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

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 microm) 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 microm-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 microm 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 microm 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.

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

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.

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

Centreline V_rbc 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, V_rbc 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.

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

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.

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

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.

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

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.