Antagonism by hemoglobin of effects induced by L-arginine in neuromuscular preparations from rats

Braz J Med Biol Res, April 2001, Volume 34(4) 549-552 (Short Communication)

Antagonism by hemoglobin of effects induced by L-arginine in neuromuscular preparations from rats

C.R. Ambiel² and Wilson Alves-Do-Prado¹

Departamentos de ¹Farmácia e Farmacologia and ²Fisiologia, Universidade Estadual de Maringá, Maringá, PR, Brasil

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References

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Abstract

Nitric oxide (NO)-synthase is present in diaphragm, phrenic nerve and vascular smooth muscle. It has been shown that the NO precursor L-arginine (L-Arg) at the presynaptic level increases the amplitude of muscular contraction (AMC) and induces tetanic fade when the muscle is indirectly stimulated at low and high frequencies, respectively. However, the precursor in muscle reduces AMC and maximal tetanic fade when the preparations are stimulated directly. In the present study the importance of NO synthesized in different tissues for the L-Arg-induced neuromuscular effects was investigated. Hemoglobin (50 nM) did not produce any neuromuscular effect, but antagonized the increase in AMC and tetanic fade induced by L-Arg (9.4 mM) in rat phrenic nerve-diaphragm preparations. D-Arg (9.4 mM) did not produce any effect when preparations were stimulated indirectly at low or high frequency. Hemoglobin did not inhibit the decrease of AMC or the reduction in maximal tetanic tension induced by L-Arg in preparations previously paralyzed with d-tubocurarine and directly stimulated. Since only the presynaptic effects induced by L-Arg were antagonized by hemoglobin, the present results suggest that NO synthesized in muscle acts on nerve and skeletal muscle. Nevertheless, NO produced in nerve and vascular smooth muscle does not seem to act on skeletal muscle.

Key words: skeletal muscle, nitric oxide, tetanic fade, L-Arginine, D-Arginine, hemoglobin

Nitric oxide (NO) is generated from L-arginine by NO-synthase (1-3). Neuronal NO-synthase is present in the sarcolemma of type II skeletal muscle fibers (4) and in the motor nerve of rats (5). It has been shown that the addition of the NO precursor L-arginine (4.7-9.4 mM) to rat neuromuscular preparations increases the amplitude of muscular contraction (AMC) in preparations indirectly stimulated at 0.2 Hz (6). In contrast, L-arginine (4.7-9.4 mM) reduces AMC when preparations previously paralyzed with d-tubocurarine are directly stimulated at low frequency (7). The facilitatory and inhibitory effects of L-arginine depend on its metabolism to NO, with actions at the pre- and postsynaptic level, respectively (7). Studies conducted at high frequencies of stimulation applied to the motor nerve have shown that L-arginine (4.7-18.4 mM) produces a dose-dependent tetanic fade followed by reduction in maximal tetanic tension (6). The tetanic fade and the reduction in maximal tetanic tension induced by L-arginine are produced by the action of NO on the motor nerve and skeletal muscle, respectively (6). The presynaptic effects observed with L- arginine reach their maximum at concentrations around 9.4 mM (6,7).

Hemoglobin may cross the wall of a large vein (8), but its diffusion through the membranes of other tissues is negligible (9). NO (authentic gas) disappears rapidly when hemoglobin is added to the bath (10), at a rate proportional to hemoglobin concentration in the bath (10). Since hemoglobin only scavenges NO released from tissues, the pre- and postsynaptic neuromuscular effects induced by NO released from skeletal muscle, motor nerve and/or vascular smooth muscle might be reduced or antagonized by previous administration of the scavenger. Therefore, the present study with hemoglobin was undertaken to determine the relative importance of NO produced from skeletal muscle, nerve and vascular smooth muscle for the neuromuscular effects induced by L-arginine.

Phrenic nerve and diaphragm muscle were isolated from Wistar rats (200-250 g) of both sexes and

mounted according to the method described by Bülbring(11). Each muscle was immersed in a 30-ml chamber containing Krebs buffer. The phrenic nerve was stimulated with a bipolar platinum electrode using a supramaximal rectangular pulse (0.2 Hz, 0.05 ms) and muscle contractions were recorded on an Ugo Basile polygraph.

In studies performed at a low frequency of stimulation, the isolated muscles were stimulated at 0.2 Hz until a steady AMC was obtained. L-Arginine (9.4 mM) was then added to the organ bath and the AMC obtained after 20 min was taken as a percentage of that observed before any drug administration. In the experiments performed with a high frequency of stimulation applied to the motor nerve, the standard rate of stimulation was 0.2 Hz. Nevertheless, stimulation at a higher (tetanic) rate was applied to the nerve for 10 s at 20-min intervals. The same experimental design was used for neuromuscular preparations previously paralyzed with d-tubocurarine and directly stimulated. The tension produced at the beginning of tetanic stimulation (A) was compared to that obtained at the end of tetanic stimulation (B). The stimulation rate (F) (180-200 Hz) required to obtain a 0.50 ratio (R = B/A) was determined for each preparation and used throughout the experiments. L-Arginine or D-arginine was then added to the organ bath and F was repeated at t = 20, 40, 60 and 80 min. Krebs buffer in the bath was replaced with drug-free Krebs buffer (4 times) at t = 90 min and the R ratio was observed at t = 100 and 120 min. The same sequences were repeated with the use of hemoglobin, except that the NO-scavenger was added 5 min before L-arginine. The R ratio obtained after drug addition was taken as a percentage of that observed before drug administration. Data were submitted to ANOVA, followed by the Bonferroni test, with the level of significance set at P<0.05. The following drugs were used: L-arginine, D-arginine and bovine hemoglobin (Sigma Chemical Co., St. Louis, MO, USA).

Confirming previous reports with L-arginine, our study showed that L-arginine (9.4 mM) increased AMC induced by low frequency of stimulation and produced tetanic fade when high frequencies (180-200 Hz) of stimulation were applied to the motor nerve (Figures 1 and 2). L-Arginine also reduced AMC (30 ± 0.02%, N = 8) and maximal tetanic tension (50 ± 0.04%, N = 6) in neuromuscular preparations previously paralyzed with d-tubocurarine and directly stimulated. The effects observed with L-arginine were stereospecific, since D-arginine (data not shown) was not effective in similar experiments. Results resemble those obtained in other studies using similar preparations and methods (6,7) and depend on the action of NO at the presynaptic (increase in AMC and tetanic fade) and postsynaptic (reduction in AMC and maximal tetanic tension) levels (6,7).

Hemoglobin did not produce any effect on AMC or on tetanic fade of preparations indirectly stimulated with pulses at low or high frequency, respectively. However, hemoglobin induced a dose-dependent (25-50 nM) reduction in the facilitatory effect induced by L-arginine when the nerve was indirectly stimulated at low frequency (Figure 1). It was also verified that 50 and 100 nM hemoglobin antagonized the tetanic fade induced by 9.4 mM L-arginine (Figure 2). On the other hand, hemoglobin neither inhibited nor reduced the postsynaptic effects induced by NO in preparations previously paralyzed with d-tubocurarine and directly stimulated. Taken as a whole, the results showed that hemoglobin only antagonized the NO-induced presynaptic effects. This suggests that NO synthesized in muscle may act on nerve and in muscle, whereas NO produced in nerve and vascular smooth muscle seems to act solely at the presynaptic level.

Acknowledgments

We are grateful to Mrs. I.L. Santos for technical support. We also thank Prof. Dr. F. Cunha for kindly supplying bovine hemoglobin.

Address for correspondence: W. Alves-Do-Prado, Laboratório de Farmacologia da Transmissão Neuromuscular, DFF, UEM, Av. Colombo, 5790, 87020-900 Maringá, PR, Brasil. Fax: [phone]. E-mail: [e-mail] or [e-mail]

Research supported by CNPq (No. 521727/96). Received April 12, 2000. Accepted January 30, 2001.

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Correspondence and Footnotes

Publication Dates

• Publication in this collection
  29 Mar 2001
• Date of issue
  Apr 2001

History

• Accepted
  30 Jan 2001
• Received
  12 Apr 2000