www.elsevier.com/locate/vph

Vascular Pharmacology 40 (2004) 205–211

Acute effects of toremifene on the vasculature of intact and

menopause-induced rats

Jorge Gonzalez-Pereza, Marıa J. Crespob,*

aDepartment of Biology, University of Puerto Rico-Rio Piedras, San Juan, Puerto RicobDepartment of Physiology, University of Puerto Rico School of Medicine, GPO Box 365067, San Juan 00936-5067, Puerto Rico

Received 25 February 2003; accepted 12 June 2003

Abstract

Clinical studies have shown that cardiovascular performance in postmenopausal women could be modified by treatment with selective

estrogen receptor modulators (SERM). However, the mechanisms by which these drugs act on the cardiovascular system have not been

elucidated. This work evaluates the effect of toremifene, a new member of the SERM family, on the vasculature of intact and

ovariectomized adult Sprague–Dawley rats. The responsiveness of rings from the thoracic aorta to norepinephrine, potassium chloride,

acetylcholine and sodium nitroprusside was assessed before and after 15 min of incubation with 1.0-AM toremifene. Toremifene displaced

the concentration–response curve for norepinephrine-induced contractions to the right in both groups of animals. Moreover, the EC50

values for the curves increased from 154F 31 to 754F 162 nM (P < .05) in intact rats and from 88F 11 to 230F 71 nM (P < .05) in

ovariectomized rats. Toremifene also reduced contractile responses to potassium chloride (10–120 mM), displacing the entire curve to the

right in both groups of animals without modifying the EC50 values. The drug shifted the concentration–response curve for the

acetylcholine-induced relaxation to the left and significantly increased Emax values (18% for ovariectomized rats vs. 16% for controls)

without affecting EC50 values in either group tested. In addition, toremifene potentiated the relaxing responses to physiological doses

(0.1–1.0 nM) of sodium nitroprusside in both groups, suggesting a direct effect at the level of the vascular smooth muscle. Acute

toremifene incubation increased basal relaxation in aortic rings from both intact and ovariectomized rats. These results suggest that

toremifene, by improving the functional status of the endothelium–smooth muscle unit, may have a beneficial effect on the cardiovascular

status of menopause-induced rats.

D 2003 Published by Elsevier Inc.

Keywords: Toremifene; Menopause; Ovariectomized rats; Endothelial dysfunction; Vascular reactivity

1. Introduction

Epidemiological studies have demonstrated that the

incidence of cardiovascular diseases increases abruptly with

the onset of menopause (de Kleijn et al., 2002; Matthews et

al., 2001). The decrease in estrogen levels that normally

occurred at menopause has been linked directly to this fact.

Some of the beneficial actions of estrogen on the cardio-

vascular system could be attributed to the interaction of this

hormone with the endothelium–smooth muscle unit in the

vasculature (Kauser and Rubanyi, 1997; Nechmad et al.,

1537-1891/$ – see front matter D 2003 Published by Elsevier Inc.

doi:10.1016/j.vph.2003.06.001

* Corresponding author. Tel.: +1-809-758-2525; fax: +1-787-753-

0120.

E-mail address: mcrespo@rcm.upr.edu (M.J. Crespo).

1998). The vascular endothelium is responsible for cardio-

vascular homeostasis and plays an important role in main-

taining blood fluidity, vascular tone and many other

physiological processes (Furchgott and Zawadzki, 1980;

Rubanyi, 1993; Davies and Hagen, 1993). When homeo-

stasis is lost, the endothelium becomes dysfunctional. The

presence of endothelial dysfunction has been confirmed in

menopausal women (Taddei et al., 1996; Staessen et al.,

1998) and in menopause-induced animal models (Riveiro et

al., 2001). Although hormone replacement therapy (HRT)

in menopausal women has been reported to improve vas-

cular function and decrease risk factors for the development

of cardiovascular complications (Higashi et al., 2001), a

recent report indicates that this therapy increases cardiovas-

cular risks in healthy menopausal women (Rossouw et al.,

2002). Clearly, the development of alternative therapies

J. Gonzalez-Perez, M.J. Crespo / Vascular Pharmacology 40 (2004) 205–211206

with compounds having estrogenic-associated properties is

needed. Administration of selective estrogen receptor mod-

ulators (SERM) has been considered as an alternative

therapy for decreasing cardiovascular events (Blum and

Cannon, 2001; Saitta et al., 2001a,b). This group of drugs

exhibits different stimulatory and/or inhibitory effect at the

estrogen receptor in different tissues (Katzenellenbogen et

al., 2001). Chronic treatment of healthy menopausal women

with SERMs (droloxifene or raloxifene) has been reported

to increase blood flow in the brachial artery (Herrington et

al., 2000; Saitta et al., 2001b) and to reduce cardiovascular

risk factors (Herrington et al., 2000; de Valk-de Roo et al.,

1999). The mechanisms involved in these actions are not

fully understood.

Toremifene, a member of the SERM family, has been

recently approved for treatment of breast cancer in the

United States (Maenpaa et al., 2000) although its cardio-

vascular profile have not been investigated. The present

study was designed to characterize the effects of acute

administration of toremifene on the endothelium–smooth

muscle unit in intact and ovariectomized adult rats. This is

the first study that investigates the effects of toremifene on

the vasculature. In this study, aortic rings from intact and

menopause-induced rats were used to answer the follow-

ing questions: (1) Does toremifene alter the sensitivity of

the vascular wall to vasoactive agents? and (2) Does

toremifene improve endothelial dysfunction present in

ovariectomized rats? Our results reveal that acute admin-

istration of toremifene significantly modifies the vascular

response to vasoactive agents in both intact and ovariec-

tomized rats.

2. Materials and methods

2.1. Animal model

Ninety female Sprague–Dawley rats (12 weeks of age;

Hilltop Lab Animals, Scottdale, PA) were used in the

present study. Animals were housed in groups of two to

three per cage. Water and food (Harlan Rodent Diet, 18%

protein) were provided ad libitum. The rats were maintained

on a normal light/dark cycle (12L:12D), with lights off at

5:00 p.m. and in a temperature-controlled room. All experi-

ments were approved by the Institutional Animal Care and

Use Committee and adhered to the guidelines established by

the National Institutes of Health and the American Veteri-

nary Medical Association.

2.2. Procedure for bilateral ovariectomy

Surgeries were performed when animals were f 3

months of age and all female rats had reached sexual

maturity. Before any surgical procedures, the animals re-

ceived a combination of ketamine (100 mg/ml) and xylazine

(100 mg/ml) solution (7:1) intraperitoneally prepared in

0.9% sterile saline solution. Bilateral ovariectomy was

performed on each of these rats under strict aseptic and

sterile conditions. Briefly, after complete anesthesia was

achieved, one patch of skin in the dorsal area was shaved

and disinfected with Betadine. The rat was placed on a

thermoelectric blanket with its tail directed toward the

investigator. The ovaries were removed through a small

incision on the dorsum of the animal. After separating the

fascia from the skin, a 7-mm incision was made in the

muscle to gain access to the peritoneal cavity. The ovarian

blood vessels were ligated and the ovaries were removed.

The muscle was then sutured and the wound was closed.

After recovery, the animals were returned to their home

cages and monitored for infections and surgical complica-

tions. Four weeks after the ovariectomies were performed,

the rats were sacrificed and the thoracic aorta was removed,

following the protocol described below.

2.3. Tissue preparation for isometric tension studies

The experimental setup and methods used in these

experiments have been described previously (Crespo,

2000). The same day of the experiment, animals were

weighed and anesthetized with a combination of ketamine

and xylazine intraperitoneally (100 mg/kg). The descending

thoracic aorta was removed and placed in Krebs bicarbonate

solution (composition in mM: NaCl 118, CaCl2 2.5, KCl 5,

MgSO4 1.1, NaHCO3 25, KH2PO4 1.2, glucose 10,

pH = 7.4). The connective tissue adjacent to the adventitia

of the aorta was carefully removed, avoiding damage to the

smooth muscle and the endothelium. Aortic rings (f 5 mm

long) were obtained from the proximal segment of each

aorta. Aortic rings were suspended horizontally between

two stainless-steel wires and mounted in a two-hook 50-ml

organ chamber (Radnoti, Monrovia, CA). The wires were

connected to a force–displacement transducer (Grass model

FT03C) and attached to a DC preamplifier (Grass model

7P1F). The signal was analyzed with a data acquisition card

(National Instruments, PC-LPM-16/PnP) and recorded with

the program LabView. The rings were subjected to a tension

of 2.0 g at rest. Our laboratory has determined that this

tension is optimal for these experiments. Once the optimal

tension was reached, the aortic rings were subjected to a 1-

h equilibration period.

2.4. Measurement of vascular contraction

To determine the effect of toremifene on receptor-depen-

dent contraction, cumulative concentration–response curves

for norepinephrine were conducted from 0.1 nM to 10 AM.

After the completion of each curve, the vessel was fully

relaxed with 1.0-AM sodium nitroprusside. The aorta was

then washed thrice and stabilized for 5 min. Following this

procedure, 1.0-AM toremifene was added to the incubation

chamber. After a 15-min incubation period with toremifene,

a new cumulative concentration–response curve for norepi-

Fig. 1. Concentration– response curves for norepinephrine-induced con-

tractions in aortic rings from intact (A) and ovariectomized (B) rats before

and after 1.0-AM toremifene incubation. The values shown are the

meansF S.E.M. of 14 experiments with intact rats and 6 experiments with

ovariectomized rats.

J. Gonzalez-Perez, M.J. Crespo / Vascular Pharmacology 40 (2004) 205–211 207

nephrine was performed in the presence of toremifene. The

contractility was expressed as the tension (g) divided by the

dry mass (mg) of the tested aortic segment. A similar

procedure was followed to determine the effect of toremi-

fene on the receptor-independent contraction induced by

potassium chloride (from 10 to 120 mM). Only one agent—

either norepinephrine or potassium chloride—was assayed

for each aortic ring to avoid confounding results due to

residual effects of the other agent.

2.5. Measurement of vascular relaxation

Concentration–response curves for acetylcholine and

sodium nitroprusside were performed to determine the

effect of toremifene on endothelium-dependent and endo-

thelium-independent relaxation. Aortic rings were precon-

tracted with 1.0-AM norepinephrine. When the maximal

contractile plateau was reached, cumulative concentration–

response curves were generated for acetylcholine (0.1 nM

to 10 AM). After the completion of each curve, the aorta

was washed and stabilized for 20 min. Subsequently, 1.0-

AM toremifene was added to the incubation chamber.

Following a 15-min incubation period, a new cumulative

concentration–response curve for acetylcholine was gener-

ated. A similar procedure was followed using sodium

nitroprusside (0.1 nM to 10 AM) to assess the effect of

toremifene on the endothelium-independent relaxation. For

each individual experiment, the relaxation was expressed as

a percentage of the relaxation achieved for each individual

concentration relative to the maximal contraction induced

by 1.0-AM norepinephrine.

2.6. Measurement of basal resting tension

Basal resting tension (g) was defined as the difference

between the tension registered in the aorta before (T0) and

after (T15) the 15-min incubation with toremifene. As

previously described, aortic rings from intact and ovariec-

tomized rats were mounted in the chamber and allowed to

stabilize. The basal resting tension (T0) was recorded and

toremifene was then added to the incubation bath at a

concentration of 1.0 AM. After the incubation period, the

tension (T15) was recorded again. A similar set of experi-

ments was conducted in the presence of 1.0 mM of the

nitric oxide synthase inhibitor NG-nitro-L-arginine (L-

NAME). The purpose of these latter experiments was to

evaluate the possible involvement of nitric oxide in basal

tension.

2.7. Statistical analysis

Results are presented as the meansF S.E.M. EC50 values

were determined by graphic analysis (GraphPad, San Diego,

CA). Statistical comparisons between groups were per-

formed with Student’s t test when only two variables were

compared and with the analysis of variance when more than

two variables were compared. Values were considered

statistically significant at P < .05.

3. Results

3.1. Effect of toremifene on vascular contractility

Fig. 1 depicts the effect of toremifene on the norepi-

nephrine-induced contraction of aortic rings from intact

and ovariectomized rats. The concentration– response

curves for the norepinephrine-induced contraction (from

0.1 nM to 10 AM) of aortic rings from both intact and

ovariectomized rats were shifted to the right in the pres-

ence of toremifene. Toremifene increased the EC50 value

fivefold in intact rats (from 154F 31 to 754F 162 nM,

n = 14, P < .05) and decreased the Emax value by 20%

(P < .05). Similarly, toremifene increased the EC50 value

in the aorta of ovariectomized rats (from 88F 11 to

230F 71 nM, n = 6, P < .05) and reduced Emax by 31%

Fig. 2. Concentration– response curves for potassium chloride-induced

contractions in aortic rings from intact (A) and ovariectomized (B) rats in

the presence and absence of toremifene (1.0 AM). The values shown are the

meansF S.E.M. of six experiments with intact rats and nine experiments

with ovariectomized rats.

Fig. 3. Concentration– response curves for acetylcholine-induced relaxa-

tions of aortic rings from intact (A) and ovariectomized (B) rats before and

after a 15-min incubation period with toremifene (1.0 AM). The values

shown are the meansF S.E.M. of eight experiments with intact rats and

eight experiments with ovariectomized rats.

J. Gonzalez-Perez, M.J. Crespo / Vascular Pharmacology 40 (2004) 205–211208

(from 0.61F 0.04 to 0.42F 0.08 g/mg tissue, n = 6,

P < .05). Similarly, the receptor-independent induced con-

traction evoked by potassium chloride (from 10 to 120

mM; Fig. 2A and B) was also modified by toremifene in

both intact (n = 6, P < .05) and ovariectomized (n = 9,

P < .05) rats. In the intact group, the Emax value decreased

by 31% in the presence of toremifene (P < .05), whereas

the reduction, although statistically significant, was only

11.5% in the ovariectomized group. Potassium chloride,

however, did not alter the EC50 values in either intact or

ovariectomized rats.

3.2. Effect of toremifene on vascular relaxation

The effect of acute administration of toremifene on the

endothelium-dependent and endothelium-independent relax-

ation was evaluated in intact and ovariectomized rats by

performing concentration–response curves for acetylcholine

and sodium nitroprusside. A complete curve was performed

by adding cumulative concentrations of acetylcholine (from

0.1 nM to10 AM) to aortic rings precontracted with 1.0-AMnorepinephrine both before and after incubation with tor-

emifene. In the intact group (Fig. 3A), the presence of

toremifene displaced the entire curve to the left without

significantly affecting the EC50 value (158F 55 nM before

and 71F 34 nM after, n = 8, P>.05). Toremifene increased

the maximal relaxation by f 16% (from 94.4F 2.3% to

113.2F 2.9%, n = 8, P < .05). Similar results were obtained

for this drug when aortic rings were used from menopause-

induced rats (Fig. 3B). Toremifene incubation shifted the

concentration–response curve for the acetylcholine-induced

relaxation to the left and increased Emax from 59.2F 4.2%

to 73.5F 4.7% (n = 8, P < .05) without affecting the EC50

values (590F 150 nM before and 1000F 380 nM after,

n = 8, P>.05).

Fig. 4A and B depict the effect of toremifene on the

endothelium-independent relaxation induced by sodium

nitroprusside (from 0.1 nM to 10 AM) in rings from intact

and menopause-induced rats. Toremifene did not modify

Fig. 4. Concentration–response curves for sodium nitroprusside-induced

relaxations in aortic rings from intact (A) and ovariectomized (B) rats

before and after treatment with toremifene (1.0 AM). The values shown are

the meansF S.E.M. of nine experiments with intact rats and five

experiments with ovariectomized rats.

Fig. 5. Basal relaxation of aortic rings from intact rats after a 15-min

incubation period with 1.0-AM toremifene in the presence and absence of L-

NAME (1 mM). Note that the basal relaxation induced by toremifene was

fully blocked by L-NAME.

Fig. 6. Basal relaxation of aortic rings from ovariectomized rats after a 15-

min incubation period with 1.0-AM toremifene in the presence and absence

of L-NAME (1 mM). Note that toremifene decreased resting tension in

ovariectomized rats although at a lesser magnitude than in intact rats. The

effect of toremifene was fully abolished by L-NAME.

J. Gonzalez-Perez, M.J. Crespo / Vascular Pharmacology 40 (2004) 205–211 209

the EC50 values in rings from either intact (34F 5.4 vs.

28F 8.7 nM before and after the drug, P>.05) or ovariec-

tomized (66F 11 vs. 68F 8.2 nM before and after tor-

emifene, P>.05) rats. The relaxing effect of low

concentrations of sodium nitroprusside (0.1 nM to 1.0

nM), however, was significantly potentiated by the pres-

ence of toremifene.

3.3. Effect of toremifene on the basal resting tension

The effect of toremifene incubation on basal resting

tension is depicted in Figs. 5 and 6. In rings from intact

rats, basal tone decreased by f 0.13 g after incubation

with toremifene for 15 min (from � 18.1F 3.2 mg before

incubation to � 130.0F 14.0 mg after incubation, n = 15,

P < .05). Similar effects were observed in rings from

menopause-induced rats (Fig. 6) where resting tension

changes from � 6.9F 4.0 to � 33.0F 10.0 mg before

and after the addition of toremifene (n= 13, P < .05). In

both cases, the basal relaxation induced by toremifene was

fully abolished by concurrent incubation with 1-mM L-

NAME. The effect of toremifene in the presence of L-

NAME in either intact or ovariectomized rats was similar

to the effect of L-NAME alone.

J. Gonzalez-Perez, M.J. Crespo / Vascular Pharmacology 40 (2004) 205–211210

4. Discussion

Toremifene modifies the vascular response to both

contractile and relaxing factors, promoting a state of

relaxation in the vessels. Acute administration of toremi-

fene depresses the contraction induced by the a1 receptor

agonist norepinephrine in rings from both intact and

ovariectomized rats. Lamb and Barna (1998) reported that

10-AM tamoxifen did not modify the contraction induced

by 10-nM norepinephrine in aortic rings from male

Sprague–Dawley rats. Unlike the latter experiments, how-

ever, we used female rats and performed a complete dose–

response curve. The EC50 for the norepinephrine-induced

contraction was dramatically enhanced after incubation

with toremifene. This finding may suggest that toremifene

has a direct action at the receptor level, although additional

experiments using membrane fractions are needed to

corroborate this possibility. Other mechanisms may be also

participating in the reduced contraction to norepinephrine,

such as alterations in the intracellular signal transduction

pathway for the a1 receptor, or in the handling of intra-

cellular calcium concentration. Indeed, tamoxifen, structur-

ally similar to toremifene, reduces calcium released by the

sarcoplasmic reticulum of cardiac cells (Kargacin et al.,

2000; Dodds et al., 2001). In addition, Dıaz (2002) has

proven that both tamoxifen and toremifene reduce basal

tone in isolated mouse duodenal muscle by inhibition of L-

type calcium channels. These alternative possibilities need

to be evaluated to elucidate the mechanisms involved in

the reduction of norepinephrine-induced vascular contrac-

tion caused by toremifene.

The finding that toremifene decreases the response to

potassium chloride in the vasculature of intact or meno-

pause-induced rats suggests that this drug modulates, to

some extent, the L-type calcium channel, thus favoring

decreased vascular contractility. Comparable results have

been reported for other members of the SERM family.

Raloxifene and tamoxifen significantly depress the recep-

tor-independent contraction elicited by potassium chloride

in aortic rings from rabbits (Figtree et al., 1999, 2000). A

similar effect has been observed with tamoxifen on the tail

artery and aortic rings of male Sprague–Dawley rats (Song

et al., 1996; Lamb and Barna, 1998). Whereas the chem-

ical structures of these SERMs differ, their effect on the

vascular contraction induced by potassium chloride is

similar. Thus, this effect appears to be a common property

of the SERM family of drugs regardless of the animal

model studied.

The decrease in resting tone promoted by toremifene

suggests that this drug interferes with the basal nitric oxide

production system. The fact that coincubation with L-

NAME fully abolished this effect confirms the involvement

of toremifene in the activation of nitric oxide pathway. L-

NAME also abolishes the relaxation induced by tamoxifen

(Figtree et al., 2000) and raloxifene (Figtree et al., 1999) in

the coronary arteries of female New Zealand rabbits. An

increase in endothelial nitric oxide synthase (eNOS) activ-

ity and/or protein expression is likely to be involved in this

process. Simoncini and Genazzani (2000), using human

endothelial cells, showed that raloxifene increases eNOS

enzymatic activity. The improvement in endothelial func-

tion observed after treatment with SERMs may be attrib-

uted to the partial agonistic properties of these drugs on the

estrogen receptors (Figtree et al., 1999, 2000). Further-

more, it has been suggested that estrogen stimulates the

eNOS on activation of the estrogen receptor a via a

nongenomic mechanism (Chen et al., 1999). In addition

to modifying basal nitric oxide production, toremifene

amplifies receptor-dependent nitric oxide synthesis. An

enhancement of the acetylcholine-induced relaxation also

has been observed in aortas from ovariectomized rats after

chronic treatment with idoxifene (Ma et al., 2000) and in

the vasculature of male spontaneously hypertensive rats

treated with raloxifene (Wassmann et al., 2002). The fact

that toremifene does not affect the EC50 values for acetyl-

choline-induced relaxation eliminates any direct effect of

the drug on the muscarinic receptors. In contrast, tamox-

ifen interacts with the muscarinic receptors in membrane

fractions from urinary bladder and myometrium (Batra,

1990). Nevertheless, further investigation is needed regard-

ing the involvement of toremifene in the observed changes

to the nitric oxide system. The amplification of endotheli-

um-independent responses observed at low doses of sodi-

um nitroprusside after acute exposure to toremifene

suggests that the drug has an effect at the smooth muscle

level. This amplification is comparable with that observed

in porcine coronary arteries that are exposed to physiolog-

ical concentrations of 17h-estradiol (Teoh et al., 1999).

The mechanism responsible for the endothelium-indepen-

dent relaxation is unknown, although this drug may alter

the cGMP levels in the vascular smooth muscle. To further

investigate this possibility, the effect of toremifene on

sodium nitroprusside-induced cGMP levels needs to be

evaluated.

In summary, this study demonstrates that acute adminis-

tration of toremifene interferes with the vascular wall of

both intact and menopause-induced rats, promoting a state

of vascular relaxation. In addition, the presence of toremi-

fene improves the endothelial dysfunction observed in

menopause-induced rats. By decreasing the responses to

contractile agonists and enhancing vascular relaxation, this

drug could contribute to diminished total peripheral resis-

tance and improved cardiovascular status in menopause-

induced rats.

Acknowledgements

This work was supported by grants from the National

Institutes of Health (RR-03051, 2 SO6 GM08224 MBRS-

SCORE and RISE Program) and a Porter fellowship from

the APS.

J. Gonzalez-Perez, M.J. Crespo / Vascular Pharmacology 40 (2004) 205–211 211

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