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1 Department of Molecular and Cellular Physiology, University of Cincinnati College of Medicine, Cincinnati, Ohio 45267; and 2 Dermatology Division, Department of Medicine, University of California at Los Angeles, Los Angeles, California 90095
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Force generated by smooth muscle cells is believed to result from the interaction of actin and myosin filaments and is regulated through phosphorylation of the myosin regulatory light chain (LC20). The role of other cytoskeleton filaments, such as microtubules and intermediate filaments, in determining the mechanical output of smooth muscle is unclear. In cultured fibroblasts, microtubule disruption results in large increases in force similar to contractions associated with LC20 phosphorylation (15). One hypothesis, the "tensegrity" or "push-pull" model, attributes this increase in force to the disruption of microtubules functioning as rigid struts to resist force generated by actin-myosin interaction (9). In porcine coronary arteries, the disruption of microtubules by nocodazole (11 µM) also elicited moderate but significant increases in isometric force (10-40% of a KCl contracture), which could be blocked or reversed by taxol (a microtubule stabilizer). We tested whether this nocodazole-induced force was accompanied by changes in coronary artery stiffness or unloaded shortening velocity, parameters likely to be highly sensitive to microtubule resistance elements. Few changes were seen, ruling out push-pull mechanisms for the increase in force by nocodazole. In contrast, the intracellular calcium concentration, measured by fura 2 in the intact artery, was increased by nocodazole in parallel with force, and this was inhibited and/or reversed by taxol. Our results indicate that microtubules do not significantly contribute to vascular smooth muscle mechanical characteristics but, importantly, may play a role in modulation of Ca2+ signal transduction.
coronary artery; isometric force; nocodazole; taxol
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INTRODUCTION |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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THE CYTOSKELETON OF ANIMAL CELLS is primarily composed of three filament systems (actin, microtubules, and intermediate filaments) as well as proteins that associate and interact with these filaments. The force generated by smooth muscle cells is believed to result from the interaction of actin and myosin filaments and is regulated through phosphorylation of the myosin regulatory light chain (LC20). However, the role of other filament systems in modifying the mechanical output of smooth muscle has received little attention.
In nonmuscle cells, such as fibroblasts and endothelial cells, microtubule disruption results in increases in contractility, which can be of similar magnitude to contractions associated with the actin-myosin system at near complete phosphorylation of LC20 (13-15). Recently, several studies (17, 25, 28) have shown that microtubule disruption increases force in intact arteries, and one study (2) reported a decrease in force. In this study, we report that treatment of coronary arteries with nocodazole, an inhibitor of microtubule assembly, significantly increases isometric force.
A model for the organization of the cytoskeletal structure, which is consistent with the increase in force concomitant with microtubule disruption in many cell types, has been termed the "tensegrity" or "push-pull" model (7, 9). In this model, microtubules are postulated to function as rigid struts opposing the force generated by myosin. Disruption of this postulated internal resistive force is predicted to allow a greater fraction of the force generated by the actin-myosin interaction to be transmitted to external structures. Although this model can explain the increase in force observed upon microtubule disruption, direct mechanistic evidence supporting this hypothesis is lacking.
We investigated possible mechanisms through which microtubule disruption might enhance contraction in arterial rings, with emphasis on the relevance of the tensegrity model to smooth muscle. To this end, we measured the effects of microtubule disruption on isometric force, stiffness, and shortening velocity in porcine coronary arteries. The latter two mechanical parameters are sensitive to mechanical resistance and/or internal opposing forces. We report few changes in these mechanical parameters, which would be anticipated to be highly sensitive to the type of structural role for microtubules envisioned by tensegrity theories.
We thus further tested whether the observed increase in force with microtubule disruption was paralleled by an increase in intracellular calcium, a key second messenger-regulating force in smooth muscle. The intracellular calcium concentration ([Ca2+]i) was increased by nocodazole, and this was reversed by taxol, which stabilizes microtubules. Our results suggest that microtubules may not significantly contribute to vascular smooth muscle mechanical characteristics but, importantly, may play a hitherto unrecognized role in modulation of Ca2+ signal transduction.
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METHODS |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Preparation of Arterial Rings
Porcine hearts, obtained shortly after slaughter, were rinsed of blood and placed in a cold (4°C) bicarbonate-buffered, physiological salt solution (PSS), which contained the following (in mM): 122 NaCl, 4.73 KCl, 25 NaHCO3, 1.19 MgCl2, 0.02 EDTA, 1.19 KH2PO4, 11.10 glucose, and 2.50 CaCl2. When aerated with 95% O2-5% CO2, the pH of the PSS was 7.3-7.4 at 37°C. The distal portions of the left anterior descending coronary artery were dissected and placed in cold PSS. Arteries were then cleaned of fat and connective tissue and cut into 5-mm segments or 1-mm circumferental strips. The arterial wall thickness was between 300 and 500 µm. For studies on endothelium-denuded arteries, the segments were everted, and both preparations were denuded of endothelium by gentle rubbing (22).Isometric Force Measurements
Arterial rings were suspended isometrically on two stainless steel posts, one of which was attached to a Kistler-Morse DSK force transducer. The rings were placed inside a water-jacketed chamber filled with 15 ml of PSS that was maintained at 37°C. The chambers were bubbled with 95% O2-5% CO2 to maintain a physiological pH of 7.4. Tension was recorded on a Linear Instrument 1200 recorder and an IBM compatible computer.After the tissues were isometrically mounted, a period of 1 h was used to equilibrate the rings at a load of 40 mN. This load was chosen on the basis of prior experiments (26) to set a tissue length in the optimal range for maximum tension development. After the equilibration period had passed, three contraction-relaxation cycles were performed using a final concentration of 80 mM KCl to ensure reproducible force responses. The stable response to 80 mM KCl was used as 100% to normalize the force responses for each ring, except as otherwise noted in the text.
The integrity of the endothelium or its removal was validated by testing for the endothelium-dependent relaxation to substance P (0.01 µM) during the second contraction-relaxation cycle. Arteries that inappropriately responded were discarded from this study.
Force-Velocity and Stiffness Measurements
Arteries were cut into 1- to 2-mm circumferential strips and mounted between glass posts with a cyanoacrylate glue. One post was connected to an AME 801 silicon strain-gauge (SensoNor) force transducer, and the other post was connected to a post whose position, and therefore the length of fiber, was controlled by Cambridge Technologies ergometer. The artery strips were bathed in MOPS-buffered PSS (MOPS-PSS) that contained the following (in mM): 140 NaCl, 4.7 KCl, 1.2 NaH2PO4, 0.02 EDTA, 1.2 MgSO4, 2.5 CaCl2, 5.5 dextrose, and 20 MOPS; pH 7.4 at 37°C. The MOPS-PSS was used for these experiments to eliminate the mechanical noise associated with the bubbling with CO2 required for a bicarbonate-based PSS.The fiber length was adjusted to set an initial resting tension of 0.3-0.4 mN, which was 10-20% of the maximum isometric force. This length is in the range for optimal force development. The artery was then subjected to several contraction-relaxation cycles and stimulated with 29 mM KCl until the force generated was reproducible. After a steady-state force was achieved, stiffness (or its inverse, compliance) was measured by imposition of rapid (<1 ms) shortening steps [5-15% of the initial length of artery strip (Lo)], and measurement of the subsequent minimum value of force was obtained. A series of eight step changes at 60-s intervals constituted an experimental set. The slope of initial linear region of the relation between the change in force and imposed length step was taken as the stiffness.
Maximum shortening velocity was determined by imposing constant shortening velocities on the artery after achievement of a stable force in response to KCl (29 mM). The force at 3 s after initiation of the ramp was measured. A series of eight different velocities at 60-s intervals were used for each individual force-velocity relation. The force-velocity relations were fitted with the Hill equation [(F + a) × (V + b) = b × (Fo + a), where F is force, Fo is the initial force of the artery, V is velocity, and a and b are variables] using a nonlinear least squares routine (Origin), and the maximum velocity (Vmax) was taken as the value at zero force.
Intracellular Calcium Measurements
[Ca2+]i was measured with the fluorescent probe fura 2-acetoxymethyl ester (AM), as previously described (21). Arterial rings were everted and mounted on a stainless steel bracket, which kept the artery isometric. The artery was incubated for 3 h at 37°C in well-stirred MOPS-PSS containing 12.5 µM fura 2-AM, 25 µg/ml pluronic F-127 (TM BASF Wyandotte), and ~2 mg/ml bovine serum albumin. After they were incubated, the tissues were rinsed in bicarbonate-buffered PSS for 30-60 min. The isometric artery ring was then attached to a Teflon mount with an inflow and outflow port and fitted into an acrylic spectrofluorimeter cuvette; the final chamber volume was 2.4 ml. The cuvette was connected to a Cole-Palmer circulating pump via polyethylene tubing and perfused (10 ml/min) with PSS at 37°C. The cuvette was placed in a water-jacketed holder maintained at 37°C and aligned so that the tissue was perpendicular to the light path of the excitation beam of a PTI Delta Scan-1 (Photon Technology International, South Brunswick, NJ) dual-wavelength spectrofluorimeter configured for front-face measurements. Fluorescence was excited at wavelengths of 340 and 380 nm, with emission measured at 510 nm. In control experiments, the background fluorescence in arteries not loaded with fura 2 was measured. The background fluoresence was not affected by the interventions used in this study. The emission intensities for the 340 and 380 nm excitation were ratioed and normalized to 0% for resting muscle and 100% for the maximum KCl-stimulated response. This normalization protocol was chosen given the uncertainties in absolute calibration (11) and the near-linear relation between the 340-to-380-nm ratio and [Ca2+]i in the 50-1,000 nM range (19). More details and comparisons of different normalization protocols have been previously reported (4).Chemicals
Chemicals were the highest grade and obtained, except where noted, from Sigma Chemical (St. Louis, MO). Nocodazole, taxol, and fura 2-AM (Molecular Probes, Eugene, OR) were all dissolved in DMSO. 9-11-Dideoxy-11
,9-epoxymethanoprostaglandin
F2
(U-46619) was dissolved in ethanol. Substance P was
dissolved in H2O. Vehicle controls for ethanol and DMSO
showed no effects as long as the volume added was 
0.1% of the bath volume.
Analysis of Data
The values given are the arithmetic means ± SE; n values for sample size represent the number of hearts from which arteries were taken. Differences between groups were assessed using a standard ANOVA or two-tailed Student's t-test for paired data as appropriate. A significance level of P < 0.05 was chosen for rejection of the null hypothesis.| |
RESULTS |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Effects on Isometric Force
Our initial experiments were conducted with maximum KCl stimulation (50 mM). The rationale here was that if microtubules provided a compressible stratum opposing actin-myosin generated force, full activation of the myosin motors would maximize the degree of load shifting upon disruption of microtubules and hence optimize the measured force response. However, in eight rings from four arteries studied (4 endothelium-denuded and 4 intact arteries), no effects of nocodazole (11.1 µM) on isometric force were observed.We then studied submaximal stimulation using 10 and 20 mM KCl and, for
receptor-mediated stimulation, 10
8 M U-46619 (a
thromboxane A2 analog). Because isometric force is often not maintained
in these coronary arteries with submaximal stimulation, paired rings
from the same artery were employed as vehicle and time controls in the
experimental design; a typical recording is shown in Fig.
1. The addition of nocodazole to these submaximal contractions elicited a slowly developing but sustained contraction, whereas controls tended to decline over the same time
period. Taxol or the vehicle (DMSO) per se had little effect. The
response to nocodazole was blunted by pretreatment with taxol; a
statistical summary of these experiments is discussed later. A
scattergram of data from these types of experiments plotting the
responses to nocodazole against the absolute level of stimulated isometric force before the nocodazole or vehicle treatment is shown in
Fig. 2. All arteries studied in this
protocol were denuded of endothelium. The control tissues declined in
force, whereas the paired arteries treated with nocodazole increased in
force. The magnitude of the responses to nocodazole was not dependent on the level of preexistent force for these submaximal stimulation conditions.
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To assess whether this response to nocodazole was mediated by
microtubules, taxol (22 µM) and a vehicle control were given 30 min
before the addition of nocodazole using a paired ring protocol similar
to that just described. These results for endothelium-denuded arteries
stimulated with 10 or 20 mM KCl are summarized in Fig. 3, A and B. Taxol
(22 µM) alone had little effect, with a trend to a slight decrease in
force that was not statistically significant. The time and vehicle
controls showed little change. Nocodazole elicited statistically
significant increases, as in the previous experiments (Fig. 2), but
importantly, after preincubation with taxol, the effects of nocodazole
were blocked. The largest increase in response to nocodazole, ~40%
of the KCl maximum contraction, was seen when the artery was pretreated
with 10 mM KCl. The increase in response to nocodazole was smaller
(5%) but significant when precontracted with higher stimulus levels
(20 mM KCl). Thus there appears to be an optimal level of stimulation
for maximization of the increases in force in response to nocodazole.
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Similar but smaller effects were observed in the absence of
stimulation. Nocodazole induced a small but statistically significant increase in force that was blocked by taxol. These results are summarized for eight pairs of endothelium-intact rings in Fig. 4A. However, as in the above
experiments with endothelium-denuded arteries, neither an intact
endothelium nor prestimulus (Fig. 4B) was necessary for
nocodazole to elicit an increase in force. The effects of nocodazole
were larger in the denuded preparations. This may be attributable to
the greater spontaneous tone in endothelium-denuded arteries
(22).
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Effects on Stiffness and Velocity
Nocodazole consistently produced increases in force, which were dependent on the level of stimulation. These results are consistent with push-pull-type mechanisms in which some level of activation of the actin-myosin force generation is required for disruption of microtubules to lead to an increase in externally measured force. If microtubules form a rigid but compressible springlike network, one would anticipate major contributions of microtubules to arterial stiffness.Stiffness.
Stiffness was measured after achieving a near-maximal, steady-state
force in response to 29 mM KCl. Rapid (<1 ms) step
changes in length were imposed, and the subsequent decrease in force
measured. Typical responses are shown in Fig.
5A. After the step was
imposed, force decreased to a peak value in ~20 ms followed by a
small increase. This behavior is typical for activated smooth muscle (20). A plot of the peak force responses against the
imposed step length change is shown in Fig. 5B. Compliance
estimated from the linear region averaged 0.057 ± 0.006 Lo/Fo (or a stiffness of 17.5 Fo/Lo). This value is comparable to
that reported for other intact smooth muscle tissues (24).
Importantly, neither nocodazole nor taxol had any effects on the artery
compliance and/or stiffness, as summarized in Fig. 5 for six arteries.
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Velocity-force relations.
If microtubules form a compressible, internal load, the maximal
shortening velocity should be particularly sensitive to disruption or
stabilization of microtubules. This is due to the hyperbolic relation
between force and velocity. For a typical smooth muscle, the
force-velocity relation would predict that loss of an internal load of
10% of the maximal isometric force would increase
Vmax by 40%. These relations were studied by
imposing constant speed decreases in length and measurement of the
consequent force response. Figure
6A shows a typical
experimental record for a single fiber composed of individual force
responses to eight imposed shortenings of varying speeds. Force can be
seen to continuously decline with the duration of shortening, similar
to the behavior reported for rat aortas (18). Thus, at
each point in time, one has a family of force-velocity relations, as
shown in Fig. 6 (inset). In subsequent experiments, the
force at 3 s was taken for all force-velocity relations. This
point in time was chosen because it provided the widest range of
measurable points over the differing experimental conditions.
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Effects on Intracellular Calcium
It is possible that the nocodazole-induced increase in force is related to a microtubule modulation of signal transduction rather than a direct mechanical effect. We investigated whether intracellular calcium, a key second messenger in smooth muscle contraction, underscored the increase in force using fura 2 ratiometric dye techniques. As shown in Fig. 7A, the increase in isometric force elicited by nocodazole is reversed by the addition of taxol. These effects on force are paralleled by those of [Ca2+]i. These data for five arteries are summarized in Fig. 8. Though relatively small in these unstimulated conditions, the effects of nocodazole and taxol on [Ca2+]i are consistent with the observed changes in force (Fig. 4).
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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A major finding of this study is that disruption of microtubules elicits an increase in isometric force in vascular smooth muscle. The effects of nocodazole were not limited to porcine coronary arteries, because similar increases in force were also observed in canine renal arteries (Kolodney and Chapnick, unpublished observations). The increase in force in response to nocodazole was dependent on the level of stimulation. The effects were greatest (40% of the maximum response to KCl) when pretreated with 10 mM KCl, were significantly decreased (5% of the maximum response to KCl) at 20 mM, and abolished at 50 mM. In the near absence of tone in unstimulated, endothelium-intact arteries (22), the effects were small (1-2% of the maximum response to KCl), whereas in endothelium-denuded arteries, nocodazole increases in force were somewhat greater (10% of the maximum response to KCl). The potentiation of agonist-induced force by microtubule disruption is consistent with some (17, 25, 28) but not all vessels studied (2).
An underlying assumption is that nocodazole specifically disrupts microtubules. Disruption of microtubules by nocodazole has been well documented in the cell literature (10). It has also been shown that taxol blocks and reverses microtubule disruption by nocodazole in fibroblasts (5). In particular, Platts et al. (25) showed that nocodazole disrupted microtubules in cultured vascular smooth muscle cells. Importantly, they also reported that nocodazole decreased microtubule density in intact arterioles in terms of reduction of fluorescently labeled microtubules. We also used confocal immunofluorescent imaging of 5-µm cryostat sections of the pig coronary artery. In control sections, tubulin was mainly present in short linear segments, whereas in nocodazole-treated cells, the tubulin was more diffusely distributed. Though qualitative, these data indicate that nocodazole alters tubulin distribution in porcine coronary arteries. In addition, we showed that pretreatment with taxol, which is known to stabilize microtubules, blocked the contractile effects of nocodazole. Because taxol is structurally and pharmacologically different from nocodazole, we feel this argues fairly strongly for the specificity of microtubule involvement.
The mechanism of modulation of vascular contractility by microtubules is of additional interest in light of models proposed for contractility in nonmuscle cells. In our study, several lines of mechanical evidence suggest that tensegrity models, in which a microtubule network opposes force produced by the actin-myosin interaction, are not applicable in smooth muscle.
Effect of Myosin Activation on Microtubule-Mediated Contraction
If microtubules mechanically opposed a fraction of myosin-generated force, the magnitude of the load borne by microtubules should increase as myosin-generated force increases. Thus the tensegrity model would predict a larger force increase upon microtubule disruption at larger levels of preexisting myosin-generated force. In contrast to this prediction, the effects of nocodazole showed little dependence on preexisting active force per se in preparations submaximally stimulated (Fig. 2). On the other hand, the stimulus level itself was important, although difficult to tease out from its relation to force. The finding that nocodazole has little effect on maximally contracted vessel segments is consistent with a Ca2+-myosin light chain kinase activation mechanism. Once maximum force is achieved, further increases in [Ca2+]i (for example, in response to nocodazole and subsequent LC20 phosphorylation) would have little effect on force.We also employed two additional types of mechanical measurements, potentially more sensitive to microtubule disruption than isometric force.
Mechanical Stiffness
In muscle biophysics, this parameter is often taken as an index of the number of attached myosin crossbridged to actin. However, in general, smooth muscle compliance is greater than that which could be solely attributable to crossbridges (24). Stiffness measured in these studies is in the range of those reported for whole arterial tissue. For our present purposes, we could not detect any changes in stiffness as a function of microtubule assembly and/or disassembly.Contraction Velocity
If microtubules functioned as rigid elastic elements mechanically in parallel with the contractile apparatus, microtubules would be expected to bear a larger load as the cells shortened and the cytoskeleton was compressed. In addition, due to the hyperbolic nature of the relation between velocity and force, changes in Vmax are amplified, i.e., relative small changes in load can produce substantial reductions in Vmax. Both these factors should combine to result in a greater sensitivity of velocity-to-microtubule status relative to that of isometric force. In contrast to this prediction of the tensegrity model, microtubule disruption affects shortening velocity in direct proportion to its effects on isometric force.On the basis of mechanical evidence, microtubules do not make a significant contribution to arterial wall mechanical properties. Thus a mechanism for modulation of contractility as envisioned in the tensegrity model would not appear to apply to vascular smooth muscle.
Effects of Endothelium
Our results indicate that the contractile effects of nocodazole are not mediated by the endothelium. In fact, both arterial segments with an intact endothelium (porcine coronary and canine renal arteries) showed a relatively smaller contraction upon microtubule disruption relative to endothelium-denuded segments. However, submaximally contracted arterial segments (with the endothelium intact) respond to microtubule disruption with a contraction equivalent to those of endothelium-denuded preparations. This result is consistent with the predictions of the tensegrity model, which would require active force generation. However, an alternative explanation for this finding is that the inhibitory activity of the endothelium on arterial basal tone (22) may mask the relatively small contractile effects of microtubule disruption.Role of Intracellular Calcium
Activation of myosin light chain kinase by Ca2+-calmodulin is an important regulatory mechanism for smooth muscle contraction (6). We demonstrated that in the absence of stimulation, microtubule disruption is associated with a small but significant increase in intracellular free Ca2+ and that taxol reverses this increase. The increase in [Ca2+]i with microtubule disruption was ~15% of the maximal KCl-stimulated increase. Importantly, the increase in [Ca2+]i was proportional to the nocodazole-stimulated force increase, supporting our hypothesis that increases in [Ca2+]i underlie the effects of nocodazole.Mechanism of Nocodazole-Stimulated Contraction
Studies of nonmuscle cells (1, 3, 7, 8, 12) indicate that microtubule polymerization and morphology are modulated in response to agonist or mechanical stimulation. Although microtubule function has mainly been studied in nonmuscle cells, our data indicate that microtubules may have a role in the regulation of smooth muscle contraction as well. Changes in cytoskeleton stiffness in cultured bovine pulmonary artery smooth muscle cells have been postulated to play a role in the control of vascular smooth muscle cell contractility and pulmonary hypertension (16). Although a load-bearing role for microtubules could possibly explain the mechanical effects of microtubule disruption, our findings are more consistent with this contraction being mediated through increasing [Ca2+]i. Any mechanism at this stage is speculative, but there is evidence for interactions between microtubules and G protein-coupled receptors (27). Our results indicate that microtubule metabolism may play a novel role in modulation of vascular smooth muscle function mediated by altered Ca2+ signaling.The potential physiological significance of the increases in force with microtubule disruption is speculative at this point. Some level of vascular tone is needed to see significant effects, thus microtubule metabolism is more likely to play a modulatory- or sensitization-type role than that of a direct contractile agonist. Whereas the changes seen were moderate, it should be kept in mind that changes in force are amplified in terms of their effect on blood flow according to Poiseuille's relation. For example, a decrease in vessel radius of 15% would reduce blood flow by 50%. Thus microtubule metabolism can play a significant role in regulation of vessel tone based on the changes in force observed in this study.
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ACKNOWLEDGEMENTS |
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This work was supported in part by the National Institutes of Health Grant 23240 and the National American Heart Association Grant 92007130.
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FOOTNOTES |
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Address for reprint requests and other correspondence: R. J. Paul, Dept. of Molecular and Cellular Physiology, Univ. of Cincinnati College of Medicine, Cincinnati, OH 45267-0576 (E-mail: Richard.Paul{at}UC.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 6 December 1999; accepted in final form 17 May 2000.
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DISCUSSION
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E) arteries. The experimental protocol and
nomenclature are similar to that shown in Fig. 
L, change in length; del represents a
change in the given parameter (del Fo, change in initial
force).
