91: 399-420. Chase, P. B., and Kushmerick, M. %.1988. Effects of pH on contraction of rabbit fast and slow skeletal muscle fibers. Biophys. J. 53: 935 - 946. Csoke, R., and Pate, E. 1985. The effects of ADP and phosphate on the contraction of muscle fibers. Biophys. J. 48:789-7923. Dawson, M. J., Gadian, D. G . , and Wilkie, D. Ha. 1978. Muscular fatigue investigated by phosphorus nuclear magnetic resonance. Nature (Lond.), 274: 861 - 866. Dawson, M. J., Smith, S., and Wilkie, D. R. 1986. The H2P04may determine cross-bridge cycling rate and force production in living fatiguing muscle. Biophys. J. 49: 268a. Dobson, G. P., Parkhouse, W. S., and Hochachka, P. W. 1987. Regulation of anaerobic ATP generating pathways in trout fasttwitch muscle. Am. J. Physiol. 253: W184-R194. Dridzic, W. R.,and Hochachka, P. W. 19916. Control of energy metabolism in fish white muscle. Am. J. Physiol. 236): 579 -582. Fabiato, A., and Fabiato, F. 1978. Effects of pH on the rnyofilaments and the sarcoplasmic reticulum of skinned cells from cardiac and skeletal muscles. S. Physiol. (Lond.), 296: 233 -255. Fitts, R. H., and Holloszy, S. 0. 19916. Lactate and contractile force in frog muscle during development of fatigue and recovery. Am. J. Physiol. 231: 430-433. Gadian, D. G., Radda, G. K . , Brown, T. K., et a/. 1981. The activity of creatine kinase in frog skeletal muscle studied by saturation-transfer nuclear magnetic resonance. Biochem. J. 194: 215 -228. G d t , R. E., Fender, K. J., Shirley, G. C., and Nosek, T. M. 1985. Contractile failure with fatigue or hypoxia: studies with skinned skeletal and cardiac muscle fibers. Biophys. %.47: 293n.
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Herzig, J. W., Peterson, J. W., Ruegg, J. C., and Solaro, R. J. 1981. Vanadate and phosphate ions reduce tension and increase crossbridge kinetics in chemically skinned heart muscle. Biochim. Biophys. Acta, 672: 191 - 196. Johnston, I. A. 1982. Biochemistry of myosins and contractile properties of fish skeletal muscle. Mol. Physiol. 2: 15 -29. Kawai, M . , and Guth, K. 1984. ATP hydrolysis rate and crossbridge kinetics in chemically skinned rabbit psoas fibers. Bisphy s. J. 49: 9ca. Kawai, M., Guth, K.. Winnikes, K., st al. 1987. The effect of inorganic phosphate on ATP hydrolysis rate and the tension transients in chemically skinned rabbit pssas fibers. Pfluegers Arch. 408: H -9. Kentish, J. C. 1984. The effects of inorganic phosphate and creatine phosphate on force production in skinned muscle from rat ventricle. J. Physiol. (Lond.), 370: 585 -604. Lowry, 0.H., Rosebrough, N. J., Harr, A. L., and Randall, A. J. 1951. Protein measurement with the folin phenol reagent. J. Biol. Chem. 893: 265 -275. Luney , D. J. E., and Gsdt , R. E. 1986. Fatigue and maximal velocity of shortening in skinned fibers from rabbit soleus muscle: effects of pH, ADP and inorganic phosphate. Biophys. J. 49: 85a. Metzer, J . M., md Moss, R. L. 1987. Greater hydrogen ion-induced depression of tension and velocity in skinned single fibers of rat fast and slow muscles. J. Bhysiol. (Lond.), 393: 72%-742. hfilligan, C. L . , and Wood, C. M. 1986. IntracelHular and extracellular acid-base status and H+ exchange with the environment after exhaustive exercise in rainbow trout. J. Exp. Biol. 123: 93-121. Nosek, T. M*, Fender, K. Y., and Godt, R. E. 8987. It is diprotonated inorganic phosphate that depresses force in skinned muscle fibers. Science Washington, D.C.), 236: 191 - 193.
Parkhouse, W. S., Dobssn, G. B., Belcastro, A. N., and Hochachka, P. W. 1987. The role sf intermediary metabolism in the maintenance of proton and charge balance during exercise. Mol. Cell. Bioehern. 77: 37 -47. Parkhouse, W. S., Dobson, G. 9.. and Hochachka, P. W. 1988. Organization of energy provision in rainbow trout during exercise. Am. J. Physiol. 254: R302-8309. Bate, E., Franks, K., Lucianai, G., and Cooke, W. 1987. The irhibition s f muscle contraction by the products of ATB hydrolysis. Biophys. J. 51: 4ca. Pierce, G. N . , and Bhalla, N. S. 1985. Mechanisms of the defect in cardiac myofibrillar function during diabetes. Am. J. Physiol. 248: EH70-E175. Sahlin, K., Edstrom, L., Sjoholm, H., and Hultman, E. 1981. Effects s f lactic acid accumulation and ATP decrease on muscle tension and relaxation. Am. J. Physiol. 240: 42121 -C126. Spriet, L. 1991 . Phosphofn~ctokinaseactivity and acidosis during short-term tetanic contractions. Can. J. Physiol. Pharrnacol. 69: 298 - 304. Tate, C. A., Van Winkle, W., and Entman, M. E. 1980. Timedependent resistance to alkaline pH of oxaiate-supported calcium uptake by sarcoplasmic reticulum. Life Sci. 27: 1453- 1464. Taussky, H. PI., and Shorr, 8 . 2953. A micro-colorimetric method determination of inorganic phosphorus. J. Biol. @hem. 202: 675 -685. Wilkie, B. 1981. Shortage of chemical he1 as a cause of fatigue: studies by nuclear magnetic resonance and bicycle ergometry. In Human muscle fatigue: physiological mechanisms. Ciba Syrnp. $2: 102- 119.
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Received April 24, 1991 PARKHOUSE, W" S. 1992. The effects s f ATP, inorganic phosphate, protons, and lactate on isolated myofibrillar ATBase activity. Can. J. Physiol. Pharmacol. 70: 1 175- H 181. The purpose of this study was to examine the effects sf lactate, protons, inorganic phosphate, and ATP on myofibrillar ATPase activity. Myofibrils were isolated from carp (Cyprinius carpks L.) fast-twitch white muscle, and myofibrillar ATPase activities were assessed under maximal activating calcium levels (pCa 4.0) at 10°C in reaction media containing metabolic profiles similar to those seen in fatiguing muscles. The Ca2'-activated ATPase activity was assessed by an ATP regenerating assay that csupled the myofibrillar ATPase to pymvate kinase and lactate dehydrogenase. This assay allowed the effects of ATP, inorganic phosphate, protons, and lactate on myofibrillar ATPase activity to be assessed. The coupled assay was found to give similar myofibrillar ATPase kinetics, with the exception of higher maximal activities, to those seen with a standard end-point assay. Myofibrillar ATPase activity was depressed by 35% when ATP concentrations were lowered to 2.5 mM. Lowering ATP levels to 0.5 mM reduced the myofibrillar ATPase activities by 85 %. Lactate had no effect on myofibrillar ATBase activities. Inorganic phosphate levels up to about 20 mM significantly decreased the myofibrillar ATPase activities, after which further increases in inorganic phosphate content had minimal effects. The changes in ATPase activities were related to total inorganic phosphate, not ts the content of diprotonated inorganic phosphate. Myofibrillar ATPase activity was highest at pH 7.5 and lowest at pH 6.0. The interactive effects of low ATP, decreased pH, and high inorganic phosphate levels were not additive, giving similar decreases in activity to those produced by increased inorganic phosphate levels alone. These results suggest that alterations in metabolites accompanying exercise may regulate myofibrillar ATPase activity and contribute to fatigue by inhibiting myofibrillar ATP hydrolysis rates. Key words: fatigue, muscle, ATPase, fish, inorganic phosphate. -
PARKHOUSE, W S . 1992. The effects of ATP, inorganic phosphate, protons, and lactate on isolated myofibrillar ATPase activity. Can. J. Physiol. Pharmacol. 78 : 1175- 1181. Le but de cette Ctude a CtC d'examiner les effets du lactate. des protons, du phosphate inorganique et de 1'ATP sur l'activiti de 1'ATPase myofibrillaire. On a is016 des myofibrilles du muscle blanc 2i contraction rapide de la c a p e (Cygrisaius carpis L.) et CvaluC les activitCs de l'ATPase dans des conditions de taux de calcium d'activation maximale (pCA 4,0), h BO°C, dans des milieux reproduisant des conditions mdtaboliques similaires B celles des muscles en fatigue. On a 6valuC l'activitd d'ATPase activCe par le Ca2+ en recourant h un dosage regCnCrateur d7ATP, lequel a coup16 l'activit6 d'ATPase myofibrillaire h la pyruvate kinase et h la 1acticodCshydrogCmse. Ce dosage a permis d'dvaluer les effets de 19ATP,du phosphate inorganique, des protons et du lactate sur l'activitC dqATPasemyofibrillaire. I1 a donnk des cinCtiques d' ATPase myofibrillaire similaires, sauf pour ce qui est des activitCs maximales plus ClevCes. LqactivitCd'ATPase myofibrillaire a diminuC de 35% lorsque les concentrations dvATPont CtC abaissdes 2i 2,5 mM et de 85 % lorsque les concentrations snt Ctk rduites h 0,5mM. Le lactate n'a pas eu d9effetsur les activitCs d' ATPase myofibrillaire. Des taux de phosphate inorganique pouvant aller jusqu'h 20 mM ont significativernent rCduit les activitCs d'ATPase myofibrillaire; d'autres augmentations de la teneur en phosphate inorganique n'ont eu que des effets minimes. Les variations des activitCs dvATPase ont CtC relikes h la teneur totale en phosphate inorganique et non pas h la teneur en phosphate inorganique diprotonk. L'activitC d'ATPase myofibrillaire a kt6 maximale au pH 7,5 et a diminuC B des activitks minimales au pH 6,O. Les effets interactifs d'une faible teneur en ATP, d'un pH rCduit et de bux ClevCs de phosphate inorganique n'ont pas CtC additifs, donnant des diminutions d'activite sirnilaires B celles provoqutes par les augmentations de phosphate inorganique uniquement. Ces rCsultats suggkrent que des altCrations au niveau des rnCtabolites lors de l'exercice pourraient rCguler l'activitt d'ATPase myofibrillaire et contribuer h la fatigue par l'inhibition des taux d9hydrolyse le 1'ATP myofibrillaire. Mots cI&s : fatigue, muscle, ATPase, poisson, phosphate inorganique.
Introduction Many investigators have examined the relationships between fatiguing muscle preparations and the accumulation of metabolic end products (Kentish 1986; Fitts and Holloszy 1974; Sahlin et al. 1981; Parkhouse et id. 1988). In these studies, decreases in ATP levels and the accumulation of lactate, prstons, and inorganic phosphate (Pi; both total and diprstonated) have been suggested to (i) inhibit the energy-yielding pathways (Spriet 199B), ( i i ) result in large ionic imbalances that would affect membrane potentids (Parkhouse et al. 1987), (iii) compete with calcium for the calcium binding sites on troponin-@ (Fabiato and Fabiato 1978; Blanchard et al. 1984), (iv) reduce the rate and (or) amount of calcium released and sequestered by the sareoplasmic reticulum (Fabiato and Fabiato 1978), and (v) alter crossbridge kinetics (Kawai et al. Printed in Canada i ImprirnC au Canada
1987; Metzer and Moss 1987; Chase and Kushmerick 1988; Brozovich et al. 1988). Most of these investigations have utilized whole muscle, skinned fibers, or whole organism models to examine the potential causitive relationships between these metabolites and skeletal muscle fatigue. The relationship between these metabolites and mysfibrillar kinetics can only be hypothesized, since these altered metabolite levels also have effects on other cellular functions. Examining the effects of these metabolites on the isolated myofibrillar complex may provide insight into their interrelationships in the fatigue process. However, it is recognized that upon addition sf ATP, isolated myofibrils form a structure that could loosely be termed a logjam s f filaments, and that the ATPase activity of this undefined structure lie somewhere between that of purified actin and myosin and that
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Time (min) PIG. 2. Typical Ca2+-stimulated (pCa 4.0) myofibrillar ATPase activities of the coupled (C) and end-point (E) assay systems calculated every 30 s for a single isolation of carp myofibrils.
Fro. 1. (A) Carp white muscle Ca2+-stimulated myofibrillar ATPase activities at 10°C, pH 7.0, and 5 mM ATP, over the pCa range 5-8 for the end-point (E) and coupled (C) assay systems. (B) Will plot of the Ca2+-stimulated rnyofibrillar ATPase activities of the E and C assay systems.
of more intact fiber systems. Acknowledging these discrepancies, these structures allow myofibrillar ATPase kinetics to be probed in an intact stable preparation containing the contractile regulatory proteins. Therefore, the purpose of this investigation was to examine the effects s f lactate, protons, Pi, and ATP on myofibrdlar ATPase activity.
Methods Animnks and muscle san~pllng Carp (Cytbas'nims cargio L.) were obtained from a local supplier (Sun Valley Trout F a m , Mission, B.C.) and were kept in fresh, aerated, dechlorinated tap water at 10- 12'C prior to the experiments. Fish were sacrificed by decapitation, and the white muscle was rapidly excised from a site posterior to the dorsal fin and freezeclamped with aluminum tongs cooled in liquid nitrogen. Samples were stored at -80°C until analysis. Myofibrkblar isolarion The fish white muscle was homogenized in 20 volumes of ice-cold relaxing buffer (39 mM sodium borate; 25 mM KC1; 5 mM EDTA; pH 7.1) for 2 X 10 s with a tissue homogenizer (Polytron PT-10) at 80% of maximal speed. This buffer was designed to relax the muscle by chelating any calcium and magnesium present in the preparation. The honmogenate suspension was then centrifuged at 1000 x g for 12 min at 4°C and the supernatant discarded. The myofibrillar pellet was then resuspended in the relaxing buffer and the procedure repeated. The resulting myofibrillar pellet was subjected to two further resuspensions and centrifugations under identical conditions, with the exception that EDTA was excluded from the ice-cold buffer (39 mM sodium borate; 25 mM KC1; pH 7.1). These procedures were found to be sufficient to remove any remining EDTA. The myofibrillar pellet was subjected to another two resuspensions and
centrifugations at 1000 x g for 12 min at 4'C in a medium consisting of 50 mM Tris, 5 mM sodium azide (NaN,), 100 mM KC1, and 0-5% Triton X-100, pH 7.4. Sodium azide and Triton X-100 were included to inhibit mitochondria1 ATPase and to remove the sarcoplasmic reticulum and ~rcolemmalATPase, respectively. This procedure was sufficient for removal of contaminating ATPases, since no differences were observed in ATPase activities when sodium azide or ouabain was included in the assay media. The myofibrillar pellet was resuspended in a final ice-cold suspension medium consisting of 150 mM KC1 and 50 mM Tris, pH 7.4, to remove any traces of sodium azide or Triton X-BOO. The suspension was then centrifuged as described previously. The myofibrillar pellet was then resuspended in 10 volumes of suspension medium and passed through glass wool to remove any large particles or clumps of myofibrils. Passing the suspension through the glass wool resulted in a loss of 20 -40% of the protein yield. Aliquots (58 p L ) of suspended myofibrillar protein were taken in duplicate for protein determination by the method of Lowry et al. (1951), and all protein concentrations are expressed relative to wet muscle. Myofibrillar ATPase activity The norm1 myofibrillar ATPase assay (Belcastro et al. 1984; Pierce and Dhalla 1985) uses a large amount of protein and measures the accumulation of P, over a given time period (usually 2 - 5 min) . This procedure will be referred to as the end-point assay and results in the lowering of ATP levels in the medium and accumulation of ADP, making it difficult to assess the effects of these metabolites on the enzyme activity itself. Furthermore, alterations in ionic strength and the addition of ATP are known to affect the ATPase activity and structure of the myofibrillar complex in the end-pont assay. The use of an ATP regenerating assay that couples myofibrillar ATPase to pyruvate b a s e (PK) and lactate dehydrogenase (EDH) maintains ATP levels constant while allowing the use of very low levels of myofibrillar protein, owing to the sensitivity of monitoring NADH production as opposed to P, production. This assay, which will be referred to as the coupled assay, has been used previously to measure sarcoplasrmic reticulum Ca2+ATPase activity (Tate et al. 1988). The validity of this assay for use in the present study was assessed by comparing the results obtained between it and the end-point assay in terms of Ca2+-activated ATPase activity, Ca2+ sensitivities, and Ca2+ cooperativity (Fig. I), and the accumulations of Pi and ADP and declines in ATP as a function of assay time (Figs. 2-4).
PARKHOUSE
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COUPLED
c2
-5 8
to,
Time (rnin)
FIG.3 . Amount of ATP hydrolyzed per assay for the coupled (C) and end-pint (E) assay systems as a function of assay run time. TABLE1. Mean control (C) and exhaustive (E) exercise metabolite levels (para01 . g-') in fish white muscle -
Rainbow trouta
Rainbow troutb
Rainbow troutC
Carp"
ATP C E ADP
e
E pi
C E Lactate C E
PH C E "Data from Parbouse et al. (1988). bData from Dobssn et al. (1987). 'Data from Miliigan and Wood (1 986). dData from Driedzic and Hochachka (1976). 'P, values are corrected for nonspecific hydrolysis of phosphocreatine (Kr)during sampling and freezing, assuming that !XI% of the total creatine pool would be in the form of PCr in control muscle. f ~ a l u e sare calculated based on the decrease in PCr and ATP.
Myofibrillar ATPase activities were assessed every 30 s and the concentrations of ATB and ADP were determined as outlined previously (Parkhouse et al. 1988). Inorganic phosphate levels were not detectable in the regenerating system by colorimetric assay and were calcuHated based on a stoichiometric relationship with ATP declines. The csncentrations of the effectors used reflect exercising values for fish white muscle (Table I). Free Ca2+ concentrations were determined with an Orion calcium-sensitive electrode by the method of Bers (1982). End-point assay Briefly, myofibrillar ATPase activity was determined in a medium containing 50 mM KC1, 20 mM Tris, 1 mM MgC12, and 208500 pglmL of mysfibrillar protein. Following a 5-min preincubatisn period at pCa 4, the reaction was started with 5 mM Mg . ATP. The reaction was terminated at 5 min by adding 20% trichloroacetic acid. The liberated Pi was determined in the protein-free supernatant (Taussky and Shsrr 1953).
Time (min)
FIG.4. Absolute change in ATP, ADP, and P, s f the end-point (E) and coupled (C) assay systems as a function of assay mn time. Coupled assay Mysfibrillar ATPase activity was determined in an ATP regenerating medium containing 50 mM KCB, 20 mM Tris, 1 mM MgCl,, 2 mM phosphoenolpymvate (PEP), 0.08 NABH, 5 mM Mg - ATP, 10 U LDH, and 10 U PK, pH 7.0. This assay medium was incubated in a thermostated cell holder for 5 min and the reaction initiated with 20 pg of myofibrillar protein. The reaction was initiated with protein in place of Mg * ATP, for ease in establishing a stable baseline. However, initiating the assay with Mg ATP resulted in no difference in ATPase activity. The reaction was followed at 340 nm on a Pye Unicam SP8880 spectrophotometer. The initial linear portion of the curve was used to determine maximal ATPase activities at pCa 4. ATPase activity was assessed in the presence and absence of sodium azide and ouabain to test for contamination by mitochondria1 and (or) sarcolemmal ATPases. All ATBase activities were corrected for nonspecific ATP hydrolysis and checked for maximal activating Ca2+ concentrations. As ionic strength has been found to affect myofibrillar ATPase activities measured with the end-point assay, the effects of ionic strength were examined in the coupled assay medium by varying KC1 contents between 56 and 150 mM. Under these conditions, no significant differences were found in the ATPase activities.
-
Data analyses Data are presented as means f SD. A Student's P-testwas used to analyze the differences in myofibrillar ATPase activities between eorresponding values of H,PQ,-. p < 0.05 was considered significant.
Results The two assays resulted in similar Ca2* sensitivities and CaB cooperativities, but the maximal Ca2*-activated ATPase activity was about 25% higher with the coupled system
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Inorganic phosphate (mM) FIG.5. Myofibrillar ATPase activity in response to altered ATP concentrations at maximal activating Ca2+ levels (pCa 4.0).
-
2
-5
0
Condition 0
10
20
30
40
Lactate (mM)
FIG.6. Myofibrillar PhTFPase activity in response to altered lactate (A) and proton (B) concentrations at maximal activating @a2' levels (pCa 4.0).
(Fig. I). In contrast to the end-point assay, the coupled assay demonstrated a linear relationship between Ca2+-activated ATPase activity and time (Fig. 2). Figure 3 shows the large differences in the amounts of ATP hydrolyzed per assay under the two assay systems, with the end-point assay hydrolyzing 10- 15 times more ATP per assay at any given time interval. Figure 4 shows the changes in assay medium ATB, ADP, and
FIG.7. Myofibrillar ATPase activity in response to altered inorganic phosphate (Pi) levels (A) and vari-ioaas conditions of ATP, pH, and Pi (B) at maximal activating Ca2+ levels (pCa 4.0). Condition A, 5 mM ATP, pH 7.8, B mM Pi; condition B, 5 mM ATP, pH 6.5, 1 mM Pi; condition C, 5 mM ATP, pH 7.0, 18 mM Pi; condition B, 5 mM ATPp pH 6.5, BO mM Pi; condition E, 2.5 mM ATP, pH 6.5, 18 mM Pi; condition F, 5 mM ATP, pH 7.0, 20 mM Pi; condition G, 2.5 mM ATP, pH 6.5, 20 mM Pi; condition H, 0.5 mM ATP, pH 6.0, 20 mM Pi.
Pi contents under the two assay conditions as a fbnction s f incubation time. The effects of ATP, lactate, pH, and Pi on myofibrillar ATPase activities are demonstrated in Figs. 5 - 7. The major findings sf this study were that ATPase activities decreased in the face of modest ATP declines; that the declines in ATPase activity were associated with total Pi content, not &Po4-, and that protons also depress the ATPase activity but that this effect is not additive to the Pi effect. Specifically, a reduction in ATP to 2.5 mM resulted in a 35 % decline in maximal Ca2+-activated myofibrillar ATPase activity. A further reduction in the incubation medium's ATB concentration to 0.5 mM was found to reduce the ATBase activity a arther 50%. Therefore, when ATP levels were very low, ATPase activity had decreased by 85 % (Fig. 5). Maximal &la2+-activatedrnysfibrillar ATPase activity was unaffected by increases in lactate concentration to 40 mM (Fig. $A). In contrast, a reduction in pH from 7.0 to 6.5 resulted in about a 35 % loss of ATPase activity (Fig. QB).A further decline in pH to 6.0 was found to reduce the ATPase activity to less than 20% of its activity at pH 7.8 (Fig. 6B).
TABLE2. MyofibriBlar ATPase versus total and diprotonated forms of inorganic phosphate (mean 9 SD)
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hsH
Total Pi CmM)
H2P04(mM>
Activity (rams1 . mg-' protein . min- ')
NOTE:H,PO,- values are calculated based on a pK of 6.81 (Gadian et al. 1981). "Significantly different from a11 other conditions ( p < 0.05).
A 1 mM Pi concentration was found to have no effect on the n~aximal Ca2+-activated my~fibrillar ATPase activity, with the activities still k i n g in the range of 400 nmol - mg-I protein . rnin-l (Fig. 7A). However, increasing the Pi levels to 10 mM resulted in approximately a 55% decline in this ATPase activity (Fig. 7A). A further increase in Pi levels to 20 mM resulted in a final ATPase activity that was less than 28% of its original level (Fig. 7A). Increasing the Pi content from 20 to 40 mM had relatively little effect on the rnyofibrillar ATPase activity. Pi primarily exists in the physiological pH range as either the mono- (HP04") or di-protonated (H2P04-) form. The reduction in ATPase activity was unrelated to changes in the concentration of the dipr~tonatedform (Table 2). At identical concentrations of total Pi (10 mM) but vastly different concentrations of H2PO4- (3.9 and 6.7 mM), myofibrillar ATPase activity was not significantly different. In contrast, at the same concentration of H2PO4- (7.8 mM) and vastly different total Pi concentrations (1 1.6 and 20 mM), the 12 and 72 f 8 nmol . mg-I ATPase activities were 165 min-I (mean f SD). A number of combinations of Pi, ATP, and pH were used to assess the interactive effects of these metabolites on the myofibrillar ATPase activity (Fig. 7B). Condition A, which uses metabolite levels representative of control muscle (pH 7.0, 5 mM ATP, 1 mM Pi), results in an activity of about 420 nmol mg-I protein - min-I. Decreasing the pH to 6.5 results in a decrease of about 35 % of the ATPase activity (condition B). Increasing the Pi concentration to 10 mM while maintaining control levels of ATP and pH resulted in about a 55 % drop in ATPase activity (condition C) . However, decreasing pH to 6.5 in addition to increasing the Pi content to 10 mM (condition D) resulted in no further lowering of the ATPase activity. Similarly, an additional decrease of ATP to 2.5 mM in addition to the changes in pH and Pi (condition E) did not further reduce the ATPase activity. Similarly to conditions C, D, and E, a fiarther reduction in pH and ATB had no fierther effect on rnyofibrillar ATPase depression (conditions G and H) than merely increasing Pi content to 20 mM alone (condition F) .
+
Biseussion Vakdity of the mg)ofibri&&ar ATPase assay The coupled assay system gave similar CaD sensitivities, CaD cooperativities, and maximal hydrolysis rates (albeit higher) to those obtained by the end-point assay system (Fig. 1). It is apparent that the coupled assay system buffered any ATP declines and ADP accumulations while minimizing the Pi increases (Figs. 2 -4). In contrast, the end-point assay results in large changes in these three metabolites. Accumulations of
lactate and protons would be stoichismetrically matched to the increased Pi levels in the coupled assay system. Ionic strength greatly affected the myofibrillar ATPase activity in the endpoint assay but had no effect on the coupled assay system. This finding, plus that of a steady hydrolysis rate with time as opposed to the oscillatory pattern observed in the end-point assay (Fig. 2), suggests that the lower protein content in the coupled assay serves to reduce the problems associated with its lack of filament arrays. Myofihrillar ATPase activity The decreased hydrolysis rate at higher Pi and proton concentrations and lower ATP levels implies that either fewer crossbridges are mobilized or cycling, or that the crossbridges cycle more slowly. The decreased hydrolysis rate was not due to a partial activation by Ca2+ because a further increase in Ca2' to pCa 4.0 did not increase the hydrolysis rate. The decreased hydrolysis rates in the presence of increased Pi do not appear to be related to changes in ionic strength, as altering the assay medium from 58 to 150 mM KC1 and including 40 mM lactate had no effect on ATPase activities. The combination of increased proton and Pi levels plus decreased ATP were ineffective in suppressing the myofibrillar ATPase activity to a greater extent than that produced by increased Pi alone. The concentrations of metabolites used in this study, with the exception of reducing pH to 6.0 and ATP to 0.5 mM to illustrate an extreme situation, reflect those values found in exercised fish (Table 1). Maximal myofibrillar ATPase activity under optimal (control) conditions resulted in activity of about 400 nmol . mg-I protein = min-l, which is similar to the values reported for fish white muscle by Johnston (1982) but 3.5 -4 times that reported for rat plantaris muscle (Bellcastro et al. 1988) when corrected for temperature (assuming a Qlo of 2). Eflects of ATP &c&iaes on rnj~ofibri&&ar ATPnse activity The shortening velocity of skinned fiber preparations is generally unaffected until ATP levels are less than 1 mM whereas the myofibrillar ATPase activity of the isolated carp myofibrils was decreased by 35 % at 2.5 mM ATP. In contrast, rat plantaris myofibrillar ATPase was relatively unaffected by a reduction of ATP to 2.5 mM (W. % . Parkhouse, unpublished observations). This finding may reflect the observation that fish white muscle does not maintain ATP levels to the same degree as mammalian muscle during increased metabolic demands such as exercise (Parkhouse et al. 1988; Dobson et al. 1987; Dawson et al. 1978). It appears that ATP may be a novel effector of fish white muscle myofibrillar ATPase activity. Efects of Pi on ATP knydrolj~sisrates Pi in muscle cells has been shown to increase in concentration as a result of fatiguing contractions (Dawson et al. 1978; Wilkie 1981; Parkhouse et al. 1988). Although Pi has consistently been found to decrease isometric tension in skinned fibers (Herzig et al. 1981; Altringham and Johnston 1985; Cooke and Pate 1985; Godt et al. 1985; Dawson et al. 1986; Kawai and Guth 1986; Kentish 1986; Kawai et al. 1987; Nosek et al. 1987; Brozovich et al. 1988), conflicting data exist regarding its effect on ATP hydrolysis rates. In the present study, myofibrillar ATPase activity was inhibited at a relatively low level of Pi (10 mM), with no hrther suppression of activity occurring beyond 20 mM Pi (Fig. 7A). Similarly to this study, Pi suppression of ATPase rate did not
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CAN. J. PHYSIOL. PHARMACOL. VOL. 70, 1992
saturate over the 6 - 14 rnM range (Kawai et al. 1987). In that study, the Pi-mediated decrease in ATPase rate was less than the decrease in tension. These levels are frequently seen in active skeletal muscle and would therefore impart some regulation on the rate of ATP turnover (Dawson et al. 1978; Wilkie 1981). The inhibitory action of Pi on isolated myofibrillar ATPase activities supports some previous findings that phosphate inhibits the ATPase activity of isometric skinned fibers (Kawai et al. 1987). However, others (Altringham and Johnston 1985; Cooke and Pate 1985; Chase and Kushmerick 1988; Luney and Godt 1986) observed no effect, while Herzig et al. (1981) found an increased effect of Pi on the maximal velocity of shortening at neutral pH.
Eficts of lactate and pH on rnyofibriilar ATPase activity During high-intensity exercise requiring anaerobic glycolytic energy production, lactate accumulation and proton production are stoichiometrically matched. This accumulation of lactate anions and protons could potentially reduce the rate of ATP hydrolysis by the myofibrillar ATPase. The present study found no effect of lactate on myofibrdlar ATPase activity. This finding is in agreement with Chase and Kushmerick (1988) who similarly found no effect of 58 mM lactate on the velocity of unloaded shortening in either rabbit psoas or soleus muscle. In contrast, proton-induced altered Ca2+ kinetics and decreased crossbridge cycling rates are proposed to contribute to the reduced tension and decreased shortening velocities, respectively (Fabiato and Fabiato 1978; Blanchard et a%.1984; Metzger and Moss 1987; Chase and Kushmerick 1988). In the present investigation, despite maximal activating Ca2+ levels, myofibrillar ATPase activity was reduced in the presence of increased proton levels. This finding is in agreement with the findings of Chase and Kushmerick (1988), Metzger and Moss (1987), and Pate et ale (1987), who found shortening velocity of skinned fibers to increase as pH was increased over the physiological pH range. BBanchard et al. (1984) also found maximal ATPase activity of isolated rabbit psoas muscle myofibrils to be depressed by 16- 15 % at pH 6.5 whereas the present investigation found ATPase activity to be lowered by 35% under similar pH conditions. Furthermore, Blanchard et al. (1984) found no further suppression of activity at pH 6.2. These differences may be attributed to the higher glycoliytic capacity and ultimately proton-producing capacity of the carp white muscle as opposed to rabbit psoas muscle. Chase and Kushmerick (1988) and Metzger and Moss (1987) similarly found shortening velocities of fast muscles to be more affected by acidic pH than slow fibers. Eflects of pH and Pion myofibrillar ATPase activiv Of further interest are the interactive effects of both increasing proton and increasing Pi levels in terms of myofibrillar ATPase activity. Complicating the interpretation of these data is the fact that decreasing pH results in an increased content of the diprotonated form of Pi. The pK of Pi is a b u t 6.81 (Gadian et al. 1981), and at a physiological pH, Pi is relatively equally distributed between the di- and mono-protonated forms. However, as the pH drops with the onset of fatigue, a greater amount of the Pi within the muscle cell would be converted to the diprotonated form (Dawson et al. 1978). In the present study the reduction in ATPase activity was unrelated to the increasing content of H2PO4-. and the effects of pH and Pi on ATPase activity did not appear to be additive. This latter finding contrasts with the findings of Chase and Kushmerick (1988) in terms of the additive effects of pH and Pi on
shortening velocities at pH 6.0, However, in that study the additive effect was only about 10%, resulting in a find suppression of shortening velocity by about 50%. The additive effect may have been dampened in this study because of the relatively greater effect of Pi alone on myofibrillar ATPase activity. Of metabolic importance is the suggestion that Pi and protons, in addition to calcium, may serve to regulate myofibrillar ATPase hydrolysis rates. In conclusion, it appears that mysfibrillar ATPase activity is inhibited by increasing proton and Pi levels and reduced substrate (ATP) availability. This reduction in myofibrillar ATPase activity may decrease shortening velocity in exercising muscle and may serve to balance ATP hydrolysis rates to ATP production rates.
This work was supported by Naturd Sciences and Engineering Research Council of Canada operating grant. W.S.P. is gratefbl to Ian MacLean for his fine technical assistance. Altringham, J. D., and Johnston, I. A. 1985. Effects of phosphate on the contractile properties of fast and slow muscle fibers from antarctic fish. J. Physiol. (Lond.), 368: 491 -500. Belcastro, A. N., Turcotte, W., Rossiter, M., et a/. 1984. Myofibril ATPase activity of cardiac and skeletal muscle of exhaustively exercised rats. Ent. J. Biochem. 16: 297 - 303. Belcastro, A. N., Parkhouse, W. S., Dobson, G . P., and Gilchrist, J. S. 1988. Influence of exercise on cardiac and skeletal muscle myofibrillar proteins. Mol. Cell. Biochem. 83: 27 - 36. Bers, D. M . 1982. A simple method for accurate determination of free calcium in CaD-EGTA solutions. Am. J. PhysioI. 242: C404 - C408. Blanchard, E. M., Ban, B. S . , and Solaro, W. J. 1984. The effect of acidic pH on the ATPase activity and troponin Ca" 'binding of rabbit skeletal myofilaments. J. Biol. Chem. 259: 3 181- 3 186. Brozovich, F. V . , Yates, L. D., and Gordon, A. M . 1988. Muscle force and stiffness during activation and relaxation: implications for the actomyosin ATPase. J. Gen. Physiol. 91: 399-420. Chase, P. B., and Kushmerick, M. %.1988. Effects of pH on contraction of rabbit fast and slow skeletal muscle fibers. Biophys. J. 53: 935 - 946. Csoke, R., and Pate, E. 1985. The effects of ADP and phosphate on the contraction of muscle fibers. Biophys. J. 48:789-7923. Dawson, M. J., Gadian, D. G . , and Wilkie, D. Ha. 1978. Muscular fatigue investigated by phosphorus nuclear magnetic resonance. Nature (Lond.), 274: 861 - 866. Dawson, M. J., Smith, S., and Wilkie, D. R. 1986. The H2P04may determine cross-bridge cycling rate and force production in living fatiguing muscle. Biophys. J. 49: 268a. Dobson, G. P., Parkhouse, W. S., and Hochachka, P. W. 1987. Regulation of anaerobic ATP generating pathways in trout fasttwitch muscle. Am. J. Physiol. 253: W184-R194. Dridzic, W. R.,and Hochachka, P. W. 19916. Control of energy metabolism in fish white muscle. Am. J. Physiol. 236): 579 -582. Fabiato, A., and Fabiato, F. 1978. Effects of pH on the rnyofilaments and the sarcoplasmic reticulum of skinned cells from cardiac and skeletal muscles. S. Physiol. (Lond.), 296: 233 -255. Fitts, R. H., and Holloszy, S. 0. 19916. Lactate and contractile force in frog muscle during development of fatigue and recovery. Am. J. Physiol. 231: 430-433. Gadian, D. G., Radda, G. K . , Brown, T. K., et a/. 1981. The activity of creatine kinase in frog skeletal muscle studied by saturation-transfer nuclear magnetic resonance. Biochem. J. 194: 215 -228. G d t , R. E., Fender, K. J., Shirley, G. C., and Nosek, T. M. 1985. Contractile failure with fatigue or hypoxia: studies with skinned skeletal and cardiac muscle fibers. Biophys. %.47: 293n.
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