University of Groningen

(Endo)cannabinoid signaling in human bronchial epithelial and smooth muscle cellsGkoumassi, Effimia

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Chapter 2

Arachidonic Acid Mediates Non-Capacitative Calcium Entry Evoked by CB1-Cannabinoid Receptor Activation in DDT1 MF-2 Smooth Muscle Cells J Cell Physiol. 2005; 205: 58-67

Dirk G. Demuth1, Effimia Gkoumassi2, Melloney J. Dröge2, Bart G. J.

Dekkers2, Henk J. Esselink2, Rutger M. van Ree1,2, Mike E. Parsons1, Johan

Zaagsma2, Areles Molleman1, S. Ad Nelemans2.

(1) School of Life Sciences, Faculty of Health and Human Sciences,

University of Hertfordshire, College Lane, Hatfield, Hertfordshire AL10 9AB,

UK.

(2) Department of Molecular Pharmacology, University Centre for Pharmacy, University of

Groningen, A. Deusinglaan 1, 9713AV Groningen, The Netherlands

______________________________________________________Chapter 2

Abstract

Cannabinoid CB1-receptor stimulation in DDT1 MF-2 smooth muscle cells

induces a rise in [Ca2+]i which is dependent on extracellular Ca2+ and

modulated by thapsigargin-sensitive stores, suggesting capacitative Ca2+

entry (CCE), and by MAP kinase. Non-capacitative calcium entry (NCCE)

stimulated by arachidonic acid (AA) partly mediates histamine H1-receptor-

evoked increases in [Ca2+]i in DDT1 MF-2 cells. In the current study both Ca2+

entry mechanisms and a possible link between MAP kinase activation and

increasing [Ca2+]i, were investigated. In the whole-cell patch clamp

configuration, the CB-receptor agonist CP 55,940 evoked a transient, Ca2+-

dependent K+ current, which was not blocked by the inhibitors of CCE, 2-APB

and SKF 96365. AA, but not its metabolites, evoked a transient outward

current and inhibited the response to CP 55,940 in a concentration-dependent

manner. CP 55,940 induced a concentration-dependent release of AA, which

was inhibited by the CB1 antagonist SR 141716A. The non-selective Ca2+

channel blockers La3+ and Gd3+ inhibited the CP 55,940-induced current at

concentrations that had no effect on thapsigargin-evoked CCE. La3+ also

inhibited the AA-induced current. CP 55,940-induced AA release was

abolished by Gd3+ and by phospholipase A2 inhibition using quinacrine; this

compound also inhibited the outward current. The CP 55,940-induced AA

release was strongly reduced by the MAP kinase inhibitor PD 98059. The

data suggest that in DDT1 MF-2 cells AA is an integral component of the CB1

receptor signalling pathway, upstream of NCCE and, via PLA2, downstream of

MAP kinase.

28

______________________________________________________Chapter 2

Introduction

Cannabinoids have been shown to stimulate specific Gi/o-protein coupled

cannabinoid receptors (Howlett et al., 1986; Matsuda et al., 1990). To date

only two receptors have been cloned, the cannabinoid CB1 and CB2 receptor.

The CB1 receptor is highly expressed within certain areas of the brain (Tsou et

al., 1998) and in some peripheral tissues (Liu et al., 2000; Wagner et al.,

2001), while the CB2 receptor is localised mainly in the immune system

(Munro et al., 1993; Schatz et al., 1997). Their activation is linked to the

modulation of certain intracellular pathways including stimulation of MAP

kinase (Bouaboula et al., 1995) and inhibition of adenylyl cyclase (Childers

and Deadwyler, 1996). Stimulation of the CB1 receptor has also been shown

to inhibit voltage-operated Ca2+ channels (L-, N- and P/Q- type) as well as to

activate inwardly-rectifying K+ channels (Gebremedhin et al., 1999; Mackie et

al., 1995; Twitchell et al., 1997).

Cannabinoid-mediated changes in intracellular Ca2+ concentration ([Ca2+]i)

have been described in both neuronal and non-neuronal preparations. In the

neuroblastoma-glioma cell hybrid, NG108-15, CB1 receptor stimulation

induces a rise in [Ca2+]i through the activation of phospholipase C (PLC) and

the subsequent release of Ca2+ from inositol 1,4,5-trisphosphate (InsP3)-

sensitive intracellular Ca2+ stores (Sugiura et al., 1996). DDT1 MF-2 smooth

muscle cells express CB1 receptors, which can be stimulated by the herbal

cannabinoid Δ9-tetrahydrocannabinol (Δ9-THC) to induce a rise in [Ca2+]i

(Filipeanu et al., 1997). Voltage-operated Ca2+ channels are absent in these

cells making them a good preparation to study receptor-mediated increases in

[Ca2+]i (Molleman et al., 1991). We have shown previously that the rise in

[Ca2+]i in response to the synthetic cannabinoid agonist, CP 55,940 is

insensitive to PLC inhibition but was entirely dependent on Ca2+ from the

extracellular space (Begg et al., 2001). However, a release of Ca2+ from

thapsigargin-sensitive stores also played a role (Begg et al., 2001). These

data supported a model for capacitative Ca2+ entry (CCE), in which initiation of

Ca2+ influx is coupled to the filling state of the intracellular Ca2+ stores

29

______________________________________________________Chapter 2

(Putney, 1990). Furthermore the MAP kinase inhibitor PD 98059 reduced the

rise in [Ca2+]i (Begg et al., 2001).

Recently, a novel mechanism for Ca2+ entry has been described which is

independent of store depletion and subsequent capacitative Ca2+ influx. This

so called non-capacitative Ca2+ entry (NCCE) pathway, through which

intracellular messengers such as arachidonic acid (AA) mediate Ca2+ influx,

has been described in a variety of cell types (Broad et al., 1999; Fiorio Pla

and Munaron, 2001; Mignen and Shuttleworth, 2000; Munaron et al., 1997).

AA but not its metabolites, was shown to induce Ca2+ channel opening (Broad

et al., 1999; Fiorio Pla and Munaron, 2001). This Ca2+ influx has since been

designated IARC (arachidonate regulated Ca2+ current) (Mignen and

Shuttleworth, 2000).

DDT1 MF-2 cells also express histamine-H1 receptors, activation of which

results in an increase in [Ca2+]i resulting primarily from an InsP3-mediated

release of Ca2+ from intracellular stores (Den Hertog et al., 1992). However, a

small contribution to the overall increase in [Ca2+]i is due to Ca2+ influx from

the extracellular space; this influx pathway was activated by AA (Van der Zee

et al., 1995). The latter pathway could also mediate the Ca2+ entry process

seen during CB1 stimulation. The putative pathways leading to AA production

have not fully been elucidated but a common mechanism for AA liberation

involves direct action of phospholipase A2 (PLA2) on phospholipids. In HEK

293 cells type IV cytosolic PLA2 has been shown to liberate AA, which in turn

stimulates IARC (Osterhout and Shuttleworth, 2000). PLA2 activity can be

regulated by MAP kinase (Kudo and Murakami, 2002) and could be the

intermediate step between MAP kinase activation and the resulting rise in

[Ca2+]i during CB1 stimulation.

In the present study, we aimed to identify the Ca2+ influx mechanism

responsible for the CBB1 receptor-induced increase in [Ca ]2+i, including the

intracellular signalling pathways involved. Generation of free AA was

observed in response to CB1 receptor activation, which may arise from the

30

______________________________________________________Chapter 2

action of PLA2 on membrane phospholipids. Our data argue against the

classical CCE model and support the possibility that in DDT1 MF-2 cells, AA is

a major component of the CB1 signalling pathway activating IARC, resulting in

NCCE.

Materials and Methods

Cell Culture - DDT1 MF-2 smooth muscle cells were cultured under standard

conditions as described previously (Molleman et al., 1989). Briefly, cells were

grown at 37°C in Dulbecco’s Modified Eagle’s Medium (DMEM),

supplemented with 10% fetal-calf serum, penicillin (50 μg/ ml), streptomycin

(50 μg/ ml) and L-glutamine (2 mM). They were grown to confluency, in an

atmosphere of 5% CO2/ 95% O2 and subsequently plated onto glass cover

slips for electrophysiological studies or into 6 well plates for AA release

studies. All culture media and supplements were obtained from Gibco (UK).

Patch Clamp - Micropipettes (Harvard Apparatus, GC150TF-10) were heat-

polished (MF-830, Narishige) and back-filled with intracellular solution (ICS, in

mM: NaCl, 5; KCl, 142; MgCl2, 1.2; Hepes, 20; glucose, 11; K-ATP, 5; Na-

GTP, 0.1; pH 7.2) to give a typical resistance of 2-7 MΩ. Membrane currents

were amplified (Axon Instruments, Axopatch 1D) and recorded at room

temperature using a digital interface (Axon Instruments, Digidata 1200) and

then viewed in real-time, on a personal computer incorporating the pCLAMP 6

software (Axon Instruments). Monitoring of the movement of the micropipette

towards the cell was performed by the application of an electrical pulse (5mV,

5ms, 100Hz), which allowed the analysis of resting leak current and

compensation of the capacitive transient. Cells were clamped at a holding

potential of –60mV, which is close to the resting membrane potential of these

cells (Kudo and Murakami, 2002). Currents were measured at a holding

potential of –30 mV and converted to pA per pF membrane capacitance to

correct for cell surface area.

Electrophysiological measurements were carried out in extracellular salt

(ECS) solution containing (mM): NaCl, 125; KCl, 6; MgCl2, 2.5; NaH2PO4, 1.2;

31

______________________________________________________Chapter 2

Hepes, 20; glucose, 11; sucrose, 67; CaCl2, 1.2; pH 7.4. All drugs were

administered in the ECS, superfused at a rate of 4 ml/ min. Post-

experimentation, the apparatus was washed with dilute hydrochloric acid (0.1

M), absolute ethanol and distilled water to ensure complete washout of

residual ligands.

[3H]Arachidonic Acid (AA) Release - AA release was measured as described

previously (23). Cells were grown to confluency in 9.6 cm2 six well plates and

labelled with 0.25 μCi of [3H]AA (Specific Activity: 60-100Ci (2.22-

3.70TBq)/mmol) in serum-free DMEM (1 ml), for 3 h at 37oC. Cells were

washed once in ECS, twice with ECS containing 1% BSA (fatty-acid free) and

once again with ECS to eliminate any unincorporated radioactivity. They were

then allowed to equilibrate for 15 minutes during which time inhibitors of the

signalling pathway under investigation could be added. Following this the cells

were incubated with an agonist for 5 minutes. Experiments ended when the

ECS was removed and [3H]AA release was determined by liquid-scintillation

counting. Inhibitors were also applied alone to determine any effects they may

have on AA efflux by their own.

Intracellular Ca2+ Measurements - [Ca2+]i was measured using fura-2

fluorometry as reported previously (Filipeanu et al., 1997). Cells in monolayer

were harvested using a cell scraper and loaded in suspension with 3 μM fura-

2 acetoxymethylester at 22°C for 45 minutes in the dark. Fluorescence was

measured at 37°C. For measurements made using CP 55,940 the area under

the curve (AUC) was calculated starting at the point of drug application (t=0)

to t=150 seconds. A dose-response curve for CP 55,940 was then

constructed from the mean of the data obtained.

Drugs - All drugs were obtained from Sigma (UK), unless stated otherwise.

CP 55,940 ((-)-cis-3-[2-Hydroxy-4-(1,1-dimethylheptyl)phenyl]-trans-4-(3-

hydroxy-propyl) cyclohexanol) and SR 141716A (N-Piperidino-5-(4-

chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-3-pyrazole-carboxamide) were

kind gifts from Pfizer (UK) and Sanofi (France) respectively. Stock solutions of

CP 55,940 and AA were prepared in ethanol when they were used for

32

______________________________________________________Chapter 2

electrophysiological experiments, shielded from light and stored at -20°C.

When measuring [3H]AA (Perkin Elmer Life Sciences, NL) efflux, CP 55,940

and SR 141716A were dissolved in DMSO. Histamine was dissolved in

distilled water and kept at 4°C. Lanthanum (III) chloride, gadolinium (III)

chloride and nickel (II) chloride solutions were freshly made each day and

were dissolved in distilled water. 2-APB (2-aminoethoxydiphenyl borate), SKF

96365 (1-[2-(4-methoxyphenyl)-2-[3-(4-methoxyphenyl)propoxy]ethyl-1H-

imidazole hydrochloride) and PD 98059 (2-(2-amino-3-methoxyphenyl)-4H-

1benzopyran-4-one) were obtained from Tocris-Cookson (UK). 2-APB and PD

98059 were dissolved in DMSO and stored at -20°C. SKF 96365 was

dissolved in distilled water. Indomethacin and phenidone solutions were made

fresh each day and were dissolved in ethanol and DMSO respectively.

Quinacrine was dissolved in DMSO and diluted in distilled water. Thapsigargin

was dissolved in DMSO and stored at -20°C.

Data Analysis - Values are expressed as means ± standard error of the mean

(S.E.M.). Comparison of pairs of treatments was made using unpaired

Student’s t-test. A one-way ANOVA test was performed, followed by a post

hoc Dunnett test to assess significant differences between control and

multiple test values. P<0.05 was considered significant.

Results CP 55,940-Evoked Increases in [Ca2+]i Activate An Outward Current in DDT1

MF-2 Cells: Role of Extracellular Calcium - An increase in [Ca2+]i, in DDT1 MF-

2 cells activates a Ca2+-dependent K+ current (IK,Ca), which can be measured

using the whole cell version of the patch clamp technique (Begg et al., 2001).

The synthetic cannabinoid receptor agonist CP 55,940 (10 μM) evoked a

transient, outward current, with a peak amplitude of 32.4 ±1.3 pA/ pF after 208

±54 seconds of administration (Fig. 1A, n=6). Histamine (10 μM), which

releases Ca2+ from InsP3-sensitive stores and induces calcium influx (Van der

Zee et al., 1995), produced a transient outward current with a peak amplitude

of 31.4 ±5.9 pA/ pF after 46.9 ±6.8 seconds of drug application (Fig. 1B, n=8).

33

______________________________________________________Chapter 2

A

B

Cur

rent

(pA

)

0 200 400

0

300

600

900

1200

Time (s)

CP 55,940

0 200 400 600

0

300

600

900

1200

1500 Histamine

Cur

rent

(pA

)

Time (s)

Figure 1. The cannabinoid receptor

agonist CP 55,940 and histamine evoke

outward currents in DDT1 MF-2 cells. A,

sample trace of the transient outward

current evoked by 10 μM CP 55,940. B,

sample trace of the transient outward

current evoked by 10 μM histamine. Bars

indicate the presence of ligands.

CP 55,940 induced a concentration-dependent (10-7-10-4 M) increase in

[Ca2+]i, which was abolished with the removal of extracellular calcium (Fig. 2 ).

Moreover, in Ca2+-free medium, higher concentrations (>10-6 M) of CP 55,940

decreased [Ca2+]i.

-10

0

10

20

30

8 7 6 5 4

-log [CP 55,940] (M)

Ca2+

i (AU

C, n

M.s

ec) (

x 10

3 )

Ca2+

Ca2+ FreeFigure 2. CP 55,940 increases

[Ca2+]i, which is dependent on Ca2+

from the extracellular space. CP

55,940 increases [Ca2+]i in a

concentration-dependent manner

(n=4). This increase is abolished in

the absence of extracellular Ca2+

(n=4). In Ca2+-free medium higher

concentrations of CP 55,940 reduce

[Ca2+]i.

34

______________________________________________________Chapter 2

Capacitative Ca2+ Entry (CCE): Role of Store-Operated Ca2+ Channels

(SOCCs) - To examine the involvement of InsP3 receptors in the cannabinoid-

induced response, the membrane permeable InsP3 receptor blocker, 2-APB,

was used (Maruyama et al., 1997). In addition to its effects on InsP3

receptors, 2-APB (10 μM) has been shown to directly block SOCCs in human

platelets as well (Dobrydneva and Blackmore, 2001) which would make it the

most suitable to establish the contribution of CCE in DDT1 MF-2 cells.

However, 2-APB (10 μM) had no effect on the outward current evoked by 10

μM CP 55,940 (110 ± 43% of control, Fig. 3A and C,) while at the same

concentration 2-APB strongly reduced the histamine response to 8.6 ±7.3 %

of control (Fig. 3B and C, P<0.01). The effect of another inhibitor of CCE, SKF

96365 (Merritt et al., 1990), on the CP 55,940-induced current was also

investigated. At a concentration of 10 μM, SKF 96365 had no effect on the

cannabinoid-induced outward current (106±19 % of control, n=6).

35

______________________________________________________Chapter 2

B

C

0

50

100

150

200

CP 55,940 2-APB +CP 55,940

Histamine 2-APB +Histamine

Peak

Cur

rent

(% o

f Con

trol)

*

A

0 200 400 600 800

0

200

400

600

800

10002-APB

CP 55,940C

urre

nt (p

A)

Time (s)

0 100 200

0

200

400

600

800

1000 Histamine2-APB

Cur

rent

(pA

)

Time (s)

Figure 3. 2-APB has no effect

on CP 55,940-evoked currents

but inhibits the currents evoked

by histamine. A, sample trace

of the effect of 2-APB on the

CP 55 940 (10 μM)-mediated

current. B, sample trace of the

effect of 2-APB on the

histamine-evoked current. Bars

indicate the presence of

ligands. C, 2-APB had no

effect on the outward current

evoked by 10 μM CP 55,940

(n=6) at a concentration that

inhibited histamine-evoked

currents (n=5).

Significant difference from

histamine control: * P<0.01.

36

______________________________________________________Chapter 2

Figure 4. AA induces an

outward current, which

inhibits the response to

CP 55,940. A, sample

trace of the transient,

outward current evoked

by 10 μM AA and the

effect of this on the CP

55,940 (10 μM)-induced

current. B, sample trace

of the outward current

evoked by 50 μM AA and

the effect of this on the

CP 55,940 (10 μM)-

evoked current. Bars

indicate the presence of

ligands. C, AA produces a

concentration-dependent

increase in membrane

current (n≥4). 10 μM AA

reduced the outward

current evoked by 10 μM

CP 55,940 (n=6) and at a

concentration of 50 μM

completely abolished the

response (n=6).

Significant difference from

0 200 400 600 800 1000

0

500

1000

1500

2000

2500

Cur

rent

(pA

)

CP 55,940Arachidonic Acid

Time (s)

Time (s)0 200 400 600 800

Cur

rent

(pA)

CP 55,940Arachidonic AcidA

B

C

0

50

100

150

200

250

AA (1µM)

AA (10 µM)

AA (50 µM)

CP55,940

+ AA (10 µM)

+ AA (50 µM)

Peak

Cur

rent

(% o

f Con

trol)

*

†

†

0

500

1000

1500

2000

2500

AA (1 μM): * P<0.01,

significant difference from

CP 55,940 control: †

P<0.0001.

37

______________________________________________________Chapter 2

Non-Capacitative Ca2+ Entry (NCCE): Role of Arachidonic Acid (AA) - AA has

pre-exposure to AA had no effect on the CP 55,940-evoked outward current

igure 5. CP 55,940 evokes a

o further test the hypothesis that AA is generated in response to CB1

been shown to produce a concentration-dependent rise in [Ca2+]i in DDT1 MF-

2 cells, with a maximal release of Ca2+ obtained at 1 mM AA (Mackie et al.,

1995). This increase in Ca2+ could therefore evoke an outward K+ current. In

the present study, AA evoked a concentration-dependent, transient outward

current indeed (Fig. 4).

If

it would suggest that the CB1 signalling pathways do not involve the

generation of AA. To examine this, DDT1 MF-2 cells were exposed to AA to

induce a transient response and as soon as membrane currents had returned

to baseline, CP 55,940 was administered. Pre-exposure to 10 μM AA

significantly inhibited the outward current induced by 10 μM CP 55,940 (30 ±

2% of control, Fig. 4A and C, P<0.0001) and at a 5 times higher concentration

(50 μM) the cannabinoid-evoked response was completely abolished (Fig. 4B

and C, P<0.0001).

-log [CP 55,940] (M)

Basal 7.5 7 6.5 6 5.5 50

20

40

60

80

100

[3 H]A

A R

elea

se (%

of B

asal

)

Frelease of [3H]AA. [3H]AA-

prelabelled DDT1 MF-2 cells were

stimulated with CP 55,940 and the

release of radiation measured and

presented as increase in the

release as % of basal level. Data

represent 8 experiments in

triplicate. Basal AA release: 16.6

±2.6 dps/well.

T

stimulation, AA release was measured in [3H]AA-prelabelled DDT1 MF-2 cells.

CP 55,940 produced a concentration-dependent efflux of AA (EC50 = 0.10 ±

38

______________________________________________________Chapter 2

0.04 μM, Fig. 5). To establish whether the production of AA was receptor-

mediated, the effect of the CB1 antagonist SR 141716A (1 μM) was studied.

CP 55,940 (1 μM) -mediated release of AA was completely prevented in the

presence of SR 141716A (Table 1).

domethacin, a non-selective cyclooxygenase inhibitor (Bakalova et al.,

Table 1. The effect of various inhibitors on [3H] arachidonic acid release (dps/well)

Control + CP 55,940 (1μM)

In

2002) and phenidone, a dual inhibitor of both cyclooxygenase and

lipoxygenase (Kim et al., 2000) were used to establish if the outward current

in response to CB1 stimulation requires the production of AA metabolites.

Pretreatment of cells with indomethacin (10 μM) had no significant effect on

the outward current evoked by 10 μM CP 55,940 (93 ± 10% of control, n=7).

Phenidone (100 μM) also had no effect on this response (114 ± 13% of

control, n=5).

vehicle (DMSO, 0.6 % ) 16.5±1.2 (20) 32.2±3.7 (30) *

SR 141716A (1μM) 21.9±2.9 (7) 22.6 ±2.1 (14)

Quinacrine (100μM) 18.9±3.0 (10) 17.4±1.6 (11)

Gd3+ (1μM) 19.1±2.0 (8) 17.4±2.0 (13)

PD 98059 (30μM) 21.3±2.0 (13) 24.5±1.3 (12)

Data are presented as mean ± sem (n riments). Sign ,

anthanum (La3+)-Sensitive and Gadolinium (Gd3+)-Sensitive Ca2+ Influx - In

umber of expe ificance level: *

P< 0.002 vs. control

L

the presence of the non-specific Ca2+ channel blocker La3+ (50 μM), the

histamine-evoked Ca2+ influx in DDT1 MF-2 cells has been shown to be

abolished, leading to a reduction of the outward current (Van der Zee et al.,

1995). This Ca2+ influx may represent a NCCE pathway as observed in other

cell lines, which is also sensitive to inhibition by La3+ (Fiorio Pla and Munaron,

2001; Mignen and Shuttleworth, 2000). Indeed La3+-sensitive Ca2+ channels

39

______________________________________________________Chapter 2

are activated during CB1 stimulation, as application of 50 μM La3+, prior to CP

55,940 (10 μM) application, abolished the CP 55,940-evoked outward current

(Fig. 6, P<0.0001). To further explore this activation of NCCE, the effect of

La3+ on AA-induced Ca2+ entry was established since AA has also been

shown to activate IK,Ca (Kirber et al., 1992). These results would help to

ascertain if AA production does indeed occur upstream of the Ca2+ entry

process. Indeed La3+ (50 μM) was able to reduce the response to 10 μM AA

to 17 ± 13% of control (Fig. 6, P<0.001).

Figure 6. La3+ inhibits the

CE in DDT1 MF-2 cells can be evoked with the sarcoplasmic-endoplasmic

0

20

40

60

80

100

120

CP 55,940 La3+ + CP 55,940

AA La3+ + AA

Peak

Cur

rent

(% o

f Con

trol)

**

*

outward current evoked by

CP 55,940 and AA. 50 μM

La3+ inhibited the AA (10

μM)-evoked outward current

(n=6) and completely

abolished the response to

10 μM CP 55,940 (n=7).

Significant difference from

CP 55,940 and AA control: *

P<0.001, ** P<0.0001.

C

reticulum Ca2+-ATPase (SERCA) inhibitor thapsigargin. Nickel (Ni2+), which

has been shown to inhibit CCE in other cell systems (Kerschbaum and

Cahalan, 1999), inhibited the Ca2+ influx component of thapsigargin in a

concentration-dependent manner (EC50: 0.8 mM, n=3, Fig. 7A,). At

concentrations ranging from 0.01 to 100 μM, neither La3+ nor Gd3+ had any

effect on the thapsigargin-induced elevation in [Ca2+]i (Fig. 7B and C), while

Ni2+ clearly inhibited the Ca2+ influx. This suggests that separate Ca2+ influx

pathways are initiated by CP 55,940 (NCCE) and thapsigargin (CCE).

40

______________________________________________________Chapter 2

Figure 7. Thapsigargin-evoked

rat aortic smooth muscle cells low concentrations (1 μM) of Gd3+ inhibited

CE, while higher concentrations (100 μM) were shown to inhibit both CCE

B

0

50

100

150

0 1000 2000 3000

Time (s)

[Ca2+

] i (nM

)

Thapsigargin

Ni2+

c

Ca2+

bad

ef

0

50

100

150

0 1000 2000 3000

Time (s)

[Ca2+

] i (nM

)

Thapsigargin

Ni2+

c

Ca2+

bad

ef

bad

ef

A

Time (s)

0

50

100

150

0 1000 2000 3000

[Ca2+

] i(n

M)

Thapsigargin

Ca2+

C

0

100

200

300

400

500

0 1000 2000 3000

Time (s)

[Ca2+

] i(n

M)

Thapsigargin

Ca2+

0

100

200

300

400

500

0 1000 2000 3000

Time (s)

[Ca2+

] i(n

M)

Thapsigargin

Ca2+

La3+

bd

ac

ef

Ni2+La3+

bd

ac

ef

Ni2+

a bc

d

a bc

d

Gd3+ Ni2+

CCE. Effects of Ni2+, La3+ and

Gd3+. Internal Ca2+ stores were

initially depleted with 1 μM

thapsigargin in Ca2+-free

medium. Re-addition of 1 mM

Ca2+ to the external milieu

produced a rise in [Ca2+]i

reflecting the activation of CCE.

A, sample trace of the effect of

Ni2+ at concentrations : 0.1, 1

and 10 μM (a), 100 μM (b), 300

μM (c), 1 mM (d), 3 mM (e), 10

mM (f). B, sample trace of the

effect of La3+ at concentrations:

0.01, 0.1, 1, 10 and 100 μM (a),

followed by addition of Ni2+ at

concentrations: 100 μM (b), 300

μM (c), 1 mM (d), 3 mM (e), 10

mM (f). C, sample trace of the

effect of Gd3+ at concentrations:

0.01, 0.1, 1, 10 and 100 μM (a),

followed by addition of Ni2+ at

concentrations: 100 μM (b), 300

μM (c), 1 mM (d). In contrast to

Ni2+ no effects on CCE were

observed for La3+ or Gd3+ (n=3).

In

C

and NCCE (Broad et al., 1999). Therefore, the effects of Gd3+ on CP 55,950

evoked membrane current and AA efflux were also investigated. Gd3+ (1 μM)

inhibited the outward current produced by 10 μM CP 55,940 to 23 ± 11% of

control (Fig 8, P<0.0001). However, the observation that 100 μM, Gd3+ had no

effect on thapsigargin induced rise in [Ca2+]i, implies that CP 55,940 activates

41

______________________________________________________Chapter 2

a Ca2+ influx pathway distinct from CCE. The Ca2+ influx generated through

the NCCE pathway in response to CP 55,940 appears to be essential for

PLA2 activation in view of the abolition of AA release in the presence of Gd3+

(1 μM, Table 1).

Figure 8. Gd3+ inhibits the

outward current evoked by CP

55,940. A, samp

effect of Gd3+

le trace of the

μM) on the CP

,940 (10 μM)-evoked outward

A Production: Role of Phospholipase A2 (PLA2) and MAP kinase -

annabinoid-induced mobilisation of AA was shown to involve the activities of

e cytoplasmic PLA2 isoenzyme in WI-38 lung fibroblasts (Wartmann et al.,

995). To explore the possibility that AA is being generated by PLA2 in DDT1

(1

B

0

20

40

60

80

100

120

CP 55,940 Gd3+ + CP 55,940

Peak

Cur

rent

(% o

f Con

trol)

*

A

0 100 200

0

200

400

600

800Gd 3+

CP 55,940

Cur

rent

(pA

)

Time (s)

55

current. Bars indicate the

presence of ligands. B, Gd3+

significantly reduces the outward

current evoked by 10 μM CP

55,940 (n=9). Significant

difference from CP 55,940

control: * P<0.0001.

A

C

th

1

MF-2 cells, the effects of a non-selective inhibitor of PLA2, quinacrine, were

investigated (Lu et al., 2001). At concentrations of 10 μM and 30 μM

quinacrine inhibited the outward current evoked by 10 μM CP 55,940 to 64

42

______________________________________________________Chapter 2

±8% of control (Fig. 9B, P<0.001) and 63 ±6% of control (Fig. 9A and B,

P<0.01) respectively.

Figure 9. Quinacrine inhibits the CP

55,940-induced outward current and

release of [3H]AA. Cells were pre-

treated with quinacrine (10-30 μM)

for 10 minutes before agonist

application. A, sample trace of the

effect of 30 μM quinacrine on the

outward current evoked by 10 μM

CP 55,940. B, Quinacrine at 10

(n=5) and 30 μM (n=9) reduced the

CP 55,940 (10 μM)-induced current.

uinacrine (100 μM) alone had no effect on membrane currents in DDT1 MF-

cells (results not shown, n=4). To add further support to the involvement of

B1-mediated activation of PLA2, the effect of quinacrine (100 μM) on CP

5,940-evoked [3H]AA release was established. Quinacrine alone had no

ffect on basal [3H]AA efflux, but the CP 55,940 (1 μM)-evoked AA release

as abolished (Table 1). CP 55,940-induced AA release is also inhibited

trongly by the MAP kinase pathway inhibitor PD 98059 (30 μM, Table 1),

upporting the hypothesis that PLA2 activation by CP 55,940 is regulated by

AP kinase.

A

B

0

50

100

150

CP 55,940 + Quinacrine(10 μM)

+ Quinacrine(30 μM)

Peak

Cur

rent

(% o

f Con

trol)

** *

0 200 400

0

300

600

900

1200

1500 CP 55,940Quinacrine

Cur

rent

(pA

)

Time (s)

Significant difference from CP

55,940 control: * P<0.01, **

P<0.001.

Q

2

C

5

e

w

s

s

M

43

______________________________________________________Chapter 2

Discussion

In the present study we further explored the signal transduction mechanisms

mediating CB1 receptor-induced increases in [Ca2+]i in DDT1 MF-2 smooth

muscle cells. Previous work has shown that the cannabinoid agonist CP

55,940 evokes a transient outward current, which is completely abolished by

the removal of Ca2+ from the bathing solution (Begg et al., 2001). Similarly, we

showed a con 2+centration-dependent increase in [Ca ]i by CP 55,940, which

as abolished by the removal of extracellular Ca2+. This suggested that the

ease in [Ca2+]i is entirely dependent on Ca2+ influx from the 2+ release from thapsigargin-sensitive stores

v ed by the

annabinoid receptor agonist CP 55,940 (Begg et al., 2001). Similarly we

w

CB1-induced incr

extracellular space, although Ca

was also shown to play a role (Begg et al., 2001). This would imply a model

for CCE, where the depletion of intracellular Ca2+ stores activates membrane-

bound SOCCs, resulting in Ca2+ influx (Putney, 1990). Interestingly, in the

current study we show that in the absence of extracellular Ca2+, higher

concentrations of CP 55,940 even decreased [Ca2+]i. This suggests that at

least part of the Ca2+ transport mechanism is still operational under these

conditions, but reversed due to the inverted driving force for Ca2+.

DDT1 MF-2 cells have been used previously to study changes in [Ca2+]i

caused by activation of histamine H1 and purine P2 receptors (Molleman et al.,

1991). It was demonstrated that activation of these receptors elevates [Ca2+]i

through an InsP3-mediated release from internal Ca2+ stores. The production

of InsP3 involves the activation of PLC. The inhibitory effect of thapsigargin on

the cannabinoid response (Begg et al., 2001) would suggest that a release of

Ca2+ from internal stores is also required to mediate this response. However,

inhibition of PLC has no effect on the outward current e ok

c

found in the current study that the InsP3 receptor antagonist, 2-APB had no

effect at all on the CP 55,940 response at a concentration that was shown to

inhibit histamine-evoked currents. In addition to its effects on the InsP3

receptors 2-APB has been shown to directly block SOCCs in human platelets

at concentrations used in the present study (Dobrydneva and Blackmore,

44

______________________________________________________Chapter 2

2001). This suggests that CCE does not mediate the cannabinoid-induced

rise in [Ca2+]i in DDT1 MF-2 cells. We also used the SOCC inhibitor SKF

96365 to support our data obtained with 2-APB. The inhibitor was found to

have no effect on the CP 55,950-evoked response, at a concentration shown

to inhibit CCE in other systems (Merritt et al., 1990). Thus, taken together, the

SKF 96365 and 2-APB data strongly argue against CCE as a mechanism for

CB1 evoked increases in [Ca2+]i.

Recently an NCCE pathway has been described to operate independently of

intracellular store depletion. In this pathway the rise in [Ca2+]i occurs via Ca2+

influx activated by intracellular messengers, including AA. AA-evoked Ca2+

influx has been described in a variety of cell types including Balb-C 3T3

mouse fibroblasts (Munaron et al., 1997), rat aortic smooth muscle cells

(Broad et al., 1999), bovine aortic endothelial cells (Fiorio Pla and Munaron,

2001) and rat astrocytes (Shivachar et al., 1996). In HEK 293 cells the

channels responsible for the AA- mediated Ca2+ influx were investigated and

e resulting membrane current was designated as IARC (Mignen and

his suggests that AA activates the same

a2+ channels that mediate Ca2+ influx during CB1 as well as H1 receptor

stimulation. In order to determine whether CB1-induced increases in [Ca2+]i

th

Shuttleworth, 2000). In addition, AA has been shown to initiate Ca2+ influx

during H1 stimulation in DDT1 MF-2 cells (Van der Zee et al., 1995),

suggesting that a NCCE pathway is operational in this cell line. This lends

support to the possibility that CB1 stimulation induces a rise in [Ca2+]i through

a similar non-capacitative pathway.

Application of AA to DDT1 MF-2 cells evoked a transient outward current,

similar to that caused by application of CP 55,940. Exogenous application of

AA has been shown to induce Ca2+ influx in cells utilising NCCE (Broad et al.,

1999; Fiorio Pla and Munaron, 2001; Mignen and Shuttleworth, 2000;

Munaron et al., 1997), including DDT1 MF-2 cells (Van der Zee et al., 1995).

Application of AA prior to histamine resulted in inhibition of the histamine-

evoked NCCE-mediated Ca2+ influx, as reflected by abolition of outward

current (Van der Zee et al., 1995). T

C

45

______________________________________________________Chapter 2

occur through the activation of an IARC-like channel, the effects of AA on the

CP 55,940-evoked current were established. AA was found to inhibit

concentration-dependently the CP 55,940-evoked response.

In support of an NCCE signalling pathway mediated by AA we observed an

increase in [3H]AA release in response to CP 55,940 application, indicating

that CB1 stimulation results in A production. This was confirmed by using the

CB

A

i

e-induced [3H]AA release (Van der Zee et al.,

995). In accordance with this we found that inhibitors of either of these two

1 antagonist SR 141716A, which was able to block the CP 55,940-evoked

AA release completely. Previous work using DDT1 MF-2 cells has shown that

SR 141716A reduces the outward currents evoked by the herbal cannabino d

Δ9-THC and by CP 55,940 (Filipeanu et al., 1997; Begg et al., 2001). Together

with the present data this suggests that CP 55,940 generates AA in a CB1

receptor-dependent manner.

In N18 mouse neuroblastoma cells (Hunter and Burnstein, 1997) and rat brain

astrocytes (Shivachar et al., 1996) Δ9-THC has been found to mobilise AA.

These effects were SR 141716A-sensitive, implying the involvement of the

CB1 receptor. The signalling implications for AA in the latter experimental

preparation were not explored further but it is interesting that AA induces Ca2+

influx in primary rat astrocyte cell cultures (Sergeeva et al., 2003). AA is

thought to activate Ca2+ channels directly in DDT1 MF-2 cells, as inhibitors of

the cyclooxygenase and lipoxygenase pathway did not affect the

characteristics of the histamin

1

pathways had no effect on the CP 55,940-evoked outward current.

Comparable observations showing that AA rather than its metabolites mediate

NCCE have also been made in other preparations (Putney, 1990; Munaron et

al., 1997; Mignen and Shuttleworth, 2000).

The non-selective Ca2+ channel blocker La3+ abolished the CP 55,940-evoked

outward current. At the same concentration La3+ has been shown to inhibit

IARC in HEK 293 cells (Mignen and Shuttleworth, 2000), bovine aortic

endothelial cells (Fiorio Pla and Munaron, 2001) and DDT1 MF- 2 cells (Van

46

______________________________________________________Chapter 2

der Zee et al., 1995). La3+ was also able to inhibit outward currents evoked by

AA almost completely, which suggests that AA production occurs upstream of

Ca2+ entry and hence Ca2+ channel activation. The small remaining part of the

outward current induced by AA in the presence of the same La3+

concentration that abolished the CP 55,940-induced response, may be

ttributed to AA -mediated release of Ca2+ from internal stores (Watson et al.,

ooth muscle cells; at low

but had no effect on CCE evoked by thapsigargin.

his may suggest that the AA-sensitive channels in DDT1 MF-2 cells are

a

2004; Van der Zee et al., 1995; Fiorio Pla and Munaron, 2001). La3+ can also

inhibit CCE (Putney, 2001), so we investigated the effects of this inhibitor on

thapsigargin-evoked CCE in DDT1 MF-2 cells.

Thapsigargin depletes Ca2+ stores by inhibiting the SERCA pumps on the

sarcoplasmic reticulum, thereby initiating Ca2+ influx (Holda et al., 1998). Ni2+

decreased Ca2+ entry evoked by thapsigargin, in a concentration-dependent

manner but interestingly La3+ had no effect on this Ca2+ influx, at a

concentration that abolished the CP 55,940-evoked current. This clearly

implicates a Ca2+ influx pathway utilised during CB1 signalling in DDT1 MF-2

cells distinct from CCE.

Gd3+, another inhibitor of Ca2+ influx, is able to distinguish between AA-

sensitive CCE and NCCE in rat aortic sm

concentrations (1 μM) Gd3+ inhibited CCE, while at higher concentrations (100

μM) both CCE and NCCE were inhibited (Broad et al., 1999). Other authors

have shown the CCE pathway to be potently inhibited by 1 μM Gd3+ (Luo et

al., 2001; Putney, 2001), but Gd3+-insensitive SOCCs are also expressed in

some experimental systems (Fernando and Barritt, 1994; Fernando and

Barritt, 1995). In DDT1 MF-2 cells Gd3+ (1 μM) reduced the outward current in

response to CP 55,940,

T

distinct from those described in rat aortic smooth muscle cells, which is also

supported by the finding that SKF 96365 which at 100 nM inhibited NCCE in

rat aortic cells (Moneer et al., 2003), at a 100 fold greater concentration has

no effect on the CP 55,940-evoked outward current in DDT1 MF-2 cells. Our

data agree with previous work showing that low concentrations of Gd3+ can

inhibit currents mediated by IARC and Ca2+ influx in response to AA application

47

______________________________________________________Chapter 2

in HEK 293 cells (Mignen et al., 2003) and rat astrocytes (Sergeeva et al.,

2003), respectively, indicating that the present Gd3+ results are consistent with

the idea that the Ca2+ influx pathway initiated during CB1 stimulation is not

capacitative.

A major property of IARC activation is that it occurs independently from

intracellular Ca2+ store depletion (Broad et al., 1999; Fiorio Pla and Munaron,

2001; Mignen and Shuttleworth, 2000). However, it was previously shown that

thapsigargin inhibited the outward current induced by CP 55,940 in DDT1

ill already activate the outward current (Begg et al., 2001),

ereby antagonising the CP 55,940-induced NCCE signal to these channels.

ctivity of both MAP kinase and cytosolic PLA2 (Wartmann et al., 1995). The

non-selective PLA2 inhibitor quinacrine reduced the CP 55,940-evoked

MF-

2 cells, suggesting that CB1-mediated Ca entry is partly dependent on

intracellular Ca release (Begg et al., 2001). Our present results, indicating a

NCCE evoked response after CP 55,940 stimulation, offer an explanation for

this apparent inhibition, since emptying Ca stores by thapsigargin

pretreatment w

2+

2+

2+

th

Reciprocal regulation of CCE and NCCE has been described in other cells

exhibiting AA-mediated Ca2+ influx (Moneer and Taylor, 2002; Moneer et al.,

2003; Mignen et al., 2001; Luo et al., 2001). In accordance with the

proposition of IARC activation after CB1 receptor stimulation, such a reciprocal

mechanism seems not to be active in DDT1 MF-2 cells since inhibition of CCE

by SKF 96365 was without effect on the CP 55,940-induced outward current.

Another objective of the current study was to examine the putative link

between MAP kinase activation and the rise in [Ca2+]I following the generation

of AA by stimulation of the CB1 receptor. Liberation of AA can occur through

the direct action of PLA2 on phospholipids. In particular cytosolic PLA2 has

been shown to mediate AA liberation, regulated by Ca2+ and requiring

phosphorylation by MAP kinase for maximal activation (Kudo and Murakami,

2002).

Experiments in fetal lung fibroblasts have suggested that a cannabinoid-

mediated increase in AA is associated with an increased phosphorylation and

a

48

______________________________________________________Chapter 2

current in DDT1 MF-2 cells and abolished the [3H]AA release. Inhibition of AA

release was also observed in the presence of the MAP kinase inhibitor PD

98059. Together, these AA release data suggest that the primary pathway for

AA production, during CB1 stimulation, involves the activation of both PLA2

and MAP kinase. Ca2+ influx through the NCCE pathway is essential for this

activation in view of the complete inhibition of AA release observed in the

presence of Gd3+.

egg et al., 2001). This is consistent with the idea that the

ain increase in [Ca2+]i of the histamine response is derived from InsP3-

The intracellular signalling pathways that evoked a release of AA in DDT1 MF-

2 cells during H1-receptor stimulation, have not been identified, although

stimulation of the receptor has been shown to induce phosphorylation of MAP

kinase, with considerable phosphorylation occurring already after one minute

(Robinson and Dickenson, 2001). This suggests an immediate action of MAP

kinase, presumably resulting in PLA2-induced release of AA, during H1

stimulation in DDT1 MF-2 cells. Previous work has shown that both the

histamine- and CP 55,940-induced currents in DDT1 MF-2 cells were inhibited

by the MAP kinase inhibitor PD 98059, but that histamine was less sensitive

to this inhibition (B

m

sensitive stores and that PD 98059 is only inhibiting the NCCE component of

the histamine response.

49

______________________________________________________Chapter 2

Ca2+Ca2+Ca2+Ca2+

CB1R

SR 141716 CP 55,940

cPLA2AAAA

Ca2+

MAP kinase PD 98059+

Thapsigargin

QuinacrineQuinacrine

2-APB

Gd3+

La3+Ni2+

2-APBSKF 96365

K+

K+

NCCE CCE

SERC

A+

+ +

+ +

IP3R

Figure 10. Model for cannabinoid-induced NCCE in DDT1 MF-2 cells. In this scheme all

experimental data presented are depicted. The pathway elicited by CB1 receptor stimulation

with CP 55,940 (bold lines) generates arachidonic acid (AA) mediated by MAP kinase-

sensitive cytosolic PLA2 activity. Release of AA induces non capacitative Ca2+ entry (NCCE)

via IARC and subsequent activation of Ca2+-dependent K+ channels. NCCE is essential for

further AA release. Relevant parts of the CCE pathway are also depicted, showing Ca2+ entry

in response to depletion of 2-APB-sensitive Ca2+ pools by thapsigargin. + indicates activation

and inhibition.

The simplified scheme as depicted in Fig.10 summarizes the main features of

the results obtained, which are consistent with the proposition that

cannabinoid signalling generates AA-induced NCCE by MAP kinase regulated

activation of cPLA2. The resulting Ca2+ influx via IARC further stimulates cPLA2

activity and AA release, and simultaneously elicits outward current.

50

______________________________________________________Chapter 2

In conclusion, we have shown for the first time that stimulation of CB1

receptors can lead to Ca2+ influx mediated by AA. This influx pathway is

distinct from CCE and instead involves the activation of IARC and hence

NCCE. Our results also provide evidence for a CB1 receptor-mediated

activation of PLA2, upstream of AA production and presumably downstream of

MAP kinase activation.

Acknowledgements – We thank Pfizer (UK) and Sanofi (France) for their kind

gifts of CP 55,940 and SR 141716A respectively, and the Netherlands

Asthma Foundation for a travel grant to RMvR.

51

______________________________________________________Chapter 2

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46. Van der Zee L., Nelemans A, Den Hertog A., 1995. Arachidonic acid is functioning as a second messenger in activating the Ca2+ entry process on H1-histaminoceptor stimulation in DDT1 MF-2 cells. Biochem. J. 305 ( Pt 3): 859-864.

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48. Wartmann M, Campbell D, Subramanian A, Burstein SH, Davis RJ, 1995. The MAP kinase signal transduction pathway is activated by the endogenous cannabinoid anandamide. FEBS Lett. 359: 133-136.

49. Watson EL, Jacobson KL, Singh JC, DiJulio DH, 2004. Arachidonic acid regulates two Ca2+ entry pathways via nitric oxide. Cell Signal. 16: 157-165.

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