α-adrenergic and neuropeptide Y Y1 receptor control of collateral circuit conductance: influence of exercise training

J Physiol 586.24 (28) pp 5983 5998 5983 α-adrenergic and neuropeptide Y Y1 receptor control of collateral circuit conductance: influence of exercise training Jessica C. Taylor 1,H.T.Yang 1, M. Harold Laughlin 1,2,3 and Ronald L. Terjung 1,2,3 1 Department of Biomedical Sciences, College of Veterinary Medicine, 2 Department of Medical Pharmacology & Physiology, College of Medicine and 3 Dalton Cardiovascular Research Center, University of Missouri, Columbia, MO 65211, USA This study evaluated the role of α-adrenergic receptor- and neuropeptide Y (NPY) Y1 receptor-mediated vasoconstriction in the collateral circuit of the hind limb. Animals were evaluated either the same day (Acute) or 3 weeks following occlusion of the femoral artery; the 3-week animals were in turn limited to cage activity (Sed) or given daily exercise (Trained). Collateral-dependent blood flows (BFs) were measured during exercise with microspheres before and after α-receptor inhibition (phentolamine) and then NPY Y1 receptor inhibition (BIBP 3226) at the same running speed. Blood pressures (BPs) were measured above (caudal artery) and below (distal femoral artery) the collateral circuit. Arterial BPs were reduced by α-inhibition (5 6 mmhg) to mmhg, but not further by NPY Y1 receptor inhibition. Effective experimental sympatholysis was verified by 5 % increases (P <.1) in conductance of active muscles not affected by femoral occlusion with receptor inhibition. In the absence of receptor inhibition, vascular conductance of the collateral circuit was minimal in the Acute group (.13 ±.2), increased over time in the Sed group (.41 ±.3; P <.1), and increased further in the Trained group (.53 ±.3; P <.2). Combined receptor inhibition increased collateral circuit conductances (P <.5), most in the Acute group (116 ± 37%; P <.2), as compared to the Sed (41 ± 6.6%; P <.1) and Trained (31 ± 5.6%; P <.1) groups. Thus, while the sympathetic influence of the collateral circuit remained in the Sed and Trained animals, it became less influential with time post-occlusion. Collateral conductances were collectively greater (P <.1) in the Trained as compared to Sed group, irrespective of the presence or absence of receptor inhibition. Conductances of the active ischaemic calf muscle, with combined receptor inhibition, were suboptimal in the Acute group, but increased in Sed and Trained animals to exceptionally high values (e.g. red fibre section of the gastrocnemius: 7mlmin 1 ( g) 1 mmhg 1 ). Thus, occlusion of the femoral artery promulgated vascular adaptations, even in vessels that are not part of the collateral circuit. The presence of active sympathetic control of the collateral circuit, even with exercise training, raises the potential for reductions in collateral BF below that possible by the structure of the collateral circuit. However, even with release of this sympathetic vasoconstriction, conductance of the collateral circuit was significantly greater with exercise training, probably due to the network of structurally larger collateral vessels. (Received 19 July 28; accepted after revision 22 October 28; first published online 3 November 28) Corresponding author R. L. Terjung: Department of Biomedical Sciences, E12 Vet. Medical Bldg, University of Missouri, Columbia, MO 65211, USA. Email: terjungr@missouri.edu Peripheral arterial insufficiency (PAI) is a common chronic disease (Stewart et al. 22) whereupon walking patients often manifest symptoms of intermittent claudication and are required to stop because of ischaemic pain. Studies involving experimental animal models of PAI (Waters et al. 24) demonstrate that pre-existing anastomoses form a collateral circuit capable of circumventing the vascular obstruction (Yang et al. 1996; Buschmann & Schaper, 1999, 2), that the conductance of this circuit increases following acute occlusion of a primary hind limb artery (Yang et al. 1996), and that the circuit exhibits a robust increase in conductance following treatments such as therapeutic growth factor delivery (Yang et al. 1996, 2a, 21) and/or enhanced physical activity (Yang et al. 199, 1995a; Lloyd et al. 21; Prior et al. 24). Improvements in collateral-dependent blood flow to the calf muscles are coincident with observations of enlarged collateral vessels DOI: 1.1113/jphysiol.28.1611

5984 J. C. Taylor and others J Physiol 586.24 that comprise the remodelled circuit (Yang et al. 1995a; Prior et al. 24). Thus, it has been reasonable to assign the improvement in function to the increased structural enlargement of the collateral vessels (Yang et al. 1995b; Prior et al. 24). Normally, blood flow delivered to active muscle is dependent upon both the structural capacity of the vascular circuit (number and size of vessels) and factors that exert vasomotor control of that structural circuit. These vascular control features involve both central, neurally derived influences that modulate vascular resistance in peripheral tissues and local influences related to tissue conditions affecting vasodilatation, as well as, the vasoresponsiveness of vessels to such signals. For example, during exercise there is an enhanced central sympathetic drive for peripheral vasoconstriction that is tempered within active muscle due to powerful local vasodilatation. This functional sympatholysis serves to redirect cardiac output to support high blood flow to the active muscles. Further, the conduit vessels proximal to the active muscle offer little vascular resistance and effectively propagate a high arterial pressure to the distal muscles. The situation is very different in peripheral arterial insufficiency where a proximal lesion can create a major upstream obstruction requiring flow to be diverted through a relatively high resistance of the collateral circuit. As a consequence, the collateral-dependent muscle of the distal limb is perfused at a reduced pressure, the extent to which depends upon the upstream collateral resistance and blood flow. It is apparent that blood flow to the ischaemic muscle is no longer determined primarily by local dilatory conditions, as is normally the case. Rather, it becomes imperative that extensive enlargement of the collateral vessels occur, if upstream resistance is to be minimized. Further, another complication becomes evident in the control of blood flow to collateral-dependent muscle. Since the collateral circuit develops from relatively small anastomoses (Buschmann & Schaper, 1999, 2), which are capable of dominant vasoconstriction responses compared to large conduit arteries (Pernow et al. 1987; Smiesko et al. 1989; Ping & Faber, 1993; Ekelund & Erlinge, 1997), and since the collateral vessels are not expected to experience powerful dilatory signals, because the upstream circuit is typically surrounded by relatively less ischaemic muscle (Yang et al. 1996, 2b, 22; Ito et al. 1997; Buschmann & Schaper, 1999), there could be a marginal reduction in vascular resistance of the collateral circuit with the increased sympathetic nerve activity during exercise. If this happened, then the collateral-dependent blood flow to the active muscle could be well below that possible by the vascular capacity of the collateral circuit plumbing itself. Sympathetic outflow increases in response to stressors which include, but are not limited to, exercise, vascular occlusion, or cold stress (Lundberg et al. 1985; Buckwalter & Clifford, 21; Buckwalter et al. ). Sympathetic neurotransmitter release increases in parallel with the intensity of the stressful stimuli (Lundberg et al. 1985; Buckwalter & Clifford, 21; Buckwalter et al. ). This increase in sympathetic outflow could be exaggerated further when two or more stressors are compounded, such as exercise coupled with tissue ischaemia (Bull et al. 1989; Bakke et al. 27). Two sympathetic receptors classes,α-adrenergic and neuropeptide Y, stand out as important candidates for restricting blood flow during exercise and/or arterial occlusion. Both α1- and α2-adrenergic receptors exhibit a robust vasoconstriction to noradrenaline (norepinephrine) (Smiesko et al. 1989), are the major contributors to basal tone in the vasculature, and are tonically activated during exercise (Buckwalter et al. 1997, 1998, 21). We have previously shown that these receptors are significantly activated in the nascent collateral circuit that is apparent upon occlusion of the femoral artery in adult rats. Inhibition of α-adrenergic receptors resulted in a significant increase in collateral circuit conductance (Taylor et al. 28). Whether the collateral circuit adapts to modify this adrenergic vasoconstriction over time post-occlusion or with the imposition of physical activity, which is known to induce significant vascular remodelling (Lloyd et al. 21; Prior et al. 24), remains to be determined. Neuropeptide Y (NPY) is stored in sympathetic nerves and co-released with noradrenaline, especially during periods of stress such as tissue injury, ischaemia or treadmill exercise (Lundberg et al. 1985; Michel, 24). This discharge of NPY can act as a potentiator resulting in NPY-induced NPY release. Acting via the constitutively expressed NPY Y1 receptor, NPY is capable of inducing potent and long-lasting vasoconstriction (Lundberg et al. 1985; Michel, 24), as well as, potentiating the constrictor effects of noradrenaline (Lundberg et al. 1985; Michel, 24). In fact, vascular occlusion has been shown to augment NPY levels in the circulation of the occluded limb, compared with the non-occluded limb (Lee et al. 23). Whether the increased sympathetic outflow and subsequently heightened release of NPY during exercise could serve to further impede the already structurally limited blood flow through the collateral circuit, is not known. The purpose of this study was to test the hypotheses: (1) that the α-adrenergic receptor-mediated vasoconstriction of the collateral circuit is lessened (a) over time post-occlusion, and (b) by exercise training, albeit at a higher vascular conductance; (2) that NPY Y1 receptor inhibition increases the vascular conductance of the collateral circuit in animals following acute occlusion of the femoral artery, in animals 3 weeks following femoral artery occlusion, and in 3-week-occluded animals that are exercise trained; and (3) that the vascular conductances of ischaemic muscle during exercise are

J Physiol 586.24 Sympathetic control of collateral vasculature 5985 not altered by α-adrenergic or NPY Y1 receptor inhibition. Methods Ethical approval The care and treatment of all animals and experimental procedures were conducted in accordance with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication no. 85-23, revised 1996) and approved by the Animal Care and Use Committee of the University of Missouri. Experimental design This study evaluated the effects of α-adrenergic and α-adrenergic plus NPY Y1 receptor inhibition on muscle and collateral circuit conductances following occlusion of the major supply artery to the distal hind limb (femoral artery), the influence of time on collateral development, and the adaptive changes induced by daily exercise, using adult ( 35 g) male Sprague Dawley (Harlan, Indianapolis, IN, USA) rats. Ligation of the femoral artery was performed surgically to reduce the blood flow capacity to the lower limb muscles by 8 85% (Yang et al. 1996, 2b). While this significantly limits the blood flow reserve to the calf muscles, there are no noticeable behavioural changes in cage activity, development of limb pathology or any appearance of necrosis. This is because the residual flow reserve is still 2- to 3-fold greater than the blood flow measured in quiescent calf muscles of anaesthetized animals (Mackie & Terjung, 1983). Thus, this experimental model of peripheral arterial insufficiency is characterized by an absence of symptoms at rest, but the development of limb ischaemia during exercise. To determine the effect of α-adrenergic and α-adrenergic plus NPY Y1 receptor inhibition on collateral-dependent blood flow during exercise, muscle blood flows were determined during treadmill exercise in animals randomly assigned to the following groups. Group 1: acute unilateral occlusion of the femoral artery; Group 2: animals that had bilateral occlusion of the femoral arteries for 3 weeks; and, Group 3: animals with bilateral occlusion of the femoral arteries that were exercised daily for 3 weeks. Rats underwent three blood flow measurements, using radiolabelled microspheres, during the second minute of each running bout on a motorized rodent treadmill, as done previously (Yang et al. 2a, 22; Prior et al. 24). Based upon previous work (Yang et al. 2a, 22; Prior et al. 24), speeds were selected for each group to establish maximal collateral-dependent BF in the calf muscles, since their exercise capacities were different (2 m min 1 for Acute group; m min 1 for Sed group; and 3 m min 1 for Trained group). The first control BF measurement was to determine peak collateral circuit capacity and tissue vascular responses without pharmacological intervention. Following the first brief exercise bout rats were given phentolamine, a non-selective α-adrenergic antagonist (2.6 mg kg 1, 66% inhibition of noradrenaline-induced increase in distal femoral artery blood pressure; n = 6). After adequate circulation of the drug ( 5 min), as determined by a stable and significant decrease ( 45 mmhg) in mean arterial pressure (MAP), a second blood flow measurement was obtained at the same treadmill speed. Finally, rats were administered BIBP 3226, a selective NPY Y1 receptor antagonist (1 mg kg 1, 6% inhibition of NPY-induced increase in MAP; n = 5), and following circulation of the drug ( 5 min) a final blood flow was obtained, again at the prior treadmill speed. Thus, assessment of NPY Y1 receptor-mediated contribution to collateral circuit resistance was evaluated under α-adrenergic inhibition, in an attempt to minimize the obfuscation of results possible with α-adrenergic receptor-induced vasoconstriction. While complete receptor blockade was not employed, based on preliminary work we did achieve the highest possible receptor inhibition that still allowed for cardiovascular control, evident by an ability to maintain arterial pressure and exercise. The above experimental design allowed for: (1) each rat to serve as its own control; (2) examination of the contribution of α-receptors in the circulation; and (3) examination of NPY Y1 contributions without fully active α-adrenergic receptors masking the results. Rats were familiarized with the treadmill by running 5 6 min day 1 for 4 5 days prior to experimentation. This familiarization with treadmill running does not induce adaptations typical of exercise training (Yang et al. 2a,b; Yanget al. 22; Prior et al. 24). Blood flow measurements were obtained during exercise in order to minimize resistance of the distal limb tissue, which is necessary to correctly determine the conductance of the upstream collateral circuit (Yang et al. 22). Calf muscle BF is not solely dependent upon the function of the collateral circuit, as % of the total vascular resistance is exerted by the vasculature distal to the collateral circuit (Yang et al. 22). Thus, an additional group of rats (n = 9) was used to evaluate the potential for remodelling of distal conduit vessels in vitro, following approximately 3 weeks of unilateral femoral artery occlusion. Animal care and exercise training protocols Rats were housed two per cage in a temperature-controlled (2 ± 1 C) animal room with a 12 h : 12 h light dark cycle. Rats were fed a standard rat chow diet and water ad libitum.

5986 J. C. Taylor and others J Physiol 586.24 On the second day following surgical ligation, rats in Group 3 began running on a motorized rodent treadmill at 2 m min 1 twice a day. As exercise tolerance improved from the initial 5 8 min bout 1, rats rapidly progressed in time to run 55 min twice per day within the 3-week period. Surgical procedures Ligation of the femoral artery. Details of the ligation procedure have been previously described (Yang & Terjung, 1993). Briefly, animals were brought to the laboratory and anaesthetized with a combination of ketamine ( mg kg 1 )andacepromazine(.5mgkg 1 ). The femoral artery was exposed distal to the inguinal ligament and dissected free from surrounding connective tissue. A ligature (3- silk) was placed around the artery ( 3 mm distal to the ligament) and secured. Catheterization for blood flow determination. For the purpose of determining blood flows three catheters were placed as follows: (1) a catheter (polyethylene-5 tubing) was inserted into the femoral artery distal to the site of occlusion for the purpose of measuring downstream collateral-dependent perfusion pressure; (2) a second catheter was placed in the left carotid artery and advanced to the aortic arch for the infusion of microspheres and pharmacological antagonists; and (3) a third catheter was inserted into the caudal (tail) artery for withdrawal of the microsphere reference blood sample and for monitoring mean arterial pressure. Caudal artery pressure was used as the perfusion pressure for all non-ischaemic tissues while distal femoral pressure was used as the perfusion pressure for the ischaemic tissues of the distal hind limb. The difference between the two pressures was used to calculate the pressure drop across the collateral circuit. Catheters were filled with heparinized saline ( IU ml 1 ), tunnelled under the skin, and exteriorized at the base of the neck (Mathien & Terjung, 199). Rats recover quickly from the anaesthesia and exhibit cage activity within a couple of hours (Yang et al. 2a,b; Lloyd et al. 21; Prior et al. 24; Taylor et al. 28). Blood flow determination. Blood flows were determined using radiolabelled microspheres ( 85 Sr or 13 Ru, 141 Ce and 46 Sc, 15 ±.1 μm diameter, New England Nuclear; Boston, MA, USA) infused during the second minute of treadmill exercise (Laughlin et al. 1982; Mathien & Terjung, 199). A well-mixed suspension of microspheres was infused into the arch of the aorta, via the carotid catheter, followed by a saline flush over approximately 2 s. Ten seconds prior to the infusion of microspheres, withdrawal of the reference blood sample began at a rate of.5 ml min 1 via the caudal artery catheter. Adequate mixing of the microspheres in the circulation was assured by comparing flows to the right and left kidney and the right and left abdominal muscles. Repeated bouts of exercise were performed with approximately 8 1 min between bouts. After completion of the third exercise bout, rats were killed with an overdose of ketamine and ace promazine via intra-arterial injection, followed by a pneumothorax. Tissue samples were taken comprising both hind limbs, the middle third of the kidneys, the diaphragm, and portions of the abdominal and psoas muscles; these samples were counted to a 1% error (Wallac 1282 CompuGamma CS, Turku, Finland) and corrected for spillover between isotope counting windows. Blood flows (ml min 1 ( g) 1 ) were calculated as: Blood flow = (.5ml min 1 CPM 1 RBS ) (CPM tissue (tissuewt) 1 ) where RBS is reference blood sample and CPM is counts per minute. Blood flows from individual tissue sections were combined to determine BF to the whole hind limb and the proximal and distal portions of the hind limb. Muscle conductances were calculated by dividing the measured BFs by the appropriate perfusion pressures, where BF is the regional or muscle-specific blood flow in ml min 1 ( g) 1 and pressure is MAP or region-specific pressure in mmhg (caudal artery or distal femoral artery, as appropriate). Similarly, conductances of the collateral circuit were calculated as the total flow through the circuit (i.e. distal hind limb blood flow) divided by the pressure drop across the circuit (i.e. caudal pressure minus distal hind limb pressure). Preparation of isolated arteries To assess the potential for adaptations in the distal vasculature, which exerts % of the total resistance in BF to the distal limb, additional rats that experienced unilateral femoral artery occlusion approximately 3 weeks earlier were anaesthetized with pentobarbital (1 mg kg 1 ) and killed by pneumothorax followed by heart excision. Hind limbs were skinned, separated from the torso and placed in a Krebs solution on ice. Each hind limb was then transferred to a dissecting dish containing ice-cold physiological salt solution (PSS) at ph 7.4 and dissected with the aid of an Olympus dissecting microscope. The saphenous and feed arteries to the medial head of the gastrocnemius and to the soleus, from each limb, were isolated free of any connective tissue and placed in a Plexiglas chamber filled with PSS. The vessels were cannulated on glass micropipettes (9 115 μm) filled with PSS-albumin (PSSA) and secured with a fine suture. Following cannulation the chambers were transferred to the stage of an inverted microscope (Nikon Diaphot

J Physiol 586.24 Sympathetic control of collateral vasculature 5987 Table 1. Body and hind limb weights (g) Chronic occlusion Acute occlusion Sedentary Exercised ANOVA P value Body weight 415 ± 6.9 4 ± 7.2 398 ± 5.1 <. Hind limb weight Total 24.2 ±.28.1 ±.33 24.7 ±.29 n.s. Proximal 15.7 ±.23 16.5 ±. 16.4 ±.19 n.s. Distal 8.3 ±.7 8.4 ±.13 8.2 ±.12 n.s. Calf muscles 2.9 ±.6 3. ±.6 2.9 ±.5 n.s. n 8 1 1 Values are mean ± S.E.M. n = number of animals per group. Hind limb sections: total = whole leg; proximal = all tissues above the knee; distal = all tissues below the knee. Calf muscles = gastrocnemius, plantaris and soleus muscles. Different from Sedentary group (P <.5). n.s., not significant. 2) attached to a video camera (Javelin Electronics, Los Angeles, CA, USA), video micrometer (Microcirculation Research Institute, Texas A & M University), and MacLab data acquisition system. Luminal diameter and pressure were monitored continuously throughout the experiment. Vessels were only included if they were free of leaks, as indicated by an ability to maintain pressure and contracted in response to 8 mm KCl. Each vessel was allowed to warm to 37 C and equilibrate at 45 cmh 2 O intraluminal pressure for 1 h. If the vessels did not exhibit spontaneous tone, as indicated by a decrease in diameter over the 1 h of pre-incubation, they were stimulated to constrict with phenylephrine until a minimum of 3% constriction. Preconstrictions were necessary prior to each vasodilatation curve for the saphenous artery (n = 9 out of 9) and gastrocnemius feed artery (n = 5 of 5), but not for the soleus feed arteries (n = of 9). To examine endothelium-dependent dilatation vessels were exposed to acetylcholine (1 9 to 1 4 M) added in whole log increments to determine the dose response relationship. The dose response to phenylephrine (1 9 to 1 4 M) was then obtained at this low luminal pressure. Following a washout period the luminal pressure was elevated to 12 cm H 2 O to assess the capacity for endothelial-independent dilatation using sodium nitroprusside (SNP; 1 9 to 1 4 M). At the end of the experiment the PSS was removed from the bath and replaced with calcium-free PSS. The vessel was allowed to remain in calcium-free PSS for 45 min with the solution changed every 15 min. To confirm that maximal calcium-free diameter had been achieved the bathing solution was removed and replaced with 1 mm caffeine dissolved in calcium-free PSS; thapsigargin was also added to prevent calcium reuptake by the sarcoplasmic reticulum. The vessel was then allowed to bathe in this solution for an additional 15 min to ensure maximal diameter. Solutions and drugs The PSS used for isolated vessel dissection and experimentation contained the following (in mm): 145. NaCl, 4.7 KCl, 2. CaCl 2, 1.17 MgSO 4, 3. 3-(N-morpholino)propanesulphonic acid (MOPS), 1.2 NaH 2 PO 4, 5. glucose, 2. pyruvate, and.2 EDTA. PSSA was composed of the same chemicals as PSS with 1 g l 1 albumin added to the solution. The 8 mm KCl contraction solution contained the following (in mm): 65 NaCl, 84.7 KCl, 2 CaCl 2, 1.17 MgSO 4, 3. MOPS, 1.2 NaH 2 PO 4, 5. glucose, 2. pyruvate, and.2 EDTA. Phenylephrine, acetylcholine and SNP were all diluted from their stock concentrations (drug dissolved in saline and frozen in useful aliquots) in PSS. Calcium-free PSS was made with the reagents above except that 2. mm CaCl 2 was omitted and 2mM EDTA was added. Caffeine was diluted in calcium-free PSS. Thapsigargin was diluted in ethanol such that the final concentration of ethanol in the bath was not more than 1%. All drugs and chemicals were purchased from Sigma (St Louis, MO, USA). Statistics Values are presented as mean± S.E.M. Main treatment effects were evaluated with two-way repeated measures analyses of variance (ANOVA) with Tukey s test for post hoc analyses, using P <.5 for statistical significance. Results Body/tissue weights, blood pressures and heart rates Typical of animals that exercise routinely, there was a small but significant decrease in body weight (P <.5) in the trained group; however, hind limb tissue weights were not different among the groups (Table 1).

5988 J. C. Taylor and others J Physiol 586.24 Table 2. Heart rates and blood pressures Caudal artery blood Distal femoral artery Heart rate pressure (mmhg) blood pressure (mmhg) Pre-exercise Exercise Pre-exercise Exercise Pre-exercise Exercise Acute (n = 8) Pre-drug 455 ± 14. 485 ± 13.6 137 ± 2.9 135 ± 4.6 45 ± 4.3 37 ± 2.4# Phentolamine 495 ± 17. 495 ± 17. 77 ± 3. ± 5. 32 ± 2.8 26 ± 2.8# Phentol + BIBP 48 ± 12.7 48 ± 9.8 79 ± 2.6 83 ±.6 2 ± 2. 19 ± 3. Sedentary (n = 1) Pre-drug 474 ± 7.5 54 ± 6. 133 ± 1.8 13 ± 1.9 5 ± 5.3 35 ± 3.1# Phentolamine 54 ± 6. 57 ± 7. 85 ± 3.2 12 ± 3.1 24 ± 3.2 21 ± 2.1# Phentol + BIBP 54 ± 7.5 57 ± 7. 85 ± 2.9 84 ± 2.3 17 ± 2.1 15 ± 1.9 Trained (n = 1) Pre-drug 47 ± 7.7 57 ± 1.4 134 ± 3. 13 ± 3.4 55 ± 5.3 4 ± 3.1# Phentolamine 513 ± 7. 513 ± 7. 71 ± 4.3 97 ± 5.3 32 ± 5.8 ± 2.8# Phentol + BIBP 5 ± 7.7 51 ± 7.7 91 ± 3.5 85 ± 3.8 24 ± 2.6 22 ± 3.5 Values are mean ± S.E.M. Main treatment effects: phentolamine (Phentol) decreases blood pressure (BP) (P <.1) and increases heart rate (P <.1); # exercise decreases distal femoral artery BP (P <.1); exercise increases caudal artery BP (P <.1). As illustrated in Table 2, there were main treatment effects for heart rate to increase during exercise (P <.1) and by the imposition of a decrease in blood pressure (P <.1) with adrenergic receptor inhibition via phentolamine. Decreases in caudal artery BP (P <.1) were fairly similar across treatment groups pre-exercise (36 47%) and significantly increased (P <.5) to approximately mmhg across groups during exercise. Added inhibition of NPY Y1 receptors did not change the pre-exercise caudal BP in the Acute and Sed groups, but did increase BP (P <.5) in the Trained group. Caudal BP was not elevated during exercise with BIBP administration, as they were with phentolamine only. Blood pressures in the distal femoral artery were well below the corresponding caudal artery pressures among the groups prior to exercise (P <.1), decreased upon administration of phentolamine (P <.1), and then decreased further with BIBP administration (P <.1). As expected with an increased flow across a resistance, BP in the distal femoral artery decreased during treadmill running (P <.1), with the greatest changes (8 15 mmhg; 18 3%) apparent in the absence of sympathetic receptor inhibition. increasing to 2.82 ±.13 ml min 1 ( g) 1 mmhg 1 ; for control and combined inhibition in the Acute and Sed groups, respectively). On the other hand, renal conductances were not elevated in the Trained group during receptor inhibition (e.g. 2.58 ±.17, 2.48 ±.33 and 1.72 ±. ml min 1 ( g) 1 mmhg 1 for control, phentolamine and combined phentolamine plus BIPP conditions), resulting in the reduced BF corresponding to the reductions in perfusion pressure. Blood flows and conductances of muscles not impacted by hind limb ischaemia BFs to the diaphragm and psoas muscles were similar across groups prior to drug administration and were fairly well maintained following α-adrenergic and NPY Y1 receptor inhibition (cf. Fig. 1) even though there were significant decreases in arterial BP. The sustained BFs were possible by vasodilatation, as evident by increased vascular conductances (P <.1) in these muscles following receptor inhibition. Thus, tonic activation of the α and NPY Y1 receptors must normally occur in these muscles during exercise. Renal BF and conductances with α-adrenergic and NPY Y1 receptor inhibition Renal BF was fairly consistent for the Acute and Sed groups across receptor inhibition conditions, at approximately 3 ml min 1 ( g) 1. These BFs were reasonably well maintained due to significant increases (P <.) in vascular conductances following receptor inhibition (e.g. 2.26 ±.29 increasing to 3.36 ±.36 and 2.23 ±.14 Blood flows and conductances of the ischaemic hind limb muscles A similar demonstration of experimental sympatholysis, as observed in the diaphragm and psoas muscles, was evident in the quadriceps of the limbs that experienced femoral artery occlusion. Note in Fig. 2 that BF of the red (high-oxidative, high vascular capacity) and white (low-oxidative, low vascular capacity) sections of the

J Physiol 586.24 Sympathetic control of collateral vasculature 5989 Figure 1. Blood flows (ml min 1 ( g) 1 ) and conductances (ml min 1 ( g 1 ) mmhg 1 ) to muscles not affected by femoral artery occlusion Significant increase in conductance with receptor inhibition (P <.1). quadriceps were reasonably well maintained following receptor inhibition, albeit at very different flow rates. BF to this region of the proximal hind limb is relatively high, typical of normal muscle at these treadmill speeds, and not collateral dependent, even though the vessels comprising the collateral circuit course through the thigh and hamstrings. The sustained BF occurred because vascular conductances of the muscle sections increased with Figure 2. Blood flows and conductances to the active high- and low-oxidative muscle fibre sections of the quadriceps muscles Significant increase in conductance with receptor inhibition (P <.1); # significantly lower blood flows (P <.1).

599 J. C. Taylor and others J Physiol 586.24 receptor inhibition. Interestingly, this was most apparent in the white quadriceps section where both a release of sympathetic vasoconstriction and enhanced recruitment of fibres within the section could have contributed to the response. On the other hand, recruitment of the motor units within the red quadriceps section is expected to be far more complete, since these red fibres are the primary fibre type recruited during treadmill running at the speeds used in this study (Dudley et al. 1982; Laughlin & Armstrong, 1982). Notably, the significant increase in conductance of the red quadriceps section, was observed only in the animals following acute ligation of the femoral artery (cf. Fig. 2). This implies that sympathetic vasoconstriction was initially greater in the animals immediately post-occlusion, as compared to animals that were kept Sed or Trained for 3 weeks. Blood flows and conductances of the ischaemic calf muscles As illustrated in Fig. 3, BFs to the calf muscles of the Acute group are markedly below those measured in similarly occluded animals (Sed), but after 3 weeks of accommodation to occlusion of the femoral artery. Further, the Trained group exhibited significantly greater BF (P <.5) than animals that remained sedentary. Thus, vascular adaptations are apparent in the control of BF to active muscles that are collateral dependent. Further, it is apparent that BFs were well maintained, within each group, in response to α-adrenergic and NPY Y1 receptor inhibition (cf. Fig. 3), even though perfusion pressures to the distal hind limb muscles were appreciably reduced with phentolamine and BIBP treatments (cf. Table 2). This was possible due to the increase in vascular conductances of the ischaemic calf muscles with receptor inhibition (P <.1). Thus, in the absence of receptor inhibition, there was a dramatic stimulus for vasoconstriction influence that kept vascular conductance well below that possible. Blood flows and conductances of the collateral circuit The significant differences among calf muscle BFs, identified above, are primarily due to the resistances of the upstream collateral circuit, the collateral circuit comprised a major fraction of the total resistance measured in distal leg BF (74.1 ± 1.87%, 78.2 ± 1.55% and 72.7 ± 2.68% for the Acute, Sed and Trained groups, respectively). It is apparent from Fig. 4 that vascular conductance of the collateral circuit is least, soon after occlusion of the femoral artery (cf. Acute group). There is a significant increase in conductance over time in the Sed group and a further increase in the Trained group. The marked increase in vascular conductance of the collateral circuit in the time following femoral artery occlusion (P <.1) is due in part to a reduced tonic influence of α-adrenergic and NPY Y1 receptor activation, since the increases in vascular conductance with combined receptor inhibition were the greatest (e.g. Acute group, 116 ± 36.8% increase; Figure 3. Blood flows and conductances to the ischaemic calf muscle during exercise following adrenergic and combined α- plus NPY Y1-receptor inhibition Significant increases in conductances with receptor inhibition (P <.1); # significantly lower BF and conductances than sedentary (Sed) and Trained groups (P <.1); significantly greater BF than Sed group (P <.1). Figure 4. Vascular conductances of the collateral circuit during exercise following α-receptor and combined α- plus NPY Y1-receptor inhibition Significantly lower conductances than Sed and Trained groups (P <.1); # significant increases in conductances with receptor inhibition (P <.1); significantly greater conductances than Sed group (P <.1).

J Physiol 586.24 Sympathetic control of collateral vasculature 5991 P <.1), as compared to the Sed (41 ± 6.6% increase; P <.1) and Trained (31 ± 5.6% increase; P <.1) groups. However, it is also clear that the peak conductance of the collateral circuit of the Acute group, in the presence of combined receptor inhibition, is well below the conductance observed in the Sed group (P <.1). Thus, it is likely that meaningful vascular remodelling occurred in the time following occlusion of the femoral artery. Similarly, conductances of the collateral circuit of the Trained group, with or without receptor inhibition were significantly greater (P <.1) than those observed for the Sed group. The structural enlargement of the collateral vessels, reported previously (Prior et al. 24), is expected to contribute to this greater vascular capacity of the collateral circuit. Indeed, the lower resistance of the collateral circuit accounts for approximately 85% of the improvement in calf muscle BF in the Trained group. The remaining 15% can be attributed to a decrease in the resistance within the distal limb vessels and musculature. Blood flows and conductances of the fibre sections comprising the ischaemic calf muscle As illustrated in Figs 5 and 6, blood flows to the individual muscle fibre sections that comprise the calf muscle were reasonably well maintained following receptor inhibition among the Acute, Sed and Trained groups, albeit at very different absolute BFs. Even though distal perfusion pressures were significantly reduced with phentolamine and BIBP administration, BFs within muscle sections and groups were maintained because of corresponding increases in vascular conductances (cf. Fig. 5 and 6). Thus, in the absence of drug intervention, sympathetic activation serves to limit blood flow by not permitting full use of the vascular capacity within each muscle section. Interestingly, the absolute BFs to muscle sections of the Acute group were exceptionally low, as compared to the corresponding muscle sections of the Sed and Trained groups. Even more striking are the markedly reduced vascular conductances of the Acute group, as compared to those of the Sed and Trained groups. However, it is apparent that the relatively low conductances observed with Acute occlusion, do not remain as similar animals evaluated 3 weeks post-occlusion exhibited high conductances. There were no further increases in muscle section conductances with Training. Interestingly, the conductances in the high vascular capacity, red-type muscle fibre sections (red gastrocnemius and soleus) are exceptionally high following dual inhibition of α-sympathetic and NPY Y1 receptors. While the conductances of the Sed and Trained white gastrocnemius section are only approximately 1/3 that of the red gastrocnemius and soleus muscle sections, the values Figure 5. Blood flows and conductances to the soleus muscle and red gastrocnemius fibre section of the calf muscles following α-receptor and combined α- plus NPY Y1-receptor inhibition Significantly lower BF and conductances than Sed and Trained groups (P <.1); # significant increases in conductances with receptor inhibition (P <.1); significantly greater pre-drug BF than Sed pre-drug group (P <.2).

5992 J. C. Taylor and others J Physiol 586.24 are relatively high for this low vascular capacity muscle section. These relatively high vascular conductances reflect the exaggerated dilatation experienced by ischaemic muscle following exposure to receptor inhibition. Conduit vessel size and vasoresponsiveness of distal supply vessels of the hind limb We sought to determine the reasons for the low vascular conductances of the active ischaemic calf muscle fibre sections immediately following occlusion of the femoral artery (Acute group). Heightened resistance from the conduit vessels distal to the occlusion, but proximal to the calf muscle, is a likely contributor. As illustrated by the compliance curves shown in Fig. 7, maximal vessel distension would be reduced at the lower intraluminal pressures (i.e. 35 mmhg; Table 2) observed immediately following occlusion of the femoral artery. In the absence of smooth muscle tone, these smaller vessel diameters of the saphenous, medial gastrocnemius and soleus feed arteries would reduce conductances by 5 %, compared to the size possible at their normal higher intraluminal pressures. Interestingly, as illustrated in Fig. 7, meaningful remodelling of the saphenous and feed arteries to the gastrocnemius and soleus muscles occurs over time, post-occlusion, as evident by their enlarged passive diameters. In fact, these enlarged diameters returned the size of these vessels, observed at the low perfusion pressure, to that initially possible at their normal higher luminal pressure. Thus the recovered ability to distend could have contributed to the increased vascular conductances observed in the Sed, as compared to the Acute groups. We did not evaluate compliance curves for the vessels from Trained animals, but expect that similar enlargements would be induced since the higher vascular conductances among individual muscle sections were similar between the Sed and Trained animals (cf. Fig. 5 and 6). On the other hand, the vasoresponsiveness of the saphenous and feed arteries to the medial gastrocnemius and soleus muscles were fairly similar among vessels obtained from non-occluded and occluded groups (cf. Fig. 8). While vasoconstriction to phenylephrine was robust among the vessels, the endothelial-dependent dilatory response to acetylcholine was fairly modest when measured at the low intraluminal pressure ( 33 mmhg) in these vessels following femoral artery occlusion (cf. Table 2). Even the capacity for endothelial-independent dilatation, determined at the higher, more normal intraluminal pressure of 88 mmhg using SNP, was not different among vessels, although the magnitude of response was the least in the soleus feed artery (Fig. 8). Thus, the increased vessel diameters remain as the primary change that is implicated in improving vascular conductance to the active ischaemic muscle post-occlusion. Figure 6. Blood flows and conductances to the plantaris muscle (mixed fibres) and white gastrocnemius fibre section of the calf muscles following α-receptor and combined α- plus NPY Y1-receptor inhibition Significantly lower BF and conductances than Sed and Trained groups (P <.1); # significant increases in conductances with receptor inhibition (P <.1); significantly increased BF with receptor inhibition (P <.1).

J Physiol 586.24 Sympathetic control of collateral vasculature 5993 Discussion The present study adds new insights into the control of blood flow to ischaemic muscle caused by peripheral arterial insufficiency, in two important ways. First, it expands our previous findings demonstrating that during exercise sympathetic vasoconstriction is present in the nascent collateral circuit immediately following onset of arterial occlusion (Taylor et al. 28). This remains following remodelling of the vessels, although it becomes tempered. Even with animals that are exercise trained, sympathetic vasoconstriction is apparent in the collateral circuit, since inhibition of sympathetic receptors (α-adrenergic and NPY Y1) increases the already greater conductance. However, the structural enlargement of the collateral circuit, observed with training (Prior et al. 24), is critical to the capacity for blood flow, since the conductance of the collateral circuit of trained animals is significantly elevated, irrespective of sympathetic inhibition. Nonetheless, this influence of sympathetic receptor activation raises the concern that collateral-dependent BF could be less than that determined by the structure of the circuit itself. The expected deficit in collateral function could be exaggerated if an ischaemic pressor response, typical of patients during exercise-induced claudication (Bakke et al. 27), were to occur during activity. Second, vascular conductances of the ischaemic muscle during exercise are appreciably below that possible by the vascular capacity of the circuit itself. This serves to exacerbate the reduction in BF caused by the upstream resistance of the collateral circuit. We demonstrate that the absence of optimal dilatation in the individual muscle fibre sections of the calf is most apparent following occlusion of the femoral artery. This lack of dilatation is not simply due to exaggerated α-adrenergic and/or NPY Y1 receptor activity, but is probably influenced by a loss of vessel distension within the distal vasculature and lessened with the vascular remodelling that occurs with increasing time post-occlusion. Collectively, these findings illustrate the importance of vascular adaptations that developed within the collateral circuit and active muscle in conditions of reduced BF caused by peripheral arterial insufficiency. Sympathetic control of the collateral circuit The influence of α-adrenergic activation on the collateral circuit, observed soon after occlusion of the primary supply artery (Taylor et al. 28), has been expanded to show that activation of NPY Y1 receptors can also play a role in restricting collateral circuit conductance. While there were main treatment effects of both phentolamine (a non-selective α-adrenergic antagonist) and BIBP 3226 (an NPY Y1 receptor antagonist) to increase conductances of the collateral circuit across groups, the influences were proportionally largest in the Acute group where BFs were determined soon after occlusion of the femoral artery. This implies that the nascent vessels, that comprise the developing collateral circuit, do not receive or are less responsive to dilatory stimuli, and/or experience a more dominant vasoconstriction influence. It is easy to understand how this may occur, since collateral BF arises from conduit vessels that circumvent the vascular obstruction (e.g. internal iliac), courses through small pre-existing anastomoses in the thigh (Yang et al. 1996; Taylor et al. 28), and re-enters conduit vessels of the lower limb for distribution to the active muscles. All of these vessels are richly innervated with α-adrenergic receptors (Ping & Faber, 1993) which could impart the α1 and α2 responses observed previously (Taylor et al. 28) and confirmed in the present study. Further, the smaller arteries and arterioles are innervated with NPY Y1 receptors (Pernow et al. 1987; Ekelund & Erlinge, 1997) which can produce a robust vasoconstriction (Michel, 24). Thus, a powerful vasoconstriction of the Diameter (μm) Diameter (μm) Diameter (μm) 7 6 5 4 3 2 45 4 35 3 2 15 5 2 15 5 Saphenous* Soleus Feed* Passive Compliance Occluded Non-occluded 1 2 3 4 5 6 7 8 9 Gastroc Feed* 1 2 3 4 5 6 7 8 9 1 2 3 4 5 6 7 8 9 Pressure (mmhg) Figure 7. Passive compliance of vessels in the absence and presence of femoral artery occlusion for 3 weeks Significant increase in vessel size due to vascular occlusion (P <.1).

5994 J. C. Taylor and others J Physiol 586.24 collateral circuit should occur, if sympathetic activation is elevated. As importantly, there may be relatively little stimulus surrounding the small collateral vessels for dilatation, as typically found to countermand neural vasoconstriction (McGillivray-Anderson & Faber, 1991; Buckwalter & Clifford, 21; Buckwalter et al. 24), since the musculature on the thigh is relatively free from ischaemia. In addition, we have previously identified a dulled endothelial-mediated dilatory response, but a retained robust capacity for vasoconstriction, associated with collateral vessels having to function at the new lower intraluminal pressure caused by the occlusion of the femoral artery (Taylor et al. 28). We have observed that collateral vessels are also refractory to flow-mediated dilatation (P. N. Colleran, unpublished observations). Thus, the conductance of the nascent collateral circuit is understandably low (Conrad et al. 1971; Lash et al. 1995; Unthank et al. 1995;Yanget al. 22), since there has been minimal time for remodelling of the vessels. There are markedly greater collateral conductances in the Sed group, irrespective of inhibitor treatment (cf. Fig. 4), as compared to the Acute group. While the structural enlargement of the collateral circuit, typical of that observed over time (Prior et al. 24) is expected to be the major reason for the increased conductance, other factors could contribute. For example, the sympathetic dominance adding to the inherent resistance of the collateral circuit is significantly lessened, as the fractional improvement in collateral conductance with receptor inhibition is well below that observed in the Acute group (cf. Fig. 4). There could be two contributors to this response: first, sympathetic activation could be less, if the central sympathetic drive were lessened over time. However, this does not appear likely since the responses to sympathetic inhibition were similar among the kidney and non-ischaemic diaphragm and psoas muscles of the Acute and Sed animals. Rather, the vasomotor balance in response to that sympathetic activation may have been altered. We have observed that significant adaptations develop within the collateral vessels which could enhance the dilatory response of the collateral circuit (P. N. Colleran, unpublished observations). Of particular note is the upregulation of the vasodilator NPY Y2 receptors in the collateral vessels that are not normally constitutively expressed. This occurs with occlusion of a supply artery and may be related to regional ischaemia (Lee et al. 23). Activation of the NPY Y2 receptor occurs via NPY3 36, the hydrolytic product of NPY by the action of dipeptidylpeptidase IV (DPPIV) (Mentlein et al. 1993). Interestingly, DPPIV mrna is also upregulated in the collateral vessel (P. N. Colleran, unpublished observations). Thus, adaptations of an enlarged vessel % Possible Constriction % Possible Dilatation % Possible Dilatation PE Saphenous Occluded 5 Non-occluded @33 mmhg 1-9 1-8 1-7 1-6 1-5 1-4 5 ACh 1-9 1-8 1-7 1-6 1-5 1-4 5 SNP @33 mmhg @88 mmhg 1-9 1-8 1-7 1-6 1-5 1-4 Log dose 5 5 PE ACh Gastroc Feed Artery @33 mmhg 1-9 1-8 1-7 1-6 1-5 1-4 @33 mmhg 1-9 1-8 1-7 1-6 1-5 1-4 5 SNP @88 mmhg 1-9 1-8 1-7 1-6 1-5 1-4 Log dose 5 PE Soleus Feed Artery @33 mmhg 1-9 1-8 1-7 1-6 1-5 1-4 5 ACh @33 mmhg 1-9 1-8 1-7 1-6 1-5 1-4 5 SNP @88 mmhg 1-9 1-8 1-7 1-6 1-5 1-4 Log dose Figure 8. Vasoresponsiveness of the saphenous artery (n = 9), and gastrocnemius (n = 5) and soleus (n = 9) feed arteries in the absence and presence of femoral artery occlusion for 3 weeks When the data points overlap, a single curve has been drawn for simplicity. Note the low intraluminal pressures, corresponding to those observed in vivo (cf. Table 2), used for phenylephrine and acetylcholine, but the higher pressure for sodium nitroprusside to evaluate the capacity for endothelial-independent dilatation.

J Physiol 586.24 Sympathetic control of collateral vasculature 5995 structure, as well as modifications in the function of the vessels, can be expected as the collateral circuit develops over time. It is likely, however, that in the absence of sustained arteriogenic stimuli, further expansion of the collateral vessels will not occur (Tronc et al. 1996). Structural enlargement of the collateral circuit, apparent by larger and more numerous vessels, is most significant when the circuit experiences a heightened stimulus for development, as can be found when flow demands through the circuit are enhanced by daily activity (Prior et al. 24). This was evident in this study where the vast majority ( 85%) of the increase in BF to the active calf muscles of the Trained animals is attributed to an increase in the vascular capacity of the collateral circuit. Even though sympathetic influence remains, the conductance of the collateral circuit is significantly greater in the Trained animals in the absence (P <.2) or in the presence of combined sympathetic receptor inhibition (P <.1), as compared to the Sed group. We interpret this to indicate that structural enlargement of the collateral circuit plays a critical role in the improved BF to the distal muscle of Trained animals. The retained influence of sympathetic innervation, however, is not surprising, since the exercise intensity during BF measurement was higher (3 m min 1 ) than that used with the Sed group ( m min 1 ). In order to measure peak collateral-dependent BF, we needed to challenge the greater exercise capacity of the trained animals. Recall that downstream resistance should be minimized, in this case through exercise, in order for the upstream series resistance of the collateral circuit to be rate-limiting. We have previously shown that exercise training, as used in this study, significantly increases the endurance capacity of animals with peripheral arterial insufficiency (Yang et al. 1991, 1995a,b). This reveals an important advantage in the function of the collateral circuit, especially at submaximal exercise conditions. It is likely that the sympathetic influence to restrain flow through the collateral circuit would be less in Trained, as compared to Sed animals, since central sympathetic drive is related to the relative intensity of exercise (Seals & Victor, 1991). Thus, as demonstrated, the Trained animals would be expected to exhibit a far greater exercise tolerance during a submaximal exercise intensity that is more easily accommodated by the conditioned animals. Further, this training adaptation should be useful during relatively modest intensity activities which demand far less of the animal s capacity. Sympathetic control of the vasculature in active ischaemic muscle A surprising finding from our previous work was the less than maximal conductances observed in the active muscles that were made ischaemic following occlusion of the femoral artery. Even though absolute BFs in collateral-dependent tissues were only approximately 1% of the normal BFs measured in the contralateral limb, vascular conductances of the calf muscle were considerably less than half those observed in the contralateral calf muscle (Taylor et al. 28). Even α-adrenergic receptor inhibition did not return the vascular conductance back to normal. The same phenomenon was observed in the present study where the conductance deficit remained following both α and NPY Y1 receptor inhibition. Interestingly, the deficits were apparent in all the muscle fibre sections, even though they possess very different vascular capacities. This finding was most profound in the Acute group, with increases in vascular conductances of the individual muscle fibre sections markedly improved with time, post-occlusion, but not further altered by training (cf. Figs 5 and 6). The 7 % loss of perfusion pressure to the calf muscles that occurs with occlusion of the femoral artery (cf. Table 2), probably contributed to this phenomenon. Immediately following occlusion of the femoral artery, there is a marked reduction in the distending pressure within the primary vessels distal to the collateral circuit (cf. Fig. 7). The smaller vessel calibres that are possible at 35 4 mmhg, even with minimal smooth muscle tone, could add an appreciable vascular resistance distal to the collateral circuit, but prior to the active muscle. It is possible that this added resistance could have reduced the vascular conductances by 5 6%, amounts that are within the range to meaningfully contribute to the findings of the Acute group. It is unknown whether the smaller arteriolar vasculature within the active muscle also contributed to this added resistance; however, these smaller arterioles typically operate at lower luminal pressures (Williams & Segal, 1992) that may not be as markedly reduced following upstream occlusion of the femoral artery. As noted, the relatively low vascular conductances within the active muscle of the Acute group did not occur in the Sed group, even though perfusion pressures to the calf muscles were similarly reduced. Insights into this response become apparent from the remodelling that occurs in the conduit vessels (i.e. saphenous, and feed arteries to the gastrocnemius and soleus muscles). All three vessels exhibited an increase in structural diameter, independent of vascular tone (cf. Fig. 7). Similar findings are evident in the vessels obtained from adipose tissue from patients with critical limb ischaemia (Hillier et al. 1999). Applying the intraluminal pressures measured in the distal vasculature of this study (cf. Table 2), it is apparent that this vessel enlargement could return vessel diameter to that possible at the higher, more normal intraluminal pressure that is present in the absence of vessel enlargement. Further, this vessel enlargement is expected to recover some loss in radial wall tension caused at the low pressure, and stretch smooth muscle