BIOCHEMICAL
Vol. 188, No. 2, 1992 October 30, 1992
NG-MONOMETHYL-L-ARGININE IN ISOLATED ARTERIAL Stephen
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS Pages 590-596
CAUSES NITRIC RINGS: TROUBLE
L. Archer
and Va’clav
OXIDE SYNTHESIS IN PARADISE*
Hampl
Division of Cardiology, Veterans Administration Medical Center University of Minnesota, Minneapolis, Minnesota 55417
Received
August
31,
and
1992
SUMMARY. Arginine analogs are commonly used as inhibitors of the synthesis of endothelium-derived relaxing factor, nitric oxide. However, their effect on nitric oxide levels is rarely measured. Using a chemiluminescence assay for nitric oxide, we found that NG-monomethylL-arginine enhanced, rather than reduced, nitric oxide synthesis in pulmonary arterial and aortic rings. NG-monomethyl-L-arginine inhibited relaxation to the endothelium-dependent vasodilator A23187 in aortic but not pulmonary arterial rings. In contrast, No-nitro-L-arginine did not stimulate nitric oxide synthesis and it inhibited relaxation to A23 187 in all rings. We conclude that NG-monomethyl-L-arginine is a partial agonist for nitric oxide synthesis. 0 1992Academic Press,Inc.
Arginine analogs, such as NG-monomethyl-L-arginine (L-NMMA) and NW-nitro-L-arginine (L-NNA), inhibit endothelium-dependent vasodilation in systemic vessels (1, 2). Few studies have documented the inhibitory effect of arginine analogs on the synthesis of nitric oxide (NO, endotheliumderived relaxing factor) (1, 3-6). Nonetheless, arginine analogs are widely utilized as “specific” inhibitors of NO synthesis. In fact, much of the knowledge of NO physiology and pathophysiology is based on experiments using L-NMMA. It is often assumed that any hemodynamic effect of arginine analogs results from NO synthesis inhibition; this may not always be true. In the present study we measured the effect of L -NMMA and LNNA on NO production by arterial rings. As the role of endothelium*Supported by the Department of Veterans Affairs, Minnesota Medical Foundation, NIH grant # lR29-HL45735-1, and the Proshek Foundation. . . brevw : L-NMMA, NG-monomethyl-L-arginine; L-NNA, Nm-nitroL-arginine. 0006-291X/92 $4.00 Copyright 0 I992 by Academic Press, All rights of reproduction in any form
Inc. reserved.
590
Vol.
188,
No.
2,
1992
BIOCHEMICAL
derived relaxing factor in the compared the effects of arginine rings.
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
pulmonary circulation is not clear, we analogs on aortic and pulmonary arterial
METHODS Abdominal aorta and second division right pulmonary artery rings were dissected from anesthetized (pentobarbital 50 mg/kg, IP) adult, male, specific pathogen free, Sprague Dawley rats (7). Rings were mounted on stainless steel wires attached to Grass UC-2 force-displacement transducers and suspended in a 75 ml bath containing Earl’s balanced salt solution (37.5OC, pH=7.43, PG2=130-140 mm Hg). The rings (2/bath) were held 1 cm apart and samples for NO analysis (2 ml) were withdrawn midway between the vessels using a gas-tight, N2-flushed syringe. Rings equilibrated for 30 minutes at resting tensions of 800 mg and 2000 mg for pulmonary arterial and aortic rings, respectively. Superoxide dismutase (2000 units) was given because it enhances NO detection (6), presumably by increasing NO survival. Protocol. All rings were primed with low dose of phenylephrine (1 O-8 M) and then given L-NMMA (lo-4 M, 16 pulmonary arterial and 16 aortic) or L-NNA (2 x 10-S M, 12 pulmonary arterial and 6 aortic). Control rings (19 pulmonary arterial and 14 aortic) received no arginine analogs. The use of phenylephrine was based on the observation that in the absence of catecholamine priming there was no demonstrable effect of the arginine analogs on vascular tone, thus making it difficult to know when “enough” analog had been given. The dose of phenylephrine was the smallest which consistently permitted a subsequent arginine-analoginduced constrictor response. Once the L-NMMA or L-NNA constriction had plateaued (after 15 minutes), rings were given norepinephrine (10-S M ) . Norepinephrine was used to preconstrict the rings since phenylephrine was an effective “primer” but, at the doses used, did not cause a sustained constriction. When the norepinephrine constriction plateaued, control and L-NNA-treated pulmonary arterial and aortic rings (n=6/group) were given sodium nitroprusside (lo-6 M). All other rings received A23187 (lo-6 M). To prove that a detectable NO signal was produced by a dose of authentic NO that elicited similar dilation as A231 87, pulmonary arterial and aortic rings (n=12/group) were given dissolved exogenous NO gas (lo-7 M) at the plateau of norepinephrine contraction at the end of the protocol. In a separate set of pulmonary arterial rings, the effects of increasing n = 12), or passively (passive stretch tension actively (by phenylephrine, to 800-2000 mg, n = lo), on vasoconstriction caused by L-NNA were compared. Pulmonary arterial rings denuded by a gentle rubbing with a the endothelium dependence of metal probe were used to study constrictions induced by L-NMMA (n = 5) and L-NNA (n = 6). NO measurement. NO was measured using a chemiluminescence assay (Sievers Research, Boulder, CO) which detects the light emitted by the interaction of ozone (03) with NO (6). Samples were injected into a vacuum chamber held at 3 mm Hg where NO was “stripped” into the head space by bubbling it with helium (12 ml/min) for 30 seconds. The gas was 591
Vol.
188,
No.
2,
1992
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
drawn into the analyzer and mixed with 03 (generated internally by electrostatic discharge) in front of a cooled Hamamatsu, red-sensitive photomultiplier tube. A red filter was interposed between the reaction chamber and the photomultiplier tube to eliminate chemiluminescence due to volatile sulfides. Calibration of NO assay. To study the effects of 02 tension and the delay from collection to measurement, on NO detection, 100 ~1 of NO was oxygenated Earl’s solution or dissolved in either deoxygenated water, oxygenated Earl’s solution from a bath containing pulmonary arterial rings. All samples were drawn up in N2-flushed, air-tight syringes. Samples (n=2/group) were measured immediately and after sitting 5 minutes at room temperature. Drugs. All reagents were from Sigma, St. Louis, MO except of LNMMA (Calbiochem, La Jolla, CA) and NO gas (Matheson, Joliet, IL). They were dissolved in normal saline except for A23187 and L-NNA (DMSO and 0.4 N HCl, respectively). Statistics. Values are the meankstandard error of the mean. A 2tailed, unpaired Student’s t-test or l-factor ANOVA with Fisher PLSD test were used as appropriate. A ~~0.05 was considered statistically significant.
RESULTS The NO analyzer was sensitive to dissolved or gaseous NO in amounts as little as 20 pmol. Samples of dissolved NO were found to be stable in an airtight syringe for at least 5 minutes at room temperature. The mean chemiluminescence signals produced at 30 seconds and 5 minutes by a 100 pl of gaseous NO were 6.73 and 6.85 mV. When the same dose of NO was dissolved in deoxygenated Earl’s solution or in a bath containing arterial rings, the 30 second and 5 minute measurements were 6.91 and 5.32 mV and, respectively, 6.39 and 5.65 mV. Exogenous NO caused a rapid fall in tension of both pulmonary arterial (-12k3 %) and aortic (-19k3 %) rings. The NO chemiluminescence signal rose from 0 to 79.0& 10.6 mV in pulmonary arterial and to 127.7k23.4 mV in aortic rings. Endogenous NO synthesis was significantly elevated in aortic and pulmonary arterial rings treated with L-NMMA, compared to control or LNNA rings, before as well as after A23187 (Fig. 1A). L-NNA did not change the NO chemiluminescence compared to control rings (Fig. 1A). The percentage of rings displaying NO signal was higher in pulmonary arterial and aortic rings given L-NMMA than in control or L-NNA rings (Fig. 1B). L-NMMA and L-NNA increased tone of phenylephrine-preconstricted pulmonary arterial and aortic rings (Table 1) while the reactivity to norepinephrine was not affected (Table 2). L-NNA caused much less pulmonary arterial constriction when the rings were not primed with phenylephrine (Table 3). Passive stretching was not as effective as 592
Vol.
188, No. 2, 1992
BIOCHEMICAL
AND BIOPHYSICAL
: m’A + E ‘6. t l--J
- x-
d
8
12.
xi .r
6.
RESEARCH COMMUNICATIONS
Control l-NMMA L-NNA t
*
5 =
J’ -
-d
E 2
4
x-r _---
0
Before After PA rings
4
Before Aortic
After rings
loo6
z a
60.
;
60.
P '5
40.
2 u)
20.
P 'C
‘/
I x . /
0
6
.
#' 7
>'
'
Before Aortic
Before After PA rings
8
,' ,'
After rings
re 1; L-NMMA increase the magnitude (A) and prevalence (B ) of nitric oxide synthesis in arterial rings. PA=pulmonary arterial. The values are meanGEM of NO level (panel A) and % of rings that produced any NO (panel B). n=lO-18/group, except for aortic L-NNA group, where n=6. “Before” and “After”=before and after A23 187 (lo-6 M) administration, respectively. *pcO.O5 compared to control rings and to rings treated with L-NNA. t pcO.05 compared to the value before A23187 in L-NMMA treated pulmonary arterial rings.
phenylephrine in “priming” the vessels prior to arginine analogs, despite the small constrictor response to phenylephrine (resting tension of 800 mg elevated by phenylephrine to 823klO mg). The increases in tension caused
TABLE
1:
Constriction of to arginine
RING
DRUG
Aortic
L-NMMA L-NNA L-NMMA L-NNA
PA
n 10 6 10 12
arterial analogs
STARTING
rings
TENSION [mg] 2308+ 100 2007f7 8 872k2 9 823k 10
in
response
% A TENSION 13f3 3Ok4 * 24k7 32k3
PA=pulmonary arterial. Values are mean&SEM. Increase in tension caused by L-NMMA (lo-4 M) or L-NNA (2x10-5 M) is expressed as % of starting tension (after priming with 10-S M phenylephrine). *pO.O5).
TABLE 3: arginine
Constrictor analogs
ENDOTHELIUM Intact Intact Denuded Intact Denuded
response depends
PRIMING None PE PE PE PE
of pulmonary arterial on phenylephrine-priming
DRUG L-NNA L-NNA L-NNA L-NMMA L-NMMA
A TENSION +146zt3 8 * +294*2 9 +282+9 2 +366+6 2 +4Ok23
rings
[mg]
to
n 10 12 6 20 5
Rings which were “primed” received 3.1x10-s M phenylephrine (PE) prior to administration of L-NNA (2x10-5M) or L-NMMA (10-d M). Denuded rings were unresponsive to acetylcholine (lo-6 M) after a gentle rubbing with a metal probe. Values are the mean&EM. *pcO.O5 compared to PE-primed rings receiving L-NNA. 594
Vol.
188,
No.
2,
1992
BIOCHEMICAL
AND
6
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
p < 0.05
z; 200 2
180 0 =160 .g 140 Zl20 a g 100 g
80
i
60
_-----_--__--__________
40 s a,
20
E 's
0
8
(il=lO)
(Il=12)
(Il=lO)
PA rings
Aortic
(n=@
rings
Relaxation to A23187 is not inhibited by L-NMMA in pulmonary arterial rings. In control pulmonary arterial (PA) and aortic rings, A23187 (10-6 M) decreased the tension by 117 mg and 346 mg, respectively, and these values are taken as 100 % for this graph (indicated by the broken line). The mean+SEM of relaxation of aortic and PA rings to A23 187 after LNMMA (lo-4 M) and L-NNA (2 x 10-S M) are shown.
DISCUSSION There are few specific inhibitors in biochemistry and hypotheses are strongest if based on direct measurements. Although the arginine analogs are frequently touted as “specific” inhibitors of NO synthesis, few studies have actually measured NO synthesis or correlated NO levels and vascular tone in physiologic experiments (1, 3-6). In the current study L- N M MA 1). This finding is not entirely unexpected promoted NO synthesis (Fig. since endothelial cells in culture are reported to be able to metabolize LNMMA to L-citrulline which, in turn, can be used as a precursor for NO synthesis (8). Our study is the first to directly confirm that L-NMMA can enhance NO synthesis. We speculate that the relative inability of L-NM MA to inhibit vasodilation induced by A23187 (Fig. 2) and acetylcholine (6) in pulmonary artery compared to aortic rings occurs because the pulmonary vasculature can metabolize L-NMMA to NO more readily than aortic endothelium. Indeed, our results show that more NO was produced in response to L-NMMA in pulmonary arterial than in aortic rings. These experiments found evidence that the arginine analogs may in arterial rings by NO-independent mechanism. elicit constriction Constriction of aortic and pulmonary arterial rings in response to arginine analogs only took place if the rings were “primed” by phenylephrine; passive increase in tension was much less effective. This finding confirms the results of Crawley et al. (9) in the isolated rat and human pulmonary of this fact could be that arteries. The possible interpretation 595
Vol.
188,
No.
2,
1992
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
elicited endothelium-derived phenylephrine-induced increase in tension relaxing factor production. Inhibition of endothelium-derived relaxing factor synthesis by the arginine analogs then allowed full expression of the However, this seems unlikely for vasoconstrictor effect of phenylephrine. at least two reasons. First, the constrictor response to norepinephrine was not significantly different between the control rings and those treated with L-NMMA or L-NNA (Table 3). Second, we did not observe any increase in NO chemiluminescence in response to phenylephrine. Increased cytosolic calcium would facilitate most types of vasoconstriction. Phenylephrine might have been acting through its ability to increase intracellular calcium. It is possible that vasoconstriction of arterial rings to L-NMMA and L- NN A occurs by an endothelium-derived relaxing factor-independent mechanism. This is supported by the persistence of I.,-NNA -induced contraction in denuded rings. The need for pharmacologic priming of rings to Conclusion: demonstrate constriction in response to arginine analogs and the persistence of L-NNA-induced contraction in denuded rings suggest that arginine analogs have a direct vasoconstrictor effect on vascular smooth muscle, unrelated to NO synthesis inhibition. L-NMMA is a partial agonist for NO synthesis. These data do not refute the NO hypothesis but illustrate the need to measure NO when possible, rather than rely on the effect of arginine analogs alone. REFERENCES 1. Rees, D.D., Palmer, R.M.J., Hodson, H.F., and Moncada, S. (1989) Br J Pharmacol 96, 418-424. 2. Ishii, K., Chang, B., Kerwin, J.F., Huang, Z.-J., and Murad, F. (1990) Eur J Pharmacol 176, 219-223. 3. Ignarro, L.J., Buga, G.M., Wood, K.S., Byms, R.E., and Chaudhuri, G. (1987) Proc. Natl. Acad. Sci. 84, 9265-9269. 4. Chen, W.Z., Palmer, R.M.J., and Moncada, S. (1989) J Vast Med Biol 1, l-6. 5. Amezcua, J.L., Palmer, R.M.J., de Souza, B.M., and Moncada, S. (1989) Br J Pharmacol 97, 1019-1024. 6. Archer, S.L., and Cowan, N.J. (1991) Circ Res 68, 1569-1581. 7. Archer, S.L., Rist, K., Nelson, D.P., DeMaster, E.G., Cowan, N., and Weir, E.K. (1990) J Appl Physiol 68, 735-747. 8. Hecker, M., Mitchell, J.A., Harris, Vane, J.R. (1990) Biochem. Biophys.
H.J., Katsura, M., Thiemermann, Res. Commun. 167, 1037-1043.
C., and
9. Crawley, D.E., Liu, S.F., Evans, T.W., and Barnes, P.J. (1990) Br J Pharmacol 101, 166-170.
596
Vol. 188, No. 2, 1992 October 30, 1992
NG-MONOMETHYL-L-ARGININE IN ISOLATED ARTERIAL Stephen
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS Pages 590-596
CAUSES NITRIC RINGS: TROUBLE
L. Archer
and Va’clav
OXIDE SYNTHESIS IN PARADISE*
Hampl
Division of Cardiology, Veterans Administration Medical Center University of Minnesota, Minneapolis, Minnesota 55417
Received
August
31,
and
1992
SUMMARY. Arginine analogs are commonly used as inhibitors of the synthesis of endothelium-derived relaxing factor, nitric oxide. However, their effect on nitric oxide levels is rarely measured. Using a chemiluminescence assay for nitric oxide, we found that NG-monomethylL-arginine enhanced, rather than reduced, nitric oxide synthesis in pulmonary arterial and aortic rings. NG-monomethyl-L-arginine inhibited relaxation to the endothelium-dependent vasodilator A23187 in aortic but not pulmonary arterial rings. In contrast, No-nitro-L-arginine did not stimulate nitric oxide synthesis and it inhibited relaxation to A23 187 in all rings. We conclude that NG-monomethyl-L-arginine is a partial agonist for nitric oxide synthesis. 0 1992Academic Press,Inc.
Arginine analogs, such as NG-monomethyl-L-arginine (L-NMMA) and NW-nitro-L-arginine (L-NNA), inhibit endothelium-dependent vasodilation in systemic vessels (1, 2). Few studies have documented the inhibitory effect of arginine analogs on the synthesis of nitric oxide (NO, endotheliumderived relaxing factor) (1, 3-6). Nonetheless, arginine analogs are widely utilized as “specific” inhibitors of NO synthesis. In fact, much of the knowledge of NO physiology and pathophysiology is based on experiments using L-NMMA. It is often assumed that any hemodynamic effect of arginine analogs results from NO synthesis inhibition; this may not always be true. In the present study we measured the effect of L -NMMA and LNNA on NO production by arterial rings. As the role of endothelium*Supported by the Department of Veterans Affairs, Minnesota Medical Foundation, NIH grant # lR29-HL45735-1, and the Proshek Foundation. . . brevw : L-NMMA, NG-monomethyl-L-arginine; L-NNA, Nm-nitroL-arginine. 0006-291X/92 $4.00 Copyright 0 I992 by Academic Press, All rights of reproduction in any form
Inc. reserved.
590
Vol.
188,
No.
2,
1992
BIOCHEMICAL
derived relaxing factor in the compared the effects of arginine rings.
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
pulmonary circulation is not clear, we analogs on aortic and pulmonary arterial
METHODS Abdominal aorta and second division right pulmonary artery rings were dissected from anesthetized (pentobarbital 50 mg/kg, IP) adult, male, specific pathogen free, Sprague Dawley rats (7). Rings were mounted on stainless steel wires attached to Grass UC-2 force-displacement transducers and suspended in a 75 ml bath containing Earl’s balanced salt solution (37.5OC, pH=7.43, PG2=130-140 mm Hg). The rings (2/bath) were held 1 cm apart and samples for NO analysis (2 ml) were withdrawn midway between the vessels using a gas-tight, N2-flushed syringe. Rings equilibrated for 30 minutes at resting tensions of 800 mg and 2000 mg for pulmonary arterial and aortic rings, respectively. Superoxide dismutase (2000 units) was given because it enhances NO detection (6), presumably by increasing NO survival. Protocol. All rings were primed with low dose of phenylephrine (1 O-8 M) and then given L-NMMA (lo-4 M, 16 pulmonary arterial and 16 aortic) or L-NNA (2 x 10-S M, 12 pulmonary arterial and 6 aortic). Control rings (19 pulmonary arterial and 14 aortic) received no arginine analogs. The use of phenylephrine was based on the observation that in the absence of catecholamine priming there was no demonstrable effect of the arginine analogs on vascular tone, thus making it difficult to know when “enough” analog had been given. The dose of phenylephrine was the smallest which consistently permitted a subsequent arginine-analoginduced constrictor response. Once the L-NMMA or L-NNA constriction had plateaued (after 15 minutes), rings were given norepinephrine (10-S M ) . Norepinephrine was used to preconstrict the rings since phenylephrine was an effective “primer” but, at the doses used, did not cause a sustained constriction. When the norepinephrine constriction plateaued, control and L-NNA-treated pulmonary arterial and aortic rings (n=6/group) were given sodium nitroprusside (lo-6 M). All other rings received A23187 (lo-6 M). To prove that a detectable NO signal was produced by a dose of authentic NO that elicited similar dilation as A231 87, pulmonary arterial and aortic rings (n=12/group) were given dissolved exogenous NO gas (lo-7 M) at the plateau of norepinephrine contraction at the end of the protocol. In a separate set of pulmonary arterial rings, the effects of increasing n = 12), or passively (passive stretch tension actively (by phenylephrine, to 800-2000 mg, n = lo), on vasoconstriction caused by L-NNA were compared. Pulmonary arterial rings denuded by a gentle rubbing with a the endothelium dependence of metal probe were used to study constrictions induced by L-NMMA (n = 5) and L-NNA (n = 6). NO measurement. NO was measured using a chemiluminescence assay (Sievers Research, Boulder, CO) which detects the light emitted by the interaction of ozone (03) with NO (6). Samples were injected into a vacuum chamber held at 3 mm Hg where NO was “stripped” into the head space by bubbling it with helium (12 ml/min) for 30 seconds. The gas was 591
Vol.
188,
No.
2,
1992
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
drawn into the analyzer and mixed with 03 (generated internally by electrostatic discharge) in front of a cooled Hamamatsu, red-sensitive photomultiplier tube. A red filter was interposed between the reaction chamber and the photomultiplier tube to eliminate chemiluminescence due to volatile sulfides. Calibration of NO assay. To study the effects of 02 tension and the delay from collection to measurement, on NO detection, 100 ~1 of NO was oxygenated Earl’s solution or dissolved in either deoxygenated water, oxygenated Earl’s solution from a bath containing pulmonary arterial rings. All samples were drawn up in N2-flushed, air-tight syringes. Samples (n=2/group) were measured immediately and after sitting 5 minutes at room temperature. Drugs. All reagents were from Sigma, St. Louis, MO except of LNMMA (Calbiochem, La Jolla, CA) and NO gas (Matheson, Joliet, IL). They were dissolved in normal saline except for A23187 and L-NNA (DMSO and 0.4 N HCl, respectively). Statistics. Values are the meankstandard error of the mean. A 2tailed, unpaired Student’s t-test or l-factor ANOVA with Fisher PLSD test were used as appropriate. A ~~0.05 was considered statistically significant.
RESULTS The NO analyzer was sensitive to dissolved or gaseous NO in amounts as little as 20 pmol. Samples of dissolved NO were found to be stable in an airtight syringe for at least 5 minutes at room temperature. The mean chemiluminescence signals produced at 30 seconds and 5 minutes by a 100 pl of gaseous NO were 6.73 and 6.85 mV. When the same dose of NO was dissolved in deoxygenated Earl’s solution or in a bath containing arterial rings, the 30 second and 5 minute measurements were 6.91 and 5.32 mV and, respectively, 6.39 and 5.65 mV. Exogenous NO caused a rapid fall in tension of both pulmonary arterial (-12k3 %) and aortic (-19k3 %) rings. The NO chemiluminescence signal rose from 0 to 79.0& 10.6 mV in pulmonary arterial and to 127.7k23.4 mV in aortic rings. Endogenous NO synthesis was significantly elevated in aortic and pulmonary arterial rings treated with L-NMMA, compared to control or LNNA rings, before as well as after A23187 (Fig. 1A). L-NNA did not change the NO chemiluminescence compared to control rings (Fig. 1A). The percentage of rings displaying NO signal was higher in pulmonary arterial and aortic rings given L-NMMA than in control or L-NNA rings (Fig. 1B). L-NMMA and L-NNA increased tone of phenylephrine-preconstricted pulmonary arterial and aortic rings (Table 1) while the reactivity to norepinephrine was not affected (Table 2). L-NNA caused much less pulmonary arterial constriction when the rings were not primed with phenylephrine (Table 3). Passive stretching was not as effective as 592
Vol.
188, No. 2, 1992
BIOCHEMICAL
AND BIOPHYSICAL
: m’A + E ‘6. t l--J
- x-
d
8
12.
xi .r
6.
RESEARCH COMMUNICATIONS
Control l-NMMA L-NNA t
*
5 =
J’ -
-d
E 2
4
x-r _---
0
Before After PA rings
4
Before Aortic
After rings
loo6
z a
60.
;
60.
P '5
40.
2 u)
20.
P 'C
‘/
I x . /
0
6
.
#' 7
>'
'
Before Aortic
Before After PA rings
8
,' ,'
After rings
re 1; L-NMMA increase the magnitude (A) and prevalence (B ) of nitric oxide synthesis in arterial rings. PA=pulmonary arterial. The values are meanGEM of NO level (panel A) and % of rings that produced any NO (panel B). n=lO-18/group, except for aortic L-NNA group, where n=6. “Before” and “After”=before and after A23 187 (lo-6 M) administration, respectively. *pcO.O5 compared to control rings and to rings treated with L-NNA. t pcO.05 compared to the value before A23187 in L-NMMA treated pulmonary arterial rings.
phenylephrine in “priming” the vessels prior to arginine analogs, despite the small constrictor response to phenylephrine (resting tension of 800 mg elevated by phenylephrine to 823klO mg). The increases in tension caused
TABLE
1:
Constriction of to arginine
RING
DRUG
Aortic
L-NMMA L-NNA L-NMMA L-NNA
PA
n 10 6 10 12
arterial analogs
STARTING
rings
TENSION [mg] 2308+ 100 2007f7 8 872k2 9 823k 10
in
response
% A TENSION 13f3 3Ok4 * 24k7 32k3
PA=pulmonary arterial. Values are mean&SEM. Increase in tension caused by L-NMMA (lo-4 M) or L-NNA (2x10-5 M) is expressed as % of starting tension (after priming with 10-S M phenylephrine). *pO.O5).
TABLE 3: arginine
Constrictor analogs
ENDOTHELIUM Intact Intact Denuded Intact Denuded
response depends
PRIMING None PE PE PE PE
of pulmonary arterial on phenylephrine-priming
DRUG L-NNA L-NNA L-NNA L-NMMA L-NMMA
A TENSION +146zt3 8 * +294*2 9 +282+9 2 +366+6 2 +4Ok23
rings
[mg]
to
n 10 12 6 20 5
Rings which were “primed” received 3.1x10-s M phenylephrine (PE) prior to administration of L-NNA (2x10-5M) or L-NMMA (10-d M). Denuded rings were unresponsive to acetylcholine (lo-6 M) after a gentle rubbing with a metal probe. Values are the mean&EM. *pcO.O5 compared to PE-primed rings receiving L-NNA. 594
Vol.
188,
No.
2,
1992
BIOCHEMICAL
AND
6
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
p < 0.05
z; 200 2
180 0 =160 .g 140 Zl20 a g 100 g
80
i
60
_-----_--__--__________
40 s a,
20
E 's
0
8
(il=lO)
(Il=12)
(Il=lO)
PA rings
Aortic
(n=@
rings
Relaxation to A23187 is not inhibited by L-NMMA in pulmonary arterial rings. In control pulmonary arterial (PA) and aortic rings, A23187 (10-6 M) decreased the tension by 117 mg and 346 mg, respectively, and these values are taken as 100 % for this graph (indicated by the broken line). The mean+SEM of relaxation of aortic and PA rings to A23 187 after LNMMA (lo-4 M) and L-NNA (2 x 10-S M) are shown.
DISCUSSION There are few specific inhibitors in biochemistry and hypotheses are strongest if based on direct measurements. Although the arginine analogs are frequently touted as “specific” inhibitors of NO synthesis, few studies have actually measured NO synthesis or correlated NO levels and vascular tone in physiologic experiments (1, 3-6). In the current study L- N M MA 1). This finding is not entirely unexpected promoted NO synthesis (Fig. since endothelial cells in culture are reported to be able to metabolize LNMMA to L-citrulline which, in turn, can be used as a precursor for NO synthesis (8). Our study is the first to directly confirm that L-NMMA can enhance NO synthesis. We speculate that the relative inability of L-NM MA to inhibit vasodilation induced by A23187 (Fig. 2) and acetylcholine (6) in pulmonary artery compared to aortic rings occurs because the pulmonary vasculature can metabolize L-NMMA to NO more readily than aortic endothelium. Indeed, our results show that more NO was produced in response to L-NMMA in pulmonary arterial than in aortic rings. These experiments found evidence that the arginine analogs may in arterial rings by NO-independent mechanism. elicit constriction Constriction of aortic and pulmonary arterial rings in response to arginine analogs only took place if the rings were “primed” by phenylephrine; passive increase in tension was much less effective. This finding confirms the results of Crawley et al. (9) in the isolated rat and human pulmonary of this fact could be that arteries. The possible interpretation 595
Vol.
188,
No.
2,
1992
BIOCHEMICAL
AND
BIOPHYSICAL
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elicited endothelium-derived phenylephrine-induced increase in tension relaxing factor production. Inhibition of endothelium-derived relaxing factor synthesis by the arginine analogs then allowed full expression of the However, this seems unlikely for vasoconstrictor effect of phenylephrine. at least two reasons. First, the constrictor response to norepinephrine was not significantly different between the control rings and those treated with L-NMMA or L-NNA (Table 3). Second, we did not observe any increase in NO chemiluminescence in response to phenylephrine. Increased cytosolic calcium would facilitate most types of vasoconstriction. Phenylephrine might have been acting through its ability to increase intracellular calcium. It is possible that vasoconstriction of arterial rings to L-NMMA and L- NN A occurs by an endothelium-derived relaxing factor-independent mechanism. This is supported by the persistence of I.,-NNA -induced contraction in denuded rings. The need for pharmacologic priming of rings to Conclusion: demonstrate constriction in response to arginine analogs and the persistence of L-NNA-induced contraction in denuded rings suggest that arginine analogs have a direct vasoconstrictor effect on vascular smooth muscle, unrelated to NO synthesis inhibition. L-NMMA is a partial agonist for NO synthesis. These data do not refute the NO hypothesis but illustrate the need to measure NO when possible, rather than rely on the effect of arginine analogs alone. REFERENCES 1. Rees, D.D., Palmer, R.M.J., Hodson, H.F., and Moncada, S. (1989) Br J Pharmacol 96, 418-424. 2. Ishii, K., Chang, B., Kerwin, J.F., Huang, Z.-J., and Murad, F. (1990) Eur J Pharmacol 176, 219-223. 3. Ignarro, L.J., Buga, G.M., Wood, K.S., Byms, R.E., and Chaudhuri, G. (1987) Proc. Natl. Acad. Sci. 84, 9265-9269. 4. Chen, W.Z., Palmer, R.M.J., and Moncada, S. (1989) J Vast Med Biol 1, l-6. 5. Amezcua, J.L., Palmer, R.M.J., de Souza, B.M., and Moncada, S. (1989) Br J Pharmacol 97, 1019-1024. 6. Archer, S.L., and Cowan, N.J. (1991) Circ Res 68, 1569-1581. 7. Archer, S.L., Rist, K., Nelson, D.P., DeMaster, E.G., Cowan, N., and Weir, E.K. (1990) J Appl Physiol 68, 735-747. 8. Hecker, M., Mitchell, J.A., Harris, Vane, J.R. (1990) Biochem. Biophys.
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