www.elsevier.com/locate/brainres

Brain Research 1006 (2004) 114–125

Research report

The synthesis and distribution of the kinin B1 and B2 receptors

are modified in the hippocampus of rats submitted to

pilocarpine model of epilepsy

Gustavo Adolfo Arganaraza,b, Jose Antonio Silva Jrc,d, Sandra Regina Perosaa,Luciana Gilbert Pessoac, Fatima Ferreira Carvalhoa, Jean Loup Bascandse,

Michael Baderf, Edivaldo da Silva Trindadeg, Debora Amadoa, Esper Abrao Cavalheiroa,Joao Bosco Pesqueroc, Maria da Grac�a Naffah-Mazzacorattia,g,*

aDepartamento de Neurologia/Neurocirurgia, Universidade Federal de Sao Paulo, Sao Paulo, Brazilb Instituto de Biofisica Carlos Chagas Filho, UFRJ, Brazil

cDepartamento de Biofısica, Universidade Federal de Sao Paulo, Sao Paulo, BrazildDepartamento de Patologia, Universidade Federal de Sao Paulo, Sao Paulo, Brazil

e INSERM, Toulouse, FrancefMax-Delbruck Center, Berlin, Germany

gDepartamento de Bioquımica, Universidade Federal de Sao Paulo, Sao Paulo, Brazil

Accepted 28 December 2003

Abstract

Kinins, a special class of polypeptides, are represented by bradykinin (BK), kallidin (Lys-BK), as well as their metabolites. The biological

actions of these polypeptides binding on their receptors (B1 and B2) have been related to inflammation process, cytokines action, glutamate

release and prostaglandins production. Usually, kinin B1 receptor is not expressed at a significant level under physiologic conditions in most

tissues, but its expression is induced by injury, or upon exposure in vivo or in vitro to pro-inflammatory mediators. The kinin B2 receptor

subtype is constitutively and widely expressed throughout the central and peripheral nervous system. These data raise the possibility for de

novo expression of those receptors during the temporal lobe epilepsy (TLE), which has been related to cell death, gliosis and hippocampal

reorganization. To correlate kinin system and TLE, adult male Wistar rats were submitted to pilocarpine model of epilepsy. The hippocampi

were removed 6 h, 5 and 60 days after status epilepticus (SE) onset. The collected tissues were used to study the expression of kinin B1 and

B2 mRNA receptors, using Real-Time PCR. Immunohistochemistry assay was also employed to visualize kinin B1 and B2 distribution in the

hippocampus. The results show increased kinin B1 and B2 mRNA levels during acute, silent and chronic periods and changes in the kinin B1

and B2 receptors distribution. In addition, the immunoreactivity against kinin B1 receptor was increased mainly during the silent period,

where neuron clusters of could be visualized. The kinin B2 receptor immunoreactivity also showed augmentation but mainly during the acute

and silent periods. Our results suggest that kinin B1 and B2 receptors play an important role in the epileptic phenomena.

D 2004 Elsevier B.V. All rights reserved.

Theme: Disorders of the nervous system

Topic: Epilepsy: basic mechanisms

Keywords: Kinin B1 and B2 receptors; Temporal lobe epilepsy; Pilocarpine; Hippocampus; Plasticity; Status epilepticus

1. Introduction

0006-8993/$ - see front matter D 2004 Elsevier B.V. All rights reserved.

doi:10.1016/j.brainres.2003.12.050

Abbreviations: BK, bradykinin; IL-1a, interleukin 1a; IL-1h, interleu-kin 1h; IL-6, interleukin 6; Lys-BK, kallidin; Lys-BK, lysil-bradykinin or

kallidin; MAPK, mitogen-activated protein kinase; Neu-N, neuronal

marker; NF-nB, nuclear factor-nB; SE, status epilepticus; TLE, temporal

lobe epilepsy; TNFa, tumor necrosis factor a

* Corresponding author. Tel.: +55-11-55-76-54-09; fax: +55-11-55-49-

47-43.

E-mail address: naffahmazz.nexp@epm.br (M.G. Naffah-Mazzacoratti).

Prolonged or recurrent seizures, which are some of the

important characteristics of epileptic syndrome, have

shown several consequences on brain cytoarchitecture.

One of great significance brain modification, which is

found in human temporal lobe epilepsy (TLE), is the

hippocampal sclerosis. Under this pathologic environ-

G.A. Arganaraz et al. / Brain Research 1006 (2004) 114–125 115

ment, neuronal cell loss is observed in the dentate gyrus,

hilus, CA1 and CA3 regions of the hippocampal forma-

tion [25]. In addition, Heineman et al. [16] also found

functional changes in glial cells, characterizing the hip-

pocampal gliosis. According to the epileptogenic glial

scar hypothesis, the reactive astrocytes, present in the

lesion areas, are able to release neurotrophic factor, which

will support the axonal sprouting and neosynaptogenesis.

These cellular events probably are involved in the further

development of a chronic hyperexcitability [39]. Thus,

the presence of mossy fiber sprouting, which is an

aberrant projection of the granule cell axons into their

own dendritic fields, has been described by several

authors [27,45]. This pathway may have an important

role in propagation and/or maintenance of hippocampal

excitability [35].

Considering that several animal models of epilepsy yield

similar patterns of hippocampal cell death, we might pre-

sume that selective neuronal loss is one the earliest conse-

quence of prolonged seizure activity, commonly mediated

by glutamate-induced excitotoxicity in temporal lobe epi-

lepsy [7,18,44].

Kinins, a special class of polypeptides, are represented by

bradykinin (BK) and kallidin (Lys-BK), which are generated

after limited proteolysis of high and low molecular weight

kininogens, through kallikreins action in the plasma and

tissues [43]. These short-living peptides are rapidly degraded

by kininases [3], originating active metabolites such as des-

Arg9-BK and des-Arg10-kallidin as well inactive products.

The kallikrein-kinin system receptors are denominated B1

and B2 and both are coupled to G proteins. The kinin B2

receptor shows high affinity for BK and Lys-BK and low

affinity for the active metabolites des-Arg9-BK and des-

Arg10-kallidin [17,26]. In contrast, the kinin B1 receptor is

preferentially activated by the active metabolites des-Arg9-

BK and des-Arg10-kallidin, possessing a low affinity for BK

and Lys-BK [28,38]. Kinin B1 receptor agonists led to

inositol phosphate generation, promoting a transient rise in

intracellular Ca2 + levels, after phospholipase C activation

[1,22]. Furthermore, stimulation of kinin B1 and B2 recep-

tors induce tissue edema and phospholipase A2 activation,

producing prostaglandins [3,11,46]. Agonists of kinin B2

receptor also induce tyrosine phosphorylation of endothelial

cells proteins [12]. In addition, kinin B1 and B2 stimulation

also activate mitogen-activated protein kinase (MAPK)

(ERK1/ ERK2) in cell culture, resulting in AP-1 transloca-

tion, modifying the immediate early gene expression [31].

On the other hand, intra-ventricular administration of brady-

kinin induces increase of serotonin and prostaglandin E2

concentration, mainly in the hypothalamus and midbrain [2].

Usually, kinin B1 receptor is not expressed at a signif-

icant level under physiologic conditions in most tissues, but

its expression is induced by injury or upon exposure in vivo

or in vitro to pro-inflammatory mediators, such as lipopoly-

saccharide and cytokines [24]. Moreover, Ni et al. [33]

showed several evidences that nuclear factor-nB (NF-nB) is

also involved in the dynamic regulation of human kinin B1

receptor gene expression, during inflammatory processes.

In contrast, kinin B2 receptor is constitutively and widely

expressed in all nervous system [5] and has been found in

the nucleus of neurons from hippocampus, hypothalamus

and cortex [9]. Nevertheless, the real function of this

receptor in neuronal nucleus is still unknown.

Bregola et al. [4] showed that endogenous kinin B1

agonist Lys-des Arg9 BK increases the glutamate overflow

in kindled rats slices (40–50%) and, to a smaller extent

(20%) in slices of kainate-treated animals, supporting the

idea that kinin B1 receptor may play a role in TLE

excitotoxicity. The authors also suggest that the relationship

between Lys-des Arg9 BK and glutamate release is not a

mere consequence of seizures, but it is associated with a

condition of latent hyperexcitability, found in epileptic

tissues. In addition, Ongali et al. [34] showed a significant

decline of kinin B2 receptor binding sites, accompanied by

an impressive increase of kinin B1 receptor binding sites

labeling in the brain of rats submitted to kindling model of

epilepsy.

Considering the experimental model of epilepsy induced

by systemic administration of pilocarpine, several authors

have shown increased hippocampal levels of prostaglandins

[13,30], increased tyrosine-phosphorylated proteins levels

[14], MAPK activation [15] and increased glutamate release

[7,44]. Since these pathways can be also activated by kinin

receptor agonists [12,31,32], the relationship between pilo-

carpine model of epilepsy and the kallikrein-kinin system

should be analyzed. Therefore, the present work was delin-

eated to verify changes in the expression levels of kinin B1

and B2 receptors in the hippocampus of Wistar rats,

submitted to pilocarpine model of epilepsy [20]. The kinin

B1 and B2 mRNA quantifications were performed using

quantitative Real-Time PCR, and the time-course of kinin

B1 and B2 receptors staining in the hippocampus of rats

during status epilepticus (SE), silent and chronic phases was

evaluated by immunohistochemistry. In addition, we also

made use of indirect double-label immunofluorescence

(confocal microscopy) in order to establish the distribution

of kinin B1 and B2 receptors within neuronal organelles.

2. Materials and methods

2.1. Wistar rats treatment

The animal experiments were performed under Institu-

tional ethical approval of protocol and all efforts were made

to minimize animal suffering. Moreover, assistance in feed

and hydration were carried out during the initial recovery

after SE establishment to improve the animal’s state and

survival.

Wistar adult male rats, weighing 250 g, were housed in

groups of three to four per cage and maintained in controlled

room temperature, humidity and light–dark cycle (12:12 h)

G.A. Arganaraz et al. / Brain Research 1006 (2004) 114–125116

with chow pellets and tap water available ad libitum. The

rats received a single dose of pilocarpine (350 mg/kg,

intraperitoneal [i.p.]). To prevent peripheral cholinergic

effects, scopolamine methylnitrate was injected subcutane-

ously at a dose of 1 mg/kg, 30 min before pilocarpine

administration. A group of animals was killed 6 h after

status epilepticus onset (6 h SE) (acute group). Another

group was killed during the seizure-free period (5 days after

SE onset) (silent group) and the last set was killed 60 days

after SE induction (period of spontaneous recurrent seiz-

ures) (chronic group). The rats that received pilocarpine but

did not develop status epilepticus, presenting only partial

seizures (partial group) were killed 6 h after pilocarpine

injection. Animals composing chronic group were sacrificed

at least 24 h after an overt seizure during the interictal

period. Saline-treated animals were sacrificed 6 h, 5 or 60

days after saline and scopolamine methylnitrate injections

and were used as control (control groups). These groups

were used to perform the quantification of kinin B1 and B2

receptors mRNA by Real-Time PCR assay (n = 3 per group)

and the spatial and temporal localization of both kinin

receptors were analyzed using immunohistochemistry

(n = 3 per group).

To localize the cellular distribution of kinin receptors, a

co-localization of kinin B1 receptor and Neu-N protein

(nuclear marker) (n = 3) as well as a co-localization of kinin

B1 and B2 receptors were performed in the hippocampal

formation of control rats (n = 3).

2.2. Quantitative real-time Taqmank PCR

Brains for biochemical evaluation of kinin B1 and B2

receptors were collected from the following groups: Group

1, composed by saline-treated rats (i.p.) (control group);

group 2, animals which received scopolamine methylnitrate

(SM) plus pilocarpine injection (350 mg/kg i.p.) and were

killed 6 h after status epilepticus onset. Animals that

received SM and pilocarpine were sacrificed either 5 (silent

period) or 60 days (chronic period) after the pilocarpine

injection. Rats from the above groups (n= 3 for each group)

were killed and their brains were isolated, dissected and the

hippocampi frozen in liquid nitrogen and stored at � 80 jC.Thawed tissue was homogenized in 1 ml TRIzol reagent

(Gibco BRL, Gaithersburg, MD) and total RNA was isolat-

ed according to the manufacturer’s instructions.

Samples were submitted to a 20 Al reaction using

TaqManR Amplification System with an ABI PRISMR7000 Sequence Detection System (Applied Biosystem,

Foster City, CA, USA). Real-Time PCR was performed

with 900 ng of cDNA for kinin B1 and B2 receptors and

100 ng of cDNA for glyceraldehyde 3-phosphate dehydro-

genase (GAPDH), used as an internal standard. Oligonucle-

otide primer and fluorogenic probe sets for Taqmank Real-

Time PCR were designed for kinin receptors and GAPDH

using Assays-by-Design Service (Applied Biosystems) to

meet all Taqmank design guidelines. Probes were synthes-

ised with a reporter dye 6-carboxyfluorescein (6-FAM)

covalently linked at the 5V end and a quencher dye 6-

carboxy-tetramethyl-rhodamine (TAMRA) was linked to

the 3Vend of the probe. Each reaction was carried out with

10 Al of Master Mix (Applied Biosystem) and 1 Al of a mix

containing two primers (18 AM each) and a probe (5 AM),

specific to mRNA of kinin B1 receptor (probe B1: 5V-CCCAAGAGGCCAAAGA-3V, forward primer: 5V-CCAGGGTTCGTCATCACTATCTG-3V, reverse primer:

5V-GCAAAAGGAAGAAGGACAAGACTAA-3V), kinin

B2 receptor (probe B2: 5V-CCGCACTGGAGAACA-3V,forward primer: 5V-CCCTTCCTCTGGGTCCTCTT-3V, re-

verse primer: 5V-CAGAACACGCTGAGGACAAAGA-3V)or GAPDH (probe: 5V-TTGGCAGCACCAGTGG-3V, for-

ward primer: 5V-GGGCAGCCCAGAACATCAT-3V, reverseprimer: 5V-CCGTTCAGCTCTGGGATGAC-3V). The cycle

conditions were: 50 jC for 2 min, then 95 jC for 10 min,

followed by 50 cycles of 95 jC for 15 s (melting step), 60

jC for 1 min (anneal/extend step).

2.3. Data analysis

Increases in the amount of reporter dye fluorescence

during the 50 cycles of amplification were monitored using

Sequence Detector software (SDS version 1.6 Applied

Biosystems). The PCR cycle when a given fluorescence

threshold is crossed by the amplification curve is considered

the first parameter to analyze mRNA expression and named

Ct. The bigger is the initial copies amount, the less will be

the Ct number. A normalized value is obtained by subtract-

ing Ct of GAPDH from Ct of kinin B1 or B2, resulting in

DCt. As it is uncommon to use DCt as a relative expression

data due to this logarithmic characteristic, the 2� DCt pa-

rameter was used to express the relative expression data

[23].

The levels of expression of kinin B1 and B2 receptors

mRNA are presented as an n-fold difference relative to

the levels expressed during different experimental model

phases were compared using the one-way ANOVA fol-

lowed by t-test. The data are presented as mean-

sF S.E.M.; p < 0.05 was considered to be statistically

significant.

2.4. Antigen retrieval and immunohistochemistry for kinin

B1 and B2 receptors

The brains were fixed in formalin solution (5%, 48 h),

then submerged in paraffin and coronal section was done.

The retrieval of kinin B1 and B2 receptors immunoreactiv-

ity, from formalin fixed paraffin-infiltrated specimens, were

obtained by a modification of the method of Shi et al. [41].

Briefly, paraffin sections (3 Am thick) were deparaffinized

and endogenous peroxidase was inactivated with 3% H2O2

in buffered saline for 15 min. Sections were then incubated

with 50 mM Tris–HCl, pH 9.5, and heated in a microwave

oven at a potency of 700 W for 4 min, followed by a 1 min-

Fig. 1. (A) Levels of expression of kinin B1 receptor mRNA in the

hippocampus of rats submitted to pilocarpine model of epilepsy by Real-

Time PCR assay. All data have been normalised for levels of GAPDH

expression within the same sample and are expressed relative to levels

detected in each experimental model phase. Data are meanF S.E.M. (n= 3

per group). (B) Levels of expression of kinin B2 receptor mRNA in the

hippocampus of rats submitted to pilocarpine model of epilepsy by Real-

Time PCR assay. All data have been normalised for levels of GAPDH

expression within the same sample and are expressed relative to levels

detected in each experimental model phase. Data are meanF S.E.M. (n= 3

per group).

G.A. Arganaraz et al. / Brain Research 1006 (2004) 114–125 117

period of interval and by an additional period of 3 min of

heating.

All the following incubations were carried out at room

temperature. Microwave-treated sections were placed in

Tris–glycine buffer (0.1 M glycine, pH 7.4) for 30 min to

quench unreacted aldehyde groups. Sections were then

incubated with 20 mM sodium phosphate buffer, pH 7.4,

0.45 M NaCl (PBS), 0.3% Triton X-100 (Triton buffer,

Sigma) and 5% defatted dry milk for 5 h. The slices were

incubated overnight with the following primary polyclonal

anti-B1 receptor antibody (1:100) or monoclonal anti-B2

receptor antibody (1:100) in blocking buffer. The anti-B1

antibody was a kindly given by J.L. Bascands from France

[40] and the anti-B2 antibody was obtained from Transduc-

tion Laboratory (Lexington, KY, USA). Experimental pro-

tocols in control tissues, in which the primary antibody was

replaced by blocking buffer, were also carried out. Sections

were washed four times with PBS and anti-B1 receptor

antibody was detected with a biotinylated goat anti-rabbit

IgG (Vector, Burlingame, CA; BA-2000) diluted 1:100 in

blocking buffer for 60 min and anti-B2 receptor antibody

was detected with a biotinylated goat anti-mouse IgG

(Vector, Burlingame, CA; BA-2000) diluted 1:100 in block-

ing buffer for 60 min. The complex was then incubated with

ABC Kit (Elite PK-6102; Vector) and visualized using 3,3V-diaminobenzidine (1 mg/ml in 1% H2O2). The sections were

then dehydrated, coverslipped and analyzed by light mi-

croscopy using bright-field illumination.

2.5. Double-label procedure

In order to verify whether kinin B1 or B2 receptors

immunoreactivity were localized near nuclear membrane

or embedded into plasma membrane of hippocampal

neurons, we employed a double-label immunofluores-

cence protocol to co-localize the kinin B1 receptor

containing cells in combination with the neuronal marker

(Neu-N). The Neu-N is a protein found in the cell nuclei.

The first primary antibody employed was a rabbit anti-B1

(polyclonal antibody), followed by the mouse anti-neuro-

nal nuclei (Neu-N) (monoclonal antibody from Chemicon

International, Temecula, CA). Thus, an indirect immuno-

fluorescence double-label procedure was used to double-

label the kinin B1 receptor plus Neu-N protein. Same

protocol was done employing anti-B1 (polyclonal anti-

body) receptor and anti-B2 (monoclonal antibody) recep-

tor as primary antibodies to co-localize kinin B1 and B2

receptors in neurons.

The animals were killed by decapitation. The brains were

removed from the skull, fixed for 30 min with a 2%

paraformaldehyde in 0.1 M sodium phosphate buffer, and

cryoprotected overnight in 30% sucrose in 0.1 M sodium

phosphate buffer (PBS, pH 7.4) at 4 jC. The brains were

sectioned at a thickness of 10 Am on a sliding knife

microtome and mounted on Silane-Prepk slides (Sigma)

and stored at � 20 jC.

Following six washes in PBS buffer, the sections were

incubated for 30 min in PBS containing 0.1 M glycine to

quench unreacted aldehyde groups. Then, the sections were

washed six times in PBS and incubated with PBS containing

0.01% saponin (Calbiochem, Bioscience, La Jolla, CA)

(PBS-saponin) and 1% bovine serum albumin (blocking

buffer) for 1 h to prevent background staining. The slices

were incubated overnight with the first primary rabbit anti-

B1 polyclonal antibody, diluted 1:100 in blocking buffer.

Sections were then washed in PBS-saponin and incubated

overnight with the second primary mouse anti-Neu-N

monoclonal antibody, diluted 1:200 in blocking buffer.

Sections were then washed in PBS-saponin and incubated

sequentially in PBS containing the anti-rabbit antibody

coupled to Alexa Fluor 594 (red) (1:300) for 1 h. Following

several washes in PBS, the sections were incubated in PBS

containing the anti-mouse antibody coupled to Alexa Fluor

488 (green) (1:300) (Molecular Probe, Eugene, OR) for 1 h.

Sections were then washed in PBS and cover-slipped with

Fluoromount-G (Electronic Microscopy Science, Fort

Washington). Different magnification images were obtained

Table 1

Real-Time RT-PCR quantification and immunoreactivity for kinin B1 and B2 receptors in the hippocampal formation

Hippocampal Control group Acute group Silent group Chronic group

formationB1 B2 B1 B2 B1 B2 B1 B2

CA1 + + ++ + ++ + + + + + + + +

CA2 + + + + + + + +

CA3 + + ++ + ++ + + + + + + + +

Hilus + + ++ + + ++ + + + ++ + + +

Granular cell + + ++ + ++ + + + + + + + +

Real-Time

RT-P CR

7.47� 10� 5F1.30� 10� 5

1.15� 10� 3F0.14� 10� 3

4.28� 10� 4F0.41�10� 4,

p< 0.0001

3.03� 10� 3F0.45� 10� 3,

p< 0.0001

4.01�10� 4F0.56� 10� 4,

p< 0.0001

1.67� 10� 3F0.16� 10� 3,

p< 0.05

6.48� 10� 5F0.83� 10� 5,

p< 0.24

3.23� 10� 3F0.45� 10� 3,

p< 0.0001

Staining intensity: + low, ++ moderate, +++ high. For comparison between two groups, a Student’s t was performed.

G.A. Arganaraz et al. / Brain Research 1006 (2004) 114–125118

by a dual channel confocal argon/krypton laser scanning and

these images were collected using appropriate emission

filters. During co-localization of B1 and Neu-N, kinin B1

receptors were stained in red while the Neu-N positive cells

Fig. 2. (I) Photomicrographs of the hippocampus processed for kinin B1 receptor im

group, (D) chronic group—scale bars: 0.3 mm. (II) Photomicrographs of CA1 regi

(A) saline-treated group, (B) 6 h SE group, (C) silent group, (D) chronic group—

were stained in green. Similar procedure was employed to

co-visualize kinin B1 (1:100) and kinin B2 (1:100) recep-

tors. The positive kinin B2 cells were stained in green and

kinin B1 positive cells were stained in red.

munohistochemistry: (A) saline-treated group, (B) 6 h SE group, (C) silent

on of hippocampus processed for kinin B1 receptor immunohistochemistry:

scale bars: 0.02 mm.

G.A. Arganaraz et al. / Brain Research 1006 (2004) 114–125 119

3. Results

3.1. Animals behavior

Reproducing previous data [6], after pilocarpine injection

the Wistar rats presented akinesia, ataxic lurching and

tremor, masticatory automatisms with myoclonus of facial

muscles and wet dog shakes persisting for 10–15 min.

These behavioral changes progressed to motor limbic seiz-

ures as previously described [6,47]. Limbic convulsive

behavior persisted for 45–60 min, evolving to SE in 75%

of Wistar rats or returned progressively to normal appearing

behavior after 3–5 h in the remaining 25% of rats. SE

persisted for 12 h without remission. Approximately 30% of

rats with SE died during this period. Surviving animals were

unresponsive to environmental stimuli, hypotonic and mo-

Fig. 3. (I) Photomicrographs in CA3 region of hippocampus processed for kinin

group, (C) silent group, (D) chronic group—scale bars: 0.02 mm. (II) Photomicrog

receptor immunohistochemistry: (A) saline-treated group, (B) 6 h SE group, (C)

tionless 24 after pilocarpine administration. Behavior

returned progressively to normal pattern, although some

aggressive response was observed when animals were

handled. This period lasted 14 days and no behavior sign

related to epileptic activity was observed (silent period).

Spontaneous seizures began to be observed after this period

(3 or 4/week) and rarely lasted longer than 50–60 s.

Spontaneous remission of these seizures was never observed

during experimental period.

3.2. Real-time PCR analysis for kinin B1 and B2 receptors

The amount of mRNA for both kinin receptors in Wistar

rats was analyzed using quantitative RNA assay. Kinin B1

mRNA showed increased expression in the group of rats

presenting 6 h of SE ( p < 0.0001) and in the silent group

B1 receptor immunohistochemistry: (A) saline-treated group, (B) 6 h SE

raphs of granule cells of the hippocampal formation processed for kinin B1

silent group, (D) chronic group—scale bars: 0.02 mm.

G.A. Arganaraz et al. / Brain Research 1006 (2004) 114–125120

( p < 0.0001) (Fig. 1A). The control and chronic groups

present lower levels of expression of mRNA for kinin B1

receptor, when compared with acute and silent groups. The

amount of mRNA for kinin B2 receptor was increased in 6

h SE group ( p < 0.0001), silent ( p < 0.05) and chronic groups

( p < 0.0001) (Fig. 1B). Animals presenting only partial

seizure showed no alterations in kinin B1 nor B2 receptors

expression levels. These data showed that the synthesis of

both receptors was induced after SE-induced injury (Table 1).

3.3. Immunoreactivity of kinin B1 and B2 receptors in the

hippocampus

The immunohistochemical analysis showed similar dis-

tribution of kinin B1 receptor in all hippocampal formation

of all studied groups (Fig. 2, panel IA–D). Regions such as

Fig. 4. (I) Photomicrographs of the hippocampus processed for kinin B2 receptor im

group, (D) chronic group—scale bars: 0.3 mm. (II) Photomicrographs of CA1 regio

(B) 6 h SE group, (C) silent group, (D) chronic group—scale bars: 0.02 mm.

CA1, CA2, CA3, hilus as well as the granular cells from the

dentate gyrus were stained. However, the staining intensity

for kinin B1 immunoreactivity was very different in the

groups studied. An increased immunoreactivity was ob-

served in CA1 and CA3 regions of rats during the acute

(6 h SE) and silent periods (Figs. 2, panel IIB,C and 3, panel

IB,C). The pyramidal neurons from CA1 and CA3 regions

were intensely stained in acute group, but this intensity was

highly increased in the CA1 and CA3 regions of the silent

group (Figs. 2, panel IIC and 3, panel IC), when compared

with saline-treated rats. Note that the CA2 region (Fig. 2,

panel IC), which shows fewer cell death after pilocarpine-

induced SE, has presented minor immunoreactivity against

kinin B1 antibody when compared with other hippocampal

areas, during the silent period. The pyramidal cells of the

hippocampal formation showed several clusters of immu-

munohistochemistry: (A) saline-treated group, (B) 6 h SE group, (C) silent

n of hippocampus processed for kinin B2 receptor: (A) saline-treated group,

G.A. Arganaraz et al. / Brain Research 1006 (2004) 114–125 121

noreactivity against kinin B1 antibody and fibers could be

visualized in CA1 regions of the hippocampus (Fig. 2, panel

IIC). Clusters of cells in the dentate gyrus (Fig. 3, panel IIC)

were also detected in the dentate gyrus, during the silent

period. The chronic period showed similar pattern of hip-

pocampal kinin B1 immunoreactivity when compared with

saline-treated group (Figs. 2, panels I and IID and 3, panels I

and IID).

The reactivity against kinin B2 receptor was also

visualized in all hippocampal formation of all groups

studied (Fig. 4, panel IA–D). The nucleus of neurons

from CA1, CA2, CA3, hilus and dentate gyrus regions

were stained, when kinin B2 antibody was employed (Figs.

4 and 5). Increased staining was found in the hippocampus

of rats from acute (6 h SE), silent and chronic groups,

Fig. 5. (I) Photomicrographs of CA3 region of hippocampus processed for kinin

group, (C) silent group, (D) chronic group—scale bars: 0.02 mm. (II) Photomicrog

receptor immunohistochemistry: (A) saline-treated group, (B) 6 h SE group, (C)

when compared with saline-treated animals. However, the

major intensity of nucleus staining was found in rats

presenting 6 h SE. (Figs. 4, panel IIB and 5, panels I

and IIB). Nevertheless, a cytoplasmatic staining of neurons

was also visualized in the CA3 regions of rats during

acute, silent and chronic periods (Fig. 5, panel IB,C,D).

The dentate gyrus showed increased nuclear staining in the

hippocampus of rats during the acute, silent and chronic

periods, when compared with saline-treated rats (Fig. 5,

panel IIB,C,D). Animals presenting only partial seizures

showed no alteration in kinin receptors expression, show-

ing that long-lasting seizures are necessary to modify the

normal pattern of expression. The Table 1 summarizes the

semi-quantitative analysis of the immunohistochemical

results.

B1 receptor immunohistochemistry: (A) saline-treated group, (B) 6 h SE

raphs of granule cells of the hippocampal formation processed for kinin B2

silent group, (D) chronic group—scale bars: 0.02 mm.

n Research 1006 (2004) 114–125

3.4. Double staining of B1 receptor and Neu-N protein and

kinin receptors in the hippocampus of Wistar rats

Fig. 6 (panels C–E) shows that kinin B1 receptor was

co-localized with Neu-N protein in the CA3 region (panel

G.A. Arganaraz et al. / Brai122

Fig. 6. Double staining for distribution analysis of kinin B1 and B2 receptors and

Neu-N in the CA3 region of hippocampal formation saline-treated rats (in green)—

region of hippocampal formation of saline-treated rats (in red)—scale bars: 20 Ahippocampal formation of saline-treated rats—scale bars: 20 Am. (D) Kinin B1 r

formation of saline-treated rats—scale bars: 50 Am. (E) Kinin B1 receptor (red) plu

rats—scale bars: 20 Am. (F) Kinin B1 receptor (red) plus kinin B2 receptor (green

scale bars: 20 Am.

C) and granular cells (panels D and E) of hippocampal

formation of saline-treated rats. The kinin B1 receptor was

visualized surrounding the nucleus of pyramidal (panel C)

and granular neurons (panels D and E). In Fig. 6 (panel F),

we can perceive the kinin B2 receptor (in green) and the

Neu-N protein in the Wistar rats hippocampus. (A) Immunoreactivity for

scale bars: 20 Am. (B) Immunoreactivity for kinin B1 receptor in the CA3

m. (C) Kinin B1 receptor (red) plus Neu-N (green) in the CA3 region of

eceptor (red) plus Neu-N (green) in the granular cells of the hippocampal

s Neu-N in the granular cells of the hippocampal formation of saline-treated

) in the granular cells of the hippocampal formation of saline-treated rats—

Fig. 7. (A) Kinin B1 receptor immunoreactivity in the cerebral temporal

cortex (6 h of SE) showing immunostaining in the pyramidal neurons

surrounding the cell nucleus and the plasma membrane—sale bars: 0.07

mm.

G.A. Arganaraz et al. / Brain Research 1006 (2004) 114–125 123

kinin B1 receptor (in red) in granular cells of saline-treated

animals, showing the co-localization of both receptors.

However, the cortical immunostaining of pyramidal neurons

(6 h of SE) showed kinin B1 receptor surrounding not only

the cell nucleus but the plasma membranes labeled by kinin

B1 antibody, showing that this receptor can be found in

different compartments of neurons (Fig. 7, panel A).

3.5. Kinin B1 and B2 antibodies specificity

During the immunohistochemical procedure, when the

primary antibodies for both kinin receptors were replaced by

blocking solution, no staining was found in the brain slices.

In addition, Shi et al. [42] using the same anti-kinin B2

receptor antibody (from Transduction Lab, Lexington) pre-

absorbed with C-terminal domain of kinin B2 receptor

showed an abolition of the staining, indicating the antibody

specificity.

4. Discussion

This work shows that during the epileptogenic process a

significant increase in both kinin B1 and B2 mRNA

receptors synthesis occurs. This event could underlie the

physiopathologic mechanism participating in the establish-

ment of an epileptic focus.

Kinin B1 mRNA level was increased during the acute

and silent periods showing that de novo synthesis of this

receptor was induced in the hippocampus of Wistar rats. The

correspondent amount of kinin B1 receptor was also in-

creased at the same periods, as visualized by immunohisto-

chemistry. These data are in agreement with those, reported

by Marceau [24] who showed that the kinin B1 receptor is

not normally found in normal tissues and its synthesis is

induced due to several stimuli. As found in the hippocampus

of saline-treated rats, kinin B1 receptor mRNA increases

after pilocarpine injection, indicating that the SE was

necessary for such induction. On the other hand, kinin B1

mRNA level was not altered during the chronic period of the

epilepsy model indicating that the occurrence of spontane-

ous and short-lasting isolated seizures is not able to induce

de novo synthesis of this receptor. However, the correspon-

dent protein level remained increased during the chronic

period, showing that kinin B1 receptor synthesis was main-

tained constant and its presence during spontaneous seizures

has remained important.

One interesting aspect observed during the silent ‘‘sei-

zure-free’’ period was the particularly significant increase in

the hippocampal kinin B1 receptor immunostaining. This

fact was clearly observed in CA1, CA3 and dentate gyrus,

where clusters of stained cells could be observed. The

hippocampal CA2 region, known to be more resistant to

cell death in TLE was less importantly stained than the

regions mentioned above. As reported by Bregola et al. [4],

the kinin B1 receptor agonist, Lys-des Arg9 BK, is able to

increase the glutamate release from hippocampal slices of

animals submitted to kainic acid or kindling models of

epilepsy. Accordingly to the authors, the increased levels

of this receptor observed in hippocampal areas, particularly

prone to status epilepticus-induced cell death, seems to

indicate that kinin B1 activation could increase the gluta-

mate release, thus facilitating the excitotoxicity and cell

death of hippocampal neurons. In addition, De Simoni et al.

[10] and Lerner-Natoli et al. [21] have reported that several

cytokines are released after long-lasting seizures such as

IL-1h, IL-6 and TNFa, supporting the hypothesis of an

inflammatory process in the hippocampus of epileptic rats.

Furthermore, the presence of these cytokines in the tissue

may indicate cell death due to necrosis and/or apoptosis.

In the rat hippocampus, kinin B1 receptor was found

surrounding the nuclear membrane, near Neu-N protein

localization either in saline-treated rats or in the epileptic

animals. However, in the cerebral cortex of animals sub-

mitted to 6 h SE, we found the immunostaining labeling the

nucleus as well as the plasma membrane of pyramidal

neurons, showing that kinin B1 receptor can be found in

different compartments of neurons, probable during differ-

ent phases of receptor action (Fig. 7, panel A).

The intracellular location of kinin B1 receptor could

mean internalization of this receptor due to desensitization

process. In opposition to this hypothesis, several compo-

nents of kallikrein-kinin system have been found into

neuronal cell [8,37], supporting the idea that these poly-

peptides may have a cytoplasmatic or nuclear function.

The kinin B2 mRNA level was also increased in the

hippocampus of Wistar rats during the acute, silent and

chronic periods. The corresponding expression of kinin B2

protein was also increased in the same tissue of the

experimental groups, when compared with saline-treated

animals. These data suggest that kinin B2 receptor, which

has been described as a constitutive protein has its synthesis

increased during the epileptic phenomena. The kinin B2

G.A. Arganaraz et al. / Brain Research 1006 (2004) 114–125124

receptor staining was observed in the cell nucleus of CA1,

CA3 and dentate gyrus cells, mainly during 6 h of SE, when

this reactivity was very intense and punctual. The silent and

chronic rats presented a nuclear immunoreactivity in pyra-

midal and granular cells but the cytoplasmatic region was

also labeled in these neurons.

The positive effect of BDNF and NGF on the expression

of kinin B2 receptor in dorsal ganglia neurons has been

reported by Lee et al. [19]. Together with the occurrence of

increased levels of trophic factors in experimental models of

epilepsy [29], these facts cannot be directly correlated but

they seem to indicate that kinin B2 receptor expression is

increased during the epileptogenic process via the release of

trophic factors.

In spite of data reported by Ongali et al. [34], where they

show a significant decline of kinin B2 receptor binding sites

in the brain of rats submitted to kindling, we report an

increased synthesis of kinin B2 receptor. This difference

may occur due to the proper physiopathology of each

epilepsy model. In addition, we do not know if kinin B2

receptor, also found near nuclear membrane is in its proper

activated state. As reported by Wolsing and Rosenbaum

[48], BK induces a rapid desensitization of its own receptor

and this mechanism would be linked to BK degradation in

the cell cytoplasm. In contrast, BK also induces an enhance-

ment of its promoter by a positive feedback [36], justifying

the increased level of this receptor during the acute, silent

and chronic period of this epilepsy model.

Taken together, these data describe for the first time

changes in kinin B1 and B2 receptors mRNA synthesis as

well as their hippocampal distribution during the acute,

silent and chronic periods of the pilocarpine model of

epilepsy, supporting the hypothesis that both kinin receptors

are related to temporal lobe epilepsy. Accordingly to this,

the use of kinin B1 and B2 receptors knockout animals in

experimental epilepsy could lead to a better understanding

of the role of kinin receptors in the mechanisms underlying

epileptogenesis.

Acknowledgements

Supported by FAPESP, CNPg, FADA, PRONEX.

References

[1] C.E. Austin, A. Faussner, H.E. Robinson, S. Chakravarty, D.J. Kyles,

J.M. Bathon, D. Proud, Stable expression of human kinin B1 recep-

tor in Chinese hamster ovary cells, J. Biol. Chem. 272 (1997)

11420–11425.

[2] S.K. Bhattacharya, P.J.R. Mohan Rao, S.J. Brumleve, S.S. Parmar,

Role of putative neurotransmitters in bradykinin-induced catalepsy in

the rat, Pharmacol. Res. 3 (1986) 162–166.

[3] K.D. Bhoola, C.D. Figueroa, K. Worthy, Bioregulation of kinin: kal-

likreins, kininogens and kininases, Pharmacol. Rev. 44 (1992) 1–80.

[4] G. Bregola, K. Varanl, S. Gessi, L. Beani, C. Bianchi, P.A. Borea, D.

Bregoli, M. Simonato, Changes in hippocampal and cortical B1 bra-

dykinin receptor biological activity in two experimental models of

epilepsy, Neuroscience 92 (1999) 1043–1049.

[5] J.B. Calixto, D.A. Cabrini, J. Ferreira, M.M. Campos, Kinins in pain

and inflammation, Pain 87 (2000) 1–5.

[6] E.A. Cavalheiro, J.P. Leite, Z.A. Bortolotto, W.A. Turki, C.

Ikonomidou, L. Turki, Long-term effects of pilocarpine in rats: struc-

tural damage of brain triggers kindling and spontaneous recurrent

seizures, Epilepsia 32 (1991) 778–782.

[7] E.A. Cavalheiro, M.J. Fernandes, L. Turski, M.G. Naffah-Mazza-

coratti, Spontaneous recurrent seizure in rats: amino acids and

monoamines determination in the hippocampus, Epilepsia 35

(1994) 1–11.

[8] M.E. Cerf, D.M. Raidoo, Immunolocalization of plasma kallikrein in

human brain, Metab. Brain Dis. 15 (2000) 315–323.

[9] E.Y. Chen, D.F. Emerich, R.T. Bartus, J.H. Kordower, B2 bradykinin

receptor immunoreactivity in rat brain, J. Comp. Neurol. 427 (2000)

1–18.

[10] M.G. De Simoni, C. Perego, T. Ravizza, D. Moneta, M. Conte, F.

Marchesi, A. Luigi, S. Garattini, A. Vezzani, Inflammatory cytokines

and related genes are induced in the rat hippocampus by limbic status

epilepticus, Eur. J. Neurosci. 12 (2000) 2623–2633.

[11] S.G. Farmer, R.M. Burch, Biochemical and molecular pharmacol-

ogy of kinin receptors, Annu. Rev. Pharmacol. Toxicol. 32 (1992)

511–536.

[12] I. Fleming, R. Busse, Tyrosine phosphorylation and bradykinin-in-

duced signaling in endothelial cells, Am. J. Cardiol. 80 (1997)

102A–109A.

[13] U. Forternn, A. Seregi, G. Hertting, Anticonvulsant effects of endog-

enous prostaglandins formed in the brain of spontaneously convulsing

gerbils, Prostaglandins 27 (1984) 913–923.

[14] M.G. Funke, D. Amado, E.A. Cavalheiro, M.G. Naffah-Mazzacoratti,

Tyrosine phosphorylation is increased in the rat hippocampus during

the status epilepticus induced by pilocarpine, Brain Res. Bull. 47

(1998) 87–93.

[15] Y.C.S. Garrido, E.R.G. Sanabria, M.G. Funke, E.A. Cavalheiro, M.G.

Naffah-Mazzacoratti, Mitogen activated protein kinase is increased in

the limbic structures of the rat brain during the early stages of status

epilepticus, Brain Res. Bull. 47 (1998) 223–229.

[16] U. Heineman, S. Gabriel, R. Jauch, K. Schulze, A. Kivi, A. Eilers, R.

Kovacs, T.-N. Lehman, Alterations of glial cell function in temporal

lobe epilepsy, Epilepsia 41 (Suppl. 6) (2000) S185–S189.

[17] J.F. Hess, J.A. Borkowski, G.S. Joung, C.D. Strader, R.W. Ransom,

Cloning and pharmacological characterization of a human bradyki-

nin (BK2) receptor, Biochem. Biophys. Res. Commun. 184 (1992)

260–268.

[18] G.M. Klan, I. Smolders, H. Lindekens, J. Manil, G. Ebinger, Y.

Michotte, Effects of diazepam on extracellular brain neurotransmitter

in pilocarpine-induced seizures in rats, Eur. J. Pharmacol. 373 (1999)

153–161.

[19] Y.J. Lee, O. Zachrisson, D.A. Tonge, P.A. Mcnaughton, Upregulation

of bradykinin B2 receptor expression by neurotrophic factors and

nerve injury in mouse sensory neurons. Molecular and cellular, Neu-

roscience 19 (2002) 186–200.

[20] J.P. Leite, Z.A. Bortolotto, E.A. Cavalheiro, Spontaneous recurrent

seizures in rats: an experimental model of partial epilepsy, Neurosci.

Biobehav. Rev. 14 (1990) 511–517.

[21] M. Lerner-Natoli, P. Montpied, M.C. Rousset, J. Bockaert, G.

Rondouin, Sequential expression of surface antigens and transcription

factor by hippocampal cells in excitotoxicity and experimental epilep-

sy, Epilepsy Res. 41 (2000) 141–154.

[22] J.K. Liao, C.J. Homcy, The G protein of the Gai and Gaq family

couple the Bradykinin receptor to release of endothelium-derived

relaxing factor, J. Clin. Invest. 92 (1993) 2168–2172.

[23] K.J. Livak, T.D. Schmittgen, Analysis of relative gene expression data

using real-time quantitative PCR and the 2(-Delta Delta C(T)) Meth-

od, Methods 25 (4) (2001) 402–408.

G.A. Arganaraz et al. / Brain Research 1006 (2004) 114–125 125

[24] F. Marceau, Kinin B1 receptors: a review, Immunopharmacology 30

(1995) 1–26.

[25] G.S. Mathern, T.L. Babb, J.P. Leite, J.K. Pretorius, K.M. Yeoman,

P.A. Kuhlman, The pathogenic and progressive features of chronic

human hippocampal epilepsy, Epilepsy Res. 26 (1996) 151–161.

[26] A.E. McEachern, E.R. Sheltol, S. Bhakta, R. Opernolt, C. Bach, P.

Zuppan, J. Fujisaki, R.W. Aldrich, K. Jarnagin, Expression cloning

of a rat bradykinin receptor, Proc. Natl. Acad. Sci. U. S. A. 88

(1991) 7724–7728.

[27] L.E.A.M. Mello, E.A. Cavalheiro, A.M. Tan, W.R. Kupfer, J.K.

Pretorius, T.L. Babb, D.M. Finch, Circuit mechanism of seizures in the

pilocarpine model of chronic epilepsy: cell loss and mossy fiber

sprouting, Epilepsia 34 (1993) 985–995.

[28] J.G. Menke, J.A. Borkowscki, K.K. Bierelo, T. Macneil, A.W.

Derreck, K.A. Schneck, R.W. Ransom, C.D. Strader, D.L. Linemeyer,

J.F. Hess, Expression cloning of a human B1 bradykinin receptor, J.

Biol. Chem. 269 (1994) 21583–21586.

[29] G. Mudo, X.H. Jiang, T. Timmusk, M. Bindoni, N. Beluardo,

Change in neutrophins and their receptor mRNA in the rat fore-

brain after status epilepticus induced by pilocarpine, Epilepsia 37

(1996) 198–207.

[30] M.G. Naffah-Mazzacoratti, M.I. Bellıssimo, E.A. Cavalheiro, Profile

of prostaglandin levels in the rat hippocampus in pilocarpine model of

epilepsy, Neurochem. Int. 27 (1995) 461–466.

[31] H. Naraba, A. Ueno, M. Yoshimura, Y. Kosugi, S. Oh-ishi, Exami-

nation of signal transduction pathway of stimulated B1 and B2 kinin

receptors. MAP kinase pathway to AP-1 translocation in HEK 293

cells, Immunopharmacology 45 (1999) 35–38.

[32] H. Neppl, H. Neuhof, F. Afflerbach, P.L.J. Neppl, A. Berghofer,

Bradykinin-induced edema formation proceeds from B2 receptor

stimulation and is potentiated by concomitantly released prostaglan-

dins, 141 (1991) 141–147.

[33] A. Ni, L. Chao, J. Chao, Transcription factor nuclear factor nB reg-

ulates the inducible expression of the human B1 receptor gene in

inflammation, J. Biol. Chem. 273 (1998) 2784–2791.

[34] B. Ongali, M.M. Campos, G. Bregola, D. Rodi, D. Regoli, G. Thibault,

M. Simonato, R. Coutere, Autoradiographic analysis of rat brain kinin

B1 and B2 receptors: normal distribution and alteration induced by

epilepsy, J. Comp. Neurol. 461 (2003) 506–519.

[35] J.M. Parent, D.H. Lowenstein, Mossy fiber reorganization in the ep-

ileptic hippocampus, Curr. Opin. Neurol. 10 (1997) 103–109.

[36] J.B. Pesquero, C.J. Lindsey, A.C.M. Paiva, D. Ganten, M. Bader,

Transcriptional regulatory elements in the rat bradykinin B2 receptor

gene, Immunopharmacology 33 (1996) 36–41.

[37] D.M. Raidoo, N. Ramchurren, Y. Naidoo, S. Naidoo, W. Miller-

Esterl, K.D. Bhoola, Visualization of bradykinin B2 receptors on

human brain neurons, Immunopharmacology 33 (1996) 104–107.

[38] D.M. Regoli, J. Barabe, Pharmacology of bradykinin and related

kinin, Pharmacol. Rev. 32 (1980) 1–46.

[39] A. Represa, J. Niquet, H. Pollard, Y. Bem-Ari, Cell death, gliosis and

synaptic remodeling in the hippocampus of the epileptic rats, J. Neu-

robiol. 26 (1995) 413–425.

[40] J.P. Schanstra, E. Bataille, M.E.M. Castano, Y. Barascud, C. Hirtz,

J.B. Pesquero, C. Pecher, F. Gauthier, J.P. Girolami, J.L. Bascands,

The B1-agonist [des-Arg10]-kallidin activates transcription factor NF-

nB and induces homologous upregulation of the Bradykinin B1-re-

ceptor in cultured human lung fibroblasts, J. Clin. Invest. 101 (1998)

2080–2091.

[41] S.R. Shi, M.E. Key, K.L. Kalra, Antigen retroviral in formalin-fixed,

paraffin-embedded tissues: an enhancement method for immunohis-

tochemical staining based on microwave oven heating of tissue sec-

tions, J. Histochem. Cytochem. 39 (1991) 741–748.

[42] B. Shi, G. Bhat, V.B. Mahesh, M. Brotto, T.M. Nosek, D.W. Brann,

Bradykinin receptor localization and cell signaling pathways used by

bradykinin and the regulation of gonadotropin-releasing hormone se-

cretion, Endocrinology 140 (1999) 4669–4676.

[43] J.-A. Silva Jr., R.C. Araujo, O. Baltatu, S.M. Oliveira, C. Tschope,

E. Fink, S. Hoffmann, R. Plehm, K.X. Chai, L. Chao, J. Chao, D.

Ganten, J.B. Pesquero, M. Bader, Reduced cardiac hypertrophy

and altered blood pressure control in transgenic rats with the hu-

man tissue kallikrein gene, FASEB J. 14 (2000) 1858–1860.

[44] I. Smolders, K. Van Belle, G. Ebinger, Y. Michotte, Hippocampal and

cerebellar extracellular amino acids during pilocarpine-induced seiz-

ures in freely moving rats, Eur. J. Pharmacol. 319 (1997) 21–29.

[45] D. Tauck, J.V. Nadler, Evidence of function mossy fiber sprouting in

hippocampal formation of kainic acid-treated rats, J. Neurosci. 5

(1985) 1016–1022.

[46] S. Tippmer, U. Quitterer, V. Kolm, A. Faussner, A. Rosher, L. Mosthaf,

W. Miller-Esterl, H. Hering, Bradykinin induces translocation of the

protein kinase C isoforms a, h and m, Eur. J. Biochem. 225 (1994)

297–304.

[47] W.A. Turski, E.A. Cavalheiro, M. Schwarz, S.J. Czuczwar, Z.

Kleinrok, L. Turski, Limbic seizures produced by pilocarpine rats:

behavioural, electroencephalographic and neuropatological study,

Behav. Brain Res. 9 (1983) 315–335.

[48] D.H. Wolsing, J.S. Rosenbaum, Bradykinin-stimulated inositol phos-

phate production in NG108-15 cells is mediated by a small population

of binding sites which rapidly desensitize, J. Pharmacol. Exp. Ther.

257 (1991) 621–633.