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. 2001 Nov 1;536(Pt 3):937–946. doi: 10.1111/j.1469-7793.2001.00937.x

Role for endothelial cell conduction in ascending vasodilatation and exercise hyperaemia in hamster skeletal muscle

Steven S Segal 1, Tonya L Jacobs 1
PMCID: PMC2278906  PMID: 11691885

Abstract

  1. Vasodilatation initiated by contracting skeletal muscle ‘ascends’ from the arteriolar network to encompass feed arteries. Acetylcholine delivery from a micropipette onto a feed artery evokes hyperpolarisation at the site of application; this signal can conduct through gap junctions along the endothelium to produce vasodilatation. We tested whether conduction along the endothelium contributes to the ascending vasodilatation that occurs in response to muscular exercise.

  2. In anaesthetised hamsters, a feed artery (resting diameter 64 ± 4 μm) supplying the retractor muscle was either stimulated by local microiontophoretic application of acetylcholine or the muscle was contracted rhythmically (once per 2 s, 1–2 min), before and after light-dye treatment (LDT) to disrupt the endothelial cells within a 300 μm-long segment located midway along the vessel. Endothelial cell damage with LDT was confirmed by the local loss of vasodilatation in response to acetylcholine and labelling with propidium iodide. Local vasodilatation in response to acetylcholine applied 500 μm proximal (upstream) or distal (downstream) to the central segment with LDT remained intact.

  3. Before LDT, vessel diameter increased by more than 30 % along the entire feed artery (observed 1000 μm upstream from the retractor muscle) in response to distal acetylcholine or muscle contractions. Following LDT, vasodilatation in response to acetylcholine and to muscle contractions encompassed the distal segment but did not travel through the region of endothelial cell damage. At the upstream site, wall shear rate (and luminal shear stress) increased more than 3-fold, with no change in vessel diameter. Thus, flow-induced vasodilatation did not occur.

  4. In response to muscle contractions, feed artery blood flow increased nearly 6-fold; this hyperaemic response was reduced by half following the loss of ascending vasodilatation.

  5. These findings indicate that rhythmic contractions of skeletal muscle can initiate the conduction of a signal along the endothelium. We propose that this signalling pathway underlies ascending vasodilatation and promotes the full expression of exercise hyperaemia.

Ascending vasodilatation entails the progression of smooth muscle relaxation from distal branches of the arteriolar network, through intermediate and proximal arteriolar segments, and into the arterial supply proximal to the muscle (Hilton, 1959; Folkow et al. 1971; Goodman et al. 1978; Williams & Segal, 1993; Welsh & Segal, 1997). This coordinated interaction within and among vascular segments is invoked by a mismatch between oxygen supply and demand during hypoxia (Granger et al. 1976) and by rhythmic contractions of skeletal muscle (Hilton, 1959; Welsh & Segal, 1997; Van Teeffelen & Segal, 2000). Independent of neural pathways (Honig & Frierson, 1976), two distinct mechanisms have been proposed to explain how vasodilatation spreads from intramuscular arterioles into the proximal arterial supply. One mechanism invokes flow-induced vasodilatation, which reflects the increase in luminal shear stress (the product of wall shear rate, WSR, and blood viscosity) acting on endothelial cells to stimulate the production of vasodilatory autacoids (Pohl et al. 1986; Koller & Kaley, 1990; Smiesko & Johnson, 1993; Koller et al. 1994; Davies, 1995). In this manner, dilatation of the distal segments will increase flow through the proximal segments to stimulate endothelial cells. An alternative mechanism entails the production of a dilatory signal that may be conducted from cell to cell along the vessel wall (Segal & Duling, 1986b; Welsh & Segal, 1998; Emerson & Segal, 2000a,b) in response to the contraction of skeletal muscle fibres (Hilton, 1959; Welsh & Segal, 1997).

Micropressure measurements indicate that the feed arteries of the hamster retractor muscle (Welsh & Segal, 1996) and of the rat soleus and extensor digitorum longus muscles (Williams & Segal, 1993) contribute up to ∼30 % of the total resistance to blood flow. In these preparations, as well as the rat spinotrapezius muscle (Lash, 1994), dilatation of feed arteries has been observed in response to rhythmic muscle contractions. In the retractor muscle, dilatation of the feed arteries increases with duty cycle (Welsh & Segal, 1997) and with motor unit recruitment (Van Teeffelen & Segal, 2000). Conversely, ascending vasodilatation is suppressed during muscle fatigue and reductions in metabolic demand (Jacobs & Segal, 2000). Indeed, the lack of feed artery dilatation has been predicted to limit muscle blood flow by maintaining high vascular resistance proximal to the arteriolar network (Segal & Duling, 1986a; Williams & Segal, 1993). Remarkably, the signalling mechanism by which vasodilatation originating within exercising skeletal muscle is transmitted upstream into the arterial supply has remained unresolved.

In vitro studies of feed arteries from the retractor muscle have demonstrated that the conduction of hyperpolarisation along the endothelial cell layer is highly effective in driving the hyperpolarisation and relaxation of surrounding smooth muscle cells (Emerson & Segal, 2000a,b, 2001). Furthermore, acetylcholine reliably evokes hyperpolarisation of endothelial cells to initiate the conduction of vasodilatation along arterioles as well as feed arteries (Welsh & Segal, 1998; Emerson & Segal, 2000a). Whereas the conduction of vasodilatation into feed arteries has been inferred in studies of functional hyperaemia (Hilton, 1959; Welsh & Segal, 1997), it has not been established whether conduction is actually evoked by muscular activity; nor has the cellular pathway of ascending vasodilatation been ascertained.

In the present study, our goal was to determine the mechanism by which vasodilatation ascends from within contracting skeletal muscle and into its feed arteries. We hypothesised that cell-to-cell conduction along the endothelium is a critical component of the integrated vasomotor response. To test this hypothesis, endothelial cells within a segment of a feed artery supplying the retractor muscle were selectively damaged using light-dye treatment (LDT; Bartlett & Segal, 2000; Emerson & Segal, 2000a). Feed artery responses to muscle contraction were evaluated before and after LDT with corresponding reference to the conduction of vasodilatation evoked by acetylcholine. Red blood cell velocity (Vrbc) and luminal diameter were evaluated concomitantly to determine whether changes in blood flow and WSR (a direct index of luminal shear stress assuming constant blood viscosity) were integral to ascending vasodilatation.

METHODS

Animal care and use

Procedures were reviewed and approved by the Institutional Animal Care and Use Committee of The John B. Pierce Laboratory in accordance with the Guide for the Care and Use of Laboratory Animals (National Research Council, USA). Male golden hamsters (∼100 g; Charles River Breeding Laboratories, Kingston, NY, USA) were anaesthetised with pentobarbital sodium (60 mg kg−1; i.p.). Surgical procedures were performed using a stereomicroscope; oesophageal temperature was maintained at ∼37 °C with conducted heat. The trachea was intubated (PE 190 tubing) to ensure airway patency and the left femoral vein was cannulated (PE 50 tubing) to allow the replacement of fluids and maintenance of anaesthesia (10 mg pentobarbital (ml isotonic saline)−1, infused at 410 μl h−1). The hamster was positioned on a transparent acrylic platform, then the right retractor muscle was prepared for observing feed arteries and secured in a 10 ml chamber integral to the platform; mean arterial (carotid) pressure was consistently 90–100 mmHg throughout experiments (Jacobs & Segal, 2000; Van Teeffelen & Segal, 2000). The completed preparation was transferred to the stage of an intravital microscope (ACM, Zeiss, Thornwood, NY, USA). Each end of the muscle was connected to a micrometre drive, with one end attached to a force transducer (Jacobs & Segal, 2000). Muscle length was adjusted to obtain peak twitch tension and the preparation was equilibrated for ∼1 h. Upon the completion of experimental procedures (duration, 3–4 h), an overdose of pentobarbital was delivered through the venous cannula.

Reagents

The muscle was superfused continuously (10 ml min−1) with fresh bicarbonate-buffered physiological saline solution (PSS, 34–35 °C; pH 7.4); effluent was aspirated continuously to maintain a constant volume and fluid level within the muscle chamber. PSS was composed of the following (mm): NaCl 131.9, KCl 4.7, CaCl2 2.0, MgSO4 1.2 and NaHCO3 18, and was equilibrated with 95 % N2 and 5 % CO2. These reagents were obtained from Sigma (St Louis, MO, USA) or J. T. Baker (Phillipsburg, NJ, USA) and were dissolved in purified deionised water (dH2O). Stock solutions were prepared weekly, stored at 4 °C, and diluted to final working concentrations on the day of an experiment. Synthetic capsaicin (Pfaltz & Bauer; Waterbury, CT, USA) was prepared fresh daily (1 mm in 100 % ethanol) and diluted 1000-fold by addition to the superfusion solution.

Light-dye treatment

Three locations were defined along a feed artery (Fig. 1A). The distal site was taken as that just external to the medial edge of the muscle. Central and proximal sites were located 500 and 1000 μm upstream from the distal site, respectively (Bartlett & Segal, 2000; Emerson & Segal, 2000a). Endothelial cells were damaged at the central site by illuminating a fluorochrome contained within the vessel lumen. For this purpose, fluorescein isothiocyanate conjugated to bovine serum albumin (FITC-BSA, ∼70 kDa; molar substitution ratio = 3:1) was injected (0.3 ml, 10 % w/v) into the femoral vein and equilibrated in the systemic circulation for 10 min. Based upon previous studies (Bartlett & Segal, 2000; Emerson & Segal, 2000a) and preliminary studies (data not shown), a segment (length, 300 μm) midway along a feed artery was epi-illuminated for 6 min through a fluorescein filter cube (Zeiss 487705; excitation, 450–490 nm; emission, > 520 nm) using a ×40 immersion objective (Zeiss; NA = 0.75); a 75 W xenon arc lamp provided light. After 5 min of recovery, the vasomotor responses to acetylcholine at the central site were re-evaluated (Fig. 1B). Illumination and recovery periods were repeated until the responses to acetylcholine delivered to the central site were abolished (Bartlett & Segal, 2000; Emerson & Segal, 2000a).

Figure 1. The effect of LDT at the central site on resting feed artery diameter and vasodilatation in response to acetylcholine.

Figure 1

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A, illustration of the experimental design. The retractor muscle (RET) is shown reflected away from the hamster with the direction of blood flow indicated along the feed artery (other vessels are omitted for clarity). Asterisks indicate sites at which diameter measurements were obtained: the distal site (D) was just external to the muscle; the central site was 500 μm upstream and centred within the segment exposed to LDT (see Methods); the proximal site (P) was 1000 μm upstream from the distal site. B, resting diameter (filled symbols) and corresponding change in diameter (open symbols; calculated as peak minus rest values) in response to the microiontophoretic application of acetylcholine (1000 nA, 500 ms) at the central site during LDT (n = 9). Each period of illumination lasted 6 min, with vessel diameter and vasodilatation evaluated after 5 min of recovery. Vasodilatation decreased progressively during LDT and was abolished following the fourth period of illumination. Maximal diameter, 98 ± 4 μm. * P < 0.001, main effect of LDT; one-way repeated measures ANOVA.

Microiontophoresis

Acetylcholine (1 m in dH2O; Sigma) was delivered abluminally using microiontophoresis (1000 nA, 500 ms) from a borosilicate glass micropipette with the tip (i.d. 1–2 μm) positioned adjacent to the vessel wall using a micromanipulator. This brief stimulus evokes transient maximal dilatation at the site of delivery with negligible tachyphylaxis (Segal et al. 1999; Emerson & Segal, 2000a). Acetylcholine evokes endothelium-dependent vasodilatation and hyperpolarisation in feed arteries; conduction of these responses confirms the integrity of the endothelium along the vessel intima (Emerson & Segal, 2000a). Phenylephrine (0.5 m in dH2O; Sigma) was delivered in an identical manner, with local vasoconstriction providing an index of smooth muscle integrity (Bartlett & Segal, 2000; Emerson & Segal, 2000a). Acetylcholine was prepared every few weeks and stored at 4 °C; phenylephrine was prepared fresh and protected from light.

Muscle stimulation

Field stimulation (0.1 ms pulse at 140 V) was performed using platinum electrodes secured to either side of the muscle chamber. Stimuli were delivered using a stimulus isolation unit (SIU5, Grass Instruments, Quincy, MA, USA), which was driven by a square-wave stimulator (S48, Grass), to depolarise motor end plates on retractor muscle fibres (Welsh & Segal, 1997; Jacobs & Segal, 2000). Before and following LDT, rhythmic contractions were implemented (400–800 ms train at 40–70 Hz; once per 2 s for 60–100 s). Based upon previous experiments (Welsh & Segal, 1997; Jacobs & Segal, 2000; Van Teeffelen & Segal, 2000), the level of activity was adjusted across preparations to consistently evoke similar dilatation of feed arteries under control conditions while minimising muscle fatigue. When normalised to muscle cross-sectional area (average muscle length = 30 mm; mass = 88 mg; Jacobs & Segal, 2000), integrated tension (g s−1) corresponded to ∼20 % duty cycle, generating 50 % of the maximum tetanic tension (Jacobs & Segal, 2000).

Criterion experiments

One feed artery was studied per hamster, as illustrated in Fig. 1A. Responses to acetylcholine and to muscle contraction were evaluated for each vessel. In three of these experiments spontaneous tone was weak and so phenylephrine was added to the PSS (10−8m). Resting tone (∼65 % of maximal diameter) and vasomotor responses during these three experiments were similar to those of the six vessels studied without phenylephrine; therefore, data were pooled. Summary data from these vessels (n = 9) are presented in Figs 1–5. The maximum diameter for each vessel was determined by the addition of sodium nitroprusside (10 μm; Sigma) to the superfusion solution.

Figure 5. The effect of LDT on Vrbc and WSR responses to muscle contractions.

Figure 5

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Centreline Vrbc and WSR were determined at the proximal site (refer to Fig. 1A) at rest and corresponding peak values were determined upon cessation of contractions (see Methods; n = 9). A, Vrbc was elevated above Rest to a similar extent by muscle contractions both pre- and post-LDT. B, the increase in WSR following LDT was nearly twice that observed before LDT. Data were analysed using one-way repeated measures ANOVA with Student-Neuman-Keuls test for post hoc comparisons. * P < 0.01, Peak vs. Rest; +P < 0.05, Post vs. Pre.

Acetylcholine was delivered to the distal site and local vasomotor responses were recorded. After recovery of the resting diameter (2–3 min), the stimulus was repeated at the distal site to confirm conduction at the proximal site. Acetylcholine was then delivered to the central site and to the proximal site to evaluate respective local responses under control conditions. Each evaluation of feed artery responses to muscle contraction entailed a separate period of contractile activity; previous experiments have verified that the entire vessel dilates to a similar extent under control conditions (Welsh & Segal, 1997; Jacobs & Segal, 2000; Van Teeffelen & Segal, 2000). Therefore, to minimise muscle activity and fatigue, control responses at the proximal site were taken to represent those of the entire vessel. The order in which responses to acetylcholine and to muscle contraction were evaluated was varied across hamsters.

Once control data were acquired, LDT was performed midway along the vessel (Fig. 1A). Following LDT, the effects of muscle contraction were evaluated at the distal site to ascertain whether feed arteries were still responsive, and at the proximal site to determine the effect of LDT on ascending vasodilatation. The preparation was allowed to recover between periods of contractile activity until resting tone was re-established (typically 2–3 min).

Evaluation of cellular integrity

The following manoeuvres were performed to determine the integrity of endothelial cells and smooth muscle cells following LDT. Responses initiated by acetylcholine at the distal site were evaluated at the proximal site to ascertain whether conduction still occurred through the central segment. Local responses to acetylcholine were evaluated at the distal site and at the proximal site to evaluate endothelium-dependent vasodilatation at the respective locations. Local responses to phenylephrine were evaluated at the central site and at the distal site to determine the effect of LDT on smooth muscle contraction.

Cellular damage was evaluated independently with propidium iodide (Molecular Probes, Eugene, OR, USA); this vital dye is normally excluded from healthy cells and permeates cells only following membrane disruption, thereby revealing the nuclei in damaged cells (Bartlett & Segal, 2000; Emerson & Segal, 2000a). Following data collection in two experiments, propidium iodide was injected (10−6m in sterile isotonic saline; 0.5 ml) via the venous catheter and allowed to circulate for at least 10 min. Dye labelling was evaluated with epi-illumination using an immersion objective and a rhodamine filter cube (Zeiss 487715; excitation, 546/12 nm; emission, > 590 nm).

Control experiments

Time controls were performed in the absence of FITC-BSA or epi-illumination (n = 2). To evaluate the non-specific effects of epi-illumination through the fluorescein filter, FITC-BSA was omitted (n = 2). To evaluate the non-specific effects of circulating FITC-BSA, epi-illumination was omitted (n = 2). These controls are in accord with those performed previously for LDT (Bartlett & Segal, 2000; Emerson & Segal, 2000a); each condition was evaluated with respect to the local and conducted vasomotor responses to acetylcholine and to muscle contractions at time points that corresponded to criterion measurements. In four additional hamsters, a role for sensory nerve fibres (Holzer, 1991) associated with feed arteries of the retractor muscle (Grasby et al. 1999) was evaluated by determining whether ascending vasodilatation was affected by exposure to capsaicin (1 μm in the superfusion solution for ∼10 min).

Data collection and analysis

Feed arteries were observed (Leitz UM 32 objective, NA = 0.20) with bright-field illumination (condenser NA = 0.32); a video image was acquired with a CCD camera (C2400, Hamamatsu, Japan) that was coupled to a video monitor (PVM 1343 MD, Sony, Japan) with a total magnification of ×860. Internal vessel diameter was measured (resolution, ∼2 μm) using a video calliper; centreline Vrbc was monitored at the proximal site using an optical Doppler velocimeter (Welsh & Segal, 1997; Segal et al. 1999). Mean red blood cell velocity (Vm) was calculated as equal to Vrbc/1.6; blood flow was calculated as equal to π(D/2)2Vm, where D = diameter; WSR was calculated as equal to 8Vm/D. Since accurate velocimeter readings were difficult to obtain between contractions, Vrbc was measured immediately upon the cessation of muscle activity, with a mean value determined for the first ∼1.5 s; these measurements in feed arteries provide our best index of the peak total blood flow entering the retractor muscle (Jacobs & Segal, 2000).

Data were acquired at 100 samples s−1 using a MacLab system (CB Sciences; Dover, NH, USA) coupled to a Macintosh IIVX computer. Data were analysed using SigmaStat software (version 2.03; SPSS, Chicago, IL, USA); the statistical tests used are given in Results and in the figure legends. Differences were accepted as statistically significant at P < 0.05. The figures were prepared using SigmaPlot (version 4.01; SPSS). Summary data are presented as means ±s.e.m.

RESULTS

Effect of LDT on resting diameter and local responses to acetylcholine

Delivery of acetylcholine at each site along the vessel increased the local diameter by ∼50 % (Fig. 1B and Fig. 2). LDT produced a slight transient increase in resting diameter following the first period of illumination that recovered during the second to fourth periods of illumination (Fig. 1B). At proximal and distal sites, resting diameter was not significantly different either pre- or post-LDT (Fig. 2). The local response to acetylcholine at the central site diminished progressively with LDT (Fig. 1B). Following LDT, local dilatations in rsponse to acetylcholine delivered at proximal and distal sites were not different from the initial control responses (Fig. 2).

Figure 2. Integrity of local responses to acetylcholine delivery at proximal and distal sites pre- and post-LDT.

Figure 2

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Acetylcholine was microiontophoresed (1000 nA, 500 ms) at the proximal site (A) or at the distal site (B) to determine the integrity of endothelium-dependent vasodilatation at those locations (n = 9) before (Pre) and after (Post) LDT. Resting diameter (Rest), peak response diameter (Peak), and the local change in diameter (Change, calculated here and in subsequent figures as peak minus rest values) were not different between sites either pre- or post-LDT. Data were analysed using one-way repeated measures ANOVA with Tukey tests for post hoc comparisons. * P < 0.001, Peak vs. Rest.

Conducted vasodilatation in response to acetylcholine

Before LDT, acetylcholine delivered at the distal site triggered dilatation that conducted along feed arteries to increase vessel diameter by ∼30 % at the proximal site (Fig. 3); Vrbc and WSR increased transiently at the onset of dilatation and returned to approximate resting values by the time vasodilatation had peaked (8–10 s from onset; data not shown), as reported previously (Segal et al. 1999). Following LDT, the conduction of vasodilatation was abolished (Fig. 3). Whereas the proximal site no longer dilated in response to acetylcholine delivered at the distal site, haemodynamic responses at the proximal site included a near-doubling of Vrbc (rest, 11 ± 2 mm s−1; peak, 21 ± 3 mm s−1; P < 0.001), WSR (rest, 1137 ± 300 s−1; peak, 2017 ± 464 s−1; P = 0.002) and blood flow (rest, 18 ± 4 nl s−1; peak, 35 ± 8 nl s−1; P = 0.006; Student's paired t tests; n = 9).

Figure 3. The effect of LDT on conducted vasodilatation in response to acetylcholine.

Figure 3

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Acetylcholine was delivered at the distal site (refer to Fig. 1A). The local response was recorded, then the stimulus was repeated at the distal site and the conducted response was recorded at the proximal site (n = 9). LDT was then performed (see Methods) and local and conducted responses to acetylcholine delivered at the distal site were re-evaluated. Refer to Fig. 2 for resting diameters and control responses to the local delivery of acetylcholine. Conducted vasodilatation averaged 67 % of the local response (P < 0.001; paired t test). LDT had no effect on the local response, yet it abolished the conducted response in each case (note the lack of error bar with no change in diameter at the conducted site post-LDT). * P < 0.001, Post vs. Pre; Mann-Whitney rank sum test.

Local responses to phenylephrine and smooth muscle cell integrity

Following LDT, the vasoconstrictor response to phenylephrine persisted at the central site (diameter change, 13 ± 3 μm) but was reduced (P = 0.001; n = 6, paired t test) by half compared to the response to phenylephrine delivered at the distal site (28 ± 2 μm). The impairment of constriction at the site of LDT is consistent with the effects of LDT extending into surrounding smooth muscle cells once endothelial cell integrity has been disrupted (Bartlett & Segal, 2000).

Integrated tension

Integrated tension produced during contractions of the retractor muscle before LDT (428 ± 74 g s−1) was not significantly different from that observed following LDT (343 ± 76 g s−1), although the mean value was reduced by ∼20 %. This change in integrated tension may account for part of the reduction in the feed artery blood flow response to muscle contractions following LDT (Fig. 4).

Figure 4. The effect of LDT on ascending vasodilatation and exercise hyperaemia.

Figure 4

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Vessel diameter and blood flow were determined at the proximal site (refer to Fig. 1A) under resting conditions (Rest) and immediately upon the cessation of contractions (Peak; see Methods; n = 9). A, LDT abolished ascending vasodilatation with no change in resting diameter. The lack of error bar for change in diameter (Change) post-LDT indicates a complete loss of ascending vasodilatation following LDT. B, the hyperaemic response to muscle contractions was reduced by half following loss of ascending vasodilatation; resting blood flow was unchanged. Repeated measures ANOVA on ranks was performed for diameter data. One-way repeated measures ANOVA was performed for blood flow data, with Tukey tests for post hoc comparisons. * P < 0.01, Peak vs. Rest; +P≤ 0.001, Post vs. Pre; ++P < 0.02, Post vs. Pre.

Ascending vasodilatation, blood flow, Vrbc and WSR

Rhythmic contractions of the retractor muscle produced dilatation along the entire feed artery; responses typically began within 10–15 s, reached steady state in 30–60 s, and the diameter recovered within 2–3 min following the cessation of contractions. At the proximal site, the diameter (rest, 59 ± 6 μm) increased by 35 % (to 80 ± 8 μm) and was associated with a 6-fold increase in blood flow above resting values (Fig. 4).

In response to muscle contractions following LDT, feed artery diameter at the distal site increased from 52 ± 4 μm to 70 ± 5 μm (P < 0.001), yet dilatation was abolished upstream from the (central) site of LDT (Fig. 4A). Nevertheless, acetylcholine delivered at the proximal site increased the local diameter by 50 % (Fig. 2A), confirming the integrity of endothelium-dependent vasodilatation at the proximal site. The loss of ascending vasodilatation following LDT was associated with a 47 % reduction in the hyperaemic response to muscle contractions (Fig. 4B), which was probably compensated for by an increase in oxygen extraction by the retractor muscle. Across muscle preparations, there was no correlation between the change in integrated tension and the change in feed artery blood flow (r = 0.12; n = 8). The Vrbc at rest was not different pre- vs. post-LDT, nor was peak Vrbc at the end of muscle contractions (Fig. 5A). However, concomitant with the loss of ascending vasodilatation was a 330 % increase in WSR at the proximal site; this increment is more than twice that observed prior to LDT (Fig. 5B).

Control experiments

Endothelial cell nuclei were labelled with propidium iodide within the illuminated segment but not elsewhere along the vessel; labelling of smooth muscle cell nuclei was negligible. Responses to acetylcholine or to muscle contractions were not affected by time alone, epi-illumination in the absence of dye, or intravascular dye in the absence of epi-illumination. These findings are consistent with previous controls for the selective effects of LDT (Bartlett & Segal, 2000; Emerson & Segal, 2000a).

Exposure to 1 μm capsaicin initially constricted the feed arteries (from a resting diameter of 45 ± 3 μm to 13 ± 8 μm; n = 4) for several minutes and was followed by sustained vasodilatation (to 87 ± 6 μm), which was taken to reflect the release of neuropeptides (e.g. calcitonin gene-related peptide and substance P) from sensory nerve fibres (Holzer, 1991; Grasby et al. 1999); resting diameter recovered within ∼45 min. A second exposure to capsaicin produced an attenuated constriction (to 37 ± 1 μm) with no after-dilatation, confirming the inhibition of sensory nerve fibres by the initial treatment (Holzer, 1991). Ascending vasodilatation in response to muscle contractions following capsaicin treatment (diameter change, 31 ± 2 μm) was identical to that preceding treatment. Vehicle alone (0.1 % ethanol) had no effect on either vessel diameter or ascending vasodilatation.

DISCUSSION

In the feed arteries of the hamster retractor muscle, hyperpolarisation initiated by acetylcholine is conducted though gap junctions between endothelial cells; myoendothelial gap junctions enable conduction along the endothelium to rapidly drive smooth muscle cell relaxation along the vessel wall (Emerson & Segal, 2000a,b, 2001). We reasoned here that if conduction along the endothelium was integral to ascending vasodilatation in response to skeletal muscle activity, then disrupting the endothelial cell pathway should prevent vasodilatation beyond the region of damage. Our findings demonstrate that both conducted vasodilatation in response to acetylcholine and ascending vasodilatation in response to muscular contraction were abolished following selective damage to endothelial cells within a segment of the vessel located midway along the conduction pathway. Furthermore, the loss of ascending vasodilatation reduced the magnitude of the hyperaemic response to muscular contractions by half, indicating that conduction along the endothelium is integral to the full expression of functional hyperaemia. These data uniquely establish a direct link between the cellular pathway that mediates conduction and a physiological role for conduction in governing muscle blood flow during exercise.

Alternative mechanisms of ascending vasodilatation

The arterial vasculature lies external to the muscle parenchyma and is thereby removed from the direct influence of the vasodilator metabolites released from active muscle fibres. Thus, a pervading question has been the nature of the signalling pathway through which vasodilatation initiated by exercise ascends from the arteriolar network into the proximal arterial supply. Whereas conduction has been implied in previous studies (Hilton, 1959; Welsh & Segal, 1997), others have shown that flow-induced vasodilatation mediates the relaxation of arterial smooth muscle (Lie et al. 1970; Pohl et al. 1986; Smiesko & Johnson, 1993). With a stable resting diameter, an increase in blood flow increases WSR (and luminal shear stress) in proportion to the velocity of flowing blood. Endothelial cells can transduce this mechanical stimulus into the production of such vasodilators as nitric oxide and prostacyclin (Koller et al. 1994; Davies, 1995). Released abluminally, these autacoids diffuse to and relax smooth muscle cells at sites where shear stress is increased.

With endothelial cell damage constrained to the central segment (Fig. 1), we reasoned that if flow-induced dilatation prevailed in feed arteries, then vasodilatation should occur at the proximal site in response to arteriolar dilatation and the elevation of flow associated with contractile activity (Pohl et al. 1986; Koller & Kaley, 1990; Smiesko & Johnson, 1993). Remarkably, the sustained elevation of WSR during exercise was without effect on feed artery diameter (Fig. 3 and Fig. 4). Furthermore, at the proximal site WSR more than doubled in response to acetylcholine delivered at the distal site, with no change in diameter (see Results). Nevertheless, robust local responses to acetylcholine (Fig. 2) confirmed that endothelium-dependent vasodilatation was unaffected along regions not exposed to LDT. Taken together, these findings indicate that flow-induced vasodilatation is not the signalling pathway for ascending vasodilatation in the feed arteries of the hamster retractor muscle.

In the light of contemporary studies of the electrical properties of smooth muscle (Burnstock & Prosser, 1960), conduction along the media was proposed as a mechanism to explain dilatation of the femoral artery in response to contractions of the gastrocnemius muscle (Hilton, 1959). However, the role of endothelial cells in mediating vasodilatation through autacoid release (Furchgott & Zawadzki, 1980) or myoendothelial coupling (Emerson & Segal, 2000b; Yashiro & Duling, 2001) had not yet been established. Recent studies of arterioles supplying the hamster cheek pouch have illustrated that the smooth muscle layer can serve as a pathway for the conduction of depolarisation and constriction as well as hyperpolarisation and dilatation, according to the particular stimulus that initiates the response (Welsh & Segal, 1998; Bartlett & Segal, 2000). In contrast, smooth muscle cells in feed arteries of the retractor muscle appear to be ineffective as a conduction pathway (Emerson & Segal, 2000a); the spread of vasoconstriction required noradrenaline release along perivascular sympathetic nerves (Welsh & Segal, 1996; Segal et al. 1999). The resting tone in these vessels is independent of sympathetic nerves (Welsh & Segal, 1996), and both functional and histochemical findings argue against a pathway for sympathetic cholinergic vasodilatation (Welsh & Segal, 1996, 1997; Grasby et al. 1999). The lack of effect of capsaicin on ascending vasodilatation further argues against a role for sensory nerves in mediating feed artery dilatation in response to the contractile activity of skeletal muscle fibres.

Functional significance of ascending vasodilatation

At rest in a normoxic environment, the venous effluent from skeletal muscle remains well saturated with oxygen (Andersen & Saltin, 1985). When arterial and venous measurements are performed across the vascular bed of a skeletal muscle or hindlimb, an increase in oxygen extraction or fall in venous PO2 is taken to reflect the dilatation of intramuscular arterioles (Granger et al. 1976; Goodman et al. 1978). The dilatation of distal arterioles increases capillary perfusion (Klitzman et al. 1982; Sweeney & Sarelius, 1989) and, thereby, the functional surface area for diffusional exchange between muscle fibres and the microcirculation embedded within the muscle parenchyma. As metabolic demand (e.g. the intensity of rhythmic contractions) increases, an increase in oxygen extraction can maintain mitochondrial respiration up to the point at which the intracellular PO2 falls below that necessary to maintain oxidative phosphorylation. When the demand for oxygen exceeds that provided by the prevailing blood flow, the net delivery of oxygen into the muscle is increased via vasodilatation ascending into the proximal arterioles and feed arteries that govern total blood flow into the muscle. While this ‘shift’ in the locus of blood flow control was originally inferred from arterial-venous measurements (Granger et al. 1976; Goodman et al. 1978), intravital microscopy has confirmed that such behaviour is an intrinsic property of the resistance network of skeletal muscle (Welsh & Segal, 1997; Jacobs & Segal, 2000).

Feed artery dilatation increases with the recruitment of motor units (Van Teeffelen & Segal, 2000) and with increments in duty cycle (Welsh & Segal, 1997). With muscle fatigue, dilatation persists in distal arterioles but is suppressed in proximal arterioles and feed arteries (Jacobs & Segal, 2000). Previously, pharmacological inhibition of feed artery dilatation was found to diminish blood flow to the retractor muscle during rhythmic contractions, despite robust arteriolar dilatation (Welsh & Segal, 1997). The present findings demonstrate that functional hyperaemia is profoundly impaired when feed artery dilatation is compromised following LDT. In view of these collective findings, we propose that the conduction pathway evoked by exercise is sensitive to the activity level of skeletal muscle and that it can be modulated by factors known to interfere both reversibly and irreversibly with the endothelium-dependent mechanisms of vasodilatation. Such integration and coordination of vasodilatation among vessel branches is consistent with a vascular network in which resistance is distributed dynamically between the distal and proximal elements.

Conclusion

In the present study, LDT abolished the ascending vasodilatation evoked by skeletal muscle contractions as well as the conducted vasodilatation evoked by acetylcholine. The consistency of disrupting conduction along feed arteries in vivo and in vitro with focal damage to the endothelium supports the hypothesis that exercise gives rise to a hyperpolarising signal that conducts along the endothelium to evoke smooth muscle cell relaxation in the manner described for acetylcholine. The present findings thereby illustrate a key role for conduction along the endothelium in promoting muscle blood flow during exercise. This signalling pathway is intrinsic to the resistance vasculature and provides a highly responsive mechanism for coordinating the dilatation of feed arteries with the metabolic demands of skeletal muscle fibres.

Acknowledgments

This research was supported by grants RO1-HL56786 and RO1-HL41026 from the National Heart, Lung and Blood Institute of the National Institutes of Health; United States Public Health Service. We are grateful to Sara J. Haug for her contributions to this work.

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