Published online before print January 22, 2004, doi: 10.1161/01.RES.0000118248.17466.B7
© 2004 American Heart Association, Inc.
Cellular Biology |
Muscarinic M2 Receptor Stimulation of Cav1.2b Requires Phosphatidylinositol 3-Kinase, Protein Kinase C, and c-Src
From the Department of Physiology and Cell Biology, University of Nevada, Reno, Nev.
Correspondence to Kathleen Keef, PhD, Department of Physiology and Cell Biology, University of Nevada, Reno, NV 89557. E-mail kathy{at}physio.unr.edu
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Abstract |
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This study investigated regulation of L-type calcium channels (Cav1.2b) by acetylcholine (ACh) in rabbit portal vein myocytes. Whole-cell currents were recorded using 5 mmol/L barium as charge carrier. ACh (10 µmol/L) increased peak currents by 40%. This effect was not reversed by the selective muscarinic M3 receptor antagonist 4-DAMP (100 nmol/L) but was blocked by the M2 receptor antagonist methoctramine (5 µmol/L). The classical and novel protein kinase C (PKC) antagonist calphostin C (50 nmol/L) abolished ACh responses, whereas the classical PKC antagonist Gö6976 (200 nmol/L) had no effect. ACh responses were also abolished by the phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002 (20 µmol/L), by the c-Src inhibitor PP2 (10 µmol/L) (but not the inactive analogue PP3), and by dialyzing cells with an antibody to the G-protein subunit Gß
. Cells dialyzed with c-Src had significantly greater currents than control cells. Current enhancement persisted in the presence of LY294002, suggesting that c-Src is downstream of PI3K. Phorbol 12,13-dibutyrate (PDBu, 0.1 µmol/L) increased currents by 74%. This effect was abolished by calphostin C and reduced by Gö6976. The PDBu response was also reduced by PP2, and the PP2-insensitive component was blocked by Gö6976. In summary, these data suggest that ACh enhances Cav1.2b currents via M2 receptors that couple sequentially to Gß, PI3K, a novel PKC, and c-Src. PDBu stimulates the novel PKC/c-Src pathway along with a second pathway that is independent of c-Src and involves a classical PKC.
Key Words: smooth muscle • L-type calcium channels • kinase • patch clamp
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Introduction |
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Cav1.2b channels (L-type Ca2+ channels) are voltage-dependent channels that open in response to membrane depolarization to allow the entry of Ca2+ into the cell. They are ubiquitously expressed in vascular smooth muscle and play a fundamental role in the regulation of tone. Agonists such as norepinephrine can increase Cav1.2b channel activity through cell depolarization. However, studies over the past 15 years have suggested that a variety of agonists can also more directly affect Cav1.2b by increasing the amount of current generated at a given voltage.1,2 The mechanisms involved in this kind of channel regulation are still not well understood and are the topic of this study.
We have previously shown that Cav1.2b currents in rabbit portal vein myocytes are increased when adrenergic ß2a receptors are stimulated with isoproterenol. This action involves both G-protein subunits, ie, effects of G
s are mediated by protein kinase A (PKA) and effects of Gß are mediated by protein kinase C (PKC).3,4 The Gß subunit and PKC are also required for AT1 receptor stimulation of Cav1.2b currents in rat portal vein myocytes.5,6 In these studies, the immediate downstream mediator of Gß was found to be phosphatidylinositol-3 kinase (PI3K).7 Indeed, dialysis of cells with activated PI3K also stimulated Cav1.2b currents, and this effect was sensitive to PKC blockade. Thus, Gß coupled to two different receptors as well as two different Gß subunits dialyzed into cells all stimulate Cav1.2b currents in a PKC-dependent manner, suggesting that the Gß/PI3K/PKC pathway may represent a ubiquitous mechanism by which agonists stimulate Cav1.2b.
Muscarinic M2 receptors (coupled to the G-protein Gi)8 have also been shown to activate a Gß/PI3K/PKC pathway in Xenopus oocytes and tracheal smooth muscle cells.9 In contrast, a recent study by Jin et al10 has suggested that M2 receptor stimulation of Cav1.2b in rabbit colonic myocytes involves the nonreceptor tyrosine kinase c-Src. c-Src is abundantly expressed in vascular smooth muscle11 and recently has been linked to agonist-induced inhibition of potassium channels.12 PKC can increase c-Src activity,13–16 and c-Src has been shown to stimulate Cav1.2b currents in vascular smooth muscle cells.17,18 In light of these observations, we hypothesized that c-Src is the downstream mediator of Cav1.2b channel stimulation in the Gß/PI3K/PKC pathway. To explore this hypothesis, we evaluated the role of Gß, PI3K, PKC, and c-Src in M2-receptor stimulation of Cav1.2b currents in rabbit portal vein myocytes. Various blockers were used to examine the actions of acetylcholine (ACh), phorbol 12,13-dibutyrate (PDBu), and c-Src on Cav1.2b currents. Our results show for the first time that M2 receptor stimulation of Cav1.2b currents is coupled to the Gß/PI3K/PKC pathway, lending support to the ubiquitous nature of this agonist-induced pathway. They additionally demonstrate that an important downstream mediator is c-Src, which either directly stimulates the channel or activates an additional second messenger to enhance Cav1.2b activity.
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Materials and Methods |
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Isolation of Rabbit Portal Vein Myocytes
Myocytes were isolated using previously described methods.4 Male albino rabbits (1.5 to 2.0 kg; Western Oregon Rabbitry, Philomath, Ore) were killed with an intravenous overdose of sodium pentobarbital (50 mg/kg). Smooth muscle cells were isolated from the portal vein. The animal use protocol was reviewed and approved by the Animal Care and Use Committee of the University of Nevada.
Electrophysiology
The patch-clamp experiments were performed as previously described.3 Inward currents were measured using an Axopatch-1D patch-clamp amplifier, digitized with a 16-bit analog to digital converter (Model DIGIDATA 1320A, Axon Instruments), and controlled by pClamp8 (Axon Instruments).
Ba2+ currents (IBa) in myocytes were measured using both the dialyzed whole cell and the perforated patch configurations. The bath solution used to record IBa was composed of (in mmol/L) NaCl 120, TEACl 10, BaCl2 5, MgCl2 0.5, glucose 5.5, CsCl 5, and HEPES 10, pH 7.40, with NaOH. Both TEACl and CsCl were used to block potassium currents. For perforated patch experiments, the composition of the pipette solution was (in mmol/L) cesium aspartate 120, TEACl 20, EGTA 1, and HEPES 20, adjusted pH 7.2 with CsOH. Amphotericin B (90 mg/mL) was dissolved with dimethylsulfoxide (DMSO), sonicated, and diluted to give a final concentration of 270 µg/mL in the pipette solution. For the dialyzing whole-cell experiments, the composition of the pipette solution was (in mmol/L) CsCl 120, TEACl 20, glucose 5.5, MgCl2 2, ATP 5, EGTA 5, and HEPES 10, pH 7.2 with CsOH.
Drugs
Collagenase type I, protease type XXVII, BSA, ACh, amphotericin B, PDBu, calphostin C, 4-diphenylacetoxy-N-methylpiperidie (4-DAMP), and methoctramine were purchased from Sigma. PP2, LY294002, chelerythrine Cl, and Gö6976 were from CalBiochem. Purified c-Src kinase dephosphorylated at the terminal inhibitory tyrosine was obtained from Upstate Biotechnology. Gß antibody (T20) was obtained from Santa Cruz Biotechnology Inc. Drugs insoluble in water were first dissolved in DMSO and then additionally diluted so that the final concentration of DMSO was <0.2%. DMSO alone at 0.2% had no effect on IBa.
Data Analysis
All experimental values are presented as mean±SEM, and n refers to the number of cells tested. Differences between the values from different groups were compared using Student’s paired and unpaired t tests and 2-way ANOVA, where appropriate. P<0.05 was considered significantly different. The IC50 for PP2 was determined by fitting the data with a nonlinear least-squares regression program (GraphPAD).
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Results |
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ACh Enhances Cav1.2b Currents via M2 Receptors and the Gß
SubunitBa2+ currents (IBa) through L-type Ca2+ channels (Cav1.2b) were recorded in freshly isolated rabbit portal vein myocytes using the whole-cell voltage-clamp technique. Currents were elicited by stepping voltage from a holding potential of -70 to 0 mV for 370 ms at 30-second intervals. These currents are blocked by nicardipine.19 Peak steady-state current amplitudes were obtained after 4 to 5 minutes (166±11.5 pA, n=36). Addition of ACh (10 µmol/L) caused a significant increase in peak IBa to 140±5% of the basal level (n=14) (Figures 1A and 1B). The increase in currents occurred without a shift in the voltage dependence of activation or inactivation (data not shown).
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) and absence (
of ACh (10 µmol/L) (n=14). D, Bar graph of summed results. ACh significantly increased IBa. Blocking M3 receptors with 4-DAMP (100 nmol/L) led to a small but significant additional increase in IBa (-M3), whereas current stimulation was reversed by blocking M2 receptors with methoctramine (5 µmol/L) (-M2). Bars plot mean peak IBa±SEM (n=9). E and F, Dialysis of cells with Gß antibody blocks ACh stimulation of Cav1.2b. E, Plot of peak IBa as a function of time in 2 cells from the same experiment using dialyzing patch pipettes. For one cell, Gß antibody was included in the pipette (
). ACh (10 µmol/L) was applied to both cells 5 minutes after attaining the whole-cell configuration. This increased current in the control cell but not in the cell dialyzed with Gß antibody (10 µg/mL). F, Bar graph plotting IBa under various conditions. ACh (n=6) significantly increased current above basal levels in control cells (n=6) but not in cells dialyzed with Gß antibody (n=7). Bars represent mean peak IBa±SEM. *P<0.05, **P<0.01, ***P<0.001, significantly different from the control value under the same experimental conditions.The predominant muscarinic receptors mediating contraction in smooth muscle are M2 and M3,20,21 and previous studies have suggested that the actions of ACh on Cav1.2b are attributable to M2 receptor stimulation.10 To ascertain whether this is the case in the portal vein, we examined the effect of the selective M3 antagonist 4-DAMP (100 nmol/L) and the selective M2 antagonist methoctramine (5 µmol/L). 4-DAMP gave rise to a small but significant increase in the ACh-induced response (140% versus 154%, n=9), whereas methoctramine significantly reduced the response (Figures 1C and 1D). These data suggest that ACh enhances Cav1.2b currents via the M2 receptor. Unless otherwise specified, the remaining experiments with ACh were carried out in the presence of 4-DAMP (100 nmol/L).
We have hypothesized that M2 receptors stimulate Cav1.2b via Gß. To provide direct evidence for the role of Gß in the actions of ACh, we tested a blocking antibody to the Gß subunit. In previous studies, we have shown that this antibody distinguishes between the Gß and Gs effects associated with adrenergic ß2a receptors in portal vein myocytes.3 Dialysis of cells with Gß antibody (10 µg/mL) entirely blocked the stimulatory effects of ACh (n=7; Figures 1E and 1F).
Cav1.2b Is Regulated by Both Novel and Classical PKCs
Previous studies have shown involvement of PKC in Gß stimulation of Cav1.2b currents4,6; therefore, we tested the effects of several PKC inhibitors on the ACh response. Calphostin C (50 nmol/L), which blocks both classical and novel PKCs, reversed the effect of ACh (n=6) (Figures 2A through 2C). However, Gö6976 (200 nmol/L), which blocks only classical PKCs,22 had no significant effect on the ACh response (n=6) (Figures 2D through 2F). These results suggest that ACh stimulates IBa via a novel PKC isoform.
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To further investigate which isoforms of PKC lead to current stimulation, we tested PDBu, which activates both classical and novel PKCs.23 Application of PDBu (100 nmol/L) consistently increased peak IBa (n=20; Figure 3) without a shift in the voltage dependence of activation or inactivation (data not shown). A steady-state response was reached after 3 to 6 minutes and remained stable thereafter (ie, up to 20 minutes). The response to PDBu was entirely blocked by calphostin C (n=11) (Figures 3A through 3C). In contrast, Gö6976 (200 nmol/L) significantly reduced but did not abolish the PDBu response (n=8; Figures 3D through 3F). These data suggest that both novel and classical PKCs are linked to simulation of IBa in portal vein myocytes.
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M2 Stimulation of Cav1.2b Involves PI3K
Studies by others suggest that the immediate downstream mediator of Gß is PI3K.6,7,24 To examine the role of PI3K in the ACh-induced response, we tested the PI3K inhibitor LY294002 (20 µmol/L). LY294002 completely reversed current stimulation induced by ACh (Figures 4A through 4C). To determine whether PI3K is upstream or downstream of PKC, PDBu (100 nmol/L) was applied to cells preexposed to LY294002 (20 µmol/L). LY294002 alone had no significant effect on IBa. Current stimulation with PDBu in the presence of LY294002 was not significantly different from the response to PDBu in the absence of LY294002 (Figures 4D through 4F), suggesting that PK3K is not downstream of PKC activation.
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M2 Stimulation of Cav1.2b Involves c-Src
We next investigated the hypothesis that the downstream mediator of the M2/Gß/PI3K/PKC pathway is c-Src. Cav1.2b in native cells seems to be basally phosphorylated by c-Src, because blockers of c-Src are known to reduce basal Cav1.2b currents.17,25 This was also the case for rabbit portal vein myocytes, because superfusion with the c-Src inhibitor PP2 (10 µmol/L) led to a 46% (n=5) reduction in peak current amplitude (Figure 5). We therefore applied PP2 first to test its effect on ACh. In the presence of PP2, the response to ACh was entirely abolished. Removal of PP2 in the continued presence of ACh revealed the stimulatory effects of ACh (Figures 5B and 5C; n=6). The inactive analogue PP3 had no effect on the ACh response (n=4). These results suggest that c-Src plays a role in the stimulatory effects of ACh on Cav1.2b. Because the potency of PP2 for different Src family members varies,26 we also examined the concentration dependence of block. Half block of the ACh response was 0.4 µmol/L PP2 (Figure 5D).
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To additionally investigate the relationship of novel PKCs to c-Src, we examined the effect of PP2 on the PDBu response. The response to PDBu in the presence of PP2 was significantly reduced but not abolished (Figure 6). The component of the PDBu response that persisted in the presence of PP2 was attributable to a classical PKC, because it was entirely abolished by Gö6976. Removal of both PP2 and Gö6976 while retaining PDBu revealed additional PDBu stimulation (n=8) (Figure 6). These data support the hypothesis that a novel PKC leads to c-Src–dependent stimulation of Cav1.2b. They additionally suggest that a classical PKC can stimulate the channel via a c-Src–independent pathway.
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c-Src Enhances Cav1.2b, and This Effect Is Independent of PI3K
We have proposed that the downstream mediator of the Gß/PI3K/PKC pathway is c-Src. To test whether c-Src can directly stimulate Cav1.2b, we applied activated c-Src to cells by including it in the pipette solution (7.5 U) and dialyzing it into the cell using the whole-cell patch configuration. In the absence of c-Src, a small amount of current run up occurred immediately after access, and a steady state was reached 2 to 3 minutes later. Thereafter, currents remained stable for 
8 minutes before run down began. When c-Src was included in the patch pipette, the currents recorded were significantly larger than those of control cells, but there was no significant shift in the I-V relationship (Figure 7). c-Src can lead to activation of PI3K.27 To investigate this possibility, we repeated some experiments in the presence of the PI3K inhibitor LY294002. Cells were bathed with LY294002 (20 µmol/L) for at least 5 minutes before the whole-cell patch configuration was attained. Thereafter, currents were recorded in cells dialyzed with c-Src. LY294002 did not significantly reduce c-Src stimulation (n=4) (Figure 7B).
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) generated currents that were significantly larger than those recorded in the absence of c-Src ( |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The present study investigated the actions of ACh on Cav1.2b currents in rabbit portal vein myocytes. ACh significantly increased currents via stimulation of muscarinic M2-receptors. Our results suggest that this response is coupled to a second messenger pathway that includes Gß
, PI3K, and a novel PKC and that the downstream mediator of the Gß/PI3K/PKC pathway is c-Src. A diagram of the proposed pathway is shown in Figure 8.
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Previous studies of rabbit colonic myocytes suggest that ACh stimulates Cav1.2b currents via M2 receptors.28 The present study concurs with this conclusion, because ACh responses were blocked with the M2 receptor antagonist methoctramine but not the M3 receptor antagonist 4-DAMP. M2 receptors are generally coupled to the G-protein Gi.8 In rabbit portal vein myocytes, the stimulatory effects of ACh seem to be mediated by the Gß subunit of Gi, because the response was abolished by dialyzing cells with a Gß blocking antibody. A known target of Gß is PI3K,29,30 and there is evidence from rat portal vein studies that Gß stimulates Cav1.2b via PI3K.6,7 We found that the response to ACh was abolished by the selective PI3K inhibitor LY294002, suggesting that PI3K participates in the M2/Gß pathway in rabbit portal vein as well. In contrast, the stimulatory effect of PDBu was not blocked by LY294002, suggesting that PKC is downstream of PI3K rather than the reverse.
At present it is unclear how PI3K activates PKC. PtdIns(3,4,5)P3, the lipid product of PI3K, targets several different second messengers, including PKC.29 Furthermore, PI3K itself has serine-kinase activity,31 and this could lead to PKC activation. The ACh/M2/Gß/PI3K pathway regulating chloride channels in oocytes was linked to PtdIns(3,4,5)P3-dependent activation of PKC
, an atypical PKC that is insensitive to both Ca2+ and DAG.9 However, this does not seem to be the case for rabbit portal vein myocytes, because current stimulation with either ACh (present study) or dialysis of cells with Gß124 is blocked with calphostin C. Calphostin C inhibits both classical and novel PKCs but not atypical PKCs.32 Furthermore, the effects of ACh (present study) and Gß4 are not blocked when selective antagonist of classical PKCs are used, leading us to the conclusion that a novel PKC is involved. PKC
has been identified in rabbit portal vein myocytes,33 so it is possible that PKC is the novel PKC isoform activated by the Gß/PI3K pathway. PI3K and PtdIns(3,4,5)P3 have been linked to activation of PKC in other studies.34–36 Interestingly, the regulation of KV currents by angiotensin II in rabbit portal vein myocytes is also suggested to involve PKC.33 Finally, another important downstream target of PtdIns(3,4,5)P3 is phosphoinositide-dependent kinase 1 (PDK1).37 PKC immunoprecipitates with PDK1,38 and some studies have suggested that PKC isozymes may be controlled by PI3K via PDK1.38,39 Taken together, our results clearly suggest a link between Gß, PI3K, and a novel PKC in the actions of ACh on Cav1.2b currents. However, additional studies are required to understand the details of this cascade.
Wang et al9 also investigated the M2 receptor pathway in Xenopus oocytes and native tracheal cells. Their results suggest that the Gß/PI3K/PKC pathway couples to the regulation of ion channels in both models and that this signaling cascade may have broad relevance to neurotransmitter signaling. Our study provides additional support for this proposal and, when considered in conjunction with studies of adrenergic ß2a receptors3 and AT1 receptors,5–7 suggests that agonist stimulation of Cav1.2b via the Gß/PI3K/PKC pathway represents a general scheme that may encompass a variety of different receptor/G-protein complexes.
Our results suggest that the downstream mediator of PKC is c-Src. C-Src is a nonreceptor tyrosine kinase that is abundantly expressed in vascular smooth muscle cells.11 The response to ACh was abolished with the c-Src inhibitor PP2, suggesting a role for this kinase in the Gß/PI3K/PKC pathway. We and others17,18 have also found that dialysis of cells with activated c-Src enhances Cav1.2b currents in vascular smooth muscle cells. PKC activation precedes c-Src activation, because current stimulation with PDBu is also reduced after c-Src inhibition with PP2. PKC has also been shown to activate c-Src in other smooth muscles and other cell types.13–16 In addition, G-protein activation has been linked sequentially to PKC and c-Src in other cells types.40–42 Finally, c-Src seems to be the downstream mediator of agonist-induced inhibition of K+ channels in coronary artery.12
At present, it is unclear how PKC activates c-Src. It is possible that other nonreceptor tyrosine kinases (ie, Pyk2 and FAK) are interposed between PKC and c-Src. Alternatively, recent studies suggest that PKC may enhance c-Src activity by stimulating a protein tyrosine phosphatase that dephosphorylates the inhibitory c-terminal tyrosine of c-Src.43 Dephosphorylation of this tyrosine will enhance c-Src activity by allowing the protein to unfold to expose its catalytic domain and undergo autophosphorylation.44 Additional studies will be needed to identify the details leading from PKC to c-Src activation.
Both classical and novel PKCs appear capable of Cav1.2b current stimulation, because the PDBu response is abolished by calphostin C and reduced by the classical PKC inhibitor Gö6976. In contrast, the ACh response is insensitive to Gö6976. As discussed above, we have proposed that stimulation of Cav1.2b by ACh is specifically coupled to activation of c-Src by a novel PKC. In a similar manner, the novel PKC component of the PDBu response is blocked by PP2, whereas the classical PKC component persists. Thus, a separate pathway must exist by which classical PKCs stimulate Cav1.2b independent of c-Src.
The IC50 for PP2 block of the ACh response is 0.4 µmol/L. PP2 and PP1 are almost identical in structure, and in vitro studies suggest that both are more potent blockers of Lck and Fyn (IC50, 5 nmol/L) than of c-Src (IC50, 170 nmol/L).45 Other IC50 values reported for block of Src activity range from 0.07 to 0.5 µmol/L11 to 2 µmol/L.25 Thus, the potency we observed for PP2 is compatible with an action on c-Src, but the possible contribution of other Src family members can not be eliminated.
The mechanism by which c-Src enhances Cav1.2b currents is still unclear and may vary between tissues. Recent studies in the rat model suggest that c-Src phosphorylates a particular tyrosine on the c-terminus of Cav1.2.17,46 However, the rabbit smooth muscle isoform of Cav1.2b cannot be regulated by this mechanism, because this phosphorylation site is absent. Several other sites with moderate and high phosphorylation potential are present at the c-terminus of the rabbit sequence. Furthermore, there is evidence that c-Src regulates Cav1.2b in other rabbit smooth muscles, ie, colon10,47 and ear artery.18,25,48 These data suggest that c-Src may still directly regulate rabbit Cav1.2b without the tyrosine phosphorylation site identified in the rat. Interestingly, the human isoform of Cav1.2b also lacks the tyrosine phosphorylation site identified in the rat, but it does contain five potential phosphorylation sequences at the c-terminus.
In summary, this study has provided evidence for a second messenger pathway initiated by M2 receptors that couples sequentially from Gß to PI3K, a novel PKC, and the tyrosine kinase c-Src. Taken together with other studies, these results suggest that this pathway may have broad relevance to the control of Cav1.2b currents mediated by a variety of different receptor/G-protein complexes in smooth muscle as well as to the regulation of other ionic conductances.
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Acknowledgments |
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This research was funded by NIH RO1 grant HL40399 to K.D.K.
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Footnotes |
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Original received October 3, 2003; revision received January 9, 2004; accepted January 13, 2004.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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1. Beech DJ. Actions of neurotransmitters and other messengers on Ca2+ channels and K+ channels in smooth muscle cells. Pharmacol Ther. 1997; 73: 91–119.[CrossRef][Medline] [Order article via Infotrieve]
2. Gollasch M, Nelson MT. Voltage-dependent Ca2+ channels in arterial smooth muscle cells. Kidney Blood Press Res. 1997; 20: 355–371.[Medline] [Order article via Infotrieve]
3. Zhong J, Hume JR, Keef KD. ß-Adrenergic receptor stimulation of L-type Ca2+ channels in rabbit portal vein myocytes involves both s and ß G protein subunits. J Physiol. 2001; 531: 105–115.
4. Zhong J, Dessauer CW, Keef KD, Hume JR. Regulation of L-type Ca2+ channels in rabbit portal vein by G protein s and ß subunits. J Physiol (Lond). 1999; 517: 109–120.
5. Macrez N, Morel JL, Kalkbrenner F, Viard P, Schultz G, Mironneau J. A ß dimer derived from G13 transduces the angiotensin AT1 receptor signal to stimulation of Ca2+ channels in rat portal vein myocytes. J Biol Chem. 1997; 272: 23180–23185.
6. Viard P, Exner T, Maier U, Mironneau J, Nurnberg B, Macrez N. Gß dimers stimulate vascular L-type Ca2+ channels via phosphoinositide 3-kinase. FASEB J. 1999; 13: 685–694.
7. Quignard JF, Mironneau J, Carricaburu V, Fournier B, Babich A, Nurnberg B, Mironneau C, Macrez N. Phosphoinositide 3-kinase mediates angiotensin II-induced stimulation of L-type calcium channels in vascular myocytes. J Biol Chem. 2001; 276: 32545–32551.
8. Eglen RM, Reddy H, Watson N, Challiss RA. Muscarinic acetylcholine receptor subtypes in smooth muscle. Trends Pharmacol Sci. 1994; 15: 114–119.[CrossRef][Medline] [Order article via Infotrieve]
9. Wang YX, Dhulipala PD, Li L, Benovic JL, Kotlikoff MI. Coupling of M2 muscarinic receptors to membrane ion channels via phosphoinositide 3-kinase and atypical protein kinase C. J Biol Chem. 1999; 274: 13859–13864.
10. Jin X, Morsy N, Shoeb F, Zavzavadjian J, Akbarali HI. Coupling of M2 muscarinic receptor to L-type Ca channel via c-src kinase in rabbit colonic circular smooth muscle. Gastroenterology. 2002; 123: 827–834.[CrossRef][Medline] [Order article via Infotrieve]
11. Oda Y, Renaux B, Bjorge J, Saifeddine M, Fujita DJ, Hollenberg MD. cSrc is a major cytosolic tyrosine kinase in vascular tissue. Can J Physiol Pharmacol. 1999; 77: 606–617.[CrossRef][Medline] [Order article via Infotrieve]
12. Alioua A, Mahajan A, Nishimaru K, Zarei MM, Stefani E, Toro L. Coupling of c-Src to large conductance voltage- and Ca2+-activated K+ channels as a new mechanism of agonist-induced vasoconstriction. Proc Natl Acad Sci U S A. 2002; 99: 14560–14565.
13. Brandt D, Gimona M, Hillmann M, Haller H, Mischak H. Protein kinase C induces actin reorganization via a Src- and Rho-dependent pathway. J Biol Chem. 2002; 277: 20903–20910.
14. Bruce-Staskal PJ, Bouton AH. PKC-dependent activation of FAK and src induces tyrosine phosphorylation of Cas and formation of Cas-Crk complexes. Exp Cell Res. 2001; 264: 296–306.[CrossRef][Medline] [Order article via Infotrieve]
15. Chang YJ, Holtzman MJ, Chen CC. Interferon--induced epithelial ICAM-1 expression and monocyte adhesion: involvement of protein kinase C-dependent c-Src tyrosine kinase activation pathway. J Biol Chem. 2002; 277: 7118–7126.
16. Ping P, Zhang J, Zheng YT, Li RC, Dawn B, Tang XL, Takano H, Balafanova Z, Bolli R. Demonstration of selective protein kinase C-dependent activation of Src and Lck tyrosine kinases during ischemic preconditioning in conscious rabbits. Circ Res. 1999; 85: 542–550.
17. Wu X, Davis GE, Meininger GA, Wilson E, Davis MJ. Regulation of the L-type calcium channel by 5ß1 integrin requires signaling between focal adhesion proteins. J Biol Chem. 2001; 276: 30285–30292.
18. Wijetunge S, Hughes AD. pp60c-src increases voltage-operated calcium channel currents in vascular smooth muscle cells. Biochem Biophys Res Commun. 1995; 217: 1039–1044.[CrossRef][Medline] [Order article via Infotrieve]
19. Ishikawa T, Hume JR, Keef KD. Regulation of Ca2+ channels by cAMP and cGMP in vascular smooth muscle cells. Circ Res. 1993; 73: 1128–1137.
20. Matsui M, Motomura D, Fujikawa T, Jiang J, Takahashi S, Manabe T, Taketo MM. Mice lacking M2 and M3 muscarinic acetylcholine receptors are devoid of cholinergic smooth muscle contractions but still viable. J Neurosci. 2002; 22: 10627–10632.
21. Ehlert FJ. Pharmacological analysis of the contractile role of M2 and M3 muscarinic receptors in smooth muscle. Receptors Channels. 2003; 9: 261–277.[CrossRef][Medline] [Order article via Infotrieve]
22. Martiny-Baron G, Kazanietz MG, Mischak H, Blumberg PM, Kochs G, Hug H, Marme D, Schachtele C. Selective inhibition of protein kinase C isozymes by the indolocarbazole Go6976. J Biol Chem. 1993; 268: 9194–9197.
23. Yanagita T, Kobayashi H, Yamamoto R, Takami Y, Yokoo H, Yuhi T, Nakayama T, Wada A. Protein kinase C and the opposite regulation of sodium channel - and ß1-subunit mRNA levels in adrenal chromaffin cells. J Neurochem. 1999; 73: 1749–1757.[CrossRef][Medline]
[Order article via Infotrieve]
24. Macrez N, Mironneau C, Carricaburu V, Quignard JF, Babich A, Czupalla C, Nurnberg B, Mironneau J. Phosphoinositide 3-kinase isoforms selectively couple receptors to vascular L-type Ca2+ channels. Circ Res. 2001; 89: 692–699.
25. Wijetunge S, Lymn JS, Hughes AD. Effects of protein tyrosine kinase inhibitors on voltage-operated calcium channel currents in vascular smooth muscle cells and pp60(c-src) kinase activity. Br J Pharmacol. 2000; 129: 1347–1354.[CrossRef][Medline] [Order article via Infotrieve]
26. Bain J, McLauchlan H, Elliott M, Cohen P. The specificities of protein kinase inhibitors: an update. Biochem J. 2003; 371: 199–204.[CrossRef][Medline] [Order article via Infotrieve]
27. Gerthoffer WT, Gunst SJ. Invited review: focal adhesion and small heat shock proteins in the regulation of actin remodeling and contractility in smooth muscle. J Appl Physiol. 2001; 91: 963–972.
28. Mayrleitner M, Chadwick CC, Timerman AP, Fleischer S, Schindler H. Purified IP3 receptor from smooth muscle forms an IP3 gated and heparin sensitive Ca2+ channel in planar bilayers. Cell Calcium. 1991; 12: 505–514.[CrossRef][Medline] [Order article via Infotrieve]
29. Wymann MP, Pirola L. Structure and function of phosphoinositide 3-kinases. Biochim Biophys Acta. 1998; 1436: 127–150.[Medline] [Order article via Infotrieve]
30. Brock C, Schaefer M, Reusch HP, Czupalla C, Michalke M, Spicher K, Schultz G, Nurnberg B. Roles of G ß in membrane recruitment and activation of p110/p101 phosphoinositide 3-kinase . J Cell Biol. 2003; 160: 89–99.
31. Hunter T. When is a lipid kinase not a lipid kinase? When it is a protein kinase. Cell. 1995; 83: 1–4.[CrossRef][Medline] [Order article via Infotrieve]
32. Gordge PC, Ryves WJ. Inhibitors of protein kinase C. Cell Signal. 1994; 6: 871–882.[CrossRef][Medline] [Order article via Infotrieve]
33. Clement-Chomienne O, Walsh MP, Cole WC. Angiotensin II activation of protein kinase C decreases delayed rectifier K+ current in rabbit vascular myocytes. J Physiol (Lond). 1996; 495: 689–700.
34. Balciunaite E, Kazlauskas A. Early phosphoinositide 3-kinase activity is required for late activation of protein kinase C in platelet-derived-growth-factor-stimulated cells: evidence for signalling across a large temporal gap. Biochem J. 2001; 358: 281–285.[CrossRef][Medline]
[Order article via Infotrieve]
35. Toker A, Meyer M, Reddy KK, Falck JR, Aneja R, Aneja S, Parra A, Burns DJ, Ballas LM, Cantley LC. Activation of protein kinase C family members by the novel polyphosphoinositides PtdIns-3,4-P2 and PtdIns-3,4,5-P3. J Biol Chem. 1994; 269: 32358–32367.
36. Moriya S, Kazlauskas A, Akimoto K, Hirai S, Mizuno K, Takenawa T, Fukui Y, Watanabe Y, Ozaki S, Ohno S. Platelet-derived growth factor activates protein kinase C through redundant and independent signaling pathways involving phospholipase C or phosphatidylinositol 3-kinase. Proc Natl Acad Sci U S A. 1996; 93: 151–155.
37. Wick KL, Liu F. A new molecular target of insulin action: regulating the pivotal PDK1. Curr Drug Targets Immune Endocr Metabol Disord. 2001; 1: 209–221.[CrossRef][Medline] [Order article via Infotrieve]
38. Le Good JA, Ziegler WH, Parekh DB, Alessi DR, Cohen P, Parker PJ. Protein kinase C isotypes controlled by phosphoinositide 3-kinase through the protein kinase PDK1. Science. 1998; 281: 2042–2045.
39. Chou MM, Hou W, Johnson J, Graham LK, Lee MH, Chen CS, Newton AC, Schaffhausen BS, Toker A. Regulation of protein kinase C by PI 3-kinase and PDK-1. Curr Biol. 1998; 8: 1069–1077.[CrossRef][Medline]
[Order article via Infotrieve]
40. Robin P, Boulven I, Desmyter C, Harbon S, Leiber D. ET-1 stimulates ERK signaling pathway through sequential activation of PKC and Src in rat myometrial cells. Am J Physiol Cell Physiol. 2002; 283: C251–C260.
41. Sayeski PP, Ali MS, Harp JB, Marrero MB, Bernstein KE. Phosphorylation of p130Cas by angiotensin II is dependent on c-Src, intracellular Ca2+, and protein kinase C. Circ Res. 1998; 82: 1279–1288.
42. Lu WY, Xiong ZG, Lei S, Orser BA, Dudek E, Browning MD, MacDonald JF. G-protein-coupled receptors act via protein kinase C and Src to regulate NMDA receptors. Nat Neurosci. 1999; 2: 331–338.[CrossRef][Medline] [Order article via Infotrieve]
43. Brandt DT, Goerke A, Heuer M, Gimona M, Leitges M, Kremmer E, Lammers R, Haller H, Mischak H. Protein kinase C
induces Src kinase activity via activation of the protein tyrosine phosphatase PTP . J Biol Chem. 2003; 278: 34073–34078.
44. Bjorge JD, Jakymiw A, Fujita DJ. Selected glimpses into the activation and function of Src kinase. Oncogene. 2000; 19: 5620–5635.[CrossRef][Medline] [Order article via Infotrieve]
45. Hanke JH, Gardner JP, Dow RL, Changelian PS, Brissette WH, Weringer EJ, Pollok BA, Connelly PA. Discovery of a novel, potent, and Src family-selective tyrosine kinase inhibitor: study of Lck- and FynT-dependent T cell activation. J Biol Chem. 1996; 271: 695–701.
46. Bence-Hanulec KK, Marshall J, Blair LA. Potentiation of neuronal L calcium channels by IGF-1 requires phosphorylation of the 1 subunit on a specific tyrosine residue. Neuron. 2000; 27: 121–131.[CrossRef][Medline]
[Order article via Infotrieve]
47. Hu XQ, Singh N, Mukhopadhyay D, Akbarali HI. Modulation of voltage-dependent Ca2+ channels in rabbit colonic smooth muscle cells by c-Src and focal adhesion kinase. J Biol Chem. 1998; 273: 5337–5342.
48. Wijetunge S, Hughes AD. Activation of endogenous c-Src or a related tyrosine kinase by intracellular (pY)EEI peptide increases voltage-operated calcium channel currents in rabbit ear artery cells. FEBS Lett. 1996; 399: 63–66.[CrossRef][Medline] [Order article via Infotrieve]
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