Effect of nitric oxide on single skeletal muscle fibres from the mouse

Abstract

1. Single skeletal muscle fibres from a mouse foot muscle were used to investigate the effects of nitric oxide on contractile function.

2. We measured force production and myoplasmic free Ca²⁺ concentration ([Ca²⁺]_i) in single fibres exposed to the nitric oxide donors S-nitroso-N-acetylcysteine (SNAC) and nitroprusside.

3. The nitric oxide donors reduced myofibrillar Ca²⁺ sensitivity, whereas [Ca²⁺]_i transients were increased during submaximal tetani. Force was largely unchanged. SNAC did not change maximum shortening velocity, the rate of force redevelopment, or force production at saturating [Ca²⁺]_i.

4. The guanylyl cyclase inhibitor LY83583 increased tetanic [Ca²⁺]_i but had no effect on Ca²⁺ sensitivity. LY83583 did not prevent the decrease in myofibrillar Ca²⁺ sensitivity in response to SNAC. The oxidizer sodium nitrite increased tetanic [Ca²⁺]_i and decreased myofibrillar Ca²⁺ sensitivity.

5. We conclude that under our experimental conditions nitric oxide impairs Ca²⁺ activation of the actin filaments which results in decreased myofibrillar Ca²⁺ sensitivity.

Nitric oxide (NO) has been identified as an important physiological messenger in a wide variety of tissues (Ignarro, 1990; Moncada, Palmer & Higgs, 1991). NO and related biologically active redox forms (included in the generic term ‘NO’) interact with their cellular targets via redox and nitrosative reactions on transition metals and nucleophilic centres, especially thiols; some of their effects are then mediated by cyclic GMP (cGMP), following binding and activation of soluble guanylyl cyclase (Ignarro, 1990; Stamler, Singel & Loscalzo, 1992; Schmidt, Lohmann & Walter, 1993; Stamler, 1994). It has recently been shown that mammalian skeletal muscle fibres express two isoforms of NO synthase, including one that may be specific for striated muscle (Kobzik, Reid, Bredt & Stamler, 1994; Kobzik, Stringer, Balligand, Reid & Stamler, 1995; Silvagno, Xia & Bredt, 1996). An early study of the effects of NO on the contractile function of rat diaphragm muscle found that inhibition of NO synthase led to increased force production whereas NO donors reduced force (Kobzik et al. 1994). NO-induced depression of force was only partially explained by cGMP-dependent signalling. The remaining force deficit was attributed to NO-induced changes in the redox state of target proteins. A common postulate in both mechanisms was that NO diminishes Ca²⁺ activation of muscle fibres.

The present study was designed to test this postulate. Ca²⁺ activation can be inhibited by decreasing Ca²⁺ transients during contraction, by decreasing Ca²⁺ sensitivity of the myofilaments, or both. We evaluated each aspect of Ca²⁺ activation by measuring force and myoplasmic free Ca²⁺ concentration ([Ca²⁺]_i) during contractions of intact single fibres from a mouse foot muscle exposed to two chemically different NO donors. Muscle fibres were also exposed to nitrite (NO₂⁻) and to an inhibitor of guanylyl cyclase to explore whether the effects of NO were mediated via redox mechanisms or were secondary to increased cGMP levels. Our results indicate that NO diminishes Ca²⁺ activation by reducing myofibrillar Ca²⁺ sensitivity, an effect produced either by exogenous NO donors or by NO₂⁻. These interventions also increased [Ca²⁺]_i transients during tetanic contractions such that force production was largely unchanged. The effects of NO were not mediated via cGMP-dependent pathways. We conclude that NO alters Ca²⁺ activation by two mechanisms, decreasing Ca²⁺ sensitivity of the myofilaments and increasing tetanic [Ca²⁺]_i transients. The effect of NO on force production depends on whether one of these mechanisms predominates.

METHODS

Male mice (NMRI strain, weight about 30 g) were killed by rapid neck disarticulation and intact, single fibres were dissected from the flexor brevis muscle of the hindlimb following a procedure described elsewhere (Lännergren & Westerblad, 1987). The isolated fibre was mounted between an Akers 801 force transducer and an adjustable holder or, in experiments with shortening, the moveable arm of a galvanometer (G120DT; General Scanning, Watertown, MA, USA). Fibre length was adjusted to that giving maximum tetanic force. The fibre was stimulated by supramaximum current pulses (duration 0.5 ms) delivered via platinum plate electrodes lying parallel to the fibre.

Solutions and drugs

Experiments were performed at room temperature (22°C). The fibre was superfused by a Tyrode solution of the following composition (mM): NaCl, 121; KCl, 5.0; CaCl₂, 1.8; MgCl₂, 0.5; NaH₂PO₄, 0.4; NaHCO₃, 24.0; glucose, 5.5; 0.2 % fetal calf serum was added to the solution. The solution was bubbled with 5 % CO₂-95 % O₂ and pH was maintained at 7.4 throughout the experiment. The S-nitrosothiol S-nitroso-N-acetylcysteine (SNAC) was prepared as a 100 mM stock by mixing equimolar amounts of N-acetylcysteine and NaNO₂ dissolved in 0.5 M HCl (Arnelle & Stamler, 1995) and used immediately. The iron nitrosyl sodium nitroprusside (NP, Sigma) (Stamler et al. 1992) was prepared as a 100 mM stock in Tyrode solution just before use. These two chemically distinct NO donors were selected because they release NO spontaneously (Feelisch, 1991; Stamler et al. 1992; Arnelle & Stamler, 1995) and have been used previously to demonstrate the effects of NO on skeletal muscle function (e.g. Kobzik et al. 1994; Murrant, Woodley & Barclay, 1994; Morrison, Miller & Reid, 1996). The guanylyl cyclase inhibitor LY83583 (6-anilino-5,8-quinolinedione) (Mülsch, Busse, Liebau & Förstermann, 1988; Kobzik et al. 1994) was purchased from Alexis Corp. (Switzerland) and prepared as a 4 mM stock. All other chemicals were obtained from Sigma unless otherwise stated. The drugs were added to the superfusing solution from freshly made, concentrated solutions to achieve the desired final concentrations: SNAC, 0.25-1 mM; NP, 1 and 5 mM; NO₂⁻ (as 1 mM NaNO₂ in Tyrode solution), 50 and 100 μM; LY83583, 5 μM; caffeine, 10 mM.

[Ca²⁺]_i measurements

[Ca²⁺]_i was measured with the fluorescent Ca²⁺ indicator indo-1 as described in the preceding paper (Andrade, Reid, Allen & Westerblad, 1998). Of the drugs used in the present study only caffeine was found to have an immediate effect on the intracellular fluorescence of indo-1; it attenuated the intensity of the fluorescence signals without affecting the ratio, in agreement with previous studies (O'Neill, Donoso & Eisner, 1990; Allen & Westerblad, 1995). Tetanic [Ca²⁺]_i and force (in kPa) were measured as the mean over a number of integral stimulation cycles with a total duration of about 100 ms (20-60 Hz) or as the mean over the last 100 ms of stimulation (70-100 Hz).

After tetanic stimulation there is a slow final phase of [Ca²⁺]_i decline. These ‘tails’ of elevated [Ca²⁺]_i were used to estimate the sarcoplasmic reticulum (SR) Ca²⁺ leak and the function of the SR Ca²⁺ pumps (Klein, Kovacs, Simon & Schneider, 1991; Westerblad & Allen, 1993). A double exponential function was first fitted to the [Ca²⁺]_i tails and from this function data points of the rate of [Ca²⁺]_i decline (d[Ca²⁺]_i/dt) vs.[Ca²⁺]_i were obtained; the [Ca²⁺]_i at rest was used to determine the zero value for d[Ca²⁺]_i/dt. The following equation was then fitted to these data points:

(1)

where A reflects the rate of SR Ca²⁺ pumping and L is the SR Ca²⁺ leak. N is a power function and was set to 4, which gave a good fit under all conditions studied (Klein et al. 1991; Westerblad & Allen, 1993).

Maximum shortening velocity

The maximum shortening velocity (i.e. the velocity at zero load, V₀) was measured with slack tests (Lännergren, 1978; Edman, 1979) in separate experiments where [Ca²⁺]_i was not measured. In these experiments, the stimulation frequency during tetani was kept constant at 100 Hz. Rapid releases of at least four different amplitudes were given in subsequent tetani and the time to take up the slack was measured. V₀ was obtained from the slope of the linear regression line in plots of the release amplitude vs. the time to take up the slack. V₀ is expressed in fibre lengths s⁻¹ and the fibre lengths ranged from 0.66 to 0.86 mm.

The exact starting point of force redevelopment after releases was often difficult to appraise. To get an objective measure, we fitted a single exponential function to the initial 40 ms of force redevelopment (starting at a point where force was clearly above the baseline) and the time to take up the slack was obtained by extrapolation to zero force. These measurements of the rate of force redevelopment after releases were also used to assess cross-bridge kinetics under isometric conditions (Brenner, 1988).

Experimental procedure

Tetanic contractions were produced at 1 min intervals. In most experiments, the stimulation frequency during tetani was kept constant at a frequency that under control conditions gave approximately 50 % of the maximum force. This frequency ranged between 20-60 Hz. At the beginning of the experiment, tetani were given until force and [Ca²⁺]_i were stable. A drug was then added and this resulted in some alteration of force and/or [Ca²⁺]_i within 1 min. After 3-5 min exposure to the drug, force and [Ca²⁺]_i were reasonably stable at a new level and measurements were performed. The drug was then washed out and force and [Ca²⁺]_i generally returned to their control values within a few minutes. Although the effects were fully reversible with all the drugs used in this study, we tried to limit experimental treatments to one per fibre, except in those experiments requiring drug combinations: SNAC and caffeine, SNAC and LY83583. Therefore, the effects, if any, were detected in fresh fibres, and the number of experiments reported in Results refers to the number of fibres used for each particular experiment. When investigating the effects of a particular donor or NO₂⁻, we started with the highest concentration reported, and decreased it by 50 % as long as the response was the same. To check if the effects observed with the donors or NO₂⁻ were dose dependent, we plotted the changes of [Ca²⁺]_i or force vs. the concentration of the compound. Linear regression on these plots never showed any significant slopes, which indicates that the effects were not dose dependent.

The force-[Ca²⁺]_i relationship was studied by producing tetani at 20-100 Hz and fitting the following Hill equation to force-[Ca²⁺]_i data points (for details see Westerblad & Allen, 1993):

(2)

where P is the relative force, P_max is the force at saturating [Ca²⁺]_i, Ca₅₀ is the [Ca²⁺]_i giving 50 % of P_max, and N is a constant which describes the steepness of the function. In this curve fitting, P_max was assumed to be 8 % higher than the force during 100 Hz control tetani (Allen & Westerblad, 1995; see also present results with caffeine).

Statistics

Values are presented as means ±s.e.m. Paired Student's t tests were used to verify group differences, and the significance level (P) was set at 0.05 throughout.

RESULTS

Figure 1 shows original records from one fibre that was exposed to 0.25 mM SNAC. This fibre was stimulated at 40 Hz and application of SNAC induced a reversible increase in [Ca²⁺]_i, while force remained almost unaffected. Twelve fibres were exposed to SNAC (5 fibres to 0.25 mM, 6 to 0.5 mM, and 1 to 1 mM). Since the results were the same regardless of concentration (see Methods), they are presented together. The response of force to SNAC showed the largest variability, with an increase of more than 10 % in three fibres (0.25, 0.5 and 1 mM SNAC, respectively) and an equally large reduction in three other fibres (all exposed to 0.25 mM SNAC). [Ca²⁺]_i during the contraction also showed some variability with one fibre (0.5 mM SNAC) displaying a minor (less than 5 %) reduction, while [Ca²⁺]_i clearly increased in seven fibres (3 fibres to 0.25 mM, 3 to 0.5 mM and 1 to 1 mM SNAC). Thus, there was a significant increase of [Ca²⁺]_i during submaximal tetani from 0.67 ± 0.03 μM in control to 0.73 ± 0.04 μM with SNAC (P < 0.05), whereas the mean force was not significantly changed by SNAC. The effects of SNAC on tetanic [Ca²⁺]_i and force were rapidly reversible and 1 min after removal of SNAC neither of them were significantly different from the control values.

Figure 1. Effect of SNAC on submaximal [Ca²⁺]_i and force.

Original records from 40 Hz tetani produced in one fibre under control conditions, after 4 min exposure to 0.25 mM SNAC, and after wash-out. The upper part of the figure shows [Ca²⁺]_i records obtained with indo-1 and the lower part shows force. The bars show the mean [Ca²⁺]_i over the period where measurements were done. The short exposure to SNAC increased tetanic [Ca²⁺]_i from 0.69 to 0.77 μM. In this fibre the increase in [Ca²⁺]_i was accompanied by a small (3 %) decrease of force (measured during the same period as [Ca²⁺]_i). Both [Ca²⁺]_i and force had returned to their control values 1 min after wash-out of SNAC.

Similar results to those with SNAC were obtained with NP and NO₂⁻ (Fig. 2). Both NP (n= 9 fibres; 5 fibres in 1 mM and 4 in 5 mM) and NO₂⁻ (n= 8 fibres; 4 fibres in 50 μM and 4 in 100 μM) significantly increased [Ca²⁺]_i during submaximal tetani while having no significant effect on force. As with SNAC, tetanic [Ca²⁺]_i and force promptly returned to the control level after wash-out of NP and NO₂⁻. N-acetylcysteine and ferricyanide, the metabolites of SNAC and NP, respectively, did not affect force or tetanic [Ca²⁺]_i (data not shown). The effects of NO are often secondary to increased levels of cGMP. We used the guanylyl cyclase inhibitor LY83583 to investigate the role of cGMP in our experiments. Exposing fibres to LY83583 (n= 8 fibres) resulted in significant increases of [Ca²⁺]_i and force during submaximal tetani (Fig. 2). When fibres were co-incubated with LY83583 and SNAC (n= 9 fibres) [Ca²⁺]_i increased significantly, but force remained unaltered (Fig. 2).

Figure 2. Nitric oxide donors increase tetanic [Ca²⁺]_i but not force.

Relative changes of tetanic [Ca²⁺]_i (A) and force (B) as a result of exposure to the drugs indicated below the columns. Results are presented as means ±s.e.m. from fixed-frequency comparisons within each fibre; n= 12 for SNAC, 9 for NP, 7 for NO₂⁻, 8 for LY83583, and 9 for SNAC + LY83583. * Significant difference from control (P < 0.05). Tetanic [Ca²⁺]_i was elevated with all the treatments: SNAC, 109 ± 3 %; NP, 107 ± 2 %; NO₂⁻, 110 ± 3 %; LY83583, 107 ± 2 %; LY83583 + SNAC, 106 ± 1 %. LY83583 also increased force (108 ± 3 %).

SNAC increased resting [Ca²⁺]_i by 7 ± 3 nM from a control value of 66 ± 5 nM (P < 0.05, n= 12). This might reflect increased SR Ca²⁺ leak or reduced SR Ca²⁺ pumping or possibly reduced myoplasmic Ca²⁺ buffering. To discriminate between increased leak and reduced pumping, we evaluated the effects of SNAC on [Ca²⁺]_i‘tails’ (see Methods) after tetanic stimulation. Figure 3 shows averaged records of [Ca²⁺]_i tails under control conditions and after exposing the same fibres to SNAC. It can be seen that SNAC caused an upward and parallel shift of the [Ca²⁺]_i tail; [Ca²⁺]_i was significantly increased by 14 ± 4 nM at 1.5 s, and 13 ± 4 nM at 2.5 s after the end of tetanic stimulation. Analysis of the SR function according to eqn (1) was performed on double exponential fits of these [Ca²⁺]_i tails (dashed lines in Fig. 3A) and showed no significant effect on the SR Ca²⁺ leak (L), whereas the rate of SR Ca²⁺ pumping (reflected by A) was reduced to about 67 % of the control value. In other words, at a given [Ca²⁺]_i, the rate of Ca²⁺ reuptake by the SR is slower in the presence of SNAC.

Figure 3. SNAC decreases the SR Ca²⁺ pumping rate.

A, averaged records from twelve fibres showing the recovery of [Ca²⁺]_i at the end of tetanic stimulation under control conditions and during exposure to SNAC. SNAC caused an upward and parallel shift of the [Ca²⁺]_i tail. B, analysis of the SR function according to eqn (1) performed on double exponential fits of the [Ca²⁺]_i tails (A, dashed lines). Control, • and dashed line; SNAC, ○ and continuous line. SNAC had no significant effect on the SR Ca²⁺ leak (L), whereas the rate of SR Ca²⁺ pumping (reflected by A in eqn (1)) was reduced to about 67 % of the control value.

Resting [Ca²⁺]_i was not altered by NP, NO₂⁻, or LY83583. Furthermore, the [Ca²⁺]_i tails after tetani did not shift in response to any of these drugs. Thus, there is no evidence that these agents affected SR Ca²⁺ leak or pumping.

Force-[Ca²⁺]_i relationship

The above results show that NO donors and NO₂⁻ increase tetanic [Ca²⁺]_i while having no effect on force. This can be explained by a reduction in the ability of the cross-bridges to generate force and/or reduced myofibrillar Ca²⁺ sensitivity. A reduction of the ability of the cross-bridges to generate force will result in decreased force at saturating [Ca²⁺]_i. To study this, we produced 100 Hz tetani in the presence of 10 mM caffeine, which results in [Ca²⁺]_i levels high enough to provide maximum cross-bridge activation (Allen & Westerblad, 1995). Figure 4 shows one experiment where caffeine was applied first in control and then after addition of SNAC. It can be seen that [Ca²⁺]_i is high in both contractions, and the forces are almost identical. Similar results were obtained in four more fibres; the force increase with caffeine was 8 ± 2 % in the control, in agreement with previous results (Allen & Westerblad, 1995), and 8 ± 2 % with SNAC. Thus, the effect of SNAC was not exerted via a reduction of the ability of the cross-bridges to produce force.

Figure 4. Effect on force production at saturating [Ca²⁺]_i.

Original records of [Ca²⁺]_i (upper traces) and force (lower traces) from 100 Hz tetani produced in the presence of 10 mM caffeine. [Ca²⁺]_i was high in both contractions and the forces were almost identical. Force during co-incubation with caffeine was 422 ± 18 kPa for control, and 420 ± 21 kPa for SNAC (n= 5).

Figure 5A shows force-[Ca²⁺]_i data points obtained in one fibre before, during and after exposure to SNAC. All data points obtained during SNAC exposure lie to the right of the control points, and Ca₅₀ (eqn (2)) increased from 0.68 μM in control to 0.81 μM with SNAC. The results from twelve fibres showed a mean increase of Ca₅₀ from 0.64 ± 0.02 μM in control to 0.74 ± 0.05 μM with SNAC (P < 0.05); hence Ca₅₀ was significantly larger (16 ± 7 %) in the presence of SNAC. The steepness of the force-[Ca²⁺]_i relationship was not affected by SNAC: N was 5.3 ± 0.6 in control and 5.9 ± 0.6 with SNAC. NP also shifted the force-[Ca²⁺]_i relationship to the right, increasing Ca₅₀ by 11 ± 2 % without altering N, very similar to the change observed with SNAC. The effect of NO₂⁻ on the force-[Ca²⁺]_i relationship was studied in two fibres, and Ca₅₀ increased 7 and 11 % from the control levels.

Figure 5. Typical change of force-[Ca²⁺]_i relationship with SNAC.

A, analysis of the force-[Ca²⁺]_i relationship in one fibre in control (•), with 0.25 mM SNAC (○), and after wash-out (▴). Curves were drawn by fitting data points in control (dashed line) and with SNAC (continuous line) to eqn (2). All the data points obtained during the exposure to SNAC lie to the right of the control points, and Ca₅₀ (eqn (2)) increased from 0.68 to 0.81 μM. This rightward shift due to SNAC was reversible with wash-out (▴). B, force-[Ca²⁺]_i relationship in one fibre in control (• and dashed line), and during co-incubation with 5 μM LY83483 and 0.5 mM SNAC (○ and continuous line). As with SNAC alone, co-incubation with LY83583 and SNAC shifted all the data points to the right of the control relationship, and Ca₅₀ increased from 0.44 to 0.57 μM.

Force-[Ca²⁺]_i curves were also constructed in experiments with application of LY83583. In these experiments both tetanic [Ca²⁺]_i and force were increased (see Fig. 2) and in accordance with this, we found no change in Ca₅₀: 0.64 ± 0.05 μM in control and 0.67 ± 0.07 μM with LY83583 (n= 7 fibres). N was altwo so not changed by LY83583. Nevertheless, when SNAC was combined with LY83583, the force-[Ca²⁺]_i relationship shifted to the right (Fig. 5B). Co-incubation with LY83583 and SNAC changed Ca₅₀ from 0.58 ± 0.7 μM in control to 0.67 ± 0.09 μM without changing N (n= 8 fibres). This increase in Ca₅₀ (15 ± 4 %) was of the same magnitude as with SNAC alone (above).

Shortening velocity

The decrease in myofibrillar Ca²⁺ sensitivity with SNAC, evidenced by the rightward shift of the force-[Ca²⁺]_i relationship and increased Ca₅₀ may be due to (1) impaired Ca²⁺ activation of the actin filaments leading to a reduction in the number of recruited cross-bridges, or (2) altered cross-bridge kinetics (Brenner, 1988). To distinguish between these two possibilities, we performed rapid releases during tetani elicited under control conditions and with SNAC present. Figure 6A shows force responses associated with a 120 μm shortening step. It is clear that the lag before force redevelopment occurs is very similar in control and with SNAC. In Fig. 6B, we have plotted the amplitude of the shortening step vs. the time required to take up the slack. The data points obtained in control and SNAC are almost identical at all amplitudes tested. Similar results were obtained in two more fibres and V₀ was 6.8 ± 1.4 fibre lengths s⁻¹ in control and 6.7 ± 1.1 fibre lengths s⁻¹ with SNAC. The rate of force redevelopment, measured in the shortest (40 μm) releases, was also similar in control and with SNAC: 49.1 ± 4.4 s⁻¹vs. 51.8 ± 5.5 s⁻¹. Thus, neither V₀ nor the rate of force redevelopment was significantly affected by SNAC, suggesting that NO did not alter cross-bridge kinetics.

Figure 6. Lack of effect on shortening velocity.

Slack tests performed in control and with 0.25 mM SNAC. A, original force records from 120 μm releases; note that the time until force redevelopment starts is very similar with (dashed line) and without SNAC (continuous line). B, plot of the amplitude of shortening vs. the time to take up the slack. There was no noticeable difference between the times measured in control (• and dashed line) and with SNAC (○ and continuous line) at any of the four release steps studied.

DISCUSSION

NO is an important signalling substance which exerts its effects via cGMP-dependent and cGMP-independent pathways that affect a wide array of physiological functions, sometimes in opposite directions (Moncada et al. 1991; Schmidt et al. 1993; Stamler, 1994). This is clearly illustrated by the present results where NO donors were found to reduce the myofibrillar Ca²⁺ sensitivity, which would tend to decrease force, and at the same time increase [Ca²⁺]_i during submaximal tetani, which would tend to increase force. Thus, force production will depend on the fine balance between these two opposing effects. This is especially true in submaximal tetani, where the force-[Ca²⁺]_i relationship is steep (see Fig. 5), and small changes of myofibrillar Ca²⁺ sensitivity or tetanic [Ca²⁺]_i may have a large effect on force. The mean results of the present study show that NO donors have no significant effect on force production in submaximal tetani, and thus, on average, the opposing effects would counterbalance each other. Previous studies confirm the variability in sub-maximal force in response to NO donors. Force in rat diaphragm has been shown to decrease slightly when exposed to NO (Kobzik et al. 1994; Morrison et al. 1996). In contrast, force production by mouse hindlimb muscles increased after 15 min of exposure to NO donors (Murrant et al. 1994; Murrant & Barclay, 1995). However, force did not change in response to the donors during the period (< 15 min) that corresponds to our own experimental protocol (Fig. 3 in Murrant et al. 1994; Fig. 2 in Murrant & Barclay, 1995). Furthermore, a recent report demonstrates that the direction of the change in force in response to NO donors depends significantly on the interval between contractions. When muscles are stimulated more frequently, force increases in response to NO; the opposite occurs when the stimulation interval increases (Murrant, Frisbee & Barclay, 1997). In combination, these data suggest that exposures to NO have complex, multifactorial effects on contractile function, as seems to be the case in more isolated preparations (see below). In this context, it is worth noting that a pilot study showed significant increases of tetanic [Ca²⁺]_i and force with NP (Reid, Allen, Lännergren & Westerblad, 1996). The report was based on data from repeated exposures to NP in only four fibres, and the effect on the force-[Ca²⁺]_i relationship was not investigated. This effect on force has proven inconsistent in later experiments and the mean results show no significant effect of NP on tetanic force.

The effects of NO donors observed in the present study were most probably due to NO and not to some non-specific drug effect since we used two NO donors with different chemical structures, the S-nitrosothiol SNAC and the iron nitrosyl NP, and they had similar effects (e.g. Figure 2). NO may influence skeletal muscle by activating guanylyl cyclase and increasing cGMP. To test for a role of this cGMP pathway, we exposed fibres to the guanylyl cyclase inhibitor LY83583. If the effects observed by application of NO donors were due to increased cGMP, inhibition of soluble guanylyl cyclase would have the opposite effect, i.e. increase myofibrillar Ca²⁺ sensitivity and reduce tetanic [Ca²⁺]_i. However, application of LY83583 increased tetanic [Ca²⁺]_i, but did not change Ca²⁺ sensitivity; consequently force was increased during submaximal tetani. When the NO donor SNAC was included, LY83583 did not prevent the rightward shift in the force-[Ca²⁺]_i relationship (Fig. 5B) and Ca₅₀ increased to the same extent as with SNAC alone. Thus, the observed decrease in myofibrillar Ca²⁺ sensitivity with the NO donors and NO₂⁻ is not consistent with a cGMP-mediated effect. Our results with LY83583 agree with results from rat diaphragm (Kobzik et al. 1994) and suggest that cGMP acts as an inhibitor of muscle contraction (Warner, Mitchell, Sheng & Murad, 1994). Because LY83583 significantly increased both [Ca²⁺]_i and force, cGMP-dependent inhibition must be prominent in the basal state.

Since the effects of the NO donors appear to be independent of cGMP, they are probably due to direct interactions with target proteins, either by oxidation of thiol residues, or by nitrosylation of nucleophilic centres. This is reinforced by the fact that NO₂⁻ and the NO donors had similar effects on [Ca²⁺]_i and force: NO₂⁻ was used because it crosses cell membranes, works mainly as an oxidant and is a poor activator of soluble guanylyl cyclase (Romanin & Kukovetz, 1988; Ye & Huang, 1990; Feelisch, 1991; Kowaluk, Chung & Fung, 1993).

Force-[Ca²⁺]_i curves produced in experiments with SNAC, NP and NO₂⁻ showed a reduction of the myofibrillar Ca²⁺ sensitivity; this was expected as the three drugs increased tetanic [Ca²⁺]_i without affecting submaximal force (Fig. 2). This reduction in myofibrillar Ca²⁺ sensitivity can be due to (1) impaired Ca²⁺ activation of the actin filaments leading to a reduction of the number of recruited cross-bridges, or (2) altered cross-bridge kinetics (Brenner, 1988). We evaluated the possible role of altered cross-bridge kinetics in experiments using SNAC. The ability of cross-bridges to produce force at saturating [Ca²⁺]_i was not affected by SNAC, as judged from experiments with caffeine (Fig. 4). Furthermore, SNAC had no effect on V₀ or on the rate of force redevelopment after a release. These data agree with previous reports on rat diaphragm (Kobzik et al. 1994; Morrison et al. 1996), and indicate that the reduced Ca²⁺ sensitivity is not due to altered cross-bridge kinetics. Thus, the reduced myofibrillar Ca²⁺ sensitivity observed in intact muscle fibres could be due to impaired Ca²⁺ binding to troponin C, or to alterations in the interaction between troponin C and the other elements of the regulatory system, i.e. troponin I, troponin T and tropomyosin. For example, a current model of the Ca²⁺-dependent interaction between troponin C and troponin I includes conformational changes (Farah & Reinach, 1995), which could be interfered with by modification of cysteine residues.

Studies on skinned skeletal muscle fibres found that NO donors decrease maximal force, Ca₅₀, V₀ and myosin ATPase activity, and concluded that NO alters the kinetics of the interaction between myosin and actin (Perkins, Han & Sieck, 1997; Galler, Hilber & Göbesberger, 1997). These cGMP-independent effects were presumably due to the oxidation of reactive thiols on the contractile proteins, particularly myosin (Perkins et al. 1997). It should be noted that there are factors inherent in more isolated systems (i.e. skinned fibres and SR vesicles; see below) that may influence the experimental results, like the use of the reductant dithiothreitol during their preparation (e.g. 10 mM in Perkins et al. 1997). This chemical would shift the native redox state of the contractile proteins to an abnormally reduced state, with unpredictable effects on function. Moreover, prolonged exposure to redox agents (≥ 20 min in Perkins et al. 1997) may result in the non-specific oxidation of thiol groups throughout the fibre, resulting in artifactual alterations in function (Crowder & Cooke, 1984). In our experiments, intact single fibres were exposed to low concentrations of NO donors for brief periods of time. We do not know the actual NO concentration that the fibres were exposed to. Moreover, we cannot measure the influence and mechanistic implications of intracellular redox-active compounds like glutathione, which will alter the response to exogenous NO (Arnelle & Stamler, 1995). Therefore it is possible that our data and the results of Galler et al. (1997) and Perkins et al. (1997) with skinned fibres represent different sides in a spectrum of responses to NO. For example, at low concentrations and/or during short exposures, NO decreases myofibrillar Ca²⁺ sensitivity. As the exposure continues, or when NO concentration increases even more, myosin is affected, decreasing ATPase activity, maximal force and V₀. This type of differential sensitivity is an attractive paradigm, as it has already been shown to occur in the SR Ca²⁺ release channel (see below).

[Ca²⁺]_i during the present type of brief tetanic contractions depends on the myoplasmic Ca²⁺ buffering, the rate of SR Ca²⁺ uptake, and the SR Ca²⁺ release (Westerblad & Allen, 1996). The increase in tetanic [Ca²⁺]_i induced by NO donors and NO₂⁻ can then be explained by changes of any of these parameters. One important myoplasmic Ca²⁺ buffer during brief tetani is the Ca²⁺-specific sites on troponin C (Westerblad & Allen, 1996). The reduction of the myofibrillar Ca²⁺ sensitivity seen during exposure to the NO donors and NO₂⁻ might indicate decreased Ca²⁺ binding to the specific sites on troponin C and hence reduced myoplasmic Ca²⁺ buffering. This might contribute to the observed increase of tetanic [Ca²⁺]_i (Figs 1 and 2).

SNAC increased the amplitude of [Ca²⁺]_i tails after tetani, which indicates a reduction in the rate of SR Ca²⁺ uptake. Slowing of the SR Ca²⁺ pumps increases tetanic [Ca²⁺]_i (Westerblad & Allen, 1994) and provides another mechanism behind the increased resting and tetanic [Ca²⁺]_i observed with SNAC. However, slowing of the SR Ca²⁺ pumps was not a general NO effect, because NP and NO₂⁻ did not alter [Ca²⁺]_i tails after tetani, or increase resting [Ca²⁺]_i. This may suggest that SNAC is a more potent NO donor, and a more effective oxidizer than NP and NO₂⁻ (Feelisch, 1991; Arnelle & Stamler, 1995). It also indicates that the myofibrils are more sensitive than the SR to these chemicals, as all had the same general effect on contractile function.

Little is known about the effect of NO on SR Ca²⁺ release in skeletal muscle. NO donors have been reported both to decrease and increase the open probability of isolated SR Ca²⁺ release channels (Mészàros, Minarovic & Zahradnikova, 1996; Stoyanovsky, Murphy, Anno, Kim & Salama, 1997). These conflicting findings may be due to a biphasic response of isolated SR Ca²⁺ release channels to NO, reflecting distinct thiol subpopulations in the channel tetramer (Aghdasi, Reid & Hamilton, 1997; Aghdasi, Zhang, Wu, Reid & Hamilton, 1997). An important caveat in the interpretation of these reports is that all start by exposing the preparation to a reducing agent like dithiothreitol or β-mercaptoethanol, changing the native redox state of the system under study. Our results indicate that NO donors and NO₂⁻ have no effect on the passive SR Ca²⁺ leak in intact fibres, which would be the equivalent to the open probability measured in isolated SR Ca²⁺ release channels. The increase in tetanic [Ca²⁺]_i detected (Figs 1 and 2) could be partially due to a small increase in voltage-activated SR Ca²⁺ release, sufficient to largely offset the concurrent decrease in myofibrillar Ca²⁺ sensitivity, and maintain force unchanged.

The guanylyl cyclase inhibitor LY83583 increased tetanic [Ca²⁺]_i without altering myofibrillar Ca²⁺ sensitivity or [Ca²⁺]_i tails after tetani. Therefore, the increase in tetanic [Ca²⁺]_i cannot be explained by reduced myoplasmic Ca²⁺ buffering or slowed SR Ca²⁺ pumping. Thus, guanylyl cyclase inhibition stimulated SR Ca²⁺ release, which agrees with the general role of cGMP as a factor that inhibits contraction (Kobzik et al. 1994; Warner et al. 1994). However, these results do not support cGMP as a mediator of the response to NO. In addition, the combination of LY83583 and SNAC gave a reduction in myofibrillar Ca²⁺ sensitivity similar to that seen with SNAC alone.

In conclusion, the effects of NO on intact single skeletal muscle fibres were independent of cGMP. NO decreased myofibrillar Ca²⁺ sensitivity and increased tetanic [Ca²⁺]_i. The balance between these two opposing influences would determine the direction of the change in force production.