Calcitonin ameliorates enhanced arterial contractility after chronic constriction injury of the sciatic nerve in rats

doi: 10.1111/j.1472-8206.2011.00934.x

O R I G I N A L

A R T I C L E

Calcitonin ameliorates enhanced arterialcontractility after chronic constriction injuryof the sciatic nerve in rats

Takeshi Yoshimuraa,b*, Akitoshi Itoa, Shin-ya Saitob, Mineko Takedaa,Hiroshi Kuriyamaa, Tomohisa Ishikawab

aLaboratory for Development Pharmacology, Pharmaceuticals Research Center, Asahi Kasei Pharma Corporation,

632-1 Mifuku, Izunokuni City, Shizuoka 410-2321, JapanbDepartment of Pharmacology, Graduate School of Pharmaceutical Sciences, University of Shizuoka, 52-1 Yada,

Suruga-ku, Shizuoka City, Shizuoka 422-8526, Japan

I N T R O D U C T I O N

Calcitonin, a polypeptide hormone secreted from the

parafollicular (C) cells of the mammalian thyroid gland

into general circulation [1,2], regulates blood calcium

concentration and bone metabolism by acting on oste-

oclasts [1,3,4] and is used clinically to treat hypercalce-

mia [5] and osteoporosis [6–8]. In addition, calcitonin

has been used to relieve pain associated with a variety of

disorders, including postmenopausal osteoporosis [9],

rheumatoid arthritis [10], metastatic cancer [11], reflex

sympathetic dystrophy [12], and knee osteoarthritis

[13]. Several studies have suggested that the serotoner-

gic system is involved in the antinociceptive effect of

calcitonin on ovariectomy-induced hyperalgesia in rats

[14–16].

Calcitonin has also been reported to improve the

peripheral circulatory disturbance in patients with Ray-

naud’s syndrome [17] and complex regional pain syn-

drome (CRPS) [18]. Enhanced vascular contractility in

response to noradrenaline appears to be related to the

interruption of circulation that occurs in these syn-

dromes [19–21]. A model of chronic neuropathic pain

induced by chronic constriction injury (CCI) of the sciatic

nerve, developed by Bennett and Xie [22], exhibits the

signs and symptoms of CRPS. It is of interest that skin

blood flow at the plantar surface of the hind paw in CCI

rats is decreased [23], and that the plantar arteries

Keywords

calcitonin,

CCI,

noradrenaline,

peripheral circulatory

disturbance,

plantar artery,

vasodilation

Received 11 June 2010;

revised 28 December 2010;

accepted 25 January 2011

*Correspondence and reprints:

[email protected].

co.jp

A B S T R A C T

In addition to its regulatory effect on bone mass, calcitonin has been shown to relieve

pain and alleviate peripheral circulatory disturbance in patients with Raynaud’s

syndrome and complex regional pain syndrome. In the present study, we investigated

whether calcitonin ameliorates diminished blood flow and enhanced arterial

contraction in response to noradrenaline in chronic constriction injury (CCI) of the

sciatic nerve in rats. Following surgically induced CCI, laser Doppler flowmetry

studies showed a significant decrease in plantar skin blood flow of the ipsilateral hind

paw compared to the contralateral side. A subcutaneous bolus injection of elcatonin

(20 U/kg), a synthetic derivative of eel calcitonin, significantly improved decreased

skin blood flow in the ipsilateral side. In vitro analysis of plantar arteries isolated from

the ipsilateral hind paw 7–13 days after the CCI procedure showed higher sensitivity

to noradrenaline than the plantar arteries from the contralateral side. Elcatonin (0.1–

10 nM) significantly reduced noradrenaline-induced contraction in the arteries of the

ipsilateral side, whereas it had little effect on those of the contralateral side. These

results suggest that calcitonin selectively ameliorates enhanced arterial contractility

in CCI neuropathic rats, thus leading to its alleviating effect on peripheral circulatory

disturbance.

ª 2011 The Authors Fundamental and Clinical Pharmacology ª 2011 Societe Francaise de Pharmacologie et de TherapeutiqueFundamental & Clinical Pharmacology 26 (2012) 315–321 315

Fund

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tal &

Cli

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y

isolated from the CCI rat hind paw show higher

sensitivity to noradrenaline [24]. Thus, we assumed

that calcitonin improves peripheral circulatory distur-

bance by reducing the enhanced noradrenaline-induced

contraction of the cutaneous arteries in CCI rats. In the

present study, we show that calcitonin selectively

ameliorates ipsilateral decreased skin blood flow and

enhanced arterial contractility in response to noradren-

aline in CCI rats.

M A T E R I A L S A N D M E T H O D S

Operative procedure

Male Sprague-Dawley rats (Charles River Laboratory

Japan Inc., Atsugi, Japan) weighing 180–300 g were

treated in accordance with the Institutional Animal Care

Committee of the Pharmaceutical Research Center of

Asahi Kasei Pharma Corporation. The rats were individ-

ually housed in a room in which the temperature was

controlled to 22 ± 1 �C and humidity to 55 ± 10%,

with a 12-h light–dark cycle and free access to food and

water.

The CCI procedure was performed according to the

method described by Bennett and Xie [22] with one

slight modification. Briefly, the right sciatic nerve was

exposed, and four loosely constrictive ligatures, using

braided silk 4-0 (Niccho Industry Co. Ltd., Tokyo, Japan),

were placed around the sciatic nerve at the mid-thigh

level in an area 5 mm in length. The incision was then

closed with braided silk sutures (2-0; Natsume Seisaku-

sho Co. Ltd., Tokyo, Japan). The completion of the CCI

operation was confirmed when plantar skin blood flow,

which was measured using a laser Doppler flowmeter

(ALF21R; Advance Co. Ltd., Tokyo, Japan), was lower on

the ipsilateral side more than 10 mL/min per 100 g

compared to on the contralateral side. Experiments were

started 7 days after the operation.

Laser Doppler flowmetry studies

Blood flow was evaluated by laser Doppler flowmetry

studies as previously described [25]. The CCI rats were

anesthetized with pentobarbital sodium (40 mg/kg)

administered intraperitoneally and placed in the prone

position on a heating pad to maintain a rectal temper-

ature of 37.5 ± 1.0 �C. The probe of the laser Doppler

flowmeter (A type; Advance Co. Ltd.) was positioned at

the prominences located on the plantar surface of the rat

hind paw. Electrical signals from the laser Doppler

flowmeter and a digital thermometer (DT-300; Inter

Medical Co. Ltd., Nagoya, Japan) were recorded using an

analog-to-digital converter (Maclab/8s; AD Instruments,

Milford, MA, USA). Each measurement was continued

for 1 min and repeated twice. We confirmed that blood

flow decreased to 0 when rats were sacrificed.

Ten CCI rats were divided into vehicle-treated and

elcatonin (eCT)-treated (Asahi Kasei Pharma Corpora-

tion, Tokyo, Japan) groups by a multivariable blocking

assignment method using the SAS software (Version 8.2;

SAS Institute, Tokyo, Japan). Nine days after undergoing

CCI, either vehicle or eCT (20 U/kg) was injected

subcutaneously into the upper back of rats at a volume

of 1 mL/kg. The eCT was dissolved in 0.1 mM sodium

acetate buffer (pH 5.5) with 0.9% sodium chloride and

0.02% bovine serum albumin. Blood flow was measured

at 0.5 and 24 h (exam 1), 1 h (exam 2), 3 h (exam 3),

and 6 and 48 h (exam 4) after eCT administration. Four

independent examinations were carried out to determine

the time course effects.

Contraction measurement

Twenty-seven CCI rats were anesthetized with diethyl

ether and then sacrificed by a sharp blow to the neck and

resultant exsanguination. The lateral plantar artery was

immediately dissected from the plantar surface of the

hind paw and conserved overnight in Krebs-Henseleit

(KH) solution (composition in mM: NaCl, 118; KCl, 4.7;

CaCl2, 2.55; MgSO4, 1.18; KH2PO4, 1.18; NaHCO3,

24.8; and glucose, 11.1) at 4 �C.

The isolated arteries were cut into ring segments about

2 mm in width. Each ring segment was horizontally

mounted on a myograph (Multi Myograph Model 610M;

Danish Myo Technology A/S, Aarhus, Denmark) with

two tungsten wires measuring 40 lm in diameter.

Isometric tension was measured and recorded using a

data analysis program (Myodaq 2.01; Danish Myo

Technology A/S). The organ chamber was filled with

KH solution, which was maintained at 37 �C and gassed

with 95% O2/5% CO2. The preparation in the organ

chamber was allowed to equilibrate for 30 min under an

optimal tension of 4–5 mN/mm and then were con-

tracted repeatedly with 80 mM of KCl-KH (composition

in mM: NaCl, 42.7; KCl, 80; CaCl2, 2.55; MgSO4, 1.18;

KH2PO4, 1.18; NaHCO3, 24.8; and glucose, 11.1) until

reproducible contractions were observed. In all experi-

ments, maintenance of the endothelium was confirmed

by carbachol (1 lM)-induced relaxation in the artery

precontracted with 100 nM of noradrenaline.

Concentration–response relationships were obtained

by cumulative additions of noradrenaline, and sensitivity

to the agonist (pD2 = )log EC50, where EC50 is the

316 T. Yoshimura et al.

ª 2011 The Authors Fundamental and Clinical Pharmacology ª 2011 Societe Francaise de Pharmacologie et de TherapeutiqueFundamental & Clinical Pharmacology 26 (2012) 315–321

agonist concentration needed to produce 50% of the

maximal response) was calculated by interpolation fit to

a logistic curve of individual concentration–response

curves using the JMP 6 software (SAS Institute).

Chemicals

Carbachol was obtained from Calbiochem (La Jolla, CA,

USA). Noradrenaline hydrogen tartrate monohydrate

and all other chemicals were obtained from Wako Pure

Chemical Industries Ltd., Osaka, Japan. All reagents were

dissolved in distilled water.

Statistical analysis

Data are presented as mean ± SEM. Statistical signifi-

cance was evaluated by the unpaired, two-tailed t test

using SAS software Version 8.2 (SAS Institute). P values

<0.05 were considered statistically significant.

R E S U L T S

Effects of elcatonin on skin blood flow

Plantar skin blood flow of the ipsilateral and contralateral

sides was comparable 5 days prior to the CCI procedure.

At 4, 7, 10, and 14 days after CCI, however, the blood

flow in the ipsilateral side decreased significantly com-

pared to that in the contralateral side (Figure 1). A

subcutaneous bolus injection of eCT (20 U/kg) at 9 days

after CCI significantly improved the decreased blood flow

in the ipsilateral side at 1, 3, 6, and 24 h after eCT

treatment; in addition, blood flow after 1, 3, and 6 h was

comparable to blood flow before CCI (Figure 2a). The

administration of eCT also induced slight increases in the

blood flow in the contralateral side at 1, 3, and 6 h after

the injection (Figure 2b).

Effects of elcatonin on noradrenaline-induced

contraction

Plantar arteries isolated from the ipsilateral hind paw

showed higher sensitivity to noradrenaline than those

from the contralateral side (Figure 3a); a significant differ-

ence was found between the pD2 values of the ipsilateral

side (7.01 ± 0.11) and contralateral side (6.60 ± 0.17;

P < 0.05). In the presence of eCT (10 nM), however,

no significant difference in the concentration–response

relationship for noradrenaline was observed between the

isolated ipsilateral and contralateral plantar arteries

(Figure 3b); pD2 values were 6.65 ± 0.13 and 6.58 ±

0.19 for the ipsilateral and contralateral sides, respectively.

The administration of eCT (10 nM) did not affect the basal

tension of arteries isolated from the ipsilateral side before

and after the application of eCT (4.92 ± 0.30 and

4.83 ± 0.32 mN, respectively; n = 11) or of the arteries

from the contralateral side before and after the applica-

tion of eCT (4.07 ± 0.21 and 4.00 ± 0.20 mN, respec-

tively; n = 9).

The effect of eCT was also evaluated in the arteries

precontracted with 100 nM of noradrenaline. As shown

in Figure 4a,c, noradrenaline-induced contraction was

not sustained and gradually declined in the arteries

isolated from both the ipsilateral and contralateral sides.

Therefore, the tension with each concentration of eCT

(Figure 4b,d) was normalized to that of the control at the

corresponding time point (Figure 4a,c). Figure 4e shows

the summarized data: eCT caused significant concentra-

tion-dependent relaxation in the plantar arteries isolated

from the ipsilateral side but caused only a small

relaxation response in the arteries isolated from the

contralateral side.

D I S C U S S I O N

Sciatic nerve ligation in rats induces clinical signs and

symptoms that mimic human conditions of neuropathic

origin. Calcitonin has been shown to improve the

peripheral circulatory disturbance in patients with

–5 0 2 4 6 8 10 12 1430

40

50

60

70

80

∗ ∗∗∗∗

Blo

od fl

ow (m

L/m

in/1

00 g

)

Time after operation (days)

Contralateral

Ipsilateral

∗

Figure 1 Chronic constriction injury (CCI) of the sciatic nerve

induced a decrease in plantar skin blood flow in rats. Plantar skin

blood flow of CCI rat hind paw in the ipsilateral (closed square) and

contralateral sides (open circle) was measured 5 days before and 4,

7, 10, and 14 days after the CCI procedure. Data for five animals

are expressed as mean ± SEM. *P < 0.05; **P < 0.01 vs. the

contralateral side.

Vasodilator effect of calcitonin in CCI rats 317


Raynaud’s syndrome [17] and CRPS [18]. In the present

study, we showed that in CCI rats, calcitonin improved

the decreased plantar skin blood flow in the ipsilateral

side. Moreover, calcitonin relaxed noradrenaline-induced

contraction in the plantar arteries isolated from the

ipsilateral side. These results suggest that calcitonin

ameliorates enhanced contractility in response to nor-

adrenaline in cutaneous arteries from the ipsilateral side

of CCI rats, leading to alleviation of peripheral circula-

tory disturbance in the neuropathic rats.

Consistent with the findings of previous studies

[23,24], we found that plantar skin blood flow of the

hind paw was decreased and that the plantar artery

became more sensitive to noradrenaline after induction

of CCI of the sciatic nerve. The plantar artery is

innervated by the tibial nerve, which originates from

the sciatic nerve [26]. Small unmyelinated axons in the

sciatic nerve, including those of the sympathetic nerves,

have been shown to be degenerated in CCI rats [27]. A

study of isolated subcutaneous arteries from CCI rats

clearly demonstrated that arteries from the ipsilateral

side are less responsive to electrical field stimulation than

arteries from the contralateral side, but are more

sensitive to a1-adrenoceptor agonists, suggesting that

sympathetic dysfunction in CCI rats consists of denerva-

tion-induced supersensitivity to catecholamines [24].

Thus, sympathetic nerve degeneration after CCI appears

to be responsible for the enhanced vascular contractility

that occurs in response to noradrenaline in the plantar

arteries of CCI rats, thereby leading to the decreased skin

blood flow seen in the neuropathic model.

Interestingly, eCT at subnanomolar concentrations

was sufficient to reduce noradrenaline-induced contrac-

tion in the plantar arteries from the ipsilateral side of CCI

rats. Several studies have reported the vascular actions

of calcitonin: salmon calcitonin (sCT) relaxes noradren-

aline-induced contraction in the common carotid artery

of the dog [28]; porcine calcitonin decreases KCl-induced

Ca2+ influx and contraction in the rat tail artery [29];

and sCT stimulates adenylate cyclase production in

rabbit renal microvessels [30]. However, these effects of

calcitonin were observed at relatively high concentra-

tions (>300 nM), which are considered to be beyond the

physiological range. Therefore, calcitonin has been

regarded to have minimal vascular action [31]. The

present study is the first to demonstrate the vasodilator

effect of calcitonin at concentrations within the physi-

ological range in vitro, although the effect was observed

only under pathological conditions.

Subcutaneous injection of eCT (15 U/kg) has been

shown to prevent bone loss in ovariectomized (OVX) rats

[32]. In addition, repeated subcutaneous administration

of eCT (20 U/kg per day) for 3 or 4 weeks has been

shown to relieve prolonged hyperalgesia associated with

ovariectomy [16]. In the present study, a subcutaneous

bolus injection of the same dose of eCT (20 U/kg)

improved decreased skin blood flow in CCI rats and the

effect continued for more than 6 h. Because the maxi-

mum plasma concentration (Cmax) and the half life in

plasma of eCT after intramuscular bolus administration

of 40 U/kg eCT in rats are estimated to be approximately

4 nM and 64 min, respectively [33], we assumed that

Cmax after the subcutaneous injection of 20 U/kg eCT in

rats would be approximately 2 nM and the plasma levels

would decline gradually. In in vitro experiments, eCT at

concentrations of 0.3 nM significantly suppressed nor-

adrenaline-induced contraction in the plantar artery

isolated from the ipsilateral side of CCI rats. Thus, the

effective concentrations of eCT in the in vitro exper-

(a) Ipsilateral

(b) Contralateral

30

40

50

60

70

80

Pre-CCI

Post-CCI

Blo

od fl

ow (m

L/m

in/1

00 g

)

30

40

50

60

70

80

0.5 1 3 6 24 48Time after injection (h)

VehicleeCT

30

40

50

60

70

80

Pre-CCI

Post-CCI

30

40

50

60

70

80

0.5 1 3 6 24 48Time after injection (h)

VehicleeCT

∗

∗∗ ∗∗

∗∗∗∗∗∗

Blo

od fl

ow (m

L/m

in/1

00 g

)

Figure 2 Effects of elcatonin (eCT) on plantar skin blood flow of

the hind paw in the ipsilateral (a) and contralateral (b) sides of CCI

rats. Blood flow was measured at 0.5, 1, 3, 6, 24, and 48 h after a

single subcutaneous injection of eCT (20 U/kg). Data for five

animals are expressed as mean ± SEM. Average blood flow (n = 40)

measured 5 days before (pre-CCI) and 7 days after CCI (post-CCI) is

shown for comparison. *P < 0.05; **P < 0.01 vs. vehicle.



iments of the present study are likely to be comparable to

the plasma concentrations of eCT after a subcutaneous

bolus injection of 20 U/kg. Interestingly, Hilton et al.

have reported poor reversibility of sCT binding to

calcitonin receptors [34]. They clearly demonstrated

that C-terminal half of sCT is sufficient to bind to

02468

101214

Tens

ion

(mN

)

02468

101214

Tens

ion

(mN

)

Ipsilateral

Contralateral

02468

101214

Tens

ion

(mN

)

10 min

10 min10 min

(a)

(c)

(b)100 nM noradrenaline

100 nM noradrenaline

100 nM noradrenaline

eCT (nM)

0.1

310.3

10

02468

101214

Tens

ion

(mN

) 100 nM noradrenaline

eCT (nM)

10 min

(d)

0.10.3 1 3 10

0.1 1 1040

50

60

70

80

90

100

110

∗

∗∗∗∗

∗∗

Con

tract

ion

(% n

orad

rena

line)

eCT (nM)

ContralateralIpsilateral

0

(e)

Figure 4 Effects of elcatonin (eCT) in isolated plantar arteries precontracted with noradrenaline. Typical traces of noradrenaline

(100 nM)-induced contraction in plantar arteries isolated from the ipsilateral (a and c) and contralateral (b and d) sides of CCI rats. eCT was

applied in a cumulative manner. (e) After normalizing to corresponding controls, concentration–response relationships for eCT-induced

relaxation were compared between the ipsilateral (closed square) and contralateral (open circle) sides. Data are expressed as mean ± SEM for

10–12 arteries. *P < 0.05; **P < 0.01 vs. the contralateral side.

1 10 100 1000 10 000

0

20

40

60

80

100

120

140C

ontra

ctio

n (%

of K

Cl)

Noradrenaline (nM)

Contralateral

Ipsilateral

(a)

1 10 100 1000 10 000

0

20

40

60

80

100

120

140

Con

tract

ion

(% o

f KC

l)

Noradrenaline (nM)

Contralateral

Ipsilateral

10 nM eCT(b)

Figure 3 Effects of elcatonin (eCT) on noradrenaline-induced contraction in the plantar arteries of CCI rats. Concentration–response

relationships for noradrenaline-induced contraction of the arteries isolated from the ipsilateral (closed circle) and contralateral sides

(open circle) in the absence (a) and presence (b) of eCT (10 nM) were compared. pD2 values for the ipsilateral and contralateral sides

were 7.01 ± 0.11 and 6.60 ± 0.17, respectively, in the absence of eCT (P < 0.05) and 6.65 ± 0.13 and 6.58 ± 0.19, respectively, in the

presence of eCT. The maximum response to 80 mM of KCl was taken as 100%. Data are expressed as mean ± SEM for 9–16 arteries.



calcitonin receptor and exhibits complete reversibility,

whereas N-terminal half is required for its irreversibility

[34]. We also confirmed that a subcutaneous bolus

injection of 20 U/kg sCT improves decreased skin blood

flow in ipsilateral side, which continued for several hours

(unpublished observation). It is important to note that

amino acid sequence of eCT is identical with sCT except

that just three residues in C-terminal are substituted.

Taken together, it is plausible to conclude that the

dissociation of eCT from calcitonin receptor is compara-

ble to that of sCT. Thus, the continuous effect of eCT on

blood flow would be because of its characteristics of slow

half-life and low dissociation rate from its receptor.

Koenigsberger et al. [35] have recently proposed a

model describing that the sensitivity to vasoconstrictor is

higher in isometric than isobaric or isotonic conditions.

This model is supported by several experimental findings

in porcine coronary [36] and rat mesenteric arteries

[35,37]. The enhanced vasoconstrictor sensitivity seems

to result from a higher arterial wall stress in isometric

conditions [35]. This model predicts that care should be

taken in the interpretation of experimental results in

isometric measurement. It is thus possible that the effect

of eCT on the isometric contraction may have been

overestimated in the present in vitro experiments.

However, because the contraction of ipsilateral and

contralateral side was compared in the same conditions,

we could still say that eCT ameliorates the augmented

vasoconstrictor response in the ipsilateral side after CCI

both in vivo and in vitro.

On the basis of the data obtained here, a possible

hypothesis is that calcitonin may alleviate the peripheral

circulatory disturbance and abnormal vascular contrac-

tility that occur in response to noradrenaline in patients

with Raynaud’s syndrome and CRPS, thereby relieving

the ischemic pain associated with peripheral circulatory

disturbance. However, several questions need to be

addressed before establishing the efficiency of calcitonin.

For example, it will be important to determine why

calcitonin has a significant effect only in vessels with

higher sensitivity to noradrenaline. In addition, it will be

necessary to identify the vascular tissues, i.e., smooth

muscle, endothelium, and nerve terminal, on which

calcitonin acts. Further experiments in immunohisto-

chemistry and with calcitonin receptor inhibitors such as

sCT [8–32] will be required to address these issues. A

recent study provided evidence for the elevated expres-

sion of calcitonin receptors in the pathological vascular

tissue of atherosclerotic rabbits [38]. In the atheroscle-

rotic rabbits, Moura et al. [29] reported that repeated

porcine calcitonin treatment suppresses the increase in

Ca2+ influx in the vascular smooth muscle. Thus,

calcitonin may lead to the development of an efficient

drug capable of improving peripheral circulatory distur-

bance by its specific action on calcitonin receptors

expressed in pathological conditions.

In conclusion, the present study provides evidence for

ameliorating effect of calcitonin on enhanced arterial

contraction through the activation of a-adrenoceptors in

CCI neuropathic rats. The vasodilator effect of calcitonin

seems to be responsible for its alleviating effect on

peripheral circulatory disturbance. Thus, calcitonin

would be expected to selectively improve decreased blood

flow in patients with peripheral circulatory disturbance

and may relieve ischemic pain associated with it.

A B B R E V I A T I O N S L I S T

CCI – chronic constriction injury

CRPS – complex regional pain syndrome

eCT – elcatonin

KH – Krebs-Henseleit

OVX – ovariectomized

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