Sympathetic nerves inhibit conducted vasodilatation along feed arteries during passive stretch of hamster skeletal muscle

J Physiol (2003), 552.1, pp. 273 282 DOI: 10.1113/jphysiol.2003.046284 The Physiological Society 2003 www.jphysiol.org Sympathetic nerves inhibit conducted vasodilatation along feed arteries during passive stretch of hamster skeletal muscle Sara J. Haug *, Donald G. Welsh * and Steven S. Segal * *The John B. Pierce Laboratory, Department of Cellular and Molecular Physiology and Department of Biomedical Engineering,Yale University, New Haven, CT 06519, USA Ascending vasodilatation is integral to blood flow control in exercising skeletal muscle and is attributable to conduction from intramuscular arterioles into proximal feed arteries. Passive stretch of skeletal muscle can impair muscle blood flow but the mechanism is not well understood. We hypothesized that the conduction of vasodilatation along feed arteries can be modulated by changes in muscle length. In anaesthetized hamsters, acetylcholine (ACh) microiontophoresis triggered conducted vasodilatation along feed arteries (diameter, 50 70 mm) of the retractor muscle secured at 100 % resting length or stretched by 30 %. At 100 % length, ACh evoked local dilatation (> 30 mm) and this response conducted rapidly along the feed artery (14 ± 1 mm dilatation at 1600 mm upstream). During muscle stretch, feed arteries constricted ~10 mm (P < 0.05) and local vasodilatation to ACh was maintained while conducted vasodilatation was reduced by half (P < 0.01). Resting diameter and conduction recovered upon restoring 100 % length. Sympathetic nerve stimulation (4 8 Hz) produced vasoconstriction and attenuated conduction in the manner observed during muscle stretch, as did noradrenaline or phenylephrine (10 nm). Inhibiting nitric oxide production (N v -nitro-l-arginine, 50 mm) produced similar vasoconstriction yet had no effect on conduction. Phentolamine, prazosin, or tetrodotoxin (1 mm) during muscle stretch abolished vasoconstriction and restored conduction. Inactivation of sensory nerves with capsaicin had no effect on vasomotor responses. Thus, muscle stretch can attenuate conducted vasodilatation by activating a-adrenoreceptors on feed arteries through noradrenaline released from perivascular sympathetic nerves. This autonomic feedback mechanism can restrict muscle blood flow during passive stretch. (Resubmitted 1 May 2003; accepted after revision 29 July 2003; first published online 1 August 2003) Corresponding author S. S. Segal: The John B. Pierce Laboratory, Yale University School of Medicine, 290 Congress Avenue, New Haven, CT 06519, USA. Email: sssegal@jbpierce.org Muscle blood flow is controlled throughout resistance networks according to the metabolic demand of muscle fibres (Granger et al. 1976; Laughlin & Armstrong, 1982; VanTeeffelen & Segal, 2000). For example, vasodilator signals arising within exercising muscle can ascend the arteriolar network into feed arteries, which are located external to the muscle they supply (Hilton, 1959; Folkow et al. 1971; Segal & Jacobs, 2001). With feed arteries providing up to half of the total resistance to muscle blood flow, ascending vasodilatation into these proximal segments is integral to the full expression of functional hyperaemia (Williams & Segal, 1993; Welsh & Segal, 1997). In feed arteries of the hamster retractor muscle, vasodilatation in response to acetylcholine (ACh) is conducted via hyperpolarization along the endothelium and into the smooth muscle layer (Emerson & Segal, 2000a,b). Remarkably, this conduction pathway may be triggered by contractile activity as well as by ACh, thereby enhancing muscle blood flow during exercise (Segal & Jacobs, 2001). The activation of sympathetic nerves during exercise (Seals, 1989) redistributes cardiac output away from inactive vascular beds to promote blood flow to contracting skeletal muscle (Rowell, 1974). In resting muscle, blood flow is diminished during passive stretch (Hirche et al. 1970; Supinski et al. 1986), an effect that has been attributed to the deformation and compression of vessels by muscle fibres (Gray & Staub, 1967; Supinski et al. 1986). However, early studies did not observe the vasculature during tissue deformation. In the hamster retractor muscle, feed arteries and arterioles were found to constrict progressively as resting muscle length was increased by 30 % and to return to their initial diameter upon restoring the original muscle length (Welsh & Segal, 1996). This reversible response to muscle stretch is explained by the activation and deactivation of perivascular sympathetic nerves, respectively. In this way, passive increases in muscle length (e.g. during shortening of an antagonistic muscle) can actively contribute to the regulation of blood

274 S. J. Haug, D. G. Welsh and S. S. Segal J Physiol 552.1 flow through a physiological range of motion (Ledvina & Segal, 1995; Burkholder & Lieber, 2001) that encompasses the length tension relationship of muscle fibres (Ramsey & Street, 1940; Welsh & Segal, 1996). Whether changes in muscle length can affect conducted vasodilatation is unknown. In the present study, we hypothesized that conduction of vasodilatation along feed arteries is modulated by changes in the resting length of skeletal muscle. Using the hamster retractor muscle preparation, vasomotor responses were evaluated at 100 % of the initial resting muscle length and during a 30 % increase in muscle length. Our findings illustrate that conducted vasodilatation is inhibited during muscle stretch and recovers when the original resting length is restored. Pharmacological interventions reveal that this reversible effect of muscle stretch is exerted through the activation of a-adrenoreceptors on smooth muscle by noradrenaline released from perivascular sympathetic nerves. METHODS Animal care All procedures were approved by the Institutional Animal Care and Use Committee of the John B. Pierce Laboratory and were performed in accordance with the Guide for the Care and Use of Laboratory Animals of the National Research Council (USA). Male Syrian golden hamsters (n = 33, 90 115 g; Charles River Breeding Laboratories, Kingston, NY, USA) were maintained at 24 C on a 14 h/10 h (light/dark) cycle and provided rodent chow and water ad libitum. Hamsters were anaesthetized with pentobarbital sodium (60 mg kg _1 I.P.) and the trachea cannulated (polyethylene tubing; PE-190). The left carotid artery was cannulated (PE-50) to monitor arterial blood pressure and the left femoral vein was cannulated (PE-50) to replace fluids and maintain anaesthesia during the experiment (10 mg pentobarbital (ml sterile saline) _1, infused at 410 ml h _1 ). Depth of anaesthesia was assessed according to the stability of blood pressure, spontaneous rate of ventilation, and lack of withdrawal to toe pinch; additional anaesthetic was infused if required. Oesophageal temperature was maintained between 35 and 38 C using a heated copper plate positioned beneath the hamster. At the end of the experimental procedures, hamsters were given an overdose of pentobarbital intravenously. Retractor muscle preparation A hamster was positioned on a transparent acrylic platform and the right retractor muscle was prepared for study (VanTeeffelen & Segal, 2003). Briefly, an incision was made through the overlying skin. Taking great care to minimize trauma, the muscle was cleared of connective tissue and visible nerve branches using microdissection under a stereomicroscope. The origin and the insertion of the muscle were clamped, the distance between the clamps was measured (± 0.1 mm), and respective ends were severed from the hamster. The muscle was reflected away from the body and positioned in a chamber (volume, 10 ml) filled with bicarbonate-buffered physiological salt solution (PSS, ph 7.4, 34 35 C) of the following composition (mm): NaCl 131.9, KCl 4.7, CaCl 2 2.0, MgSO 4 1.2, NaHCO 3 18; equilibrated with 95 % N 2 5 % CO 2. Muscle clamps were secured to micrometer spindles, resting length was established, and the completed preparation was transferred to the stage of an intravital microscope (modified ACM; Zeiss, Thornwood, NY, USA) where it equilibrated for ~60 min. The muscle was superfused continuously (10 ml min _1 ) with fresh PSS while the effluent was removed with a vacuum line to maintain a constant fluid level and a stable optical image. Surgical procedures required 3 4 h and preparations were then stable for ~ 5 h. Video microscopy and data acquisition Feed arteries and arterioles were observed using bright-field illumination (Zeiss ACH/APL condenser, numerical aperture (NA) = 0.32; Leitz UM32 objective, NA = 0.20). The optical image was coupled to a video camera (C2400; Hamamatsu, Japan) and monitor (PVM 1343 MD; Sony, Japan) at total magnification of w 860. Internal diameter was measured with a video calliper at the widest point of the vessel lumen or column of red blood cells (resolution, 2 mm). Muscle tension was recorded using a load beam (LCL-1136; Omega, Stamford, CT, USA; 0 100 g ± 0.1 g) mounted in series with a muscle clamp. Data were acquired at 40 Hz using a MacLab system (AD Instruments, Australia) coupled to a personal computer. Each vessel studied demonstrated brisk and reversible dilatation in response to sodium nitroprusside (10 mm) delivered in the superfusion fluid. Microiontophoresis Micropipettes with a tip internal diameter of ~1 mm were prepared from borosilicate glass capillaries and filled with ACh (1 M in dh 2 O; Sigma). A micropipette was secured in a micromanipulator and connected via an Ag AgCl wire to a constant current source (Micro-Iontophoresis Programmer, Model 160; World Precision Instruments, Sarasota, FL, USA); a Pt electrode secured along the edge of the muscle chamber served as the reference electrode. The micropipette was held in a micromanipulator secured to the acrylic platform that contained the retractor muscle preparation, enabling all components to be moved as a unit. Thus, with the tip of the micropipette positioned adjacent to the distal end of a feed artery (Fig. 1), responses were observed along the vessel without disturbing the micropipette. Retaining current (0.1 0.2 ma) was adjusted to prevent leakage (indicated by vasodilatation) of ACh from the micropipette tip. Based upon the stimulus response relationship to ACh microiontophoresis, the ejection current and duration were set to 1 ma and 1 s, respectively. This stimulus was sufficient to elicit a maximal response at the local site of stimulus delivery and did not produce tachyphylaxis. Changing muscle length Two common reference lengths for skeletal muscle are the resting length in situ (L IS ) and the optimal length for active tension production (L O ). Separate experiments were performed at each respective resting muscle length. L IS was measured in the hamster and L O was determined based on peak active tension during supramaximal field stimulation (140 V, 0.1 ms, 40 Hz; model S48, Grass Instruments, West Warwick, RI, USA) of the muscle between a pair of Pt electrodes (VanTeeffelen & Segal, 2003). The micrometer spindle at each end enabled the muscle to be passively stretched and shortened without displacement of feed arteries (Fig. 1). Only one reference length (L IS or L O ) was studied for a given muscle. The time required to stretch the muscle to 130 % of its reference length was varied from 1 to 10 min with no effect on results. Each muscle length was studied for up to 2 h in a given preparation.

J Physiol 552.1 Sympathetic nerves modulate feed artery conduction 275 Perivascular nerve stimulation (PNS) A stimulating microelectrode was prepared by filling a micropipette (tip diameter, ~3 mm) with 0.9 % NaCl, securing it in a micromanipulator, and positioning its tip adjacent to a primary (1A) arteriole within the muscle at a distance of ~2 mm from the ACh micropipette in order to minimize electrical interference. The microelectrode was connected via an Ag AgCl wire to the cathode of a stimulator (model S48, Grass Instruments) and the anode was connected to the Pt reference electrode. Perivascular nerves were stimulated continuously (100 V, 1 ms, 4 8 Hz) for at least 5 min prior to first ACh stimulus. Pharmacological reagents Reagents, their final concentrations, and sources were: N v -nitro- L-arginine (L-NA, 50 mm), L-noradrenaline (10 nm), phentolamine (1 mm for L IS, 10mM for L O ; a non-selective a-adrenoreceptor antagonist), L-phenylephrine (10 nm), and sodium nitroprusside (SNP, 10 mm) were all from Sigma; capsaicin (100 nm; to deplete sensory nerve terminals) was from Pfaltz & Bauer, Inc. (Waterbury, CT, USA), prazosin HCl (1 mm; a selective a 1 -adrenoreceptor antagonist) from Research Biochemicals, Inc. (Natick, MA, USA) and tetrodotoxin (TTX, 1 mm; to inhibit action potentials) from Calbiochem-Novabiochem Ltd (San Diego, CA, USA). Reagents were prepared as concentrates in sterile 0.9 % NaCl, diluted in fresh PSS to the final working concentration, and equilibrated in the muscle chamber for ~5 min with the exception of L-NA and capsaicin, which were equilibrated for at least 30 and 15 min, respectively. With capsaicin, the feed artery first constricted and then dilated maximally. Once baseline diameter recovered (~1 h), capsaicin was reapplied; with no observable change in vessel diameter, neurotransmitter stores were taken to be depleted (Segal & Jacobs, 2001). The interaction between muscle stretch and conducted vasodilatation was then re-evaluated during superfusion with control PSS. Experimental protocol Muscle lengths were studied in random order across hamsters. Therefore, a muscle was set at 100 % reference length (L IS or L O ) or 130 % of reference length during equilibration following surgery. Vasodilatation to ACh microiontophoresis was recorded at the local site of delivery (i.e. distance = 0) and at 400, 800, 1200 and 1600 mm upstream from the stimulus along the feed artery (Segal et al. 1999). A separate stimulus was used for each observation. Following an ACh stimulus, the vessel typically recovered to resting diameter in ~30 s and ~2 min elapsed between consecutive stimuli. The stimulus site and locations of measurements remained constant throughout an experiment and the order in which responses at respective sites were recorded was varied across experiments. Once all responses were recorded at a given muscle length, the muscle was either passively stretched by 30 % from L IS or L O or was passively shortened to L IS or L O using the micrometer spindles. The muscle was re-equilibrated (~15 min) in fresh PSS, and vasomotor responses to ACh re-evaluated as above. At the end of each experiment, SNP was added to determine maximal vessel diameter. Perivascular nerve stimulation and pharmacological treatments are presented in context. Data analysis and statistics The magnitude of local and conducted responses was calculated as the difference between the peak response diameter to ACh and the preceding baseline diameter. Pooled data (mean ± S.E.M.) were analysed (SigmaStat, v.2.03; SPSS, Chicago, IL, USA) with respect to muscle length using two-way analysis of variance (ANOVA) with repeated measures, unless otherwise indicated. When significant F ratios were obtained from ANOVA, Tukey post hoc analyses were performed. Differences were considered statistically significant with P < 0.05. Linear regression (least-squares) was performed on the vasomotor responses at conducted sites and a slope and intercept calculated. RESULTS Mean arterial pressure averaged ~90 mmhg and remained stable throughout experimental procedures. For muscles studied at 100 % and 130 % L IS, the resting diameter of feed arteries averaged 67 ± 3 and 56 ± 2 mm (n = 16, P < 0.05; paired t test), respectively. For muscles studied at 100 % and 130 % L O, corresponding values were 54 ± 4 and 45 ± 4 mm (n = 17, P < 0.05; paired t test). Robust spontaneous Figure 1. Experimental protocol for muscle stretch and conduction A, schematic diagram illustrating hamster retractor muscle with feed artery giving rise to arteriolar networks (not to scale). B,representative records of conducted vasodilatation observed at 400 mm (site 1) and at 1600 mm (site 2) upstream from ACh stimulus (1 ma, 1 s delivered at time 0). During muscle stretch, diameter change was maintained at site 1 and attenuated by half at site 2. Values of 100 % and 130 % refer to muscle length (L O ) as described in Methods. Note vasoconstriction during muscle stretch.

276 S. J. Haug, D. G. Welsh and S. S. Segal J Physiol 552.1 vasomotor tone was confirmed in all experiments by local dilatation (typically > 30 mm) to ACh; peak diameters (104 ± 5 and 87 ± 4 mm for vessels studied at L IS and L O, respectively) were not significantly different from maximal values recorded during exposure to SNP. When normalized to maximal diameter to account for size differences among vessels, resting tone and reactivity were similar between experiments based on L IS and those based upon L O (Table 1). Moving the ACh micropipette ~100 mm away from the vessel eliminated all responses to ACh, confirming that vasodilatation at remote sites was not attributable to diffusion or convection of ACh in the superfusion solution. Muscle stretch consistently produced constriction of feed arteries (Table 1) that reversed spontaneously when muscles were restored to their original resting length. This effect of muscle stretch was mimicked by the application of noradrenaline, phenylephrine, or L-NA (Table 1). When muscles were held at 100 %, reference muscle lengths were 32 ± 1 mm for L IS (n = 16) and 24 ± 1 mm for L O (n = 17). When muscles were stretched to 130 %, respective lengths were 42 ± 1 and 31 ± 1 mm. This difference in muscle lengths between sets of experiments is due to corresponding differences in the design of muscle clamps: those based on L IS used clamps which attached to the ends of the muscle, while experiments based on L O used clamps which required 3 5 mm of tissue at each end of the muscle for attachment. Nevertheless, the effect of muscle stretch on resting tone or conducted vasodilatation was independent of whether L IS or L O was used as the reference length (Figs 2 5). Muscle stretch attenuates conducted vasodilatation Muscle stretch significantly attenuated the conduction of vasodilatation at distances > 400 mm (Figs 1 and 2). For experiments based on L IS, local vasodilatation was even

J Physiol 552.1 Sympathetic nerves modulate feed artery conduction 277 greater during muscle stretch and may be attributed to the reduction in resting diameter (Fig. 2A; n = 16); this enhanced local response to ACh was not observed for experiments based upon L O (Fig. 2B; n = 17). For all experiments, conducted vasodilatation at 400 mm was not affected by changes in muscle length. Therefore, to account for variability between preparations in the resting diameter of the feed arteries (Table 1) and to more clearly illustrate the effects of muscle stretch on conduction, data were normalized to conducted responses at 400 mm and are shown in Fig. 2C and D. In all cases, the attenuation of conducted vasodilatation during muscle stretch was reversed completely upon restoration of 100 % resting muscle length. Summary data are also normalized in subsequent figures to illustrate the effects of experimental interventions. Local vasodilatation to ACh was not impaired under any condition. Perivascular sympathetic nerves attenuate conduction via a-adrenoreceptor activation At 100 % of L IS or L O, PNS consistently attenuated conducted vasodilatation along the feed artery as compared to responses recorded under control conditions (Fig. 3A and B; total n = 7). Addition of noradrenaline (Fig. 3C) or phenylephrine (Fig. 3D) to the superfusion solution produced constriction similar to that observed with muscle stretch (Table 1) and attenuated conducted vasodilatation to the same extent (total n = 15). With the muscle stretched to 130 % of L IS or L O, phentolamine restored resting diameter and conducted responses to values recorded with the muscle at 100 % of reference length (Figs 4A and B; total n = 11). A similar effect was confirmed with prazosin: at 1600 mm upstream from the ACh stimulus, conduction was 15 ± 2 mm at L O, 8 ± 1 mm at 130 % L O, and 12 ± 1 at 130 % L O with prazosin (n = 3). Figure 2. Muscle stretch attenuates conducted vasodilatation A and B indicate the absolute change in diameter along feed artery at sites upstream from ACh stimulus. A represents experiments based upon in situ resting length, L IS (n = 16). The slope of the line describing the decay in the amplitude of conducted vasodilatation between 400 and 1600 mm along feed arteries (expressed as: mm diameter change (mm vessel length) _1 ) increased from _0.0054 ± 0.0024 at 100 % of L IS to _0.0115 ± 0.0024 at 130 % of L IS. B represents experiments based upon optimal length for tension production, L O (n = 17). The slope of the line (defined as in A) describing conducted vasodilatation increased from _0.0053 ± 0.0017 at 100 % L O to _0.0086 ± 0.0012 at 130 % L O. C and D illustrate the data in A and B, respectively, with values at each conducted site normalized to the change in diameter at the 400 mm site. Vessel diameters are in Table 1. * Significant difference from response at 130 % (P < 0.05).

278 S. J. Haug, D. G. Welsh and S. S. Segal J Physiol 552.1 Phentolamine also prevented the attenuation of conducted responses induced by the addition of noradrenaline (Fig. 4C; n = 5) or phenylephrine (Fig. 4D; n = 4). Inhibition of adrenoreceptors had no effect on resting diameter or conduction at 100 % muscle length (data not shown). Vasoconstriction with L-NA does not attenuate conduction Each of the previous interventions was associated with a decrease in resting diameter. To test whether vasoconstriction itself could explain the attenuated conducted response, local and conducted vasodilatation to ACh were recorded in the presence or absence of L-NA. In the presence of L-NA with the muscle at 100 % of either reference length, feed arteries constricted to same extent as observed with PNS or muscle stretch (Table 1). Nevertheless, conducted vasodilatation was not different from that observed under control conditions (Fig. 5). Further evidence for a specific action of perivascular sympathetic nerves The addition of TTX to the superfusate abolished the ability of muscle stretch to constrict feed arteries and to attenuate the conducted response (n = 7, Table 1). Pretreatment with capsaicin had no effect on the attenuation of conducted vasodilatation induced by muscle stretch: observed at 1600 mm, conducted vasodilatation was 13 ± 1 mm at L O, 6±1mm at 130 % L O, and 6 ± 1 at 130 % L O following capsaicin (n = 3). DISCUSSION The primary finding of this study is that changing the resting length of skeletal muscle can modulate the conduction of vasodilatation along feed arteries which control muscle blood flow. We show that the inhibition of conduction during muscle stretch is attributable to the activation of a- Figure 3. Perivascular nerve stimulation (PNS) and adrenoreceptor agonists attenuate conducted vasodilatation Conducted vasodilatation was attenuated during PNS with muscles held at 100 % of L IS (A, n = 4) or at 100 % of L O (B, n = 3). Stretching muscles to 130 % of respective reference lengths attenuated conduction in the manner recorded during PNS. At 100 % of L IS, noradrenaline (NA, 10 nm) attenuated conducted vasodilatation (C, n = 5). At 100 % of L O, phenylephrine (PE, 10 nm) attenuated conducted vasodilatation (D, n = 10). Data are normalized as in Fig. 2. Vessel diameters are in Table 1. * Significant difference from responses at 100 % during PNS or with noradrenaline or phenylephrine (P < 0.05). + Significant difference from responses at 130 % (P < 0.05).

J Physiol 552.1 Sympathetic nerves modulate feed artery conduction 279 adrenoreceptors through the release of noradrenaline from perivascular sympathetic nerves. Conversely, returning the muscle to its original resting length restores conducted vasodilatation. Electrophysiological studies have demonstrated that the vasodilatory responses observed here reflect the conduction of hyperpolarization from cell to cell through gap junction channels along endothelium and into the surrounding smooth muscle cell layer (Emerson & Segal, 2000a,b). Complementary experiments indicate that this signalling pathway is activated in response to the contractile activity of the retractor muscle (Segal & Jacobs, 2001). The present observations are therefore the first to indicate that cell-to-cell signalling along the resistance vasculature is sensitive to changes in muscle length. The effect of muscle stretch on conducted vasodilatation increased with distance along feed arteries. To quantify this relationship, linear regression analyses were performed on vasomotor responses at respective sites. When increasing muscle length from 100 % to 130 % of either L IS or L O, the slope of the regression nearly doubled (Fig. 2), indicating greater decay of conducted vasodilatation with muscle stretch. Thus, at 100 % muscle length, vasodilatation will conduct > 3 mm along a feed artery before there is a 50 % reduction in the conducted response observed at 400 mm from the ACh stimulus. This persistence of conduction along feed arteries is consistent with previous observations in vivo (Segal et al. 1999) and in vitro (Emerson & Segal, 2000b; Emerson et al. 2002). In contrast, the same response initiated during muscle stretch would travel < 2 mm before decaying by half. This inhibition of conducted vasodilatation can effectively restrict muscle blood flow (Segal & Jacobs, 2001). Figure 4. Attenuation of conducted vasodilatation during muscle stretch is inhibited by blockade of a-adrenoreceptors During muscle stretch, addition of phentolamine (1 mm for L IS (A, C);10 mm for L O (B, D)) blocked the attenuation of conducted vasodilatation whether 100 % L IS (A, n = 5) or L O (B, n = 6) was used for initial reference length. Phentolamine also prevented the attenuation of conducted responses induced by noradrenaline (C, n = 5) or phenylephrine (D, n = 4). Data are normalized as in Fig. 2. Vessel diameters are given in Table 1. * Significant difference from response at 130 % (P < 0.05).

280 S. J. Haug, D. G. Welsh and S. S. Segal J Physiol 552.1 Vasoconstriction was maintained throughout the period of muscle stretch and reversed upon returning to 100 % resting length or by inhibiting the effect (or release) of noradrenaline during muscle stretch. Thus, reduction and recovery of resting diameter accompanied the inhibition and restoration of conduction, respectively. In order to test whether vasoconstriction alone could impair conduction, vasomotor tone was increased to a similar level by an independent mechanism: inhibiting the constitutive release of NO. Under the latter conditions, the diameter change with conduction along feed arteries was not different from control, in accord with previous results (Segal et al. 1999). In arterioles, vasoconstriction induced through elevating PJ in the superfusion solution was also without affect on conducted vasodilatation (Kurjiaka & Segal, 1995). Our present finding that topical application of noradrenaline or phenylephrine mimicked the effects of muscle stretch supports the hypothesis that sympathetic neuroeffector activity inhibits conducted vasodilatation (Kurjiaka & Segal, 1995) by a mechanism other than through an increase in vasomotor tone. Figure 5. Vasoconstriction with L-NA does not attenuate conducted vasodilatation With muscles at 100 % of L IS (A, n = 4) or of L O (B, n = 4), vasoconstriction with L-NA to the same extent as that observed with 130 % muscle stretch had no effect on conducted vasodilatation. Data are normalized as in Fig. 2. Vessel diameters are given in Table 1. * Significant difference from responses at 130 % (P < 0.05). Multiple mechanisms may modulate conducted vasodilatation through perivascular sympathetic nerve activity. First, noradrenaline can depolarize smooth muscle (Nelson et al. 1988), which would antagonize the conduction of hyperpolarization and diminish vasodilatation at remote sites. Second, activation of a-adrenoreceptors may trigger a signalling pathway that modulates gap junction channels, thereby affecting cell-to-cell coupling. For example, protein kinase C, activated via a 1 -adrenoreceptor stimulation (Exton, 1985), can regulate gap junction channels by phosphorylating connexin 43 (Lampe et al. 2000; Bowling et al. 2001), which is expressed in the endothelium of the retractor feed artery (R. Looft-Wilson & S. Segal, unpublished observations). Since smooth muscle cells appear to be poorly coupled in these vessels, while endothelial coupling is robust (Emerson & Segal, 2000b), we hypothesize that modulation of myoendothelial coupling (Emerson & Segal, 2000a) may be integral to physiological regulation of conducted vasodilatation by perivascular sympathetic nerves. The third possibility is that sympathetic nerve activity is changing the resistive properties of the cells comprising the vessel wall, thereby enhancing the dissipation of hyperpolarization with distance along the vessel. While each of these mechanisms could explain the inhibitory effect of muscle stretch on conducted vasodilatation, they are not mutually exclusive. Thus, further experiments are required to resolve the mechanism(s) by which conducted vasodilatation is modulated through the activation of perivascular sympathetic nerves. Stretch of skeletal muscle has been shown to promote neurotransmitter release from the motor end plate of skeletal muscle fibres (Turkanis, 1973; Hamill & Martinac, 2001) through integrin signalling (Chen & Grinnell, 1995). In our system, feed arteries did not undergo physical strain during muscle stretch. Therefore neurotransmitter release along the feed artery is not due to direct mechanical action at the nerve varicosity. Further, the finding that TTX abolished the effect of muscle stretch indicates that action potentials travelling along perivascular nerves underlie the effects of muscle stretch on feed artery diameter (Welsh & Segal, 1996). Muscle stretch has been shown to produce mechanical stimulation of afferent nerve endings that can reflexively elicit sympathetic outflow (Stebbins et al. 1988). However, in the retractor, we have excluded a neural reflex as mediating the effects of muscle stretch on perivascular sympathetic nerves (Welsh & Segal, 1996). Further, a role for sensory nerves is excluded by finding that capsaicin did not alter the inhibitory effect of muscle stretch on conducted vasodilatation. These findings collectively implicate a unique mechanotransduction sequence for sympathetic nerve activation by muscle stretch through a signalling pathway that remains to be defined.

J Physiol 552.1 Sympathetic nerves modulate feed artery conduction 281 The anatomically defined range of motion readily encompasses a 30 % change in muscle fibre length (Ledvina & Segal, 1995; Welsh & Segal, 1996; Burkholder & Lieber, 2001). Previous experiments have demonstrated that the effect of passively stretching the retractor muscle on sympathetic vasoconstriction increases with muscle length through this range of motion (Welsh & Segal, 1996). Perusal of the present data indicates that the effect of muscle stretch on conducted vasodilatation is also graded. For example, measurements of peak active tension in the retractor muscle indicate that L IS approximates 85 % of L O (Welsh & Segal, 1996). Thus, the absolute increase in muscle length is greater when muscles are stretched to 130 % of L O as compared to 130 % of L IS and the inhibition of conducted vasodilatation was consistently greater under the former condition (Figs 2 5). Nevertheless, the ability of muscle stretch to significantly attenuate conducted vasodilatation under respective conditions attests to the reproducibility of our findings across experiments. We therefore conclude that, in addition to resting diameter (Welsh & Segal, 1996), cell-to-cell communication along feed arteries can be actively modulated throughout physiological changes in the resting length of skeletal muscle. In summary, we found that muscle stretch attenuated conducted vasodilatation along feed arteries controlling blood flow to the hamster retractor. This effect of muscle stretch was mimicked by perivascular nerve stimulation, noradrenaline, or phenylephrine and inhibited by antagonists of sympathetic neurotransmission. 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