Effect of Endothelin-1 on Actomyosin ATPase Activity
Implications for the Efficiency of Contraction
Abstract Endothelin is a powerful inotropic peptide that increases isometric force in isolated papillary muscle and the extent of shortening in isolated single cardiac myocytes. Its mechanism of action has been variously attributed to increased Ca2+ activation, increased Ca2+ sensitivity of the contractile proteins, and increased intracellular pH, but the physiological function of the changes in cardiac performance remains obscure. In this study, the effects of endothelin-1 on both force development and the kinetics of contraction have been examined. Isometric force, actomyosin ATPase activity, and unloaded shortening velocity were measured. The effects were dose dependent. From 1 to 50 pmol/L endothelin-1 did not alter force development in isolated trabeculae with intact endothelial cells, but actomyosin ATPase activity was increased. Between 100 pmol/L and 10 nmol/L endothelin-1 raised isometric force, decreased actomyosin ATPase activity, and decreased unloaded shortening velocity. The reduction in ATPase activity was progressively enhanced as sarcomere length was increased from 1.9 to 2.4 μm. These results indicate that the effects of endothelin-1 on the force of contraction and the rate of ATP hydrolysis are not tightly coupled and are changed in the opposite directions by endothelin-1 over most of its effective-dose range. This raises the possibility that endothelin-1 may increase the economy of contraction. A novel function of endothelin may be the modulation of the efficiency of contraction, particularly when increased preload raises the contractile work of the heart.
Endothelin is a small peptide first identified as increasing the contraction of smooth muscle, but it has subsequently been found to be one of the most powerful inotropic agents.1 It is present in the culture medium of endothelial cells2 and in the coronary venous effluent of isolated hearts3 in concentrations high enough to affect cardiac muscle myocytes. The synthesis, storage, and release of endothelin appear to involve a mechanism that balances the rate of work performed by the heart with the rate of energy supplied to the heart.4 As the oxygen tension in the myocardium rises, production and release of endothelin by coronary vascular endothelial cells increase. These observations suggest that endothelin is in some way involved in the relation between inotropism and energy metabolism.
The mechanism of action of endothelin on cardiac contractility is not clear. Several different studies5 6 7 8 have concluded that there are multiple effects, some of which are dose related. The effect at low concentrations (0 to 50 pmol/L) appears to depend on the level of activity of endothelial cells in the preparation.4 The sensitivity of the preparation to endothelin is increased in the absence of endothelial cells. Endothelial cells may buffer the effect of added endothelin by releasing nitric oxide or other material that opposes the action of endothelin.9 10 11 In isolated single cardiac myocytes, 1 to 50 pmol/L endothelin raises the velocity of shortening and increases the extent of shortening,5 6 while in isolated thin trabeculae treated with Triton X-100 to disrupt endothelial cells, small increases in force are produced.4 When endothelial cells are intact, no significant change in force occurs in rat heart until 50 to 100 pmol/L endothelin, although a small increase of about 10% has been observed in rabbit papillary muscle between 10−2 and 10 pmol/L.7 12 There is evidence of a transition from one pattern of response to another in the range of concentrations between those that increase the amount of shortening of single myocytes and those that increase isometric force in intact trabeculae. At concentrations of 100 to 1000 pmol/L, Kelly et al5 observed that endothelin had little or no effect on shortening in about half of their preparations, and we have found13 two different patterns of response of actomyosin ATPase activity to endothelin in the same concentration range. Kelly et al5 also made the intriguing observation that prior exposure to β-adrenergic agonist blocked the effect of endothelin at these concentrations.
The molecular bases for these changes are not well understood. Evidence for increase in inward Ca2+ current and increased Ca2+ sensitivity of the contractile proteins from intracellular alkalinization exists,14 15 but these mechanisms cannot explain all of the characteristics of the increase in force. To distinguish among several possible ways in which endothelin might function, it is necessary to examine not only force but also the kinetic parameters of the contraction related to cycling of force generators. In the simple two-state model of crossbridge cycling (see “Discussion”), the transition to the force-generating state and the detachment from the thin filament are the rate-limiting steps, respectively, for the rate of ATP hydrolysis and unloaded shortening velocity. For these reasons, we have measured the effects of endothelin on ATPase activity and velocity as well as force. Over a large part of the dose range in which endothelin is effective, the peptide raises force and lowers actomyosin ATPase activity. This should result in an increase in the economy of energy use in the maintenance of tension. A novel and potentially important physiological function of endothelin in cardiac muscle may be the regulation of efficiency of contraction.
Materials and Methods
Endothelin-1 was obtained from Sigma Chemical Co and BQ-123 from Peninsula Laboratories and Sigma.
Measurement of Developed Force
Male Wistar rats, weighing between 150 and 600 g, were killed by cervical dislocation in accordance with AALAC guidelines. The heart was rapidly removed and two thin papillary muscles or trabeculae were isolated from the endocardial surface of the right ventricle. Trabeculae were chosen for study on the basis of the ease with which they could be removed and attached to the force transducer without producing damage except at the cut ends. Trabeculae were selected only if their radii did not exceed 0.3 mm, their length was at least 3 mm, and they were free from the wall of the ventricle except at their ends. Considerable care was taken to avoid contact of dissection tools with the surface of the tissue bundle to avoid damage to endothelial cells and to produce preparations that had intact endothelium. Trabeculae with visible irregularities or trabeculae that displayed spontaneous contraction at any time during the experiment were rejected. The range of cross-sectional areas of the trabeculae included in this study was 0.05 to 0.1 mm2 (mean±SD=0.06±0.008).
After transfer to the bathing solution, one end of each papillary muscle was attached to a Cambridge Model 401 force transducer (Cambridge Technology, Inc), and the mass of ventricular tissue at the other end was snared by a loop of thin stainless steel wire attached to the arm of a small scanning motor (General Scanning, Inc). The resonance frequency of the system with the bundle mounted was ≈800 Hz. Muscles were suspended in a horizontal position in a chamber containing Krebs’ solution (in mmol/L: NaCl 118, KCl 4.8, KH2PO4 1.0, MgSO4 1.2, NaHCO3 25, CaCl2 2.5, and glucose 11.1, buffered at pH 7.4) bubbled continuously with 95% O2 and 5% CO2 through a fritted glass in the chamber. The bubbling provided a sufficient rate of mixing so that the performance of the muscles was not diffusion limited. The volume of fluid in the chamber was 30 mL, which was >104 times the volume of the tissue. The chamber was cleaned with acid between experiments to eliminate the possibility of residual material binding to the glass. Once the bundle was mounted, supramaximal stimulation through platinum wires imbedded in the chamber walls was begun at 0.2 Hz, using square pulses of 0.1 millisecond’s duration. The bundle was stretched until sarcomere length was ≈2.2 μm, the length at which developed force was maximal. Stimulation continued for 90 to 120 minutes to allow the bundle to stabilize. The average maximum isometric force developed by the trabeculae was 35±11 mN/mm2 (mean±SD). When the force reached a stable level, the length of the papillary muscle was measured, the concentration of Ca2+ was reduced to 1.25 mmol/L, and 30 minutes later the bathing solution was replaced with one containing endothelin.
At the end of the experiment, the trabeculae were fixed with glutaraldehyde, sectioned, and sarcomere length was measured. Cross-sectional areas were estimated through the light microscope at the end of each experiment and confirmed by measurement in sections of glutaraldehyde-fixed tissues.3
Actomyosin ATPase Activity
Actomyosin ATPase activity was measured in cryostat sections of quickly frozen hearts using quantitative histochemistry. The detailed description of the method has already been published.16 After animals had been killed by acceptable procedures, the hearts were removed quickly, washed in Krebs’ solution, and immediately frozen in isopentane precooled with liquid nitrogen. Multiple serial sections were cut from each heart and picked up on coverslips in an order that prevented bias in selection of sections for different protocols. Uniformly thick sections were cut, with the temperature in the microtome set between −22°C and −26°C, and ATPase activity was assayed within a short time. Briefly, the tissue sections were exposed in succession to preincubation and incubation solutions. Inorganic phosphate liberated by the ATPase activity of actomyosin in the incubation solution was immediately trapped and then quantitatively replaced by the opaque salt cobalt sulfide in a two-step process. The optical density of the cobalt sulfide was measured, without systematic bias in selection of regions of the tissues measured. For each protocol, at least 10 tissue sections from each heart were measured. When the effect of endothelin-1 was studied, the drugs were added to both the incubation and preincubation solutions.12 For dose-response curves, an entire series of measurements was made on sections from each heart so that comparisons could be made among the same cells. The final reaction product remained sharply localized to myosin, and the density was proportional to the amount of ATP split. It was measured by digitizing the image of the section and calculating the average density over the cells with a program that eliminated the intercellular and intracellular spaces. Complete dose-response curves were generated for each of 12 different hearts, using serial sections to determine the effects of different concentrations of endothelin. Multiple areas randomly chosen on multiple sections were measured at each concentration of endothelin.
Quickly frozen ventricles preserve ultrastructure very well, including the contractile filament lattice, and when sectioned at 4 μm (one third of a cell diameter) at low temperature, the interiors of all cells are open. Therefore, the concentrations of Ca2+, Mg2+, ATP, Pi, etc, can be carefully controlled in the microenvironment of the contractile filaments. Portions of sarcolemma with functioning receptors and enzymes remain. With diffusion distances from the residual membranes to myofibrils very similar to the distance out of the section, messenger molecules that bind to cell contents such as protein kinase A (PKA) and protein kinase C (PKC) are effective in modifying myofibrils. In the histochemical measurement of actomyosin ATPase activity, the cells shorten a small amount from the resting sarcomere length during the first few seconds of application of the contraction solution and then maintain the same length. Since the ATPase activity is measured over 10 minutes, the rate of ATP splitting is determined during isometric contraction and can be compared with the force developed at the same sarcomere length during an isometric contraction.
The sarcomere length was measured at the end of the assay using the opaque reaction product accumulated in the A bands to identify the sarcomere pattern. The reaction product from ATP splitting was retained very close to the enzymatic sites on the thick filament and defined the sarcomere length. Mean sarcomere length was calculated from the length of at least 10 consecutive sarcomeres in at least five different regions of the section and averaged. Sarcomere lengths were uniform within a given preparation and could vary among different preparations. The standard deviation for sarcomere length within a given preparation never exceeded 0.1 μm. The reproducibility of sarcomere length in the same area between serial sections was within 2%. In the determination of sarcomere length, it was necessary to make a correction for the shrinkage of filaments produced by exposure to ethanol after the ATP splitting had been terminated. From measurements of sarcomere lengths with phase contrast and Nomarski differential interference microscopy in other frozen sections at each stage in the assay, it was ascertained that sarcomeres shrink by 14% in the steps of the assay after the release of Pi by ATP hydrolysis. Almost all of this shrinkage occurs during the exposure to absolute ethanol.
Among the advantages of this technique are the stability and reproducibility (±2%) of the ATPase activity. Serial sections provide excellent controls because they sample mostly the same cells. The ATPase assay was carried out under conditions in which Ca2+ concentration was not limiting, thereby eliminating the degree of activation as a variable.
Effect of Sarcomere Length on Response to Endothelin-1
The trabeculae were suspended with a tie around one end and another around the center of the bundle, so that when tension was produced by pulling on the ties, half of the bundle was stretched, while the other half remained unstretched. After suspension, the tension on the half of the trabecula between the ties was increased by an amount that would produce sarcomere lengths from 1.9 to 2.4 μm. Since it was not possible to freeze these preparations rapidly when mounted on the transducer, because of the short distance between attachment sites, they were not mounted on the transducer. The bundles were left quiescent in oxygenated Krebs’ solution for 30 minutes and then quickly frozen in nitrogen-cooled isopentane. After freezing, the bundles were mounted longitudinally on the cryostat and sectioned so that portions of both halves of the bundle were included in individual sections. Actomyosin ATPase activity was measured by quantitative histochemistry. This technique has subcellular spatial resolution and can distinguish the ATPase activity in stretched cells from that in unstretched cells. Sarcomere length was measured as described above.
Maximum Velocity of Unloaded Shortening
Maximum velocity of unloaded shortening was measured by the slack-length method.17 The muscle bundle was released during a contraction by a series of different length steps that were large enough to eliminate tension and produce slack in the bundle. The time taken for tension to reappear was the time required to take up the slack produced by the length step. The slope of the line relating the size of the length step to the time until tension reappears is equal to the maximum velocity of shortening. Any systematic errors that might influence the value obtained from a single measurement, such as end compliance, were removed by using multiple length steps of different sizes. Visualization of the preparation through the microscope was an added check that the muscle was in the same position at the precise points at which tension reappeared. Care was taken to operate over sarcomere lengths at which the effect of length on velocity of shortening was small. Extensive control studies have already been carried out to define this relationship. Velocity was measured in 1.25 mmol/L Ca2+ Krebs’ solution after equilibration at 20°C for 45 minutes to increase the duration of the twitches.
On five successive twitches, shortening steps of 10%, 12%, 14%, 15%, and 16% of the muscle length were imposed on the tissue, with each step beginning when the twitch force had reached ≈80% of maximum. The release was complete within 2 milliseconds. Steps of these lengths were sufficient to completely unload the bundle. Both the force and motor position signals were digitized at 0.0004 Hz and stored on disk. The time required for the papillary muscle to begin redeveloping force was then determined from the digitized records. The unloaded velocity of shortening was determined from the linear regression of the length change imposed versus the time to begin redeveloping force.
After a series of three control sets of releases, each set consisting of five releases, the solution in the chamber was changed to one containing either 1 or 10 nmol/L endothelin at the same Ca2+ concentration and temperature. Thirty minutes after the exposure to endothelin, another series of releases was imposed to determine the effect of endothelin on unloaded shortening velocity.
Measurement of Isoforms of Myosin Heavy Chain
The relative amounts of the isoforms of myosin heavy chain were measured in parallel studies by nondenaturing polyacrylamide gel electrophoresis.
Values were compared using Student’s t test paired for the changes in ATPase activity and unloaded velocity of shortening due to endothelin or isoproterenol, and unpaired for the effect of endothelin on ATPase activity at different sarcomere lengths.
Effect of Endothelin on Isometric Force
The force generated during isometric contraction was increased by 0.2 to 10 nmol/L endothelin (Figs 1⇓ and 2⇓). No change occurred at lower concentrations, and the maximum effect was produced by 1 to 10 nmol/L. The amplitude of the increase in force depended on the age of the animal (Fig 3⇓). The largest increases, >150%, occurred in the youngest animals, which were about 5 weeks old. As the age of the animals increased, there was a sharp decline in the effect of endothelin on the generation of force, and by 4 months of age the response of the trabeculae was small, equal to 10% to 20% of control isometric force. The relative shape of the dose-response curve remained the same as the animals aged and their weights increased (data not shown). Takanashi and Endoh7 have reported that the change in force by addition of endothelin follows a similarly shaped dose-response curve in isolated trabeculae from older rats (350 g). The maximum increase reached in their study was ≈25%.
Effect of Endothelin on Actomyosin ATPase Activity
ATPase activity was measured in cryostat sections of quickly frozen rat hearts by quantitative histochemistry to detect whether any changes were due to the establishment of different populations of cells, or even sarcomeres, or to a uniform alteration of all sarcomeres in all cells of the cardiac tissue. The same concentrations of endothelin that increased isometric force had the opposite effect on actomyosin ATPase activity (Fig 4⇓). The maximum decrease in the rate of ATP hydrolysis was about 10%, and it was observed at 1 to 10 nmol/L endothelin (n=23, P<.01). Mean sarcomere length was 2.0 μm. The average ATPase activity within sarcomeres was uniform. From the data, however, it was not possible to distinguish between a 10% decrease in ATPase activity of each force generator and a larger change in only some force generators uniformly distributed among the sarcomeres. Low concentrations of endothelin that did not alter isometric force did increase ATPase activity. Although these low concentrations have been reported to increase the amount and velocity of shortening in isolated, freely suspended cardiac myocytes,5 6 a change in force was not directly measured in those studies. The addition of 1 μmol/L BQ-123, an endothelin receptor A antagonist, prevented any significant change in ATPase activity from the subsequent addition of endothelin (n=5), showing that the decrease in ATPase activity was initiated by the binding of endothelin to its normal receptor in the membrane. Since the effect of specific inhibition of the endothelin receptor A was complete with BQ-123, no other inhibitor was used.
The extent of the decrease in the ATPase activity produced by endothelin was the same in animals over the entire age range studied. No significant difference was detected in hearts from animals of different ages (data not shown). The effect of endothelin on actomyosin ATPase activity, unlike the effect on isometric force, did not vary with age.
Effect of Sarcomere Length on Actomyosin ATPase Activity
A greater efficiency of contraction occurs in hearts enlarged from chronic hypersecretion of growth hormone18 and chronic overload,19 and decreases in actomyosin ATPase activity have been reported in dilated or enlarged hearts, resulting from increased preload or afterload.18 19 20 The effect of increase in length of cardiac myocytes on the response of the contractile system to endothelin was examined by comparing the change in ATPase activity produced by endothelin in stretched and unstretched segments of isolated cardiac trabeculae.
Increasing sarcomere length in the absence of endothelin decreased actomyosin ATPase activity, as expected from the decrease in overlap of thick and thin filaments (Fig 5⇓). Since ATPase activity was measured under conditions in which Ca2+ concentration was not limiting, the measurement was not influenced by the increase in Ca2+ sensitivity and Ca2+ release from the sarcoplasmic reticulum that an increase in sarcomere length causes in intact cells. A significant change in the response of actomyosin ATPase activity to endothelin occurred as sarcomere length was increased from 1.9 to 2.4 μm (n=8, P<.05). Addition of 100 pmol/L endothelin produced a progressively greater decline in ATPase activity as sarcomere length was increased from 1.9 to 2.4 μm. This concentration of endothelin was used in these experiments because it is the lowest concentration that gave a large and near-maximum change in ATPase activity. Therefore, it would likely be the most informative concentration in evaluating physiological function. The addition of 1 μmol/L BQ-123 prevented any significant change in ATPase activity from the addition of endothelin in stretched and unstretched myocytes (n=5).
Velocity of Shortening
In some of the muscles in which the isometric force was also measured, the effect of endothelin on the maximum velocity of unloaded shortening was measured using the slack-length technique (Fig 6⇓). In nine trabeculae, 10 nmol/L endothelin decreased the maximum velocity of shortening by 11±3% (P<.03) while increasing isometric force by 47±9% (P<.01). The results with 1 nmol/L endothelin were not significantly different from those with 10 nmol/L (n=4). There was no relation between the magnitude of the change in unloaded velocity and the age of the animal or between the size of the increase in force and the magnitude of the change in velocity. Since internal viscosity would be related to force, it could not have been the cause of the change in velocity. In three of the nine experiments, 0.1 μmol/L isoproterenol was applied 30 to 60 minutes after the removal of endothelin to assure that the established effect of β-adrenergic agonists could be observed. Isoproterenol increased force by 58±17% and velocity of unloaded shortening by 17±6%.
Several conclusions can be drawn from the response of cardiac muscle to a broad range of concentrations of endothelin: (1) Actomyosin ATPase activity is increased at very low concentrations of endothelin, whereas force is unchanged. This is the concentration range that produces an increase in the velocity and extent of shortening in isolated myocytes.5 6 (2) Actomyosin ATPase activity is decreased at somewhat higher concentrations, whereas force is increased. (3) The magnitude of the decrease in ATPase activity increases with increasing sarcomere length from 1.9 to 2.4 μm. (4) Force and ATPase activity can be independently modified by endothelin. (5) Force and unloaded shortening velocity are changed in opposite directions in the same preparation by the same concentration of endothelin.
The most novel potential consequence of the effects of endothelin on cardiac muscle is a reduction in the energy cost for maintaining tension. An increase in force concurrent with a reduction in the rate of ATP splitting would mean that less ATP is hydrolyzed to generate a given level of force, and the economy of conversion of biochemical to mechanical energy would be greater (economy is defined as the tension-time integral divided by the chemical energy expended21 ). This is particularly relevant to the contraction of the heart, as most of the chemical energy in a cardiac contraction is expended on the maintenance of tension.21 Since maintenance of tension in cardiac muscle requires most of the energy used during a contraction, 0.5 to 10 nmol/L endothelin should increase the efficiency of cardiac contraction. The increase in economy would become progressively greater with longer sarcomere lengths, making endothelin potentially more effective when the heart dilates and its work per contraction increases. A decrease in actomyosin ATPase activity due to a change in myosin isoforms occurs with chronic dilatation and hypertrophy as a result of altered gene expression,22 but the response of ATPase activity to endothelin is much faster, providing the cardiac cell with the possibility of a rapidly responding posttranslational mechanism for modifying the economy of the contraction that becomes more active as the workload on the heart increases.
In these studies of the effect of endothelin on the development of force and the rate of ATP hydrolysis, the two measurements were made on different tissues. The measurements of developed force and unloaded shortening velocity were, however, made on the same preparations under the same conditions. For a rigorous demonstration of a change in efficiency, force development and the rate of energy use should ideally be measured on the same preparation at the same time using either myothermic methods with intact cells or the rate of ATP hydrolysis with the enzyme-coupled technique that allows simultaneous monitoring of force and ATP concentration. The major concern in making force and ATPase measurements on different preparations is the possibility of differences in the sarcomere lengths at the time of each experiment. That concern has been addressed in the design of the experiments. Endothelin reduced ATPase activity at all sarcomere lengths between 2.0 and 2.4 μm. The sarcomere length in the preparation used for the force measurements was 2.2 μm. Therefore, even if as much as a ±10% change in sarcomere length during an isometric twitch were to occur (a very unlikely occurrence with a stiff transducer and carefully suspended tissue), the preparation would still be in the range of sarcomere length at which endothelin had been shown to decrease ATPase activity.
In the measurement of ATPase activity, the concentration of Ca2+ was not limiting. Therefore, there was no possibility of variation in the number of active crossbridges from effects other than changes in the contractile proteins themselves. The linear decline in actomyosin ATPase activity with increasing sarcomere length was analogous to what occurs for maximal Ca2+-activated or tetanic force. This shows that in the absence of endothelin, changes in sarcomere length and transverse distance between thick and thin filaments did not alter the cycling of crossbridges overlapped by thin filaments.
Endothelin can change force without changing ATPase activity, change ATPase activity without changing force, or change ATPase activity and force in opposite directions. The ability of endothelin to regulate force and actomyosin ATPase activity separately should facilitate modulation of efficiency.
Developed force can be enhanced by an increase in the number of cycling crossbridges or the unitary force of each crossbridge or by an increase in the fraction of the cycle spent in the tension-producing state (duty cycle). ATPase activity can be changed by altering the number of cycling crossbridges or the rate constants in the crossbridge cycle. In a simple two-state model of muscle contraction, crossbridges exist in either a weakly bound and non–force-producing or a tightly bound and force-producing state.23 24 Two rate constants, a and b, respectively, control the transitions into and out of the force-producing state. In this model, the rate-limiting step for ATP hydrolysis is the rate of transition from the weakly bound to the strongly bound state (a). For the velocity of shortening, the rate at which crossbridges developing negative force detach from actin (b) is limiting. The duty cycle is proportional to a/(a+b). According to this model, the kinetics of contraction can be changed by modifying either a or b, and the efficiency of the contraction can be modified by altering a/(a+b). If a and b are changed proportionally, the ATPase activity and velocity of unloaded shortening are altered, but the time-averaged force generation is unchanged. If the crossbridge cycles more rapidly, it uses more ATP, but it still develops force during the same fraction of the time and thus does not alter total force. A disproportional change will alter the duty cycle and force. Slower cycling decreases the rate at which ATP must be split to produce and maintain a given level of force. Force can change independently of kinetics by increasing the unitary force per crossbridge. A change in the number of active crossbridges, which could result from changes in the level of Ca2+ activation or the Ca2+ sensitivity of the contractile system, will alter force and ATPase activity proportionately. Of the three possible mechanisms for increasing force, only one, an increase in the duty cycle, seems reasonable, in view of the simultaneous decrease in ATPase activity. An increase in the number of cycling crossbridges or in the unitary force per crossbridge is unlikely to account for the decrease in the rate of ATP hydrolysis.
It is not clear how endothelin decreases the rate of ATPase activity and presumably crossbridge cycling. Some help may be provided, however, by new work studying changes in crossbridge position in isolated natural thick filaments from rat heart produced by phosphorylation of C protein.25 Phosphorylation of C protein in isolated thick filaments by PKA causes a 3-nm extension of the actin attachment site on the crossbridge, bringing the crossbridge right to the surface of the thin filament. It is reasonable that this more favorable position of the crossbridge increases the probability of forming a force-generating link to the thin filament, thus increasing the rate of cycling and ATP hydrolysis. In this mechanism, one might expect the effect to be sensitive to the distance between thick and thin filaments and therefore sarcomere length. The combination of the lack of effect of sarcomere length or distance between thick and thin filaments on ATPase activity in the absence of endothelin and the sensitivity to sarcomere length in the presence of endothelin is significant. It suggests that changes in crossbridge conformation and position may be a crucial part of the mechanism of endothelin’s effect on ATPase activity just as they appear to be in the effect of β-adrenergic agonist on ATPase activity.
The α-adrenergic activity of norepinephrine on contractility is very similar to that of endothelin. It increases PKC activity, increases force development, and decreases unloaded shortening velocity.26 27 28 Particularly relevant is the similar biphasic effect on ATPase activity. Low concentrations raise enzymatic activity, and optimal concentrations for enhancement of force lower ATPase activity.29 PKC lowers ATPase activity. Although it produces the same pattern of phosphorylation in C protein as PKA, it causes a different pattern of phosphorylation in troponin I.30 The result of phosphorylation of C protein from PKA or PKC may be to make the crossbridge sensitive to the distance from its attachment site on actin, and the different patterns of phosphorylation of troponin I from PKA and PKC may determine whether a given crossbridge position is more or less favorable for attachment. This is a clearly testable hypothesis.
At low concentrations of endothelin, the cycling rate of crossbridges produces a faster contraction, with no change in maximum force. At higher concentrations, the cycling rate decreases and force increases, resulting in greater efficiency. While both effects may be relevant physiologically, the most important consequence and possibly the most useful function of the mechanism would be the modulation of the efficiency. Force increase without greater energy utilization should be possible, with the only apparent sacrifice being a slower development of force. This is an alternative to the inotropism of β-adrenergic stimulation, which increases force but also increases the cycling rate of crossbridges and therefore decreases economy. The myocardium contains two separate mechanisms for modulating the kinetics and efficiency of contraction. One enhances efficiency; the other, velocity.
The response of actomyosin ATPase activity, measured under conditions in which Ca2+ is not limiting, remains the same over an age range that produces widely different effects on force. The difference in the effects of aging on the response of force production and ATPase activity to endothelin suggests that a change in the number and not the kinetics of cycling of crossbridges occurs, possibly from a change in either Ca2+ sensitivity or the concentration of activating Ca2+.
The mechanism for the difference in effects at low and intermediate concentrations of endothelin is not clear. The rate of phosphoinositide hydrolysis is stable until 100 pmol/L and then increases fivefold between 100 pmol/L and 10 nmol/L,8 so it is possible that the increase in force and/or the decrease in ATPase activity are related to increased PKC stimulation of the hydrolysis. There are insufficient data to say whether the receptors or messengers are different or whether there are interactions with other regulatory systems, as suggested by the work of Kelly et al,5 who found that exposure to β-adrenergic agonists blocks the ability of very low concentrations of endothelin to alter the contraction of single cardiac myocytes.
This research was supported by National Institutes of Health grant HL RO1 16010 to Dr Winegrad.
- Received December 11, 1995.
- Accepted February 13, 1996.
- © 1996 American Heart Association, Inc.
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