Coupling of M2 Muscarinic Receptors to Membrane Ion
Channels via Phosphoinositide 3-Kinase
and Atypical Protein Kinase
C*
Yong-Xiao
Wang,
Prasad D. K.
Dhulipala,
Lei
Li,
Jeffrey L.
Benovic
, and
Michael I.
Kotlikoff§
From the Department of Animal Biology, School of Veterinary
Medicine, University of Pennsylvania, Philadelphia, Pennsylvania
19104 and the
Kimmel Cancer Institute, Thomas Jefferson
University, Philadelphia, Pennsylvania 19107
 |
ABSTRACT |
We report a novel signaling pathway linking
M2 muscarinic receptors to metabotropic ion channels.
Stimulation of heterologously expressed M2 receptors, but
not other Gi/Go-associated receptors (M4 or
2c), activates a calcium- and
voltage-independent chloride current in Xenopus oocytes. We
show that the stimulatory pathway linking M2 receptors to
these chloride channels consists of G
stimulation of
phosphoinositide 3-kinase
(PI-3K
), formation of
phosphatidylinositol 3,4,5-trisphosphate (PIP3), and
activation of atypical protein kinase C (PKC). The chloride current is
activated in the absence of M2 receptor stimulation by the
injection of PIP3, and PIP3 current activation
is blocked by a pseudosubstrate inhibitory peptide of atypical PKC but
not other PKCs. Moreover, the current is activated by injection of
recombinant PKC
at concentrations as low as 1 nM.
M2 receptor-current coupling was disrupted by inhibiton of
PI-3K and by injection of 
binding peptides, but it was not
affected by expression of dominant negative p85 cRNA. We also show that
this pathway mediates M2 receptor coupling to metabotropic
nonselective cation channels in mammalian smooth muscle cells, thus
demonstrating the broad relevance of this signaling cascade in
neurotransmitter signaling.
 |
INTRODUCTION |
M2 muscarinic receptors mediate numerous cellular
functions including presynaptic regulation of transmitter release by
neurons in the brain and autonomic nervous system and postsynaptic
control of the heart, smooth muscle, and secretory cells. Stimulation of M2 receptors in heart cells opens inward rectifier
K+ channels through a direct interaction between released G
protein 
subunits and channel proteins (1-6), but the signaling
pathways linking M2 receptors to ion channels in nerve,
smooth muscle, and secretory cells are poorly understood.
Hormone-stimulated phosphoinositide 3-kinase
(PI-3K)1 plays an important
role in cell growth, adhesion, and survival and in actin assembly (7).
The identification of the G
-stimulated PI-3K
(8-10) extends
the potential range of processes mediated by PI-3K to G protein-coupled
receptors, although specific physiological processes mediated by
PI-3K
have not been identified. One potential target of lipid second
messengers generated by PI-3K are atypical protein kinase C enzymes
(aPKCs), which lack a diacylglycerol binding site and are activated
in vitro by phosphatidylinositol phosphates (11-14). Here
we show that stimulation of heterologously expressed M2
receptors, but not other Gi/Go-linked
receptors, opens endogenous metabotropic chloride channels in
Xenopus oocytes by activation of PI-3K
, generation of
PIP3, and stimulation of aPKC. We also show that this
signaling pathway mediates physiological coupling between
M2 receptors and nonselective cation channels in mammalian
smooth muscle cells.
 |
EXPERIMENTAL PROCEDURES |
Xenopus Oocyte Procedures--
Surgical removal of oocytes was
performed in the laboratory of Dr. Peter Drain in accordance with a
protocol approved by the University of Pennsylvania Animal Care and Use
Committee. Oocyte defolliculation, injection, and dual-electrode
voltage clamp were as described previously (15). Currents were
amplified (OC-725C, Warner Instruments, Hamden, CT), filtered at 200 Hz
(
3 dB; 8-pole Bessel filter, model 902, Frequency Devices, Haverhill,
MA), digitized at 1 kHz (TL125, Axon Instruments, Foster City, CA), and
monitored and simultaneously stored on disk (Axotape, Axon
Instruments). All currents shown were leak-subtracted using identical
voltage paradigms before exposure to mACH. Pipettes with resistances
between 0.5 and 1 megaohm were filled with 3 M KCl.
Extracellular bath solution was (concentrations in mM): 115 NaCl, 2.8 KCl, 1 MgCl2, and 10 Hepes. Intracellular
injection of all substances consisted of 50-nanoliter volumes, and the
indicated concentrations assume a 20-fold dilution in the oocyte
cytosol (1 µl volume). Injections were made 10 min before oocyte
stimulation. Antibodies directed against specific G
subunits were
injected at the obtained concentration (1:20 final titer). Solution
changes were made by washing the bath with at least 25× bath volume (1 ml).
Preparation of cRNAs--
cRNA was prepared using the mMessage
mMachine kit (Ambion). Plasmid DNAs were linearized with appropriate
restriction enzymes, and cRNAs were synthesized using the appropriate
RNA polymerase. The integrity of the cRNAs was tested on ethidium
bromide-stained agarose gels, and concentrations were estimated by
spectrophotometry. The
P85 construct (16) in PGEX was obtained from
Dr. M. Kasuga and subcloned in pBlueScript KS+ (Stratagene).
M2, M3, and M4 clones were kindly
provided by Dr. E. Peralta and Dr. T. Morelli.
Patch-clamp and Myocyte Dispersion--
Equine trachealis tissue
was obtained in accordance with protocols approved by the University of
Pennsylvania Animal Care and Use Committee. Cell isolation, whole cell
recording, and agonist application were as described previously (17).
Seals were formed with 3-5-megaohm pipettes, and cells were dialyzed
with the following (concentrations in mM): 130 CsCl, 1.2 MgCl2, 1 MgATP, 0.1 EGTA, and 10 Hepes, pH 7.3. The bath
solution was (concentrations in mM): 125 NaCl, 5 KCl, 1 MgSO4, 1.8 CaCl2, 10 glucose, and 10 Hepes, pH
7.4. Cells were allowed to adhere to a glass coverslip, and recordings
in relaxed cells were made at room temperature. Following break-in,
cells were dialyzed for 5 min before activation of currents by
application of the muscarinic agonist using a puffer pipette.
Chemicals--
Chelerythrine, GF109203X, Gö 6976, and
cPKC pseudosubstrate peptide were obtained from Calbiochem and
PIP3 and PKC
from Biomol. aPKC and QEHA peptides were
custom synthesized. Bovine transducin
was kindly provided by Dr.
H. E. Hamm.
 |
RESULTS AND DISCUSSION |
Heterologous expression of M2 receptors in
Xenopus oocytes indicated that receptor stimulation
activates a novel metabotropic chloride current. The muscarinic agonist
methacholine (mACH) activated only a sustained inward current in
oocytes injected with M2 receptor cRNA and recorded in
calcium-free conditions, whereas a transient inward current was
observed in oocytes expressing Gq/11-coupled M3
receptors (Fig. 1A). The
transient current was shown to be the ubiquitous endogenous
calcium-activated chloride current, as it was blocked by chelating
intracellular calcium or by inhibiting calcium release with heparin,
whereas activation of the M2 current could be obtained
repeatedly in calcium-free solutions and was not affected by
intracellular calcium chelation or by blockade of calcium release.
These currents were identified in early original experiments
characterizing muscarinic acetylcholine receptor subtypes in
Xenopus oocytes (18), although the sustained current was not
isolated and was reported to be cation selective. Ion substitution experiments clearly identified the M2 current as anion
selective, with a selectivity sequence of I
> Cl
> isethionate, whereas substitution of more than 90%
of the sodium for Tris had no effect on current reversal potential
(Fig. 1, B and C). The chloride current activated
following M2 receptor binding was voltage-independent, and
no measurable current was available in the absence of M2
stimulation (no shift in background current observed with anion
substitution), indicating that receptor binding is required for channel
opening.

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Fig. 1.
M2 receptors activate an
endogenous chloride current in Xenopus oocytes.
A, application of 50 µM mACH to oocytes
injected with M2 or M3 cRNA elicited markedly
different responses. The M2 current response was slowly
activating, sustained during muscarinic stimulation, and could be
activated repeatedly in nominally Ca2+-free solution
(inset shows second response in same oocyte). Neither
calcium chelation by incubation for 4 h with 50 µM
1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid
(BAPTA) nor block of intracellular calcium release by
preinjection of heparin (10 mg/ml) altered the M2 current.
Conversely, stimulation of M3-expressing oocytes activated
the transient, calcium-activated chloride current, which could not be
repeatedly activated in Ca2+-free solution and was blocked
by BAPTA incubation and heparin injection. B, the
M2 current is anion-selective. Cation substitution
(equimolar Tris-Cl (TrisCl) substituted for NaCl in bath
solution) did not alter the current-voltage relationship of the
M2 current, but anion substitution (sodium isethionate and
NaI for NaCl in the bath solution) markedly altered the magnitude and
reversal potential of the current, as predicted for an anion-selective
current. Currents shown are from voltage clamp steps to between 90
and 60 mV in 10-mV increments (Vh = 60 mV),
imposed during activation of the M2 current. Current
families were obtained before and after changing bath solution from the
control to the test solution. Figure shows control currents for Tris-Cl
experiment only. Changes in background current were negligible in test
solutions. Each experiment shown was performed in at least 5 oocytes.
C, current-voltage relationships for the experiments shown
in B, indicating a relative anion permeability sequence of
I > Cl > isethionate and no
shift with cation substitution.
|
|
To examine the linkage between M2 receptors and the novel
chloride current, we used antibodies, specific peptides, dominant negative constructs, and enzyme inhibitors to disrupt receptor-effector coupling. M2 receptor-chloride current coupling was blocked
by preinjection of antibodies directed against G
i or
G
o, but not G
q, proteins (Fig.
2, A and B).
Anti-G
i1/G
i2 and
anti-G
i3/G
o antibodies blocked 83 ± 3% (n = 6) and 52 ± 4% (n = 6)
of the current, respectively. Whereas M2 signaling was
clearly coupled by Gi/Go proteins, the
M2 current was not activated by heterologously expressed
adrenergic
2C receptors or muscarinic M4
receptors, which coupled weakly to intracellular calcium release and
the associated calcium-activated chloride current (Fig. 2C).
These receptors also preferentially associate with
Gi/Go proteins (19, 20), indicating that the
signaling pathway leading to activation of the M2 chloride
current discriminates between receptors signaling through pertussis
toxin-sensitive G proteins. Moreover, whereas both M2 and
M4 receptors are capable of activating inward rectifying K+ channels through 
proteins (21), the stimulatory
pathway linking M2 receptors and chloride channels
effectively distinguishes between these closely related receptors.

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Fig. 2.
Activation of the metabotropic chloride
current is not common to all Gi/Go-linked
receptors. A and B, injection of
subtype-specific antibodies directed against
Gi/Go proteins inhibited muscarinic
receptor-chloride current coupling with no effect on the M3
current, whereas anti-Gq antibodies had no effect on the
M2 current, but blocked M3 calcium release and
the attendant current. C, stimulation of heterologously
expressed inhibitory G protein-coupled M4 and
2C receptors resulted in weak stimulation of calcium
release (relative to M3 receptors) but did not activate the
calcium-insensitive M2 current. Activation of the
transient, calcium-activated chloride current by M3,
M4, and 2C receptor stimulation in
calcium-free solutions is shown. Current was not evoked in oocytes
incubated in
1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid
(BAPTA, 50 µM for 4 h), as shown for the
M4 receptor experiment. Oocytes were injected with
equivalent cRNA concentrations. Stimulation of muscarinic and
adrenergic receptors was with 50 µM mACH and 10 µM norepinephrine, respectively. Data summaries show
mean ± S.E. for indicated number of oocytes. *, indicates
significance by one-way analysis of variance.
|
|
Protein kinase C (PKC) molecules that are activated by diacylglycerol
and calcium following stimulation of phospholipase C by G
protein-coupled receptors have been implicated in M2
receptor-ion channel coupling (22-25). M2 coupling to the
novel chloride current was disrupted by exposure of oocytes to the
nonselective protein kinase C inhibitor chelerythrine. However,
GF109203X and Gö 6976 (not shown), more selective inhibitors of
several conventional and novel PKC isoforms, had no effect on current
activation (Fig. 3, A and
B). None of the protein kinase C inhibitors affected M3 coupling to phospholipase C, and conversely,
M2 receptor coupling was not affected by phospholipase C
inhibition (Fig. 3A). aPKCs that are activated by
phosphatidylinositol 4,5-diphosphate, PIP3, and
cis-fatty acids have been implicated as effectors in
mitogen, apoptotic, and contractile signaling (11-13, 26, 27),
although involvement of aPKCs in ion channel signaling has not been
reported. We examined the role of protein kinase C subtypes in
M2 receptor coupling using peptides that selectively bind
and inhibit conventional PKCs or aPKCs. Preinjection of aPKC
pseudosubstrate inhibitory peptides (28), but not cPKC inhibitory
peptides (29), inhibited the M2 chloride current coupling,
suggesting that aPKC activation is necessary for
M2-chloride channel coupling.

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Fig. 3.
M2/chloride current
receptor-effector coupling is mediated by protein kinase C. A, M2 coupling to the chloride current was
inhibited by the nonselective PKC antagonist chelerythrine (6.6 µM), but not by GF109203X (200 nM), which
selectively blocks several conventional and novel
(c/n) PKCs with high affinity (41), or by a
pseudosubstrate peptide for conventional PKCs (up to 500 µM) corresponding to amino acids 19-31 of PKC . An
atypical PKC inhibitory peptide corresponding to conserved residues in
the pseudosubstrate inhibitory region of aPKCs (27) significantly
inhibited coupling when injected to a final concentration of 50 µM. M3 receptor coupling to the
calcium-activated chloride current was not affected by PKC inhibition
but was abrogated by inhibition of phospholipase C. All substances were
microinjected before stimulation with 50 µM mACH.
B, average data for M2 PKC experiments shown in
A. *, indicates significance by one-way analysis of
variance; pep, peptide.
|
|
We confirmed the role of aPKC in receptor-effector coupling by direct
injection of aPKC into the cytosol (Fig.
4). Injection of recombinant PKC
activated the chloride current in a concentration dependent fashion,
with currents observed at final concentrations of PKC
as low as 1 nM. Moreover, injection of phosphatidylinositol 3,4,5-trisphosphate (PIP3), which stimulates PKC
in vitro (12), activated the current. Current activation was
specific to PIP3 injection. Phosphatidylinositol
4,5-diphosphate, phosphatidic acid, phosphatidylcholine, linolenic
acid, phosphatidylserine, and arachidonic acid failed to induce any
current (data not shown). PIP3 and PKC
currents were
indistinguishable from that observed following M2 receptor
stimulation in several respects. First, PIP3 and PKC
activated an anion selective current with a permeability sequence of
I
> Cl
> isethionate and a
current-voltage relationship identical to the M2 current.
Second, the current was sustained in the absence of extracellular
calcium following activation by either agent and was insensitive to
calcium chelation. Third, currents activated by M2 receptor
stimulation and by PIP3 or PKC
injection were not
additive. Following activation of the current by 50 µM
mACH, little or no further current was elicited by the injection of PIP3 or PKC
(n = 6), and
PIP3 or PKC
, current activation abrogated subsequent
mACH current (n = 5) (Fig. 4A). Finally, the
aPKC pseudosubstrate inhibitor blocked both the M2 and
PIP3-induced chloride currents (Fig. 4, A and
B). PIP3 has been shown to stimulate novel
PKC-
and PKC-
(30) as well as atypical PKC
activity, and the
block of PIP3 currents by the aPKC pseudosubstrate peptide
indicates the specific nature of PKCs mediating current activation.
Moreover, neither diacylglycerol analogues nor phorbol esters activated the current (although 1-oleoyl-2-acetyl-sn-glycerol weakly activated the calcium-activated chloride current), and M2
receptor-chloride current coupling was not affected by prior exposure
to these agents (Fig. 4A). Thus pharmacologic and peptide
inhibitors that are selective for aPKCs blocked activation of the
M2 chloride current, and lipid activators of aPKCs (but not
known activators of conventional and novel PKCs) and PKC
evoked the
current in the absence of M2 receptor stimulation,
indicating that stimulation of aPKC is both necessary and sufficient to
open M2-coupled chloride channels.

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Fig. 4.
The M2 chloride current is
activated by PIP3 and atypical PKC. A,
top, injection (V) of PIP3 (10 µM) induced a slowly activating, sustained chloride
current (left). If injected after the chloride current was
induced by mACH (50 µM), PIP3 had no effect
(center). The current was blocked by preinjection of the
aPKC inhibitory peptide (inhib pep, right).
Middle, PKC injection induced a chloride current with
similar slow activation kinetics in a
concentration-dependent fashion (left and
center). The current was not available following muscarinic
stimulation (right). Bottom, neither OAG (10 µM) nor phorbol 12-myristate 13-acetate (PMA,
3 µM) activated the chloride current (although OAG weakly
stimulated the calcium-activated chloride current), and their
application had no effect on stimulation of the chloride current by
mACH. Symbol (V) indicates point of injection. B,
average currents evoked in oocytes from the same batches. *,
indicates significantly different from PIP3 injection
alone.
|
|
Hormone-stimulated PI-3K activity results in the formation of
PIP3, which has been implicated as a second messenger in a
wide variety of cellular processes such as glucose transport, actin rearrangement, chemotaxis, and apoptosis (see Ref. 7). Activation of
chloride channels by PIP3 suggested the involvement of
PI-3K in the stimulatory pathway linking M2 receptor
stimulation to chloride channel opening. Consistent with such a
signaling cascade, the covalent PI-3K inhibitor wortmannin blocked the
M2-stimulated current (Fig.
5), with no effect on M3
coupling (not shown). Two groups of hormone-stimulated (class I) PI-3Ks
have been identified based on activation by tyrosine kinase or by G
protein-coupled signaling pathways. The former kinases are heterodimers
composed of a catalytic p110 subunit that is stimulated following
interaction with a p85 or p55 regulatory subunit containing SH2 and SH3
domains, whereas G protein-stimulated PI-3 kinase activity occurs
through the direct activation of p110
by the binding of G
subunits (9, 10, 31-33). To determine the identity of the PI-3K
involved in M2 coupling to aPKC, we used a peptide fragment
of adenylyl cyclase 2 (QEHA peptide) containing a putative G
binding motif, which blocks G
signaling to several protein
targets (34) and has been shown to inhibit 
stimulation of
PI-3K
in vitro (33). The QEHA peptide inhibited coupling
between M2 receptors and chloride channels by as much as
80%. Concentration-dependent inhibition of M2
receptor-effector coupling by the peptide was quite similar to that
observed for PI-3K
(33) (Fig. 5, A and B). The
peptide had no effect on PIP3 activation of the current,
indicating that the block is upstream of PIP3 formation
(data not shown). Similarly, purified bovine transducin
proteins,
which bind free G
subunits with high affinity, strongly inhibited
M2-chloride channel coupling (Fig. 5C).
Injection of proteins to a final concentration of approximately 5 µM inhibited the current by 77.5 ± 5.3%
(n = 12), whereas injection of boiled transducin
proteins was without effect. We also expressed a dominant negative p85
cRNA (
p85), which has been shown to prevent the activation of
p85-regulated PI-3K (16). Expression of
p85 or wild-type p85 had no
effect on M2 signaling when cRNAs were injected at
concentrations up to 4-fold higher than M2 receptor cRNA.

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Fig. 5.
M2 receptor-chloride coupling
occurs through G -stimulated
PI-3K . A, mACH (50 µM) induced similar currents in oocytes expressing
M2 receptors alone or M2 receptors and a
dominant negative P85 construct ( P85), whereas injection of a
peptide encoding a region of adenylyl cyclase 2 that binds G
(QEHA peptide, 500 µM) or transducin (5 µM) markedly inhibited the M2 current. In the
experiment shown, the P85 construct was injected at a 3.6-fold
higher concentration than the M2 cRNA (1.8 µg/µl P85
and 0.5 µg/µl M2). B, mean data from
experiments described in A. Wort, wortmannin.
C, concentration-dependent inhibition of the
M2 current by the QEHA peptide relative to the current
evoked by mACH alone in paired experiments.
|
|
Finally, we sought to determine whether the PI-3K·aPKC coupling
pathway was involved in physiological M2 neurotransmission. Release of acetylcholine from vagal nerves stimulates M3
and M2 receptors on smooth muscle cells at neuromuscular
junctions in the lung, bladder, viscera, and some vessels. Stimulation
of M2 receptors on isolated myocytes activates a sustained
cation current (Icat) that mediates slow
excitatory postsynaptic potentials (35-38), although the coupling
process is poorly understood. In voltage-clamped single tracheal smooth
muscle cells, dialysis of the aPKC pseudosubstrate peptide selectively
abolished Icat without affecting the large calcium-activated chloride current (ICl(Ca))
that is associated with M3-mediated calcium release (see
Ref. 37) (Fig. 6), indicating that aPKC
activation is necessary for M2 coupling to
Icat in smooth muscle. As shown, dialysis of the
cPKC peptide had no effect on coupling. M2 coupling to
Icat, but not M3 coupling to
ICl(Ca), was also disrupted by dialysis of the
QEHA peptide and by the PI-3 kinase inhibitor wortmannin, indicating
that key elements of the signaling pathway linking M2
receptors to chloride channels in Xenopus oocytes are
required for coupling of these receptors in mammalian cells. It should
be noted, however, that coupling of muscarinic receptors to
nonselective cation is an example of signaling convergence in which
discrete signals generated by the simultaneous stimulation of the
M2 and M3 receptors are required for current
activation (37, 39, 40). That is, current activation requires the
release of intracellular Ca2+, although such release is not
itself sufficient for channel opening without simultaneous
M2 receptor engagement (37). Not surprisingly, dialysis of
tracheal myocytes with PIP3 did not result in the activation of Icat (data not shown). Taken
together, however, these data indicate that the
M2·PI-3K
·aPKC coupling pathway likely underlies
physiological postsynaptic muscarinic signaling in smooth muscle.

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Fig. 6.
M2 receptor coupling to
nonselective cation channels in smooth muscle is mediated through
PI-3K and atypical PKC. A,
single equine tracheal myocytes were voltage-clamped
(Vh = 60 mV) and dialyzed for 5 min with cPKC
peptide (500 µM), aPKC peptide (500 µM),
QEHA peptide (500 µM), or wortmannin (1 µM)
before exposure to mACH. Muscarinic activation of the nonselective
cation current (Icat, the noisy, sustained, low
amplitude current in top trace) was blocked by aPKC, QEHA,
and wortmannin, but not by cPKC. The transient, calcium-activated
chloride current was unaffected; the full chloride current amplitude
(often >1 nA) is not shown, to enable Icat
comparison. B, average amplitude of the nonselective cation
current in experiments as shown in A. *, indicates
significance by one-way analysis of variance.
|
|
In summary, we demonstrate a novel signaling pathway linking
M2 receptors to metabotropic ion channels. Receptor binding
results in the stimulation of the G
-regulated PI-3K
, formation
of PIP3, and activation of aPKC. This signaling pathway
leads to the opening of a novel, second messenger-activated chloride
current in Xenopus oocytes and mediates activation of
nonselective cation channels in smooth muscle cells. These findings
define a novel signaling cascade linking G protein-coupled receptors to
membrane ion channels and provide further insight into the intricate
role of lipid second messengers in receptor signaling.
 |
ACKNOWLEDGEMENTS |
We thank Drs. H. E. Hamm, T. Morielli,
E. Peralta, and M. Kasuga for providing reagents.
 |
FOOTNOTES |
*
This work was supported by National Institutes of Health
Grants HL45239 and HL41084 (to M. I. K.) and a grant from the
American Heart Association (to Y.-X. W.).The costs of publication of this article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
§
To whom correspondence should be addressed: Dept. of Animal
Biology, University of Pennsylvania, 3800 Spruce St., Philadelphia, PA
19104-6046. Tel.: 215-898-2839; Fax: 215-573-6810; E-mail: mik{at}vet.upenn.edu.
 |
ABBREVIATIONS |
The abbreviations used are:
PI-3K, phosphoinositide 3-kinase;
PIP3, phosphatidylinositol 3,4,5-trisphosphate;
PKC, protein kinase C;
aPKC, atypical PKC;
cPKC, conventional PKC;
mACH, methacholine;
GF109203X, bisindolylmaleimide I.
 |
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