University of Groningen
(Endo)cannabinoid signaling in human bronchial epithelial and smooth muscle cells
Gkoumassi, Effimia
IMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to
cite from it. Please check the document version below.
Document Version
Publisher's PDF, also known as Version of record
Publication date:
2007
Link to publication in University of Groningen/UMCG research database
Citation for published version (APA):
Gkoumassi, E. (2007). (Endo)cannabinoid signaling in human bronchial epithelial and smooth muscle cells
s.n.
Copyright
Other than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of the
author(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons).
Take-down policy
If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately
and investigate your claim.
Downloaded from the University of Groningen/UMCG research database (Pure): http://www.rug.nl/research/portal. For technical reasons the
number of authors shown on this cover page is limited to 10 maximum.
Download date: 18-06-2017
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-receptorevoked 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 CB1 receptor-induced increase in [Ca2+]i, including the
B
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 heatpolished (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; NaGTP, 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. Postexperimentation, 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 (2.223.70TBq)/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 fura2 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
hydroxy-propyl)
((-)-cis-3-[2-Hydroxy-4-(1,1-dimethylheptyl)phenyl]-trans-4-(3cyclohexanol)
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)-4H1benzopyran-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 MF2 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
1200
Figure 1. The cannabinoid receptor
CP 55,940
agonist CP 55,940 and histamine evoke
Current (pA)
900
outward currents in DDT1 MF-2 cells. A,
600
sample trace of the transient outward
300
current evoked by 10 μM CP 55,940. B,
0
sample trace of the transient outward
0
200
current evoked by 10 μM histamine. Bars
400
indicate the presence of ligands.
Time (s)
B
Histamine
1500
Current (pA)
1200
900
600
300
0
0
200
400
600
Time (s)
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.
Ca
2+
i
(AUC, nM.sec) (x 103)
30
Ca2+
Figure 2. CP 55,940 increases
Ca2+ Free
[Ca2+]i, which is dependent on Ca2+
from the extracellular space. CP
20
55,940
increases
[Ca2+]i
concentration-dependent
10
in
a
manner
(n=4). This increase is abolished in
the absence of extracellular Ca2+
(n=4). In Ca2+-free medium higher
0
concentrations of CP 55,940 reduce
[Ca2+]i.
-10
8
7
6
-log [CP 55,940] (M)
34
5
4
______________________________________________________Chapter 2
Capacitative Ca2+ Entry (CCE): Role of Store-Operated Ca2+ Channels
(SOCCs) - To examine the involvement of InsP3 receptors in the cannabinoidinduced 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
A
1000
Current (pA)
800
CP 55,940
2-APB
Figure 3. 2-APB has no effect
on CP 55,940-evoked currents
but inhibits the currents evoked
600
by histamine. A, sample trace
400
of the effect of 2-APB on the
200
CP 55 940 (10 μM)-mediated
0
current. B, sample trace of the
0
200
400
600
800
indicate
Current (pA)
Histamine
1000
800
Peak Current (% of Control)
on
the
the
presence
of
effect on the outward current
evoked by 10 μM CP 55,940
600
(n=6) at a concentration that
400
inhibited
200
histamine-evoked
currents (n=5).
Significant
0
100
200
Time (s)
200
150
100
50
*
0
CP 55,940 2-APB + Histamine 2-APB +
CP 55,940
Histamine
36
2-APB
ligands. C, 2-APB had no
2-APB
0
C
of
histamine-evoked current. Bars
Time (s)
B
effect
difference
from
histamine control: * P<0.01.
______________________________________________________Chapter 2
Figure 4. AA induces an
2500
Current (pA)
A
CP 55,940
Arachidonic Acid
outward
inhibits the response to
1500
CP 55,940. A, sample
1000
trace
transient,
effect of this on the CP
200
2500
Current (pA)
the
by 10 μM AA and the
400
600
800
55,940 (10 μM)-induced
Time (s)
Arachidonic Acid
current. B, sample trace
of the outward current
CP 55,940
evoked by 50 μM AA and
2000
the effect of this on the
1500
CP
1000
evoked
55,940
(10
μM)-
current.
Bars
indicate the presence of
500
ligands. C, AA produces a
0
concentration-dependent
0
200
400
600
800
1000
increase
Time (s)
in
membrane
current (n≥4). 10 μM AA
250
Peak Current (% of Control)
of
outward current evoked
500
0
C
which
2000
0
B
current,
*
reduced
the
outward
current evoked by 10 μM
200
CP 55,940 (n=6) and at a
concentration of 50 μM
150
completely abolished the
response (n=6).
100
Significant difference from
AA (1 μM): * P<0.01,
†
50
significant difference from
†
0
CP
55,940
control:
†
P<0.0001.
AA
(1µM)
AA
(10 µM)
AA
(50 µM)
CP
55,940
+ AA
+ AA
(10 µM) (50 µM)
37
______________________________________________________Chapter 2
Non-Capacitative Ca2+ Entry (NCCE): Role of Arachidonic Acid (AA) - AA has
been shown to produce a concentration-dependent rise in [Ca2+]i in DDT1 MF2 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 pre-exposure to AA had no effect on the CP 55,940-evoked outward current
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).
[3H]AA Release (% of Basal)
100
Figure 5. CP 55,940 evokes a
80
release
of
[3H]AA.
[3H]AA-
prelabelled DDT1 MF-2 cells were
60
stimulated with CP 55,940 and the
release of radiation measured and
40
presented
as
increase
in
the
release as % of basal level. Data
represent
20
8
experiments
in
triplicate. Basal AA release: 16.6
±2.6 dps/well.
0
Basal
7.5
7
6.5
6
5.5
5
-log [CP 55,940] (M)
To further test the hypothesis that AA is generated in response to CB1
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 receptormediated, 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).
Indomethacin, a non-selective cyclooxygenase inhibitor (Bakalova et al.,
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).
Table 1.
The effect of various inhibitors on [3H] arachidonic acid release (dps/well)
Control
+ CP 55,940 (1μM)
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 (number of experiments). Significance level: * ,
P< 0.002 vs. control
Lanthanum (La3+)-Sensitive and Gadolinium (Gd3+)-Sensitive Ca2+ Influx - In
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).
Peak Current (% of Control)
120
Figure 6. La3+ inhibits the
outward current evoked by
100
CP 55,940 and AA. 50 μM
La3+ inhibited the AA (10
80
μM)-evoked outward current
(n=6)
60
and
completely
abolished the response to
40
*
10 μM CP 55,940 (n=7).
Significant difference from
20
CP 55,940 and AA control: *
**
P<0.001, ** P<0.0001.
0
CP 55,940
La3+ +
CP 55,940
AA
La3+ + AA
CCE in DDT1 MF-2 cells can be evoked with the sarcoplasmic-endoplasmic
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
A
150
a
[Ca2+]i (nM)
CCE. Effects of Ni2+, La3+ and
Ni2+
Ca2+
b
c
Gd3+. Internal Ca2+ stores were
d
100
initially depleted with 1 μM
e
f
thapsigargin
in
Ca2+-free
medium. Re-addition of 1 mM
50
Ca2+
Thapsigargin
produced
0
0
1000
2000
to
the
a
external milieu
rise
in
[Ca2+]i
reflecting the activation of CCE.
3000
A, sample trace of the effect of
Time (s)
Ni2+ at concentrations : 0.1, 1
and 10 μM (a), 100 μM (b), 300
B
[Ca2+]i (nM)
150
Ca2+
La3+
μM (c), 1 mM (d), 3 mM (e), 10
Ni2+
a
b
c
100
mM (f). B, sample trace of the
d
effect of La3+ at concentrations:
e
f
0.01, 0.1, 1, 10 and 100 μM (a),
followed by addition of Ni2+ at
50
concentrations: 100 μM (b), 300
Thapsigargin
0
μM (c), 1 mM (d), 3 mM (e), 10
0
1000
2000
3000
mM (f). C, sample trace of the
Time (s)
effect of Gd3+ at concentrations:
0.01, 0.1, 1, 10 and 100 μM (a),
C
Gd3+
Ca2+
500
Ni2+
a
[Ca2+]i (nM)
400
b
followed by addition of Ni2+ at
concentrations: 100 μM (b), 300
c
d
300
μM (c), 1 mM (d). In contrast to
Ni2+ no effects on CCE were
observed for La3+ or Gd3+ (n=3).
200
100
Thapsigargin
0
0
1000
2000
3000
Time (s)
In rat aortic smooth muscle cells low concentrations (1 μM) of Gd3+ inhibited
CCE, while higher concentrations (100 μM) were shown to inhibit both CCE
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).
A
CP 55,940
800
Figure
Gd 3+
Gd3+
inhibits
the
outward current evoked by CP
600
Current (pA)
8.
55,940. A, sample trace of the
effect of Gd3+ (1 μM) on the CP
400
55,940 (10 μM)-evoked outward
200
current.
Bars
indicate
the
presence of ligands. B, Gd3+
0
0
100
200
significantly reduces the outward
current evoked by 10 μM CP
Time (s)
55,940
difference
B
from
Significant
CP
55,940
control: * P<0.0001.
120
Peak Current (% of Control)
(n=9).
100
80
60
*
40
20
0
CP 55,940
Gd3+ + CP 55,940
AA Production: Role of Phospholipase A2 (PLA2) and MAP kinase Cannabinoid-induced mobilisation of AA was shown to involve the activities of
the cytoplasmic PLA2 isoenzyme in WI-38 lung fibroblasts (Wartmann et al.,
1995). To explore the possibility that AA is being generated by PLA2 in DDT1
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.
A
1500
Current (pA)
1200
Figure 9. Quinacrine inhibits the CP
CP 55,940
Quinacrine
55,940-induced outward current and
release of [3H]AA. Cells were pre-
900
treated with quinacrine (10-30 μM)
600
for
10
minutes
before
agonist
application. A, sample trace of the
300
effect of 30 μM quinacrine on the
0
0
200
400
Time (s)
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.
B
Significant
150
Peak Current (% of Control)
55,940
difference
control:
*
from
P<0.01,
CP
**
P<0.001.
100
**
*
50
0
CP 55,940
+ Quinacrine + Quinacrine
(10 μM)
(30 μM)
Quinacrine (100 μM) alone had no effect on membrane currents in DDT1 MF2 cells (results not shown, n=4). To add further support to the involvement of
CB1-mediated activation of PLA2, the effect of quinacrine (100 μM) on CP
55,940-evoked [3H]AA release was established. Quinacrine alone had no
effect on basal [3H]AA efflux, but the CP 55,940 (1 μM)-evoked AA release
was abolished (Table 1). CP 55,940-induced AA release is also inhibited
strongly by the MAP kinase pathway inhibitor PD 98059 (30 μM, Table 1),
supporting the hypothesis that PLA2 activation by CP 55,940 is regulated by
MAP kinase.
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 concentration-dependent increase in [Ca2+]i by CP 55,940, which
was abolished by the removal of extracellular Ca2+. This suggested that the
CB1-induced increase in [Ca2+]i is entirely dependent on Ca2+ influx from the
extracellular space, although Ca2+ release from thapsigargin-sensitive stores
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 membranebound 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 evoked by the
cannabinoid receptor agonist CP 55,940 (Begg et al., 2001). Similarly we
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
the resulting membrane current was designated as IARC (Mignen and
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 histamineevoked NCCE-mediated Ca2+ influx, as reflected by abolition of outward
current (Van der Zee et al., 1995). This suggests that AA activates the same
Ca2+ channels that mediate Ca2+ influx during CB1 as well as H1 receptor
stimulation. In order to determine whether CB1-induced increases in [Ca2+]i
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 AA production. This was confirmed by using the
CB1 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 cannabinoid
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 histamine-induced [3H]AA release (Van der Zee et al.,
1995). In accordance with this we found that inhibitors of either of these two
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
attributed to AA -mediated release of Ca2+ from internal stores (Watson et al.,
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 AAsensitive CCE and NCCE in rat aortic smooth muscle cells; at low
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, but had no effect on CCE evoked by thapsigargin.
This may suggest that the AA-sensitive channels in DDT1 MF-2 cells are
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 MF2 cells, suggesting that CB1-mediated Ca2+ entry is partly dependent on
intracellular Ca2+ 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 Ca2+ stores by thapsigargin
pretreatment will already activate the outward current (Begg et al., 2001),
thereby antagonising the CP 55,940-induced NCCE signal to these channels.
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 cannabinoidmediated increase in AA is associated with an increased phosphorylation and
activity of both MAP kinase and cytosolic PLA2 (Wartmann et al., 1995). The
non-selective PLA2 inhibitor quinacrine reduced the CP 55,940-evoked
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+.
The intracellular signalling pathways that evoked a release of AA in DDT1 MF2 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 (Begg et al., 2001). This is consistent with the idea that the
main increase in [Ca2+]i of the histamine response is derived from InsP3sensitive stores and that PD 98059 is only inhibiting the NCCE component of
the histamine response.
49
______________________________________________________Chapter 2
CP 55,940
SR 141716
CB1R
Quinacrine
NCCE
+
Ca2+
cPLA2
AA
MAP kinase
+
+
Ca2+
Ca2+
K+
Gd3+
La3+
+
PD 98059
Thapsigargin
SE
RC
A
AA
+
CCE
Ca2+
IP3R
+
Ca2+
Ni2+
2-APB
SKF 96365
2-APB
K+
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 kinasesensitive 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
References
1. Bakalova R, Matsuura T, Kanno I, 2002. The cyclooxygenase inhibitors indomethacin
and Rofecoxib reduce regional cerebral blood flow evoked by somatosensory
stimulation in rats. Exp. Biol. Med. (Maywood. ) 227: 465-473.
2. Begg M, Baydoun A, Parsons ME, Molleman A, 2001. Signal transduction of
cannabinoid CB1 receptors in a smooth muscle cell line. J. Physiol 531: 95-104.
3. Bouaboula M, Poinot-Chazel C, Bourrie B, Canat X, Calandra B, Rinaldi-Carmona M,
Le Fur G, Casellas P, 1995. Activation of mitogen-activated protein kinases by
stimulation of the central cannabinoid receptor CB1. Biochem. J. 312 ( Pt 2): 637-641.
4. Broad LM, Cannon TR, Taylor CW, 1999. A non-capacitative pathway activated by
arachidonic acid is the major Ca2+ entry mechanism in rat A7r5 smooth muscle cells
stimulated with low concentrations of vasopressin. J. Physiol 517 ( Pt 1): 121-134.
5. Childers SR, Deadwyler SA, 1996. Role of cyclic AMP in the actions of cannabinoid
receptors. Biochem. Pharmacol. 52: 819-827.
6. Den Hertog A, Hoiting B, Molleman A, van den Akker J, Duin M, Nelemans A, 1992.
Calcium release from separate receptor-specific intracellular stores induced by
histamine and ATP in a hamster cell line. J. Physiol 454: 591-607.
7. Dobrydneva Y, Blackmore P, 2001. 2-Aminoethoxydiphenyl borate directly inhibits
store-operated calcium entry channels in human platelets. Mol. Pharmacol. 60: 541552.
8. Fernando KC, Barritt GJ, 1994. Characterisation of the inhibition of the hepatocyte
receptor-activated Ca2+ inflow system by gadolinium and SK&F 96365. Biochim.
Biophys. Acta 1222: 383-389.
9. Fernando KC, Barritt GJ, 1995. Characterisation of the divalent cation channels of the
hepatocyte plasma membrane receptor-activated Ca2+ inflow system using lanthanide
ions. Biochim. Biophys. Acta 1268: 97-106.
10. Filipeanu CM, de Zeeuw D, Nelemans SA, 1997. Delta9-tetrahydrocannabinol
activates [Ca2+]i increases partly sensitive to capacitative store refilling. Eur. J.
Pharmacol. 336: R1-R3.
11. Fiorio Pla A, Munaron L, 2001. Calcium influx, arachidonic acid,and control of
endothelial cell proliferation. Cell Calcium 30: 235-244.
12. Gebremedhin D, Lange AR, Campbell WB, Hillard CJ, Harder DR, 1999. Cannabinoid
CB1 receptor of cat cerebral arterial muscle functions to inhibit L-type Ca2+ channel
current. Am. J. Physiol 276: H2085-H2093.
13. Holda JR, Klishin A, Sedova M, Huser J, Blatter LA, 1998. Capacitative Calcium
Entry. News Physiol Sci. 13: 157-163.
14. Howlett AC, Qualy JM, Khachatrian LL, 1986. Involvement of Gi in the inhibition of
adenylate cyclase by cannabimimetic drugs. Mol. Pharmacol. 29: 307-313.
15. Hunter S. A. and Burnstein, S. H. (1997) Receptor mediation in cannabinoid stimulated
arachidonic acid mobilisation and anandamide synthesis. Life Sci. 60, 1563-1573.
16. Kerschbaum HH, Cahalan MD, 1999. Single-channel recording of a store-operated
Ca2+ channel in Jurkat T lymphocytes. Science 283: 836-839.
52
______________________________________________________Chapter 2
17. Kim HC, Jhoo WK, Bing G, Shin EJ, Wie MB, Kim WK, Ko KH, 2000. Phenidone
prevents kainate-induced neurotoxicity via antioxidant mechanisms. Brain Res. 874:
15-23.
18. Kirber MT, Ordway RW, Clapp LH, Walsh JVJr, Singer JJ, 1992. Both membrane
stretch and fatty acids directly activate large conductance Ca2+-activated K+ channels
in vascular smooth muscle cells. FEBS Lett. 297: 24-28.
19. Kudo I, Murakami M, 2002. Phospholipase A2 enzymes. Prostaglandins Other Lipid
Mediat. 68-69: 3-58.
20. Liu J, Gao B, Mirshahi F, Sanyal AJ, Khanolkar AD, Makriyannis A, Kunos G, 2000.
Functional CB1 cannabinoid receptors in human vascular endothelial cells. Biochem.
J. 346 Pt 3: 835-840.
21. Lu XR, Ong WY, Halliwell B, 2001. The phospholipase A2 inhibitor quinacrine
prevents increased immunoreactivity to cytoplasmic phospholipase A2 (cPLA2) and
hydroxynonenal (HNE) in neurons of the lateral septum following fimbria-fornix
transection. Exp. Brain Res. 138: 500-508.
22. Luo D, Broad LM, Bird GS, Putney JWJr, 2001. Mutual antagonism of calcium entry
by capacitative and arachidonic acid-mediated calcium entry pathways. J. Biol.
Chem. 276: 20186-20189.
23. Mackie K, Lai Y, Westenbroek R, Mitchell R, 1995. Cannabinoids activate an inwardly
rectifying potassium conductance and inhibit Q-type calcium currents in AtT20 cells
transfected with rat brain cannabinoid receptor. J. Neurosci. 15: 6552-6561.
24. Maruyama T, Kanaji T, Nakade S, Kanno T, Mikoshiba K, 1997. 2APB, 2aminoethoxydiphenyl borate, a membrane-penetrable modulator of Ins(1,4,5)P3induced Ca2+ release. J. Biochem. (Tokyo) 122: 498-505.
25. Matsuda LA, Lolait SJ, Brownstein MJ, Young AC, Bonner TI, 1990. Structure of a
cannabinoid receptor and functional expression of the cloned cDNA. Nature 346: 561564.
26. Merritt JE, Armstrong WP, Benham CD, Hallam TJ, Jacob R, Jaxa-Chamiec A, Leigh
BK, McCarthy SA, Moores KE, Rink TJ, 1990. SK&F 96365, a novel inhibitor of
receptor-mediated calcium entry. Biochem. J. 271: 515-522.
27. Mignen O, Shuttleworth TJ, 2000. I(ARC), a novel arachidonate-regulated,
noncapacitative Ca2+ entry channel. J. Biol. Chem. 275: 9114-9119.
28. Mignen O, Thompson JL, Shuttleworth TJ, 2001. Reciprocal regulation of capacitative
and arachidonate-regulated noncapacitative Ca2+ entry pathways. J. Biol. Chem. 276:
35676-35683.
29. Mignen O, Thompson JL, Shuttleworth TJ, 2003. Ca2+ selectivity and fatty acid
specificity of the noncapacitative, arachidonate-regulated Ca2+ (ARC) channels. J.
Biol. Chem. 278: 10174-10181.
30. Molleman A, Nelemans A, Den Hertog A, 1989. P2-purinoceptor-mediated membrane
currents in DDT1 MF-2 smooth muscle cells. Eur. J. Pharmacol. 169: 167-174.
31. Molleman A, Nelemans A, van den Akker J, Duin M, Den Hertog A, 1991. Voltagedependent sodium and potassium, but no calcium conductances in DDT1 MF-2
smooth muscle cells. Pflugers Arch. 417: 479-484.
53
______________________________________________________Chapter 2
32. Moneer Z, Dyer JL, Taylor CW, 2003. Nitric oxide co-ordinates the activities of the
capacitative and non-capacitative Ca2+-entry pathways regulated by vasopressin.
Biochem. J. 370: 439-448.
33. Moneer Z, Taylor CW, 2002. Reciprocal regulation of capacitative and noncapacitative Ca2+ entry in A7r5 vascular smooth muscle cells: only the latter operates
during receptor activation. Biochem. J. 362: 13-21.
34. Munaron L, Antoniotti S, Distasi C, Lovisolo D, 1997. Arachidonic acid mediates
calcium influx induced by basic fibroblast growth factor in Balb-c 3T3 fibroblasts. Cell
Calcium 22: 179-188.
35. Munro S, Thomas KL, bu-Shaar M, 1993. Molecular characterization of a peripheral
receptor for cannabinoids. Nature 365: 61-65.
36. Osterhout JL, Shuttleworth TJ, 2000. A Ca2+-independent activation of a type IV
cytosolic phospholipase A2 underlies the receptor stimulation of arachidonic aciddependent noncapacitative calcium entry. J. Biol. Chem. 275: 8248-8254.
37. Putney JWJr, 2001. Pharmacology of capacitative calcium entry. Mol. Interv. 1: 8494.
38. Putney JWJr, 1990. Capacitative calcium entry revisited. Cell Calcium 11: 611-624.
39. Robinson AJ, Dickenson JM, 2001. Activation of the p38 and p42/p44 mitogenactivated protein kinase families by the histamine H1 receptor in DDT1 MF-2 cells. Br.
J. Pharmacol. 133: 1378-1386.
40. Schatz AR, Lee M, Condie RB, Pulaski JT, Kaminski NE, 1997. Cannabinoid
receptors CB1 and CB2: a characterization of expression and adenylate cyclase
modulation within the immune system. Toxicol. Appl. Pharmacol. 142: 278-287.
41. Sergeeva M, Strokin M, Wang H, Ubl JJ, Reiser G, 2003. Arachidonic acid in
astrocytes blocks Ca2+ oscillations by inhibiting store-operated Ca2+ entry, and
causes delayed Ca2+ influx. Cell Calcium 33: 283-292.
42. Shivachar AC, Martin BR, Ellis EF, 1996. Anandamide- and delta9tetrahydrocannabinol-evoked arachidonic acid mobilization and blockade by
SR141716A [N-(Piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4 -methyl1H-pyrazole-3-carboximide hydrochloride]. Biochem. Pharmacol. 51: 669-676.
43. Sugiura T, Kodaka T, Kondo S, Tonegawa T, Nakane S, Kishimoto S, Yamashita A,
Waku K, 1996. 2-Arachidonoylglycerol, a putative endogenous cannabinoid receptor
ligand, induces rapid, transient elevation of intracellular free Ca2+ in neuroblastoma x
glioma hybrid NG108-15 cells. Biochem. Biophys. Res. Commun. 229: 58-64.
44. Tsou K, Brown S, Sanudo-Pena MC, Mackie K, Walker JM, 1998.
Immunohistochemical distribution of cannabinoid CB1 receptors in the rat central
nervous system. Neuroscience 83: 393-411.
45. Twitchell W, Brown S, Mackie K, 1997. Cannabinoids inhibit N- and P/Q-type calcium
channels in cultured rat hippocampal neurons. J. Neurophysiol. 78: 43-50.
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.
47. Wagner JA, Jarai Z, Batkai S, Kunos G, 2001. Hemodynamic effects of cannabinoids:
coronary and cerebral vasodilation mediated by cannabinoid CB1 receptors. Eur. J.
Pharmacol. 423: 203-210.
54
______________________________________________________Chapter 2
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.
55