INTRODUCTION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

THE MYOGENIC RESPONSE is an inherent ability of smooth muscle to respond to increased force. In arterial blood vessels, the myogenic response is manifested as a sustained constriction following luminal pressure elevation. The time course of a vascular myogenic constriction occurs in several phases. In most vessels, a rapid elevation in luminal pressure produces an immediate distention. The amount of distension is variable and depends on the level of initial tone (10). After the distension phase, a contraction begins to develop. In large vessels, the delay before vascular smooth muscle (VSM) contraction varies from many seconds in arteries to a fraction of a second in arterioles (17). The amount of constriction is also greater in arterioles (3, 24). In some vessels, the constriction is biphasic, with an exaggerated initial phase that is independent of the rate of pressure rise, followed by a partial relaxation to a steady-state phase (9, 10). The steady-state constriction appears to be maintained almost indefinitely (18) and accounts for the basal tone of resistance vessels in almost every organ.

Implicit in the concept of a myogenic constriction is the assumption that VSM shifts to a higher activation state. Johnson (16) stated this assumption clearly in his landmark chapter in the Handbook of Physiology, basing his conclusion on length-tension analyses of in vivo arterioles from cat mesentery (15). Specifically, enhanced activation associated with myogenic tone was predicted to explain the progressive, leftward shift of calculated arteriolar wall tension with pressure. Whereas Johnson's initial analyses required arteriolar pressures to be extrapolated from measurements of systemic arterial pressure, direct measurements of arteriolar pressure in subsequent experiments confirmed Johnson's predictions (6).

However, analyses of the myogenic response in vivo are subject to concerns about modulation by parenchymal cell metabolites and endothelial cell vasoactive factors because changes in oxygen delivery and flow necessarily accompany changes in perfusion pressure (8). Thus it is important that these protocols be repeated in isolated arterioles where the endothelium is denuded and/or where changes in flow are prevented. Even so, such evidence would only qualitatively support the concept of an increased VSM activation state. A more rigorous test of this idea would be to demonstrate that increased myogenic tone reflects an increase in maximal velocity of shortening (V_max) of arteriolar smooth muscle. However, measurements of VSM force-velocity relationships have only been possible, to date, on larger blood vessels that lack significant myogenic responsiveness (1, 11, 14, 20, 21).

In this study, we tested the hypothesis that myogenic activation of arterioles is associated with an increase in unloaded shortening velocity of arteriolar smooth muscle. To make these measurements, we performed isotonic release protocols on isolated, cannulated segments of second-order (2A) hamster cheek pouch arterioles using a servo-control system to regulate wall tension. These vessels have been studied extensively in vitro and generate ~50% myogenic tone (relative to maximal passive diameter), which is comparable to 2A tone observed in vivo. Furthermore, maximal length-tension relationships for these vessels have been previously determined (4). As an additional aspect of this study, we compared how enhanced myogenic tone and agonist-induced tone might differentially alter the force-velocity relationship of arteriolar smooth muscle.

	METHODS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Isolated arteriole preparation. Golden Syrian hamsters (120-180 g) were anesthetized with intraperitoneal injections of pentobarbital sodium (60 mg/kg). One cheek pouch from each animal was cleaned, excised, and placed in 4°C physiological saline solution containing 1 g/100 ml albumin. All animal protocols conformed to institutional guidelines.

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Microscope system. Each vessel segment was transilluminated using a Zeiss IM-35 inverted microscope with a heat-filtered, 100-W tungsten light source, filtered at 515 nm. The 2A branch was visualized at high magnification using a Leitz ×75 oil immersion objective (numerical aperature = 1.0) and bright-field (numerical aperature = 0.63) condenser. The vessel image was displayed on a video monitor using a Newvicon camera, and internal diameter was measured manually with a video micrometer. Pressure and diameter recordings were digitized using a Macintosh 8100/80 computer and MIO-16x data acquisition card (National Instruments; Austin, TX). Experiments were videotaped using a videotimer signal so that arteriolar diameter transients could be measured at high time resolution (1/5 real time) during replay of the videotape. To synchronize diameter and pressure measurements on replay, a video multiplexer was used to record pressure onto the audio track of the videotape. The analog output from the multiplexer was redigitized in synchrony with the replayed diameter signal.

Servo control of pressure and wall tension. To provide computer control over the pressurization system, the following modifications were made. Two static pressure reservoirs used for initial equilibration of the vessels were connected through three-way valves to Ling vibration pumps (model V203; Herts, UK). These pumps were driven by custom-made power amplifiers and allowed servo control of vessel pressure. Pressures at the pumps were measured using Statham P23 Db pressure transducers connected to a custom-built carrier amplifier. Output from this unit was compared with a command pressure from the computer by a custom-built servo amplifier. In initial experiments, tuning low-pass filters on each pressure channel optimized the frequency response and gain of the system. The response was confirmed to be flat to at least 20 Hz by servo-null measurements of pressure at the vessel midpoint.

Protocols. After equilibration and development of spontaneous myogenic tone under pressurization to 44 mmHg from the static reservoir, pressure control was switched over to the computer and set to 40 mmHg. Software programs to control this and subsequent protocols were written using LabView (National Instruments). After verification that the system was working properly, i.e., that there were no leaks and no oscillations in the servo-control system, at least one 2-min pressure step to 90 mmHg was performed while recording internal diameter to check for myogenic constrictions. Pressure was then returned to 40 mmHg, and the preparation was allowed to stabilize for 10-15 min. 2As developed ~43% basal tone at 40 mmHg (Table 1), which is greater than that previously reported (see Fig. 3d in Ref. 4). Of 17 arterioles attempted, one was discarded for failing to develop myogenic tone, and one was discarded due to excessive spontaneous vasomotion.

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Off-line analysis. The typical diameter response to an isotonic release was a rapid (<200 ms), initial collapse of the arteriole, presumably reflecting recoil of VSM series-elastic elements (23). This was followed by a period of force development (constriction) that lasted 1-10 s. After this, some vessels would begin to myogenically dilate, but at some isotonic release levels, the pressure required to control the desired level of tension was too low (<20 mmHg) to elicit a myogenic response (see Fig. 3d in Ref. 4). Accurate measurements of the diameter changes during the first few seconds following isotonic release were critical for calculation of initial isotonic shortening velocity. Videotapes of the experiment were replayed off-line in slow motion so that the diameters could be remeasured using the video micrometer (several times in some cases) and the computer could subsequently digitize its output. The replayed diameter records were used for subsequent data analysis, whereas the initial (on-line) diameter measurements were necessary for control of wall tension. However, there was almost always very close agreement between the two. Automated diameter tracking devices did not prove sufficiently accurate to track internal diameter because any glitches in tracking led to large spikes in the servo-controlled pressure system. Any errors in the online measurement would obviously generate errors in the pressure servo. However, pressure compensations were typically rather small (a few mmHg, as shown in Fig. 2), and associated errors would not have had a major effect on the shortening velocity measurements.

Normalization procedures. V'_max was expressed as shortening in micrometers per second or in lengths per second. For the later case, the actual change in diameter was normalized to L_o for that vessel.

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View larger version (24K): [in this window] [in a new window]   Fig. 1.   Illustration of a myogenic constriction for a representative second-order arteriole (2A) on a tension-radius plot. Inset: raw pressure (P) and diameter (D) traces. Inverted "Y" shape in the center of the larger graph represents 220 paired diameter (D) and pressure (P) points converted to tension, normalized to passive diameter (Lo), and plotted against L/Lo, where L is diameter at each point in time. Numbers associated with these points indicate the time course of the response and correspond to the numbers in the inset. Open circles, passive tension-diameter plot for this 2A. Dashed line, calculated maximal active tension curve for this vessel as described in METHODS (see Eq. 2). Differences between point 1 and Lo and between point 3 and Lo represent amount of tone at 40 and 90 mmHg, respectively. Ratio of initial tension (Ti) to maximal active tension (Tmax) at each pressure represents "fractional tension" (as summarized in Table 1) of the 2A at the two different levels of myogenic tone.

	RESULTS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Myogenic constriction represented on a tension-radius plot. One goal of these experiments was to test whether increased myogenic tone is associated with an enhanced activation of arteriolar smooth muscle. The first indication that this occurs is apparent in the myogenic behavior of a representative 2A as plotted on a length-tension curve (Fig. 1). The raw pressure and diameter data are shown as a function of time following a pressure step from 40 to 90 mmHg (see inset). The open arrows (Fig. 1) in the center of the diagram illustrate the time-dependent sequence of the myogenic constriction. At pressure equalling 40 mmHg, the radius equaled 0.6 L_o (point 1). When pressure was rapidly increased to 90 mmHg, the vessel distended to a radius of 0.68 L_o (point 2), then constricted to a minimum of 0.4 L_o (point 3), and maintained a steady-state constriction at that diameter. For reference purposes, the passive radius-tension relationship for the same 2A is also plotted (open circles) as is the maximal active tension curve (dashed line) calculated from Davis and Gore (4). It is apparent that the myogenic constriction is associated with a leftward shift of the radius-tension relationship for the arteriole. The solid arrows in Fig. 1 indicate the relative degree of myogenic tone, where tone is defined as the difference between the passive diameter at 40 mmHg (L_o) and the normalized steady-state diameter (D_i/L_o). Thus this 2A developed ~40% tone at a pressure equalling 40 mmHg and ~60% tone at a pressure equalling 90 mmHg. Also shown in Fig. 1 are vertical lines that project from the two steady-state diameters to denote the intersections with the maximal active tension curve. The ratio of T_i to T_max at each diameter may perhaps be a superior index of "tone," as defined by VanBavel et al. (25). However, in most previous myogenic studies, the term "tone" refers to a difference in the active versus passive diameter. Therefore to avoid confusion, we employ the term "fractional tension" to refer to the T_i-to-T_max ratio under different conditions. With the use of this criterion, the 2A shown in Fig. 1 had a fractional tension equalling 0.19 at 40 mmHg and 0.49 at 90 mmHg, respectively. The average values for tone and fractional tension at both pressures and after NE are summarized in Table 1.

Isotonic release protocol. Changes in pressure and diameter as a function of tension during two isotonic releases are shown in Fig. 2. The baseline diameter of this arteriole was 24-27 µm at a pressure equalling 40 mmHg. In response to a sudden drop in wall tension from the calculated control value to 20% of control, there was a rapid initial collapse of the vessel (<0.2 s), which presumably represented the recoil of series elastic components (SEC) (26). After the recoil was a 2- to 3-s shortening phase until the arteriole suddenly began to dilate at ~1.8 s after release. The myogenic dilation (induced by the low pressure required to reduce afterload to 0.2) was variable in magnitude and time of onset from vessel to vessel, but never interfered with accurate determinations of the rate of initial shortening. After reequilibration for about 1.5 min at a pressure equalling 40 mmHg, a second release was performed on this vessel, to afterload equalling 0.6. As can be seen from Fig. 2, right, the phase of passive collapse is not clearly discernable, but the initial rate of shortening is lower than for the previous release. Also, the magnitude of the secondary myogenic dilation is greater (dilating above the control diameter; Fig. 2, left) presumably because the vessel is more myogenically active at the pressure required to achieve an afterload of 0.6 (~30 mmHg) compared with the pressure required to achieve an afterload of 0.2 (~15 mmHg).

View larger version (23K): [in this window] [in a new window]   Fig. 2.   Example showing two isotonic releases performed on an isolated 2A. Near the beginning of the trace (time ~6 s), the system is switched from pressure control mode (P = 40 mmHg) to tension control mode. Shortly after this switch, tension is suddenly released to 20% of its control value (left) for ~5 s. The sudden drop in pressure and tension is associated with a rapid (<0.2 s) fall in tension that corresponds to recoil of the series elastic component. After this, a shortening phase commences, lasting ~4 s. At time ~11 s, the arteriole begins to dilate, demonstrating that it is myogenically responsive. This dilation is cut short by return of the tension to control. After ~90 s of recovery at P = 40 mmHg, tension is released to 60% of control (right). Same phases are present in the diameter trace, with the series elastic components (SEC) recoil phase being barely discernable. In this case, the secondary myogenic dilation brings diameter above the control level.

Repeatability of diameter changes during identical isotonic releases. To verify that reliable and repeatable changes in diameter could be recorded during isotonic releases, a series of nearly identical release protocols were repeated on a single 2A, as shown in Fig. 3. This arteriole was allowed to develop myogenic tone at a pressure of 40 mmHg. The servo system was switched to tension control mode, and the initial computed wall tension was used as a reference point. Tension was then released to an afterload equivalent to 20% of this value for ~14 s while recording diameter (in slow motion on video playback). The servo system was switched off, pressure was returned to 40 mmHg, and the vessel was allowed to regain myogenic tone. This procedure was repeated two times. The three resulting releases were then superimposed for comparison. The time courses of the diameter changes following isotonic release are remarkably consistent, especially over the first 2-3 s after release (Fig. 3). Note that there are some differences in the baseline tension for each trial due to slight variations in the amount of myogenic tone developed during each recovery period. Also, note that a partial myogenic relaxation occurred (t = 9 s) in one of the trials but that it was inconsequential to the measurement of initial shortening velocity. Importantly, the absolute diameter changes during the isotonic shortening phase (2.0-2.5 µm) were well within the resolution of the microscope system.

View larger version (20K): [in this window] [in a new window]   Fig. 3.   Repeatability of diameter shortening phase during isotonic release protocols. This 2A was released three times from control to 20% of control tension. The first two releases were 20 min apart, and the third release was 1 min after that. Traces are superimposed and displayed as the diameter change from control. Magnitude of SEC recoil phase varied from 0.5 to 1.0 µm. Velocities for the first 1 s of the shortening phases were 0.55, 0.91, and 0.76 µm/s. Note slight myogenic dilation that occurs in one protocol at time ~9 s.

Analysis of data from a series of isotonic releases. To assess the force-velocity relationship for each arteriole, tension was released to a series of different afterloads. Each release, upon completion of the force change and SEC recoil, was associated with a shortening phase in which the rate of shortening slowed progressively with time (Figs. 2 and 3). When the logarithm of (1  fractional shortening) was plotted against time for isotonic releases to various afterloads, the data for the first 2 s of each shortening phase were almost always well fit by a first-order exponential function (not shown).

View larger version (13K): [in this window] [in a new window]   Fig. 4.   Method of data analysis is illustrated. A: diameter changes for 5 isotonic releases (as recorded on playback of the videotape in slow motion and with corrected time scales). Note progressive increase in magnitude of SEC recoil and progressive increase in initial velocity of shortening as afterload is released to smaller fractions of control tension. Possible exceptions to the latter statement are the very similar initial velocities for releases to 0.1 and 0.3. Myogenic dilations of various magnitudes occur at time ~11 s for three of the releases. Four other releases were performed on the same vessel (data not shown for clarity). B: force-velocity plot of nonnormalized data for all 9 isotonic releases. These data were fit to find the Hill coefficients, a and b (see text for details), and the coefficients were then used to draw a hyperbolic curve through the data and to determine maximal velocity (V'max) from the y-intercept. C: force-velocity plot of same data after normalization of shortening velocity to Lo for this vessel. Lengths/s = (µm · s1 · L) · 100.

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Comparison of active and passive tensions. The above procedure is commonly used to determine unloaded shortening velocity on isolated strips of muscle. A possible concern with using it to analyze isotonic shortening data from arterioles, which contain significant amounts of nonforce-generating connective tissue, would be if passive tension made a significant contribution to total wall tension under these conditions and thereby contaminated measurements of active shortening. To address this issue, we compared the range of vessel radius changes that occurred during isotonic releases with the passive tension-radius relationship obtained for each vessel at the end of the experiment, when active tone had been eliminated by calcium-free bath solution containing nitroprusside. An example of this analysis is shown in Fig. 5, where the open circles represent the passive tension-radius relationship and some of the raw isotonic release data for the same vessel are plotted as individual points.

View larger version (22K): [in this window] [in a new window]   Fig. 5.   Tension-radius plot to show relationships among active tension, passive tension, tone and fractional tension for a representative 2A subjected to isotonic releases at P = 40 mmHg, 90 mmHg, and after norepinephrine (NE). Open circles, passive tension-radius curve; dotted line, calculated maximal active tension curve (from Eq. 2). Clusters of points are data for single isotonic releases (to different afterloads) from initial tensions associated for the three conditions; each initial release point is enclosed in an open square. Open arrows, release (vertical arrow) and shortening (horizontal arrow) phases for isotonic release after equilibration at 90 mmHg. Solid vertical lines, reference points for illustrating the relative amount of tone associated with the three conditions and also indicate maximum active tension that corresponds to the initial diameter for each condition. Ratio of Ti/Tmax (fractional tension) was used to normalize shortening data for these three trials and other data for the other arterioles as shown in Figs. 7 and 8.

Comparison of force-velocity plots for vessels with low and high myogenic tone. Both raw and normalized shortening velocities for each isotonic release protocol from each arteriole were compiled into a spreadsheet for analysis of group data. The first method of analysis is shown in Fig. 6, where the force-velocity relationships were compared for vessels equilibrated at low myogenic tone (pressure = 40 mmHg; n = 12) or high myogenic tone (pressure = 90 mmHg; n = 12). For this analysis, the data were pooled by afterload, the means were calculated, and then Hill coefficients were computed from nonlinear fits of the transformed data. There is clear separation between the data sets, as indicated by comparisons of the group data at each afterload. Shortening velocities at the three lowest afterloads, 0.3, 0.2, and 0.1, were all significantly higher in arterioles with higher initial myogenic tone (ANOVA, P < 0.05). The curves through the data sets represent the best fits of the data to the Hill equation. Determinations of V'_max from the y-intercepts suggest that V'_max is substantially higher for arterioles with higher myogenic tone.

View larger version (17K): [in this window] [in a new window]   Fig. 6.   Comparison of force-velocity relationships for arterioles with low myogenic tone (filled triangles; P = 40 mmHg) and high myogenic tone (open triangles; P = 90 mmHg). For this analysis, data were grouped according to afterload, the means fit to the Hill equation, and values of V'max determined from the y-intercepts for each of the two data sets. Lengths/s = (µm · s1 · L) · 100.

Analysis of data after normalization to maximal isometric force. As mentioned previously, force-velocity data are typically normalized to some force index such as maximal isometric tension. To do this, each data point was normalized to maximal active tension at the respective diameter, as diagrammed in Fig. 5. For example, from the point at which isotonic releases were performed at a pressure of 90 mmHg, this arteriole had 58% initial tone (1.0-0.42), T_i = 78 dyn/cm, an estimated maximal active tension of 135 dyn/cm (from Eq. 2), and therefore a fractional tension of 0.58. The vertical open arrow in Fig. 5 indicates the release phase and the horizontal open arrow indicates the shortening phase when tension was released to 10% of its initial value. Single releases to other afterloads for the pressure at 40 mmHg and NE protocols are also shown to indicate the representative relationships among active and passive tension, tone, and fractional tension relative to the dimensional changes that occurred during the isotonic releases. It should be clear that passive tension is minimal during the isotonic releases.

View larger version (25K): [in this window] [in a new window]   Fig. 7.   Comparison of force-velocity relationships for arterioles with low myogenic tone (filled triangles; P = 40 mmHg) and high myogenic tone (open triangles; P = 90 mmHg). Shortening velocity was normalized to Lo and force was normalized to Ti/Tmax. Force intercepts were determined by the average initial Ti/Tmax values for each data set: 0.22 for P40 and 0.56 for P90. Lengths/s = (µm · s1 · L) · 100.

Comparison of force-velocity plots for vessels with and without NE. To test whether agonist-induced activation of arteriolar smooth muscle would produce a similar shift in the force-velocity relationship, we studied a third group of vessels treated with NE at a pressure of 40 mmHg. The concentration of NE was titrated to produce an additional constriction roughly comparable to the level of myogenic tone seen at a pressure of 90 mmHg. These data, after normalization to maximal active tension, are shown in Fig. 8, along with the data set for arterioles equilibrated at the same pressure in the absence of NE. The curves represent the best fits of the two data sets to the Hill equation. Shortening velocities at almost all afterloads were significantly higher for arterioles constricted with NE, and determinations of V'_max from the y-intercepts suggest that V'_max was substantially higher for arterioles treated with NE. For the NE data, the average initial T_i/T_max, and hence the force intercept, was at fractional tension equaling 0.26. 

View larger version (20K): [in this window] [in a new window]   Fig. 8.   Comparison of force-velocity relationships for arterioles with low myogenic tone (filled triangles; P = 40 mmHg) and NE-induced tone (open triangles; P = 40 mmHg). Shortening velocity was normalized to Lo and force was normalized to Ti/Tmax. Force intercepts were determined by the average initial Ti/Tmax values for each data set: 0.22 for P40 and 0.26 for NE. Lengths/s = (µm · s1 · L) · 100.

	DISCUSSION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

The results of our study constitute the first experimental evidence that increases in myogenic tone reflect an enhanced V_max of VSM. Although previous studies of arteriolar mechanics (13, 15) and of myogenic signaling pathways (5, 25) indicate that arteriolar smooth muscle is indeed activated by pressure, V_max is perhaps the best quantitative parameter by which to assess the degree of this activation. V_max has not previously been measured in true arterioles. In addition to a shift in V'_max with pressure, NE-induced tone was also associated with an increase in V'_max; however, the characteristics of the two respective force-velocity curves were different. Myogenic tone produced roughly parallel increases in velocity and force, whereas NE produced increases in velocity at low force. Therefore, myogenic and NE-induced activation of arteriolar smooth muscle are associated with different mechanical states of arteriolar smooth muscle, which is likely a reflection of different underlying signaling pathways activated by the two stimuli.

Consideration of technical issues. Our conclusions are subject to consideration of several technical issues related to the unique way in which these experiments were performed. One issue is the accuracy of diameter measurements. Because the diameter changes that occur during isotonic releases are only a few microns in magnitude, we chose an objective/condenser system based on the compromise between optical quality and working distance. Given the objective, condenser, and wavelength of light used in our system, the theoretical resolution of this system is 0.38 µm (see Ref. 12, p. 115). Of course, light scattering above and below the focal plane raises this value, an effect that can be rather large when visualizing arterioles in intact tissue (12). However, the use of an isolated arteriole, in the absence of parenchymal tissue and adventitia, minimizes degradation of the image due to light scattering. We estimate that diameter changes of 0.5 µm could clearly be resolved under the conditions of our experiments (4). Moreover, for determination of V'_max, the most critical measurements of shortening velocity are those required for isotonic releases to the lowest afterloads, e.g., 0.1 and 0.2, and it is at these afterloads that the absolute magnitude of shortening is greatest, usually well over 0.5 µm and sometimes over 2 µm (Figs. 2-4). Therefore, we have the most confidence in accurate diameter measurements for the most critical protocols.

Comparison with previous measurements of V_max. How do our estimates of V'_max compare with measurements from other smooth muscle? Comparisons are necessarily limited by the fact that we measured V'_max of partially activated arteriolar smooth muscle, whereas most other studies have measured V_max of maximally activated smooth muscle. We obtain V'_max values between 0.04 and 0.08 lengths/s for arterioles with low and high myogenic tone, respectively (Table 2). Estimates of V_max for other smooth muscles are 0.61 lengths/s in the Bufo stomach (26), 0.51-0.53 lengths/s in the rat portal vein (14, 21), 0.12 lengths/s in the hog carotid (11), and 0.013 lengths/s in the rat caudal vein (20). With the exception of the latter study, our estimates of V'_max for arterioles are, predictably, at the low end of the reported range. However, in the most directly relevant study, Boels et al. (1) reported a V_max of 0.041 lengths/s for coronary "microarteries" (140 µm ID), which is reasonably close to ours (Table 2). However, the unloaded shortening velocity for maximally activated 2As remains to be determined.

Comparison of effects of myogenic- versus agonist-induced tone. Our results show that high levels of myogenic tone and NE produce comparable increases in V'_max. However, pressure-induced tone (Fig. 7) is associated with smaller increases in velocity (4.2 to 8.3 lengths/s; ~100% increase) than force (0.22 to 0.56 T_i/T_max; ~250% increase), whereas NE-induced tone (Fig. 8) is associated with larger increases in velocity (4.2 to 7.5 lengths/s; ~80% increase) than force (0.22 to 0.26 T_i/T_max; ~20% increase). The differences likely reflect underlying differences in the mechanical states produced by pressure and NE. The classical interpretation of the force-velocity relationship is that an increase in isometric force represents an increase in the number of active cross bridges, whereas an increase in V_max represents an increase in the cycling rate of active cross bridges (19). Thus the differences in the force-velocity curves produced by pressure and NE lead to the conclusion that myogenic tone is associated with an increase in both the number and cycling rate of active cross bridges.