254
Selective Guanylyl Cyclase Inhibitor Reverses
Nitric Oxide-Induced
Vasorelaxation
Linda J. Olson, Edward T Knych Jr, ThomasC Herzig, JamesG Drewett
Downloaded from http://hyper.ahajournals.org/ by guest on June 15, 2017
Abstract Effects of a novel soluble guanylyl cyclase mhtbrtor, lH-[1,2,4]oxadtazolo[4,3-a]qumoxal~n-l-one
(ODQ), were
characterized on guanylyl cyclase activity m cytosohc fraction of
COS-7 cells overexpressmg the (Y, and PI subunits of the rat
soluble enzyme ODQ was a noncompetmve mhtbttor of soluble
guanylyl cyclase with respect to Mn’+ or Mn’+-GTP and was a
mixed competrttve/noncompetltlve mhrbltor wtth respect to mtnc
oxide (NO) donation ODQ (10 pmol/L) reduced deta nonoatestimulated cGMP productron m COS-7 cells overexpressmg soluble guanylyl cyclase and m rat aorttc vascular smooth muscle
cells ODQ did not mhrbtt parttculate forms of the enzyme rat
guanylyl cyclase-A, -B, or -C, did not block NO synthase, and
did not auto-oxrdtze deta nonoate-donated NO m the presence
of cells at physrologrcal pH Therefore, ODQ IS a selective mhrbttor of soluble guanylyl cyclase Using ODQ m isolated aorttc
rmg preparations, we tested the hypothesis that soluble guanylyl
cyclase mediates vasorelaxant acttvtty associated with NO Phenylephrme (100 nmol/L)-precontracted,
isolated rat aortas were
relaxed m a concentratton-dependent manner by deta nonoate
(001 to 100 pmol/L) and mtroglycerm (001 to 300 hmol/L)
ODQ (10 pmol/L) attenuated deta nonoate- and mtroglycermmediated relaxatron of contracted aortas ODQ had no effect on
natrmrettc peptrde-, S-bromo-cGMP-,
rsoproterenol-, or brmakahm-mediated aorttc relaxation These results support the hypothesis that soluble guanylyl cyclase mediates vasorelaxant
acttvtty associated with mtnc oxide (Hypertension. 1997;29
[part 2]:254-261.)
Key Words l cychc GMP l endothelmm-dertved relaxing
factor l guanylyl cyclase l mtrtc oxide l mtrtc oxtde
synthase l natrmrettc peptrdes l vasodrlatatton
N
vascular relaxation and lower cGMP concentrations as-
‘itrtc oxide is now known to be the maJor endothehum-dertved
relaxmg
factor I* Several
va-
sodilators, mcludmg bradykmm, acetylcholme,
and serotonm, are thought to have activity by virtue of
then effects to strmulateendothelial cell NOS. 12Several
studiesover the years have suggestedthat the vasodilatory actions of NO are mediatedby increasesm cGMP’-3
NO and severalprecursorsof the radical activated soluble
forms of guanylyl cyclase,l-4 and membranepermeable
analogsof cGMP vasodilated blood vessels15 In addltron, m most studiesNO-stimulated production of cGMP
correlates with the NO-mediated vasodilation However,
NO-induced smoothmuscle relaxation hasbeen drssociated from increasesm cGMP in somestudies.4.6.7
Potential explanations for the latter observation are that NO
also stimulates guanylyl cyclase activity in cell types
other than vascular smooth muscle (ie, adventrtia, fibroblasts, autonomic nerves, and/or endothelmm), that current assaysfor vascular contractile responsesare significantly more sensitive than the measurementof cGMP
concentrations wnhm cellular compartmentsassociated
with vasoactivny, and that NO may mediatevascular effects independentof guanylyl cyclase stimulation
Several groups have used either methylene blue or
LY83583 as sGC mhibitors to focus on the latter posabihty by attempting to dissociateNO-mediated smooth
muscle relaxation from increases in enzyme activatton 1,~ Both of these compounds appear to block the
From the Department
of Pharmacology
and Toxicology
(L .I 0,
J G D ), Department
of Physiology
(T C H ), and the Cardiovascular
Research Center, Medical College of Wtsconsm (Mrlwaukee), and
the Department
of Pharmacology,
University
of Minnesota-Duluth
School of Medicine
(E T K )
Reprmt requests to James G Drewett,
PhD, Department
of Pharmacology and Toxicology,
Medical College of Wlsconsm,
8701 Watertown Plank Rd, Milwaukee,
WI 53226.4801
0 1997 American
Heart Assoclatlon,
Inc
sociated with vasoacttve agents that stimulate
NOS or NO
donors, including mtroglycerm, sodium mtroprusstde,
and 3-morpholmo-sydnommme (ie, SIN-l) Therefore,
methylene blue and LY83583 have been used to reach
the concluston that sGC mediatesthe vasodllatory effect
of NO. This concluston IS questionable becauseboth
methylene blue and LY83583 are not truly selective sGC
mhibitors, and then effects on the NO/sGC system are
likely completely or partially explained by mdnect actions Methylene blue is now known not only to inhibit
NOS 1011but also to lower NO concentrattonsby the production of superoxide amon ~3 LY83583 was first reported to reduce cGMP concentrattonsm the guinea pig
lung by an unknown mechanismnot mvolvmg a reduction of sGC activity 14Regardless,several studies over
the last decadehave usedit to form conclusionsabout the
relattonshtp of NO- and sGC-associatedcellular events,
including vasorelaxatton m aortrc rings 8.9An early report
demonstratedthat LY83583 blocked the production of
endothelmm-derived relaxing factor, 15and a very recent
study hasdemonstratedthat, like methyleneblue, LY83583
also can act as an mhibnor of NOS m rat cerebellar homogenatesii The latter study,” as well asothersi6.17has
revealed more parallels between methylene blue and
LY83583, mcludmg the production of superoxtde anion,
to explain the effects of the latter compound to decrease
NOS activity or auto-oxtdize NO Therefore, it is tmpossible to use LY83583 and/or methylene blue to drfferentlate between direct mhibmon of sGC activity (te, effects on the receptor) or reductions m NOS or NO
concentrations (te, effects on hgand concentrations).
Garthwaite et al18recently reported the ldentificatton
of a potent, selective mhibrtor of NO-sensitive guanylyl
cyclase called ODQ They demonstratedthat ODQ mhrbned purified NO-stimulated sGC activity m a noncompetitive manner with respect to the substrateGTP and
3lson et al Selective Inhibitor of Guanylyl Cyclase
Selected Abbreviations and Acronyms
8-bromo-cGMP = 8-bromo-guanosme 3’ 5’-monophosphate
ANP = type A natrnrrettc peptrde
BNP = type B natrrurettc peptrde
CNP = type C natnurettc pepttde
GC-A, -B, -C = guanylyl cyclase-A, -B, -C
LY83583 = 6-amhno-5,8-qumohnedrone
NO(S) = nitric oxide (synthase)
ODQ = lH-[ 1,2,4]oxadrazolo[4,3-nlqulnoxalm-lone
sGC = soluble guanylyl cyclase
Downloaded from http://hyper.ahajournals.org/ by guest on June 15, 2017
further determined that ODQ did not inactivate reactive
NO, did not produce superoxideamons,and did not block
the sGC-independenteffect of NO to induce macrophage
cytotoxicity. 18ODQ blocked NO-donor stimulation of
sGC m intact endothehalcells and had no effect on ANP
stimulation of particulate guanylyl cyclase asassessed
by
measurementof cGMP production by radioimmunoassay.18ODQ also did not inhibit forskolin-stimulated adenylyl cyclase m endothehalcells 18Taken together, these
initial resultssuggestedthat ODQ waspotentially a much
better reagent for assessingthe involvement of sGC m
NO effects than either methylene blue or LY83583
In the present study, we characterized the effects of
ODQ on Mn’+-, Mn’+-GTP-, and deta nonoate19-stimulated sGCs m the cytoplasmic fraction from COS-7
cells overexpressmg the LY~and pi subunits of the rat
enzyme (COS-7 sGC cells) Furthermore, we also examined the effect of ODQ m cells overexpressmg particulate forms of the enzyme GC-A, GC-B, and GC-C,
which act as receptors for ANP, CNP, and heat-stable
enterotoxm, respectively Followmg this characterization, we used ODQ m rat isolated aortic rings to test the
hypothesis that sGC mediates vasorelaxant activity associated with NO donation
Methods
General
Unless specified m the followmg text, common laboratory reagents used m this study were purchased from either Sigma
Chemical Co or Fisher Sctenttfic All protease mhtbttors were
from Sigma Chemical Co as well unless otherwise noted
Cell Culture and Transfection
Cells were mamtamed m a humidified 95% an-5% CO2 waterJacketed incubator (Nuarre) at 37°C. HEK-293 cells overexpressmg stable transfectants of rat GC-A, GC-B, and GC-C (HEK293 GC-A, -B, and -C cells, respectively) were kindly provided
by Dr David L Garbers (Howard Hughes Medical Instttute, Umverstty of Texas Southwestern Medical Center [Dallas]) and cultured as described *a22
COS-7 cells were cultured as previously described23 and
transfected with the a, and PI subunits of sGC by the DEAE/
dextran method 24The rat (Y, and PI subunit cDNAs m the mammalian expressron vector (pCMV5) were also provided by Dr
D.L Garbers 25 The expression of active sGC requires the cotransfectron of both (Y and p subunits 25-29Cells were cotransfected with 5 pg of each subumt Mock-transfected cells were
transfected with 10 pg pCMV, Cells were allowed to grow for
48 hours before experrmentatton Expression of both subunits
was confirmed by deta nonoate (nonoate) (Cayman Chemtcal)stimulated production of cGMP m whole cells or guanylyl cyclase actrvtty m soluble fractions from these cells
255
Rat Aortic Vascular Smooth Muscle Cells
Cells were isolated according to the method of Dtgho et al 30
Male Sprague-Dawley rats (300 to 400 g) were given a lethal
mtraperttoneal mlectron of sodium pentobarbrtal(50 mg/kg), and
a thoracotomy was performed to remove the full-length thoracrc
aorta. These mampulatrons were performed m accordance with
the Medical College of Wtsconsm Animal Care Commrttee
gmdelmes Ring explants (1 to 1 5 mm thick) were placed mto
24-well tissue culture plates (Falcon) contammg Dulhecco’s
modified Eagle’s medium (Grbco Life Technologtes) supplemented with 10% defined fetal bovine serum (Hyclone Lab) Explants were removed 3 to 4 days later, and the cells remammg
on the dish were grown to confluence Growth medium was the
same as that above during explantatton with the following supplements 2 mmol/L L-glutamme, 100 U/mL pemcillm, and 70
pmol/L streptomycm (Grbco) The smooth muscle identity of
these cultures was verified morphologrcally (te, htll and valley
growth pattern) and by tmmunohtstochemrcal stammg for both
the presence of smooth muscle-specific cu-actm and the absence
of factor VIII antigen Immunohrstologrcal staining verified that
the cells were more than 97% smooth muscle The cells were
used m passages 4 to 6
Bovine Coronary Artery Endothelial
Cells
Endothehal cells were kindly provided by Dr Wtlham B
Campbell (Medical College of Wrsconsm [Milwaukee]) The tsolatron and culturing condmons of these cells have been prevrously
descrtbedsr and were followed throughout these studies
Whole-Cell
cGMP Production
Studies
Experiments testing ODQ effects (Tocrts Cookson) on parttculate guanylyl cyclases were examined m HEK-293 GC-A, -B,
and -C cells overexpressmg each of these enzymes Those testing
the effects of ODQ on sGC were performed m both COS-7 sGC
cells and rat aorttc vascular smooth muscle cell primary cultures
All experiments were performed m two patred 24-well plates
(Falcon or Sarstedt) One plate received ODQ treatment and the
other ODQ vehicle (ethanol) Before mmatron of experiments,
confluent plates of cells were rinsed twice with 5 mL PBS (pH
7 4, 37°C). Cells were premcubated at 37°C for 10 minutes with
0 25 mmol/L rsobutylmethylxanthme and ODQ vehicle or 10
bmol!L ODQ m HEPES (25 mmoI/L)-buffered (pH 7.4) Dulbecco’s modified Eagle’s medium (Medical College of Wrsconsm Tissue Culture Facthty) Premcubatron medium was removed
and replaced with fresh buffer of the same components with the
appropriate guanylyl cyclase agonist for 5 minutes at 37°C Four
paired experiments were performed per set of ODQ vehicle- and
ODQ-treated plates HEK-293 GC-A, -B, and -C cells were
treated with vehicle (water) and increasing concentratrons of
ANP, CNP, and heat-stable enterotoxm, respectively COS-7
sGC cells and rat aortrc vascular smooth muscle cells were treated
with nonoate vehicle (0 01 mol/L NaOH) and increasing concentrations of the NO donor or tts by-product, ethylamme (Aldrich
Chemical Co). Spectfically, vehicle or one of five concentrattons
of agonist was given per well (ANP, CNP, heat-stable enterotoxin vehicle, lo-“, lo-“, lo-*, 10m7, or 10d6 mol/L, nonoate/
ethylamme. vehicle, 10m6, lo-‘, 10e4, lo-‘, or lo-‘mol/L)
After
the 5-minute mcubatton period, the supernatants were aspirated,
and the guanylyl cyclase reaction was terminated by the addition
of 0 5 mL of 1 mol/L perchloric acid Antiserum for cGMP was
graciously supplied by Dr D L Garbers 32 Samples contammg
cGMP were subJected to alumina-Dowex ton-exchange column
punficatron~2 and radtormmunoassay as previously described z734
Resultant samples were counted on a Packard Cobra II AutoGamma Counter
Guanylyl Cyclase Assay
COS- 7 sGC Cells
Two days after transfectron, COS-7 sGC cells were harvested
and resuspended m 10 mmol/L HEPES (pH 7 4) 150 mmol/L
256
Hypertension Vol29, No 1, Part 2 January 1997
Downloaded from http://hyper.ahajournals.org/ by guest on June 15, 2017
NaCl, 1 mmol/L EDTA, 1.5 bmol/L aprotmm, 10 hmol/L E-64
(Boehrmger Mannhetm Biochemicals), and 0 1 pmol/L PMSF
After homogemzahon, cytosohc proteins were isolated by centrifugation (15 OOOg, 30 minutes, 4°C) Guanylyl cyclase activtty
was determmed m 100~PL reactions containing 5 pg soluble protern,35 40 mmol/L triethanolamme HCl, pH 7 5, 0.25 mmol/L
isobutylmethylxanthme, 2 mmol/L D’IT, 1 pmol/L PMSF, and 1
pmol/L pepstatm A In kmettc studies, 5-minute reactions were
performed m the presence or absence of ODQ (10 pmol/L) at
37°C containing the varied or fixed concentrattons of Mn*‘,
Mn*+-GTP, and/or nonoate as described m “Results” and m Table 1 The synthesis of cGMP was vertfied to be linear over the
5-mmute course of the experiment. Reactions were terminated,
and cGMP was purified and quantified as described above
For assays quantifying the conversion of radiolabeled GTP to
cGMP, the reaction mixture also contamed 1 $1 [c?P]GTP
(ICN Radiochemtcals) and either 600 nmoVL Mn2+ per 100
nmol/L GTP or 1 1 mmol/L Mn*+ per 1 mmol/L GTP Proteins
were premcubated (10 mmutes, 37°C) either m the presence of
10 pmol/L ODQ or vehicle (ethanol) before the addition of Mn*+GTP Reacttons were terminated after 5 minutes by the addition
of 0 5 mL 110 mmol/L zmc acetate and 0 5 mL 110 mmol/L
sodium bicarbonate Samples were centrifuged (lOOOg, 5 mmutes), and supernatants were purified over alumina columns 36
Eluates containing [32P]cGMP were subJected to scmtillation
counting on Packard Instrument Tri-Carb 2 1OOTR
Rat Aortas
Male Sprague-Dawley rats (300 to 400 g) were given a lethal
mtraperttoneal inJectron of sodmm pentobarbital (50 mg/kg)
These mampulahons were performed m accordance with the
Medical College of Wtsconsm Animal Care Committee gmdelmes For each expertment, three full-length thoracic aortas were
obtained, and the fascia was carefully removed from each vessel
Aortas were homogemzed on me m 5 mL buffer containing 25
mmol/L triethanolamme HCl (pH 7 6), 10 mmol/L NaCl, 5
hmol/L aprotmm, 10 pmol/L E-64, and 0 1 pmol/L PMSF Guanylyl cyclase assays were performed m 100 PL of the above buffer
containing 20 pg protein35 and 0 25 mmol/L isobutylmethylxanthme as descrtbed above for COS-7 sGC cells Samples were
premcubated with ODQ vehicle or 10 pmol/L ODQ before the
addttton of nonoate vehicle or 1 mmol/L nonoate to stimulate the
enzyme for 5 minutes Reactions were stopped, samples punfied,
and cGMP levels determined as described above
NOS Activity
Samples contammg 0 1 mg lysate protem35 from transiently
transfected COS-7 sGC cells or endothehal cells were premcubated (10 minutes, 37°C) m the presence of 10 pmol/L ODQ, 0 1
mmol/L NW-mtro-L-argmme,37 0 1 mmol/L methylene blue,10 or
respective vehicle treatment NOS activity was measured by
momtormg the converston of L-[‘HI-argmme to L-[3H]-cttrullme
as prevtously described 31Reactions were terminated, and L-[~H]cmullme was purified as described 31 The levels of L-[3H]-cttrullme were determined by scmttllation countmg (Packard Tn-
Carb 2100TR) Data were expressed as femtomoles of L-[~H]cmullme formed per milhgram of lysate protein per minute
Vasorelaxation
Studies
Male Sprague-Dawley rats (300 to 400 g) received a lethal
mtraperttoneal mJection of sodmm pentobarbital (50 mg/kg)
These mampulattons were performed m accordance with the
Medical College of Wtsconsm Animal Care gmdelmes Thoracic
aortas were removed and nngs prepared m 5-mL tissue baths as
previously described 33.38Contractile responses were monitored
using Grass mstrumentatton as described 3138
After the premcubation and precontractions, one rmg of each
pair received ODQ vehicle (ethanol) and the other rmg received
ODQ (10 pmol/L) 15 minutes before the addition of the LY,selective adrenoceptor agonist phenylephrme (100 nmol/L) The
rings were allowed to maxtmally contract over the next 15
minutes before the addttton of vehicle and increasing cumulative
concentrations of nonoate (1O-8 to 3X10-’ mom), ANP (lo-”
to lo-’ mol/L), CNP (lo-” to 10m6 mol/L), mtroglycenn (lo-’
to 3X 10m6mol/L), tsoproterenol (lo-* to lo-’ mom), brmakahm
(lo-” to lo-$ mol/L), 8-bromo-cGMP (lo-’ to 3~ 10m4 mol/L,
ICN Biochemicals), or the nonoate by-product ethylamme (IO-*
to lo-“ mol/L) every 4 minutes or until a plateau was reached
followmg each addition of relaxant agomst Because nonoate
must be stored m 0 01 mol/L NaOH to avoid hydrolysis, appropriate dtlutions of the NO donor m Krebs’ buffer were made
approximately 1 minute before addition to the tissue baths to
ehmmate the potential for aikalmrzatton of the bathing medium
and associated nonspecific effects Nitroglycerm was manufactured by Parke-Davis, and btmakahm was manufactured by
Merck KGaA Co39 and graciously provided by Dr Garrett J
Gross (Medical College of Wisconsm [Milwaukee]) 40
In the presence of paired ODQ vehicle- and ODQ-treated aortic rmgs, we also tested not only for potenttal desensttization to
phenylephrme-induced contractions over time but also for possible time-dependent nonoate-vehicle effects on contractile activity Two sets of paired rings were contracted with phenylephrme
with or without nonoate-vehicle treatment (ie, appropriate ddutions of the nonoate storage solutton, 0 01 mol/L NaOH, akm to
those above for nonoate treatment) over a 65-mmute penod every
4 to 8 minutes to approximate the time necessary for complete
responsiveness to each nonoate addition durmg typical expenmentation Nonoate (1 mmol/L) was added to the paired vessels
after the completion of these time-dependent experiments to verify the capacity of each rmg to respond to the NO donor
Auto-oxidation
of NO
NO measurements were performed using a commercially
available Clark-type electrode &o-NO, World Precision Instruments) and documented on a strip-chart recorder The electrode
was calibrated daily using actdtfied potassium mtnte as the NO
donor accordmg to the manufacturer’s mstructrons All measurements were made under constant stnrmg The effects of ODQ or
the superoxide generating system xanthme/xanthme oxtdase were
studied using a small plastic vial containing 2 mL Krebs’ bicar-
TABLE 1. Effect of ODQ on Kinetic Parameters
of Guanylyl Cyclase
Activity in the Cytosolic Fraction From COS-7 Ceils Transiently
Transfected With a1 and p, Subunits of Rat Soluble Enzyme
ODQ Vehicle
F~xecl
Mn*’
Mn*‘-GTP
Mn2+/Mn2+-GTP
Varled
Mn’+-GTP
Mn2+
Nono
KIPI
50+10
84506
81 -c2
V max
0 4920 06
061+006
0 3220 04
10 pmollL
Km
60”20
70+10
185558
ODQ
V max
017t003’
023+002*
020c006’
”
3
4
7
V,, (nmol cGMP formed/mln/mg
protein) and K,,, bmol/L) were calculated by doublereciprocal plots 4’ Values are meantSE,
n, number of Independent
assays Mn*+ 125
pmol/L, Mn’+-GTP
2 to 50 pmol/L, Mn2+-GTP 2 mmoVL, Mn2+, 5 to 75 hmol/L, Mn2+/Mn2+GTP 125/12 5 pmol/L, nonoate 0 05 to 1 mmol/L Pawed t test comparison of ODQ vehicle
(ethanol) vs 10 pmol/L ODQ, *PC 05
Olson et al
1.0
0.6
0
(+) ODQ
tt
0.6
0.4
0.2
0.0
VEH
B
C
70
{
ii
0
4
40
g
20
30
0
10
n1
VEH
LOG[Nono~~el
-6
(m-&L)
-3
(-) ODQ
Downloaded from http://hyper.ahajournals.org/ by guest on June 15, 2017
-6
-5
LOG[Nonoate]
-4
-3
(mol/L)
-2
FIQ 1 Effect of ODQ (10 PmoVL) on whole-cell
deta nonoate
(nonoate)-strmulated
cGMP productron
In primary
cultures of rat
aottrc vascular
smooth
muscle
cells (A) and In COS-7
cells
overexpressing
the (Y, and p, subunits
of rat soluble
guanylyl
cyclase (6) Data points are mean-t-SE
(-) ODQ indicates
ODQvehicle treatment,
(+) ODQ, ODQ treatment,
VEH, nonoate-vehlcle treatment
For comparison
of each nonoate
treatment
vs
VEH, “P< 05, **PC 01, ***P< 001
bonate buffer (pH 7 4)x8 and 2x10’ COS-7 cells or a rat aorttc
ring The electrode was allowed to equlhbrate after which nonoate (50 pmol/L) was added After the electrode signal restabtltzed m the presence of nonoate, ODQ (10 pmol/L), xanthme (10
,umol/L), xanthme oxldase (5 U/mL), or a combmatlon of xanthme (10 pmol/L) and xanthme oxtdase (5 U/mL) were added,
and changes m NO concentration were recorded
Data Analyses
The effect of ODQ on agonist-stimulated cGMP productton
m whole cells and agomst- or &bromo-cGMP-induced
relaxation of precontracted rat aorttc rings was assessed by comparison of control (agonist vehicle) to each treatment concentration by Dunnett’s modtficatton of Student’s paired I test
The effect of ODQ over the full concentratton-response
curve
was assessed for each agomst or 8-bromo-cGMP
by repeated
measures ANOVA
K,,, and V,,, values from the kinetic assays of enzyme activity
from the cytoplasmtc fraction of COS-7 sGC cells were determined by tradtttonal double-reciprocal conversions 41 Stattsttcal
compartsons between ODQ-treated and ODQ vehicle-treated
preparations for both K,,, and V,,,, values were made using Student’s paired t test
In all figures and tables, astensks denote the statisttcal slgnificance (*P< 05, **P< 01, ***P< 001) of a specific treatment when compared with a matched control group (te, vehicle
treatment)
Selective Inhibitor
of Guanylyl
Cyclase
257
tton-dependent (ANOVA P<.OOOl) increase in cGMP
production with an EC50 value of approximately 30
pmol/L. In aorttc cells (Frg lA), no greater effect was
obtained after 10 mmol/L nonoate treatment (n=2,
L.J.O., T C.H., J.G.D., unpublished data, 1996), similar
to the results in COS-7 sGC cells (Fig 1B) Therefore,
m both cell types, 1 mmol/L nonoate resulted m a maximal effect on cGMP production We choseto use a loPmollL concentratton of ODQ in these studies on the
basis of previous results demonstrating that concentration to be maxtmally effective. 18 Pretreatment with
ODQ reduced the abrlrty of nonoate to mcreasecGMP
levels in COS-7 cells (Fig 1B) and completely blocked
the nonoate effect m the vascular smooth muscle cells
(Fig 1A) (both by ANOVA, P< 0001) We also found
that ODQ completely prevented maximal nonoate (1
mmol/L) stimulation of guanylyl cyclase activity in soluble fractions from rat thoracrc aorta, consistent with
the results in aortrc smoothmuscle cell cultures (L.J.0 ,
J.G.D., unpublished data, 1996).
Nonoate1sknown to hydrolyze mto equalmolar amounts
of NO and ethylammeat neutralPH.rgAs a control for a byproduct effect, ethylamme(10e7to 10e3mol/L) was tested
for any effect on sGC acttvtty. It was found to be without
effect (L-J 0, J.G.D., unpublisheddata, 1996).
Effect of ODQ on Nonoate- and NitroglycerinMediated Relaxation of PhenylephrinePrecontracted Rat Aortas
Theseexperimentswereperformedto determmewhether
ODQ blocked the vasorelaxantactivity of two NO donors,
nonoateand mtroglycerm. The summarydatafrom several
experimentsassessmg
the effect of ODQ on nonoate-mduced relaxation are shown m Fig 2. In the absenceof
ODQ pretreatment,nonoate( 10e7to 10e4mol/L) resulted
in a concentratton-dependentrelaxation of phenylephrmeprecontractedrat aorta(I&,= 1 pmol/L, ANOVA, P< 0001)
Complete relaxation was observed on reaching a final
nonoate concentration of 10e4mol/L in the absenceof
ODQ. After ODQ (10 pmol/L) treatment, the IC50 for
nonoate (ANOVA, P< 0001) was increasedby 30-fold,
and the maximal effect of nonoate was attained at lo-’
mol/L and reduced by about 75%. No further relaxation
---*--
(+) ODQ
Results
Effect of ODQ on NO-Donor-Mediated
of cGMP Production
Stimulation
were destgnedto compare the effects on nonoate-mediatedcGMP productton m both rat
aorttc vascular smooth muscle cells (Fig 1 A) and COS-7
sGC cells (Fig IB) In both rat aortic vascular smooth
muscle cells (Fig IA) and COS-7 sGC cells (Fig lB),
nonoate (10T5 to 10m3mol/L) resulted m a concentraThese experiments
LOG[Nonoate]
FIG 2.
(mol/L)
Effect of ODQ (10 pmol/L) to reverse deta nonoate (nonoat+Induced
relaxatron
of 100 nmol/L phenylephnne-precontracted rat aorta Data pornts are mean&SE
For all concentrations, 3x1 Om5 mol/L and lower, n=l2,
for those 1 Om4 mol/L and
greater,
n=3
(-) ODQ indicates
ODQ-vehicle
treatment,
(+)
ODQ, ODQ treatment,
and VEH, nonoate-vehrcle
treatment
For
comparison
of each nonoate-treatment
vsVEH, *PC 05, **PC 01,
***p< 001
258
Hypertension
Vol29,
No 1, Part 2
January 1997
Downloaded from http://hyper.ahajournals.org/ by guest on June 15, 2017
was observed at 3~10~~ mol/L nonoate (n=3) These
results are consistentwith a mixed competittve/noncompetittve antagonismof NO effects in the rat aorta. The
sameODQ concentration also blocked the ability of mtroglycerm to relax phenylephrme-precontracted aorta
in a similar manner (n=3, unpublisheddata, 1996). Neither the nonoate by-product,‘9 ethylamme (lo-’ to 10m3
mol/L), nor the nonoate vehicle (ethanol) had any effect
on basal- or phenylephrme-induced rat aortic contractions (L.J O., J.G.D , unpublished data, 1996; Ftg 2)
The nonoate-mediatedrelaxation in the ODQ-treated
vessel also was not simply due to time-dependent loss
of contractile acttvtty, nonoate-vehicle effect, or desensitization to phenylephrme. Control experiments
were performed whereby phenylephrme was added to
paired ODQ-treated and ODQ vehicle-treated aortic
rings The associated phenylephrme-induced contractions were maintained with or without nonoate-vehicle
treatment for the approximate 65-minute duration of the
experiments m these studies (n=3). In both variations,
addition of 1 mmol/L nonoate at the end of this time
period resulted in complete relaxation of the ODQ vehicle-treated ring and partial relaxation (=20% to 25%)
of the ODQ-treated vessel (n=3), akm to the results of
Ftg 2
Effect of ODQ on sGC Activity in the Cytosolic
Fraction From COS-7 sGC Cells
It hasbeenprevtously shownthat maximal rat lung sGC
acttvation is obtainedin the presenceof excessMn2+ over
GTP 42For kmetic analysisof an enzyme systemrequiring
an excessconcentrationof cation over nucleottde,the system can be viewed as having two substrates.a metalnucleottdechelate, Mn’+-GTP, and the metal ion, Mn2+ 43
Chnsmanet a142have prevtously demonstratedthat sGC
is such an enzyme. Similar methodsto those of the latter
study42were used m the followmg experiments assessing enzyme activation in the cytoplasmic fraction from
COS-7 sGC cells
These studies were performed to determine whether
ODQ was a competitive and/or noncompetitive mhibitor
of sGC with respect to NO, Mn2+, and Mn”‘-GTP K,,,
and V,,, values of sGC for Mnzf and Mnzf-GTP were
determined at near-saturatingconcentrattons42of Mn’+GTP (2 mmol/L) and Mn*+ (125 ymol/L), respectively,
in the presenceand absenceof ODQ (Table 1) In both
variations, ODQ had a stattstically stgmficant effect to
lower V,,, approximately threefold with no effect on K,,,
(Table 1) The NOS mhtbttor, N“‘-mtro-L-argmme (0 1
mmol/L) did not alter these kinetic reactions (L J 0 ,
J G D , unpublisheddata, 1996). These resultsindicate a
noncompetttive mhibttion with respectto both metal-nucleotide chelate or Mn2+ independentof the presenceof
NO. Km and V,,, values of sGC for NO were determined
m the presenceand absenceof ODQ with fixed concentrations of Mn2+ (125 pmol/L) and GTP (12 5 pmollL)44
(Table 1) As shown in Table 1, ODQ significantly lowered the V,,, and increasedthe K,,, (approxtmately twofold) of the enzyme for nonoate-donatedNO. The latter
result arguesthat ODQ is a mixed competittve/noncompetitive inhibitor with respectto NO donation. In all three
variations, the synthesisof cGMP was linear over the 5minute course of the experiment. Subsequent expenments also demonstratedthat NOS activity was not detected m COS-7 sGC cells (seebelow, Table 2).
TABLE 2. Effects of ODQ, N”-Nitro-L-Arginme,
and
Methylene Blue on NOS Actwity In Whole-Cell Lysates
From Bovine Coronary Artery Endothelial Cells
NOS Actwty,
fmol
cltrulhne
formed/mg
lysate
proteidmln
InhIbitor
ODQ (10 pmol/L)
N”-nltro+-arginlne
(0 1 mmol/L)
Methylene
blue (0 1 mmol/L)
Values are meanzSE
(+), lnhkbltor
treatment,
for n=4 (-) lndlcates
and ND, not detectable
I-1
(+I
247214
222klO
24015
22827
ND
ND
InhIbItor
vehicle
treatment,
Lack of ODQ Effect on NOS Activity in Endothelial
Cells and Absence of NOS Activity in COS-7
sGC Cells
The purpose of these experiments was to test for an
effect of ODQ on NOS acttvity m endothehalcells and m
COS-7 sGC cells Table 2 showsthat NW-mtro+argmme
and methylene blue (0 1 mmol/L each) completely block
the conversion of [3H]-L-argmmeto [3H]-L-cttrullme m
bovine coronary artery endothehal cells. ODQ (10
pmol/L), however, doesnot effect the samereaction,demonstrating that it doesnot modulateNO generation NOS
activity was not detectablem COS-7 sGC cells (Table 2)
Effect of Xanthine/Xanthine
Oxidase and ODQ on
the Auto-oxidation of Nonoate-Donated NO
These studiesdetermmedwhether ODQ could oxidize
NO m the presenceof COS-7 cells or rat aortic rmgs at
physiological pH In both of thesepreparations,the effects
of the superoxtde generating system xanthmelxanthme
oxidase and ODQ were examined Followmg treatment
with 50 ,umol/L nonoatem the presenceof xanthmelxanthme oxidase vehicle, the concentration of NO generated
was 168215 nmol/L (n=3) or 266+47 nmol/L (n=2) m
the presenceof COS-7 cells or aorta, respechvely Combined treatment with xanthme (10 pmol/L) and xanthme
oxidase (5 U/mL) lowered the levels of nonoate-donated
NO from those obtained m the presenceof nonoate and
xanthme/xanthine oxidase vehicle (above) to 49.4?9.8%
for COS-7 cells (n=3) and 22 123 2% (n=2) for aortic
rings In contrast, ODQ (10 pmol/L) did not result m reductions of NO concentrations,which were 105 l+l 3%
(n=3) and 107 820 2% (n=2) of the control levels
reached m the presenceof both 50 pmol/L nonoate and
xanthme/xanthmeoxidasevehicle (above) for COS-7 cells
and rat aortic rmgs, respectively Xanthme (10 pmol/L) or
xanthme oxidase (5 U/mL) alone had no effect on the
amount of nonoate-donatedNO m the presenceof COS-7
cells or aorttc rmgs (L J.O., E T.K., J G D , unpublished
data, 1996)
Lack of ANP-, CNP-, and Heat-Stable EnterotoxinStimulated cGMP Production in HEK-293 Cells
Expressing Stable Transfectants of GC-A, -B,
and 4, Respectively
Theseexperimentswere designedto test whether ODQ
had any effect on the membrane-associated
guanylyl cyclasesGC-A, -B, or -C ANP, CNP, and heat-stableenterotoxm (10-l’ to lo+ mol/L) result in concentrationdependent (ANOVA, P<.OOOl) sttmulation of cGMP
production m HEK-293 GC-A (Fig 3A), -B (Fig 3B), and
-C (Fig 3C) cells, respectively. ODQ pretreatmenthad no
Olson et al
s
5
A
1
”
Selective Inhibitor of Guanylyl Cyclase
T
---a.--
VEH
(-) ODQ
(+)ODQ
-10
-9
LOG[ANP]
-6
$~ollL-;l
fz
0
0
05
i
---*--
ANP (6)
ANPtODQ
(6)
-
CNP (5)
CNP+ODQ
(5)
1 ---*--
0.0’VEHr
-
VEH
-10
LOG$NP]
-(“,oIi;
-6
C
2
f
Downloaded from http://hyper.ahajournals.org/ by guest on June 15, 2017
---A---
is
f
259
(+)
O,,Q
-10
-9.5
-9
-a 5
LOG[peptlde]
L
-5
-7.5
(mol/L)
;
-7
\
-6.5
-6
FIG 4
Absence
of ODQ (10 pmol/L)
effect on ANP and CNP
natnuretrc
peptrde-induced
relaxation
of phenylephnne
(100
nmol/L)-precontracted
rat aorta.
ANP/CNP
indicates
pepttde+ODQ
vehicle treatment,
ANP/CNP+ODQ,
peptrde+ODQ
treatment,
and VEH, natnuretic
peptrde-vehicle
treatment
N In
parentheses
for each treatment
Data points are mean?SE
For
comparison
of each peptide
concentratron
vs VEH, *PC 05,
**p< 01, ***PC 001
1
ment did not affect the ability of 8-bromo-cGMP
the precontracted vessel (Fig 5, ANOVA)
VEH
FIG 3
Absence
socrated
guanylyl
enterotoxrn
(STa,
cells overexpressing
respectrvely
Data
vehicle treatment,
vehicle treatment
-10
-9
LOG[STa]
-6
-7
(mol/L)
-6
of ODQ (10 fimol/L)
effect on membrane-ascyclases
ANP- (A), CNP- (B), and heat-stable
C)-strmulated
cGMP
productron
in HEK-293
stable transfectants
of GC-A, -B, and -C,
potnts are mean?SE
(-) ODQ indicates
ODQ(+) ODQ, ODQ treatment,
and VEH, peptrde-
effect by ANOVA on the abihty of ANP, CNP, or heatstable enterotoxin to stimulate the membrane-associated
guanylyl cyclases GC-A, -B, or -C (Fig 3A through C,
respectively), which act as the receptors for these pepttdes.
Lack of ODQ Effect on Natriuretic Peptide-Induced
Relaxation of Phenylephrine-Precontracted
Rat Aorta
These studies tested the ability of ODQ to block the
effect of two known vasorelaxant pepttdes, ANP and CNP,
which act as agonists of the membrane-associated guanylyl cyclases GC-A and GC-B. Both ANP ( low9 to lo-’
mol/L, I(&,=3 nmol/L; Fig 4) and CNP (lo-* to 10e6
mol/L, I&,=30 nmol/L; Fig 4) resulted m concentrationdependent vasorelaxanon of precontracted rat aorta (ANOVA,
P<.OOOl) similar to those published previously from this
laboratory 33 The vasorelaxant effects of both ANP and
CNP were unaffected by pretreatment with ODQ (Fig 4,
ANOVA, P<OOOl).
to relax
Lack of ODQ Effect on Isoproterenol- and
Bimakalim-Mediated
Relaxation of PhenylephrinePrecontracted Rat Aortas
To further examine the specifictty of ODQ for blocking
NO-mediated vasorelaxation, we also tested the quinoxalm derivative for any antagomsttc effect on two other
known vasodilatory agents that signal their cellular responses independent of guanylyl cyclase activatron ODQ
(10 pmol/L) was found to have no effect on the vasorelaxation facilitated by the ,&selective adrenoceptor agonist
isoproterenol45 and the ATP-sensitive Kt channel opener
brmakalim39240(L.J.O., J G D , unpublished data, 1996).
Discussion
Nonoate resulted m a concentration-dependent stimulation of cGMP production m rat aortlc vascular smooth
muscle cells and COS-7 sGC cells The ability of nonoate
to increase cGMP synthesis m these cells was sigmficantly
attenuated by ODQ pretreatment, consistent with a potential mhibrtory effect of the qumoxalm-derivative on sGC 18
Lack of ODQ Effect on SBromo-cGMP-Mediated
Relaxation of Phenylephrine-Precontracted
Rat Aorta
These experiments tested whether ODQ blocked cGMPmediated vasorelaxatton downstream of guanylyl cyclase.
Fig 5 shows that the membrane-permeable cGMP analog
Sbromo-cGMP
(10m7 to 3X10e4 mom) resulted in a
concentration-dependent relaxation of phenylephrme-contracted rat aortas (IC&=30 pmol/L; ANOVA, P< 0001).
ODQ pretreatment of a paired aortic ring m each experi-
VEH
FIG 5
-7
-6.6
-6
-5 5
LOG[B-bromo-oGMP]
-5
-4.6
-4
-3.5
(mol/L)
Absence
of ODQ (10 pmol/L) effect on 8-bromo-cGMPmediated
relaxation
of phenylephnne
(100 nmoVL)-precontracted
rat aorta. Data pornts are mean+SE
(-) ODQ Indicates
ODQvehicle treatment,
(+) ODQ, ODQ treatment;
and VEH, 8-bromocGMP-vehrcle
treatment
260
Hypertension
Vol29,
No 1, Part 2
January 1997
Downloaded from http://hyper.ahajournals.org/ by guest on June 15, 2017
More specifically, ODQ completely blocked nonoate effects m the aortic cellsand slgmficantly shiftedthe nonoate
concentration-responsecurve m the COS-7 sGC cells to
the right and suppressedthe maximal effect. ODQ therefore presenteda mixed competltlve/noncompetitlve antagonism.Nonoateandnitroglycerin, anotherNO donor, were
likewise found to relax phenylephrme-precontractedrat
aorta m a concentration-dependentmanner ODQ caused
a sm-ularsignificant nghtward shift of the nonoate-responsecurve and an attenuation of the maxlmal effect,
mdlcatmg a competitlve/noncompetltive mhlbltlon of the
vasorelaxant effect of the NO donor This result 1sconsistent with the observationson whole-cell cGMP productlon
m this study and a previous study m isolatedintact bovine
pulmonary arteries46
Thesedata led to a further charactenzatlonof ODQ effects on the NO/sGC/cGMP slgnalmgcascadein both the
COS-7 sGC cells andrat aortas.ODQ could have resulted
m the observed effects on cGMP concentrations by dlrectly mhlbltmg sGC or lowenng donated-NO concentratlons by the generationof superoxideamon Kmetlc studies on sGC activity m the cytosollc fraction from
COS-7 sGC cells demonstratedthat ODQ is a mixed competitive/noncompetitive inhibitor of the enzyme with respect to nonoate stimulation m the presence of fixed
concentrationsof Mn2+ and Mn2+-GTP This result 1sconsistentwith the pharmacologicalactivity of ODQ on both
nonoate-mediatedcGMP production m whole cells and
aortic vasorelaxation and with a recent report that ODQ
acts m part by binding to the heme-moiety46Additionally,
we consideredwhether the noncompetitive effect of ODQ
on sGC activity was mediatedby competition with metal
ion- or nucleotide-substrateKinetic studieswith a saturating concentration of Mn2’-GTP or Mn*+ revealed that
ODQ wasa noncompetitive mhibltor with respectto varying concentrationsof Mn2+ or Mn’+-GTP, respectively, m
the absenceof NO donatlon or NOS activity Taken together, our kmetlc data suggestthat ODQ can modulate
enzyme activity both by effects on the NO bmchngsite and
by interacting with a region other than that associatedwith
NO, metal, or nucleotlde bmdmg47
The resultsof the presentstudy with ODQ also yield a
clearer conclusion than those previously associatmgsGC
with vasorelaxationusing LY83583 and/or methyleneblue
For over the last decade,both compoundshave beentouted
assGCinhibitors They arenow known to be nonspecificm
then mhibltory effects on the NO/cGMP signaltransduction
pathway, mcludmgthe machvationof reactiveNO~2J6J7
and
the mtibition of endothehum-derivedrelaxmg factor generation’ or NOS activity in the presentstudy, as well asothers 10~1
Moreover, LY83583 also may reducecGMP levels
m cells by an unknown mechamsmnot associatedwith reductionsof sGC activity I4 We found that ODQ doesnot
m&bit NOS activity and doesnot auto-oxi&ze NO m the
presenceof COS-7 cells or aortic rmgs.The latter result 1s
consistentwith the previousreport demonstratingthat ODQ
doesnot oxldlze NO m the absenceof cells and doesnot
attenuateNO-mediatedmacrophagetoxlclty 18
The effects of ODQ on vasorelaxationand cGMP production arealsoselectivefor NO. ODQ chdnot affect ANPandCNP-inducedaortlc vasorelaxatlonor ANP-, CNP-, and
heat-stable enterotoxm-stimulated cGMP production m
HEK-293 GC-A, -B, and -C cells, respectively GC-A,
GC-B, and the related heat-stable enterotoxm receptor
GC-C would all be potential targets for ODQ, since they
are highly conservedm the catalytic region in comparison
to sGC 1In the presentstudy, ODQ alsodid not attenuate
the relaxation evoked by either lsoproterenol,a P-selective
adrenoceptoragonist,45or bimakahm, an ATP-sensitive
K+ channelopener39.40
The former 1sthought to relax vascular smoothmuscleby increasingadenylyl cyclaseactlvlty45and the latter by hyperpolmzatlon resemblingthat of
endothehum-derivedhyperpolarlzmg factor 3~4~~48
ODQ alsodoesnot have any apparenteffect on vascular
activity of cGMP downstreamof cyclase activauon (le,
protein kmase or phosphochesterasestimulation) This
conclusion 1sbasedon the novel observationsthat ODQ
IS unable to block membrane-permeablecGMP analogand CNP-mediatedvasorelaxatlon m the absenceof the
phosphodlesterasemhlbitor, lsobutylmethylxanthme. A
vasorelaxant role for GC-B has been previously established m that a monoclonalantibody specific to the extracellular region of GC-B blocks CNP bmdmg, CNP-medlated production of cGMP, and CNP-inducedrelaxation of
phenylephrme-contractedrat aorta 33
Interestingly, ODQ doesnot fully antagonizenonoatemediatedaortic relaxation but completely ablatesthe NOdonor-stimulatedcGMP production even at maximally efficacious concentrations (ie, 1 mmol/L) in rat aortic
vascular smoothmusclecells and m soluble protem fractions from rat aorta, respectively Similar results usmg
ODQ m bovine pulmonary artenes led to the suggestlon
of sGC-dependentand sGC-independentmechanismsfor
NO-mediated vascular relaxation 46 The data from the
presentstudy could be interpreted to fit this dichotomy as
well. However, perhapsthe assayof NO-donor effects on
vascular smoothmuscle reactivity 1smore sensltlvethan
the current measurementsof intracellular cGMP concentrations, renderinga clear conclusionof two separatepathways impossibleat this time Future studiesmay be able
to addressthis issuemoreJudiciously with comparisonsof
NO-donor activity m isolated vesselsfrom wild-type versus sGC-geneknockout animals, if and when the latter
becomeavailable
The resultsof the presentstudy clearly demonstratethat
ODQ can inhibit sGC activation independentof the presence of NO donors or NOS activity and that ODQ neither
modulatesthe vasculareffectsof membranepermeable
cGMP
analogsnor auto-oxidizes NO. These observationschssoelate the activities of ODQ from effects on NO concentrations and downstreamcGMP-mechatedcellular events(eg,
activation of cyclic nucleotlde-sensitiveprotem kmasesor
cyclic nucleotide-mediated activation of ion channels),
further supportmga &rect effect of the qumoxahn denvatlve to inhibit the purified enzyme l8.47
Thesedatasupport
the hypothesisthat sGC mediatesvasorelaxantactlvlty associatedwith NO donors ODQ ~111likely becomean lmportant tool m attempting to differentiate between sGCdependentand -Independenteffects of NO
Acknowledgments
This work was supported by Amencan Heart Assoclatlon,
Wisconsm Affiliate, Grant-m-Aid 96-GB-58 (Dr Drewett) and
the 1995 Ruth Salta Junior Investigator Award (Dr Drewett) from
the Natlonal Heart Foundation, a program of the American Health
Assistance Foundation The authors also thank Dr David L Garbers and the Howard Hughes Me&Cal Institute for providing the
cGMP antibody, the HEK-293 cells overexpressmg stable transfectants of the membrane-associated guanylyl cyclases (GC-A,
-B, and -C), and the cDNA clones of the rat cr, and PI soluble
guanylyl cyclase subumts, Dr W&am B Campbell for provldmg
Olson et al
the bovine coronary
artery endotheltal
cells, Dr Garrett J Gross
for provrdmg
btmakalrm
and mtroglycerm, and Dr Begonia
Y
Ho for the crlttcal reading of this manuscrrpt
References
Downloaded from http://hyper.ahajournals.org/ by guest on June 15, 2017
1 Drew&t JG, Garbers DL. The family of guanylyl cyclase-receptors,
then hgands and functtons Endocrme Rev 1994.15 135-162
2 Cohen RA, Vanhoutte PM Endothehum-dependent
hyperpolanzatton beyond nitric oxide and cychc GMP
Ctrculanon
1995,92
3337-3349
Waldman SA, Murad F Cychc GMP synthesis and function Pharmacol Rev 1987,39 163-196
Nakatsu K, Diamond J Role 01 cGMP m relaxation of vascular and
other smooth muscle Can J Physlol Pharmacol
1989,67 25 l-262
Lmcoln TM, Cornwell TL Intracellular
cychc GMP receptor proteins FASEB J 1993.7 328-338
Bono T, Koibuchi Y, Sata N, Funuchi A, Nishn M, Yamamoto T,
Man J, Kohsaka M, Ohtsuka M Vasorelaxant
mechanism of the new
vasoddator, FK409 Eur J Phatmacol
1993,246 205-212
I Bolotma VM, NaJibi S, Palacmo JJ, Pangano PJ, Cohen RA Nitric
oxide directly activates calcmm-dependent
potassmm channels m
vascular smooth muscle cells Nature (Lond) 1994,368 850-853
8 Diamond J Effects of LY83583, nordihydroguaiarettc
acid, and qmnacrme on cychc GMP elevation and mhibmon of tension by muscanmc agonists m rabbit aorta and left atrium Can J Physzol Pharmacol 1987,65 1913-1917
9 Malta E, Macdonald PS, Dusting GJ Inhibition of vascular smooth
muscle relaxation by LY83583
Naunyn Schmzedebergs Arch Pharmacol 1988,337 459-464
10 Mayer B, Brunner F, Schmidt K Inhibition of nitric oxide synthests
by methylene blue Btochem Pharmacol
1993,45 367-374
11 Luo D, Das S, Vincent SR Effects of methylene blue and LY83583
on neuronal nitric oxide synthase and NADPH-diaphorase
Eur J
Pharmacol
1995,290 247-251
12 Wolm MS, Cherry PD, Rodenburg JM, Messma EJ, Kaley G Methylene blue mhibtts vasodilation of skeletal muscle arterioles to acetylcholme and mtnc oxide via the extracellular
generation of superoxide amon J Pharmacol Exp Thrr 1990,254 872-876
13 Brune B, Schmidt K-U, Ullnch V Activation of soluble guanylate
cyclase by carbon monoxide and mhtbmon by superoxide amon Eur
JB~ochem
1990,192 683-688
WS, Flemh JH
14 Schmidt MJ, Sawyer DB, Truex LL, Marshall
LY83583
an agent that lowers intracellular
levels of cychc guanosme 3’ 5’-monophosphate
I Phnrmacol
Exp Ther 1985,232
764-769
15 Mulsch
A, Busse R, Lrebdu
S, Forstermann
U LY83583
interferes with the release of endothelmm-derived
relaxing factor
and mhibits soluble guanylate
cyclase J Pharmacol
Exp Ther
1988,247 283-288
16 Mulsch A, Luckhoff A, Pohl U, Busse R, Bassenge E LY83583
(6-amlmo-5,8-qumolmedione
blocks mtrovasodtlator-mduced
cychc
GMP Increases and mhtbmon
of platelet aggregation
Naunyn
Schmezdebergs Arch Pharmacol
1989,340 119-125
17 Cherry PD, Omar HA, Farrell KA, Stuart JS, Wolm MS. Superoxide
anion mhibits cGMP-associated
bovme pulmonary
arterial relaxation Am / Physzol 1990,25 H1056-H1062
18 Garthwaite
J, Southam E, Boulton CL, Nielsen EB, Schmidt K,
Mayer B Potent and selective mhtbmon of nitric oxide-sensitive
guanylyl cyclase by 1&[1,2,4]0xadiazolo[4,3-a]qmnoxalm-l-one
Mel Pharmacol
1995,48 184-188
19, Hrabie JA, Klose JR, Wmk DA, Keefer LK New mtnc oxrde-releasing zwitterions
derived from polyammes
J Org Chem 1993,
58.1472-1476
20 Potter LR, Garbers DL Dephosphorylation
of the guanylyl cyclaseA receptor causes desensmzatton
J Btol Chem 1992,267 1453114534
21 Chnsman TD, Schulz S, Potter LR, Garbers DL Seminal plasma
factors that cause large elevattons m cellular cychc GMP are C-type
natrmretic peptides J Blol Chem 1993,268 3698-3703
22 Vaandrager AB, Schulz S, De Jonge HR, Garbers DL Guanylyl cyclase C is an N-linked
glycoprotem
receptor that accounts for
multiple heat-stable enterotoxm-bmdmg
proteins m the intestine
J Bzol Chem 1993,268*2174-2179
23 Thompson DK, Garbers DL Dommant negatrve mutations of the
guanylyl cyclase-A receptor J Bzol Chem 1995,270 425-430
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
SelectiveInhibitor of Guanylyl Cyclase 261
Krtegler M DNA transfer In Gene Transfer and Expresston
New
York, NY Stockton Press, 1990 99-100
Yuen PST, Doohttle LK, Garbers DL Dominant negative mutants of
the mtnc oxide-sensitive
guanylyl cyclase J B~ol Chem 1994,269
791-793
Harteneck C, Koeslmg D, Soling A, Schultz G, Bohme E Expression
of soluble guanylate cyclase catalytm acttvtty requires two enzyme
subunits FEES Lett 1990,272 221-223
Nakane M, Arm K, Sahekr S, Kuno T, Buechler W, Murad F Molecular cloning and expression of cDNAs coding for soluble guanylate cyclase from rat lung J Bzol Chem 1990;265 16841-16845
Harteneck C, Wedel B, Koeslmg D, Malkewitz
J, Bohme E, Schultz
G Molecular cloning and expressron of a new or-subunit of soluble
guanylyl cyclase FEBS Left 1991,292 217-222
Buechler WA, Nakane M, Murad F Expression of soluble guanylate
cyclase activity requires both enzyme subunits Bzochem Btophvs Rey
Commun 1991,174 351-357
Dtgho CA, Grammas P, Giacomelh F, Wiener J Angiogenesis in rat
aorta nng explant cultures Lab Invest 1989.60 523-53 1
Campbell WB, Gebremedhm
D, Pratt PF, Harder DR Identtficatton
of epoxyeicosatnenom
acids as endothelmm-derived
hyperpolarizmg
factors Czrc Res 1996,78 415-423
Hansborough
JR, Garbers DL Sodmm-dependent
activation of sea
urchin spermatazoa by speract and monensm. J B1o1 Chem 1981,
256 2235-2241
Drewett JG, Fendly BM, Garbers DL, Lowe DG Natnuretm pepttde
receptor-B (guanylyl cyclase-B) mediates C-type natnuretic peptlde
relaxation
of precontracted
rat aorta J Bcol Chrm
1995,270
4668-4674
Olson LJ, Lowe DG, Drewett JG Novel natrmrettc pepttde receptor/
guanylyl cyclase A-selective agonist mhibtts angiotensm II- and forskolm-evoked
aldosterone syntheses m a human zona glomerulosa
cell-line h401 Pharmacol
1996,50 430-435
Bradford MM A rapid and sensitive method for the quantttatton of
microgram quantmes of protein utihzmg the prmctple of protein-dye
binding. Anal Bzochem 1976,72 248-253
Schulz S, Green CK, Yuen PST, Garbers DL Guanylyl cyclase IS a
heat-stable enterotoxm receptor Cell 1993,63 941-948
Ishu K, Chang B, Kerwm JF Jr, Huang Z-J, Murad F N”-mtro-Largmme a potent mhtbitor of endothehum-denved
relaxing factor
formation
Eur J Pharmacol
1990.76 219-223
Drewett JG, Trachte GJ, Marchand GR Atnal natrmretic factor mhibits adrenergtc and punnergm neurotransmtsston
m the rabbit isolated vas deferens J Pharmacol Exp Ther 1989,248 135-142
van Woerkens LJ, Baas NR, van der Giessen WJ, Verdouw PD Cardiovascular effects of the novel potassmm channel opener bimakahm
m conscious pigs after myocardtal
mfarctron
a comparative
study
with mcorandtl Cardrovasc Drugs Ther 1992,6 409-417
Gross GJ, Mel DA, Schulz JJ, Mizumura
T Criteria for a mediator
or effector of myocardial precondmonmg
do KATP channels meet
the requirements7
BUSK Res Cardzol 1996,91 31-34
Lmeweaver
H, Burk D The determmatron
of enzyme dtssociation
constants J Am Chem Sot 1934,56 658-666
Chnsman TD, Garbers DL, Parks MA, Hardman JG Charactertzation
of particulate and soluble guanylyl cyclases from rat lung J B1o1
Chem 1975,250 374-381
Cleland WW Steady-state kmetics In Boyer PD, ed The Enzymes,
Vol 2 3rd ed New York, NY Academic Press, 1970 l-65
Ktmura H, Mittal CK, Murad F Activation of guanylate cyclase from
rat liver and other tissues by sodium aztde J Bzol Chem 1975,
250 8016-8022
Hoffman BB, Letkow~tz RJ Catecholammes, sympathomtmetic
drugs,
and adrenergm receptor antagonists, In Hardman JG, Limbud LE,
Mohnoff PB, Ruddon RW, Gilman AG, eds Goodman and Gzlman’s
The Pharmacologwal
Basrs of Therapeutrcs
9th ed New York, NY
McGraw-Hill,
1996 199-248
Brunner F, Schmtdt K, Nielsen EB, Mayer B Novel guanylyl cyclase
inhibitor potently mhlbtts cyclic GMP accumulation
m endothehal
cells and relaxation of bovine pulmonary
artery J Pharmacol Exp
Ther 1996,277 48-53
Schrammel A, Behrends S, Schmidt K, Koeslmg D, Mayer B Characterization of lH-]1,2,4]oxadiazolo[4,3-a]qmnoxahn-l-one
as a hemesite mhibttor of mtnc oxtde-senstttve
guanylyl cyclase MoZ Pharmacol 1996.50 l-5
Standen NB, Quayle JM, Davies NW, Brayden JE, Huang Y, Nelson
MT Hyperpolanzmg
vasodilators activate ATP-sensitive
K+ channels m artenal smooth muscle Science 1989,245 177-180
Selective Guanylyl Cyclase Inhibitor Reverses Nitric Oxide-Induced Vasorelaxation
Linda J. Olson, Edward T. Knych, Jr, Thomas C. Herzig and James G. Drewett
Downloaded from http://hyper.ahajournals.org/ by guest on June 15, 2017
Hypertension. 1997;29:254-261
doi: 10.1161/01.HYP.29.1.254
Hypertension is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231
Copyright © 1997 American Heart Association, Inc. All rights reserved.
Print ISSN: 0194-911X. Online ISSN: 1524-4563
The online version of this article, along with updated information and services, is located on
the World Wide Web at:
http://hyper.ahajournals.org/content/29/1/254
Permissions: Requests for permissions to reproduce figures, tables, or portions of articles originally
published in Hypertension can be obtained via RightsLink, a service of the Copyright Clearance Center, not
the Editorial Office. Once the online version of the published article for which permission is being requested
is located, click Request Permissions in the middle column of the Web page under Services. Further
information about this process is available in thePermissions and Rights Question and Answer document.
Reprints: Information about reprints can be found online at:
http://www.lww.com/reprints
Subscriptions: Information about subscribing to Hypertension is online at:
http://hyper.ahajournals.org//subscriptions/