INTRODUCTION

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THE MECHANICAL PERFORMANCE of skeletal muscle during repetitive contractions can be indicative of the match between the metabolic demands of the muscle fibers and the supply of substrate (2, 3) and/or metabolite washout (7). The active hyperemic response of contracting muscle is one mechanism that enables high levels of mechanical output over prolonged periods of time, through provision of this supply and washout, during repetitive muscle activation such as exercise. One agent that may play a role in the hyperemic process is the endothelium-derived relaxing factor, presently identified as nitric oxide (NO) (29). NO has been shown to be synthesized in the endothelial cells lining the vasculature, through the activity of a constitutive nitric oxide synthase (NOS) enzyme that acts to produce NO from the precursor L-arginine (28). Once produced, NO acts directly on the vascular smooth muscle to cause relaxation, and there is an attendant drop in resistance as the vessel luminal diameter increases. With respect to skeletal muscle vasculature, it has been shown that the effects of the NO pathway are significant in muscles with a high percentage of oxidative fibers (17), specifically those characterized as slow-oxidative and fast-oxidative glycolytic.

The stimuli that may initiate this response within the muscle vasculature include elevated intravascular shear forces produced by increased flow of blood within the vessels (29) and extravascular shear forces produced by repeated compression of the vessels within the contracting muscle (21), perhaps as a product of intramuscular pressure (P_IM) development (4). Evidence for this latter possibility has been demonstrated in rhythmically squeezed rabbit arterial segments that produced elevated cGMP levels in platelets circulating through the segments as an index of NO production (21). Furthermore, a recent study in the canine gastrocnemius-plantaris (GP) muscle has shown production of high intramuscular stress (1,600 mmHg) during brief tetanic muscle contractions in situ (4), suggesting the propensity for development of large extravascular shear forces acting on the endothelium of the muscle vasculature in this muscle group.

However, studies in several different species and models have produced varied results and a lack of consensus on whether NO plays a significant role in the active hyperemic response (8, 11, 12, 14, 17-19, 21, 27, 30, 34). We believe that three major factors have been responsible for this discrepancy. One is that twitch or low-demand contractions have been utilized in many studies (8, 12, 19, 34), which may not adequately test the capacity of the response (2, 14). Another has been pretreatment with a systemic bolus of competitive NOS inhibitors (19, 30), which may complicate interpretation of results in the contracting muscle vascular bed because of unquantifiable metabolism and sequestration in nonmuscle tissue beds throughout the body. Finally, in some studies, the administered NOS inhibitor effective dose obtained in the resting state was not increased proportionately to account for the increased blood flow during muscle contractions (12, 34). Consideration of these factors suggests that the minimal role reported for NO requires further examination, based on the facts that shear stress is a known strong potentiator of NO production by endothelial cells (29) and deendothelialization of the GP muscle vessels results in significantly attenuated hyperemic responses (27). Furthermore, the fiber type composition of the canine GP muscle is highly oxidative (45% fast-twitch fatigue resistant, 55% slow-twitch fatigue resistant; Ref. 22), which might predispose it toward significant NO-dependent responses (17).

Thus the purpose of this study was to determine whether NO plays a significant role in control of vascular resistance (R_v) during repetitive tetanic contractions in the canine GP muscle in situ with the use of a previously published contraction paradigm that reliably results in achievement of high P_IM development, blood flow, and oxygen uptake (O₂) (2-4, 10). We hypothesized that inhibition of NO production through local administration of blockers would result in significantly increased R_v, if NO played a significant role in this response under these conditions. Rejection of the hypothesis would indicate that even these high-intensity contractions have no effect on the NO system within the canine GP muscle vasculature, suggesting regulation of the resistance response by other, non-NO-related factors.

	METHODS

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General methods. Mongrel dogs of both sexes (10-15 kg), obtained and housed in accordance with the University of Pittsburgh Institutional Animal Care and Use Committee, were utilized in these studies. Initial anesthesia was induced with pentobarbital sodium (30 mg/kg iv), followed by maintenance doses of 60 mg. The animals were ventilated through an endotracheal tube with a Harvard respirator, and end-tidal CO₂ was maintained at 4.5-5%. Body and muscle temperatures were maintained at 37-39°C. The muscle group studied was the GP with isolated circulation surgically prepared as described previously (2-4, 10). Anticoagulation of the blood was achieved with heparin (2,600 U/kg). The sciatic nerve was dissected and cut, and the distal stump placed in an electrode holder. The calcaneus tendon was freed, cut, and clamped, and the muscle was anchored as described previously (2-4, 10).

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Muscle mechanics. Muscle force development during contractions was monitored with a pneumatic lever specifically designed for the canine GP muscle (13). The force transducer on the lever was calibrated by hanging known weights onto the tendon clamp attachment hook, with the lever perpendicular to the floor. During setup of the lever, precautions were taken to minimize flexing and movement of the apparatus with subsequent tetanic contractions (3, 4). Repetitive isometric tetanic contractions (50 imp/s, 0.2-ms pulse width × 4-V pulse amplitude; 200-ms train duration, 1 contraction/s) were produced at L_o, which was determined as the length at which tension of prior twitch contractions (0.2-ms pulse, 4 V) was maximal (102 ± 9 g/g, n = 8 muscles). The activation stimulus train resulted in peak tetanic active tension (T_act) development of 540 ± 59 g/g (n = 8) and a twitch/tetanic ratio of 0.19 ± 0.01. The tetanic contraction repetition rate was chosen as one that reliably results in high and O₂ in this preparation (2, 3, 10). Passive tension (T_pass) at L_o was 55 ± 10 g/g. During contraction trials, the T_pass baseline was monitored as an index of incomplete relaxation to determine whether manipulations of NO produced measurable alterations under these conditions.

Resting muscle experiments. Just before the tests of resting muscle responses, muscles were stimulated to produce repetitive isometric tetanic contractions (1/s), over a period of 3-5 min, to allow determination of the contraction-induced level of hyperemia and decrement in R_v with no drugs. A systemic indomethacin bolus (0.5 mg/kg iv) was given immediately after the brief contraction trial to block contribution of prostaglandins in the response during the subsequent NO synthesis blockade (18, 23). This dose was chosen based on pilot experiments (n = 2) that indicated that a 5 mg/kg systemic dose resulted in elevation of MAP to a level that was considered nonphysiological (200 mmHg), whereas 0.5 mg/kg produced only mild elevation of MAP to 120-140 mmHg. This treatment may have produced less than a total blockade of prostaglandin synthesis but was considered reasonable with regard to the normal range of systemic MAP.

Contracting muscle protocol and experiments. L-NAME and L-ArgSA dose-response experiments performed during repetitive tetanic contractions followed the same approach outlined in Resting muscle experiments, except that the achieved during the brief preinfusion contraction trial was used as the perfusion pump set point for during the contractions. As above, the syringe pump flow rate was 10% of the roller pump rate, typically 5-6 and 50-60 ml/min, respectively, and drug doses and flows were chosen to achieve concentrations of millimoles of drug per liter of blood within the blood delivered to the muscle. In all experiments, during the preinfusion trial with spontaneous , R_v in the resting muscle and during subsequent contractions averaged ~500 and 100 PRU (P < 0.05), respectively, demonstrating a significant drop in resistance with induction of the active hyperemic response. The high resting R_v level was always reestablished during the rest period between the preinfusion and infusion trials, suggesting a robust autoregulatory response. Beginning at minute 3 of contractions under subsequent pump perfusion control, L-NAME or L-ArgSA was constantly infused from the syringe pumps. This time was chosen because prior tetanic contraction experiments in this preparation have shown that maximal and O₂ occur from 3 to 5 min of repetitive contractions with the use of this stimulus paradigm (2, 3, 10). Respective NOS inhibitor dosages were increased at 3- to 5-min intervals during the remainder of the contraction trials.

	RESULTS

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Cardiovascular characteristics of the dogs were similar to those obtained in prior experiments that used this preparation (systemic arterial pH = 7.38 ± 0.11, arterial PO₂ = 104 ± 16 Torr, arterial PCO₂ = 35 ± 4 Torr) (2, 3). No significant changes were noted in any of these arterial variables throughout the experiments; therefore, all animals were considered to be similar with regard to systemic oxygenation and acid-base status.

Responses in resting muscle. Results from ACh-infusion experiments in resting muscles are shown in Fig. 1. R_v displayed a dose-dependent reduction with increasing ACh levels, demonstrating a significant endothelium-dependent response (18) under these conditions. SNP (0.2-1.0 µg/ml) produced similar results, with decrements ranging from 200 to 500 PRU. These experiments indicated that an endothelium-independent response (18) could also be elicited within this preparation at rest.

View larger version (16K): [in this window] [in a new window]   Fig. 1.   ACh dose-response relationship (means ± SE) of resting gastrocnemius-plantaris (GP) muscle vasculature (n = 3 muscles). Resistance decreased as a function of increasing ACh dose, when muscle blood flow () was maintained at resting levels. Decreases in resistance with highest ACh dose at rest were similar in magnitude to those during contractions with no ACh. PRU, peripheral resistance units. * P < 0.05 compared with 10 mM ACh; v P < 0.05 compared with dose = 0.

Responses in contracting muscle. In the ten experiments in which O₂ was evaluated, averaged 0.29-0.40 and 1.34-1.46 ml · min¹ · g¹ with the muscle at rest and contracting, respectively (Table 2). The similarity of within-group values during contractions, compared between spontaneous and pump-controlled conditions with no blockers, indicates that the initial target was achieved by using the perfusion pump. O₂ averaged 21-27 µl · min¹ · g¹ with the muscle at rest and was similar between spontaneous and pump-controlled conditions (Table 2). O₂ averaged 200-228 µl · min¹ · g¹ during repetitive tetanic contractions and was also similar between conditions. Infusion of blockers L-ArgSA and L-NAME (1.0 mM) produced no significant drops in O₂ within the brief (3-min) time window of drug infusion evaluated in these experiments. These values reflect a 10-fold increase in O₂ from rest, indicating achievement of a high metabolic rate in conjunction with the active hyperemic response resultant of the tetanic contraction regime. for all other experiments described below averaged 1.2-1.5 ml · min¹ · g¹ during contractions.

View this table: [in this window] [in a new window]   Table 2.   , O2, and MAP at rest and during contractions

View larger version (20K): [in this window] [in a new window]   Fig. 2.   Dose-response relationship (means ± SE) obtained in contracting muscles for nitric oxide synthase (NOS) inhibitors L-argininosuccinic acid (L-ArgSA; solid bars, n = 4 muscles) and NG-nitro-L-arginine methyl ester (L-NAME; open bars, n = 8 muscles). P < 0.05, L-NAME compared with respective initial value with no drug;  < 0.05, L-ArgSA compared with respective initial value with no drug.

View larger version (15K): [in this window] [in a new window]   Fig. 3.   Muscle vascular resistance (Rv) changes due to hyperosmotic solution administration (120 mM) during contractions with simultaneous L-ArgSA infusion in 1 muscle. , Rv at rest (1st symbol) and with 3 min of contractions during spontaneous (2nd symbol); , Rv values with perfusion pump set up in place. A high resting Rv was reestablished after 30-min recovery period after contractions with spontaneous , and low Rv was reachieved with 3 min of contractions during pump control of set at the spontaneous hyperemic level. Contractions alone, no drug infusion; L-Arg, L-arginine (1,270 mosM/kg); D-Arg, D-arginine (1,270 mosM/kg). L-Lysine solution was 1,432 mosM/kg; sucrose solution was 1,082 mosM/kg. Lowermost , achievement of low Rv due to hyperosmotic vasodilatory effects; uppermost , initial achievement followed by reestablishment of high Rv with continued L-ArgSA infusion and subsequent discontinuation of each hyperosmotic compound (each infused for 3-5 min).

View larger version (33K): [in this window] [in a new window]   Fig. 4.   Changes in Rv during contraction trials with L-ArgSA (means ± SE). First set of bars on left, average Rv measured in each trial during contractions with no drugs infused; other bar sets, results with constant 1.0 mM L-ArgSA infusion. Time-control trials (open bars, n = 4 muscles) show increasing Rv with no opposing nitric oxide agonist. Stepwise increments in simultaneously delivered nitric oxide precursor L-arginine (0.5, 1.0, 10 mM) did not reverse Rv increase produced with L-ArgSA (hatched bars, n = 4 muscles), with exception of 120 mM. v P < 0.05, 120 mM L-arginine compared with all prior Rv values in this trial. Stepwise increments in simultaneously delivered sodium nitroprusside (SNP) reversed L-ArgSA-mediated Rv increase (solid bars, n = 6 muscles; except for 2.0 and 3.0 µg/ml, n = 4 muscles). * P < 0.05, 1.0 µg/ml SNP vs. matching time-control value; P < 0.05 compared with respective Rv value with contractions alone (i.e., no drug). Timing and perfusion protocol are identical for each trial, as described in text.

View larger version (20K): [in this window] [in a new window]   Fig. 5.   Top: decrements (means ± SE) in active tension (Tactive) with administration of 1.0 mM L-ArgSA beginning at 3 min of repetitive contractions (, n = 7 muscles). , Tactive in control experiments with no antagonist (n = 3 muscles). Bottom: simultaneous change in active peak intramuscular pressure (PIM) during contractions in same L-ArgSA experiments as measured within zone II, i.e., in muscle "belly" or central region of medial head of GP muscle. * P < 0.05 compared with value at time = 0 min.

View larger version (23K): [in this window] [in a new window]   Fig. 6.   Top: increments (means ± SE) in passive tension (Tpass) with administration of 1.0 mM L-ArgSA during contractions (, n = 7 muscles). , Baseline Tpass in control experiments with no antagonist (n = 3 muscles). Bottom: simultaneous change in passive PIM during contractions in same L-ArgSA experiments, shown as increase from PIM baseline. Zones I, II, and III are popliteal origin, muscle belly, and insertion region near calcaneus tendon of medial head of GP muscle, respectively. * P < 0.05 compared with value at time = 0 min.

	DISCUSSION

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The main finding of this study was that NO appears to play a role in control of R_v at rest and during repetitive tetanic contractions within the canine GP muscle in situ. NO synthesis inhibitors, delivered directly into the muscle vasculature, resulted in a significant increase in R_v during electrically stimulated contractions of high force and metabolic rates. The competitive, reversible NO inhibitor L-NAME produced statistically significant increments of R_v during contractions that were reversible with L-arginine. The noncompetitive, irreversible NO inhibitor L-ArgSA produced large increments in R_v that were not reversible with L-arginine. This increase in R_v with L-ArgSA was reversed by administration of SNP, presumably through direct donation of NO to vascular smooth muscle, thus bypassing the endothelial NO blockade. During the administration of L-ArgSA, muscle performance was measurably affected, demonstrating a significant decline in T_act and P_IM production and a significant increase in T_pass and baseline P_IM that was not reversible with L-arginine.

Comparisons with other studies of hyperemia. The results of this study agree with those of Hussain et al. (18), in which L-ArgSA was used as a NO synthesis blocker, and NO was reported to play a significant role (22-41%) in the alteration of R_v in the canine diaphragm in situ during twitch contractions (2/s). Our results are also consistent with those of Sagach et al. (27), in which loss of the "functional" hyperemic response was observed with endothelial blockade in the contracting canine GP muscle in situ. Finally, our data are consistent with studies in which L-NAME decreased the diameter of arterioles mainly in contracting, as opposed to resting, hamster cremaster muscles (16).

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Interpretation of R_v at rest. The resting muscle experiments indicate that NO production is significant even when no contractions are occurring. Furthermore, the results indicate that the competitive, reversible inhibitor L-NAME was less effective than the noncompetitive, irreversible blocker L-ArgSA in blocking this production (Table 1). One possible explanation for this difference is that the concentration gradient for L-arginine favors release from skeletal muscle into the circulation by a factor of 6.5-fold (9), thus presenting a competitive outward flux that would have to be overcome by L-NAME for it to be most effective. However, it should be noted that, although the magnitude of increment of R_v in resting muscle with 1 mM L-ArgSA was significant and eventually similar to that observed with contractions, it occurred over an extended time range (10-20 vs. 3 min). Figure 7 graphically shows the difference in the evolution of the R_v response with L-ArgSA-induced NO blockade during the critical timing window of initial L-ArgSA administration. Taken together, the above data suggest that the rate of NO production was slower in resting muscle compared with contracting muscle. However, we cannot rule out the possibility that these differences might be due to the fact that more L-ArgSA was delivered to more vascular beds within the contracting muscle.

View larger version (22K): [in this window] [in a new window]   Fig. 7.   Rv changes (means ± SE) as a function of time in resting () and contracting () muscles infused with 1 mM L-ArgSA during constant pump perfusion are shown for initial 3-min drug infusion window. Total Rv change due to L-ArgSA for resting and contracting muscles was 104 and 165 PRU, respectively, over 3 min. Initial drop of Rv in contracting muscles was 350 PRU because of vasodilation under pump-perfusion control.

Interpretation of R_v during contractions. The results of the present study suggest several possible interpretations with respect to the influence of NO on R_v. One interpretation is that the rate of NO production by the endothelium within this muscle group during rhythmic high-force contractions is so great that an irreversible blocker of NO production (L-ArgSA) given directly over time is necessary to counter these effects. This interpretation is suggested by Figs. 3 and 4. L-Arginine, given in low, matching, and higher doses, did not reverse this effect; its reversal required direct donation of NO to the smooth muscle or induction of a hyperosmotic vasodilatory effect through a separate pathway. The data of Hirai et al. (17), which show a significant role of the NO pathway in determination of in exercising muscles with high-oxidative fiber type composition, support this interpretation. Also, similar to the present study, a two- to threefold difference in magnitude of effect between locally administered L-ArgSA and the competitive inhibitor L-NAME has been reported previously for the perfused, repetitively contracting diaphragm muscle (18). By the above interpretation, the significant but less robust results with L-NAME might be expected, because higher doses of a competitive inhibitor would be required to dampen a strongly potentiated NO signal resulting from intense muscular contractions (16). Therefore, there are precedents, to which we are presently inclined, for the above interpretation of the present data.

Comparisons with other studies of muscle performance. Prior studies of effects of NO on in situ muscle performance have documented a variety of results. One study of the GP muscle showed that L-NAME increased force development of twitches at 1 and 2 twitches/s, but not at 4 or 6 twitches/s (19), whereas another found no effects of L-NMMA on force development of twitches at 4 twitches/s (8), suggesting some consistency of effects as a function of stimulation frequency. The addition of NO, through administration of S-nitroso-N-acetyl-penicillamine, produced, on average, 3-6% greater fatigue in force than did N-acetyl-penicillamine alone during repetitive twitches of 1.5 and 4/s and tetanic contractions of 40/min, but no effects with 0.5 twitch/s and 12/min tetanic contractions (25). Again, this heterogeneity of response was attributed to stimulation frequency differences but is difficult to compare with the present results because we utilized 60/min tetanic contractions and did not add SNP alone. The absence of these alterations in muscular performance with L-NAME in the present study may have been due to either dosing insufficient to overcome gradients for entry into the skeletal muscle fibers (9) or to intracellular quantities insufficient to compete with native L-arginine (9, 26). Studies have shown that a plasma concentration of 3.0 mM is necessary to saturate the entry mechanism for L-arginine into skeletal muscle (6). Assuming that L-NAME follows this pathway and must compete with intracellular L-arginine for NOS, a reduced or absent response with 1.0 mM L-NAME might be expected in muscle in situ.

Critique of methods. The design of the experiments in the present study included local administration of NO antagonists and agonists directly into the GP muscle vasculature during contractions with controlled . Under these conditions, the time response of the increase in R_v to L-ArgSA was rapid (3 min) and ever increasing with continued administration (Fig. 4). This rapid and sensitive behavior of the vasculature necessitated the use of brief timing windows (3-5 min) during drug administration. However, we found that strict regulation of muscle arterial was not simple to achieve under these conditions of manual pump control because of the capacity of the muscle vasculature to autoregulate under changing conditions of pressure and metabolic demand (31). For instance, when the arterial supply pump was set to maintain and allow arterial pressure to fluctuate as a function of R_v, we observed that the response with L-ArgSA was strong enough to cause a small but significant drop in , even though the roller pump speed setting was unchanged (Table 2). Figure 8 shows this effect, which was not significant for the case of L-NAME. The drop in probably was the result of the intense vasoconstriction produced by L-ArgSA, also evidenced as the significant increase of MAP shown in Table 2. Attempts to rectify this drop in through minor manual adjustments of the pump speed control were abandoned because of the delay between the adjustment and response, typically resulting in repeated overshoot of the target and a miss of the timing window. Thus the approach we employed likely resulted in smaller R_v changes than might have otherwise been observed, because more strict rectifications and maintenance of would have produced even greater MAP elevations and subsequently greater increases of R_v during NO blockade. Although these results show a significant role for NO in the control of R_v during tetanic contractions of the GP muscle group, they likely underestimate this response because of the limitations of our experimental technique.

View larger version (21K): [in this window] [in a new window]   Fig. 8.   changes in individual muscles with 1 mM NOS blocker infusion during contractions under pump-perfusion control show significant decrease in flow in all muscles with L-ArgSA administration. For each muscle, time period between each datum with no drug (contractions) and datum with blocker is 3 min. n = 5 Muscles per blocker; n.s., not significantly different. Mean blood flow values for each case are as shown in Table 2.