Coupling of M2 Muscarinic Receptors to Membrane Ion Channels via Phosphoinositide 3-Kinase gamma  and Atypical Protein Kinase C*

Yong-Xiao Wang, Prasad D. K. Dhulipala, Lei Li, Jeffrey L. BenovicDagger , and Michael I. Kotlikoff§

From the Department of Animal Biology, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104 and the Dagger  Kimmel Cancer Institute, Thomas Jefferson University, Philadelphia, Pennsylvania 19107

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

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 alpha 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 Gbeta gamma stimulation of phosphoinositide 3-kinase gamma  (PI-3Kgamma ), 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 PKCzeta at concentrations as low as 1 nM. M2 receptor-current coupling was disrupted by inhibiton of PI-3K and by injection of beta gamma 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
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

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 beta gamma 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 Gbeta gamma -stimulated PI-3Kgamma (8-10) extends the potential range of processes mediated by PI-3K to G protein-coupled receptors, although specific physiological processes mediated by PI-3Kgamma 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-3Kgamma , 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
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

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 Galpha 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 Delta 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 PKCzeta from Biomol. aPKC and QEHA peptides were custom synthesized. Bovine transducin alpha  was kindly provided by Dr. H. E. Hamm.

    RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

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.


View larger version (19K):
[in this window]
[in a new window]
 
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 Galpha i or Galpha o, but not Galpha q, proteins (Fig. 2, A and B). Anti-Galpha i1/Galpha i2 and anti-Galpha i3/Galpha 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 alpha 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 beta gamma proteins (21), the stimulatory pathway linking M2 receptors and chloride channels effectively distinguishes between these closely related receptors.


View larger version (19K):
[in this window]
[in a new window]
 
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 alpha 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 alpha 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.


View larger version (14K):
[in this window]
[in a new window]
 
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 alpha . 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 PKCzeta activated the chloride current in a concentration dependent fashion, with currents observed at final concentrations of PKCzeta as low as 1 nM. Moreover, injection of phosphatidylinositol 3,4,5-trisphosphate (PIP3), which stimulates PKCzeta 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 PKCzeta currents were indistinguishable from that observed following M2 receptor stimulation in several respects. First, PIP3 and PKCzeta 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 PKCzeta 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 PKCzeta (n = 6), and PIP3 or PKCzeta , 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-epsilon and PKC-eta (30) as well as atypical PKCzeta 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 PKCzeta 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.


View larger version (18K):
[in this window]
[in a new window]
 
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, PKCzeta 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 p110gamma by the binding of Gbeta gamma 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 Gbeta gamma binding motif, which blocks Gbeta gamma signaling to several protein targets (34) and has been shown to inhibit beta gamma stimulation of PI-3Kgamma 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-3Kgamma (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 alpha  proteins, which bind free Gbeta gamma 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 alpha  proteins was without effect. We also expressed a dominant negative p85 cRNA (Delta p85), which has been shown to prevent the activation of p85-regulated PI-3K (16). Expression of Delta 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.


View larger version (17K):
[in this window]
[in a new window]
 
Fig. 5.   M2 receptor-chloride coupling occurs through Gbeta gamma -stimulated PI-3Kgamma . A, mACH (50 µM) induced similar currents in oocytes expressing M2 receptors alone or M2 receptors and a dominant negative P85 construct (Delta P85), whereas injection of a peptide encoding a region of adenylyl cyclase 2 that binds Gbeta gamma (QEHA peptide, 500 µM) or transducin alpha  (5 µM) markedly inhibited the M2 current. In the experiment shown, the Delta P85 construct was injected at a 3.6-fold higher concentration than the M2 cRNA (1.8 µg/µl Delta 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-3Kgamma ·aPKC coupling pathway likely underlies physiological postsynaptic muscarinic signaling in smooth muscle.


View larger version (16K):
[in this window]
[in a new window]
 
Fig. 6.   M2 receptor coupling to nonselective cation channels in smooth muscle is mediated through PI-3Kgamma 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 Gbeta gamma -regulated PI-3Kgamma , 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.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
  1. Logothetis, D. E., Kurachi, Y., Galper, J., Neer, E. J., and Clapham, D. E. (1987) Nature 325, 321-326[CrossRef][Medline] [Order article via Infotrieve]
  2. Wickman, K. D., Iniguez-Lluhi, J. A., Davenport, P. A., Taussig, R., Krapivinsky, G. B., Linder, M. E., Gilman, A. G., and Clapham, D. E. (1994) Nature 368, 255-257[CrossRef][Medline] [Order article via Infotrieve]
  3. Reuveny, E., Slesinger, P. A., Inglese, J., Morales, J. M., Iniguez-Lluhi, J. A., Lefkowitz, R. J., Bourne, H. R., Jan, Y. N., and Jan, L. Y. (1994) Nature 370, 143-146[CrossRef][Medline] [Order article via Infotrieve]
  4. Huang, C. L., Slesinger, P. A., Casey, P. J., Jan, Y. N., and Jan, L. Y. (1995) Neuron 15, 1133-1143[Medline] [Order article via Infotrieve]
  5. Slesinger, P. A., Reuveny, E., Jan, Y. N., and Jan, L. Y. (1995) Neuron 15, 1145-1156[Medline] [Order article via Infotrieve]
  6. Kunkel, M. T., and Peralta, E. G. (1995) Cell 83, 443-449[Medline] [Order article via Infotrieve]
  7. Toker, A., and Cantley, L. C. (1997) Nature 387, 673-676[CrossRef][Medline] [Order article via Infotrieve]
  8. Stephens, L., Smrcka, A., Cooke, F. T., Jackson, T. R., Sternweis, P. C., and Hawkins, P. T. (1994) Cell 77, 83-93[Medline] [Order article via Infotrieve]
  9. Thomason, P. A., James, S. K., Casey, P. J., and Downes, C. P. (1994) J. Biol. Chem. 269, 16525-16528[Abstract/Free Full Text]
  10. Stoyanov, B., Volinia, S., Hanck, T., Rubio, I., Loubtchenkov, M., Malek, D., Stoyanova, S., Vanhaesebroeck, B., Dhand, R., and Nurnberg, B. (1995) Science 269, 690-693[Medline] [Order article via Infotrieve]
  11. Ono, Y., Fujii, T., Ogita, K., Kikkawa, U., Igarashi, K., and Nishizuka, Y. (1989) Proc. Natl. Acad. Sci. U. S. A. 86, 3099-3103[Abstract]
  12. Nakanishi, H., Brewer, K. A., and Exton, J. H. (1993) J. Biol. Chem. 268, 13-16[Abstract/Free Full Text]
  13. Liscovitch, M., and Cantley, L. C. (1994) Cell 77, 329-334[Medline] [Order article via Infotrieve]
  14. Nishizuka, Y. (1995) FASEB J. 9, 484-496[Abstract/Free Full Text]
  15. Nara, M., Dhulipala, P. D., Wang, Y. X., and Kotlikoff, M. I. (1998) J. Biol. Chem. 273, 14920-14924[Abstract/Free Full Text]
  16. Hara, K., Yonezawa, K., Sakaue, H., Ando, A., Kotani, K., Kitamura, T., Kitamura, Y., Ueda, H., Stephens, L., and Jackson, T. R. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 7415-7419[Abstract]
  17. Wang, Y.-X., and Kotlikoff, M. I. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 14918-14923[Abstract/Free Full Text]
  18. Fukuda, K., Kubo, T., Akiba, I., Maeda, A., Mishina, M., and Numa, S. (1987) Nature 327, 623-625[CrossRef][Medline] [Order article via Infotrieve]
  19. Coupry, I., Duzic, E., and Lanier, S. M. (1992) J. Biol. Chem. 267, 9852-9857[Abstract/Free Full Text]
  20. Felder, C. C. (1995) FASEB J. 9, 619-625[Abstract/Free Full Text]
  21. Gadbut, A. P., Riccardi, D., Wu, L., Hebert, S. C., and Galper, J. B. (1996) J. Biol. Chem. 271, 6398-6402[Abstract/Free Full Text]
  22. Mochida, S., and Kobayashi, H. (1988) Neurosci. Lett. 86, 201-206[Medline] [Order article via Infotrieve]
  23. Bernheim, L., Beech, D. J., and Hille, B. (1991) Neuron 6, 859-867[CrossRef][Medline] [Order article via Infotrieve]
  24. Marsh, S. J., Trouslard, J., Leaney, J. L., and Brown, D. A. (1995) Neuron 15, 729-737[Medline] [Order article via Infotrieve]
  25. Cantrell, A. R., Ma, J. Y., Scheuer, T., and Catterall, W. A. (1996) Neuron 16, 1019-1026[CrossRef][Medline] [Order article via Infotrieve]
  26. Lozano, J., Berra, E., Municio, M. M., Diaz-Meco, M. T., Dominguez, Sanz, L., and Moscat, J. (1994) J. Biol. Chem. 269, 19200-19202[Abstract/Free Full Text]
  27. Gailly, P., Gong, M. C., Somlyo, A. V., and Somlyo, A. P. (1997) J. Physiol. (London) 500, 95-109[Abstract]
  28. Dominguez, I., Diaz-Meco, M. T., Municio, M. M., Berra, E., Garcia, de Herreros, A., Cornet, M. E., Sanz, L., and Moscat, J. (1992) Mol. Cell. Biol. 12, 3776-3783[Abstract]
  29. House, C., and Kemp, B. E. (1987) Science 238, 1726-1728[Medline] [Order article via Infotrieve]
  30. Toker, A., Meyer, M., Reddy, K. K., Falck, J. R., Aneja, R., Aneja, S., Parra, A., Burns, D. J., Ballas, L. M., and Cantley, L. C. (1994) J. Biol. Chem. 269, 32358-32367[Abstract/Free Full Text]
  31. Stephens, L. R., Eguinoa, A., Erdjument-Bromage, H., Lui, M., Cooke, F., Coadwell, J., Smrcka, A. S., Thelen, M., Cadwallader, K., Tempst, P., and Hawkins, P. T. (1997) Cell 89, 105-114[Medline] [Order article via Infotrieve]
  32. Tang, X., and Downes, C. P. (1997) J. Biol. Chem. 272, 14193-14199[Abstract/Free Full Text]
  33. Leopoldt, D., Hanck, T., Exner, T., Maier, U., Wetzker, R., and Nurnberg, B. (1998) J. Biol. Chem. 273, 7024-7029[Abstract/Free Full Text]
  34. Chen, J., DeVivo, M., Dingus, J., Harry, A., Li, J., Sui, J., Carty, D. J., Blank, J. L., Exton, J. H., Stoffel, R. H., Inglese, J., Lefkowitz, R. J., Logothetis, D. E., Hildebrandt, J. D., and Iyengar, R. (1995) Science 268, 1166-1169[Medline] [Order article via Infotrieve]
  35. Benham, C. D., Bolton, T. B., and Lang, R. J. (1985) Nature 316, 345-347[Medline] [Order article via Infotrieve]
  36. Byrne, N. G., and Large, W. A. (1987) Br. J. Pharmacol. 92, 371-379[Abstract]
  37. Wang, Y. X., Fleischmann, B. K., and Kotlikoff, M. I. (1997) Am. J. Physiol. 273, C500-C508[Abstract/Free Full Text]
  38. Large, W. A., and Wang, Q. (1996) Am. J. Physiol. 271, C435-C454[Abstract/Free Full Text]
  39. Inoue, R., and Isenberg, G. (1990) J. Physiol. (London) 424, 73-92[Abstract]
  40. Pacaud, P., and Bolton, T. B. (1991) J. Physiol. (London) 441, 477-499[Abstract]
  41. Martiny-Baron, G., Kazanietz, M. G., Mischak, H., Blumberg, P. M., Kochs, G., Hug, H., Marme, D., and Schachtele, C. (1993) J. Biol. Chem. 268, 9194-9197[Abstract/Free Full Text]


Copyright © 1999 by The American Society for Biochemistry and Molecular Biology, Inc.