activation of b2 bradykinin receptors by neurotensin

Activation of B2 bradykinin receptors by neurotensin

Tae-Ju Park, Kyong-Tai Kim*

Department of Life Science, Division of Molecular and Life Science, Pohang University of Science and Technology, Pohang, 790-784, South Korea

Received 11 June 2002; accepted 4 November 2002

Abstract

Many previous reports suggested that relatively high concentrations of neurotensin were required to exert its effects on neurotransmitter

secretion. The neurotensin binding sites, which recognize high concentrations of neurotensin, were characterized in rat pheochromocytoma

(PC12) cells. When PC12 cells were treated with neurotensin, [3H]norepinephrine secretion and elevation of cytosolic calcium were evoked

at EC50 values of 59F 4 and 37F 7 AM, respectively. Both calcium release and inositol 1,4,5-trisphosphate (IP3) production induced by

neurotensin suggested involvement of phospholipase C. Experiments with simultaneous or sequential treatment with neurotensin and

bradykinin suggested that neurotensin and bradykinin act on the same binding sites. Furthermore, both inhibition of bradykinin- and

neurotensin-induced calcium rises by bradykinin receptor antagonists with similar IC50 values and receptor binding analysis using

[3H]bradykinin confirmed that neurotensin directly binds to B2 bradykinin receptors. The data suggest that neurotensin binds and activates

the B2 bradykinin receptors.

D 2002 Elsevier Science Inc. All rights reserved.

Keywords: Neurotensin; B2 bradykinin receptors; Phospholipase C; Xenopsin

1. Introduction

Neurotensin is a tridecapeptide isolated from both mam-

malian brain and the gastrointestinal tract [1,2]. This peptide

displays a wide spectrum of biological activities, including

hypothermia, antinociception, modulation of dopamine neu-

rotransmission, stimulation of anterior pituitary hormone

secretion, modulation of the digestive tract and the cardio-

vascular system, and growth promotion (for review, see

Refs. [3–5]). The diverse functions of neurotensin are

believed to be evoked by its interaction with specific

neurotensin receptors, and this interaction has been shown

to modulate intracellular second messengers, including

inositol phosphates, calcium, cGMP, and cAMP. So far,

three subtypes of neurotensin receptors have been purified

and cloned. Two of them belong to the family of seven

transmembrane-spanning, G protein-coupled receptors: one

has high-affinities (Kd = 0.1–0.3 nM) for neurotensin and

the other has low affinities (Kd = 3–5 nM) for the peptides.

The third neurotensin receptors spans the membrane only

once and is identical to the protein called sortilin. This

receptor showed both low (Kd = 0.3 nM) and high (Kd = 10

nM) affinities, depending on the presence of furin [6].

The possible presence of another neurotensin binding site

has been suggested on the basis of pharmacological data

such as neurotensin-induced SR48692-insensitive hypother-

mic and analgesic responses in the mouse and rat [3,4].

Furthermore, relatively high concentrations of neurotensin

were required to elicit norepinephrine release from rat

hypothalamic slices [7], potentiation of high K+-induced

release of endogenous dopamine from rat striatal slices [8],

formation of inositol phosphates in bovine adrenal medul-

lary cells [9], and dopamine release from rabbit striatum

[10]. These observations suggest the probable existence of a

novel neurotensin binding site with much lower affinity for

neurotensin than the previously reported neurotensin recep-

tors.

Rat pheochromocytoma (PC12) cells have been reported

to express mRNA for neurotensin when treated with various

stimuli including nerve growth factor, dexamethasone, acti-

vators of adenylyl cyclase, and lithium [11,12]. In addition,

stimulation of the splanchnic nerve by hypoglycemia in vivo

evoked a dramatic and long-lasting (2 weeks) increase in

neurotensin levels in the rat adrenal medulla [13]. Therefore,

0898-6568/02/$ - see front matter D 2002 Elsevier Science Inc. All rights reserved.

doi:10.1016/S0898-6568(02)00136-5

Abbreviations: PLC, phospholipase C; [Ca2+]i, cytosolic free Ca2+

concentration; IP3, inositol 1,4,5-trisphosphate.

* Corresponding author. Tel.: +82-54-279-2297; fax: +82-54-279-

2199.

E-mail address: [email protected] (K.-T. Kim).

www.elsevier.com/locate/cellsig

Cellular Signalling 15 (2003) 519–527

upon such external stimulation, a large amount of intra-

cellular neurotensin can be synthesized and released from

the cells to act on binding sites located on the plasma

membrane in an autocrine or paracrine manner. We tested

the possibility that PC12 cells could respond to neurotensin

and found that micromolar concentrations of neurotensin

evoked catecholamine secretion via phospholipase C (PLC)-

dependent [Ca2 +]i rise. However, the characteristics of the

neurotensin binding sites on PC12 cells differed from those

of previously characterized neurotensin receptors in terms of

the effective concentration of neurotensin and the potency of

neurotensin-related peptides. Strikingly, we found that neu-

rotensin directly activates B2 bradykinin receptors. In addi-

tion, we confirmed that the activation of the B2 bradykinin

receptors by neurotensin also occurs in bovine adrenal

chromaffin and human neuroblastoma cells.

2. Materials and methods

2.1. Materials

Neurotensin, neurotensin-(1–8), neurotensin-(8–13),

acetyl-neurotensin-(8–13), neurotensin-(1–11), xenopsin,

and bradykinin were purchased from Sigma (St. Louis,

MO). Neuromedin N and HOE140 were obtained from

Research Biochemical International (Natick, MA). Neuro-

tensin-(9–13) and [des-Arg10]HOE140 were purchased

from Bachem California (Torrance, CA). Fura-2/pentaace-

toxymethylester was obtained from Molecular Probes

(Eugene, OR). [3H]Norepinephrine and [3H]IP3 were pur-

chased from NEN Life Science Products. [3H]Bradykinin

was obtained from Amersham Pharmacia Biotech. The

selective, non-peptide antagonist SR48692 was kindly pro-

vided by Dr. D. Gully (Sanofi Recherche, Toulouse,

France). The anti-histamine agent levocabastine was kindly

provided from Janssen Pharmaceutica (Beerse, Belgium).

2.2. Cell culture

PC12 cells were grown in RPMI 1640 (Life Technolo-

gies, Grand Island, NY) supplemented with 10% (v/v) heat-

inactivated bovine calf serum (Hyclone, Logan, UT), 5%

(v/v) heat-inactivated horse serum (Hyclone), and 1% (v/v)

antibiotics (Life Technologies) in a humidified atmosphere

of 5% CO2/95% air at 37 jC. The culture medium was

changed every 2 days, and the PC12 cells were subcultured

weekly.

2.3. Measurement of [3H]norepinephrine secretion

The release of catecholamines from PC12 cells was

measured by a previously reported method [14] with some

modification. In brief, PC12 cells were transferred to 24-

well plates at a density of 5� 105 cells per well. The plates

had been coated with rat tail collagen [15]. After a 1-day

stabilization the cells were loaded with [3H]norepinephrine

(1 ACi/ml; 68 pmol/ml) during an incubation in serum-free

RPMI medium containing 0.1 mM ascorbic acid for 1 h at

37 jC in 5% CO2/95% air. The cells were washed with

Locke’s solution three times and then stablized in Locke’s

solution for 20 min. Then the cells were incubated again

with fresh Locke’s solution for 10 min to measure basal

secretion of [3H]norepinephrine. After two measurements of

basal secretion, the cells were incubated for 10 min in

Locke’s solution containing the drug under test. The

medium was then removed from each well and centrifuged

at 2000� g for 30 s to exclude the presence of detached

PC12 cells. Residual catecholamines in each well were

extracted from the cells by adding 0.1 N HCl. Scintillation

cocktail was then added to the supernatant and cell extract.

Radioactivity was measured in a scintillation counter and

the amount of [3H]norepinephrine secreted was expressed as

percentage of total [3H]norepinephrine content.

2.4. [Ca2+]i measurement

Cytosolic free Ca2 + concentration ([Ca2 +]i) was deter-

mined by using the fluorescent Ca2 + indicator fura-2 as

reported previously [16]. In brief, PC12 cell suspensions

were incubated in fresh serum-free RPMI 1640 medium

containing fura-2/AM (3 AM) and sulfinpyrazone (250 AM)

for 40 min at 37 jC under continuous stirring. The cells

were then washed with Locke’s solution containing sulfin-

pyrazone (250 AM) and left at room temperature until use.

Fluorescence ratios were measured by an alternative wave-

length time scanning method (dual excitation at 340 and 380

nm; emission at 500 nm).

2.5. Quantification of inositol 1,4,5-trisphosphate

Inositol 1,4,5-trisphosphate (IP3) concentration in the

cells was determined by competition assay with [3H]IP3 as

reported previously [17]. In brief, to determine agonist-

evoked IP3 production, PC12 cells were stimulated with

agonists for the indicated periods of time. The reaction was

terminated by aspirating the medium off and adding 15%

(wt/vol) ice-cold trichloroacetic acid (TCA) containing 10

mM EGTA to the cells. The cells were left on ice for 30 min

to extract the water-soluble inositol phosphates. TCA was

removed by extraction with diethyl ether. The final extract

was neutralized with 200 mM Tris and its pH adjusted to

about 7.4. Assay buffer (0.1 M Tris buffer containing 4 mM

EDTA and 4 mg/ml bovine serum albumin), [3H]IP3 (0.1

ACi/ml), and IP3 binding protein prepared from bovine

adrenal cortex were added to the cell extract. The mixture

was incubated for 15 min on ice and then centrifuged at

2000g for 10 min. Water and scintillation cocktail were

added to the pellet to measure radioactivity. IP3 concen-

tration in the sample was determined based on a standard

curve and expressed as picomoles per microgram of protein

in the soluble cell extract with TCA.

T.-J. Park, K.-T. Kim / Cellular Signalling 15 (2003) 519–527520

2.6. Determination of intracellular cAMP

Production of cAMP in intact cells was determined by

measuring the formation of [3H]cAMP from [3H]adenine

nucleotide pools as reported previously [18]. For the cAMP

content measurement, cells were transferred to collagen-

coated six-well plates and grown to confluency. The intra-

cellular ATP pool was labelled in fresh culture medium

supplemented with [3H]adenine (2 ACi/ml) for 24 h at 37

jC. Cells were then washed twice with Locke’s solution and

incubated with fresh Locke’s solution containing appropri-

ate drugs for 30 min at room temperature. Stimulation was

terminated by aspirating off the reaction buffer and adding 1

ml per well 5% (vol/vol) trichloroacetic acid containing 1

AM non-radioactive cAMP. The samples were then kept on

ice for 30 min. The trichloroacetic acid extract was trans-

ferred to Eppendorf tubes and centrifuged to yield a clear,

cell debris-free supernatant. [3H]cAMP and [3H]ATP were

separated by sequential chromatography on Dowex

AG50W-X4 (200–400 mesh) cation exchanger and a neu-

tral alumina column. The [3H]ATP fraction was obtained

from the Dowex column by elution with 2 ml of distilled

water. Then the column was eluted with 3.5 ml of distilled

water, and this eluate was loaded onto the alumina column.

The alumina column was washed with 4 ml of imidazole

solution (0.1 M, pH 7.2), and the eluates were collected

into scintillation vials containing 15 ml scintillation fluid

to count the radioactivity of the [3H]cAMP. Production

of cAMP production was expressed as [3H]cAMP/

([3H]ATP+[3H]cAMP)� 1000.

2.7. [3H]bradykinin binding assay

Binding of [3H]bradykinin to intact PC12 cells was

measured by a previously reported method [19] with some

modification. In brief, PC12 cells were transferred to colla-

gen-coated 12-well plates at a density of 1�106 cells per

well. After stabilization for 12–18 h, the cells were washed

with ice-cold Locke’s solution and incubated with 10 nM

[3H]bradykinin (specific activity, 71 Ci/mmol) and the

indicated concentrations of bradykinin or neurotensin for

90 min at 4 jC. Then the cells were washed three times with

ice-cold Locke’s solution. Finally, the cells were lysed and

scraped into 0.5 ml of 5% trichloroacetic acid, and the

radioactivity was measured by liquid scintillation counting.

Nonspecific binding, determined by coincubation with 10

AM bradykinin, amounted to less than 20% of total binding,

and was routinely subtracted from the total binding. The

binding data were analysed and expressed as percentage of

specific binding.

2.8. Statistical analysis of data

All quantitative data were expressed as meansF SEM.

EC50 and IC50 values were calculated with the Microcal

Origin for Windows program.

3. Results

3.1. [3H]norepinephrine secretion and [Ca2+]i rise induced

by neurotensin

The effect of neurotensin on catecholamine secretion was

investigated in PC12 cells. When the cells were treated with

neurotensin at concentrations of up to 1 AM, no significant

change in [3H]norepinephrine secretion was detected (data

not shown). However, neurotensin at concentrations above

10 AM induced [3H]norepinephrine secretion in a concen-

tration-dependent manner with a half maximal effective

concentration (EC50) of 59F 4 AM (Fig. 1). Maximal

secretion of [3H]norepinephrine (8.01F 0.45%) was

obtained with 500 AM neurotensin.

Since the increase in [Ca2 +]i plays an essential role in

catecholamine secretion by PC12 cells [20,21], we exam-

ined the effect of neurotensin on the level of [Ca2 +]i. At up

to 1 AM concentration, neurotensin did not cause any

significant increase in [Ca2 +]i (Fig. 2A), which correlates

with the result obtained for the [3H]norepinephrine secre-

tion. However, as expected from the results of the [3H]nor-

epinephrine secretion, micromolar concentrations of

neurotensin induced a rise in [Ca2 +]i in a concentration-

dependent manner with a similar EC50 (37F 7 AM) (Fig.

2B). When PC12 cells were stimulated with 100 AM neuro-

tensin, the [Ca2 +]i increased rapidly up to a peak after which

it slowly decreased (Fig. 2A). Maximal increase in [Ca2 +]i(175F 12 nM) was also obtained with 500 AM neurotensin.

These data suggest that the neurotensin-induced responses

cannot be attributed to the previously characterized high-

and low-affinity neurotensin receptors in view of the effec-

tive concentrations. Therefore, PC12 cells might express

neurotensin binding sites that have an extremely low affinity

for neurotensin.

Fig. 1. Neurotensin-evoked [3H]norepinephrine secretion by PC12 cells.

[3H]norepinephrine-loaded PC12 cells were treated with the indicated

concentrations of neurotensin for 10 min. Secreted [3H]norepinephrine is

expressed as percent of total [3H]norepinephrine. Three separate experi-

ments were done and each point is the meanF SEM (bars).

T.-J. Park, K.-T. Kim / Cellular Signalling 15 (2003) 519–527 521

3.2. PLC-dependent [Ca2+]i rise by neurotensin

Because previously known high-affinity neurotensin

receptors activate phospholipase C (PLC) [22,23], we tested

whether the neurotensin-evoked responses in PC12 cells are

also mediated by PLC. As shown in Fig. 3A, 100 AMneurotensin evoked a significant [Ca2 +]i rise in the absence

of extracellular calcium, thus indicating Ca2 + release from

intracellular calcium stores. One minute after the neuro-

tensin treatment, when the level of the cytosolic calcium

returned to the basal level, re-application of 4 mM CaCl2into the external medium induced a subsequent Ca2 + influx,

possibly through Ca2 + release-activated calcium channels

(CRACs). The involvement of PLC in the neurotensin-

induced responses was confirmed by measuring inositol

1,4,5-trisphosphate (IP3). When PC12 cells were treated

with 100 AM neurotensin, the concentration of IP3 increased

rapidly, reaching maximum 30 s after the addition of neuro-

tensin, and then decreasing rather slowly (Fig. 3B). Our

measurements of cellular IP3 support the conclusion that

neurotensin at micromolar concentrations induces [Ca2 +]irise and [3H]norepinephrine secretion by activation of PLC.

Many kinds of PLC-coupled receptors are known to

induce Ca2 + release from thapsigargin-sensitive Ca2 + stores

Fig. 3. PLC-mediated responses evoked by neurotensin. (A) Neurotensin-

evoked [Ca2 +]i rise can be divided into two parts by removal and

reintroduction of extracellular calcium. In the absence of extracellular

calcium, the intracellular calcium rise evoked by 100 AM neurotensin was

measured. When extracellular calcium was removed, 10 AM EGTA was

added to the experimental solution in order to preclude the possibility that

any small amount of calcium was left in the extracellular space. After a 1-

min wait, 4 mM CaCl2 was added to the extracellular medium. (B) PC12

cells were stimulated with 100 AM neurotensin, and the cellular

concentration of IP3 was determined at the indicated times. The results of

three separate experiments were reproducible. Each point is the mean -

F SEM (bars). (C) PC12 cells were stimulated with 1 AM thapsigargin and

subsequently treated with 100 AM neurotensin or 70 mM K+. The results of

three separate experiments were reproducible. Typical Ca2 + transients are

presented.

Fig. 2. Neurotensin-evoked [Ca2 +]i rise in PC12 cells. (A) Fura-2-loaded

PC12 cells were treated with 1 and 100 AM neurotensin as indicated. The

experiments were performed more than ten times independently and the

results were reproducible. Typical Ca2 + transients are presented. (B)

Concentration dependence of the neurotensin-evoked [Ca2 +]i rise. Fura-2-

loaded PC12 cells were treated with increasing concentrations of neuro-

tensin, and the peak heights of stimulation were compared. Three separate

experiments were done. The results were reproducible. Each point is the

meanF SEM (bars).


[24]. Thapsigargin has been known as an inhibitor of

microsomal Ca2 +/ATPase, thus inducing Ca2 + release from

internal Ca2 + stores without generation of IP3 and subse-

quent Ca2 + influx through CRACs [25]. In order to test

whether neurotensin also induced Ca2 + release from thap-

sigargin-sensitive Ca2 + stores, we investigated the interac-

tion between neurotensin and thapsigargin. When the Ca2 +

rise was induced by thapsigargin, a subsequent treatment

with neurotensin did not elicit another Ca2 + increase (Fig.

3C). In contrast, high K+-evoked [Ca2 +]i rise, which is

mediated by voltage-sensitive calcium channels [18] with-

out involvement of PLC or CRACs, was not affected by the

thapsigargin pretreatment (Fig. 3C). The data thus suggest

that neurotensin triggers Ca2 + release from thapsigargin-

sensitive Ca2 + stores by producing IP3.

Since high-affinity neurotensin receptors were reported

to be coupled to adenylyl cyclase, either positively [26] or

negatively [27], we also tested whether neurotensin affected

the activity of adenylyl cyclase. The basal level of cAMP

was not affected by pretreatment of the cells with micro-

molar neurotensin (Table 1). In addition, increases of cAMP

induced by forskolin and CGS21680, activators of adenylyl

cyclase and A2A adenosine receptors, respectively [18],

were not affected by the neurotensin treatment, either. The

results suggest that the neurotensin binding sites are not

linked to adenylyl cyclase in PC12 cells.

3.3. Correlation between neurotensin- and bradykinin-

induced responses

Because bradykinin receptors have also been known to

be linked to PLC and to induce Ca2 + release from thapsi-

gargin-sensitive Ca2 + stores in PC12 cells [21,24], we

studied the interaction between neurotensin- and bradyki-

nin-induced responses. First, we investigated bradykinin-

induced [3H]norepinephrine secretion and [Ca2 +]i rise. As

shown in Fig. 4A and B, bradykinin evoked [3H]norepi-

nephrine secretion and [Ca2 +]i rise in a concentration-

dependent manner with an EC50 of 2 and 11 nM, res-

pectively. Maximal responses were obtained with 5 AMbradykinin. These results are in good agreement with our

previous data [20]. We then tested whether the neurotensin-

and bradykinin-induced responses occurred independently

of each other or whether they shared some signalling path-

way. Maximal (5 AM) and submaximal (10 nM) concen-

trations of bradykinin were chosen. As shown in Fig. 4C,

when PC12 cells were simultaneously treated with 10 nM

bradykinin and 100 AM neurotensin, [3H]norepinephrine

secretion was similar to that induced by one of the two

agonists alone, suggesting that the signalling pathways for

these two receptors are not independent. [3H]Norepinephr-

ine secretion induced by simultaneous treatment with 5 AMbradykinin and 100 AM neurotensin was similar to that

induced by 5 AM bradykinin alone. The experiments meas-

uring [Ca2 +]i gave the same results (Fig. 4D). Since sub-

maximal as well as maximal concentrations of bradykinin

were used in the experiments, the possibility can be ruled

out that the full activation of PLC by bradykinin precluded

PLC from further activation by neurotensin. Instead, the

results can be explained by assuming that neurotensin and

bradykinin acted on the same receptor. In other words, we

Table 1

Lack of neurotensin effect on cAMP production

Reagent cAMP production

None 33F 4

Neurotensin 37F 9

Forskolin 323F 48

Forskolin + neurotensin 322F 55

CGS21680 326F 41

CGS21680+ neurotensin 340F 61

[3H]Adenine-loaded PC12 cells were incubated with 100 AM neurotensin, 3

AM forskolin, and 3 nM CGS21680 either alone or combination for 30 min.

The cAMP levels were measured as described in Materials and expressed as

[3H]cAMP/([3H]ATP+[3H]cAMP)� 1000. Data are meanF SEM values.

Fig. 4. Simultaneous treatment of PC12 cells with neurotensin and

bradykinin. (A) [3H]Norepinephrine-loaded PC12 cells were treated with

increasing concentrations of bradykinin (BK) for 10 min. Three separate

experiments were done and each point is the meanF SEM (bars). (B) Fura-

2-loaded PC12 cells were treated with increasing concentrations of

bradykinin and the peak heights of stimulations were compared. Four

separate experiments were done. The results were reproducible. Each point

is the meanF SEM (bars). (C) Neurotensin (NT, 100 AM)- or bradykinin

(10 nM or 5 AM)-induced secretion of [3H]norepinephrine was compared to

that induced by simultaneous treatment with neurotensin and bradykinin.

Three separate experiments were done and each point is the meanF SEM

(bars). (D) Neurotensin (NT, 100 AM)- or bradykinin (10 nM or 5 AM)-

induced [Ca2 +]i rise was compared to that induced by simultaneous

treatment with neurotensin and bradykinin. Three separate experiments

were done and each point is the meanF SEM (bars).


can hypothesize that 100 AM neurotensin corresponds to 10

nM bradykinin in terms of its effects on catecholamine

secretion and calcium increase. In such a case, simultaneous

treatment with 100 AM neurotensin and 10 nM bradykinin

can be equal to a treatment with 20 nM bradykinin. From

the results in Fig. 4A and B, we can analogize that there is

little significant difference between the 10- and 20-nM

bradykinin-induced responses, which is in good agreement

with lack of an additive response when treating the cells

simultaneously with submaximal concentrations of the two

agonists, as shown in Fig. 4C and D. In line with this logic,

the effects induced by simultaneous treatment with 100 AMneurotensin and 5 AM bradykinin should not be additive.

Therefore, the results suggest the possibility that both

bradykinin and neurotensin act on the same receptors.

We then tested whether neurotensin- and bradykinin-

induced responses desensitized each other. When PC12

cells were first treated with increasing concentrations of

bradykinin, the subsequent neurotensin-induced [Ca2 +]i rise

became gradually less (Fig. 5A). Pretreatment of the cells

with 100 nM bradykinin completely blocked neurotensin-

induced responses. In reverse, the bradykinin-induced

[Ca2 +]i rises also gradually decreased when the cells were

first treated with increasing concentrations of neurotensin

(Fig. 5B). This suggests that the two responses desensitized

each other.

We used bradykinin receptor antagonists in order to test

whether bradykinin receptors truly mediate the neurotensin-

induced responses. Two kinds of bradykinin receptor antag-

onists were used: HOE140 and [des-Arg10]HOE140 as

antagonists for B2 and B1 receptors, respectively. As shown

in Fig. 6, when cells were treated with HOE140, subsequent

[Ca2 +]i rises induced by neurotensin and bradykinin were

inhibited in a concentration-dependent manner at similar

half maximal inhibitory concentrations (IC50) of 4.4F 1.4

and 4.4F 1.0 nM, respectively. Pretreatment of cells with

[des-Arg10]HOE140 also inhibited [Ca2 +]i rises induced

subsequently by neurotensin or bradykinin, but with the

much higher IC50 values of 2.5F 0.2 and 4.2F 1.3 AM,

respectively. These results, which are in good agreement

with a previous report by Nardone et al. [19], clearly suggest

that B2 bradykinin receptors are responsible for the brady-

kinin-induced responses in PC12 cells. More important and

striking is the conclusion that neurotensin, like bradykinin,

exerts its function by acting on B2 bradykinin receptors.

Fig. 5. Desensitization of neurotensin- or bradykinin-evoked [Ca2 +]i rise.

(A) After PC12 cells were first treated with the indicated concentrations of

bradykinin (BK, closed square), the cytosolic calcium rise induced by 100

AM neurotensin was measured (closed circle). Three separate experiments

were done and the results were reproducible. Each point is the meanF SEM

(bars). (B) After PC12 cells were first treated with the indicated

concentrations of neurotensin (closed circle), the cytosolic calcium rise

induced by 10 nM bradykinin was measured (closed square). Three separate

experiments were done and the results were reproducible. Each point is the

meanF SEM (bars).

Fig. 6. Effects of bradykinin receptor antagonists on neurotensin- or

bradykinin-evoked [Ca2 +]i rise. PC12 cells were treated with the indicated

concentrations of bradykinin receptor antagonists for 3 min and then

stimulated with neurotensin (100 AM) or bradykinin (BK, 10 nM). The

peak heights of the responses were compared. Three separate experiments

were done and the results were reproducible. Each point is the meanF SEM

(bars). Open square, bradykinin treatment; closed square, neurotensin

treatment; open circle, bradykinin treatment in the presence of HOE140;

closed circle, neurotensin treatment in the presence of HOE140; open

triangle, bradykinin treatment in the presence of [des-Arg10]HOE140;

closed triangle, neurotensin treatment in the presence of [des-

Arg10]HOE140.


3.4. Inhibition of [3H]bradykinin binding by neurotensin

We performed the [3H]bradykinin binding study in order

to confirm that micromolar concentrations of neurotensin

can bind to B2 bradykinin receptors. As shown in Fig. 7,

bradykinin competed with [3H]bradykinin for binding to

PC12 cells in a concentration-dependent manner with an

IC50 of 9.9 nM. As expected based on our pharmacological

studies, neurotensin at micromolar concentrations signifi-

cantly inhibited [3H]bradykinin binding in a dose-dependent

manner. Neurotensin at 100 AM displaced [3H]bradykinin

binding by 61.8F 6.7%, which roughly corresponds to the

relative extents of catecholamine secretion and calcium

increase induced by 100 AM neurotensin (67.9F 1.5%

and 65.6F 2.2%, respectively), compared to the maximal

response by bradykinin (Fig. 4C and D). Therefore, it seems

that displacing ability of neurotensin correlates with its

effect on catecholamine secretion and calcium increase. In

contrast, angiotensin II, an 8-amino acid peptide which

bears no relation to bradykinin receptors, did not inhibit

[3H]bradykinin binding at up to 1 mM concentrations.

These results, therefore, directly suggest that micromolar

concentrations of neurotensin specifically bind to B2 bra-

dykinin receptors.

3.5. Activation of B2 receptors by neurotensin in other cells

It could be argued that the activation of B2 bradykinin

receptors by neurotensin was due to the plasticity of PC12

cells. We, therefore, tested whether this phenomenon also

occurred in other cells expressing B2 receptors. Bovine

adrenal chromaffin cells [28] and human neuroblastoma

cells [24,29] are known to express functional B2 receptors.

In chromaffin cells neurotensin and bradykinin evoked

[Ca2 +]i rises, and these effects were inhibited by HOE140

pretreatment (Fig. 8A). Interestingly, in contrast to the

results obtained with PC12 cells, the [Ca2 +]i rise induced

by 100 AM neurotensin was not completely inhibited by 100

nM HOE140, a concentration that completely blocked the

10 nM bradykinin-induced response. In human neuroblas-

toma SK-N-BE(2)M17 cells neurotensin and bradykinin

evoked significant [Ca2 +]i rises and the effects were

inhibited by 100 nM HOE140 with similar potency (Fig.

8B). These results together suggest that the activation of B2

bradykinin receptors by micromolar concentrations of neu-

rotensin is a common signalling pathway in B2 receptor-

expressing cells with no relation to the cellular plasticity of

PC12 cells.

3.6. Effects of neurotensin analogs

Neurotensin analogues have been widely used in the

characterization of neurotensin receptors [30,31]. We exam-

ined the effects of neurotensin analogs to compare the

pharmacological characteristics of the neurotensin binding

sites on PC12 cells with the previously known neurotensin

receptors of other cells. When PC12 cells were treated with

neurotensin analogues at concentrations of up to 1 AM, none

of them elicited a rise in [Ca2 +]i (data not shown). Strik-

ingly, at 100 AM (even at 500 AM), neurotensin analogues

which have been known to mimic the effect of neurotensin,

including neurotensin-(8–13) and acetyl-neurotensin-(8–

13), did not induce any significant [Ca2 +]i rise (Table 2).

Neuromedin N and neurotensin-(9–13), which also bind to

previously characterized neurotensin receptors, had also no

effect on PC12 cells. Only xenopsin evoked a [Ca2 +]i rise in

a manner similar to neurotensin. These results suggest that

Fig. 7. Competition between bradykinin or neurotensin and [3H]bradykinin

for binding to the B2 receptors. PC12 cells were incubated with 10 nM

[3H]bradykinin and various concentrations of bradykinin (BK) or neuro-

tensin for 90 min on ice. Displacement of [3H]bradykinin binding by

unlabelled bradykinin (closed square), and neurotensin (closed circle) is

presented. Incubation of cells with 1 mM angiotensin II (closed triangle) did

not prevent the binding of [3H]bradykinin. Nonspecific binding was

determined in the presence of 10 AM unlabeled bradykinin. Four separate

experiments were done and the results were reproducible. Each point is the

meanF SEM (bars).

Fig. 8. Activation of B2 receptors by neurotensin in other cells. Fura-2-

loaded bovine adrenal chromaffin (A) and human neuroblastoma SK-N-

BE(2)M17 cells (B) were stimulated with neurotensin (100 AM) or

bradykinin (BK, 10 nM) in the absence or presence of HOE140 (100 nM).

The peak heights of the responses are compared. Three separate experi-

ments were done and the results were reproducible. Each point is the

meanF SEM (bars).


among neurotensin analogues neurotensin and xenopsin

specifically activate B2 receptors.

4. Discussion

Our results clearly show that micromolar concentrations

of neurotensin activate B2 bradykinin receptors, thereby

inducing calcium increase and catecholamine secretion in

PC12 cells. It is unlikely that the neurotensin-evoked

responses at micromolar concentrations were nonspecific

events. Both [3H]norepinephrine secretion and [Ca2 +]i rise

were saturated at 500 AM neurotensin and even decreased a

little at 800 AM. In addition, neurotensin analogues, except

for xenopsin, did not evoke any [Ca2 +]i rise at concentra-

tions of up to 500 AM, suggesting the specificity for the

action of neurotensin. Furthermore, the neurotensin-evoked

[Ca2 +]i rise was completely inhibited by thapsigargin pre-

treatment. Our results suggest that the neurotensin-evoked

responses are specifically mediated by PLC eliciting Ca2 +

release from thapsigargin-sensitive Ca2 + stores and Ca2 +

influx through CRACs.

The neurotensin binding sites on PC12 cells are differ-

ent from previously reported neurotensin receptors based

on the following two arguments. First, they have an

extremely low affinity for neurotensin (EC50, 59F 4 AM)

compared to the previously known high- and low-affinity

neurotensin receptors in other tissues (Kd, less than 10 nM)

[3–5]. The other argument lies in the pharmacological

characteristics of neurotensin analogues. Previous struc-

ture–activity studies in other laboratories [30,31] suggest

that the major determinants of the biological activity of

neurotensin are located in its C-terminal region. Represen-

tative agonists of neurotensin receptors such as neuro-

tensin-(8–13) [31,32], acetyl-neurotensin-(8–13) [22,30],

neuromedin N [31,33], and neurotensin-(9–13) [22,30,32],

however, were ineffective in PC12 cells, with only one

exception: xenopsin, which mimicked the effect of neuro-

tensin. The results taken together indicate that neurotensin-

binding sites on PC12 cells are entirely different from the

known neurotensin receptors in terms of their pharmaco-

logical characteristics.

Our results clearly show that the neurotensin binding

sites are B2 bradykinin receptors. The two experimental

results, the similar effects of bradykinin receptor antago-

nists on neurotensin- and bradykinin-induced responses

and the displacement of [3H]bradykinin binding by neuro-

tensin, clearly confirms that the neurotensin-induced

responses are mediated by B2 bradykinin receptors. Since

calcium increase induced by the activation of the B2

bradykinin receptors in PC12 cells was insensitive to

pertussis toxin (data not sown), consistent with a previous

report [34], it is highly likely that neurotensin induces

calcium increase by activating B2 bradykinin receptors that

are coupled to Gq/11 and PLC [35]. In addition, similar

effects of neurotensin were found in bovine adrenal

chromaffin and human neuroblastoma cells, considerably

excluding the possible involvement of intermediate factors

or cellular plasticity of PC12 cells. In addition, closer

investigation revealed some differences among PC12,

chromaffin, and human neuroblastoma cells in the extent

of B2 receptor activation by bradykinin and neurotensin

and the antagonism by HOE140. These differences may

reflect the existence of pharmacological subtypes of B2

receptors displaying different affinities for bradykinin ana-

logues [36,37].

Comparison of the effects of neurotensin fragments and

analogues (Table 2) suggests some interesting features of the

activation of B2 bradykinin receptors by neurotensin. Sim-

ple comparison of neurotensin and bradykinin seems to

suggest that proline at the seventh and the tenth residue

position of neurotensin may play an important role in

providing a structural framework by causing turns in the

peptide. In addition, aromatic amino acids located to the

right of the proline in the 10th position may also be essential

for the recognition of the active sites on B2 bradykinin

receptors. However, xenopsin, which lacks one of the pro-

lines, was still effective, suggesting that the activation of B2

bradykinin receptors by neurotensin cannot be explained by

the simple analogy of the amino acid sequences among

peptides. Furthermore, both neurotensin-(1–11) and neuro-

tensin-(8–13) had no effect, suggesting that activation of B2

bradykinin receptors by neurotensin requires the entire

sequence of amino acids. This stands in dramatic contrast

to the previously characterized neurotensin receptors which

can be activated by neurotensin-(8–13), a C-terminal frag-

ment of neurotensin. Therefore, the three-dimensional con-

formation, rather than the primary amino acid sequence,

may be critical for the activation of B2 bradykinin receptors.

It may be assumed that peptides like neurotensin and

xenopsin share a certain conformational motif that may

interact with B2 bradykinin receptors. Since high concen-

trations of neurotensin and xenopsin were required for the

activation and since the maximal responses induced by

neurotensin were much smaller than those induced by

bradykinin, the binding site of neurotensin and xenopsin,

Table 2

Effects of neurotensin analogues on [Ca2 +]i rise

Reagent Amino acid sequence Net [Ca2 +]i rise

Neurotensin E-L-Y-E-N-K-P-R-R-P-Y-I-L 96F 4

Neurotensin-(8–13) R-R-P-Y-I-L NS

Acetyl-neurotensin-(8–13) Acetyl-R-R-P-Y-I-L NS

Neurotensin-(9–13) R-P-Y-I-L NS

Neurotensin-(1–8) E-L-Y-E-N-K-P-R NS

Neurotensin-(1–11) E-L-Y-E-N-K-P-R-R-P-Y NS

Neuromedin N K-I-P-Y-I-L NS

Xenopsin E-G-K-R-P-W-I-L 40F 3

Bradykinin R-P-P-G-F-S-P-F-R

PC12 cells were stimulated with neurotensin or its analogues (100 AMeach). Changes in cytosolic calcium concentration were measured as

described in Materials and methods. Data are meanF SEM values.

NS means no significant [Ca2 +]i rise.


when they interact with B2 bradykinin receptors, may differ

from that of bradykinin. More careful analyses including

determination of the binding sites of neurotensin and xen-

opsin and comparison of the three dimensional conforma-

tion of bradykinin, neurotensin, and xenopsin will be

necessary for the understanding of the neurotensin binding

to the B2 bradykinin receptor.

Acknowledgements

This work was supported by grants from the Korea

Science and Engineering Foundation (KOSEF), the Brain

Neurobiology Research Program, the National Research

Laboratory Program of the Ministry of Science and

Technology (MOST) and Brain Korea 21 program of the

Ministry of Education.

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