Impaired Coronary Endothelial Vasorelaxation in a Preclinical Model of Peripheral Arterial Insufficiency.

The present study was designed to determine whether adult swine with peripheral artery insufficiency (PAI) would exhibit vascular dysfunction in vessels distinct from the affected distal limbs, the coronary conduit arteries. Moreover, we sought to evaluate the effect of exercise training on coronary vasomotor function in PAI. Eighteen female healthy young Yucatan miniature swine were randomly assigned to either occluded exercise trained (Occl-Ex, n=7), or occluded-sedentary (Occl-Sed, n=5), or non-occluded, non-exercised control (Non-Occl-Con, n=6) groups. Occl-Ex pigs were progressively trained by running on a treadmill (5days/week, 12 weeks). The left descending artery (LAD) and left circumflex (LCX) coronary arteries were harvested. Vasorelaxation to adenosine diphosphate (ADP), bradykinin (BK), and sodium nitro-prusside (SNP) were assessed in LAD's; while constrictor responses to phenylephrine (PE), angiotensin II (Ang II), and endothelin-1 (ET-1) were assessed in LCX's. Vasorelaxation to ADP was reduced in LADs from Occl-Sed and Occl-Ex pigs (P<0.001) as compared to Non-Occl-Con pigs; however, Occl-Ex pigs exhibited partial recovery (P<0.001) intermediate to the other two groups. BK induced relaxation was reduced in LADs from Occl-Ex and Occl-Sed pigs (P<0.001), compared to Non-Occl-Con, and exercise modestly increased responses to BK (P<0.05). In addition, SNP, PE, Ang II, and ET-1 responses were not significantly different among the groups. Our results indicate that 'simple' occlusion of the femoral arteries induces vascular dysfunction in conduit vessels distinct from the affected hindlimbs, as evident in blunted coronary vasorelaxation responses to ADP and BK. These findings imply that PAI, even in the absence of frank atherogenic vascular disease, contributes to vascular dysfunction in the coronary arteries that could exacerbate disease outcome in patients with peripheral artery disease. Further, regular daily physical activity partially recovered the deficit observed in the coronary arteries.

Significant, but limited collateral blood flow increases occur with prolonged training in rats with femoral artery occlusion.

This study examined the capacity of collateral dependent blood flow induced by a prolonged treadmill training program, as compared to a low collateral resistance model created by femoral artery to vein (A-V) shunt. Sprague-Dawley rats, with bilateral femoral artery occlusion were confined to cage activity (Sed, n=9) or trained by daily treadmill exercise (Tr, n=15; up to ≈350 min/d) for 15 weeks. Another set of animals received a femoral A-V anastomosis in one limb and treated with (n=4) or without VEGF(165) (n=9) infusion for 2 weeks. The contralateral side was used as control. Blood flow (BF) was measured with isotope labeled microspheres. Maximal calf muscle BF increased by 15 week training (up to 100±5.0 ml x min(-1) x 100g(-1) (p<0.05); 0.71±0.04 ml x min(-1) x 100g(-1) x mmHg(-1)), a response better (20-25%) than the less demanding training programs used previously. In contrast, femoral A-V shunt with VEGF(165) increased calf muscle conductance to 1.70±0.3 ml x min(-1) x 100 g(-1) x mmHg(-1) that is similar to blood flows observed in non-occluded rats during maximal running. Our data indicate that the collateral circuit development is related to the driving stimulus and that exercise training, does not provide a maximal stimulus for adaptation that is possible. Nonetheless, exercise training results in profound increases in exercise capacity associated with this enhanced collateral blood flow. Our results illustrate that vascular adaptations can be much greater when physiologically induced stimuli are enhanced at the time of therapeutic angiogenesis.

Training-induced vascular adaptations to ischemic muscle.

Peripheral arterial insufficiency is a progressive degenerative disease associated with an increased morbidity and mortality. It decreases exercise tolerance and often presents with symptoms of intermittent claudication. Enhanced physical activity is one of the most effective means of improving the life of affected patients. While this occurs for a variety of reasons, vascular remodeling can be an important means for improved oxygen exchange and blood flow delivery. Relevant exercise-induced signals stimulate angiogenesis, within the active muscle (e.g. hypoxia), and arteriogenesis (enlargement of pre-existing vessels via increased shear stress) to increase oxygen exchange and blood flow capacity, respectively. Evidence from pre-clinical studies shows that the increase in collateral blood flow observed with exercise progresses over time of training, is accompanied by significant enlargement of isolated collateral vessels, and enhances the responses observed with angiogenic growth factors (e.g. VEGF, FGF-2). Thus, enhanced physical activity can be an effective means of enlarging the structure and function of the collateral circuit. Interestingly, disrupting normal NO production (via L-NAME) eliminates this increase in collateral blood flow induced by training, but does not disturb the increase in muscle capillarity within the active muscle. Similarly, inhibiting VEGF receptor kinase activity eliminates the increase in collateral-dependent blood flow, and lessens, but does not eliminate, angiogenesis within the calf muscle. These findings illustrate distinctions between the processes influencing angiogenesis and arteriogenesis. Further, sympathetic modulation of the collateral circuit does not eliminate the increase in collateral circuit conductance induced by exercise training. These findings indicate that structural enlargement of the collateral vessels is essential to realize the increase in collateral-dependent blood flow capacity caused by exercise training. This raises the potential that meaningful vascular remodeling can occur in patients with intermittent claudication who actively participate in exercise training.

Protecting the cellular energy state during contractions: role of AMP deaminase.

AMP deaminase activity (AMP->IMP+NH3) is the entry reaction to the purine nucleotide cycle. In skeletal muscle, excessive energy demands during contractions leads to a net production of ADP, because ATP hydrolysis exceeds ADP rephosphorylation. Elevations in ADP increase AMP, via the myokinase reaction. This accumulation of ATP hydrolysis products should lead to a catastrophic reduction in the energy state of the myocyte. The removal of AMP to IMP in times of excessively high energy demands have been hypothesized as essential to protect the energy state of the cell. While AMP deamination leads to a net loss of adenine nucleotides (principally, as ATP), the viability of the myocyte is preserved. Following these demanding contraction conditions, the concentration of IMP of fast-twitch muscle is rapidly reduced, typically with the return of the muscle adenine nucleotide content (ATP + ADP + AMP) to pre-contraction levels. While these observations are generally observed for fast-twitch skeletal muscle and consistent with the hypothesis, there has been no direct experimental evaluation. In the AK1 (-/-) mouse, there is a markedly reduced accumulation of AMP, during conditions of excessive contractile activity. Rather, there is a high ADP concentration, approaching 1.5 mM, that remains unbound 'free' within the muscle. This contributes to an inordinate reduction in the ATP/ADP ratio. At the same time, PCr hydrolysis is nearly complete leading to a large increase in orthophosphate. In combination, this leads to an exceptional decline in the free energy of ATP hydrolysis. This is projected to impair Ca(2+) handling by the sarcoplasmic reticulum and slow cross-bridge cycling rate. The outcome should be slowed contraction characteristics and possible contracture. While some contractile changes were observed, there was a remarkable ability of the muscle to function under these challenging energetic conditions. Thus, it is not essential that the AMP deaminase reaction be operating during intense contraction conditions. This helps explain why patients deficient in AMP deaminase do not always exhibit an impaired muscle function.

Arteriogenesis: role of nitric oxide.

Arteriogenesis is an important process for adapting the pre-existing circuit of vessels into functional collateral conduits for delivery of oxygen enriched blood to tissue distal to occlusion of a large, peripheral conduit artery. Recent evidence has shown that arteriogenesis is regulated by nitric oxide (NO), angiogenic factors and shear stress. NO significantly impacts vasomotor tone to enhance conductance of the newly recruited collateral arteries, and this effect is augmented by exercise training prior to arterial occlusion. NO-mediated increases in vascular conductance allows for greater collateral dependent blood flow to the tissue distal to occlusion. NO production is also critical to the efficacy of therapeutic arteriogenesis achieved by delivery of exogenous angiogenic growth factors (VEGF, FGF-2) or by exercise training. The critical role of NO in therapeutic arteriogenesis is independent of NO-mediated changes in vascular conductance and implies a central role in arteriogenic signaling events. Maintenance, or improvement, of NO production and signaling, such as with regular exercise, may improve endothelial cell function and thus may help preserve the arteriogenic potential of preexisting collateral networks.

Arteriogenesis and angiogenesis in rat ischemic hindlimb: role of nitric oxide.

Nitric oxide (NO) has been implicated in both collateral expansion (arteriogenesis) and capillary growth (angiogenesis). Exercise training increases collateral-dependent blood flow to tissues at risk of ischemia and enhances capillarity in active skeletal muscle. Exercise also acutely elevates NO. Thus we assessed the role of NO in training-induced arteriogenesis and angiogenesis. These studies utilized a rat model of peripheral vascular disease (bilateral femoral artery ligation). Untreated rats (control) and rats treated with the NO synthase inhibitor N(omega)-nitro-L-arginine methyl ester (L-NAME; 65-70 mg x kg(-1) x day(-1), via drinking water) were divided into sedentary or exercise-trained subgroups. After approximately 3 wk, L-NAME treatment had elevated preexercise mean arterial pressure approximately 39-58%, confirming NO synthesis inhibition. The training program (treadmill exercise twice per day, 20-25 m/min, 15% grade, approximately 18 days) increased collateral-dependent blood flow to the distal hindlimb, with the greatest increase (approximately 59%) in the calf (P < 0.001). This increase was inhibited by L-NAME. In contrast, the training-induced increase in muscle capillarity was not blocked by L-NAME. Thus arteriogenesis and angiogenesis appear to differ in their requirement for NO.

Influence of ribose on adenine salvage after intense muscle contractions.

The influence of ribose supplementation on skeletal muscle adenine salvage rates during recovery from intense contractions and subsequent muscle performance was evaluated using an adult rat perfused hindquarter preparation. Three minutes of tetanic contractions (60 tetani/min) decreased ATP content in the calf muscles by approximately 50% and produced an equimolar increase in IMP. Effective recovery of muscle ATP 1 h after contractions was due to reamination of IMP via the purine nucleotide cycle and was complete in the red gastrocnemius but incomplete in the white gastrocnemius muscle section. Adenine salvage rates in recovering muscle averaged 45 +/- 4, 49 +/- 5, and 30 +/- 3 nmol. h(-1). g(-1) for plantaris, red gastrocnemius, and white gastrocnemius muscle, respectively, which were not different from values in corresponding nonstimulated muscle sections. Adenine salvage rates increased five- to sevenfold by perfusion with approximately 4 mM ribose (212 +/- 17, 192 +/- 9, and 215 +/- 14 nmol. h(-1). g(-1) in resting muscle sections, respectively). These high rates were sustained in recovering muscle, except for a small (approximately 20%) but significant (P < 0.001) decrease in the white gastrocnemius muscle. Ribose supplementation did not affect subsequent muscle force production after 60 min of recovery. These data indicate that adenine salvage rates were essentially unaltered during recovery from intense contractions.

Purine salvage to adenine nucleotides in different skeletal muscle fiber types.

Rates of purine salvage of adenine and hypoxanthine into the adenine nucleotide (AdN) pool of the different skeletal muscle phenotype sections of the rat were measured using an isolated perfused hindlimb preparation. Tissue adenine and hypoxanthine concentrations and specific activities were controlled over a broad range of purine concentrations, ranging from 3 to 100 times normal, by employing an isolated rat hindlimb preparation perfused at a high flow rate. Incorporation of [(3)H]adenine or [(3)H]hypoxanthine into the AdN pool was not meaningfully influenced by tissue purine concentration over the range evaluated (approximately 0.10-1.6 micromol/g). Purine salvage rates were greater (P < 0.05) for adenine than for hypoxanthine (35-55 and 20-30 nmol x h(-1) x g(-1), respectively) and moderately different (P < 0.05) among fiber types. The low-oxidative fast-twitch white muscle section exhibited relatively low rates of purine salvage that were approximately 65% of rates in the high-oxidative fast-twitch red section of the gastrocnemius. The soleus muscle, characterized by slow-twitch red fibers, exhibited a high rate of adenine salvage but a low rate of hypoxanthine salvage. Addition of ribose to the perfusion medium increased salvage of adenine (up to 3- to 6-fold, P < 0.001) and hypoxanthine (up to 6- to 8-fold, P < 0.001), depending on fiber type, over a range of concentrations up to 10 mM. This is consistent with tissue 5-phosphoribosyl-1-pyrophosphate being rate limiting for purine salvage. Purine salvage is favored over de novo synthesis, inasmuch as delivery of adenine to the muscle decreased (P < 0.005) de novo synthesis of AdN. Providing ribose did not alter this preference of purine salvage pathway over de novo synthesis of AdN. In the absence of ribose supplementation, purine salvage rates are relatively low, especially compared with the AdN pool size in skeletal muscle.

VEGF(121)- and bFGF-induced increase in collateral blood flow requires normal nitric oxide production.

The angiogenic proteins basic fibroblast growth factor (bFGF; FGF-2) and vascular endothelial growth factor 121 (VEGF(121)) are each able to enhance the collateral-dependent blood flow after bilateral femoral artery ligation in rats. To study the effect of nitric oxide (NO) synthase (NOS) inhibition on bFGF- or VEGF(121)-induced blood flow expansion, the femoral arteries of male Sprague-Dawley rats were ligated bilaterally, and the animals were given tap water [non-N(G)-nitro-L-arginine methyl ester (L-NAME) group; n = 36] or water that contained L-NAME (L-NAME group; 2 mg/ml, n = 36). Animals from each group were further divided into three subgroups: vehicle (n = 12), bFGF (5 microg x kg(-1) x day(-1), n = 12), or VEGF(121) (10 microg x kg(-1) x day(-1), n = 12). Growth factors were delivered via intra-arterial infusion with osmotic pumps over days 1-14. On day 16, after a 2-day delay to permit clearance of bFGF and VEGF from the circulation, maximal collateral blood flow was determined by (85)Sr- and (141)Ce-labeled microspheres during treadmill running. L-NAME (approximately 137 mg x kg(-1) x day(-1)) for 18 days increased systemic blood pressure (approximately 26%, P<0.001). In the absence of L-NAME, collateral-dependent blood flows to the calf muscles were greater in the VEGF(121)- and bFGF-treated subgroups (85 +/- 4.5 and 80 +/- 2.9 ml x min(-1) x 100 g(-1), respectively) than in the vehicle subgroup (49 +/- 3.0 ml x min(-1) x 100 g(-1), P<0.001). In the presence of NOS inhibition by L-NAME, blood flows to the calf muscles were essentially equivalent among the three subgroups (54 +/- 3.0, 56 +/- 5.1, and 47 +/- 2.0 ml x min(-1) x 100 g(-1) in the bFGF-, VEGF(121)-, and vehicle-treated subgroups, respectively) and were not different from the blood flow in the non-L-NAME vehicle subgroup. Our results therefore indicate that normal NO production is essential for the enhanced vascular remodeling induced by exogenous bFGF or VEGF(121) in this rat model of experimental peripheral arterial insufficiency. These results imply that a blunted endothelial NO production could temper vascular remodeling in response to these angiogenic growth factors.

Prior exercise training increases collateral-dependent blood flow in rats after acute femoral artery occlusion.

We evaluated whether prior training would improve collateral blood flow (BF) to the calf muscles after acute-onset occlusion of the femoral artery. Exercise training was performed in the absence of any vascular occlusion. Adult male Sprague-Dawley rats ( approximately 325 g) were kept sedentary (n = 14), limited to cage activity, or exercise trained (n = 14) for 6 wk by treadmill running. Early in the day of measurement, animals were surgically prepared for BF determination, and the femoral arteries were occluded bilaterally. Four to five hours later, collateral BF was determined twice during treadmill running with the use of (141)Ce and (85)Sr microspheres: first, at a demanding speed and, second, after a brief rest and at a higher speed. The absence of any further increase in BF at the higher speed indicated that maximal collateral BF was measured. Prior training increased calf muscle BF by approximately 70% compared with sedentary animals; however, absolute BF remained below values previously observed in animals with a well-developed collateral vascular tree. Thus prior training appeared to optimize the use of the existing collateral circuit. This implies that altered vasoresponsiveness induced in normal nonoccluded vessels with exercise training serves to improve collateral BF to the periphery.

Efficacy and specificity of bFGF increased collateral flow in experimental peripheral arterial insufficiency.

Angiogenic growth factors could prove to be useful in managing peripheral arterial insufficiency. The present study was designed to evaluate the dose response of basic fibroblast growth factor (bFGF), the efficacy of critical routes and dosing regimens, and the specificity of action in rats with peripheral arterial insufficiency. Bilateral ligation of femoral arteries greatly reduces blood flow capacity to the calf muscles but does not impair resting flow needs. Collateral blood flow to calf muscles was determined 16 days postocclusion, during treadmill running, with (85)Sr and (141)Ce microspheres, in blinded-randomized trials that included intra-arterial and intravenous infusions and subcutaneous injections of recombinant human bFGF. Peak blood flow of 75-80 ml. min(-1). 100 g(-1) for calf muscle was observed at a bFGF dose of 5 microg. kg(-1). day(-1) (ia for 14 days) compared with 50 ml. min(-1). 100 g(-1) for vehicle groups. Similar increases in collateral blood flow were observed with short-term or prolonged and continuous or intermittent delivery of bFGF by any route. Collateral blood flows were similar in corresponding muscles across both limbs. Vascular remodeling induced by bFGF required attendant vascular occlusion, inasmuch as vessels in the normal nonoccluded vascular tree were unresponsive to circulating bFGF. Improvement in collateral blood flow with exogenous bFGF is robust, amenable to short-term administration, and requires vascular occlusion to be effective.

American College of Sports Medicine roundtable. The physiological and health effects of oral creatine supplementation.

Creatine (Cr) supplementation has become a common practice among professional, elite, collegiate, amateur, and recreational athletes with the expectation of enhancing exercise performance. Research indicates that Cr supplementation can increase muscle phosphocreatine (PCr) content, but not in all individuals. A high dose of 20 g x d(-1) that is common to many research studies is not necessary, as 3 g x d(-1) will achieve the same increase in PCr given time. Coincident ingestion of carbohydrate with Cr may increase muscle uptake; however, the procedure requires a large amount of carbohydrate. Exercise performance involving short periods of extremely powerful activity can be enhanced, especially during repeated bouts of activity. This is in keeping with the theoretical importance of an elevated PCr content in skeletal muscle. Cr supplementation does not increase maximal isometric strength, the rate of maximal force production, nor aerobic exercise performance. Most of the evidence has been obtained from healthy young adult male subjects with mixed athletic ability and training status. Less research information is available related to the alterations due to age and gender. Cr supplementation leads to weight gain within the first few days, likely due to water retention related to Cr uptake in the muscle. Cr supplementation is associated with an enhanced accrual of strength in strength-training programs, a response not independent from the initial weight gain, but may be related to a greater volume and intensity of training that can be achieved. There is no definitive evidence that Cr supplementation causes gastrointestinal, renal, and/or muscle cramping complications. The potential acute effects of high-dose Cr supplementation on body fluid balance has not been fully investigated, and ingestion of Cr before or during exercise is not recommended. There is evidence that medical use of Cr supplementation is warranted in certain patients (e.g.. neuromuscular disease); future research may establish its potential usefulness in other medical applications. Although Cr supplementation exhibits small but significant physiological and performance changes, the increases in performance are realized during very specific exercise conditions. This suggests that the apparent high expectations for performance enhancement, evident by the extensive use of Cr supplementation, are inordinate.

IMP degradative capacity in rat skeletal muscle fiber types.

During contractions, when the rate of ATP hydrolysis exceeds that of ADP phosphorylation, inosine 5'-monophosphate (IMP) accumulates in skeletal muscle. If the cellular energy balance is not promptly restored, subsequent purine degradation to inosine via 5'-nucleotidase can occur, a process that is most robust in the slow-twitch red, as compared to fast-twitch, skeletal muscle. We measured the distribution of 5'-nucleotidase activity among membrane-bound and soluble fractions of fiber specific skeletal muscle sections and found most (80-90%) of the total 5'-nucleotidase activity to be membrane-bound. The 5' IMP nucleotidase activity present in the soluble fraction of muscle extracts differs among fiber types with slow-twitch red > fast-twitch red > mixed fibered > fast-twitch white. Experiments testing the substrate dependence of IMP and AMP dephosphorylation by the soluble fraction of muscle extracts revealed a lower Km toward IMP (approximately 0.7-1.5 mM) than AMP (1.9-2.8 mM). Among skeletal muscle fiber sections, the soluble 5'-nucleotidase activity present in slow-twitch red muscle extracts had the highest substrate affinity, the highest activity with IMP as substrate, and an estimated catalytic efficiency (Vmax/Km) that was > 3-fold higher than calculated for fast-twitch muscle extracts. This is likely due to the Mg2+ dependent cytosolic 5' IMP nucleotidase isoform, since immunoprecipitation experiments revealed 3-4 times more activity in slow-twitch red than in fast-twitch red or fast-twitch white fibers, respectively. These finding are consistent with the previously recognized in vivo pattern of nucleoside formation by muscle where the soleus demonstrated extensive inosine formation at a much lower IMP content than fast-twitch red or fast-twitch white muscle fiber sections.

Alterations in AMP deaminase activity and kinetics in skeletal muscle of creatine kinase-deficient mice.

Alterations in the competency of the creatine kinase system elicit numerous structural and metabolic compensations, including changes in purine nucleotide metabolism. We evaluated molecular and kinetic changes in AMP deaminase from skeletal muscles of mice deficient in either cytosolic creatine kinase alone (M-CK-/-) or also deficient in mitochondrial creatine kinase (CK-/-) compared with wild type. We found that predominantly fast-twitch muscle, but not slow-twitch muscle, from both M-CK-/- and CK-/- mice had much lower AMP deaminase; the quantity of AMP deaminase detected by Western blot was correspondingly lower, whereas AMP deaminase-1 (AMPD1) gene expression was unchanged. Kinetic analysis of AMP deaminase from mixed muscle revealed negative cooperativity that was significantly greater in creatine kinase deficiencies. Treatment of AMP deaminase with acid phosphatase abolished negative cooperative behavior, indicating that a phosphorylation-dephosphorylation cycle may be important in the regulation of AMP deaminase.

Exercise training enhances basic fibroblast growth factor-induced collateral blood flow.

This study evaluated whether daily exercise would enhance the peripheral collateral vessel development found in response to exogenous basic fibroblast growth factor (bFGF) infusion. After bilateral femoral occlusion, male Sprague-Dawley rats (approximately 325 g) received intra-arterial infusions of either bFGF (1 microg/day; n = 15) or carrier solution (n = 13) via osmotic pumps for 2 wk. Subgroups of each treatment were kept sedentary (cage activity) or trained by walking at 20 m/min at 15% grade, two times a day, 5 days/wk for 4 wk. Training markedly increased citrate synthase activity in the active muscle (P < 0.001). Muscle function and blood flows (85Sr microsphere) were evaluated using an isolated hindquarter perfused at 100 mmHg via the abdominal aorta. The significant increase in blood flow to the entire hindlimb in the sedentary animals, caused by bFGF infusion (P < 0.05), was further increased (P < 0.01) in the bFGF-trained group. The quantitatively largest increases in blood flows were observed in the collateral-dependent tissues of the distal hindlimb. Blood flows to the entire calf muscle group increased approximately 140% in carrier-trained (P < 0.001), approximately 180% in bFGF sedentary (P < 0.001), and approximately 240% in the bFGF-trained (P < 0.001) groups compared with the carrier sedentary group. The increases in collateral blood flow were functionally important, as improvements in calf muscle performance correlated with measured blood flows. Our results demonstrate that exogenous bFGF administration in combination with a moderate-intensity exercise program greatly increases collateral-dependent blood flow and improves muscle performance. That physical activity enriched the bFGF response is consistent with the hypothesis that hemodynamic factors are important contributors to collateral vessel enlargement.

Molecular and kinetic alterations of muscle AMP deaminase during chronic creatine depletion.

We examined a possible mechanism to account for the maintenance of peak AMP deamination rate in fast-twitch muscle of rats fed the creatine analog beta-guanidinopropionic acid (beta-GPA), in spite of reduced abundance of the enzyme AMP deaminase (AMPD). AMPD enzymatic capacity (determined at saturating AMP concentration) and AMPD protein abundance (Western blot) were coordinately reduced approximately 80% in fast-twitch white gastrocnemius muscle by beta-GPA feeding over 7 wk. Kinetic analysis of AMPD in the soluble cell fraction demonstrated a single Michaelis-Menten constant (Km; approximately 1.5 mM) in control muscle extracts. An additional high-affinity Km (approximately 0.03 mM) was revealed at low AMP concentrations in extracts of beta-GPA-treated muscle. The kinetic alteration in AMPD reflects increased molecular activity at low AMP concentrations; this could account for high rates of deamination in beta-GPA-treated muscle in situ, despite the loss of AMPD enzyme protein. The elimination of this kinetic effect by treatment of beta-GPA-treated muscle extracts with acid phosphatase in vitro suggests that phosphorylation is involved in the kinetic control of skeletal muscle AMPD in vivo.

Triacylglycerol synthesis in the different skeletal muscle fiber sections of the rat.

Triacylglycerol (TG) synthesis rates of low-oxidative fast-twitch white and high-oxidative fast- and slow-twitch red skeletal muscle fiber sections of adult rats were measured by the incorporation of perfusate-derived palmitate into the neutral lipid fraction by use of a perfused hindquarter preparation under high-flow conditions. The perfusion medium consisted of 95% O2-5% CO2 Krebs-Henseleit buffer, pH 7.4, containing 5 g/100 ml bovine serum albumin, 100 microU/ml insulin, 5 mmol/l glucose, amino acids, and added fatty acids (FA), including 0.1 microCi/ml [3H]palmitate. FA incorporation was linear with time. TG synthesis rates correlated (r > or = 0.90) with the oxidative capacity of each of the different fiber type sections and increased in proportion to the perfusate FA concentration (0.25, 0.5, 1.0, 1.5, and 2.0 mmol/l). TG synthesis rates among different muscle fiber sections were related (r > or = 0.90) to perfusate flow during high-flow conditions; however, this was not causal, because TG synthesis rates within each muscle fiber section were independent of flow rate when experimentally varied over an approximately threefold range. Thus the relatively high TG synthesis rates observed in the high-oxidative muscle sections are not uniquely related to their high-flow capacities but are inherent to the TG synthesis process, probably events associated with FA uptake and/or capacity of the TG synthesis pathway.

Basic fibroblast growth factor increases collateral blood flow in rats with femoral arterial ligation.

The potential for exogenous infusion of basic fibroblast growth factor (bFGF) to increase collateral blood flow to dependent tissue was quantified in adult male rats with peripheral arterial insufficiency. Occlusion of the femoral artery at a proximal site did not infringe on resting blood flow to the distal hindlimb muscle, but did remove the blood flow reserve. Blood flow to the hindlimb muscles was measured with radiolabeled microspheres using an isolated hindlimb preparation perfused in the descending aorta (Krebs-Henseleit bicarbonate, 5% albumin medium containing red blood cells [40% hematocrit]) at 100 mm Hg. Calf muscle blood flow changed modestly (approximately equal to 50%) with infusion of only the carrier (heparin/saline), increased markedly over the first 2 weeks of bFGF infusion (1 microgram/d into the femoral artery), but did not change further with infusion for 4 weeks. Waiting 2 weeks after 1 week of bFGF infusion did not further increase the intermediate improvement in blood flow. The improved collateral blood flow and increased muscle capillary density likely contributed to the enhanced muscle performance observed during nerve stimulation in situ. X-ray films of arterial casts identified an expansion of upper thigh vessels that likely served as collaterals. In animals with peripheral arterial insufficiency, short-term exogenous infusion of bFGF is effective at inducing vascular expansion that is sufficient to improve the flow reserve of dependent distal tissue and enhance muscle function. This raises the expectation that a similar response in patients with peripheral arterial insufficiency would significantly improve morbidity, including the symptoms of intermittent claudication.

IMP reamination to AMP in rat skeletal muscle fiber types.

Inosine 5'-monophosphate (IMP) reamination in skeletal muscle fiber sections of the rat hindlimb was studied. High IMP concentrations were established during ischemic contractions in each fiber section: 3.1, 2.8, or 0.6 mumol/g in the fast-twitch white (FTW), fast-twitch red (FTR), and slow-twitch red (STR) muscle sections, respectively. Thereafter blood flow was restored and stimulation was discontinued to allow reamination of IMP. After 0, 2, 5, 10, 15, or 20 min of recovery, muscle sections were freeze-clamped and analyzed for metabolite contents. IMP was nearly fully reaminated after 10 and 20 min of recovery in STR and FTR muscles, respectively. Reamination in TW fibers was delayed and slower, with only 50% of the IMP reaminated after 20 min of recovery. Significant recovery (approximately 75%) of phosphocreatine occurs in each fiber section before the onset of reamination. Reamination was also evaluated after high-speed treadmill running with or without inhibition of reamination by hadacidin. Running resulted in large accumulations of IMP in FTW and FTR fibers (3.5 and 1.4 mumul/g, respectively); IMP in FTR fibers was higher with hadacidin treatment. Reamination after running was much greater in FTR than in FTW fibers and was associated with recovery of phosphocreatine. After running, the purine degradation products inosine and hypoxanthine were increased in FTW and FTR fibers in normal and hadacidin-treated animals. Plasma inosine, hypoxanthine, and urate increased after exercise; concentrations continued to increase if reamination was inhibited by hadacidin. These results demonstrate that when muscle IMP is increased, subsequent degradation and loss of purines occur. Rapid reamination should minimize the quantity of purine lost from muscle and limit the metabolic cost of replenishing purines by the de novo synthesis or salvage pathways.

Creatine analogue beta-guanidinopropionic acid alters skeletal muscle AMP deaminase activity.

Dietary supplementation of the creatine analogue beta-guanidinopropionic acid (beta-GPA) decreases in vitro skeletal muscle AMP deaminase (AMP-D) activity in rats. Downregulation of AMP-D activity was progressive and greater in fast-twitch muscles (70-80%) than in the slow-twitch soleus muscle (approximately 50%). The loss in AMP-D activity had little effect on inosine 5'-monophosphate accumulation in mixed-fiber muscle with intense tetanic contractions. In contrast, inosine 5'-monophosphate formation was evident earlier in fast-twitch red and white fiber sections of creatine-depleted animals during intense twitch contractions, indicating that fast-twitch muscle of beta-GPA-treated rats buffers decreases in the ATP/ADPfree ratio via deamination, even though AMP-D activity is less. Isoforms of skeletal muscle AMP-D mRNAs in mixed-fiber muscle were not altered by feeding beta-GPA for up to 9 wk. Creatine depletion did not alter total immunoreactivity; however, a redistribution of AMP-D immunoreactivity from primarily an approximately 80-kDa form toward lower apparent molecular mass species (approximately 60 and approximately 56 kDa) was observed. Posttranslational changes in AMP-D appear related to changes in activity.