Contractile proteins of vascular smooth muscle: Effects of hydrogen and alkali metal cations on actomyosin adenosinetriphosphatase activity

MICROVASCULAR

RESEARCH

1,344-353

(1969)

Contractile Proteins of Vascular Smooth Muscle: Effects of Hydrogen and Alkali Metal Cations on Actomyosin Adenosinetriphosphatase Activity1 R. A. MURPHY Department OfPhysiology, University of Virginia School of Medicine, Charlottesville, Virginia 22901 Received May 7,1969

This investigation continues recent studies of the ionic dependencies for ATPase activity of the actomyosin isolated from hog carotid arteries. The response to the concentration of H+, Li+, Na+, KC, Rb’, and Cs+ were determined and compared with those of myosin B isolated from rabbit skeletal muscle. The arterial contractile protein ATPase activity has a biphasic pH dependence, with an optimum at pH 5.2 and a smaller peak at pH 7.5, in contrast to the single optimum at pH 6.5 for skeletal muscle actomyosin. Although the enzymatic activity of skeletal muscle actomyosin is very sensitive to the total ionic strength, earlier reports of differences in ATPase activity in the presence of various alkali metal cations were not confirmed. The ATPase activity of arterial proteins was insensitive to changes in the ionic strength in the 0.04416 range under a variety of conditions. No evidence was obtained in support of the hypothesis that transmembrane Na+-K+ exchanges during activity could stimulate vascular smooth muscle contraction by a direct potentiation of the actomyosin ATPase activity by Na+.

Only a decade ago, any discussion of the biochemistry of vascular smooth muscle had to depend on inferences and extrapolations from studies of other smooth muscles or striated muscle (Mommaerts, 1959). Since then, a number of significant investigations have led to the isolation and partial characterization of the contractile proteins from arterial smooth muscle. These studies (reviewed by Bohr, Filo, and Guthe, 1962; Weber and Riiegg, 1966; and Somlyo and Somlyo, 1968) have shown that vascular smooth muscle (a) has a single contractile system for both phasic and tonic responses, and (b) this system, based on f-actin and myosin, is functionally analogous to that of striated muscle. It is therefore feasible to examine the responses of arterial actomyosin to obtain a better understanding of the contractile properties of vascular smooth muscle. This approach, which has proven so fruitful in cardiac and skeletal muscle physiology, isolates the actual contractile event from the various membrane, excitation-contraction coupling, and metabolic processes. In terms of the contractile proteins a relaxed muscle can be depicted as one in which there is no interaction between the myosin and actin components of the contractile system (top, Fig. 1). Similarily, a contracting muscle may be represented as an interaction between the two contractile proteins (bottom, Fig. 1) in which the protein complex acts as a transducer, converting chemical energy derived from ATP hydrolysis into a mechanical response. Control over the system is achieved by regulation of the degree of actin-myosin interaction. Without discussing the controversial areas concerning the mechanisms by which this regulation is achieved, a great 1 Presented as part of the Symposium on Vascular Smooth Muscle held at the Microcirculatory Meetings, April 1969. 344

ARTERIAL

CONTRACTILE

34.5

PROTEINS

deal of work on the contractile proteins of striated muscles (reviewed by Perry, 1960 and 1967; Gergely, 1966; Gibbons, 1968) has shown that the alterations in the ionic environment indicated in Fig. 1 can favor the contractile event. By systematically varying the indicated parameters, and looking at the response either in terms of the chemical energy input (ATPase activity) or the mechanical event (e.g., superprecipitation), one can gain a better understanding of the regulation of the final contractile event and develop an insight into the nature of this chemomechanical transducer. t

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In the studies to be described, we have systematically investigated the dependence of the arterial actomyosin ATPase activity on the concentration of hydrogen ions and of the alkali metal cations (Li+, Na+, K+, Rb+, and Cs+). These results, along with those of previous studies on the Caz+, Mg2+, and ATP ion concentration dependencies (Murphy, Bohr, and Newman, 1969), provide new information on (a) the differences between vascular smooth muscle actomyosin and the contractile proteins obtained from other muscles ; and (b) the extent to which the physiological responses of arterial smooth muscle may depend on factors which directly influence the contractile system. MATERIALS AND METHODS Actomyosin. The contractile proteins were extracted from hog carotid arteries at low ionic strength as previously described (Murphy et al., 1969) using the selective precipitation technique of Riiegg, Strassner, and Schirmer, 1965. Four arterial actomyosin preparations, dissolved in 0.6 M KC1 and diluted 50 % v/v with glycerol, were pooled and stored at -20°C until used in this study. Actomyosin yields, after extraction and purification, were typically 3.5 mg purified actomyosin per gram cleaned artery (wet weight). The skeletal actomyosin (myosin B) was isolated from rabbit back and leg muscles

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following standardized procedures (Murphy et nl., 1969). Protein concentrations were determined by the micro-Kjeldahl method using the conventional protein calculation factor of 6.25. ATPase Actbity Determinations. The majority of the experiments were conducted at 25°C with a protein concentration of 0.3 mg actomyosin/ml. Details of the ionic conditions are given in the Results, but calculations of ionic strength took into account all ion additions and the major ionic complexes formed. Enzymatic reactions were initiated by the addition of ATP, and were terminated by the addition of an equal volume of 2 % trichloroacetic acid. ATP was obtained as the disodium salt, and solutions were purified of trace amounts of divalent metal cations by passage through a Dowex-50 column (Seidel and Gergely, 1963). In the studies of the alkali metal cation dependencies, care was taken to ensure that only K+ was added to the reaction media with the hydrogen ion buffers and the ATP. Buffers were obtained as the free acids or hydrochlorides and neutralized with KOH. ATP was converted to the K’ salt by passage through a Dowex50 column prepared in the K’ form. ATPase activity was calculated after measuring inorganic phosphate liberation by the method of Rockstein and Herron (1951). Since the ATPase activity of arterial actomyosin is very low, special precautions were taken to ensure accuracy: (1) Glassware was washed with a phosphate-free detergent and rinsed exhaustively with distilled and deionized water; (2) dilute ATP solutions were freshly prepared before use to avoid high background phosphate levels due to spontaneous hydrolysis; (3) unknown samples and the various reagents involved with the calorimetric phosphate assay were taken and dispensed with automatic dilutors or automatic pipettes; (4) color development was read in a double-beam spectrophotometer (Coleman Hitachi 124) using a flow-cell with a l-ml sample chamber permitting thorough washout. Hydrogen Zon Buj%rs. Since actomyosin ATPase activity is sensitive to both pH and ionic strength, all hydrogen ion buffers were used at concentrations giving identical pH-stabilizing capacities. The concentration of the buffer bearing a net charge was calculated so that ionic strength could be adjusted to the desired value. The methods used for preparing hydrogen ion buffers have been previously described (Murphy and Koss, 1968). The various hydrogen ion buffers used in these studies (histidine, imidazole, morpholinopropanesulfonic acid or MOPS, and Tris) have been previously shown to have no direct effects on the ATPase activity of myosin B isolated from skeletal muscle (Murphy and Koss, 1968), and were used at concentrations necessary to provide a buffering value of -10 mM H+/pH unit at the indicated pH values. Thus, in all reactions, the hydrogen ion buffer would limit the drop in pH to 0. I of a pH unit if 1 mM H+ were to be added to the reaction solution. The temperature was controlled to +O. 1“C of the indicated values to avoid effects of temperature on the buffers as well as on the enzyme. Solutions. Chemicals were of reagent grade and the solutions were prepared using distilled and deionized water. All the alkali metal cations were obtained as the chloride salt, and their concentrations in stock solutions were checked by chloride titration using mercuric nitrate with diphenylcarbazone indicator (Shales and Shales, 1941). Magnesium chloride stock solutions were standardized by titration with ethylenediaminetetraacetic acid (EDTA) at pH 9 with Eriochrome Black T indicator. Calcium chloride solutions were similarly standardized by a replacement titration using MgEDTA (Flaschka, 1952). ATP stock solutions, which were kept frozen until use, were standard-

ARTERIAL

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347

ized after Dowex treatment by measuring their absorbence at 257 nm (pH 2) with 14,700used as,the value for the molar absorbency index. RESULTS If rigid precautions are taken to avoid changes in the ionic strength or buffering capacity of the test solutions (Murphy and KOSS,1968),the pH dependencefor myosin B isolated from skeletal muscle shows a single broad optimum between pH 6 and 7 under the conditions used (Fig. 2, top panel). In contrast, the arterial actomyosin ATPase activity is maximal around pH 5.2 and has a secondsmall peak at pH 7.5 (Fig. 2, bottom panel). The activity of the arterial proteins was measured under conditions differing from those of the skeletal proteins (Fig. 2) in order to achieve higher rates of ATPase activity. This difference did not significantly affect the shape of the arterial Skeletal

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FIG. 2. Graphs of pH dependence of the basic rate of skeletal (4-8 min after ATP addition) and arterial (2-22 min) actomyosin ATPase activity. Buffers used were histidine, imidazole, and Tris at concentrations required to produce a buffering value of -20 for skeletal actomyosin and -10 for the arterial actomyosin. Other conditions: 0.3 mg protein/ml; KC1 as required to give an ionic strength of 0.1; 5 mM MgATP (skeletal) or 10 mM MgC& plus 1 mM ATP (arterial); 0.1 mM CaCl, ; and 25°C (skeletal) or 37°C (arterial). The points represent mean ATPase activities at each pH value (N = 6 or 12 for skeletal and 3 or 6 for arterial actomyosin; reflecting the fact that at some pH values points were obtained with two different buffers).

actomyosin pH dependencecurve, as it is very similar to the curve obtained in an earlier study in which much lower rates of ATPase activity were measured using conditions comparable to those used for the skeletal muscle actomyosin in Fig. 2 (Bohr et al., 1962). The similarity between the arterial actomyosin pH dependencecurves in Fig. 2 and the earlier report (Bohr et al., 1962)is, in part, fortuitiously a result of the insensitivity of arterial actomyosin to changesin ionic strength (seebelow) which varied with the pH in the earlier study. The actomyosin ATPase activity of striated muscle is known to be very sensitive to changesin the ionic strength, and there is evidenceof differencesin the responsedepending on the kind of alkali metal cations contributing to the ionic strength (see Discus-

348

MURPHY

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FIG. 3. Initial rates of skeletal (O-l min) and arterial (O-15 min) actomyosin ATPase activity as a function of alkali metal cation (MeCl) concentration. Conditions: 0.3 mg protein/ml, 18 mM MOPS (pH 7.0, buffer value, -lo), 0.1 mM CaCl*, 5 mM MgCIZ, 5 mM (skeletal) or 0.5 mM (arterial) ATP, at 25°C. The K+ concentration was 34.5 mM (skeletal) or 25.5 mM (arterial) at p = 0.04, and the indicated molar concentrations of the various alkali metal chlorides (MeCl) were added to obtain higher ionic strengths. The lines connect the mean values of ATPase activity at each ionic strength for all alkali metal cations tested (N = 10 for skeletal actomyosin, N = 5 for arterial actomyosin).

sion). In the present study, no differences among Li+, Na+, K+, Rb+, or Cs+ could be detected in the rate of myosin B ATPase activity of proteins isolated from skeletal muscle (Fig. 3, upper panel). The most striking observation, however, is the virtual independence of arterial actomyosin ATPase activity to changesin the ionic strength (Fig. 3, lower panel). Since a variety of factors affect actomyosin ATPase activity (Fig. I), one explanation for the lack of response of arterial smooth muscle actomyosin to changing salt concentration could be an overall suppression of activity by an unfavorable combination of the other ionic concentrations. Figure 4 shows the arterial actomyosin ATPase activity as a function of the NaCl and Arterial

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FIG. 4. Effect of NaCl and KC1 (MeCl) concentration on initial rates of arterial actomyosin ATPase activity under ionic conditions favoring increased ATP hydrolysis. Conditions not shown on graph: 0.3 mg protein/ml, 18 mM MOPS (pH 7.0, buffering value = -10) or 24 mM histidine (pH 5.5 buffering value = -lo), K+ present at p = 0.04 was 8.8 mM (pH 5.5) or 11.0 mM (pH 7.0), 0.1 mM CaCl,, 25°C. Each point is the average of two determinations.

ARTERIAL

CONTRACTILE

Arterial

349

PROTEINS

Actomyosin

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FIG. 5. Effect of substituting NaCl for KC1 on the initial rate of arterial actomyosin ATPase activity at various ionic strengths. Conditions: 0.3 mg protein/ml, 18 mM MOPS (pH 7.0, buffering value = -lo), 0.1 mM CaCl,, 0.5 mM ATP plus 5 mM MgC12 or 1 mM ATP plus 10 mM MgC&, 25°C. Besides the indicated NaCl or KC1 concentrations, 25.4 mM K+ was present (as additions with the protein, ATP, and MOPS plus that used to adjust the ionic strength). Each point represents the average of two determinations.

KC1 concentrations under two circumstances which are known to increase levels of ATPase activity: (1) decreasing the pH to 5.5 (Fig. 2) and (2) increasing the Mg and ATP concentrations. At pH 5.5 the ATPase activity rises with increasing ionic strength (Fig. 4, dashedlines). However, the responseis not in the direction typical of the skeletal muscle contractile proteins under favorable ionic conditions and is of little physiological significance at this low pH. Doubling the MgClz and ATP concentrations did potentiate the ATPase activity, but the more favorable ionic conditions did not lead to any ionic strength dependence. Under the conditions yielding an increased ATPase activity, there appears to be a slightly higher activity in the presenceof Na+ (Fig. 4, solid circles) than in the presence of K+ (Fig. 4, open circles). Differences of this magnitude are difficult to substantiate Arterial

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FIG. 6. Effect of substituting NaCl for KC1 on the initial rate (04 min) of arterial actomyosin ATPase activity at varying levels of enzymatic activity. Conditions: 0.3 mg protein/ml, 18 mM MOPS (pH 7.0, buffering value = -lo), 0.1 mM CaC12, 10 mu MgC12, ATP as indicated on graph, 25°C. The reaction mixtures contained 11 mM K+ plus the KC1 added along with NaCl (when the KC1 concentration was 0.04 M) to give a total ionic strength of 0.08. Each point is the average of two determinations. 13

350

MURPHY

due to the very low specific activity of the arterial enzyme and systematic errors which arise between test runs. However, by substituting varying amounts of NaCl for KC1 in the same experimental run (where the NaCl plus KC1 concentration remains constant) one can measure any potentiation by Na+ with greater precision. Experiments of this type, carried out at three different total concentrations of NaCl plus KC1 (Fig. 5) clearly show no difference in the response to Na+ relative to that achieved in the presence of K+. A similar lack of Na’ stimulation of ATPase activity was obtained in a series of experiments in which the ATP concentration was altered at a constant MgC12 concentration so as to produce varying degrees of activation of the arterial actomyosin ATPase activity (Fig. 6). DISCUSSION It is difficult to draw conclusions concerning the effects of individual ionic factors on the response of actomyosin systems for two principal reasons. The first is that two types of ATPase activity are possible in purified contractile protein preparations (i.e., that of myosin alone, and the activity of the actin-moderated myosin enzyme or actomyosin). Only the latter activity, which is Mg-activated (although trace amounts of Ca2+ are required when the tropomyosin-troponin complex is present [Ebashi, 19681) has direct physiological significance. While the actomyosin ATPase activity is usually distinguishable from the Ca-activated myosin ATPase activity in skeletal muscle proteins, one cannot neglect the possibility that arterial myosin alone produces significant ATP hydrolysis during an experiment. The second difficulty is that many different parameters affect actomyosin ATPase activity (Fig. l), and an enormous effort is necessary to completely characterize their individual effects. Since one cannot duplicate the intracellular ionic conditions with certainty, experimental results may not yield information directly applicable to in vivo behavior. In this investigation, we have worked on the premise that a combination of variables which are relatively consistent with present concepts of the intracellular environment of vascular smooth muscle, and which are favorable for the response of the well-studied skeletal muscle proteins, would be satisfactory for testing the ionic dependencies of arterial actomyosin. These factors render interpretation of the biphasic pH dependence (Fig. 2) first reported by Bohr et al. (1962) as uncertain, The results shown in Fig. 2 rule out the likelihood that technical artifacts produced this result, and furthermore show that the biphasic pH dependence persists in different combinations of environmental ionic conditions. It is possible that the behavior of the isolated actomyosin at pH 7.5 represents the in vivo situation. On the other hand, the in vivo pH may be somewhat acid or the ionic conditions might differ in such a way as to shift the actomyosin pH optimum to more neutral values. In this event, the degree of activation of the isolated enzyme at pH 5.2 might be more relevant to the behavior of vascular smooth muscle. The potentiating effect of a decrease in ionic strength on the ATPase activity of myosin B isolated from skeletal muscle (Fig. 3) has long been known (Hasselbach, 1952; Maruyama and Ishikawa, 1964; Fanburg, Finkel, and Martonosi, 1964; Eisenberg and Moos, 1966). Skeletal muscle actomyosin ATPase activity at a single ionic strength has been reported to be stimulated by various alkali metal cations in decreasing order Na+ > K’ N Rb+ > Li+ > Cs+, when each of the cations were present at a concentration of 24 mM (Katz et at., 1966). Although the effect was not large in that study, it differs

ARTERIAL CONTRACTILE PROTEINS

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from our finding of no significant changes in ATPase activity measured in the presence of the various cations. The contrasting results may reflect our use of myosin B preserved in 50 % glycerol instead of the freshly prepared synthetic actomyosin tested by Katz et al. (1966). The pertinent conclusion from both studies, however, is that any potentiating effect caused by Na+ is without physiological importance in skeletal muscle since there is little evidence for changes in the low intracellular Nat concentration during activity of this tissue (Katz, 1967). In the case of cardiac actomyosin, a greater stimulation of ATPase activity by Na+ substitution for K+ may be observed (Katz et al., 1966). Since many procedures producing positive inotropic effects in cardiac muscle are associated with an exchange of intracellular K+ for Na +; it is conceivable that part of the increased cardiac contractility may result from Nat stimulation of the contractile proteins (Katz et al., 1966). However, considerations of the magnitude of the K’-Nat exchange which occur in vivo compared with those necessary for significant effects on the isolated proteins make the physiological significance of this process uncertain in cardiac muscle (Katz, 1967). Alternative hypotheses for positive inotropic effects associated with an increase in intracellular Na+ are increased Ca*+ influx (Langer and Brady, 1968), or an inhibitory effect by Na+ on Ca2+ uptake by the cardiac sarcoplasmic reticulum @tam and SonnerYblick, 1968). A number of studies indicate that the intracellular Na+ concentration of vascular smooth muscle cells is very high. Estimates running from one-fifth to one-half of the intracellular K+ concentration (Villamil et al., 1968; Schoffeniels, 1967; Schoffeniels et al., 1966; Garrahan et al., 1965; Hagemeijer et al., 1965) have been obtained. These values are subject to question, however, due to problems arising from the presence of large amounts of bound sodium in the arterial wall (Headings et al., 1960); and other measurements in the sheep carotid artery suggest that the Na+ permeability and content is low (Keatinge, 1967). If the higher estimates of intracellular Na+ concentration are correct, a transmembrane Na+-K+ exchange of this magnitude would potentiate the contractile response if the arterial actomyosin showed the same differences with respect to Na’ and K+ reported for cardiac actomyosin (Katz et al., 1966). Our results have shown no justification for the supposition that the arterial actomyosin system is potentiated by Na+. In fact, we have found little evidence of any dependence of arterial actomyosin ,4TPase activity on ionic strength in the 0.04-O. 16 range (although at high ionic strengths the dissolved proteins show a high rate of Ca2+-stimulated ATP hydrolysis characteristic of myosin). This is the first instance in which a qualitative as well as a quantitative difference between arterial and skeletal muscle contractile protein behavior has been observed in our laboratory. For the reasons discussed above, interpretation of these findings must be made with caution, although they were repeated using different ionic conditi’ons. As more information becomes available on the contractile proteins of arterial smooth muscle, we shall be able to evaluate their role in terms of the tissue contractility. In this respect, the individualities of the arterial actomyosin when compared to skeletal muscle proteins are of particular interest. ACKNOWLEDGMENTS The adviceand support of Dr. D. F. Bohr and the technical assistance of Mrs. Mildred Smythers and Miss Julie Schmeidlin were major contributions to this work. Portions of this study were supported by an NSF Institutional Grant for Science to the University of Virginia, an NIH Health Science Advance

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Award (NIH 5-S04-FR 06001), NIH Grant HE-03756, and a grant from the Michigan Heart Association. Hygrade Food Products of Richmond, Virginia generously provided the carotid arteries.

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STAM, A. C., JR..,AND SONNENBLICK, E. H. (1968). Old and new observations in excitation-contraction coupling. Proc. Intern. Union Physiol. Sci. 6, 139. VILLAMIL, M. IT., RE~TORI, V., BARAJAS, L., AND KLEEMAN, C. R. (1968). Extracellular space and the ionic distribution in the isolated arterial wall. Am. J. Physiol. 214,1104. WEBER, H. H., AND R&GG, J. C. (1966). The contractile fine structure of vertebrate smooth muscle. Med. Col. Virginia Quart. 2,12.