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MUSCLE CELL BIOLOGY AND CELL MOTILITY
Cardiovascular Division, Department of Internal Medicine, University of Virginia Health System, Charlottesville, Virginia
Submitted 7 March 2007 ; accepted in final form 21 June 2007
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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cytoskeleton; hysteresivity; latch hypothesis; vascular smooth muscle
In other types of smooth muscle, there are data suggesting that systems beyond crossbridge phosphorylation are involved in force regulation. One system involves stimulus-induced tyrosine phosphorylation of paxillin and resultant actin polymerization (reviewed in Ref. 4 and 38). Most of the smooth muscle work on stimulus-induced paxillin phosphorylation and actin polymerization was done in airway smooth muscle (reviewed in Ref. 12). Paxillin was first found to be phosphorylated on tyrosine during acetylcholine-induced contraction of intact trachealis smooth muscle (25). The time course of paxillin tyrosine phosphorylation in trachealis was similar to the time course of contraction (41), suggesting potential regulation of contraction. Reductions in extracellular Ca2+ attenuated acetylcholine-induced increases in crossbridge phosphorylation and contraction but did not alter acetylcholine-induced increases in paxillin tyrosine phosphorylation (21), suggesting that 1) paxillin tyrosine phosphorylation is not regulated by Ca2+ or crossbridge phosphorylation and 2) paxillin phosphorylation alone is not sufficient for trachealis contraction. Carbachol increased the F-actin content of cultured human airway smooth muscle (35), a process involving G protein mediated activation of rho (16). In intact trachealis, contractile stimulation increased actin polymerization as measured by an increase in F-actin content (20). These findings are consistent with the hypothesis that stimulus-induced paxillin phosphorylation could potentially modulate trachealis contraction by altering actin polymerization.
A basal level of actin polymerization appears to be necessary for the initiation of contraction in tracheal smooth muscle. Cytochalasin B and D, agents that reduce actin polymerization, attenuated carbachol-induced increases in [Ca2+]i, crossbridge phosphorylation, and contraction in bovine trachealis (37). However, another group found that latrunculin-A, another agent that inhibits actin polymerization, reduced force without altering crossbridge phosphorylation in canine trachealis (20). These data suggest two mechanisms for short-term actin inhibition to inhibit contraction: 1) reduced [Ca2+]i/crossbridge phosphorylation and/or 2) reduced actin polymerization. On the basis of a strategy of antisense, siRNA, and mutant proteins, Gunst and colleagues (see Ref. 34 for review) showed that long-term (days) interference with some of the proteins in the actin polymerization pathway (FAK, paxillin, CrkII, Cdc42, and N-WASp) interfered with actin polymerization and contraction without altering crossbridge phosphorylation values measured 5 min after activation. These experiments showed that long-term interference with actin polymerization can attenuate contraction despite stimulus-induced increases in Ca2+ and crossbridge phosphorylation.
Less is known about stimulus-induced paxillin phosphorylation and actin polymerization in arterial smooth muscle. In cells cultured from aorta, angiotensin II induced paxillin tyrosine phosphorylation and increased actin-containing stress fibers (39), a process that may involve c-Src (17). In intact freely floating mesenteric artery, norepinephrine induced a slow increase in paxillin tyrosine phosphorylation that was maximal at 2.5 min (24, 42). This result suggests that paxillin tyrosine phosphorylation could increase slower than force; however, arterial length was not controlled and short length is known to attenuate paxillin phosphorylation in airway smooth muscle (32, 33). Cytochalasin D attenuated rat pulmonary artery contraction (1). In cerebral arteries, cytochalasin D reduced myogenic tone and decreased actin polymerization, while jasplakinolide, an agent that stabilizes F actin, increased myogenic tone and increased actin polymerization (7). Crossbridge phosphorylation was not measured in either of these studies, so the mechanism responsible for inhibition of contraction is not known. We found that sustained histamine stimulation of swine carotid artery increased F-actin content (19); however, it is not known whether histamine-induced actin polymerization was necessary for the contraction.
Oscillatory changes in muscle length during contraction can provide information about the dynamic processes present in the cytoplasm. For example, the real part of stiffness (also known as the storage modulus G') is felt to be a measure of the number of attached crossbridges (22). Fredberg et al. (5, 18) suggest that the biophysical measures hysteresivity, noise temperature, and phase angle can also provide information about the physical properties (rheology) of the cell. Noise temperature can be understood within the theory of soft glassy rheology: "glasses are substances with the structural disorder of a liquid but the stiffness of a solid" (13). Glasslike stiffness is felt to originate from interactions between neighboring molecules that tend to trap each molecule in an energy well. In a glass, "structural rearrangements become limited by long-lived microconfigurations in which the system becomes trapped" (36). Thermal energy and intermolecular interactions can cause a molecule to "hop" out of its energy well, a process that will reorganize the microconfiguration of energy wells in the system (13, 36). Noise temperature is proposed to be the quantitative level of nonthermal "random agitation (noise) (that) can excite a metastable element, causing it to hop out of its well, in turn triggering secondary rearrangements and hopping events that ripple through the system" (13). A noise temperature of 1 indicates that the system approximates a Hookean elastic solid with no internal friction, no "hopping," and no rearrangements. A noise temperature of 2 indicates that the system approximates a Newtonian viscous fluid (18), with internal friction, frequent "hopping," and flow, i.e., frequent rearrangement. Therefore, the noise temperature of a smooth muscle can viewed as measure of whether a tissue is more "fluid" (a high noise temperature) or "solid" (a low noise temperature). Noise temperature is closely related to phase angle and hysteresivity (9, 13). A noise temperature of 1 corresponds to a phase angle of 0 and a hysteresivity of 0.
In intact bovine trachalis, hysteresivity, a measure of the area of the length-force loop during length oscillation, was low at rest, high during the rapid phase of contraction, and low during the sustained phase of contraction (11). In isolated trachealis cells, Fredberg et al. (2, 9) measured the length-force response when magnetic beads attached to the cell membrane were oscillated in a magnetic field. Noise temperature (the slope of relation between the real part of stiffness and oscillatory frequency) and phase angle (the imaginary part of stiffness also known as the loss modulus G'') changed with treatments that altered the biophysical characteristics of these cells. Specifically, they found 1) increased noise temperature with cyclic nucleotides and agents that reduced actin polymerization and 2) decreased noise temperature with contractile agonists and agents that increased actin polymerization.
This study had two goals. The first goal was to determine whether stimulus induced Y118 paxillin phosphorylation and actin polymerization could have a role in the initial or sustained phases of swine carotid artery contraction, a result that could have implications in the latch hypothesis. The second goal was to measure noise temperature, hysteresivity, and phase angle during the initial and sustained phases of swine carotid artery contraction to evaluate the rheology of arterial smooth muscle contraction. Swine carotid arterial tissues were depolarized with high K+ or stimulated with histamine and the biochemical (Y118 paxillin polymerization, crossbridge phosphorylation, and actin polymerization) and mechanical [noise temperature, hysteresivity, phase angle, real stiffness (G'), and stress] response evaluated.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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Biochemical measurements. Y118 paxillin and crossbridge (Ser19-MRLC) phosphorylation was determined in swine common carotid artery rings mounted isometrically at 1.0 Lo as described above. They were then treated for the same time as the F actin and stiffness experiments described below. Rings were then frozen in acetone dry ice, and homogenized in 1% SDS, 10% glycerol (vol/vol), and 20 mM dithiothreitol (0.22 ml/mg tissue dry wt) as described (29).
The level of Y118 paxillin phosphorylation was determined by loading tissue homogenates on two 10% SDS electrophoresis gels, followed by immunoblotting on PVDF for total paxillin (1:500 primary antibody from Zymed Laboratories no. 03-6100 and 1:15,000 secondary antibody from Amersham no. NA931V) and Y118 phosphorylated paxillin (1:1,000 primary antibody from Biosource no. 44-722G and 1:15,000 secondary antibody from Amersham, no. NA934V). In preliminary experiments, the antibody for Y31 phosphorylated paxillin (Biosource no. 44-720G) did not immunostain swine carotid artery homogenates. To minimize blotting and detection errors, all homogenates (including the unstimulated control) from the rings originating from the same artery were loaded on the same gel. Phosphorylation for each ring was calculated as the ratio of Y118 phosphorylated paxillin to total paxillin immunostaining. Phosphorylation for each ring was then normalized to the level of Y118 phosphorylation in the unstimulated control with the control value = 1.0.
The level of crossbridge (Ser19-MRLC) phosphorylation was determined by isoelectric focusing and immunoblotting as described (29). Three dilutions of homogenates were loaded to ensure that the enhanced chemiluminescence detection system was in the linear range (28). Phosphorylation is reported as mol Pi/mol protein.
Actin polymerization.
The relative amount of filamentous actin (F-actin) vs. total actin in swine carotid arterial rings was determined with a commercial kit (BK037) from Cytoskeleton (Denver, CO). Lysis/F-actin stabilization buffer contained 50 mM piperazine-N,N'-bis(2-ethanesulfonic acid), 50 mM NaCl, 5 mM MgCl2, 5 mM EGTA, 5% glycerol, 0.1% Nonidet P-40, 0.1% Triton X-100, 0.1% Tween 20, and 0.1% 
-mercapto-ethanol, pH 6.9, at 30°C. Immediately before use of lysis/F-actin stabilization buffer, 10 µl/ml of 100 mM ATP and 10 µl/ml of protease inhibitor cocktail, composed of (in µg/ml) 1 pepstatin, 1 leupeptin, 10 benzamidine, and 500 tosyl arginine methyl ester, was added. Arterial rings were mounted, pharmacologically treated as described above, and then frozen with tongs cooled in liquid nitrogen. The frozen carotid was weighed and pulverized in a liquid nitrogen-chilled ceramic mortar and pestle. The resultant powder was divided equally between two chilled 1.5-ml Eppendorf tubes (the assay was performed in duplicate to increase accuracy). Fifty microliters per milligram per tissue wet weight of Lysis/F-actin stabilization buffer with ATP and protease inhibitor cocktail (prewarmed at 30°C) was added to each tube. The tubes were then immediately vortexed and incubated at 30°C for 10 min. They were then immediately centrifuged at 16,000 g for 1 h at room temperature in an Eppendorf 5415C microcentrifuge. The supernatants were pipetted off the pellets, placed in empty 1.5-ml Eppendorf tubes, and then put on ice. The pellets were resuspended to the same volume as the supernatants using ice-cold nanopure water plus 1 µM cytochalasin D. The pellet samples were left on ice (with vortexing every 15 min) for 1 h to dissociate F-actin. One-half- and one-quarter-strength dilutions of each pellet and full strength supernatant were then separated on 12% SDS-polyacrylamide gels, blotted to nitrocellulose, immunostained with a rabbit commercially produced polyclonal anti-actin antibody (AAN01, Cytoskeleton) and detected with enhanced chemiluminescence. Images were analyzed with UnScanIt software. Intensities were corrected for dilution and the amount of F-actin as a percent of total actin calculated. The dilutions were corrected for offset and saturation errors as we have done with analysis of crossbridge phosphorylation (29, 40).
Stiffness, noise temperature, and hysteresivity measurements.
Both ends of swine carotid tissue strips were mounted in 1.2-mm-diameter aluminum foil cylinders with cyanoacrylate glue. One cylinder was attached to an adjustable length stationary rod and the other cylinder to the lever arm of a model 310B dual mode lever operated by Dynamic Muscle Control software (Aurora Scientific, Aurora, ONT, Canada). After the tissue length was set to Lo as described above, tissues were treated pharmacologically and at various times oscillated with sine wave changes in length (0.5% Lo at 0.3, 1, 3, 10, and 30 Hz) and the resulting change in force measured (9, 10). Force values were normalized as stress values (force per cross-sectional area, in N/m–2). G' was calculated as the peak-to-peak change in stress observed with 10-Hz oscillations (this was valid because calculated phase angles were all less than 10°, i.e. cos 10° = 
1). Noise temperature was calculated as 1 + (the least-squares regression slope of ln G' as a function of ln oscillation frequency) as described in RESULTS and in Fig. 1. Hysteresivity was calculated from plots of the change in stress as a function of change in length during 1-Hz oscillations (described in RESULTS and Fig. 2). We calculated hysteresivity as the difference between the zero length intercepts of stress during the stretch and release phases of the oscillation. Hysteresivity was then normalized to mean stress during the oscillation so that units are fractional. Phase angle (the imaginary part of stiffness also known as the loss modulus G'') was calculated using MATLAB (MathWorks, Natick, MA) from the relative delay in the zero intercept of the force oscillation compared with the zero intercept of the length oscillation (with 360° as a full cycle).
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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The relative slope of G' vs. frequency is best seen on the normalized plot of raw data (Fig. 1, right). Tissues at rest and those depolarized with 109 mM K+ for 1 min had a steeper dependence of normalized G' on frequency when compared with tissues depolarized for 25 min. These data show that noise temperature (slope) was higher in tissues at rest and depolarized for 1 min compared with tissues depolarized for 25 min.
Measurement of hysteresivity in intact swine carotid artery. When a sinusoidal length change is imposed on one end of a muscle, a plot of force (stress) vs. length typically exhibits an hysteresis loop. Hysteresivity can be measured as the area inside the loop or the height of the loop at its midsection (9). Figure 2 shows representative tracings from one of the tissues studied in Fig. 1. The lower tracings are the measured stress and the upper tracings are stress normalized to a value of 5, both plotted as a function of change in length. For this study, hysteresivity was measured as the height of the loop, specifically the stress during the stretch (increasing length) minus the stress during the release (decreasing length) at the midpoint (zero, the vertical line in Fig. 2) of the sinusoidal length change normalized to mean stress. Hysteresivity was higher in tissues at rest and depolarized with 109 mM K+ for 1 min compared with tissues depolarized for 25 min (Fig. 2).
Relation between noise temperature and hysteresivity. There was a significant linear dependence of hysteresivity on noise temperature in swine carotid tissues at rest, with K+ depolarization, and with histamine stimulation (Fig. 3, top; r2 = 0.36, P < 0.001). There was more variability in hysteresivity in resting tissues (Fig. 3, top, open squares). There was also a significant linear dependence of phase angle (the imaginary part of stiffness also known as the loss modulus G'') on noise temperature in swine carotid tissues at rest, with K+ depolarization, and with histamine stimulation (Fig. 3, bottom; r2 = 0.43, P < 0.001). These data suggest that noise temperature, hysteresivity, and phase angle were similar measures in swine carotid artery, as predicted by Fabry et al. (9).
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G' is an estimate of the number of attached crossbridges. There was a significant linear dependence of G' on stress in tissues at rest, depolarized, or stimulated with histamine (r2 = 0.96, P < 0.001; Fig. 6, top). These data suggest that the amount of force per attached crossbridge was not altered during the initial or sustained phase of contraction.
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A detailed time course of a maximal K+ contraction. Figure 7 shows a full characterization of the time course of a 109 mM K+ induced swine carotid artery contraction. Initially, high K+ induced rapid increases in crossbridge phosphorylation, G', and stress (crossbridge phosphorylation was significantly increased by 20 s and G'/stress by 40 s). Crossbridge phosphorylation, G', and stress reached near maximal values during the first minute of contraction. In the first minute of contraction, there were no significant changes in Y118 paxillin phosphorylation, the relative amount of F actin, or noise temperature/hysteresivity/phase angle. With sustained stimulation, i.e., >1 min, crossbridge phosphorylation decreased to intermediate values, while G' and stress remained high. There was a significant increase in Y118 paxillin phosphorylation by 3 min, a significant increase in the relative amount of F actin by 10 min, and a significant decrease in noise temperature and hysteresivity by 90 and 120 s, respectively. These data show a clear separation of force development from changes in Y118 paxillin phosphorylation, actin polymerization, noise temperature, and hysteresivity.
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DISCUSSION |
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Our data differs from that found in airway smooth muscle, in which the time course of paxillin tyrosine phosphorylation paralleled the time course of the contraction (41). There are clear physiological differences between airway and arterial smooth muscle. Airway smooth muscle is on the extreme continuum of smooth muscle in that it does not have a fixed length-tension relation (3). In contrast, the length-tension relation of arterial smooth muscle is less plastic. It is possible that the variable length-tension relation observed in airway smooth muscle requires a more plastic actin cytoskeleton, so that a stimulus-activated actin polymerization pathway is required for the initial contraction. Such a system may not be required in arterial smooth muscle.
There are several possible hypotheses for the role of stimulus-induced actin polymerization that occurs only during the sustained phase of smooth muscle contraction. First, it is possible that actin polymerization is required only to create a cytoskeletal structure necessary for subsequent contraction. Once the cytoskeletal structure is formed, further stimulus-dependent actin polymerization can occur but is not required for the sustained phase of arterial contraction. This hypothesis suggests no role for stimulus-induced actin polymerization in the sustained contraction. Second, it is possible that stimulus-dependent actin polymerization is only required during stimulus-dependent sustained contraction. This would suggest that actin polymerization is not necessary to create the cytoskeletal structure that is required for subsequent contraction. We think this possibility is unlikely given that 70% of actin is polymerized (F actin) in resting smooth muscle (Fig. 7 and Ref. 19). Third, it is possible that the actin polymerization pathway is required to both to create a cytoskeletal structure necessary for subsequent contraction and for the sustained phase of stimulus-dependent contraction, i.e., latch. This hypothesis predicts that interference with stimulated actin polymerization would abolish the latch phenomenon, i.e., if stimulus-induced actin polymerization were inhibited, then force would decreased in parallel with decreasing crossbridge phosphorylation during a sustained maximal contraction. Finally, it is possible that the actin polymerization pathway is required to create a cytoskeletal structure necessary for subsequent contraction and these proteins modulate, but are not required for, the sustained phase of stimulus-dependent contraction (latch). This hypothesis would imply that force is maintained during latch by both attached dephosphorylated crossbridges, i.e., latchbridges and stimulus-dependent actin polymerization.
Measurement of noise temperature and hysteresivity in intact swine carotid artery. The second goal was to measure noise temperature, hysteresivity, and phase angle during the initial and sustained phases of swine carotid artery contraction to evaluate the rheology of arterial smooth muscle contraction. As described in the introduction, noise temperature, hysteresivity, and phase angle are rheologic measures of the "fluidity" of a tissue (18). We clearly show no significant change in noise temperature, hysteresivity, and phase angle during the initial force development phase of a maximal contraction (Figs. 5 and 7). Therefore, it appears that the increase in crossbridge phosphorylation, G', and stress observed during with initial activation were not associated with a change in smooth muscle rheology.
In contrast, the sustained phase of a rapid arterial smooth contraction was associated with significant decreases in noise temperature, hysteresivity, and phase angle (Figs. 5 and 7). These data suggest that the smooth muscle behaved more like a "solid" during the sustained phase of contraction when crossbridge phosphorylation fell to intermediate levels and actin was becoming more polymerized. When arterial smooth muscle was slowly contracted, the increase in force was paralleled by decreases in noise temperature, hysteresivity, and phase angle (Fig. 8). These data suggest that the transition to a "solid" rheology, i.e., with a low noise temperature, 1) was not associated with crossbridge interactions (no correlation with crossbridge phosphorylation or stress: Figs. 5 and 6), 2) was time dependent (Figs. 7 and 8), 3) correlated with Y118 paxillin phosphorylation (Fig. 5, 7, and 9), and 4) correlated with increased actin polymerization (Fig. 7). It is possible that stimulated actin polymerization may be partly responsible for this transition to a more "solid" rheology. Supporting this contention was the finding that an inhibitor of actin polymerization, latrunculin-A, significantly increased noise temperature and hysteresivity in histamine stimulated swine carotid artery (Table 1). Our finding that latrunculin-A increased noise temperature in intact swine carotid artery parallels the finding that cytochalasin-D increased noise temperature in multiple types of isolated cultured cells (9).
Previously, noise temperature had only been measured with beads attached to the plasma membrane of isolated cells (2, 10). This study shows that noise temperature can be measured in intact tissues. Values of noise temperature measured in intact smooth muscle (this study) were approximately one-half of those measured in isolated cells (2, 10), suggesting that measurement of noise temperature at the tissue ends is different than measurement of noise temperature at the plasma membrane (2, 10). This difference could represent contributions to noise temperature from connective tissue in the intact tissue and/or differences from measurement in the axial dimension in intact tissues vs. rotational measurements in the isolated cell. As predicted (9), noise temperature, hysteresivity, and phase angle appeared to be similar measurements in the depolarized and histamine stimulated swine carotid artery (Fig. 3). This similarity suggests these measurements are robust. There was more variability in the hysteresivity measurement in resting tissues, perhaps because stress was low in resting tissues so that force measurement errors in resting tissues were amplified with normalization, e.g., Fig. 2.
The noise temperature, hysteresivity, and phase angle response did not differ when tissues were depolarized with K+ or stimulated with histamine. This result suggests that some mechanism common to depolarization or histamine (but not crossbridge phosphorylation) is responsible for changing the rheology of smooth muscle to a more "solid"-like state during the sustained phase of contraction. Therefore, any mechanisms specific to depolarization, e.g., higher Ca2+, or specific to histamine stimulation, e.g., protein kinase C activation or myosin phosphatase inhibition, are unlikely to be responsible for the observed changes in the rheology of smooth muscle.
The significant dependence of the relative amount of F actin and noise temperature on Y118 paxillin phosphorylation (Fig. 9) are consistent with the hypothesis that sustained depolarization and histamine stimulation increased Y118 paxillin phosphorylation which then caused actin polymerization, a process associated with a "solidification" of swine arterial smooth muscle. While this correlation existed for these experiments, it must be emphasized that rheology can be altered by multiple other factors that alter intermolecular interactions which then change the energy microconfiguration of the muscle. Additional research is required to further understand both the importance and regulation of stimulus-induced changes in noise temperature and hysteresivity in arterial smooth muscle.
Implications of noise temperature results for the latch phenomenon. As noted, these data suggest a potential role for Y118 paxillin phosphorylation-induced actin polymerization and/or a "solid" rheology in the sustained (latch) phase of contraction. In intact bovine trachalis, Fredberg et al. (11) found that hysteresivity, a measure of the area of the length-force loop during length oscillation, was low at rest, high during the rapid phase of contraction, and low during the sustained phase of contraction. These results led Fredberg et al. to hypothesize that tracheal smooth muscle was "solid" at rest when crossbridge cycling was low, became more "fluid" during force generation when crossbridge cycling was fast, and then became more "solid" during the sustained contraction when crossbridge cycling was intermediate. Their results differ from our results in which noise temperature, hysteresivity, and phase angle were high at rest, high during the rapid phase of contraction, and only low during the sustained phase of contraction (Figs. 5 and 7). Our data suggests that arterial smooth muscle was "fluid" both at rest and during rapid force generation, suggesting that the increase in crossbridge interactions associated with force development was not associated with a change in the microconfiguration of energy wells in the system. Arterial smooth muscle only became "solid" during the sustained contraction. This difference suggests a clear difference in the rheology of tracheal and arterial smooth muscle.
Do these findings suggest that the latch phenomenon is caused by Y118 paxillin phosphorylation-induced actin polymerization and development of a "solid" rheology? The latch phenomenon is characterized by 1) maintained force with reduced crossbridge phosphorylation, velocity, and ATP utilization (14, 23, 30, 45), 2) an invariance of stiffness and yield stress despite different crossbridge phosphorylation values (Fig. 5 and 7 and Ref. 31), and 3) a requirement for crossbridge interactions for force development, i.e., myosin kinase inhibitors abolish contraction despite partly preserved Ca2+ transients (44). The current dynamic crossbridge model of latch predicts that accumulating dephosphorylated crossbridges also explain all of these characteristics of the latch phenomenon (14, 15, 30). If regulated actin polymerization were to explain the latch phenomenon, it would also need to explain these characteristics of the latch phenomenon. Therefore, we cannot yet conclude that stimulated actin polymerization is able to explain the latch phenomenon. We plan further studies to test the importance of stimulus-dependent actin polymerization and changes in tissue rheology in the sustained phase of contraction.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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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.
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REFERENCES |
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, unstimulated; 
, 1 min of depolarization; and 
, 25 min of depolarization). Left, ln real stiffness (G') as a function of ln frequency (mean ± 1 SE). Right, raw normalized ln G' as a function of ln frequency (G' was normalized so that real stiffness at 0.3 Hz = 1). The slope of the G'-frequency relationship, termed noise temperature, was higher in unstimulated tissues and in tissues stimulated for 1 min, compared with tissues stimulated for 25 min (
, 
).
