| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Department of Pharmacology and Physiology, University of Rochester, Rochester, New York 14642
 |
ABSTRACT |
|---|
 TOP
 ABSTRACT
 INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
|
|---|
To investigate the relationship between skeletal
muscle metabolism and arteriolar dilations in the region local to
contracting muscle fibers as well as dilations at remote arteriolar
regions upstream, we used a microelectrode on cremaster muscle of
anesthetized hamsters to stimulate four to five muscle fibers lying
approximately perpendicular to and overlapping a transverse arteriole.
Before, during, and after muscle contraction, we measured the diameter of the arteriole at the site of muscle fiber overlap (local) and at a
remote site ~1,000 µm upstream. Two minutes of 2-, 4-, or 8-Hz
stimulation (5-10 V, 0.4-ms duration) produced a significant dilation locally (8.2 ± 2.0-, 22.5 ± 2.4-, and 30.9 ± 2.1-µm increase, respectively) and at the remote site (4.2 ± 0.8, 11.0 ± 1.1, and 18.9 ± 2.7 µm, respectively). Muscle
contraction at 4 Hz initiated a remote dilation that was unaffected by
15-min micropipette application of either 2 µM tetrodotoxin, 0.07%
halothane, or 40 µM 18-
-glycyrrhetinic acid between the local and
upstream site. Therefore, at the arteriolar level, muscle contraction
initiates a robust remote dilation that does not appear to be
transmitted via perivascular nerves or gap junctions.
cell coupling; microcirculation; exercise; blood flow; metabolic coupling
| |
INTRODUCTION |
|---|
|
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
|
|---|
IN SKELETAL MUSCLE, changes in metabolism lead to closely related changes in blood flow, presumably via the vasodilatory actions of products of muscle contraction, including, but not limited to, metabolites, inorganic ions, purines, etc. Gorczynski et al. (9) demonstrated that this coupling can be extremely local in nature by showing that contraction of a few muscle fibers directly under an arteriole produced dilation in that vessel in the immediate vicinity of the contracting muscle fibers. Clearly, such a very local increase in vessel diameter will not be effective in matching blood flow to metabolism in a wider tissue region unless there are mechanisms that facilitate coordination of vasodilatory responses. One such mechanism for extension of local vasoactive responses to wider vascular regions is the conducted response, a gap junctionally mediated spread of vasoactive signal along the blood vessel wall (20). Gap junctional communication may occur between vascular smooth muscle cells, endothelial cells, or both (15, 24, 26). It has been established that, in response to locally applied ACh, the transmitted dilatory signal primarily consists of electrotonic spread of hyperpolarization (24, 26, 28); other studies have shown that electrical and mechanical events in the blood vessel wall can be disassociated (27).
Berg et al. (3) and Cohen et al. (5) linked this intravascular communication pathway to muscle contraction by demonstrating that contraction of muscle fibers lying under capillary endothelial cells initiates a signal that is transmitted to upstream arterioles causing them to dilate. These remote dilations are dependent on signals transmitted along the blood vessel wall via gap junctions (5) and represent a putative mechanism for recruiting capillaries and redistributing blood flow during increased skeletal muscle activity. Subsequent to capillary recruitment, further increases in tissue blood flow must be served by decreased arteriolar resistance. Many aspects of the mechanisms by which arterioles dilate in response to muscle contraction are unknown, in particular, whether arterioles are capable of translating contraction-induced dilations (i.e., via a conducted response) over larger vascular regions.
In the present study, we reexamined the direct local vasodilatory response to muscle fiber contraction described by Gorczynski et al. (9) to investigate whether the local arteriolar dilations are associated with remote upstream dilations similar to the arteriolar dilations described when the local muscle contraction is associated with capillaries (3, 5). We stimulated muscle fiber bundles approximately perpendicular to and overlapping a transverse arteriole and observed local dilation of the arteriole in the region of the contracting muscle fibers. We also observed a dilation ~1,000 µm upstream from the site of muscle contraction, along the same arteriole. Further investigation into the nature of the signal transmission pathway indicated that the contraction-induced upstream dilation was not dependent on perivascular nerves or gap junctions. Our study demonstrates that arterioles have the capacity to transmit dilatory signals upstream in response to muscle contraction and that the transmission pathway by which the remote upstream site dilates is not typical of a conducted response, i.e., a signal communicated between cells of the blood vessel wall by electrotonic spread of hyperpolarization via gap junctions. Our findings indicate that arterioles can respond to muscle contraction and play a role in the initiation and spread of a dilatory signal throughout the microvasculature. Such coordination of responses would contribute significantly to the integrated response to exercise by facilitating spread of dilation over large areas and thus allowing many microvessels to respond in a coordinated manner to the increase in muscle metabolism.
| |
METHODS |
|---|
|
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
|
|---|
Preparation and Muscle Fiber Stimulation
Adult male Golden hamsters (100-130 g) were anesthetized with pentobarbital sodium (70 mg/kg ip) and tracheotomized. Catheters were placed in the right femoral artery (to monitor mean arterial pressure) and right femoral vein [for supplemental pentobarbital sodium as needed during the surgery and for constant infusion (10 mg/ml saline, 0.56 ml/h) throughout the experimental protocol]. The right cremaster was prepared for in situ microscopy as previously described (1, 17). Briefly, the cremaster was isolated, cut longitudinally, separated from the testis and epididymis, and gently spread over a semicircular lucite platform using insect pins to secure the edges and maintain tension on the muscle, being careful not to stretch the preparation (in this tissue layout, sarcomere length is typically between 2.4 and 2.6 µm). During surgery and experimental protocols, the muscle was superfused with a bicarbonate-buffered salt solution containing (in mM) 131.9 NaCl, 4.7 KCl, 2.0 CaCl2, 1.2 MgSO4, and 30 NaHCO3 equilibrated with gas containing 5% CO2-95% N2 (pH 7.35-7.45). Hamster esophageal temperature was maintained at 37°C via convective heat. Cremaster muscle temperature was maintained at 34.0°C by heating the superfusion solution. After the surgery, all preparations were allowed to equilibrate for 45-60 min before data collection. The tissue was visualized by transillumination with a xenon arc lamp, and the microvasculature was observed with a Leitz Laborlux microscope using a ×25 long working-distance objective (numerical aperature 0.22). The microscope image was displayed via charge-coupled device camera (Dage MTI CCD72S) on a Sony monitor and recorded on a [3/4]-in. videotape (Sony, VO-9600). Final magnification of the site was ×1,420. Diameter measurements were reproducible to ±0.3 µm, which is ~1-2% of the expected diameter.We observed transverse arterioles (Fig.
1) of ~50-µm maximal diameter.
Transverse arterioles were identified as previously described
(22). Briefly, vessels were identified according to their
proximal and distal connections. Capillaries were initially identified
and were traced back to their inflow arteriole. The inflow arteriole
was traced back to its vessel of origin and this arteriole (the branch)
was traced back to its arteriole of origin. If more than two branches
arose from this vessel, it was considered a transverse arteriole
[transverse arterioles typically have 3-8 branches
(4)]. Our only selection criterion required that muscle fibers associated with the transverse arteriole run approximately perpendicular to the vessel. This architecture is common and can be
found in all areas of the tissue preparation. Sites for observation were selected in the central region of the tissue.
|
Muscle fiber bundles (4-5 fibers) were stimulated directly using a platinum wire microelectrode (tip diameter ~25 µm) placed onto muscle fibers running approximately perpendicular to the arteriole and positioned at least 1,000 µm away from the site of the arteriole-muscle fiber intersection. The ground electrode was placed in the superfusate around the outer rim of the tissue support pedestal. We stimulated muscle fibers via a square-wave pulse of 0.4 ms, 4-10 V at frequencies of either 2, 4, or 8 Hz (Grass S48 stimulator, Quincy, MA) (see Experimental Protocols for further detail).
The overall state of vascular responsiveness in each preparation was
assessed. Only the data collected on preparations that clearly
displayed arteriolar constriction to 10% oxygen and dilation to
10
4 M adenosine were kept for further analysis (~4% of
the preparations were discarded). After each protocol, maximal
arteriolar diameters were recorded after at least 2 min of superfusion
of the preparation with 104 M adenosine.
Experimental Protocols
Presence of local and upstream dilations initiated by muscle contraction. We defined the site where the arteriole crossed the contracting muscle fibers as the local site and a site ~1,000 µm upstream from the contracting muscle fibers, along the same arteriole, was defined as the upstream site (Fig. 1). Arteriolar diameter at the local site was continuously recorded 1 min before muscle stimulation, during 2 min of muscle contraction, and for 2 min of recovery after stimulation. After 2 min, this protocol was repeated while the upstream site was observed (n = 14). Preliminary observations showed that there was no difference in the magnitude of either the local or the upstream response if the local site was observed first or if the upstream site was observed first. We chose to use high magnification to optimize the resolution for diameter measurement; thus the local and upstream sites could not be observed simultaneously and had to be observed in sequence. To investigate the relationship between the rate of muscle stimulation (or the amount of local dilation) and the magnitude of the upstream response, we recorded local and upstream responses to 2-, 4-, and 8-Hz muscle contraction (n = 7). To test whether the remote dilation initiated by 2 min of 4-Hz muscle contraction was bidirectional along the length of the arteriole, we recorded the local dilation to muscle contraction and then contracted the muscle bundle again while a site ~1,000 µm downstream of the local site was observed (n = 14).
Decay of the dilatory signal from the local to an upstream remote site is characteristic of pharmacologically induced conducted responses (7). We looked for this typical decay in the magnitude of the dilation of the contraction-induced response by measuring the arteriolar dilation to 4-Hz muscle contraction locally and at sites ~500 and 1,000 µm upstream (n = 9). To ensure that the upstream response was not the result of either tissue movement during muscle stimulation or diffusion of metabolites from contracting skeletal muscle fibers, we took advantage of the "disperse" arrangement of muscle fibers that occurs in cremaster muscle. The cremaster consists of multiple layers of skeletal muscle fibers that run at various angles with respect to each other, and it is therefore possible to identify groups of muscle fibers in the region of the selected arteriole that do not intersect with it. If mechanical movement due to contraction (or diffusion of metabolites from the contracting fibers) is the primary cause of the remote dilation, then stimulation of these "unassociated" muscle fiber bundles located ~1,000 µm away should give the same dilatory response at the upstream site on the selected arteriole as when we stimulate a similar bundle of muscle fibers intersecting the arteriole at the downstream location (see Fig. 1). The unassociated muscle fibers were stimulated for 2 min at 4 Hz while the diameter of the upstream site was observed (n = 10). To further eliminate a role for passive diffusion of metabolites from the local contraction site in the upstream response, we ensured that upstream observation sites were always at least 500 µm from the contracting muscle fibers, well beyond the distance that a small metabolite can diffuse during 2 min (3). We did not observe any local or upstream dilation when the stimulating electrode was raised off the muscle fibers (therefore not causing muscle contraction) but was still immersed in superfusate, thus eliminating the possibility that electrical spread from the stimulating electrode through the superfusate may be affecting the arteriolar diameter. Because of the geometry of the cremaster vascular architecture, it is possible that the muscle fibers chosen for stimulation could lie not only under the arteriole being investigated but also under associated capillaries. According to earlier work (3), muscle contraction under the capillaries can initiate remote upstream dilations that might contribute to the dilation in the observed arteriole. We therefore investigated how much influence, if any, a conducted response from the capillaries contributed to the dilation observed at the local site. We recorded the dilatory response at the local site to 4-Hz muscle stimulation and then micropipette applied (see below for details) a gap junction inhibitor, halothane (0.07%, n = 5), previously shown to block contraction-induced arteriolar dilation initiated at the capillaries (5). Halothane was applied across the transverse arteriole at the site of the first branch downstream from the local site for 10 min before and during reassessment of the local dilation induced by 4-Hz muscle contraction. We found no difference in the magnitude of the local dilatory response with and without halothane; thus any dilatory contribution from downstream capillaries was considered negligible. Earlier studies also support this conclusion. Using a similar muscle contraction protocol (2 min, 4 Hz), Berg et al. (3) contracted muscle fibers under capillaries and observed no significant dilation at the transverse arteriolar level. This, together with controls described in the preceding paragraph, also supports the conclusion that no significant dilatory signal came from other microvascular elements situated remotely in the network.Nature of the pathway connecting local and upstream dilations. All pharmacological agents were applied via micropipette as previously described (8, 18). Once filled, the micropipette tip was placed ~15-25 µm from the vessel wall, in the signal transmission pathway between the local and the upstream observation sites, at least 500 µm from either site. The micropipette was pressurized via a water manometer, and flow of micropipette contents was achieved by raising the pressure in the manometer (8, 18). Fluorescein isothiocyanate-dextran (100 µM) was added to each pipette solution so that brief epifluorescence could be used to verify flow from the pipette and the flow direction of the pipette contents. Care was taken to ensure that pipette contents flowed approximately perpendicular to the arteriole under observation and that neither the local arteriolar observation site, the upstream site, nor the contracting muscle fiber bundle was exposed to the pipette contents. Care was also taken to ensure that the tissue movement that occurred during contraction did not impede flow of the micropipette contents. After the 45- to 60-min equilibration period, we recorded the arteriolar diameter at the local site and at the upstream site before during and after 2 min of 4-Hz muscle contraction as described earlier (control data). After 10 min of drug application between the local and the upstream sites, local and upstream diameter measurements were repeated during 4-Hz muscle contraction. Pipette flow was then stopped and after 20-min washout, diameters at the local and upstream sites were again recorded during 4-Hz muscle stimulation (recovery measurements).
We used tetrodotoxin (TTX) to block fast sodium channels and hence to inhibit any perivascular nerve component of the signal transmission from the local to the upstream sites (3, 19). Local and upstream diameters to 2 min of 4-Hz muscle contraction were recorded during control conditions, with micropipette application of 2 × 106 M TTX between the local and upstream sites and after
recovery from TTX (n = 8). To verify the efficacy of
the delivered TTX concentration to block action potentials, we
micropipette applied TTX to contracting skeletal muscle fibers; TTX was
observed to eliminate all muscle contraction within 30 s of
application. Muscle contraction returned within 1 min of TTX removal.
At the local site, muscle contraction under the arteriole initially
results in a very local dilation, where the arteriole appears fusiform
in shape. The dilation then appears to spread over the length of the
vessel. To investigate whether the environmental factors associated
with this initial, local dilation [i.e., mechanical changes (cell
stretch) or hemodynamic changes (wall shear stress, flow, pressure,
etc.)], we used pipette application of 106 M or
105 M sodium nitroprusside (SNP, n = 9)
at the local site to produce local dilations that fell within the range
of diameter changes over a similar area of vessel as those produced by
2-, 4-, and 8-Hz muscle contraction. If the environmental changes
associated with the local dilation produced by muscle contraction were
responsible for the remote dilation, then we should expect to see a
remote dilation upstream regardless of what mechanism was used to
initiate this local dilation. To ensure that the dilation to SNP
mimicked the wall shear stress environment of the initial local
dilation to muscle contraction, we used fluorescently labeled red blood cells (3) to measure wall shear stresses at the local site after 30 s of 4-Hz muscle stimulation and after 30 s of SNP
(n = 6).
To test whether the transmission of the dilation from the local site to
the upstream site was dependent on gap junctions, we micropipette
delivered either 0.07% halothane (n = 8), 600 mOsm
sucrose (n = 8), or 40 µM 18--glycerrhitinic acid
(-GA, n = 5) to the arteriole in the transmission
pathway between the local and upstream sites (for number of
observations see Table 1). Local and
upstream diameters after 2 min of 4-Hz muscle contraction were recorded
during control conditions, with blocker present and after recovery from
the blocker. We confirmed the efficacy of each gap junction blocker
used in this study by demonstrating their ability to block an
ACh-induced conducted response. Micropipette application of
104 M ACh to an arteriole causes a local dilation and
initiates an upstream dilation that can be attenuated by gap junction
inhibitors, such as sucrose, octanol, carbon dioxide, and halothane
(8, 20). We used micropipette application of
104 M ACh for 2 min at the local site to produce both a
local dilation and an upstream conducted dilation at a similar
arteriolar site to that used in the muscle contraction experiments
(Fig. 1). Control, blocker [0.07% halothane (n = 5),
600 mOsm sucrose (n = 6), or 40 µM -GA
(n = 5)], and recovery data were collected as in the muscle contraction protocol.
|
Materials
TTX (Calbiochem) was dissolved in distilled water, and 104 M stock was stored at 
80°C. TTX was further
diluted in superfusate just before use. -GA (Sigma Chemical, St.
Louis, MO) was made fresh daily by dissolving in DMSO and diluting to
40 µM in superfusate [final DMSO concentration was 0.04%, to avoid
direct effects of the solvent (5)]. Halothane (Ayerst
Laboratories) solution was prepared fresh daily; 1 ml of halothane was
equilibrated with 9 ml of superfusate for 2 h. Given that the
solubility of halothane in aqueous solution is ~0.35%, the
superfusate was then diluted to the working concentration just before
application. All other reagents were obtained from Sigma Chemical and
dissolved in superfusate.
Data Analysis and Statistics
Generally, only one arteriole per preparation was used to collect data. When protocols were shorter (~15 min), one to three observations, each on different arterioles, were made per preparation. The number of animals used in each protocol was at least five. The reported number of observations (n) always refers to the number of arterioles observed.All experiments were videotaped and analyzed off line. Arteriolar lengths and diameters were measured via video calipers generated by a modified video analyzer (Colorado Video, model 321) using a videotaped stage micrometer for calibration. All data are reported as means ± SE. Group means were compared by a paired Student's t-test, and time course data were analyzed with a repeated-measures ANOVA. When the ANOVA identified significant differences, means between conditions were further analyzed using Dunnett's post hoc analysis (21).
| |
RESULTS |
|---|
|
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
|
|---|
Presence of Local and Upstream Dilations Initiated by Muscle Contraction
Two minutes of 4-Hz muscle stimulation under the arteriole induced a near-maximal dilation locally and a dilation of approximately one-half this magnitude upstream (Fig. 2A). Local resting diameter was 22.4 ± 1.9 µm before the 76.4 ± 2.4% increase after muscle contraction (maximum diameter 52.2 ± 1.5 µm). At 707.1 ± 94.6 µm upstream, the resting diameter was 26.6 ± 2.2 µm before a 33.1 ± 7.2% contraction-induced increase (maximum diameter 54.0 ± 2.4 µm). Locally, diameter changed steadily over ~90 s before reaching peak values (Fig. 2B). Compared with the local response, the upstream response was slightly delayed in its initiation (~10 s) before increasing steadily throughout 2 min of contraction. Note that the onset of the upstream response appears "slow" compared with previously reported ACh-induced conducted responses (20); however, this comparison is inappropriate because ACh-induced responses are the product of rapid application of high concentrations of ACh, whereas the vascular response to muscle contraction is most likely due to slow buildup of dilator over time. Both the local and upstream diameters returned to baseline within the first 2 min of recovery from muscle contraction (data not shown). Not all of the data in Fig. 2A could be used to obtain time course information, because tissue movement due to muscle contraction made time courses at the local site difficult to obtain; upstream time courses were more frequently obtainable. Thus Fig. 2B represents paired local and upstream responses (n = 12) obtained from all control data sets within this study.
|
We confirmed that the observed remote responses were not due to mechanical movement of the tissue from the contracting muscle fibers by stimulating fibers that were located ~1,000 µm away from the remote site but not associated with the selected arteriole (see METHODS). During this contraction, mean diameter of the remote site (18.1 ± 2.1 µm) was unchanged from rest (17.8 ± 1.9 µm).
Not only does the local dilation to 4-Hz muscle contraction transmit to upstream sites but also it appears to be transmitted downstream of the local site as well. However, the response is smaller than the dilation observed upstream. The local dilation to 2 min of 4-Hz muscle contraction was 14.0 ± 1.5 µm and resulted in a dilation of 4.6 ± 0.9 µm observed 997.1 ± 57.5 µm downstream (rest 18.6 ± 1.2 µm and maximal 48.9 ± 3.2 µm diameters locally; rest 15.1 ± 1.3 µm and maximal 41.0 ± 3.3 µm diameters downstream). The magnitude of the downstream dilation is significantly smaller than the magnitude of the upstream dilation. Whether both the upstream and the downstream spread of dilation are transmitted through similar mechanisms is unknown, because humoral delivery of dilatory substances from the local contraction site to the downstream site cannot be ruled out with this geometry in this protocol.
We tested whether the upstream dilation was proportional to the
magnitude of the local contraction (or the rate of local metabolism) by
observing the local and upstream responses to 2-, 4-, and 8-Hz muscle
contraction (Fig. 3). Locally, the
resting diameter of 18.7 ± 3.2 µm was increased significantly
to 26.9 ± 3.3, 43.7 ± 5.4, and 49.6 ± 4.7 µm by 2-, 4-, or 8-Hz, respectively (maximum diameter 50.7 ± 4.9 µm). The
upstream site (1,034 ± 66.0 µm from contracting fibers)
increased significantly from 23.7 ± 2.8 µm at rest to 27.8 ± 2.6, 36.9 ± 3.9. and 42.4 ± 4.8 µm by 2-, 4-, or 8-Hz
muscle contraction, respectively (maximum diameter 52.9 ± 4.2 µm). The upstream dilation is approximately proportional to the
magnitude of the local contraction in that the upstream site dilation
is approximately one-half of the magnitude of the local dilation at all
stimulation frequencies.
|
In a separate set of arterioles, we looked for decay of the upstream
dilation along the length of the observed vessel. Two minutes of 4-Hz
muscle contraction produced a 25.0 ± 2.6-µm dilation locally
(rest diameter at this site was 25.8 ± 1.4-µm, maximum diameter
57.5 ± 3.6 µm), a 15.6 ± 2.3-µm dilation at a site
670 ± 34.3 µm upstream (midvessel) from the local site (rest
diameter 29.0 ± 2.3 µm, maximum diameter 59.5 ± 3.6 µm), and a 13.0 ± 1.9-µm dilation at a site 1,467.8 ± 56.1 µm upstream from the local site (rest diameter 28.8 ± 2.6 µm, maximum diameter 56.3 ± 3.6 µm). The change in diameter
during 2 min of muscle stimulation for the two upstream sites is shown
in Fig. 4. Note that the responses are
similar in magnitude; thus the signal does not decay over the length of
arteriole observed. There is, however, a significant delay in the onset
of dilation between the two upstream sites.
|
Nature of the Pathway Connecting Local and Upstream Dilations
We applied TTX between the local and upstream site to inhibit any perivascular nerve activity in the conduction pathway and found that TTX failed to attenuate the remote upstream dilation (Fig. 5). For vessel baseline and maximum diameters, see Table 1.
|
To explore the contribution of local environmental factors associated
with the local dilation in initiating the upstream response we applied
either 106 M or 105 M SNP for 2 min at the
local site and recorded diameter changes at the local and upstream
sites. SNP was used to mimic the magnitude and rates of local dilation
to muscle contraction within the range of dilations that were observed
to produce upstream responses at 2, 4, and 8 Hz (Fig. 3). Neither the
percent increase in arteriolar diameter with local 4-Hz muscle
contraction (52.2 ± 10.3%) or SNP (76.3 ± 28.3%) nor the
relative drop in wall shear stress during the dilation produced by
muscle stimulation (63.1 ± 8.6%) or SNP (79.7 ± 8.0%)
were different from each other over the initial 30 s. But, unlike
muscle contraction, there was no upstream dilation associated with the
local SNP-induced dilation (Fig. 6). This suggests that factors associated with the generation of a local dilation do not themselves contribute significantly to the initiation of the remote response.
|
We tested whether the local dilation was transmitted upstream via gap
junctions. We measured control local and upstream dilations to 4-Hz
muscle stimulation and then applied either sucrose, halothane, or
-GA between the local and upstream sites. Sucrose caused a large
dilation at the site of pipette application that was maintained throughout the duration of its application; however, this dilation did
not change the resting diameter of either the local or upstream observation sites significantly (Table 1). Halothane application caused
a smaller and more transient dilation of the arteriole that did not
affect the resting diameters of the local or upstream observation
sites. -GA had no noticeable effect on arteriolar diameter at the
site of application. Neither halothane nor -GA inhibited the
contraction-induced upstream response (Fig.
7). To ensure that these gap junction
blockers would indeed block a gap junction-dependent response, we used
2 min of 104 M ACh application to the local site
to induce a local and a conducted dilation. Both halothane and -GA
significantly attenuated the upstream dilation initiated by 2 min of
ACh application. In contrast, sucrose attenuated both the
contraction-induced upstream response as well as the ACh-induced
upstream response (Fig. 7). Note in Fig. 7 that although the magnitude
of the local dilations differs slightly between groups (different
vessels), it does not differ significantly within a group (local,
blocker, and recovery data from the same vessels). Also note in Fig. 7
that in each group, the upstream dilation initiated by 4-Hz muscle
contraction is consistently approximately one-half the magnitude of the
local dilation.
|
We used 137 mM KCl between the local and the upstream site to block any
conducted hyperpolarization that might be involved in the signal
transmission pathway. KCl caused a constriction at the site of
application in four of the eight arterioles studied, but neither local
nor upstream resting diameters were affected after 10 min of KCl
application (Table 1). Figure 8 shows
that KCl had no effect on peak diameter changes at the upstream site after 4-Hz muscle contraction.
|
Venular-arteriolar diffusion has been documented between paired veins and arterioles whereby venular diffusion of dilatory substances has been observed to dilate the paired arteriole (11). Although the transverse arteriole has no paired venule running adjacent to it, there are often venules that run across the arteriole. We sought to investigate whether venular-arteriolar diffusion of dilatory substances was the cause of the upstream dilation induced by local muscle contraction. If the venous system was delivering dilatory substances to the upstream site, then the magnitude of the upstream dilation should correlate with the distance of the upstream site to the nearest venule. We chose 25 experiments at random to reanalyze. From images of the upstream observation site taken at low power (×10), we identified venules in close approximation to the upstream site by transillumination and plotted this distance (range 12-200 µm) against the magnitude of the upstream dilation and found no correlation between these two factors (r2 = 0.09).
| |
DISCUSSION |
|---|
|
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
|
|---|
This study shows that skeletal muscle fiber contraction associated with arterioles not only causes a local arteriolar dilation but also initiates a signal in the arteriole that is transmitted upstream resulting in a remote upstream dilation. Our data indicate that the transmitted signal is different either from the signal initiated by muscle contraction at the capillary level or from signals initiated pharmacologically by ACh at the arteriolar level.
Vasodilation of arterioles at the site of local muscle contraction has been described previously (9), but no major dilation beyond the contracting cells was observed; however, low-power objectives were used, and it was suggested by the authors that higher resolution would be necessary to study any upstream response directly. Using a very similar technical approach that produced similar local responses to those of Gorczynski et al. (9), we were indeed able, using higher resolution, to observe a remote upstream dilation initiated by muscle contraction. We could not induce the same remote dilation when muscle fibers that did not intersect with the test arteriole were stimulated at a similar distance from the upstream site. Thus we ruled out both tissue movement and diffusion of dilatory substances from active muscle as mechanisms to produce the remote response. The persistence of signal transmission during TTX exposure indicates that the upstream dilation is also independent of neural mechanisms.
We investigated whether factors associated with the local vasodilation were themselves enough to initiate the remote upstream dilation. Muscle contraction produces a local dilation that relates to contraction frequency (Fig. 3), and, independent of the magnitude or the rate of the dilation that occurred locally, an upstream response was always produced. We used SNP to mimic the local dilation to muscle contraction within the range of dilations observed to produce upstream responses and found that the dilation initiated by SNP was unable to initiate an upstream dilation. Thus we cannot ascribe the initiation of the upstream dilation to hemodynamic or mechanical factors associated with local dilation itself. This does not, however, rule out any role that such factors might play in the modification or the maintenance of the response over the 2 min of muscle stimulation. We also found no correlation between the magnitude of the upstream dilation and its distance to the nearby venules, thus ruling out venular-arteriolar diffusion of vasoactive substances as a primary cause of the upstream dilation. As neither tissue movement, diffusion, perivascular nerves, hemodynamic changes, nor venular-arteriole diffusion appear to be involved in the production of the upstream dilation, we conclude that the dilatory signal induced by muscle contraction is being transmitted from the local to the upstream site through the arteriolar wall.
Intravascular communication within the vessel wall has been studied extensively using ACh- or phenylephrine-induced conducted responses, and these responses have been associated with conducted changes in membrane potential (24, 26). In our study, given that 137 mM KCl in the transmission pathway did not interfere with the conduction of the dilation, hyperpolarization does not appear to be a major component of the contraction-induced upstream dilation, although a role for local endothelial-derived hyperpolarizing factor (EDHF) release cannot be ruled out (2).
Gap junctional communication between endothelial cells, vascular smooth
muscle cells, or both, has been identified as a pathway through which
the transmission of a conducted response induced by ACh may travel
(20). Each of the three gap junction blockers used in this
study were able to attenuate the conducted response induced by ACh, but
only sucrose was able to attenuate the contraction-induced conducted
response, implying that the two transmit signals to upstream sites via
different mechanisms. The differential effects of the gap junction
blockers may be attributable to the different modes of action of the
gap junction inhibitors, differences that may reflect their ability to
completely block specific connexins, to block either dye or electrical
coupling or both, to alter conductance states of gap junctions, to
exert nonspecific effects, etc. In particular, note that sucrose caused
a vasodilation at the site of application, whereas -GA and halothane
did not, suggesting that it initiated nonspecific effects that might
have contributed to the attenuation of the ACh-induced and
contraction-induced remote dilations. Given the potential nonspecific
effects of sucrose and the inability of either -GA or halothane to
attenuate the contraction-induced upstream dilation, we conclude that
the transmission of the contraction-induced response upstream is not
primarily a gap junctionally mediated event.
That the contraction-induced remote dilation appears unlikely to primarily involve gap junctions in its transmission not only makes this signal different from ACh-induced conducted responses but also from contraction-induced remote dilations initiated at the capillary level (3, 5). Most obviously, in our study, the vessels overlying the contracting muscle fibers contain both vascular smooth muscle and endothelial cells, whereas in the studies of Berg et al. (3) and Cohen et al. (5), the capillary overlying the contracting fibers consists primarily of endothelial cells. Muscle fiber contraction under capillaries, therefore, most likely stimulates endothelial cells and initiates a signal that is transmitted through endothelial cells eventually to cause upstream dilation where vascular smooth muscle is present. When skeletal muscle cells are stimulated under an arteriole, any signal from contracting muscle will first encounter the vascular smooth muscle cells; hence the signal that causes the remote upstream dilation may be primarily based in a vascular smooth muscle-mediated transmission system. Recent studies indicate that vascular smooth muscle cells and endothelial cells in small arterioles may act either as a syncytium (15, 26, 28) or independently (19, 23, 24). Here we raise the possibility that transmitted signals arising through vascular smooth muscle cells may be different from those initiated through endothelial cells.
If a signal transmitted along the vessel wall is not coupled via gap junctions, what are other potential mechanisms of transmission? Paracrine signaling could be involved. Paracrine relationships are often viewed as slow due to the many steps required, i.e., production of the signaling molecule, release, diffusion, and binding of the molecule to adjacent cells, second messenger production by this cell, etc. However, rapid paracrine signaling is well known (most obviously, via nitric oxide or EDHF). The lack of decay of our contraction-induced upstream dilation (Fig. 4) supports the idea of a regenerating signal rather than an electrotonic signal decaying over the length of the vessel. Interestingly, a recently proposed model for intracellular calcium signaling in glial cells (6) contains key elements that may be relevant to the contraction-induced remote dilation that we describe here, including gap junctional communication of different molecular signals (e.g., ions, second messengers) as well as paracrine signaling between cells. Such a model could explain many of the elements of the contraction-induced upstream dilations observed at both the arteriolar level (this study) and the upstream dilations induced by muscle contraction under capillaries (3, 5).
Mechanisms that result in local arteriolar dilation initiated by muscle contraction and mechanisms that transmit this local dilation upstream have clear relevance to our understanding of the coupling between blood flow and metabolism in exercising muscle. Arterioles function both to modulate blood flow through a tissue and to control capillary recruitment. An increase in functional capillary density, via capillary blood flow redistribution and recruitment of capillaries, is a significant early response to increased muscle metabolism (10, 12-14). Recent studies (3, 5) have shown that capillary recruitment is subserved by a signal initiated at the capillaries and conducted via gap junctions to the terminal arteriolar bed. Subsequent to capillary recruitment, further increases in tissue blood flow must be served by decreased arteriolar resistance. The remote dilation described in our study would contribute to the arteriolar resistance changes observed in exercising muscle that serve to maintain increased blood flow. Clearly, in an intact tissue, multiple signaling pathways will together sum to produce the overall blood flow response. Our study identifies one pathway by which vascular responses in a particular region of muscle can be matched locally to the skeletal muscle fiber activity despite the heterogeneous blood flow distribution that is typical of the dispersed motor unit distribution that is found in mixed muscles (16).
In summary, we demonstrated that muscle contraction under arterioles not only produces a local arteriolar dilation but also initiates a signal that is transmitted upstream to cause remote dilations. The transmission of this upstream signal does not appear to involve perivascular nerves or gap junctions and is therefore different from pharmacologically induced conducted dilations at the arteriolar level and different from the skeletal muscle contraction-induced upstream responses initiated at the capillary level. We propose a model involving local paracrine signaling to account for transmission of this dilatory response along the vessel wall. This signal could be between endothelial cells, between vascular muscle cells, or between the two cell types. Regardless of how the signal is transmitted from the local to the upstream site, the spread of dilatory signal that is initiated by muscle contraction at the arteriolar level seems likely to have the capacity to aid in the coordination of muscle metabolism and blood flow during exercise.
| |
ACKNOWLEDGEMENTS |
|---|
We thank Patricia A. Titus for skilled technical assistance and Ken Cohen for helpful discussion.
| |
FOOTNOTES |
|---|
This study was supported by National Heart, Lung, and Blood Institute Grant RO1-HL-56574 and American Heart Association Fellowship 9820063T.
Address for reprint requests and other correspondence: I. H. Sarelius, Dept. Pharmacology/Physiology, Univ. of Rochester, Box 711, Medical Center, Rochester, NY 14642 (E-mail: ingrid_sarelius{at}urmc.rochester.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 7 December 1999; accepted in final form 13 June 2000.
| |
REFERENCES |
|---|
|
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
|
|---|
1.
Baez, S.
An open cremaster muscle preparation for the study of blood vessels by in vivo microscopy.
Microvasc Res
5:
384-394,
1973[Web of Science][Medline].
2.
Beny, J-L.
Endothelial and smooth muscle cells hyperpolarized by bradykinin are not dye coupled.
Am J Physiol Heart Circ Physiol
258:
H836-H841,
1990
3.
Berg, BR,
Cohen KD,
and
Sarelius IH.
Direct coupling between blood flow and metabolism at the capillary level in striated muscle.
Am J Physiol Heart Circ Physiol
272:
H2693-H2700,
1997
4.
Berg, BR,
and
Sarelius IH.
Functional capillary organization in striated muscle.
Am J Physiol Heart Circ Physiol
268:
H1215-H1222,
1995
5.
Cohen, KD,
Berg BR,
and
Sarelius IH.
Remote arteriolar dilations in response to muscle contraction under capillaries.
Am J Physiol Heart Circ Physiol
279:
H1916-H1923,
2000.
6.
Charles, A.
Intercellular calcium waves in glia.
Glia
24:
39-49,
1998[Web of Science][Medline].
7.
Delashaw, JB,
and
Duling BR.
Heterogeneity in conducted arteriolar vasomotor response is agonist dependent.
Am J Physiol Heart Circ Physiol
260:
H1276-H1282,
1991
8.
Frame, MDS,
and
Sarelius IH.
L-Arginine-induced conducted signals alter upstream arteriolar responsivity to L-arginine.
Circ Res
77:
695-701,
1995
9.
Gorczynski, RJ,
Klitzman B,
and
Duling BR.
Interrelations between contracting striated muscle and precapillary microvessels.
Am J Physiol Heart Circ Physiol
235:
H494-H504,
1978
10.
Hargreaves, D,
Egglington S,
and
Hudlicka O.
Changes in capillary perfusion induced by different patterns of activity in rat skeletal muscle.
Microvasc Res
40:
14-28,
1990[Web of Science][Medline].
11.
Hester, RL.
Venular-arteriolar diffusion of adenosine in hamster cremaster microcirculation.
Am J Physiol Heart Circ Physiol
258:
H1918-H1924,
1990
12.
Honig, CR,
Odoroff CL,
and
Frierson JL.
Capillary recruitment in exercise: rate, extent, uniformity, and relation to blood flow.
Am J Physiol Heart Circ Physiol
238:
H31-H42,
1980
13.
Honig, CR,
Odoroff CL,
and
Frierson JL.
Active and passive capillary control in red muscle at rest and in exercise.
Am J Physiol Heart Circ Physiol
243:
H196-H206,
1982
14.
Klitzman, B,
Damon DN,
Gorzynski RJ,
and
Duling BR.
Augmented tissue oxygen supply during striated muscle contraction in the hamster.
Circ Res
51:
711-721,
1982
15.
Little, TL,
Xia J,
and
Duling BR.
Dye tracers define differential endothelial and smooth muscle coupling patterns within the arteriolar wall.
Circ Res
76:
498-504,
1995
16.
Murrant, CL,
and
Sarelius I.
Coupling of muscle metabolism and muscle blood flow in capillary units during contraction.
Acta Physiol Scand
168:
531-541,
2000[Web of Science][Medline].
17.
Sarelius, IH,
Damon DN,
and
Duling BR.
Microvascular adaptations during maturation of striated muscle.
Am J Physiol Heart Circ Physiol
241:
H317-H324,
1981.
18.
Sarelius, IH,
and
Huxley VH.
A direct effect of atrial peptide on arterioles of the terminal microvasculature.
Am J Physiol Regulatory Integrative Comp Physiol
258:
R1224-R1229,
1990
19.
Segal, SS,
and
Beny J-L.
Intracellular recording and dye transfer in arterioles during blood flow control.
Am J Physiol Heart Circ Physiol
263:
H1-H7,
1992
20.
Segal, SS,
and
Duling BR.
Conduction of vasomotor responses on arterioles: a role for cell-to-cell coupling?
Am J Physiol Heart Circ Physiol
256:
H838-H845,
1989
21.
Snedecor, GW,
and
Cochran WG.
Statistical Methods (8th ed.). Ames, IA: Iowa State University Press, 1989.
22.
Sweeney, TE,
and
Sarelius IH.
Arteriolar control of capillary cell flow in striated muscle.
Circ Res
64:
112-120,
1989
23.
Welsh, DG,
Jackson WF,
and
Segal SS.
Oxygen induces electromechanical coupling in arteriolar smooth muscle cells: a role for L-type Ca2+ channels.
Am J Physiol Heart Circ Physiol
274:
H2018-H2024,
1998
24.
Welsh, DG,
and
Segal SS.
Endothelial and smooth muscle cell conduction in arterioles controlling blood flow.
Am J Physiol Heart Circ Physiol
274:
H178-H186,
1998
25.
Wiedeman, MP.
Blood flow through terminal arterial vessels after denervation of the bat wing.
Circ Res
22:
83-89,
1968
26.
Xia, J,
and
Duling BR.
Electromechanical coupling and the conducted vasomotor response.
Am J Physiol Heart Circ Physiol
269:
H2022-H2030,
1995
27.
Xia, J,
and
Duling BR.
Patterns of excitation-contraction coupling in arterioles: dependence on time and concentration.
Am J Physiol Heart Circ Physiol
274:
H323-H330,
1998
28.
Xia, J,
Little TL,
and
Duling BR.
Cellular pathways of the conducted electrical response in arterioles of hamster cheek pouch in vitro.
Am J Physiol Heart Circ Physiol
269:
H2031-H2038,
1995
This article has been cited by other articles:
|
|
 |
|
  C. de Wit Different pathways with distinct properties conduct dilations in the microcirculation in vivo Cardiovasc Res, October 31, 2009; (2009) cvp340v2. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 B. E. Carlson, J. C. Arciero, and T. W. Secomb Theoretical model of blood flow autoregulation: roles of myogenic, shear-dependent, and metabolic responses Am J Physiol Heart Circ Physiol, October 1, 2008; 295(4): H1572 - H1579. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. P. Veliz, F. G. Gonzalez, B. R. Duling, J. C. Saez, and M. P. Boric Functional role of gap junctions in cytokine-induced leukocyte adhesion to endothelium in vivo Am J Physiol Heart Circ Physiol, September 1, 2008; 295(3): H1056 - H1066. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. C. Hocking, P. A. Titus, R. Sumagin, and I. H. Sarelius Extracellular Matrix Fibronectin Mechanically Couples Skeletal Muscle Contraction With Local Vasodilation Circ. Res., February 15, 2008; 102(3): 372 - 379. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. L. Armstrong, A. K. Dua, and C. L. Murrant Time course of vasodilation at the onset of repetitive skeletal muscle contractions Am J Physiol Regulatory Integrative Comp Physiol, January 1, 2007; 292(1): R505 - R515. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. C. Jantzi, S. E. Brett, W. F. Jackson, R. Corteling, E. J. Vigmond, and D. G. Welsh Inward rectifying potassium channels facilitate cell-to-cell communication in hamster retractor muscle feed arteries Am J Physiol Heart Circ Physiol, September 1, 2006; 291(3): H1319 - H1328. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. L. Murrant Stimulation characteristics that determine arteriolar dilation in skeletal muscle Am J Physiol Regulatory Integrative Comp Physiol, August 1, 2005; 289(2): R505 - R513. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. T. Kurjiaka, S. B. Bender, D. D. Nye, W. B. Wiehler, and D. G. Welsh Hypertension attenuates cell-to-cell communication in hamster retractor muscle feed arteries Am J Physiol Heart Circ Physiol, February 1, 2005; 288(2): H861 - H870. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. Duza and I. H. Sarelius Increase in endothelial cell Ca2+ in response to mouse cremaster muscle contraction J. Physiol., March 1, 2004; 555(2): 459 - 469. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. Duza and I. H. Sarelius Conducted dilations initiated by purines in arterioles are endothelium dependent and require endothelial Ca2+ Am J Physiol Heart Circ Physiol, June 5, 2003; 285(1): H26 - H37. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. L. Murrant and I. H. Sarelius Multiple dilator pathways in skeletal muscle contraction-induced arteriolar dilations Am J Physiol Regulatory Integrative Comp Physiol, April 1, 2002; 282(4): R969 - R978. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. D Cohen and I. H Sarelius Muscle contraction under capillaries in hamster muscle induces arteriolar dilatation via KATP channels and nitric oxide J. Physiol., March 1, 2002; 539(2): 547 - 555. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Visit Other APS Journals Online |
) and
the remote upstream site (
) during 2 min of 4-Hz muscle
contraction (n = 12).
) and a second site further upstream 1,467.8 ± 6.1 µm from the local site (
