Characterization of proteinase-activated receptor 2 signalling and expression in rat hippocampal neurons and astrocytes

Neuropharmacology 50 (2006) 714e725www.elsevier.com/locate/neuropharm

Characterization of proteinase-activated receptor 2 signalling andexpression in rat hippocampal neurons and astrocytes

Trevor J. Bushell a,*, Robin Plevin a, Stuart Cobb b, Andrew J. Irving c

a Department of Pharmacology and Physiology, Strathclyde Institute for Biomedical Sciences, University of Strathclyde,27 Taylor Street, Glasgow G4 0NR, UK

b Neuroscience and Biomedical Systems, Institute of Biomedical and Life Sciences, University of Glasgow, Glasgow G12 8QQ, UKc Division of Pathology and Neuroscience, Ninewells Hospital and Medical School, University of Dundee, Dundee DD1 9SY, UK

Received 31 May 2005; received in revised form 31 October 2005; accepted 29 November 2005

Abstract

Proteinase-activated receptors (PARs1e4) have recently been identified as the molecular entity underlying the cellular effects of serine pro-teinases. In the present study we have investigated PAR2 signalling, expression and desensitization using cultured and acute slice preparations.Trypsin, SLIGRL and 2f-LIGKV-OH, agonists for PAR2, induced a transient increase in intracellular Ca2þ levels in both neurons and astrocytes,via activation of the phospholipase C/IP3 pathway. Furthermore, a single application of trypsin, but not SLIGRL nor 2f-LIGKV-OH, leads toprolonged desensitization of PAR2 responses. PAR2 immunoreactivity was observed in neurons (glutamatergic and GABAergic) and astrocyteswithin cultures and acute slices, with prominent labelling in neuronal somata and proximal dendrites. Functionally, cultured neurons which ex-hibited the highest levels of PAR2 labelling, also exhibited the largest Ca2þ signals upon PAR2 activation. Given the importance of Ca2þ signal-ling in hippocampal synaptic plasticity and neurodegeneration, PAR2 may play a key modulatory role in these processes.� 2005 Elsevier Ltd. All rights reserved.

Keywords: Hippocampus; Proteinase-activated receptor; Calcium; Signalling; Expression

1. Introduction

Proteinase-activated receptors (PARs) belong to theG-protein-coupled receptor family (Macfarlane et al., 2001)and are thought to mediate the cellular effects of thrombinand trypsin. Four receptors (PAR1e4) have been cloned thusfar (Vu et al., 1991; Nystedt et al., 1994; Ishihara et al.,1997; Kahn et al., 1998; Xu et al., 1998), which are uniquein their activation, as ligand-binding in the classical sense isnot required, rather the cleavage of the N-terminus by a pro-tease reveals a ‘‘tethered-ligand’’, which then binds and acti-vates the receptor (for reviews see Coughlin, 2000;Macfarlane et al., 2001). Selective peptide agonists have

* Corresponding author. Tel.: þ44 141 548 2856; fax: þ44 141 552 2562.

E-mail address: [email protected] (T.J. Bushell).

0028-3908/$ - see front matter � 2005 Elsevier Ltd. All rights reserved.

doi:10.1016/j.neuropharm.2005.11.024

been developed for PAR1, 2 and 4, which are analogous tothe peptide sequence of the tethered-ligand revealed uponproteolytic cleavage of the N-terminus (Scarborough et al.,1992; Hollenberg et al., 1997; Kawabata et al., 1999).

Recent studies have identified important roles for PARs inthe CNS, not only in disease states such as neurogenic inflam-mation, hyperalgesia, neurodegeneration, but also in nocicep-tion (Smith-Swintosky et al., 1997; Hoogerwerf et al., 2001;Vergnolle et al., 2001; Coelho et al., 2002; Gao et al., 2002;Junge et al., 2003). To date, few studies have investigatedthe function of PARs on central neurons despite studies reveal-ing the presence of PAR1e4 in both neurons and glia of theCNS (Weinstein et al., 1995; D’Andrea et al., 1998; Niclouet al., 1998; Striggow et al., 2001; Riek-Burchardt et al.,2002; Wang et al., 2002; Sorensen et al., 2003). PAR1 hasbeen shown to potentiate NMDA responses in the hippocam-pus (Gingrich et al., 2000) and may play a vital role in

715T.J. Bushell et al. / Neuropharmacology 50 (2006) 714e725

neuropathological damage (Junge et al., 2003). Chronic activa-tion of PAR2 induces neuronal cell death in hippocampal cul-tures and is associated with increased intracellular Ca2þ levels([Ca2þ]i) (Smith-Swintosky et al., 1997). Upregulation ofPAR1, PAR2 and their potential activators, thrombin andtrypsin, have also been shown in disease states such asAlzheimer’s disease (Choi et al., 1995), HIV encephalitis(Boven et al., 2003), HIV dementia (Noorbakhsh et al.,2005) and following acute focal ischaemia (Jin et al., 2005).PAR4 activation can also drive tau hyperphosphorylation andaggregation, which is implicated in Alzheimer’s disease (Suoet al., 2003). Taken together these studies suggest that PARsplay an important role in neuromodulation and neurotoxicityin the CNS.

In the present study, we have investigated PAR2 signalling,expression and desensitization in cultured hippocampal andacute slice preparations, using Ca2þ-imaging and immunohis-tochemical techniques. We show that activation of PAR2 incultured cells leads to the release of Ca2þ from intracellularstores through the phospholipase C/IP3 signalling pathway.Desensitization of PAR2 following activation by trypsin, butnot selective peptide agonists, inhibits further activation ofthe receptor. Furthermore, we show that PAR2 immunoreactiv-ity is associated with a range of cells, including glutamatergicand GABAergic neurons and astrocytes. We conclude that themarked effects of PAR2 activation on Ca2þ signalling may in-fluence both synaptic plasticity and neurotoxicity in thehippocampus.

2. Methods

2.1. Cell culture

Primary rat hippocampal cultures were prepared as described previously

(Shanley et al., 2002). Briefly, rat pups (1e3 days old) were killed by cervical

dislocation and decapitation, following UK Home Office guidelines, and the

brain removed. The hippocampi were then dissected out, chopped and treated

with enzymes (protease type X and XIV, 0.5 mg/ml for 40e50 min). The hip-

pocampi were then washed, dissociated by trituration and plated onto dishes or

coverslips previously coated with poly-D-lysine (0.1 mg/ml). Cultures were in-

cubated in serum replacement media (SR2, Sigma) in a humidified atmosphere

of 5% CO2 at 37 �C. Cells were used experimentally from 3 to 20 days in vitro

(DIV).

2.2. Ca2þ-imaging

All imaging experiments were performed on a digital epifluorescence im-

aging system (Universal Imaging, Downingtown, PA) mounted on a Zeiss Ax-

ioskop FS microscope using a 40� objective. Prior to experiments, mixed

cultures (containing both neurons and astrocytes) were loaded with the

Ca2þ-sensitve dye, fura-2 AM (6 mM, 40e60 min, room temperature). Ratio-

metric images (350/380 nm) were obtained from the somata of neurons and

astrocytes every 2e5 s. Experiments were performed on cultures continually

perfused (1.5 ml/min) with HEPES-buffered saline (HBS) consisting of (in

mM): NaCl 130, HEPES 10, KCl 5.4, CaCl2 1.0, MgCl2 1.0, D-glucose 25,

pH 7.4, room temperature, unless otherwise stated. Tetrodotoxin (0.5 mM)

was included in the perfusate in order to block synaptically driven Ca2þ tran-

sients. All compounds investigated in this study were added via the perfusate.

Cells were identified as either neurons or astrocytes based on their morpholog-

ical characteristics and their response to depolarization with high extracellular

potassium (25 mM), with neurons included in the analysis only if responses to

PAR agonists were clearly uncontaminated by astrocytic responses. Experi-

ments were performed on cells taken from at least two separate cultures ob-

tained from different rats with data being calculated as changes in

fluorescence ratio (ratio units).

2.3. Immunohistochemistry

Primary hippocampal cultures were fixed in ice-cold 4% paraformaldehyde

for 20 min, except for the GAP43 staining where neurons were fixed in meth-

anol at �20 �C. The cultures were then washed with HBS and permeabilized

with 0.01% Triton X-100 for 10e20 min. Primary antibodies for PAR2 (B5,

Al-Ani et al., 1995; Seatter et al., 2004) were then applied to the cultures

for 1 h (room temperature). The cultures were washed and a fluorescent sec-

ondary antibody (Alexa 488 conjugated donkey anti-rabbit) applied for

30 min (room temperature). For dual labelling studies, monoclonal anti-

MAP2 (somato-dendritic marker), anti-GAP43 (axonal marker) and anti-

GAD67 (GABAergic cell marker) antibodies were utilized, with donkey

anti-mouse Cy3 used for the fluorescent secondary antibody. Where necessary

(anti-GAP43), non-specific labelling was blocked by prior incubation with me-

dium containing 10% milk powder (15e20 min). Acute hippocampal slices for

immunohistochemical studies were obtained from rats transcardially perfused

with 4% paraformaldehyde following anaesthesia. Following overnight post

fixation, 60 mM hippocampal sections were cut using a vibratome, washed

with phosphate buffered saline (PBS) and incubated in a 50% ethanol solution

for 30 min. The sections were washed further in 0.3 M PBS and incubated

overnight in antibodies against PAR2 and parvalbumin diluted in 0.3 M PBS

and 0.3% Triton X-100. Sections were incubated in the relevant secondary an-

tibody (Alexa 488 or Cy3) for 2 h at room temperature. Sections were then

washed and mounted on glass coverslips using vectashield mountant for imag-

ing. A laser scanning confocal imaging system (Zeiss LSM 510) was used for

image acquisition. Laser lines of 488 and 543 nm were used to excite Alexa

488 and Cy3, respectively. Dual labelling images were obtained in multi-

tracking mode using a 15 s scan speed. In studies where the neuronal identity

was established following Ca2þ imaging, cells were fixed and permeabilized in

situ, then treated with anti-GAD67 for 60 min followed by treatment with anti-

mouse Cy3 for 30 min. The objective was carefully backed off during this

procedure, but the relative position of the cells on the stage was maintained.

The field of cells was then brought back into focus and a Cy3 digital epi-

fluorescence image captured.

2.4. Materials

All compounds were made as 1000� stock solutions and diluted accord-

ingly in the perfusate. U73122, 2-APB and CPA and antibodies against

MAP2 and GAP-43 were obtained from Sigma-Aldrich (UK). 2-APB and

CPA stock solutions were made in dimethyl sulfoxide (DMSO), with the final

bath concentration of DMSO being a maximum of 0.1%. This concentration of

DMSO did not alter Ca2þ levels when applied alone or prior to, and in the

presence of, PAR agonists. Pertussis toxin was obtained from Calbiochem,

UK. Antibodies for GFAP and GAD67 were obtained from Chemicon (UK).

Alexa488 and Cy3 secondary antibodies were obtained from Molecular Probes

(Cambridge Bioscience, UK) and Jackson Laboratories (Stratech Scientific,

UK) respectively. The PAR2 antibody was a gift from M. Hollenberg, Univer-

sity of Calgary. Kowa Ltd, Japan, donated PAR2 activating peptides, which

were all non carboxy-amidated, with solutions prepared fresh each day.

All data are expressed as a mean � SEM unless otherwise indicated, where

n ¼ the number of neurons or astrocytes investigated. Statistical significance

was determined using paired or unpaired Student’s t tests where appropriate,

with P < 0.05 taken as significant.

3. Results

3.1. Cellular localization of PAR2 in the hippocampus

PAR2 expression in the hippocampal cultures and slices wasstudied using a previously characterized antibody selective

716 T.J. Bushell et al. / Neuropharmacology 50 (2006) 714e725

for PAR2 (Al-Ani et al., 1995; Seatter et al., 2004). In cul-tured neurons, strong PAR2 immunolabelling was presenton proximal processes and soma, where it was clearly asso-ciated with both plasma membrane and intracellular sites(Fig. 1A and B). To study the subcellular localization of neu-ronal PAR2, dual labelling experiments were performed

using antibodies against MAP-2 (somato-dendritic marker)and GAP-43 (axonal marker). Co-localization of PAR2 andMAP-2 was observed (Fig. 1C), whereas GAP-43 positiveneurons lacked PAR2 immunoreactivity (data not illustrated).Finally, PAR2 labelling was observed in astrocytes. Co-stainingof PAR2 with the glial marker, GFAP, revealed a more diffuse,

Fig. 1. Cellular localization of PAR2 in the hippocampus. (A) z-projection of a series of confocal images taken every 0.89 mm, illustrating the distribution of PAR2

immunoreactivity throughout the cell. Note PAR2 (green) is localized in neuronal soma (S) and processes (P) as well as more diffuse staining in astrocytes (G). (B)

Single, zoomed, confocal section from (A) illustrating PAR2 labelling associated with the plasma membrane (arrows). (C) Dual labelling of PAR2 (green) and

MAP-2 (red) reveals a somato-dendritic distribution of the receptor. (D) Dual labelling of PAR2 (green) with GFAP (red) reveals a perinuclear localization of

PAR2 in astrocytes. (E)e(G) Single confocal sections of MAP-2 (red; F) and PAR2 (green; E) labelling in hippocampal slices with the merged image illustrated

in (G). (H)e(J) Single confocal sections illustrating parvalbumin (green; I; arrows) and PAR2 (red; H) labelling in hippocampal slices with the merged image (J)

revealing cells expressing both markers (arrows). Images (A)e(D) are taken from 13e20 DIV hippocampal cultures and (E)e(J) from hippocampal slices prepared

from13 to 15 day old rat pups, with areas of co-localization appearing yellow. Scale bars, 20 mm in (A)e(D) and 100 mm in (E)e(J).

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vesicular PAR2 staining, which was concentrated at perinuclearregions (Fig. 1D). A similar expression pattern for PAR2 wasevident when examined in fixed, acute hippocampal sections.In agreement with immunolabelling in cultured neurons,PAR2 labelling reveals a somato-dendritic localization asobserved in excitatory pyramidal neurons (Fig. 1EeG) andparvalbumin-positive interneurons in area CA1 (Fig. 1HeJ).Furthermore, PAR2 co-localized with GFAP, indicating thepresence of PAR2 in glia of the hippocampal slice (data notillustrated).

3.2. Pharmacology of PARs expressed on hippocampalneurons and astrocytes

Application of trypsin (80 nM), a proposed endogenous ag-onist for PAR2 and PAR4, resulted in an increase in intracel-lular Ca2þ levels ([Ca2þ]i) in the majority of neurons andastrocytes (13e20 DIV; Fig. 2C and D). In 88 � 7% of neu-rons, trypsin exposure was associated with a transient increasein [Ca2þ]i (mean, peak response ¼ 0.35 � 0.05 ratio units;n ¼ 50). These responses were comparable in magnitudewith those mediated by established Gq/11-coupled receptors(group I mGlu; not illustrated). [Ca2þ]i responses were also

observed in astrocytes (0.39 � 0.03 ratio units; n ¼ 18;92 � 6% responding; P > 0.05 compared to trypsin in neu-rons). To determine whether these increases were due to theactivation of either PAR2 or PAR4, or both in combination, se-lective peptide agonists for these receptors were investigated.SLIGRL (80 mM), a peptide agonist for PAR2, evoked repro-ducible [Ca2þ]i responses in both neurons and astrocytes(13e20 DIV), following a minimum of 10 min recovery peri-od between applications (Fig. 2). In 87 � 5% of neurons,SLIGRL exposure was associated with an increase in[Ca2þ]i (0.15 � 0.02 ratio units; n ¼ 49; P < 0.001 comparedto trypsin in neurons; Fig. 2C). Significantly larger [Ca2þ]i

responses were observed in astrocytes (0.26 � 0.02 ratio units;n ¼ 18; 93 � 4% responding; P < 0.001 compared to SLIGRLin neurons; Fig. 2D). Concentrationeresponse curves forSLIGRL revealed EC50 values of 14 � 2 mM and 10 � 2 mMin neurons and astrocytes respectively (n ¼ 7 neurons orastrocytes for each concentration tested; Fig. 2A and B). Toconfirm the presence of PAR2, the selective peptide agonist,2-furoyl-LIGKV-OH (2f-LIGKV-OH; 80 mM; Ferrell et al.,2003), also produced a robust increase in [Ca2þ]i in neurons(0.23 � 0.03 ratio units; n ¼ 23; P < 0.01 compared to trypsinin neurons; Fig. 2C) and astrocytes (0.38 � 0.03; n ¼ 14;

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Fig. 2. Characterization of PARs present in hippocampal cultures. (A,B) Concentration response curves for SLIGRL in neurons and astrocytes respectively. (C) The

PAR2 agonists, SLIGRL (80 mM), 2f-LIGKV-OH (80 mM) and trypsin (80 nM), as well the PAR1 selective agonist, TFLLR (80 mM), elicit increases in neuronal

[Ca2þ]i. The PAR4 selective peptide, GYPGQV (80 mM) was ineffective. (D) A similar pharmacological profile was observed in astrocytes, although a larger in-

crease in [Ca2þ]i was observed. Compounds were applied for the times indicated by the bars above the graph.

718 T.J. Bushell et al. / Neuropharmacology 50 (2006) 714e725

P < 0.01 compared to 2f-LIGKV-OH in neurons; Fig. 2D).These robust increases in [Ca2þ]i following PAR2 agonist ap-plication were also observed in the presence of the glutamatereceptor antagonists, NBQX (20 mM) and DL-AP5 (100 mM;data not shown), suggesting that the elevated [Ca2þ]i was in-deed due to direct activation of PAR2 on neurons rather thandue to glutamate release from the activation of astrocyticPAR2. In contrast to activation of PAR2, GYPGQV (80 mMto 200 mM), a peptide agonist for PAR4, did not increase[Ca2þ]i in any neurons tested (n ¼ 45). Previous studieshave revealed the presence of PAR1 in the hippocampuswhen investigated in the acute slice preparation (Gingrichet al., 2000). In the present study, application of the selectivepeptide agonist, TFLLR (80 mM), produced robust increases in[Ca2þ]i in 98 � 1% of neurons (0.35 � 0.03 ratio units;n ¼ 26; P > 0.05 compared to trypsin in neurons; Fig. 2C)and 92 � 6% of astrocytes (0.55 � 0.07 ratio units; n ¼ 12;P < 0.001 compared to TFLLR in neurons; Fig. 2D).

To determine whether there was a differential expression ofPAR2 depending on days in vitro, SLIGRL responses werealso studied in young hippocampal cultures (3e5 DIV). Ro-bust increases in [Ca2þ]i were also observed in the youngerhippocampal cultures (0.18 � 0.02 ratio units; n ¼ 16;Fig. 3B) although significantly fewer neurons respondedthan with 13e20 DIV (27 � 5% and 87 � 5% respectively,P < 0.01; for example see Fig. 3A). In contrast, astrocytesresponded to SLIGRL equally in both younger (0.37 � 0.02ratio units; n ¼ 12; 91 � 5% of all astrocytes responded;Fig. 3C) and older cultures (0.26 � 0.02 ratio units; n ¼ 18;93 � 4% of astrocytes responded; Fig. 3C). Similarly, markedresponses to the PAR1 agonist, TFLLR were seen in themajority of neurons (0.36 � 0.02 ratio units; n ¼ 41) andastrocytes (0.52 � 0.04 ratio units; n ¼ 24) in all ages investi-gated (Figs. 2 and 3).

3.3. Signalling pathways underlying PAR2-mediatedincreases in [Ca2þ]i

Given that PAR2 elevates [Ca2þ]i levels, we characterizedthe intracellular signalling mechanism underlying this effectin 13e20 DIV cultures. Initially, the source of the [Ca2þ]i sig-nal associated with PAR2 activation was investigated. To testwhether this reflected influx of extracellular Ca2þ or releasefrom internal stores, we used Ca2þ-free external solution con-taining 100 mM EGTA. Following a 10 min exposure of cul-tures to this Ca2þ-free medium, application of either trypsinor SLIGRL resulted in a transient increase in neuronal[Ca2þ]i (0.12 � 0.02 ratio units; n ¼ 15; Fig. 4B), whichwas only modestly reduced relative to control responses ob-tained in normal Ca2þ-containing medium (0.15 � 0.02 ratiounits; n ¼ 49; 20 � 5% reduction, P < 0.05, Fig. 4E). In-creases in astrocytic [Ca2þ]i following PAR2 activation werecomparable in the presence and absence of external Ca2þ

(control, 0.26 � 0.02, n ¼ 18; Ca2þ-free, 0.24 � 0.03,n ¼ 15; P > 0.05). To explore further the role of internalCa2þ stores, we used the endoplasmic reticulum Ca2þ-ATPaseinhibitor cyclopiazonic acid (CPA, 30 mM) to deplete

intracellular Ca2þ stores (Beck et al., 2004). Treatment of cellswith CPA for 10 min evoked an initial peak increase in the lev-els of [Ca2þ]i, which partially recovered to a level above base-line values for the duration of CPA exposure, suggestingactivation of a store-operated Ca2þ channels (SOCCs). How-ever, responses to trypsin or SLIGRL in the continued pres-ence of CPA were largely abolished, even though steadystate [Ca2þ]i levels in the continued presence of CPA were be-low those of peak control responses to PAR2 agonists. It isnoteworthy that modest increases in Ca2þ influx, comparablein magnitude to the Ca2þ elevations observed with CPA, canenhance GPCR-linked Ca2þ release in hippocampal neurons(Rae et al., 2000). Overall, CPA inhibited PAR2 responses in

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Fig. 3. Neuronal responses to PAR2 agonists increase with the age of the cul-

ture. (A) Examples of SLIGRL (80 mM) and trypsin (80 nM) failing to elicit an

increase in [Ca2þ]i in 3e5 DIV cultured neurons. In contrast, the PAR1 ago-

nist, TFLLR (80 mM), elicits an increase in [Ca2þ]i in the majority of neurons

tested. (B) In neurons where SLIGRL (80 mM) and trypsin (80 nM) do elicit

increases in [Ca2þ]i, the increases were similar to those observed in cultures

13e20 DIV. (C) Summary revealing substantially fewer neurons respond to

SLIGRL (80 mM, -) in 3e5 DIV cultures than in 13e20 DIV. No differences

were observed with TFLLR (80 mM, ) in neurons at all ages tested. Both

SLIGRL (80 mM) and TFLLR (80 mM) elicited increases in [Ca2þ]i in astro-

cytes at all ages tested (**P < 0.01).

719T.J. Bushell et al. / Neuropharmacology 50 (2006) 714e725

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Fig. 4. PAR2 activation increases [Ca2þ]i through the PLC/IP3 pathway. (A) Multiple applications of SLIGRL (80 mM) elicit robust increases in [Ca2þ]i. (B)

SLIGRL (80 mM) responses are observed in the absence of external Ca2þ and are inhibited by the SERCA pump inhibitor, CPA. (C) The PLC inhibitor,

U73122, significantly inhibits the increases in [Ca2þ]i observed following SLIGRL (80 mM) application. (D) 2-APB, an IP3 receptor antagonist/SOCC blocker,

significantly inhibits SLIGRL (80 mM) induced increases in [Ca2þ]i. (E) SLIGRL increases in [Ca2þ]i were partially inhibited by removing external Ca2þ, whereas

inhibitors of the PLC/IP3 pathway, CPA, U73122 and 2-APB significantly inhibited SLIGRL-induced increases in [Ca2þ]i. (*P < 0.05, **P < 0.01).

both neurons and astrocytes by 97 � 4% (P < 0.01, Fig. 4Band E). The signalling pathway responsible for the elevationin [Ca2þ]i was further investigated using the phospholipaseC inhibitor, U73122 (Capogna et al., 2003), and the proposedIP3 receptor antagonist/ SOCC antagonist, 2-aminoethoxydi-phenylborate (2-APB; Braun et al., 2001). Application ofeither U73122 (1 mM, 15 min, Fig. 4C and E) or 2-APB(20 mM, 10 min; Fig. 4D and E) resulted in a marked inhibi-tion of [Ca2þ]i responses to SLIGRL in neurons (87 � 4%reduction, n ¼ 14, P < 0.01 and 91 � 5% reduction, n ¼ 17,P < 0.01, respectively). Similar effects were also observedon astrocytic responses (results not shown), indicating thatPAR2 is signalling via the classical PLC-IP3 pathway tomobilize Ca2þ from intracellular stores in these cells. Nextwe used pertussis toxin (PTX) to explore the involvement ofG-protein subunits in this effect. Application of SLIGRLfollowing treatment with PTX (100e200 ng/ml, 24 h), pro-duced a robust increase in [Ca2þ]i (0.14 � 0.02 ratio units;

n ¼ 26) indicating no involvement of Gi/Go but is consistentwith a pathway involving Gq/G11 G-protein subunits. In paral-lel studies using the same hippocampal cultures, PTX (100e200 ng/ml, 24 h), inhibited cannabinoid receptor reduction ofCa2þ oscillations induced by reducing external Mg2þ levels(data not shown).

3.4. Selective desensitization of PAR2 responsesfollowing trypsin application

As described earlier, the application of trypsin andSLIGRL, agonists for PAR2, results in a transient [Ca2þ]i re-sponse in both neurons and astrocytes. A differential activationand desensitization of PAR2 by tethered ligands and solublepeptide agonists has been suggested (Al-Ani et al., 2002).We investigated this possibility with regard to the PAR2s pres-ent in cultured hippocampal neurons. The differing activationof the receptor was not evident from the current experiments

720 T.J. Bushell et al. / Neuropharmacology 50 (2006) 714e725

as both trypsin (80 nM) and SLIGRL (80 mM) produced robustelevations in [Ca2þ]i, upon initial application. However, differ-ences in desensitization were apparent upon multiple applica-tions of the two agonists. Previous studies in expressionsystems have highlighted the need to use high concentrationsof agonists to study the desensitization of PARs (Kawabataet al., 1999). Hence we have used a concentration of SLIGRLthat is near maximal on its doseeresponse curve. Followingan initial application of SLIGRL (80 mM), further multipleapplications of SLIGRL (80 mM), 2f-LIGKV-OH (80 mM) ortrypsin (80 nM) produced subsequent elevations of [Ca2þ]i

(Figs. 2, 3 and 4). In contrast, after obtaining [Ca2þ]i

responses following an initial application of trypsin (80 nM),subsequent multiple applications of trypsin (80 nM) orSLIGRL (80 mM) failed to elevate [Ca2þ]i for up to 2 h follow-ing wash (Fig. 5A and B). Furthermore, no evidence of cross-desensitization between PAR1 and PAR2 was evident asmarked increases in [Ca2þ]i were observed on the applicationof the PAR1 peptide agonist, TFLLR, following desensitiza-tion of PAR2 by trypsin (Fig. 5B) or multiple applications ofPAR2 selective peptides (data not shown).

We further sought to determine whether the observed PAR2desensitization following trypsin application was due to inter-nalization of the receptor. No differences in the subcellularlocalization of immunolabelling for PAR2 was observedbetween control cultures and those treated with trypsin(0.25e2 h), indeed strong membrane-associated immunoreac-tivity was detected under both conditions and trypsin exposurewas not associated with an increase in vesicular PAR2 expres-sion (Fig. 5C). These data suggest that within this time frame,there is no clear translocation or internalization of PAR2following trypsin activation.

3.5. Enhanced PAR2 responses on interneuronsin culture

From our immunohistochemical studies it was apparent thatthe degree of PAR2 labelling varied between cultured neurons.In young cultures (3e5 DIV), a number of soma were devoidof any labelling whereas some neurons appeared to expressvery high levels of immunoreactivity. The levels of neuronalPAR2 expression generally increased with time in cultureand, in agreement with the Ca2þ imaging data, the majorityof older neurons (>13 DIV) were strongly immunopositivefor PAR2. Within these older cultures, however, there was stilla population of neurons with noticeably higher levels of PAR2immunolabelling. To establish whether the non-uniform distri-bution of PAR2 could be assigned to particular neuronal sub-type, dual labelling experiments were carried out usinga monoclonal antibody raised against GAD67 (GABAergicneurons). In the majority of neurons that were strongly immu-nopositive for PAR2, co-localization with GAD67 wasobserved (Fig. 6AeC). Thus, 14/16 cells that were GAD pos-itive demonstrated strong PAR2 immunolabelling; whereasonly 4/74 GAD negative cells had similar levels of PAR2expression (data from three cultures, 3e15 DIV). To deter-mine whether this increased PAR2 expression in GABAergic

neurons was also evident in the slice preparation, we per-formed co-labelling experiments with, parvalbumin (PV),a marker for a subset of hippocampal GABAergic neurons.Neuronal PAR2 expression was observed in both PV positiveand PV negative neurons, although labelling is similar inboth neuronal subtypes (Fig. 1EeJ). To determine whetherthe more intense immunolabelling observed in culturedGABAergic neurons correlated with a larger response toPAR2 activation, Ca2þ-imaging experiments were performed,followed by direct immunohistochemical characterization ofthe cells under investigation. Neurons, which were identifiedas GABAergic (GAD67 positive cells; Fig. 6D and E), elicitedmuch larger increases in [Ca2þ]i following PAR2 activation bytrypsin (80 nM) than putative glutamatergic neurons (GADpositive cells, 0.16 � 0.04 ratio units, n ¼ 4; GAD negativecells, 0.05 � 0.01, n ¼ 14, P < 0.01; Fig. 6F and G). Further-more, application of the group I mGlu agonist, (S )-DHPG(50 mM), elicited robust increases in [Ca2þ]i in cells that dem-onstrated small responses to SLIGRL. These data indicate thatthese reduced responses were not indicative of an inability ofthese neurons to respond to the activation of Gq-coupledreceptors.

4. Discussion

In this study we have demonstrated the pharmacology, sig-nalling pathways and characteristics of desensitization ofPAR2-mediated Ca2þ responses in neurons and astrocytes ofthe hippocampus. We have also demonstrated the cellularlocalization of PAR2 immunolabelling within these cell types.

4.1. Pharmacology of PARs present in hippocampalcultures

We have shown that activation of PAR1 and PAR2 inhippocampal cultures using the selective peptide agonists,TFLLR and SLIGRL respectively, leads to a marked increasein [Ca2þ]i in both neurons and astrocytes. These findingsare in agreement with previous reports demonstrating thefunctional expression of PAR1 and PAR2 in hippocampalneurons (Smith-Swintosky et al., 1997; Gingrich et al.,2000). Additionally, an increase in [Ca2þ]i following the appli-cation of the selective PAR2 peptide agonist, 2f-LIGKV-OH(Ferrell et al., 2003), further verified the presence of PAR2.Despite immunohistochemical evidence for the expression ofPAR4 in the hippocampus (Striggow et al., 2001) and a previousstudy reporting Ca2þ responses in astrocytes following PAR4agonist application (Wang et al., 2002), we found that applica-tion of the selective PAR4 agonist, GYPGQV, at concentrationswhich activate the receptor in expression systems and in vitrotissue preparations (Xu et al., 1998; Hollenberg et al., 1999),failed to increase [Ca2þ]i indicating that either the receptorwas not present or does not couple to Ca2þ signalling in thehippocampal cultures used in the current study. Due to lackof specific peptide agonists, the function of PAR3 in thesehippocampal cultures was not investigated.

721T.J. Bushell et al. / Neuropharmacology 50 (2006) 714e725

Time (min)0 20 40 60 80 200 225 250

trypsin

SLIGRL

TFLLR

trypsin

0.10ratio units

A

trypsin

SLIGRL

trypsin

Time (min)0 20 40 60 80

SLIGRL

B

0.10ratio units

C

control trypsin

Fig. 5. Desensitization of PAR2 by trypsin but not selective peptide agonists. (A) Desensitization of PAR2 following the application of trypsin (80 nM), but not the

peptide agonist, SLIGRL (80 mM). (B) Desensitization of PAR2 following the application of trypsin (80 nM) lasts for up to 2 h. Cross-desensitization of PAR1 does

not occur as TFLLR (80 mM) elicits robust increases in [Ca2þ]i. (C) Treatment with trypsin (80 nM, 1 h) does not affect the subcellular localization of PAR2

immunoreactivity. Scale bars, 20 mm.

4.2. Signalling pathways downstream of PAR2 activation

PAR2 activation was associated with a transient increase in[Ca2þ]i in both hippocampal neurons and astrocytes. Previousstudies have also shown that activation of PAR2 leads to theelevation of [Ca2þ]i in hippocampal cultures (Smith-Swintoskyet al., 1997) and cultured astrocytes (Ubl et al., 1998), howeverthe signalling mechanisms underlying this effect have not been

elucidated. Here, we show that the increase in [Ca2þ]i is due torelease of Ca2þ from intracellular stores as their depletion fol-lowing treatment with the SERCA inhibitor, CPA (Beck et al.,2004), inhibited PAR2 responses. In addition, inhibition ofPLC, using the inhibitor U73122 (Capogna et al., 2003), com-pletely abolished PAR2 elevations in [Ca2þ]i. The compound2-APB, which is a proposed antagonist of the IP3 receptorand a blocker of SOCCs (Braun et al., 2001), also blocked

722 T.J. Bushell et al. / Neuropharmacology 50 (2006) 714e725

Res

pons

e to

tryp

sin

(ratio

uni

ts)

0.00

0.05

0.10

0.15

0.20

0.25

**

GAD+ve

GAD-ve

5 mins

0.05ratio units

1

1

2

12 2

A

D E

F G

B C

Fig. 6. High levels of PAR2 expression found in GABAergic neurons are associated with increased Ca2þ signalling. (A) Levels of PAR2 expression (green) are

heterogeneous in neuronal cultures. (B) Following dual labelling with GAD67 (red), the neuron expressing the highest levels of PAR2 was GABAergic. (C) The

combined image illustrates the areas of co-localization (yellow). (D,E) Identification of neurons 1 and 2 as GABAergic and glutamatergic respectively, by staining

with anti-GAD67. (F) Prior to immunohistochemical identification, functional studies reveal that neuron 1 elicited the largest increases in [Ca2þ]i following ap-

plication of trypsin (80 nM). (G) The largest increases in [Ca2þ]i are observed in GAD67 (GABAergic) neurons. Scale bars, 20 mm.

PAR2 responses. It is possible that PAR2-mediated Ca2þ re-lease is also associated with some activation of SOCCs. How-ever, this is likely to make only a small contribution to theobserved Ca2þ rise as the temporal profile and magnitude ofPAR2 responses in Ca2þ-free and normal Ca2þ-containingmedium were similar.

Evidence exists for PAR coupling to both the Gq and Gi/Go

G-protein subunits, which may be dependant on the

preparation under study (Babich et al., 1990; Brass et al.,1991; Hung et al., 1992; Baffy et al., 1994; Offermannset al., 1994, 1997; Vaidyula and Rao, 2003). In our culturedneurons, the Gi/Go G-protein subunit inhibitor, PTX, has no ef-fect on increases in [Ca2þ]i following PAR2 activation, indi-cating that the activation of PAR2 leads to elevated [Ca2þ]i

predominantly through the Gq, PLC and IP3 pathway. Aprevious study revealed that in hippocampal CA1 neurons,

723T.J. Bushell et al. / Neuropharmacology 50 (2006) 714e725

responses to NMDA were potentiated in the presence of PAR1agonists (Gingrich et al., 2000). This is unlikely to contributeto the increase in [Ca2þ]i in the present study as robust re-sponses to PAR2 agonists were obtained in the presence ofglutamate receptor antagonists (DL-AP5, 100 mM andNBQX, 20 mM). Taken together, these data indicate that acti-vation of PAR2 in cultured hippocampal neurons and astro-cytes leads to an increase in [Ca2þ]i through the Gq/PLCpathway.

4.3. Desensitization of PAR2 responses

Activation of PAR2 by trypsin leads to the desensitizationof the receptor (Bohm et al., 1996; Cocks et al., 1999;Dery et al., 1999; DeFea et al., 2000; Seatter et al., 2004).Studies indicate that prolonged activation of the receptorleads to desensitization by N-terminus receptor cleavage, pro-tein kinase C mediated termination of signalling and/or inter-nalization (Macfarlane et al., 2001). PAR2 desensitization(but not PAR1) was observed in the current study followingexposure to trypsin, where subsequent applications of eithertrypsin or SLIGRL failed to elicit further elevations in[Ca2þ]i. Immunolabelling studies suggest that internalizationis unlikely to be responsible for the observed desensitizationas exposure to trypsin for up to 2 h produced no differencesbetween PAR2 localization in control and treated neurons. Inparticular, we observed no apparent translocation of PAR2away from the plasma membrane or increase in the appear-ance of PAR2 within intracellular vesicles. This is in starkcontrast to the rapid receptor internalization seen in previousstudies, where desensitization and resensitization were ob-served in expression systems and peripheral tissue withinthese time periods (Bohm et al., 1996; Dery et al., 1999;DeFea et al., 2000; Seatter et al., 2004). GPCR internalizationis mediated by a conserved mechanism despite the diversityof receptors identified (Kohout and Lefkowitz, 2003). Indeed,G-protein coupled receptor kinase (GRK) and b-arrestin arecritical factors for PAR1 and PAR2 receptor internalization(Ishii et al., 1995; Dery et al., 1999). The finding that inter-nalization in hippocampal neurons is slower than that ob-served in expression systems is common to several GPCRS,for example CB1 cannabinoid receptors and m-opioid recep-tors (Coutts et al., 2001; Bushell et al., 2002). Thus, althoughreceptor internalization is unlikely to be responsible for thetrypsin induced desensitization of the [Ca2þ]i response inthe current study, it remains to be investigated whetherlong-term exposure to PAR2 agonists does indeed lead to theinternalization of the receptor. Thus, the finding that an initialapplication of trypsin produces a rapid desensitization to sub-sequent applications of both trypsin and SLIGRL, suggests thatN-terminus cleavage and ‘‘tethered’’ ligand binding is likely toprevent further activation of the receptor. Furthermore, in con-trast to previous studies, we also observe reproducible re-sponses to brief periods of exposure to SLIGRL suggestingthat these conditions do not result in a marked loss of function-al cell surface receptors or receptor uncoupling.

4.4. Localization of PAR2 in the hippocampus

The presence of PARs 1e4 in hippocampal neurons hasbeen shown previously in both primary and organotypic cul-tures and within brain slices (Weinstein et al., 1995; Smith-Swintosky et al., 1997; D’Andrea et al., 1998; Niclou et al.,1998; Striggow et al., 2001; Riek-Burchardt et al., 2002;Wang et al., 2002; Sorensen et al., 2003). Glial immunolabel-ling for PARs 1e4 has also been observed in primary culturedastrocytes (Wang et al., 2002; Sorensen et al., 2003). In agree-ment with these studies, we saw extensive labelling for PAR2in both the neurons and glia of the hippocampus. At the sub-cellular level, neuronal PAR2 was primarily associated withthe cell soma and proximal dendrites, where it was expressedat the plasma membrane and intracellular sites. In contrast, wedid not observe any detectable axonal expression of PAR2immunoreactivity, suggesting that PAR2 is unlikely to directlymodulate synaptic transmission presynaptically. PAR2 immu-nolabelling in both this and previous studies has revealedheterogeneity in neuronal staining (Smith-Swintosky et al.,1997). Our dual labelling experiments suggest that PAR2 isexpressed on both glutamatergic and GABAergic neurons incultured and slice preparations. However, the highest levelsof expression in hippocampal cultures were observed in GA-BAergic neurons. The heterogeneity of PAR2 expression be-tween glutamatergic and GABAergic neurons in culture wasnot evident when investigated in slice preparations and mayreflect a physiological response of the neurons to the culturepreparation/environment. That said, increased expression ofPAR2 is reflected in the functional studies where GABAergicneurons in culture demonstrated markedly larger [Ca2þ]i re-sponses to PAR2 activation than putative glutamatergic neu-rons. Since PAR2 is upregulated following inflammatory andischaemic insults (Nystedt et al., 1996; Striggow et al.,2001; Jin et al., 2005) and in certain disease states (Knightet al., 2001; Ferrell et al., 2003; Noorbakhsh et al., 2005),the link between expression and function may be valuablein determining the consequence of PAR2 upregulation in thesecircumstances.

4.5. Expression of PAR2 varies with time in culture

Activation of PAR2 leads to a marked increase in [Ca2þ]i inthe majority of neurons and astrocytes in mature cultures (>13DIV). Interestingly, when younger cultures (3e5 DIV) wereinvestigated, the number of neurons that responded to PAR2activation was dramatically reduced. This is also reflected inthe lower proportion of cells expressing PAR2 immunoreactiv-ity in these cultures. Although strong PAR2 labelling were ob-served in interneurons at this time, the DIV change in PAR2expression was not due to a difference in the proportion of in-terneurons (which was consistently between 5 and 20%). Thisprofile in culture was not characteristic of all PARs as activa-tion of PAR1 elicited increases in [Ca2þ]i in the majority ofneurons investigated, both in young and old cultures. Inaddition, the number of astrocytes responding the PAR2 activ-ation was similar in both young and old cultures.

724 T.J. Bushell et al. / Neuropharmacology 50 (2006) 714e725

Immunohistochemical studies revealed similar levels of astro-cytes PAR2 labelling in both the older cultures and youngercultures. A previous study has shown the presence of PAR2throughout embryogenesis in mice (Jenkins et al., 2000), butthese data suggests that the receptor, although present, is notcoupled to PLC or is not fully functional in the younger cul-tures. Further investigation into the importance of this DIVprofile and its relevance in vivo are required.

5. Conclusion

Taken together, the data presented confirm the presenceof PAR2 in the hippocampus and reveal that PAR2 activationelicits an increase in [Ca2þ]i which is mediated by the classicalPLC/IP3 signalling pathway. Desensitization of PAR2 responsesfollowing activation by trypsin, the selective peptides SLIGRLand 2f-LIGV-OH, inhibited further activation of the receptor.These data provide the basis to further investigate the potentialrole of PAR2 in modulating synaptic plasticity and neurodege-neration in the hippocampus. Moreover, the finding thatincreased expression correlates with increased functionalresponses warrants further investigation into its role in inflam-matory conditions that affect the CNS, which are proposed toplay an important factor in many neurological diseases.

Acknowledgements

We would like to thank D. O’Malley for preparation of thecultures, M. Hollenberg for his kind gift of the PAR2 antibodyand Kowa Ltd for supplying the PAR2 peptides. This workwas partially funded by Tenovus Scotland and the BBSRC.

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