- Email: [email protected]
Glyceryl trinatrate increases platelet cyclic GMP after metabolism in fibroblasts
Ewopeatl
0
ADONIS
200 ( 1991) 1X- 178 Science Publishers B.V. 0014~2999/91/$03.50
Journal of Pharmacology,
1991 Elsevier
175
001429999100526U
EJP 20862
Short communication
Michaela Kuhn ‘, Anita Otten I, Jiirgen C. Friilich ’ and Ulrich Fiirstermann ’
Department
of Clinical Pharmacology,
and ’ Ahbort Laboratories.
Hannow
Deparmetu
Medical School, D-3000 Hatmorer.
L*
F. R G.
47s. Abbott Park, IL 60064, U.S.A.
Received 26 April 1991.accepted 14 May 1991
Glyceryltrinitrate had no direct effect on cyclicGMP levels in platelets.However, in the presence of fibroblasts (RFL-6 cells), glyceryl trinitrate increased platelet cyclic GMP up to 7.8 fold (11.5 fold in the presence of superoxide dismutasel Fibroblasts alone had no effect. Endothelial cells alone increased platelet cyclic GMP up to 4.1 fold (9.6 fold in the presence of superoxide dismutase), but glyceryl trinitrate had no additional effect. Thus fibroblasts, but not endothelial cells, meiabolize glyceryl trinitrate to a nitric oxide-like material that stimulates platelet soluble guanylyl cyclase. Nitric oxide (NO); cGMP; Platelets; Endothelial cells (cultured); Fibroblasts (RFL-6 ce!ls): EDRF (endothelium-derived factor)
1. Introduction Organic nitrates like glyceryl trinitrate induce vascular smooth muscle relaxation by activating soluble guanylyl cyclase, thereby increasing cyclic GMP. Intracellular biotransformation of glyceryl trinitrate to be an essential prerequisite for soluble
appears
guanylyl cyclase activation. In vascular smooth muscle, postulated mechanisms include the formation of nitric oxide (NO) or NO-containing agents like S-nitrosothiols as the active species (Ignarro et al., 1981; Brien et al.. 1986). NO is also the active principle of endotheliumderived relaxing factor (EDRF). In addition to its vasodilator properties, EDRF is an inhibitor of platelet aggregation (Alheid et al., 1987). Both effects are mediated by an activation of soluble guanylyl cyclase and an increase in cyclic GMP levels in the target cells. While the vasodilator action of glyceryl trinitrate (and related compounds) is well characterized, its effect on platelet is still under debate. In contrast to vascular smooth muscle cells, platelet do not seem to contain the enzymes that convert organic nitrates to their active species. The present study was designed to investigate whether glyceryl trinitrate metabolizing cells of
Correspondence to: M. Kuhn, Niederskhsisches Institut fir PeptidForschung. Feodor-iynen Str. 5. D-3000 Hannover 61. F.R.G. * Recipient of a scholarship from the German Heisenherg Foundation.
relaxi,lg
the vessel wall can release the active principle cf the drug in quantities sufficient to increase cycle GMP levels in adjacent platelets.
2. Materials and methods 2.1. Cdl cdture
Bovine thoracic aortas were obtained from the slaughterhouse. Endothelial cells were scraped off the intimal surface after incubation with collagenase (0.025% ia saline, Gibco, Karlsruhe, FRG). Cells were grown in gelatin-coated culture flasks with RPM1 and Ml99 (1 : I) medium (both Gibco) containing 15°C(v/v) fetal calf serum (Boehringer, Mannheim, FRG). Confluent cells were subcultured with collagenase (0.2% in saline) and seeded into gelatin-coated 6-well plates (Nunclon delta, Nunc, Denmark) (Kuhn et al., 1991). Rat fetal lung fibroblasts (RFL-6, ATCC. Rockville. MD) were cultured as described previously iFtirstermann et al., 1990). Experiments were performed with first passage bovine aortic endothelial cells (BAE cells) and RFL-6 cells of passages 7-9. Both types of cells were grown to confluence in 6-well plates (about 10’ cells/well). Prior to experimentation, the culture media were removed and the cells were washed three times with 1 ml of phosphate-buffered saline (PBS) of the following composition (mM): Na’ 149.8. K’ 4.2. Mg” 0.5, Ca’+ 0.7, Cl- 141.9, HPOI- 6.5. H,PO; 1.5, glucose 5.6; pH 7.4.
\l;iti5(ic;il
Ic,\\cd fcrcnce
significance
I>! t hc tot
with
lCishc:
an aniblYsis
p otectc’d
of variance
for comp;trison of Jifftxnt
tit‘3 of 0.05 or Icss wcrl: considcrcd
The cyclic GMP
conknl
means. P val-
significant.
of M J;:B 12.944 (0.1 mM,
70 minbpretrcatct18tCdplaklcls
was 0.4 rt 0.04 pmol/lU”
5i-
Platelets alone
I
3 -i
2-
0 YehlClC
fOl-
lca~t-significant-dif-
GTN
SO0
SOD+GTN
m MB
5
Platelets and fibroblasts .
cells (n = 8) and remained bation period. trinitratc
Exposure
and/or
30 U/mol
had no significant tent.
unchanged during the incuof platelets
effect on platelet of platelets
with
cyclic GMP
con-
the inhibitor
Co-incubation
with RFL-h cells had no effect on the
of
resting cyclic GMP level of platelets. In the presence of
soluble yuanylyl cyclase. methylene blue (IO PM. 30 mink also had no significant effect on platelet cyclic
RFL-6 dells, 1 mM glyceryl trinitrate increased platelet cyclic GMP levels significantly by 3.1 fold. 5.5 fold and 7.X fold after 5. IO and 30 min of incubation (n = 4)
GMP
Pretreatment
to I mM glyccryl
SOD for 5. 10 and 30 min
(n = 8) (fig. la).
(P < 0.03). platelet
SOD
(40 U/ml)
cyclic GMP
alone
had no effect
levels but potentiated
on
the effect of
glyceryl trinitrate. In the presence of SOD. glyceEl trinitrate induced 5.1 fold. 6.1 fold and 11.5 fold increases in platelet cyclic GMP 5,
I
min of incubation
Platelets and endothelial cells
nith
I
the effect of glyce~l
mink
levels after 5.
1IJ and
30
(n = 3) (P < 0.05. when compared
Pretreatment
trinitrate
of platelets
alone at 5 and 30
with
methylene
blue
( 10 PM. 30 min) had no effect on the basal cyclic GMP levels of the platelets (data not shown). but prevented the fibroblasts-mediated stimulatoy effect of glycc~l trinitrate
SOD
SOD+GlN
, GTN MB
,
(n = 3. P < 0.05) (fig. lb).
BAE cells increased platelet cyclic GMP levels b) 3.2 fold. 3.3 fold and 1.1 fold after 5. IO and 30 min of co-incubation.
respectively
tn = 1. P < 0.05).
Glgce~l
trinitratt: (I mM) had no additional effect. SOD (10 U/ml) potsntiatrd the effect of BAE cells on platelets to increase platelet cyclic GMP levels by 6.3 fold. 8.9 fold and 9.6 fold. rcspecti\.ely. after 5. 10 and 311min of 2b
SW
Platelets and endothelial cells and NNA 4-
co-incubation (n = 1) (P < 0.05). Pretreatment of platclcts Lvith methylrnc blue (IO FM. 30 min) prevcntcd the stimulating effect of BAE cc!ls on platelet cyclic GMP content irrespective of the absence or prescncc of glyceryl trinitratc (n = 1 each. P < 0.05) (fig. 21). N”-Nitro-L-arginine (0.1 mM. 30 min) completely
prevented
the stimulatory
effect of BAE
(with and Lvithout SOD) on platelet (n = 1) (fig. 3). N”-Nitro-L-&nine
cells
cyclic GMP levels had no direct
cffcct on the cyclic GMP content of platelets or on the fibroblasts-mediated cffcct of glycc~l trinitrate on platclcts (data not shown).
4. Discussion 111the prcscnt study. glyccryl trinitratc had no direct effect on the cyclic GMP content of platelets. c:-cn \vhcn SOD
\vas added.
study by Gcrzcr
Thii
is in agrccmcnt
lvith a
ct al. f 19X8). who found only marginal
cffccts of organic nitrates on the soluble guanvlvl c?‘clasc activity of platelets. Thus. in contrast to \,ascular smooth muscle. platelets do not seem to contain the enzymes that convert organic nitrates to their activespecies (presumably
NO).
In conclusion.
our results suggest that fibroblasts.
but not cndothelial cells, metabolize glyceryl Frinitrate and release active mokcu!es (probably NO or a related compound) that can inhibit the function of neighboring platelets by increasing platelet cyclic GMP
Their
rmdts
o&sin~d
\vith
r&c
cells are very similar
fo Fhose ~~bt~~~ned \vEth isolated blood wssels. suggesting ;I simik t?ioFrsnsfllrmaFion of glyct’rj’i Frinitrate in RR.-h
c&s
prcent
study some eftlus of cyclic GMP occurred after
and vascular smooth muscle cells. In the
~~~~b~~Fi~~~ of
RF!_-6 cdlb n ith plyce~l trinitratc
(data
eferences Alheld.
molecuks.
Brien.
increasing
GhlP
conten’
Prinitrcitt’.
This
BAE
of platelets incrcaw
cyclic GMP
cells incremx.l
levels the
in
cyclic
in the absence of glyce~4
iv;?!, enhanced by SOD
and
~~b~bit~d by methykne blue. Prctrcatment of BAE co!fs with N”-nitric-L-arginine. an inhibitor of EDRF/
Ml synthesis from L-arginine. the st~~i~l~ti~~n
also completely blocked of platelet cyclic GMP formation. These
inhibitor experiment indicatr that BAE ceils stimulate et cyclic GMP Irvelr; through 3 basal release of ,I
trinttratc
had
no
effect
on
levels in the presence of BAE celts: in fact. it even inhibited slightly the effect of RF,/!W on Fhc platelets. The mechanism behind inhibition is unclear. but it is not mediated by a gibceryi trinitrate-induced
increase in cyclic GMP
in
BAE cells (we Kuhn et al.. 1991). Our results indicate that bi~~Fra~sformaFion is considerably lower in BAE c&
than in fibrobiasts.
which is in accordance with
rariicr reports (Bennet et al.. 1989). Indeed. only a marginal release of cyclic GMP was observed after incubation of BAE cells with glyceryl trinitrate, even AE cells contain soluble guanylyl cyChase that can be activated by sodium nitroprusside. wbkh rckastzs iriO spontaneously (Kuhn et al., 1991).
relarinp
factor
from cultured
lYX7. Endothelium-de-
human
endotheliai
hibits a,cgregaIitrn of human platele13. Thromb. Bennett.
thereby
‘cl.. J.C‘. Friilich and U. Fiirslermann.
rlvrd
n&~t shown). indicating a conversion of glycevl trinitraFe to its active metaboiite. Our espcriments with ptstekts suggest that fibroblasts release the active ~~~gh~~r~~g platelets. ~.~~-~~~~b~~Fi~~n with
levels.
E.M..
Nahai\u
D.C.
Leirman.
and F. Murad.
malion
of glyceryl
\aric>us cultured J.F..
H.
trinitrate
B.E.
hlc G.S.
trinitrate
tlccur% concurrently U..
Schmidl.
hlark5.
L.D.
hl.
T.H 19x6,
and
in NIE-115
and calmodulin.
Sreedon.
J.S. Pollock.
F.
Murad.
K.
relaxing
neuroblastoma
Ischii,
H.H.H.W.
Hormone-induced ouidecalcium
3X. 7.
W. Siess and J.M.
platelet
aggregation
Heim.
19X8. Direct
SIN-I.
and various
and soluble
guanylate
cyclase
Res. 52. I I.
L.J.. H. Lipplon.
J.C. Edwards.
and C.A.
Gruetter.
smooth muscle relaxalion \ide
J.
factor/nitric
nitrates
P.J. Kadowitz
K.
of glycetyl
cells requires
of the effects of nitroprusside.
Ignarro.
Bennet,
of rahhit aorta.
1990,
comparison
activity. Thromh.
in
250. 316.
B.M.
with relaxation
Mol. Pharmacol.
G.. B. Karrenbrock. on
accumulation
Exp. Ther.
Biotranslormation
of rndothelium-derived
like material
K.
2.17. 611%
Gorsky.
Heller
hio\!nthesih
Cierrrr.
Laughlin.
Enp. Ther.
Kawamoto.
and cyclic GMP
cell lines. J. Pharmacol.
and
Ftintermann.
J.H.
hetween biotransfor-
1YXY. Relationship
Nakat~~
Pharmscol.
Schriider.
cells in-
Res. 47. 561.
and nitric
oxide:
sothiolr. as aclive
W.H.
hy organic
evidence
Baricos. A.L. Hyman.
19X1. Mechanism nitrates.
of vascular
nitrites.
for the involvement
inlermediates.
J. Pharmacol.
nitroprusof S-nitro-
Exp. Ther.
218.
731). Kar\er.
V. and K. Rebch. 198% Are cyclic nucleotides
inilialion Biochim. Kuhn.
M..
<:f
Otten.
J.C. Friilich
cyclic GMP
aorlic endothelial
J.E..
R. Brazdil.
actions
of
relaxing
factor/nitric
involved in the lymphocytes?. 1991. En-
oxide from bovine
Exp. Ther.
B.K. Biocee and R. Jordan. phosphodieslerase
M&B
inhibition
?2.Y4H. MY-5445
methyl-3-isohutyl-X-tmethylamino)xantine. 75
human
and U. Fiirstermann,
cells. J. Pharmacol.
selective cyclic GMP laxsnt
of
and cyclic AMP do not regulate the release
of endothelium-derived Souness.
activation
Biophys. Acta X46. 216. A.
dothelial
mitogenic
256, 677. 19X9. Role of in the myore-
vinpocetine
and
Br. J. Pharmacol.
I98,
0
ADONIS
200 ( 1991) 1X- 178 Science Publishers B.V. 0014~2999/91/$03.50
Journal of Pharmacology,
1991 Elsevier
175
001429999100526U
EJP 20862
Short communication
Michaela Kuhn ‘, Anita Otten I, Jiirgen C. Friilich ’ and Ulrich Fiirstermann ’
Department
of Clinical Pharmacology,
and ’ Ahbort Laboratories.
Hannow
Deparmetu
Medical School, D-3000 Hatmorer.
L*
F. R G.
47s. Abbott Park, IL 60064, U.S.A.
Received 26 April 1991.accepted 14 May 1991
Glyceryltrinitrate had no direct effect on cyclicGMP levels in platelets.However, in the presence of fibroblasts (RFL-6 cells), glyceryl trinitrate increased platelet cyclic GMP up to 7.8 fold (11.5 fold in the presence of superoxide dismutasel Fibroblasts alone had no effect. Endothelial cells alone increased platelet cyclic GMP up to 4.1 fold (9.6 fold in the presence of superoxide dismutase), but glyceryl trinitrate had no additional effect. Thus fibroblasts, but not endothelial cells, meiabolize glyceryl trinitrate to a nitric oxide-like material that stimulates platelet soluble guanylyl cyclase. Nitric oxide (NO); cGMP; Platelets; Endothelial cells (cultured); Fibroblasts (RFL-6 ce!ls): EDRF (endothelium-derived factor)
1. Introduction Organic nitrates like glyceryl trinitrate induce vascular smooth muscle relaxation by activating soluble guanylyl cyclase, thereby increasing cyclic GMP. Intracellular biotransformation of glyceryl trinitrate to be an essential prerequisite for soluble
appears
guanylyl cyclase activation. In vascular smooth muscle, postulated mechanisms include the formation of nitric oxide (NO) or NO-containing agents like S-nitrosothiols as the active species (Ignarro et al., 1981; Brien et al.. 1986). NO is also the active principle of endotheliumderived relaxing factor (EDRF). In addition to its vasodilator properties, EDRF is an inhibitor of platelet aggregation (Alheid et al., 1987). Both effects are mediated by an activation of soluble guanylyl cyclase and an increase in cyclic GMP levels in the target cells. While the vasodilator action of glyceryl trinitrate (and related compounds) is well characterized, its effect on platelet is still under debate. In contrast to vascular smooth muscle cells, platelet do not seem to contain the enzymes that convert organic nitrates to their active species. The present study was designed to investigate whether glyceryl trinitrate metabolizing cells of
Correspondence to: M. Kuhn, Niederskhsisches Institut fir PeptidForschung. Feodor-iynen Str. 5. D-3000 Hannover 61. F.R.G. * Recipient of a scholarship from the German Heisenherg Foundation.
relaxi,lg
the vessel wall can release the active principle cf the drug in quantities sufficient to increase cycle GMP levels in adjacent platelets.
2. Materials and methods 2.1. Cdl cdture
Bovine thoracic aortas were obtained from the slaughterhouse. Endothelial cells were scraped off the intimal surface after incubation with collagenase (0.025% ia saline, Gibco, Karlsruhe, FRG). Cells were grown in gelatin-coated culture flasks with RPM1 and Ml99 (1 : I) medium (both Gibco) containing 15°C(v/v) fetal calf serum (Boehringer, Mannheim, FRG). Confluent cells were subcultured with collagenase (0.2% in saline) and seeded into gelatin-coated 6-well plates (Nunclon delta, Nunc, Denmark) (Kuhn et al., 1991). Rat fetal lung fibroblasts (RFL-6, ATCC. Rockville. MD) were cultured as described previously iFtirstermann et al., 1990). Experiments were performed with first passage bovine aortic endothelial cells (BAE cells) and RFL-6 cells of passages 7-9. Both types of cells were grown to confluence in 6-well plates (about 10’ cells/well). Prior to experimentation, the culture media were removed and the cells were washed three times with 1 ml of phosphate-buffered saline (PBS) of the following composition (mM): Na’ 149.8. K’ 4.2. Mg” 0.5, Ca’+ 0.7, Cl- 141.9, HPOI- 6.5. H,PO; 1.5, glucose 5.6; pH 7.4.
\l;iti5(ic;il
Ic,\\cd fcrcnce
significance
I>! t hc tot
with
lCishc:
an aniblYsis
p otectc’d
of variance
for comp;trison of Jifftxnt
tit‘3 of 0.05 or Icss wcrl: considcrcd
The cyclic GMP
conknl
means. P val-
significant.
of M J;:B 12.944 (0.1 mM,
70 minbpretrcatct18tCdplaklcls
was 0.4 rt 0.04 pmol/lU”
5i-
Platelets alone
I
3 -i
2-
0 YehlClC
fOl-
lca~t-significant-dif-
GTN
SO0
SOD+GTN
m MB
5
Platelets and fibroblasts .
cells (n = 8) and remained bation period. trinitratc
Exposure
and/or
30 U/mol
had no significant tent.
unchanged during the incuof platelets
effect on platelet of platelets
with
cyclic GMP
con-
the inhibitor
Co-incubation
with RFL-h cells had no effect on the
of
resting cyclic GMP level of platelets. In the presence of
soluble yuanylyl cyclase. methylene blue (IO PM. 30 mink also had no significant effect on platelet cyclic
RFL-6 dells, 1 mM glyceryl trinitrate increased platelet cyclic GMP levels significantly by 3.1 fold. 5.5 fold and 7.X fold after 5. IO and 30 min of incubation (n = 4)
GMP
Pretreatment
to I mM glyccryl
SOD for 5. 10 and 30 min
(n = 8) (fig. la).
(P < 0.03). platelet
SOD
(40 U/ml)
cyclic GMP
alone
had no effect
levels but potentiated
on
the effect of
glyceryl trinitrate. In the presence of SOD. glyceEl trinitrate induced 5.1 fold. 6.1 fold and 11.5 fold increases in platelet cyclic GMP 5,
I
min of incubation
Platelets and endothelial cells
nith
I
the effect of glyce~l
mink
levels after 5.
1IJ and
30
(n = 3) (P < 0.05. when compared
Pretreatment
trinitrate
of platelets
alone at 5 and 30
with
methylene
blue
( 10 PM. 30 min) had no effect on the basal cyclic GMP levels of the platelets (data not shown). but prevented the fibroblasts-mediated stimulatoy effect of glycc~l trinitrate
SOD
SOD+GlN
, GTN MB
,
(n = 3. P < 0.05) (fig. lb).
BAE cells increased platelet cyclic GMP levels b) 3.2 fold. 3.3 fold and 1.1 fold after 5. IO and 30 min of co-incubation.
respectively
tn = 1. P < 0.05).
Glgce~l
trinitratt: (I mM) had no additional effect. SOD (10 U/ml) potsntiatrd the effect of BAE cells on platelets to increase platelet cyclic GMP levels by 6.3 fold. 8.9 fold and 9.6 fold. rcspecti\.ely. after 5. 10 and 311min of 2b
SW
Platelets and endothelial cells and NNA 4-
co-incubation (n = 1) (P < 0.05). Pretreatment of platclcts Lvith methylrnc blue (IO FM. 30 min) prevcntcd the stimulating effect of BAE cc!ls on platelet cyclic GMP content irrespective of the absence or prescncc of glyceryl trinitratc (n = 1 each. P < 0.05) (fig. 21). N”-Nitro-L-arginine (0.1 mM. 30 min) completely
prevented
the stimulatory
effect of BAE
(with and Lvithout SOD) on platelet (n = 1) (fig. 3). N”-Nitro-L-&nine
cells
cyclic GMP levels had no direct
cffcct on the cyclic GMP content of platelets or on the fibroblasts-mediated cffcct of glycc~l trinitrate on platclcts (data not shown).
4. Discussion 111the prcscnt study. glyccryl trinitratc had no direct effect on the cyclic GMP content of platelets. c:-cn \vhcn SOD
\vas added.
study by Gcrzcr
Thii
is in agrccmcnt
lvith a
ct al. f 19X8). who found only marginal
cffccts of organic nitrates on the soluble guanvlvl c?‘clasc activity of platelets. Thus. in contrast to \,ascular smooth muscle. platelets do not seem to contain the enzymes that convert organic nitrates to their activespecies (presumably
NO).
In conclusion.
our results suggest that fibroblasts.
but not cndothelial cells, metabolize glyceryl Frinitrate and release active mokcu!es (probably NO or a related compound) that can inhibit the function of neighboring platelets by increasing platelet cyclic GMP
Their
rmdts
o&sin~d
\vith
r&c
cells are very similar
fo Fhose ~~bt~~~ned \vEth isolated blood wssels. suggesting ;I simik t?ioFrsnsfllrmaFion of glyct’rj’i Frinitrate in RR.-h
c&s
prcent
study some eftlus of cyclic GMP occurred after
and vascular smooth muscle cells. In the
~~~~b~~Fi~~~ of
RF!_-6 cdlb n ith plyce~l trinitratc
(data
eferences Alheld.
molecuks.
Brien.
increasing
GhlP
conten’
Prinitrcitt’.
This
BAE
of platelets incrcaw
cyclic GMP
cells incremx.l
levels the
in
cyclic
in the absence of glyce~4
iv;?!, enhanced by SOD
and
~~b~bit~d by methykne blue. Prctrcatment of BAE co!fs with N”-nitric-L-arginine. an inhibitor of EDRF/
Ml synthesis from L-arginine. the st~~i~l~ti~~n
also completely blocked of platelet cyclic GMP formation. These
inhibitor experiment indicatr that BAE ceils stimulate et cyclic GMP Irvelr; through 3 basal release of ,I
trinttratc
had
no
effect
on
levels in the presence of BAE celts: in fact. it even inhibited slightly the effect of RF,/!W on Fhc platelets. The mechanism behind inhibition is unclear. but it is not mediated by a gibceryi trinitrate-induced
increase in cyclic GMP
in
BAE cells (we Kuhn et al.. 1991). Our results indicate that bi~~Fra~sformaFion is considerably lower in BAE c&
than in fibrobiasts.
which is in accordance with
rariicr reports (Bennet et al.. 1989). Indeed. only a marginal release of cyclic GMP was observed after incubation of BAE cells with glyceryl trinitrate, even AE cells contain soluble guanylyl cyChase that can be activated by sodium nitroprusside. wbkh rckastzs iriO spontaneously (Kuhn et al., 1991).
relarinp
factor
from cultured
lYX7. Endothelium-de-
human
endotheliai
hibits a,cgregaIitrn of human platele13. Thromb. Bennett.
thereby
‘cl.. J.C‘. Friilich and U. Fiirslermann.
rlvrd
n&~t shown). indicating a conversion of glycevl trinitraFe to its active metaboiite. Our espcriments with ptstekts suggest that fibroblasts release the active ~~~gh~~r~~g platelets. ~.~~-~~~~b~~Fi~~n with
levels.
E.M..
Nahai\u
D.C.
Leirman.
and F. Murad.
malion
of glyceryl
\aric>us cultured J.F..
H.
trinitrate
B.E.
hlc G.S.
trinitrate
tlccur% concurrently U..
Schmidl.
hlark5.
L.D.
hl.
T.H 19x6,
and
in NIE-115
and calmodulin.
Sreedon.
J.S. Pollock.
F.
Murad.
K.
relaxing
neuroblastoma
Ischii,
H.H.H.W.
Hormone-induced ouidecalcium
3X. 7.
W. Siess and J.M.
platelet
aggregation
Heim.
19X8. Direct
SIN-I.
and various
and soluble
guanylate
cyclase
Res. 52. I I.
L.J.. H. Lipplon.
J.C. Edwards.
and C.A.
Gruetter.
smooth muscle relaxalion \ide
J.
factor/nitric
nitrates
P.J. Kadowitz
K.
of glycetyl
cells requires
of the effects of nitroprusside.
Ignarro.
Bennet,
of rahhit aorta.
1990,
comparison
activity. Thromh.
in
250. 316.
B.M.
with relaxation
Mol. Pharmacol.
G.. B. Karrenbrock. on
accumulation
Exp. Ther.
Biotranslormation
of rndothelium-derived
like material
K.
2.17. 611%
Gorsky.
Heller
hio\!nthesih
Cierrrr.
Laughlin.
Enp. Ther.
Kawamoto.
and cyclic GMP
cell lines. J. Pharmacol.
and
Ftintermann.
J.H.
hetween biotransfor-
1YXY. Relationship
Nakat~~
Pharmscol.
Schriider.
cells in-
Res. 47. 561.
and nitric
oxide:
sothiolr. as aclive
W.H.
hy organic
evidence
Baricos. A.L. Hyman.
19X1. Mechanism nitrates.
of vascular
nitrites.
for the involvement
inlermediates.
J. Pharmacol.
nitroprusof S-nitro-
Exp. Ther.
218.
731). Kar\er.
V. and K. Rebch. 198% Are cyclic nucleotides
inilialion Biochim. Kuhn.
M..
<:f
Otten.
J.C. Friilich
cyclic GMP
aorlic endothelial
J.E..
R. Brazdil.
actions
of
relaxing
factor/nitric
involved in the lymphocytes?. 1991. En-
oxide from bovine
Exp. Ther.
B.K. Biocee and R. Jordan. phosphodieslerase
M&B
inhibition
?2.Y4H. MY-5445
methyl-3-isohutyl-X-tmethylamino)xantine. 75
human
and U. Fiirstermann,
cells. J. Pharmacol.
selective cyclic GMP laxsnt
of
and cyclic AMP do not regulate the release
of endothelium-derived Souness.
activation
Biophys. Acta X46. 216. A.
dothelial
mitogenic
256, 677. 19X9. Role of in the myore-
vinpocetine
and
Br. J. Pharmacol.
I98,