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Substance P increases cyclic GMP levels on coronary postcapillary venular endothelial cells
Life Sciences, Vol. 53, pp. PL 229-234 Printed in the USA
Pergamon Press
SUBSTANCE P INCREASESCYCLIC GMP LEVELS ON CORONARY POSTCAPILLARY VENULAR ENDOTHEUAL CELLS
M. Ziche 1, L. Morbidelli, A. Parenti, S. Amerini, H.J. Granger*, and C.A. Maggi** Department of Pharmacology, University of Florence, Florence, Italy *Microcirculation Research Institute, Department of Medical Physiology, Texas A&M University Health Science Center, College Station, Texas, USA **Pharmacol. Dept., A. Menarini Pharmaceuticals, Florence, Italy (Submitted June 18, 1993; accepted July 19, 1993; received in final form July 26, 1993)
Abstract. The vasodilating effect of substance P (SP) at the microvascular level is endotheliumdependent. In the present study we evaluated whether SP activates nitric oxide (NO) production by venular endothelial cell. We evaluated NO activation by measuring cyclic GMP levels in cultured endothelial cells isolated from coronary postcapillary venules of bovine origin (CVEC). Our results indicate that 5 min exposure of CVEC to 10 nlM SP doubled basal cyclic GMP levels. Cell treatment with the NO synthase inhibitor L-NMMA reduced the basal levels of cyclic GMP and abolished the effect of SP but did not modify the increase in cyclic GMP in response to exogenous NO. These data indicate that a) microvascular endothelium responds in an autocrine fashion to NO with increased cyclic GMP levels, b) SP activates cyclic GMP pathway through NO production.
Substance P (SP) has been proposed as a main mediator of neurogenic inflammation leading to vasodilation and increased vascular permeability (1). The vasorelaxant response to SP is dependent on the presence of endothelium and is mediated by NK1 receptors (2, 3). Endotheliumdependent relaxation has been clearly demonstrated to be caused by an endothelium-derived relaxing factor identified as nitric oxide (NO) (4, 5). We have reported that SP promotes angiogenesis in vivo and in vitro (6, 7). Recently we also reported that NO promotes endothelial cells-proliferation in vitro (8). In the present study we evaluated whether SP activates NO production at the endothelial cell level. We evaluated NO activation by measuring cyclic GMP levels in cultured endothelial cells isolated from coronary postcapillary venules of bovine origin (CVEC) (9). These cells were selected because postcapillary venules are believed to be a main site of action of SP in producing increase in vascular permeability and are actively involved in the angiogenesis process (1 O, 1 1 ). Materials and Methods Materials: Substance P was from Peninsula. Sodium nitroprusside (SNP) was used as NOgenerating drug. SNP, the NO synthase inhibitor NC°-monomethyI-L-arginine indomethacin, 3'-isobutyl-l-methylxantine
(L-NMMA),
(IBMX), prostaglandin E1 (PGE1) and superoxide
1Marina Ziche, MD, Department of Pharmacology, University of Florence, Viale Morgagni 65, 50134 Florence Italy 0024-3205/93 $6.00 + .00 Copyright © 1993 Pergamon Press Ltd All rights reserved.
PL-230
Substance P on Cyclic GMP I_~vcls
dismutase (SOD) were purchased from Sigma.
Vol. 53, No. 14, 1993
Tri-n-octylamine and 1,1,2 trichloro-
trifluoroethane were purchased from Aldrich. Fresh solutions of SNP, L-NMMA and SOD were prepared before each experiment as 10 mM solutions in PBS. Indomethacin (10 mM) was prepared in absolute ethanol and then diluted in PBS. IBMX (10 mM) was dissolved in 0.01 M NaOH and then diluted in PBS. Cell culture reagents were from Gibco. Fetal bovine serum (FBS) was from Hyclone. Cell culture plastic was from Costar. Cell line and culture conditions: Coronary venular endothelial cells (CVEC) were isolated from bovine heart as previously described (9). The endothelial identity of the cells was determined by immunofluorescence detection of factor VIII-related antigen and by electron microscopy. Cells were cloned and maintained in culture in Dulbecco's modified Eagle's medium (DMEM) supplemented with 2 mM sodium pyruvate, 2 mM L-glutamine, 100 pg/ml heparin, antibiotics (100 U/ml penicillin, 100 pg/ml streptomycin, 0.25 pg/ml amphotericin) and 20% FBS. Cells were cloned and each clone was subcultured up to a maximum of PS passages. Passages between 15 and 20 were used in these experiments. Cyclic GMP measurements: To evaluate cyclic GMP and AMP accumulation CVEC were plated in 100 mm diameter dishes and allowed to grow to 90% confluence (about 5x106 final cell number). Stimulation was carried out in PBS with calcium and magnesium. Cell monolayers were pretreated for 30 min with 10 pM indomethacin and 50 pM IBMX, to block cyclo-oxygenase and phosphodiesterase activity, respectively. Some dishes were further treated with 200 pM LNMMA for 1 h. Where required, SOD (60 U/ml) was used. After removal of inhibitors cells were stimulated with test substances for 5 min. At the end of incubation cells were washed and scraped off in 1 ml ice-cold PBS. After centrifugation at 1,500 rpm for 5 min, pellets were dissolved in 500 /~1 of ice-cold
TCA (10%, wt/vol) and the samples were stored at -20 ° C. TCA was
neutralized with 0.5 M tri-n-octylamine dissolved in 1,1,2 trichloro-trifluoroethane. The levels of cyclic GMP and cyclic AMP in the aqueous phase were measured
by commercially
available radioimmunoassays in duplicate with iodinated tracers from Amersham, following the procedure previously reported (11 ). Cell proteins were assayed by the method of Bradford (12). The cyclic GMP and cyclic AMP contents of each dish were expressed as fmol/mg protein/lO 6 cells. Statistical analysis: Results are expressed as means+sem for (n) experiments in duplicate. Differences between groups were tested for significance by Student's t-test for paired and unpaired data and P value of <0.05 was taken as significant. Results The ability of CVEC to respond to NO by increasing cyclic GMP levels was assessed by exposing CVEC to the NO-donor drug, sodium nitroprusside (SNP) (13). After 30 min incubation with
Vol. 53, No. 14, 1993
Substance P on Cyclic G M P Levels
PL-231
IBMX (50 pM) and indomethacin (10 pM), to block phosphodiesterase and cyclo-oxygenase activity, respectively, the cells were exposed to SP and SNP for 5 rain. The concentrations used were 10 pM for SNP and 10 nM for SP, respectively,
since
maximal biological activity was
obtained in capillary endothelial cells at these concentrations (6, 7, 8). In resting conditions
cyclic GMP levels in CVEC were 124.6_+2 fmol/mg protein/106 cells. Cyclic GMP was doubled cyclic GMP
when CVEC were exposed to 10 pM of SNP. Following 5 min exposure to 10 nM SP
levels increased by 6 1 % (Fig. 1). When SOD was added to the incubation medium to prolong NO half-life,
cyclic GMP levels induced by SP and SNP were further increased.
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SNP 10
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FIG.1 Effect of SP on cyclic GMP levels in CVEC. Cells were exposed to SP for 5 min. SNP was used as a direct source of NO to challenge cyclic GMP activation on CVEC. SOD (60 U/ml) was added to prolong NO half-life (dashed bars). Data are means+sere obtained from 3 experiments in duplicate. * P
PL-232
Substance P on Cyclic GMP Levels
Vol. 53, No. 14, 1993
produced by exogenous administration of NO, obtained with SNP, was not affected by L-NMMA treatment (Fig. 2).
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SNP 10 pM
FIG.2 Cyclic GMP levels in CVEC exposed to SP and to SNP following L-NMMA treatment. Pretreatment with L-NMMA (200 pM) (dashed columns) was done for 1 h. Data are means+sem from 3 experiments run in duplicate. ** P
vs 2 1 6 + 4 8
fmol/mg
protein/1 06 cells). Discussion
The increase in vascular permeability produced by SP occurs at the postcapillary level (1) and has been related to local NO production (15). The cells used in this study were
obtained
from the microvascular endothelium which is the site of action of SP and of a large number of inflammatory and immune mediators actively involved in the healing process (1 O, 11 ). In the vascular tree, NO is mainly released by endothelial cells and exerts its effect by activating guanylate cyclase on smooth muscle cells thus favouring vasoretaxation (16).
Vol. 53, No. 14, 1993
Substance P on Cyclic GMP Levels
PL-233
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SP 10 nM
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FIG.3 Cyclic AMP levels in CVEC exposed to SP and to SNP. Data are expressed as means_+sem from 3 experiments run in duplicate. * P
cyclic GMP production induced by SP
indicating NO production as mediator of the response to SP. Other reports suggested that SP-
PL-234
Substance P on Cyclic GMP levels
Vol. 53, No. 14, 1993
induced endothelium-dependent vasodilation and plasma extravasation in vivo could be modulated by NO production at the microvascular level (15). Our results indicate for the first time that cyclic GMP pathway can be directly activated by SP in postcapillary endothelial cells. We also detected a slight increase in intracellular cyclic AMP levels following SP exposure, suggesting that adenylate cyclase could also be activated by SP in CVEC. Since NK1 receptors have been indicated as responsible for SP effects at the microvascular level (6, 7), it is interesting to speculate whether the cyclic GMP pathway could be a specific transduction mechanism of SP for the NK1 receptor in endothelial cells. More experiments utilizing selective NK1 receptor antagonists and agonists are needed to clarify this point.
Acknowledqments This work was supported by funds from Italian Ministry for the University and for Scientific and Technological Research and by the National Research Council of Italy. A. Parenti was a recipient of a Menarini fellowship. References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16.
F. LEMBECK and P. HOLZER, Naunyn-Schmiedeberg's Arch. Pharmacol. 310 176-183 (1979). P. D'ORLEANS-JUSTE, S. DION, G. DRAPEAU and D. REGOLI, Eur. J. Pharmacol. 125 37-44 (1985). P. D'ORLEANS-JUSTE, S. DION,J. MIZRAHI and D. REGOLI, Eur. J. Pharmacol. 114 9-21 (1985). L.J. IGNARRO, G.M. BUGA, K.S. WOOD, R.E. BYRNS and G. CHAUDHURI, Proc. Natl. Acad. Sci. USA 84 9265-9269 (1987). R.M.J. PALMER, A.G. FERRIGE and S. MONCADA, Nature 327 524-526 (1987). M. ZlCHE, L. MORBIDELLI, M. PACINI, P. GEPPETTI, G. ALESSANDRI and C.A. MAGGI, Microvasc. Res. 40 264-268 (1990). M. ZICHE, L. MORBIDELLI, P. GEPPETFI, C.A. MAGGI and P. DOLARA, Life Sci. 48 PL7PL11 (1991). M. ZICHE, L. MORBIDELLI, E. MASINI, H.J. GRANGER, P.GEPPETTI and F. LEDDA, Biochem. Biophys. Res. Commun. 792 1198-1203 (1993). M.E. SCHELLING, C.J. MEININGER, J.R. HAWKER and H.J. GRANGER, Am. J. Physiol. 2 5 4 H1211-H1217 (1988). L. MORBIDELLI, A. PARENTI, H.J. GRANGER, M. ZICHE and F. LEDDA, Pharmacol. Res. 25 150-151 (1992). E. MASINI, P.F. MANNAIONI, A. PISTELLI, D. SALVEMINI and J. VANE, Biochem. Biophys. Res. Commun. 177 1178-1182 (1991). M. BRADFORD, Anal. Biochem. 72 248-253 (1976). M. FEELISH and E.A. NOACK, Eur. J. Pharmacol. 139 19-30 (1987). R.M.J. PALMER, D.D. REES, D.S. ASHTON and S. MONCADA, Biochem. Biophys. Res. Commun. 153 1251-1256 (1988). S.R. HUGHES, T.J. WILLIAMS and S.D. BRAIN, Eur. J. Pharmacol. 191 4 8 1 - 4 8 4 (1990). L.J. IGNARRO, R.G. HARBINSON, K.S. WOOD and P.J. KADOWlTZ, J. Pharmacol. Exp.Ther. 2 3 7 893-900 (1986).
Pergamon Press
SUBSTANCE P INCREASESCYCLIC GMP LEVELS ON CORONARY POSTCAPILLARY VENULAR ENDOTHEUAL CELLS
M. Ziche 1, L. Morbidelli, A. Parenti, S. Amerini, H.J. Granger*, and C.A. Maggi** Department of Pharmacology, University of Florence, Florence, Italy *Microcirculation Research Institute, Department of Medical Physiology, Texas A&M University Health Science Center, College Station, Texas, USA **Pharmacol. Dept., A. Menarini Pharmaceuticals, Florence, Italy (Submitted June 18, 1993; accepted July 19, 1993; received in final form July 26, 1993)
Abstract. The vasodilating effect of substance P (SP) at the microvascular level is endotheliumdependent. In the present study we evaluated whether SP activates nitric oxide (NO) production by venular endothelial cell. We evaluated NO activation by measuring cyclic GMP levels in cultured endothelial cells isolated from coronary postcapillary venules of bovine origin (CVEC). Our results indicate that 5 min exposure of CVEC to 10 nlM SP doubled basal cyclic GMP levels. Cell treatment with the NO synthase inhibitor L-NMMA reduced the basal levels of cyclic GMP and abolished the effect of SP but did not modify the increase in cyclic GMP in response to exogenous NO. These data indicate that a) microvascular endothelium responds in an autocrine fashion to NO with increased cyclic GMP levels, b) SP activates cyclic GMP pathway through NO production.
Substance P (SP) has been proposed as a main mediator of neurogenic inflammation leading to vasodilation and increased vascular permeability (1). The vasorelaxant response to SP is dependent on the presence of endothelium and is mediated by NK1 receptors (2, 3). Endotheliumdependent relaxation has been clearly demonstrated to be caused by an endothelium-derived relaxing factor identified as nitric oxide (NO) (4, 5). We have reported that SP promotes angiogenesis in vivo and in vitro (6, 7). Recently we also reported that NO promotes endothelial cells-proliferation in vitro (8). In the present study we evaluated whether SP activates NO production at the endothelial cell level. We evaluated NO activation by measuring cyclic GMP levels in cultured endothelial cells isolated from coronary postcapillary venules of bovine origin (CVEC) (9). These cells were selected because postcapillary venules are believed to be a main site of action of SP in producing increase in vascular permeability and are actively involved in the angiogenesis process (1 O, 1 1 ). Materials and Methods Materials: Substance P was from Peninsula. Sodium nitroprusside (SNP) was used as NOgenerating drug. SNP, the NO synthase inhibitor NC°-monomethyI-L-arginine indomethacin, 3'-isobutyl-l-methylxantine
(L-NMMA),
(IBMX), prostaglandin E1 (PGE1) and superoxide
1Marina Ziche, MD, Department of Pharmacology, University of Florence, Viale Morgagni 65, 50134 Florence Italy 0024-3205/93 $6.00 + .00 Copyright © 1993 Pergamon Press Ltd All rights reserved.
PL-230
Substance P on Cyclic GMP I_~vcls
dismutase (SOD) were purchased from Sigma.
Vol. 53, No. 14, 1993
Tri-n-octylamine and 1,1,2 trichloro-
trifluoroethane were purchased from Aldrich. Fresh solutions of SNP, L-NMMA and SOD were prepared before each experiment as 10 mM solutions in PBS. Indomethacin (10 mM) was prepared in absolute ethanol and then diluted in PBS. IBMX (10 mM) was dissolved in 0.01 M NaOH and then diluted in PBS. Cell culture reagents were from Gibco. Fetal bovine serum (FBS) was from Hyclone. Cell culture plastic was from Costar. Cell line and culture conditions: Coronary venular endothelial cells (CVEC) were isolated from bovine heart as previously described (9). The endothelial identity of the cells was determined by immunofluorescence detection of factor VIII-related antigen and by electron microscopy. Cells were cloned and maintained in culture in Dulbecco's modified Eagle's medium (DMEM) supplemented with 2 mM sodium pyruvate, 2 mM L-glutamine, 100 pg/ml heparin, antibiotics (100 U/ml penicillin, 100 pg/ml streptomycin, 0.25 pg/ml amphotericin) and 20% FBS. Cells were cloned and each clone was subcultured up to a maximum of PS passages. Passages between 15 and 20 were used in these experiments. Cyclic GMP measurements: To evaluate cyclic GMP and AMP accumulation CVEC were plated in 100 mm diameter dishes and allowed to grow to 90% confluence (about 5x106 final cell number). Stimulation was carried out in PBS with calcium and magnesium. Cell monolayers were pretreated for 30 min with 10 pM indomethacin and 50 pM IBMX, to block cyclo-oxygenase and phosphodiesterase activity, respectively. Some dishes were further treated with 200 pM LNMMA for 1 h. Where required, SOD (60 U/ml) was used. After removal of inhibitors cells were stimulated with test substances for 5 min. At the end of incubation cells were washed and scraped off in 1 ml ice-cold PBS. After centrifugation at 1,500 rpm for 5 min, pellets were dissolved in 500 /~1 of ice-cold
TCA (10%, wt/vol) and the samples were stored at -20 ° C. TCA was
neutralized with 0.5 M tri-n-octylamine dissolved in 1,1,2 trichloro-trifluoroethane. The levels of cyclic GMP and cyclic AMP in the aqueous phase were measured
by commercially
available radioimmunoassays in duplicate with iodinated tracers from Amersham, following the procedure previously reported (11 ). Cell proteins were assayed by the method of Bradford (12). The cyclic GMP and cyclic AMP contents of each dish were expressed as fmol/mg protein/lO 6 cells. Statistical analysis: Results are expressed as means+sem for (n) experiments in duplicate. Differences between groups were tested for significance by Student's t-test for paired and unpaired data and P value of <0.05 was taken as significant. Results The ability of CVEC to respond to NO by increasing cyclic GMP levels was assessed by exposing CVEC to the NO-donor drug, sodium nitroprusside (SNP) (13). After 30 min incubation with
Vol. 53, No. 14, 1993
Substance P on Cyclic G M P Levels
PL-231
IBMX (50 pM) and indomethacin (10 pM), to block phosphodiesterase and cyclo-oxygenase activity, respectively, the cells were exposed to SP and SNP for 5 rain. The concentrations used were 10 pM for SNP and 10 nM for SP, respectively,
since
maximal biological activity was
obtained in capillary endothelial cells at these concentrations (6, 7, 8). In resting conditions
cyclic GMP levels in CVEC were 124.6_+2 fmol/mg protein/106 cells. Cyclic GMP was doubled cyclic GMP
when CVEC were exposed to 10 pM of SNP. Following 5 min exposure to 10 nM SP
levels increased by 6 1 % (Fig. 1). When SOD was added to the incubation medium to prolong NO half-life,
cyclic GMP levels induced by SP and SNP were further increased.
500
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400
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.$
300
=k ffJJff/~
0 L. Q.
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|
200
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Basal
SP 10 nM
SNP 10
pM
FIG.1 Effect of SP on cyclic GMP levels in CVEC. Cells were exposed to SP for 5 min. SNP was used as a direct source of NO to challenge cyclic GMP activation on CVEC. SOD (60 U/ml) was added to prolong NO half-life (dashed bars). Data are means+sere obtained from 3 experiments in duplicate. * P
PL-232
Substance P on Cyclic GMP Levels
Vol. 53, No. 14, 1993
produced by exogenous administration of NO, obtained with SNP, was not affected by L-NMMA treatment (Fig. 2).
300
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SP 10 nM
SNP 10 pM
FIG.2 Cyclic GMP levels in CVEC exposed to SP and to SNP following L-NMMA treatment. Pretreatment with L-NMMA (200 pM) (dashed columns) was done for 1 h. Data are means+sem from 3 experiments run in duplicate. ** P
vs 2 1 6 + 4 8
fmol/mg
protein/1 06 cells). Discussion
The increase in vascular permeability produced by SP occurs at the postcapillary level (1) and has been related to local NO production (15). The cells used in this study were
obtained
from the microvascular endothelium which is the site of action of SP and of a large number of inflammatory and immune mediators actively involved in the healing process (1 O, 11 ). In the vascular tree, NO is mainly released by endothelial cells and exerts its effect by activating guanylate cyclase on smooth muscle cells thus favouring vasoretaxation (16).
Vol. 53, No. 14, 1993
Substance P on Cyclic GMP Levels
PL-233
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FIG.3 Cyclic AMP levels in CVEC exposed to SP and to SNP. Data are expressed as means_+sem from 3 experiments run in duplicate. * P
cyclic GMP production induced by SP
indicating NO production as mediator of the response to SP. Other reports suggested that SP-
PL-234
Substance P on Cyclic GMP levels
Vol. 53, No. 14, 1993
induced endothelium-dependent vasodilation and plasma extravasation in vivo could be modulated by NO production at the microvascular level (15). Our results indicate for the first time that cyclic GMP pathway can be directly activated by SP in postcapillary endothelial cells. We also detected a slight increase in intracellular cyclic AMP levels following SP exposure, suggesting that adenylate cyclase could also be activated by SP in CVEC. Since NK1 receptors have been indicated as responsible for SP effects at the microvascular level (6, 7), it is interesting to speculate whether the cyclic GMP pathway could be a specific transduction mechanism of SP for the NK1 receptor in endothelial cells. More experiments utilizing selective NK1 receptor antagonists and agonists are needed to clarify this point.
Acknowledqments This work was supported by funds from Italian Ministry for the University and for Scientific and Technological Research and by the National Research Council of Italy. A. Parenti was a recipient of a Menarini fellowship. References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16.
F. LEMBECK and P. HOLZER, Naunyn-Schmiedeberg's Arch. Pharmacol. 310 176-183 (1979). P. D'ORLEANS-JUSTE, S. DION, G. DRAPEAU and D. REGOLI, Eur. J. Pharmacol. 125 37-44 (1985). P. D'ORLEANS-JUSTE, S. DION,J. MIZRAHI and D. REGOLI, Eur. J. Pharmacol. 114 9-21 (1985). L.J. IGNARRO, G.M. BUGA, K.S. WOOD, R.E. BYRNS and G. CHAUDHURI, Proc. Natl. Acad. Sci. USA 84 9265-9269 (1987). R.M.J. PALMER, A.G. FERRIGE and S. MONCADA, Nature 327 524-526 (1987). M. ZlCHE, L. MORBIDELLI, M. PACINI, P. GEPPETTI, G. ALESSANDRI and C.A. MAGGI, Microvasc. Res. 40 264-268 (1990). M. ZICHE, L. MORBIDELLI, P. GEPPETFI, C.A. MAGGI and P. DOLARA, Life Sci. 48 PL7PL11 (1991). M. ZICHE, L. MORBIDELLI, E. MASINI, H.J. GRANGER, P.GEPPETTI and F. LEDDA, Biochem. Biophys. Res. Commun. 792 1198-1203 (1993). M.E. SCHELLING, C.J. MEININGER, J.R. HAWKER and H.J. GRANGER, Am. J. Physiol. 2 5 4 H1211-H1217 (1988). L. MORBIDELLI, A. PARENTI, H.J. GRANGER, M. ZICHE and F. LEDDA, Pharmacol. Res. 25 150-151 (1992). E. MASINI, P.F. MANNAIONI, A. PISTELLI, D. SALVEMINI and J. VANE, Biochem. Biophys. Res. Commun. 177 1178-1182 (1991). M. BRADFORD, Anal. Biochem. 72 248-253 (1976). M. FEELISH and E.A. NOACK, Eur. J. Pharmacol. 139 19-30 (1987). R.M.J. PALMER, D.D. REES, D.S. ASHTON and S. MONCADA, Biochem. Biophys. Res. Commun. 153 1251-1256 (1988). S.R. HUGHES, T.J. WILLIAMS and S.D. BRAIN, Eur. J. Pharmacol. 191 4 8 1 - 4 8 4 (1990). L.J. IGNARRO, R.G. HARBINSON, K.S. WOOD and P.J. KADOWlTZ, J. Pharmacol. Exp.Ther. 2 3 7 893-900 (1986).