Peroxynitrite and nitric oxide differ in their effects on pig coronary artery smooth muscle

Mandeep Walia2, Sue E. Samson1 , Tracey Schmidt1, Kelly Best2, Melinda Whittington1 and

Chui Yin Kwan1, Ashok K. Grover1,2

Departments of Medicine1 and Biology2, HSC 4N41 McMaster University, 1200 Main Street

West

Hamilton, Ontario, Canada L8N3Z5.

Running Title: Peroxynitrite generators and coronary artery

Corresponding Author:

Dr. A.K. Grover

Department of Medicine, HSC 4N41, McMaster University, 1200 Main Street West

Hamilton, Ontario L8N 3Z5 Canada

Phone: 905-525-9140 x22238

Fax: 905-522–3114

E-mail: groverak@mcmaster.ca

Copyright 2002 by the American Physiological Society.

AJP-Cell Articles in PresS. Published on November 13, 2002 as DOI 10.1152/ajpcell.00405.2002

Abstract

Peroxynitrite generated in arteries from superoxide and NO may damage their function.

Here we compare the effects of peroxynitrite and peroxynitrite/NO generating agents SIN-1 (3-

morpholinosydnonimine hydrochloride), SNAP (S-nitroso-N-acetylypenicillamine), SNP

(sodium nitroprusside) and NONOate (Spermine NONOate) on pig coronary artery. De-

endoethelialized artery rings were pretreated with these agents and then washed before

examining their contractility. The pretreatment with all the agents (200 FM) results in a

decrease in the force of contraction in response to the sarco/endoplasmic Ca2+ pump (SERCA)

inhibitor cyclopiazonic acid (CPA): SNAP > NONOate $ peroxynitrite $SIN-1 > SNAP.

Pretreatment with SNAP, NONOate or SIN-1 also inhibits the force of contraction produced with

30 mM KCl, with SNAP being the most potent. Including catalase plus superoxide dismutase

during the preincubation has no effect. Including an NO-scavenger (2-(4-carboxyphenyl)-4,4,5,

5-tetramethylimidazoline-1-oxyl-3-oxide) or a guanylate cyclase inhibitor (1H-

[1,2,4]Oxadiazolo[4,3-a]quinoxalin-1-one) partially protects against SNAP. Pretreatment of

cultured cells with peroxynitrite but not with SNAP inhibits the Ca2+ transients produced in

response to CPA. Pretreating isolated membrane vesicles with peroxynitrite inhibits the Ca2+

uptake due to the SERCA pump, with all the other agents being less effective. Thus

peroxynitrite and NO both inhibit the CPA induced contractions in de-endothelialized artery

rings - peroxynitrite by damage to the SERCA pump and NO possibly by a step downstream

from the increase in cytosolic Ca2+.

Keywords: ATPase, free radicals, oxidative stress, vascular diseases, ischaemia-reperfusion.

INTRODUCTION

Reactive oxygen species (ROS) are pivotal as intermediates in mitochondrial function and

as signalling molecules in many physiologically important processes (5,14,22,25). Their rates of

formation and dissolution are rigorously controlled. The enhanced production of ROS during

ischaemia-reperfusion and in vascular pathologies such as atherogenesis and inflammation is

implicated in cardiovascular damage (1,9,22). The damage caused by ROS to transport processes

may disturb homeostasis in vascular smooth muscle and endothelium. Such damage is not

uniform to all transport processes. Ca2+-dependent K+-channels in rat cerebral artery smooth

muscle cells are more sensitive to damage by ROS than are voltage sensitive Ca2+ channels (6).

In pig coronary artery smooth muscle, the sarco/endoplasmic reticulum Ca2+ (SERCA) pump is

more sensitive to damage by peroxide and superoxide than the plasma membrane Ca2+ (PMCA)

pump (16,19). Consequently, contractions to angiotensin II and to the SERCA pump inhibitors

thapsigargin and cyclopiazonic acid (CPA) are more sensitive to such damage by pretreatment of

the arteries with peroxide and superoxide than are the contractions to membrane depolarization

by KCl or endothelins (12,20). The damage may also be tissue dependent, e.g., smooth muscle

may be more sensitive to damage by peroxide than endothelium (18). Individual physiological

processes within each tissue may also differ in their susceptibility to different species of ROS.

Peroxynitrite differs from ROS such as peroxide, superoxide and hydroxyl radicals

(3,24,33). It can modify proteins by nitrosylation of thiols and tyrosine residues in addition to the

protein and lipid oxidation pathways used by other ROS. The effects of peroxynitrite on the pig

coronary artery have not been reported. Peroxynitrite is produced in arteries from nitric oxide

and superoxide generated by macrophages during injury or atherogenic inflammatory responses.

It may also be produced in the arteries during ischaemia-reperfusion (1,3,24,30). In the isolated

perfused rat heart, it induces both vasodilation and impaired vascular relaxation (41). Here, we

set out to determine the effects of peroxynitrite on pig coronary artery smooth muscle at three

different levels of organization: contractility in artery rings, cytosolic Ca2+ ([Ca2+]i) transients in

cultured cells and oxalate-stimulated-azide insensitive Ca2+ uptake in the SERCA enriched

subcellular membrane fraction. The effects of peroxynitrite can only be determined by adding a

bolus of this agent since it is unstable at the physiological pH. There are no agents that can

slowly generate only peroxynitrite. Most peroxynitrite generating agents produce mixtures of

various ROS including NO. NO generating agents may also cause S- and O-nitrosylation of

proteins although it is not clear if they are all equally effective in doing so (5,24). Therefore, we

also compared the effects of peroxynitrite with different peroxynitrite/NO-generating agents.

EXPERIMENTAL METHODS

Contractility Experiments: Pig hearts were obtained from Maple Leaf Meats (Burlington,

Canada) and placed immediately in an ice cold physiological saline solution (20). The left anterior

descending coronary artery was dissected, de-endothelialized and placed in a Krebs’ solution

bubbled with 95% O2 - 5% CO2. The Krebs’ solution contained (in mM): 115 NaCl, 5 KCl, 22

NaHCO3, 1.7 CaCl2, 1.1 MgCl2, 1.1 KH2PO4, 0.03 EDTA, and 7.7 glucose. From the middle

region of each artery, 3 mm long rings were cut and hung under 3 g tension in 4 ml organ baths at

37 E C for contraction as previously described (20). The maximum force of contraction was

determined for each artery ring by adding 3 M KCl to a final concentration of 60 mM. The rings

were then washed five times with 5 ml Krebs’ solution over a total period of 20 min and then

allowed to equilibrate for another 20 min. The force of contraction with 30 mM KCl (20) was

examined and the tissues were washed again. Aliquots of peroxynitrite (Calbiochem) under

nitrogen were stored at -80EC and used within 3 months. The peroxynitrite concentration was

determined by its absorbance at 302 nm (extinction coefficient = 1670 M-1cm-1) in 0.1M NaOH

immediately before use. SNAP (S-nitroso-N-acetylypenicillamine) and SNP (sodium

nitroprusside) were prepared in chilled Krebs’ solution just before use. NONOate (Spermine

NONOate), CPTIO: 2-(4-carboxyphenyl)-4,4,5, 5-tetramethylimidazoline-1-oxyl-3-oxide and

SIN-1 (3-morpholinosydnonimine hydrochloride) were prepared as concentrated stock solutions

in water and stored at -80 EC. Concentrated stock solutions of CPA and ODQ (1H-

[1,2,4]Oxadiazolo[4,3-a]quinoxalin-1-one) were prepared in dimethylsulfoxide and stored at -80

EC. Aliquots of the frozen solutions were thawed only once. In all the instances aliquots of 4 Fl

were added to the organ baths containing 4 ml of Krebs’ solution. Peroxynitrite or other agents

were added and the tissues were incubated for another 30 min. They were then washed again five

times with 5 ml Krebs’ solution over a total period of 45 min and allowed to equilibrate for

another10 min. Their contraction to 30 mM KCl was examined for 20 min, the tissues washed

again over a 30 min period and then used for monitoring their contraction with 10 FM CPA.

[Ca2+] i measurements: Smooth muscle cells in passage 4 were cultured on cover slips and used

for the [Ca2+] i measurement experiments. Isolation and characteristics of these cells have been

previously described (15,20). These cells express smooth muscle actin, contract to A23187 and

show increase in [Ca2+] i in response to CPA, thapsigargin, angiotensin II or endothelin-1 but not

to membrane depolarization by KCl. The cover slips coated with cultured cells were removed

from the culture medium and rinsed with a solution containing (in mM): 115 NaCl, 5.8 KCl, 2

CaCl2, 0.6 MgCl2, 12 glucose, 25 HEPES-Na, pH 7.4 at 37EC. These were then incubated in this

buffer at 37EC for 30 min in the presence of 200 FM peroxynitrite or other agents, washed 3

times to remove these agents, loaded with FLUO 3/AM and then used for [Ca2+] i measurement.

The total time period between the end of the pretreatment with any agents and the [Ca2+] i

measurements was 90 min.

Ca2+ uptake in isolated membranes: Subcellular membrane fraction F3 enriched in SR

was isolated from smooth muscle. Isolation of these membranes and their characteristics have

been previously reported (16,19,21). The membranes were incubated with peroxynitrite or other

agents at 37EC and then 1 mM DTT was added to quench any remaining peroxynitrite or peroxide

produced from the decomposition. The samples were placed on ice until use. Ca2+ uptake was

carried out as described earlier (16,19). The final reaction mixture consisted of creatine kinase

(70 units/ml) and the following concentrations (in mM) of the other components 100 KCl, 5 Na-

azide, 5 MgCl2, 5 ATP, 10 creatine phosphate, 1 EGTA, 0.85 CaCl2 (plus trace amounts of

45Ca), 30 imidazole-HCl (pH 6.8 at 37EC), 0.33 DTT, 0 or 5 K-oxalate (with F3 fraction) and 0 or

5 K-phosphate (with F2 fraction, pH 6.8 at 37EC). This resulted in 5 µM free [Ca2+] as described

earlier (16,19).

Nitroblue tetrazolium reduction assay: Reduction of nitroblue tetrazolium was used to monitor

superoxide generation as described earlier (23). Briefly, 75 µl aliquots of each agent were

prepared and pipetted into a 96 well plate on ice and 175 µl of ice-cold nitroblue tetrazolium

solution was added with or without catalase and superoxide dismutase. The final reaction

mixtures contained: 200 µM of peroxynitrite or other specified agent, 1.5 mM nitroblue

tetrazolium, 0 or 200 units each of catalase and superoxide dismutase in the HEPES-Krebs’

solution described above for the contractility experiments. Absorbance of the samples at 595 nm

was monitored immediately, then the foil-wrapped samples were placed in a 37EC oven and

absorbance monitored again after 30, 60 and 90 min. The difference in the absorbance changes

with and without catalase plus superoxide dismutase after 30 min were used to express the

activity.

Data Analysis: Curve fitting was carried out by nonlinear regression using the FigP software

(BioSoft, Ancaster, Canada). Statistical significance was determined using the Student’s t-test.

All experiments were replicated 3-5 times.

RESULTS

Effects of pretreatment with peroxynitrite and NO-generating agents on contraction: De-

endothelialized arteries in Krebs’ solution contract when challenged with the SR Ca2+ pump

inhibitor CPA (31) or when membranes are depolarized with high concentrations of KCl. The

mean force of contraction produced by 30 mM KCl is 23.9 ± 1.2 mN (mean ± SEM, 100 tissues)

while that with 10 FM CPA, it is 4.9 ± 0.4 mN (100 tissues). In order to compare the damage to

the artery rings by various agents with that of peroxide and superoxide reported previously, we

used the same protocols as described earlier and given in the Experimental Methods (20). This

protocol includes extensive washing to remove from the organ baths any agents and their

decomposition products used during the pretreatment. This was done to ensure that these

substances would not directly interfere in the subsequent measurements. Fig. 1 shows the

concentration-dependence of the effects of pretreatment with peroxynitrite, SNAP or NONOate.

The pretreatment with peroxynitrite does not alter the force of contraction produced with 30 mM

KCl but SNAP or NONOate inhibit the contraction with 30 mM KCl with SNAP (r2 = 0.9454, IC50

= 5.7 ± 0.9 FM) being more potent than NONOate ( r2 =0 .9366, IC50 = 47 ± 15 FM) (Fig. 1A).

Pretreatment with these agents decreases the force of contraction produced with CPA with the

following order of potency: SNAP ( r2= 0.9145, IC

50 = 1.6 ± 0.5 FM) > NONOate (r2 =0.9719, IC50 = 32 ± 8 FM) > peroxynitrite (r2 = 0.9935, IC50

= 87 ± 6 FM) (Fig. 1B). We also determined the effects of single concentrations of SNP and

SIN-1. Pretreatment with 200 FM SNP decreased the force of contraction with KCl and CPA to

76 ± 9 and 61 ± 16 % of the control, respectively (data not shown). The corresponding values with

SIN-1 were 62 ± 11 and 32 ± 10 %. The rank order of effectiveness of these agents (at 200 FM)

for the CPA contraction is SNAP > NONOate $ peroxynitrite $ SIN-1 > SNP. This order is

similar but not identical to that obtained for the contractions with KCl (SNAP $ NONOate > SIN-

1 > SNP = peroxynitrite). Since, peroxynitrite was added as a bolus in the presence of 5-fold

molar excess of NaOH, we also examined the effects of pretreatment with NaOH up to 1000 FM.

Preteatment with NaOH does not significantly affect the force of contraction (p > 0.05). For

example, in the tissues pretreated with 1000 FM NaOH, the force of contraction with KCl is 96 ±

8 % of the control and with CPA it is 86 ± 11 %.

Effects of pretreatment with various agents with catalase plus superoxide dimutase on contraction:

To ascertain which of these agents produced measurable amounts of superoxide, we used an assay

based on change in the absorbance of NBT upon reduction. We incubated these agents with NBT

with or without excess catalase plus SOD . SIN-1 shows the largest amount of SOD sensitive

reduction of NBT and peroxynitrite is next (Table 1). The change in the absorbance of NBT with

SNAP, NONOate and SNP was no different from the water used as a control.

Pretreatment with peroxide and superoxide is known to inhibit the contractions to CPA

(20). Therefore, we considered the possibility that the hydrogen peroxide or superoxide produced

by decomposition of these agents may cause the inhibition of the contractions observed in Fig. 1.

We have shown previously that catalase plus SOD can overcome the effect of superoxide, and that

catalase alone can abolish the effect of hydrogen peroxide in this system (11,16). Hence, we

examined the effects of preincubation with SIN-1, peroxynitrite and SNAP with or without excess

catalase plus SOD. Fig. 2 shows that peroxynitrite inhibited only the CPA induced contractions

(p > 0.05) and all the other agents significantly decreased the force of contractions with KCl and

CPA (p < 0.05). Catalase plus SOD had no effect (p > 0.05) indicating that the observed effect is

primarily due to peroxynitrite and not due to its decomposition products.

Further experiments on the effects of SNAP on force of contraction: Since SNAP has the highest

potency, we considered the possibility that a stable decomposition product of SNAP was

responsible for the inhibition. To test this hypothesis, we incubated a stock solution of SNAP in

Krebs’s overnight at 37 E C and then pretreated the artery rings with it. Whereas a precincubation

with 10 FM freshly prepared SNAP decreases the force of contraction with KCl to 35 % of the

control and with CPA to 7 % of the control, in 3 tissues pretreated with the decomposed SNAP, the

force of contraction is 95 % of the control with KCl and 103 % with CPA. Thus the decomposed

SNAP is ineffective. This loss of ability of SNAP to inhibit the force of contraction indicates that a

metastable product of SNAP (such as NO) is responsible for the inhibition. Since SNAP is known

to generate NO we next conducted an experiment in which CPTIO (2-(4-carboxyphenyl)-4,4,5, 5-

tetramethylimidazoline-1-oxyl-3-oxide) was used to quench the NO generated by SNAP (32). In

preliminary experiments we established that CPTIO was active in that it inhibited the endothelium

dependent relaxation, thereby showing that CPTIO can effectively quench the NO produced by the

endothelium. We next examined if CPTIO could quench the NO produced by SNAP and thus

overcome its inhibition of the CPA induced contraction in de-endothelialized artery rings. We

preincubated the de-endothelialized artery rings with 10 FM SNAP with and without 50 FM

CPTIO (data not shown). Including this concentration of CPTIO in the preincubation mixture does

not overcome the effect of 10 FM SNAP on the force of contraction produced by 30 mM KCl or

by CPA. We then preincubated the de-endothelialized artery rings with 10 FM SNAP with and

without 200 FM CPTIO. It alleviates the inhibition by 10 FM SNAP significantly (p < .05) but

only partially on the force of contraction produced with KCl or CPA (Fig. 3).

The established mode of action of NO on smooth muscle is stimulation of guanylate

cyclase (24) and hence we next examined the effects of the guanylate cyclase inhibitor 1H-

[1,2,4]Oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) (38). In a preliminary experiment we

determined that ODQ at 10 FM completely inhibits the endothelium dependent relaxation in this

tissue but preincubating de-endothelialzed artery rings with ODQ does not affect the force of

contraction with KCl or CPA (data not shown). We next preincubated the de-endothelialized

artery rings with 10 FM SNAP with and without 10 FM ODQ (Fig. 4A). Including ODQ in the

preincubation mixture completely overcomes the effect of 10 FM SNAP on the force of

contraction produced by 30 mM KCl. However, its effect is only partial when the CPA induced

contraction is examined (Fig. 4B).

Effects of peroxynitrite pretreatment on [Ca2+] i transients: Smooth muscle cells cultured from the

coronary artery show an increase in [Ca2+] i transients when challenged with 10 FM CPA. When

cells are pretreated with 200 FM peroxynitrite and then washed as described in the Experimental

Methods, a challenge with CPA gives a smaller increase in [Ca2+] i transients (p < 0.05, Fig. 5).

Including catalase plus SOD with peroxynitrite does not alter its effects (data not shown).

However, a similar pretreatment with 200 FM SNAP does not significantly diminish the CPA

induced [Ca2+] i transients (p > 0.05, Fig. 5) suggesting that peroxynitrite and SNAP act via

different mechanisms. This result is in contrast to the contractility experiments where pretreatment

with this concentration of SNAP decreases the force of contraction with CPA by > 90 % (Fig. 1).

Effects of pretreatment with various agents on Ca2+-uptake in isolated membrane vesicles: We next

examined the possibility that the pretreatment with these agents may damage the SERCA pump in

the the SR-enriched fraction F3 isolated from pig coronary artery smooth muscle. The subcellular

fractionation and Ca2+ uptake properties of F3 have been characterized previously (17,21). With

these membranes, one can use an ATP-regenerating system to obtain Ca2+ uptake which is linear

with time for up to 2 h. The uptake due to the SR in this fraction is ATP-dependent, azide

insensitive, stimulated by oxalate that prevents back flux of Ca2+, and is inhibited by CPA. In

preliminary experiment we observed that pretreating the membranes with 100 FM peroxynitrite

gave similar values for inhibition of the Ca2+ uptake as those observed for the CPA-induced

contractions with 200 FM peroxynitrite. Therefore, we compared the effects of pretreating the

membranes with 100 FM of each of these agents on the Ca2+ uptake. Fig. 6 shows that: (a)

pretreating the F3 membrane vesicles with 100 FM peroxynitrite results in an 80 % inhibition of

the Ca2+ uptake, (b) a similar pretreatment with 100 FM SNAP or SNP leads to marginal inhibition

of the uptake and 100 FM NONOate or SIN-1 do not inhibit the uptake significantly (Fig. 6), and

(c) the inhibition of the uptake by the pretreatment with any of the agents is not overcome by

including catalase plus SOD during the pretreatment. Thus the rank order for inhibition appears to

be peroxynitrite >> SNP $SNAP > NONOate > SIN-1.

DISCUSSION

The Results show that: (a) pretreating the artery rings with peroxynitrite and NO-

generating agents decreases the force of contraction in the following decreasing order of

effectiveness:

SNAP > NONOate $ peroxynitrite $ SIN-1 > SNP with CPA and SNAP $ NONOate > SIN-1

with KCl (no inhibition with SNP and peroxynitrite), (b) the force of contraction with none of the

agents is significantly altered when catalase + SOD are included during the pretreatment, (c) the

effectiveness of SNAP is decreased with CPTIO or ODQ, (d) pretreatment of cultured cells with

peroxynitrite but not with SNAP decreases the CPA induced Ca2+i -transients, and (e) in isolated

membranes, the Ca2+ uptake due to the SERCA pump is inhibited predominantly by pretreatment

with peroxynitrite with the order of effectiveness of the different agents being peroxynitrite >>

SNP $SNAP $ NONOate = SIN-1. The Discussion focuses on the methods used in this study,

comparison of these results with the literature, a proposed model to explain the results and possible

biological implications of these findings.

The agents used during the preincubation or their decomposition products may react with

the reagents used subsequently in the assays. To avoid this possibility, we washed the tissues after

the treatment with the agents used for the pretreatment and before adding any other reagents. We

have shown earlier that including catalase plus SOD prevents the effects of superoxide and

peroxide on artery rings, cultured cells and isolated membranes (11,23). Thus protocols used in

the study would monitor the damage produced by the agents during pretreatment on contractility

or other properties rather than their effects when present during the course of the assays. This

distinction is important in comparing this work with the literature. Since one can not obtain pure

PM or SR membranes from the coronary artery smooth muscle, we used the effect of oxalate on

the inhibition of back flux of Ca2+ to identify the activity of the SERCA pump (16,17,21). The

membrane fractions used in this study and the characteristics of the oxalate-stimulated Ca2+ uptake

have been established previously (21). Thus the methods for monitoring contractility, [Ca2+] i and

SR Ca2+ pump activity in isolated membranes have been previously validated using pig coronary

artery (15,20).

We first conducted the work on artery rings with peroxynitrite. However, the damage

produced in this study required high micromolar concentrations of this agent. Since in these

experiments the peroxynitrite is extremely short lived but in vivo peroxynitrite may be generated

constantly during ischaemia-reperfusion or inflammation like responses during atherosclerosis, we

then used the peroxynitrite generating agent SIN-1 (23,24). Since all peroxynitrite generating

agents also produce NO, we also used several NO generating agents. None of the NO generating

agents produce only one species of NO and even NO gas is converted to other species and/or

oxidized to nitrites and nitrates under aerobic conditions (34). The various agents not only produce

NO at different rates but may also produce different species of NO (2,23,24,35). Predominant

species generated by some of the agents are NONOate and SNP (NO.), SNAP (NO+) and Angeli’s

salt (NO-) (24). We tested SNAP and NONOate and they were both able to produce relaxation in

the de-endothelialized arteries which had been precontracted with KCl . However, in inhibiting

the contractility SNAP was the most potent, even more potent than SIN-1 and peroxynitrite. In the

NBT reduction assay, only SIN-1 and peroxynitrite showed any production of superoxide

indicating that SNAP, NONOate and SNP generated predominantly various species of NO.

Thus we verified under our experimental conditions that SIN-1 produces significantly more

superoxide than SNAP. SNAP is in fact a very potent NO release agent. It produces NO at a rate

several hundred times faster than SIN-1 (23,24). SNAP activates guanylate cyclase at lower

concentrations than spermine-NONOate and the rate of NO release from SNAP is much faster,

than from the NONOate (2,24). The various agents not only produce NO at different rates but may

also produce different species of NO (2,23,24,35). Even when NO generated as a gas may form

different species depending not only on the aqueous environment but also in a tissue dependent

manner (10,34). In addition S-nitrosothiols like SNAP decomposes spontaneously in solution to

their disulfide forms while generating NO. However, decomposed SNAP by itself was not

effective though one can not rule out that the ability of SNAP to generate simultaneously NO and

disulfide groups that would allow it to act differently than other agents.

We have shown earlier that the pretreatment with peroxide or superoxide causes a decrease

in: (a) the SR Ca2+ pump activity in the coronary artery, (b) Ca2+ -transients produced by CPA in

cultured smooth muscle cells, and (c) tissue contractility to CPA and to agents such as angiotensin

II which depend on the mobilization of the SR Ca2+ (18,20). Peroxide and superoxide act simply

in oxidation reactions presumably involving thiol groups (3,24). However, the action of

peroxynitrite or SIN-1 did not occur via decomposition into superoxide and peroxide as their

effects were insensitive to the inclusion of catalase plus SOD during the pretreatment.

Peroxynitrite has been shown to cause nitrosylation of thiol groups and tyrosine residues

(27,36,40). Such nitrosylation of SERCA2a in slow twitch skeletal muscle decreases its enzymatic

activity (42). Therefore, the most likely mechanism appears to be inactivation of the SR Ca2+

pump by peroxynitrite directly or through SIN-1. A direct effect of peroxynitrite on the contractile

machinery or on voltage dependent Ca2+ channels is thus ruled out since contraction with 30 mM

KCl was not affected. However, one cannot rule out that pathways such as store depletion

dependent Ca2+ channels are also affected by the treatment.

Several effects of NO have been reported on vascular smooth muscle. An NO mediated

increase in store depletion dependent Ca2+ entry has been reported in some studies and decrease in

others (8,13). NO binding to heme group of guanylate cyclase activates it thus producing an

increase in cellular cGMP levels (2,24). NO mediated decrease in vascular reactivity has been

proposed to occur by various mechanisms (7,26,28,37). Our results show that pretreatment with

SNAP inhibits the contractions produced by KCl and CPA. The inhibition is not affected by

CPTIO at 50 FM but it can be overcome partially with 200 FM CPTIO indicating the involvement

of NO. However, even the high concentration of CPTIO used here does not completely overcome

the effects of SNAP possibly because SNAP produces NO very rapidly (2). SNAP produces

NO+. NO+ and NOC are both quenched with CPTIO although NO- produced by Angeli’s salt is

not quenched effectively with this agent (10,29). Since these experiments required pretreatment at

high concentrations of NO for a prolonged period under aerobic conditions, one can not rule out

the conversion of the initially generated NO into other products including the nitroxyl ion.

Gaseous NO under aerobic conditions can be converted to products which no longer react with

CPTIO (34). The effect of SNAP is also overcome in part by the guanylate cyclase inhibitor ODQ

suggesting that cGMP plays a key role in this pathway. However, 10 FM ODQ does not

completely overcome the effect of SNAP even though IC50 for guanylate inhibition of ODQ is 84

nM (38). This may be because ODQ may not as readily cross the cell membranes as NO, NO has

been converted into other products or another pathway is involved in the observed effects of

SNAP. Finally, the pretreatment of cultured cells with SNAP does not produce a decrease in Ca2+

transients in response to CPA. Together, these results suggest that SNAP acts by a step

downstream of Ca2+ mobilization.

Physiological Implications: The effects of peroxynitrite and NO may be important in aging,

ischaemia-reperfusion, inflammation, atherosclerosis and spesis (5,14,22,25). SERCA2a in rat

twitch skeletal muscle shows that nitrosylated tyrosine residues increase with aging (42). Such

data are not currently available for the coronary artery. Damage to the SERCA pump in this tissue

would mean a loss of reactivity to agents such as angiotensin and α-adrenergic agents that depend

on mobilization of the SR Ca2+ pools (4,20). NO signalling pathways are of central importance in

both the maintenance of vascular homeostasis and the progression of vascular disease. Multiple

interactions of NO with other pathways can lead to either vascular protection or potentiation of

inflammatory vascular injury (33). During sepsis, an increase in [Ca2+ ]i may be an early event

responsible for cellular injury. This would normally lead to an increased contractile force in the

affected areas (39). A decrease in the Ca2+ sensitivity of the contractile apparatus would thus

provide a useful protection.

We conclude that peroxynitrite and NO both inhibit the CPA induced contractions in de-

endothelialized artery rings - peroxynitrite by via damage to the SERCA pump and NO at a step

downstream from a change in [Ca2+ ]i as one of the possibilities. Both types of interactions have

important pathophysiological ramifications.

Abbreviations: CPA: cyclopiazonic acid, CPTIO: 2-(4-carboxyphenyl)-4,4,5, 5-

tetramethylimidazoline-1-oxyl-3-oxide, DTT: dithiothreitol, EGTA: ethylene glycol-bis-(β-

aminoethyl ether)-N,N,N’,N’ tetraacetate, NONOate (Spermine NONOate, diazeniumdiolates),

ODQ: 1H-[1,2,4]Oxadiazolo[4,3-a]quinoxalin-1-one, PM: plasma membrane, PMCA: PM Ca2+

pump, ROS: reactive oxygen species, SIN-1: 3-morpholinosydnonimine hydrochloride, SNAP: S-

nitroso-N-acetylypenicillamine, SNP: sodium nitroprusside, SR: sarco/endoplasmic reticulum,

SERCA: SR Ca2+ pump, SOD: superoxide dismutase.

Acknowledgements: The authors thank Dr. N. Narayanan of University of Western Ontario for

critical comments on the manuscript. This work was funded by a grant -in-aid from Heart &

Stroke Foundation of Ontario (Grant # T-4815). AKG received a Career Investigator Award from

the Heart & Stroke Foundation of Ontario.

Figure Legends

Fig. 1. Inhibition of contractility by preatment of de-endothelialized artery rings with different

concentrations of peroxynitrite, SNAP and NONOate. In control tissues, force of contraction

produced with 30 mM KCl is 23.9 ± 1.2 mN (mean ± SEM, 100 tissues). A. Force of contraction

produced with KCl by tissues pretreated the agents specified in the figure, was expressed as

percent of this value. B. The force of contraction with 10 FM CPA, in control tissues is 4.9 ± 0.4

mN (100 tissues) and values for the CPA induced contraction in treated samples are expressed as a

percent of this control. The values are mean ± SEM of several tissues for each concentration of an

agent. The total number of tissues for the curves were: 113 for peroxynitrite, 107 for SNAP and

60 for NONOate. The data were fitted with a simple inhibition model. In A and B, at each

concentration tested the order of inhibition produced by the pretreatment was SNAP > NONOate >

peroxynitrite.

Fig. 2. Contractility experiments with catalase + SOD included during pretreatment. Artery rings

were preincubated with 10 FM SNAP, 200 FM of peroxynitrite (peroxyn), 200 FM NONOate or

200 FM SIN-1, and tested as in Fig. 1. Catalase + SOD was included during preincubation where

shown. A total of 243 tissues were used in all the groups in this experiment. All the values are

percent of control carried out in parallel without any agents added during the preincubation. Mean

± SEM percent values are given.

Fig. 3. Contractility experiments with 200 FM CPTIO included during pretreatment. Artery rings

were preincubated with 10 FM SNAP and tested as in Fig. 1. 200 FM CPTIO was included

during preincubation where shown. The number of tissues used were: 22 (control), 44 (SNAP) and

21 (SNAP + CPTIO). All the values are percent of control carried out in parallel without any

agents added during the preicubation. Mean ± SEM percent values are given.

Fig. 4. Contractility experiments with ODQ included during pretreatment. Artery rings were

preincubated with 10 FM SNAP and tested as in Fig. 1. 10 FM ODQ was included during

preincubation where shown. The number of tissues used were: 25 (control), 19 (SNAP) and 17

(SNAP + ODQ). All the values are percent of control carried out in parallel without any agents

added during the preincubation. Mean ± SEM percent values are given.

Fig. 5. Effects of pretreatment with 200 FM peroxynitrite (peroxyn) or 200 FM SNAP on CPA

induced increase in [Ca2+] i. The values are mean ± SEM from 34 replicates for control, 13 for

peroxynitrite and 9 for SNAP. ‘*’ indicates that the peroxynitrite treatment inhibits the increase in

[Ca2+]i significantly (p < 0.05). SNAP does not produce a significant inhibition ( p > 0.05).

Fig. 6. Ca2+ uptake by SR enriched membranes pretreated with different agents. Membranes

treated without any additives showed an uptake of 804 ± 18 nmol/mg protein. Values for the Ca2+

uptake for the samples treated with the various additives are expressed as percent of this control

value and are mean ± SEM of 6 replicates. Treatment with peroxynitrite significantly (p < 0.05)

inhibited the uptake.

Table 1. SOD sensitive nitroblue tetrazolium reduction

Additive Change in absorbancy

Water 0.029 ± 0.009 (11)

Peroxynitrite (200 FM) 0.127 ± 0.015 (6)

SIN-1 (200 FM) 0.400 ± 0.063 (3)

SNAP (200 FM) 0.015 ± 0.003 (6)

SNP (200 FM) -0.007 ± 0.007 (6)

Nonoate (200 FM) -.018 ± 0.007 (6)

NaOH (200 FM) -0.014 ± 0.008 (6)

Note: The values are mean ± SEM of the number of replicates shown in parentheses.

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0 . 1 1 1 0 1 0 0 1 0 0 0

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