695

Activation of Contraction and ATPase Activity inIntact and Chemically Skinned Smooth Muscle of Rat

Portal VeinDependence on Ca++ and Muscle Length

Anders Arner and Per HellstrandFrom the Department of Physiology and Biophysics, University of Lund, Lund, Sweden

SUMMARY. The mechanical manifestations of muscle contraction (force development or short-ening) are accompanied by an increased turnover of chemical energy (ATPase activity, JATP)- Inintact rat portal veins activated by high potassium medium to produce graded contractions atdifferent levels of extracellular calcium, a linear dependence of oxygen consumption on force wasfound. The slope of the relation (metabolic tension cost) was higher during early stages thanduring late stages of contraction, possibly reflecting a transient high crossbridge ATP turnoverrate. Chemically skinned (Triton X-100) rat portal vein preparations were used to study the energyturnover of the smooth muscle contractile system under constant activation. In these preparations,JATP increased on activation by calcium in the presence of 1 fiM calmodulin, remained constant formaintained contractures, and decreased promptly on relaxation. Force declined with each repeatedcontraction at optimal calcium level (10™" M), but the relation between force and JATP remainedinvariant and agreed with that of the intact muscle during the transient of high ATP turnover.Calcium activation in the range 10"9 to 10"4 5 M caused a progressively steeper (nonlinear) increasein JATP with force. Length-force relations showed a lower relative force at muscle lengths belowL,, in the intact than in the skinned tissue, possibly indicating depression of excitation-contractioncoupling. The slope of the relation between JATP and force was lower when length was variedthan when calcium was varied. At a length where no external force was produced, the activatedmuscle had a significantly higher JATP than while relaxed (10"9

M Ca++). Together with previousresults showing calcium dependence of the force-velocity relation, the present study indicates aninfluence of calcium on crossbridge kinetics in smooth muscle. (Circ Res 53: 695-702, 1983)

THE relation between force and ATPase activity insmooth muscle is influenced by several factors, someof which may relate to the activation process andsome, possibly, to characteristics of the actin-myosininteraction (cf review by Paul, 1980). During devel-opment of isometric force in K+ contractures in ratportal vein and swine carotid artery, there is a periodof transiently increased energy turnover (Arner andHellstrand, 1980; Hellstrand and Paul, 1983; Paulet al., 1983). These studies correlate with observa-tions of transient variations in the maximal short-ening velocity (Uvelius and Hellstrand, 1980; Dillonet al., 1981). Mechanical and metabolic findings mayreflect a potential for regulation of the kinetics ofcrossbridge interaction which may be unique forsmooth muscle. The development of preparationsdevoid of functional plasma membrane ('chemicalskinning') has opened up new approaches for in-vestigation of the contractile apparatus and its reg-ulation (e.g., Filo et al., 1965; Endo et al., 1977;Gordon, 1978; Saida and Nonomura, 1978; Cassidyet al., 1981; Peterson, 1980, 1982a, 1982b; Sparrowet al., 1981). These studies have shown that skinnedsmooth muscle preparations produce graded iso-

metric force in the range of Ca++ concentrations ofabout 10~7 to 10"5 M, and that the activation isaccompanied by increased phosphorylation of themol wt 20,000 myosin light chains. The Ca++ sen-sitivity is markedly increased by the addition ofexogenous calmodulin, which is involved in theactivation by Ca++ of the myosin light chain kinaseactivity (e.g., Adelstein and Eisenberg, 1980).

The question arises whether Ca++ can influenceaspects of the crossbridge interaction other than thedevelopment of isometric force. A study of the dy-namic mechanical properties of chemically skinnedguinea pig taenia coli and rat portal vein (Amer1982a, 1982b, 1983) showed that the force-velocityrelation of the skinned preparation agrees with thatof the intact muscle and revealed a marked Ca++

dependence of the maximal shortening velocity.This suggests that Ca++ regulates not only the num-ber of activated crossbridges, but, possibly, also,kinetic properties of their interaction. Since eachcrossbridge cycle is supposed to be associated withbreakdown of a specific amount of ATP, both ofthese aspects are reflected by the ATPase activity. Ageneral relationship between actomyosin ATP turn-

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over, shortening speed, and energetic tension costin smooth muscle has been proposed by Riiegg(1971). It is, however, possible that quite differentsteps in the crossbridge interaction cycle are rate-limiting for isometric and isotonic contractile activ-ity. The present study attempts to characterize fur-ther the factors influencing chemomechanical trans-duction in vascular smooth muscle. We have deter-mined ATP turnover of intact and chemicallyskinned rat portal veins under conditions of gradedcontractile activation by Ca++. In addition, thelength-tension relation was studied, as well as theATPase activity in the skinned preparation, whenactive force is changed by changing muscle length.Some of the results have been reported in prelimi-nary form (Hellstrand and Arner, 1982).

Methods

PreparationPortal veins were dissected from male Sprague-Dawley

rats weighing 200-300 g, cut open, and mounted longi-tudinally for isometric force registration under a preloadof about 5.0 mN representing approximate optimal length(Lo). Force was measured by Grass FT03 force transducers,and registrations were made on a Grass polygraph. Allexperiments were done at room temperature (21-23°C).The lengths (5-7 mm) of all muscles were measured atthe end of the experiment by using a dissection microscopeequipped with an ocular micrometer. Wet weight (1-3 mg)was determined by weighing on a Cahn electrobalanceafter brief blotting on filter paper. Metabolic rates arereported normalized to tissue wet weight. Contractile forcewas related to tissue cross-sectional area, calculated as-suming a density of 1.05 mg/mm3.

O2 Consumption of Intact Portal VeinsFor measurement of O2 consumption, the vessels were

mounted in an apparatus consisting of a glass chamberwith an attached polarographic O2 electrode, as describedpreviously (Hellstrand and Paul, 1983), except that astainless steel stopper with an O-ring seal against thechamber was used instead of a glass stopper. The volumeof the chamber used in the present experiments was 0.25ml. Force registration was by means of a 6 X 0 silk threadleaving the chamber via a stainless steel tube (length 32mm, i.d. 0.6 mm) through the stopper on which the musclewas mounted. The chamber contents were stirred by amagnetic stirring bar, and the whole arrangement, includ-ing tubing and solution reservoir, was submersed in cir-culating water for temperature control (22°).

The portal veins were mounted in a Tris-buffered so-lution of the following composition in ITIM: NaCl, 120;KC1, 6.0; MgCl2, 1.2; CaCl2, 2.5; glucose, 11.5; Na2Ca-versenate, 0.026; and tris(hydroxymethyl)aminomethane(Trizma Base, Sigma Chemical Co.) 23, titrated to pH 7.4.To reduce bacterial contamination, penicillin G (100 mg/liter) and streptomycin (300 mg/liter) were added to thesolution. The solution was equilibrated with air. Muscleswere preincubated overnight under 5 mN preload in a 50-ml organ bath before they were mounted in the chamberfor O2 measurements. The following sequence of meas-urements were made.

1. Nominally Ca++-free medium (composition asabove, except that no CaCl2 was added) 15 minutes.

2. High K+, Ca++-free medium (100 HIM NaCl ex-changed for KG) 15 minutes. The muscles remained re-laxed in this medium.

3. Addition of Ca++ by injection of 2-6 1̂ concentratedCaCI2 into the chamber. Measurements continued for 30minutes before the protocol was repeated from step 1 foranother Ca++ concentration (2.5, 0.1, 0.2, 0.3, 0.4, 2.5, 10HIM).

With this protocol, registrations were obtained of basalO2 consumption in the relaxed muscle (step 1), as well ascontinuous record of O2 consumption and isometric forcein the depolarized vein during relaxation (step 2) andcontraction (step 3). The injection of a small amount ofCaCl2 into the chamber caused minimal disturbance ofthe O2 record and allowed a high rime resolution inmeasurement of O2 consumption during the initial stagesof the contracrure. The P07 in the chamber never de-creased below 120 mm Hg. The first conditioning contrac-rure in 2.5 ITIM Ca++ was not used in the subsequentanalysis.

Chemically Skinned PreparationsThe portal veins used in the chemical skinning experi-

ments were mounted in normal medium, as above, exceptthat no antibiotics were added. After a 2-hour accommo-dation, they were chemically skinned by overnight treat-ment with a solution containing 1% Triton X-100 (Gordon,1978; Arner, 1982b). After the detergent treatment, themuscles were rinsed for 2 hours in a detergent-free me-dium before being transferred to a "relaxing solution' ofthe following composition: N-tris(hydroxymethyl)methyl-2-aminomethane sulfonic acid (TES) 30 ITIM; dithioery-thritol, 0.5 ITIM; phosphoenolpyruvate, 5 ITIM; calmodulin,1 /JM (gift from Dr. Eva Thulin, Division of PhysicalChemistry 2, Chemical Centre, University of Lund, Lund,Sweden); and pyruvate kinase, 20 U/ml. Free Mg"1"* andMgATP, 2 and 3.2 ITIM, respectively, except as noted, wereadjusted by adding Na2ATP and MgCl2. Ionic strengthwas adjusted to 0.15 with KC1 and pH to 6.9 with KOH.A free-Ca++ concentration of 10 M was obtainedby adding appropriate amounts of K2CaEGTA andK2EGTA while keeping the total EGTA concentration at 4ITIM. "Activating solutions" of varying Ca++ concentrationswere prepared by mixing the relaxing solution with oneof similar composition but with a Ca++ concentration of10"4 5M. Free ion concentrations and ionic strength of themedia were calculated by means of the method describedby Fabiato and Fabiato (1979) with the equilibrium con-stants given by Fabiato (1981). Ca++ concentrations arereported here as their negative logarithms (pCa). In ex-periments in which sodium azide and ouabain were used,pH was checked after addition of the drugs.

The skinned preparations were mounted in plastic cupsfixed on vertical stands rotating at 0.5 Hz for stirring. Theincubation volume was 0.3 ml. Usually four muscles weremounted simultaneously. The cups were exchanged at 5-or 10-minute intervals, and, at the end of experimentsthey were frozen, together with solution blanks, andstored at —80°C until analysis.

Analysis of ATP BreakdownThe analysis of ATP breakdown was performed essen-

tially as described by Peterson (1980). In principle, theADP released by the muscle into the incubation mediumis rephosphorylated to ATP by the action of the phos-phoenolpyruvate and pyruvate kinase present. As a result,pyruvate is liberated in a stoichiometric relation to the

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rephosphorylated ADP. The assay for pyruvate was per-formed by NADH-linked enzymatic analysis in a FarrandA-4 fluorimeter, as described by Lowry and Passonneau(1972). After the final fluorimeter reading in the pyruvateassay, myokinase (5 U/ml) was added and a furtherreading made to determine AMP (2 ADP produced in themyokinase reaction per AMP present). Usually, AMP re-lease by the muscle accounted for 0 to 5% of the totalobserved ATP breakdown.

StatisticsStatistical evaluations were made by Student's f-test for

paired and unpaired observations. Values are reported asmeans ± SEM, with the number of observations withinparentheses.

ResultsThe intact portal veins used for measurement of

O2 consumption (JOj) were preequilibrated in thebuffer medium overnight. In preliminary experi-ments, where muscles were equilibrated for only 2hours before measurements, active force was higherthan in the present series of experiments, but therelation between force and Jo2 was the same. How-ever, the overnight preincubation was adopted dueto the long duration of the O2 measurements (7-8hours). With this procedure, reproducible Jo2 in re-laxed muscles was obtained throughout the subse-quent metabolic measurements. Jo2 in Ca++-free nor-mal and high K+ medium was 0.091 ± 0.010 and0.091 ± 0.010 jtmol/min per g, respectively (n = 6).The time courses of force and suprabasal Jo, (AJo2 =increase in Jo2 above that in high-K+ Ca++-free me-dium) in contractures elicited by addition of 0.4 and2.5 mM Ca++ are shown in Figure 1. At the lowerCa++ concentration (upper panel), force graduallyincreases during the 30-minute contraction. In con-trast, AJo2 is transiently increased during the first 5-10 minutes, and then declines toward approximately50% of the peak value. On return to Ca++-freenormal medium, force and Jo2 return to baselinelevels. AJo2 could not be followed with certaintyuntil about 5 minutes after the solution exchange.After this point in time, there was no change in Jo2in the relaxed state. The limitation on the timeresolution of Jo2 measurements does not apply tothe initial period of contraction, since Ca wasadded without solution exchange (see Methods). Atthe higher Ca++ concentration (lower panel), forcerises quickly to an early peak value and then declinessomewhat before it is gradually restored. AJo2 showsa transient increase which is more rapid and pro-nounced than at the lower Ca++ level.

Jo2 values can be converted into ATP turnover(JATP) by using a factor of 6.4 (Hellstrand and Paul,1983). hi relaxed muscles, JATP is thus obtained as0.58 ^mol/min per g. Values for suprabasal ]ATP(AJATP) of contracted muscles are shown in Figure 2.The open squares show JATP measured at 5 minutesafter initiation of contraction plotted against theactive stress at this point in time. The open circlesshow data obtained in the stable phase at the end

pmo<mtnig

0.04

Jo,

0 00 — x—' t 5 "tn t

pmolmiring

006"

A J,'o2

, S mln ,

FIGURE 1. Time course of active force and suprabasal O2 consumption(]aj during a relaxation-contraction-relaxation cycle in intact portalvein. Muscle initially in Ca**-free high K+ depolarizing medium andthen contracted by addition of 0.4 mu(upper panel) or Z5 mMflowerpanel) Ca**. Relaxation in Ca**-free normal medium.

of the 30-minute period. For both sets of data, thepoints fall on straight lines with correlation coeffi-cients of 0.99. The slope of the line relating JATP toforce at 5 minutes is 1.8 times that of the line in thestable phase.

The time course of ATP utilization during a sus-tained contracture of a chemically skinned portalvein activated at pCa = 4.5 in the presence of 1 ^Mcalmodulin is shown in Figure 3. The measuredvalues of ATP breakdown (JATP) are placed in themiddle of the measurement periods (crosses). It isseen that activation results in about a 2-fold increasein JATP, compared with the relaxed state, and thatthis increase is maintained for as long as contractioncontinues. When JATP was measured in six musclesduring four subsequent 10-minute periods at pCa =4.5, values of 0.28 ± 0.03, 0.31 ± 0.06, 0.31 ± 0.04,and 0.31 ± 0.04 /xmol/g per min were obtained.There was thus no transient increase in JATP of theskinned muscle during the early stages of contrac-tion. Using the 'Ca-jump' method, the rate of ten-sion development in skinned preparations can beincreased (Peterson, 1982b). The present data do notexclude the possibility that a transient high JATpcould be present under such conditions. It is possiblethat an increased JATP during the development oftension could result from the changing mechanical

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min«g

0 A-

0.3-

0.2-

0 1-

0 0

A JATP

10 15mm

FIGURE 2. Suprabasal ATP turnover (AJATP) calculated from A/o, vs.isometric stress (P) in intact portal veins (oven symbols, n = 6).Squares show values measured at 5 minutes after initiation of con-traction (regression line slope A]A7r/P = 0-0250, r = 0.994), whereasopen circles show values at plateau of contraction (25-30 minutes,A/ATP/P = 0.013Z r = 0.988). Filled circle indicates mean values ofstress and A/ATT in skinned portal veins at pCa = 4.5 (n = 30).

state alone when Ca++ level and myosin phophor-ylation are already optimal.

Although the preparation shown in Figure 3 re-turned to the same JATP on relaxation as that meas-ured before contraction, there was sometimes amarkedly lower JATP in the final relaxation period. Acorresponding finding in hog carotid artery has beenreported by Peterson (1980). Several factors may beresponsible for the ATPase activity measured in therelaxed preparation. The possibility that the assaywas influenced by pyruvate released by the musclewas excluded, since no pyruvate could be detectedwhen the muscles had been incubated in the absenceof phosphoenolpyruvate and pyruvate kinase. Fur-thermore, no ATPase activity was detected afteromission of MgCl2 (free-Mg and MgATP = 0) or

JATP

FIGURE 3. Time course of active stress and ATP turnover (7ATP,crosses) during a relaxation (pCa = 9)—contraction (pCa •= 4.5)—relaxation (pCa = 9) cycle in a skinned portal vein. The marks on thecontinuous force record indicate exchange of incubation cups.

Na2ATP (MgATP = 0) in the incubation media. Thisshows that the substrate for the measured basalATPase activity is MgATP provided by the ATP-regeneraring system in the medium. Since ATPasesother than those of the contractile proteins may bepresent, the mitochondrial inhibitor sodium azide (1and 5 nut) and the Na+/K+ pump inhibitor ouabain(1 MM) were tested. Neither basal (pCa = 9) normaximally activated (pCa = 4.5) JATP could be re-duced with either treatment. No O2 consumptioncould be detected in skinned muscles.

The mean values of isometric force and JATP atpCa = 4.5 obtained in all experiments of the presentinvestigation are inserted into Figure 2 (filled circle,n = 30) to allow a comparison with the intact prep-aration. This point, representing the maximally ac-tivated skinned preparation, falls close to the regres-sion line corresponding to measurements during thetransient of high Jo2 in the intact muscle.

The force developed by the skinned muscles islower than that of the intact muscles, as alreadydiscussed (Arner, 1982b, 1983). The force develop-ment and Ca++ sensitivity in the presence of 1 HMcalmodulin observed in the present study agreedwith those obtained earlier. By electron microscopyof the skinned portal veins fixed in buffered glutar-aldehyde, the preparations were seen to be uniformthroughout, with disrupted cell membranes, nuclei,and mitochondria, indicating that there probablywere no regions of the preparations that had escapedskinning (B. Uvelius, personal communication).

To characterize the conditions for study of theenergetics of the skinned portal vein, JATP and forcemeasurements were made during repeated expo-sures to pCa = 4.5 in four muscles. The results areas shown in Figure 4. As discussed above, basal JATPin pCa = 9 (filled squares) is high, initially, but isseen to settle to a constant value after the first

"ATP

«£*!

FIGURE 4. Repeated contractures in skinned portal veins (n = 4). ]Ajrin relaxed muscles (pCa = 9, thin line on time axis) shown by squares.Stress (P) and JATR during contractures (pCa = 4.5, heavy line on timeaxis) shown by circles and triangles, respectively. Inset shows supra-basal ATP turnover (A/ATP «= /ATP in pCa = 4.5 minus /ATP in pCa =9 after contraction) plotted against stress (P). Line fitted by linearregression, r =» 0.996.

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contraction. Force (filled circles) and JATP duringcontraction (filled triangles) both fall with each sub-sequent contraction. As shown in the inset, supra-basal JATP (AJATP) decreases linearly with force. Theresults show that repeated contractions do not givereproducible force, and hence this investigation wasdesigned to use one contraction in each skinnedmuscle. Based on the results of the experiment, wealso chose consistently to evaluate AJATP relative tothe stable JATP value in the relaxed muscle aftercontraction.

Figure 5 demonstrates the relation between JATPand developed force in the skinned portal vein atdifferent Ca++ levels, as indicated by the pCa valuesinserted in the figure. The experiments were per-formed by stepwise increase in Ca++ at 20-minuteintervals. JATP of the relaxed muscle (pCa = 9) wasobtained after the contraction. The relation betweenforce and JATP is clearly nonlinear. At pCa = 6.25,force is 60%, whereas suprabasal JATP (AJATP) is only23% of the maximal value at pCa = 4.5. Thus, theenergetic cost of tension maintenance is lower in theregion of lower Ca++/force levels.

Force and JATP may be altered, not only by chang-ing levels of activation by Ca++, but, also, by chang-ing muscle length at constant Ca++ level. In the leftpanel of Figure 6 are shown values of active stressin intact portal veins (open circles) mounted at dif-ferent lengths relative to Lo and activated directlyby high K+ medium (2.5 mM Ca++) after a 2-hourpreincubation in normal medium. A clear length

JATP

0.3-

0.2-

0.1

0.0 J

\ 5

i 76

ao

1-4-I6 26

10mm

FIGURE 5. ATP turnover (/A-nJ vs. active stress (P) of skinned prepa-rations (n •= 8-12) at different Ca+* concentrations. The Ca** levelsare shown as pCa.

PmNmen rm>Q

4a 04

L, 04 06

FIGURE 6. Length dependence of active stress (P, open circles, intact;filled circles, skinned portal veins; n = 6) and /ATp (crosses, skinnedportal veins; n = 6). Intact muscles activated in high K* medium, 2.5rnMCa**. Skinned muscles activated at pCa = 4.5. Intact preparationswere mounted at different lengths relative to optimal length (Lo) andthen skinned at the same length, L, denotes shortened length whereno active force was produced. In the right panel, stress values areshown normalized to stress at £«,.• = P < 0.05, •* = P < 0.01, ns =not significantly different.

dependence of active stress above and below LQ isdemonstrated. The active stress at Lo in this serieswas higher than in the experiments shown in Figure2, possibly due to the difference in protocol. Afterthe contractions at the respective length, the muscleswere chemically skinned overnight while beingmaintained at the same length. At 1.3 Lo, passivestress fell by about 50% (from 113 to 67 mN/mm2)during the overnight treatment, but at the othermuscle lengths, it remained constant (at Lo about 14mN/mm2). Determinations of active force at 1.3 Lowere difficult to obtain, due to the high passivestress. Active stress of the skinned muscles activatedat pCa = 4.5 (closed circles, Fig. 6) shows a lengthdependence, although the absolute magnitudes ofstress are lower than in the intact muscles. Therelative reduction of force at lengths below Lo ismore prominent in the intact than the skinned prep-arations, as evident from the normalized length-tension relations shown in the right panel of Figure6. When relaxed at pCa = 9, all groups of skinnedmuscles incubated at the different lengths had sim-ilar JATP (about 0.17 jzmol/min per g). Values ofAJATP at the different muscle lengths are shown bythe crosses in Figure 6. At muscle lengths below Lo,there is a tendency for a reduced AJATP, although nosingle value is significantly different from AJATP atLQ. The reduced force at 1.3 Lo is not associated withreduced AJATP- Instead, the mean value of AJATP ishigher than at Lo, although not significantly differ-ent. One possible explanation for the dissociationbetween force and JATP at 1.3 LQ could be the altereddiffusion conditions at the longer muscle length.The fact that basal JATP was the same as at the otherlengths, and the long incubation periods used,would, however, speak against this possibility. Val-ues of AJATP obtained in muscles activated at shortlengths giving no isometric tension (*L/) are also

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0.2-

0.1

0.0

JATPU H L ,

4.5

4.5

0 10 mNmm2

FIGURE 7. ATP turnover (JATW VS. active stress (P) in relaxed (pCa =9) and contracted (pCa « 4.5) skinned portal veins (n = 8). Thepreparations were first activated at optimal length for force develop-ment (LJ, and then shortened while maintained in contracted state.Measurements of JATP were made at a muscle length at which noactive stress is produced (LJ.

shown in the figure. They do not differ from thevalues obtained at finite muscle length.

The experiments described in Figure 6 were doneon separate sets of muscles at each length, due tothe limitations on reproducibility that were de-scribed, above, in connection with Figure 4. In orderto more accurately detect a possible variation of JATPwith muscle length, the following experiment wasundertaken. Portal veins were contracted at pCa =4.5 for 10 minutes, and then shortened so that theydeveloped no active force, while being kept in theactivating solution for a further 10 minutes. Theresults are shown in Figure 7. Whereas the activatedmuscles had a significantly lower (P < 0.001) JATP atslack length than at Lo, JATP in the relaxed state atpCa = 9 was lower still (P < 0.001). This demon-strates that muscle length influences JATP, as well asactive force, but that reduction of force developmentby shortening the muscle is not sufficient to reduceJATP at maximal activation to that of the relaxedstate.

DiscussionA linear dependence of energy turnover on iso-

metric force has been a consistent finding in intactstriated and smooth muscles (Paul, 1980). In theintact rat portal vein, the slope of the relation be-tween Jo2 and force is dependent on the mechanicalcharacteristics of contraction (Hellstrand, 1977). Inphasic spontaneous contractions of short duration,the relation was steeper than in tonic high K+-

induced contractures. In later investigations by Ar-ner and Hellstrand (1980) and by Hellstrand andPaul (1983), the initial stages of a maintained highK+ contracture were shown to be associated with ahigher rate of ATP turnover than were the laterstages. Similarly, a lower maximal shortening veloc-ity (Vmax) in maintained vs. phasic contractions wasdemonstrated by Uvelius and Hellstrand (1980). Apossible mechanism to account for this variability inshortening velocity and tension-dependent metab-olism is that the rate of crossbridge turnover cansomehow be regulated in the smooth muscle, allow-ing a specific force maintenance to be associatedwith different rates of ATP turnover. Murphy andassociates (Dillon et al., 1981) have suggested thatthe rate of crossbridge turnover is regulated by thestate of phosphorylation of the mol wt 20,000 my-osin light chains. The myosin light chain kinasewhich catalyzes the phosphorylation process is reg-ulated by the Ca++-calmodulin complex (e.g., Adel-stein and Eisenberg, 1980). It is thus possible thatCa++ can be involved in the regulation of crossbridgecycling rate, not simply as an on-off switch, but ina more complex way. An influence of Ca++ oncrossbridge cycling rate is suggested by the findingthat the maximal shortening velocity of the skinnedportal vein increases with increasing Ca++ (Arner,1983).

The purpose here was to investigate aspects ofcrossbridge turnover estimated from the ATP fluxand isometric force. Transients in Jo2 were demon-strated during slowly developing contractures elic-ited in depolarized portal veins at 22°C by additionof different amounts of extracellular Ca++ (Fig. 1).These findings correspond to those previously re-ported (Hellstrand and Paul, 1983), although thetime course is slower, due to the lower temperature.The metabolic tension cost estimated at peak andsteady state JOj differed by a factor of 1.8 (Fig. 2).This difference in tension cost corresponds well tothe observed variation in V,™,* during the course ofcontraction (Uvelius and Hellstrand, 1980; Dillon etal., 1981). The experiments on skinned muscles re-ported here could bear on the possibility that intra-cellular Ca++ variation is a mechanism underlyingthe alterations in mechanical and metabolic rates. Inchemically skinned portal veins, activated at con-stant Ca++ force and JATP both remained at constantlevels throughout the contraction (Fig. 3). The force-velocity relation has also been shown to be the sameat early and late stages of the force plateau in theskinned portal vein (Arner, 1983). Thus, when Ca++

is held constant, which is feasible in the skinnedpreparation, no transients in mechanical or meta-bolic kinetics are detected.

Despite reduced force with repeated contractions,which is a common problem in studies on skinnedsmooth muscles, basal JATP (after the first contrac-tion) as well as the relation between AJATP and force(tension cost), remained invariant (Fig. 4). Basal JATP

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was about one-third of that estimated from Jo2in the intact preparation. The cause of the basalATPase activity in the skinned relaxed muscle isunknown. Since azide or ouabain did not reducethis ATP turnover, it is unlikely that mitochondrialor Na+/K+ pump activity contributes. The elevatedJATP of some muscles in relaxing solution initiallyafter skinning suggests that some energy-dependentprocess, perhaps related to Ca++ handling, is oper-ating but is successively inhibited. The recent dem-onstration of regenerative Ca^ release in prepara-tions briefly skinned with saponin (Haeusler et al.,1981; Saida, 1982) suggests a mechanism for Ca++

sequestration, although the present overnight treat-ment with Triton X-100 would be expected to de-stroy intracellular Ca++ stores much more drasti-cally.

A nonlinear relation between JATP and active stressappears when Ca++ is varied in the skinned prepa-ration (Fig. 5). A similar dissociation between JATPand force at supramaximal Ca++ seems to be presentin skinned taenia coli, as recently reported (Guthand Mrwa, 1982; Hellstrand and Arner, 1983). Sev-eral processes may contribute to the ATPase acti-vation by increasing Ca++. If myosin-actin interac-tion is the main site of ATP turnover, the resultssuggest a nonlinear relation between total cross-bridge turnover and developed force. At higherCa+ , each crossbridge cycle would then be lessefficient in producing force, perhaps as a result ofan increased cycling rate. In addition to crossbridgeinteraction, however, energy-dependent Ca++ trans-location, as discussed above, may occur when Ca++

is added. It is notable that a significant accumulationof Ca++ into mitochondria of saponin-skinned rabbitportal-anterior mesenteric vein at pCa = 5 has beenfound by Somlyo et al. (1982) using electron probeanalysis. At pCa = 6, which gave almost maximalforce, no such Ca++ accumulation was seen. Thiswould seem to be a less likely explanation for theCa++ -induced ATPase in the present Triton-skinnedpreparations. Moreover, azide did not reduce JATP ofmuscles activated at pCa = 4.5. Phosphorylation ofmuscle proteins is another possible cause of ATPusage. Based on myosin content of the portal veinand the rate of dephosphorylation estimated in themodel study on taenia coli by Peterson (1982a), therate of ATP usage associated with phosphorylation-dephosphorylation of myosin at steady state is onthe order of 0.003 jtmol/min per g, and thus negli-gible in this context. An interesting conclusion to bedrawn from the curvilinear relation between JATPand force with varied Ca++ is that the decreasedforce in repeated contractions is unlikely to becaused by Ca++ desensitization, since the AJATP/Prelation is linear when force decreases in successivecontractions (Fig. 4). The force and AJATp obtainedin our total material of skinned portal veins maxi-mally activated at pCa = 4.5 falls close to the regres-sion line representing the relation between JATp and

force in the living muscle early in the contractionduring the transient of high energy turnover (Fig.2). The contractile energetics of the skinned musclewhen maximally activated by Ca++ thus correspondsto the peak rather than to the steady state metabolicturnover rate during a sustained contracture in theliving muscle. Together with the curvilinear relationbetween force and JATP (Fig. 5), this could agree withthe possibility that variations in intracellular Ca++

are responsible for the observed variability in met-abolic and mechanical kinetics via an influence oncrossbridge turnover rate.

The structural basis of the length-force relation insmooth muscle is as yet unclear, although its generalsimilarity to that of skeletal muscle implies a slidingfilament mechanism of contraction (Murphy, 1980).Figure 6 (right panel) demonstrates that the skinnedportal vein at lengths below U is capable of devel-oping higheT relative force than the intact muscle atthe same length. A similar discrepancy between thelength-force relations of intact and skinned skeletalmuscle has been described (Schoenberg and Podol-sky, 1972). It is thus possible that, in smooth as instriated muscle, an inactivation of contraction atshort muscle lengths is present due to a depressionof excitation-contraction coupling (Taylor and Ru-del, 1970). The rate of ATP turnover of the skinnedportal veins decreased at lengths below Lo (Fig. 6,left panel), although, relative to active force, thedecrease is much less prominent than when Ca++ isvaried (cf Fig. 5). At 1.3 Lo, AJATp was not signifi-cantly different from the value at Lo, but—if any-thing—seemed to be larger, whereas force was re-duced by 44% relative to Lo. The technical problemsassociated with the stretched preparation are dis-cussed in Results, although the possibility cannot beexcluded that an acceleration of energy turnover ispresent in the stretched skinned musde. This resultcontrasts, however, with intact vascular smoothmuscle, where Jo2 has been found to decrease atlengths above U, (Paul and Peterson, 1975).

In skinned muscles shortened during a maximallyactivated contraction, there is a statistically signifi-cant reduction of JATP (Fig. 7). However, the com-ponent of ATP turnover associated in this way withforce development is smaller than the change in JATPon activation from pCa = 9 to pCa = 4.5 at Lo. Thiscould suggest the presence of Ca++-induced ATPaseactivity not related to force development as dis-cussed above. However, myosin-actin interaction inthe fully shortened muscle cannot be excluded apriori on the basis of no external force output, sinceinternal resistive forces might be of importance. Inintact and chemically skinned hog carotid artery,Pfitzer et al. (1982) report a linear decrease in stiff-ness with force as the muscle was shortened. In theskinned preparations, the stiffness-force relation,when extrapolated to zero force, indicated someresidual stiffness, which could suggest internal forcegeneration in the shortened activated muscle or,

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alternatively, the presence of attached non-force-generating crossbridges. Non-actin-activated myo-sin ATPase activity could possibly contribute to JATPof the activated shortened muscle if myosin-actininteraction is indeed eliminated in this state. Influ-ence of Ca++ on ATPase activity of smooth musclemyosin in the absence of actin has been observed inbiochemical studies (Gorecka et al., 1976, Suzuki etal., 1978).

This study has demonstrated that the variation incrossbridge turnover rate inferred from the mechan-ical and metabolic kinetics of intact vascular smoothmuscle can be reproduced in skinned preparationsat controlled Ca++ levels. The length-force relationof the intact smooth muscle may contain a compo-nent of deactivarion at lengths below L,,.

Supported by the Swedish Medical Research Council (project 14x-28) and the Medical Faculty, University of Lund.

Address for reprints: Per Hellstrand, M.D., Department of Physi-ology and Biophysics, University of Lund, Solvegatan 19, S-223 62Lund, Sweden.

Received January 6, 1983; accepted for publication September 9,1983.

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actin-myosin-ATP interaction. Annu Rev Biochem 49: 921-956Arner A (1982a) Energy turnover and mechanical properties of

smooth muscle. Acta Physiol Scand [Suppl] 505: 1-62Arner A (1982b) Mechanical characteristics of chemically skinned

guinea-pig Taenia coli. Pfluegers Arch 395: 277-284Amer A (1983) Force-velocity relation in chemically skinned rat

portal vein: Effects of Ca2+ and Mg2+. Pfluegers Arch 397: 6-12

Arner A, Hellstrand P (1980) Contraction of the rat portal veinin hypertonic and isotonic medium: Rates of metabolism. ActaPhysiol Scand 110: 69-75

Cassidy PS, Kerrick WGL, Hoar PE, Malencik DA (1981) Exoge-nous calmodulin increases Ca2+ sensitivity of isometric tensionactivation and myosin phosphorylation in skinned smoothmuscle. Pfluegers Arch 391 115-120

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Endo M, Kitazawa T, Yagi S, lino M, Kakuta Y (1977) Someproperties of chemically skinned smooth muscle fibers. InExcitation-Contraction Coupling in Smooth Muscle, edited byR Casteels, T Godfraind, JC Ruegg. Amsterdam, North-Hol-land, pp 199-209

Fabiato A (1981) Myoplasmic free calcium concentration reachedduring twitch of an intact isolated cardiac cell and duringcalcium-induced release of calcium from the sarcoplasmic re-ticulum of a skinned cardiac cell from the adult rat or rabbitventricle. J Gen Physiol 78: 457-497

Fabiato A, Fabiato F (1979) Calculator programs for computingthe composition of the solutions containing multiple metalsand ligands used for experiments in skinned muscle cells. JPhysiol (Paris) 75: 463-505

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INDEX TERMS: Vascular smooth muscle • Ca** • Metabolism• Tension cost • Chemical skinning • Length-tension relation

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A Arner and P Hellstrandmuscle of rat portal vein. Dependence on Ca++ and muscle length.

Activation of contraction and ATPase activity in intact and chemically skinned smooth

Print ISSN: 0009-7330. Online ISSN: 1524-4571 Copyright © 1983 American Heart Association, Inc. All rights reserved.is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231Circulation Research

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