Gbetagamma-mediated prostacyclin production and cAMP-dependent protein kinase activation by endothelin-1 promotes vascular smooth muscle cell hypertrophy through inhibition of glycogen synthase kinase-3

Endothelin-1 (ET1) is a vasoactive peptide that stimulates hypertrophy of vascular smooth muscle cells (VSMC) through diverse signaling pathways mediated by G(q)/G(i)/G(13) heterotrimeric G proteins. We have found that ET1 stimulates the activity of cAMP-dependent protein kinase (PKA) in VSMC as profoundly as the G(s)-linked beta-adrenergic agonist, isoproterenol (ISO), but in a transient manner. PKA activation by ET1 was mediated by type-A ET1 receptors (ETA) and recruited an autocrine signaling mechanism distinct from that of ISO, involving G(i)-coupled betagamma subunits of heterotrimeric G proteins, extracellular signal-regulated kinases ERK1/2, cyclooxygenase COX-1 (but not COX-2) and prostacyclin receptors. In the functional studies, inhibition of PKA or COX-1 attenuated ET1-induced VSMC hypertrophy, suggesting the positive role of PKA in this response to ET1. Furthermore, we found that ET1 stimulates a Gbetagamma-mediated, PKA-dependent phosphorylation and inactivation of glycogen synthase kinase-3 (GSK3), an enzyme that regulates cell growth. Together, this study describes that (i) PKA can be transiently activated by G(i)-coupled agonists such as ET1 by an autocrine mechanism involving Gbetagamma/calcium/ERK/COX-1/prostacyclin signaling, and (ii) this PKA activation promotes VSMC hypertrophy, at least in part, through PKA-dependent phosphorylation and inhibition of GSK3.     

A novel mechanism of G protein-dependent phosphorylation of vasodilator-stimulated phosphoprotein

Vasodilator-stimulated phosphoprotein (VASP) is a major substrate of protein kinase A (PKA). Here we described the novel mechanism of VASP phosphorylation via cAMP-independent PKA activation. We showed that in human umbilical vein endothelial cells (HUVECs) alpha-thrombin induced phosphorylation of VASP. Specific inhibition of Galpha13 protein by the RGS domain of a guanine nucleotide exchange factor, p115RhoGEF, inhibited thrombin-dependent phosphorylation of VASP. More importantly, Galpha13-induced VASP phosphorylation was dependent on activation of RhoA and mitogen-activated protein kinase kinase kinase, MEKK1, leading to the stimulation of the NF-kappaB signaling pathway. alpha-Thrombin-dependent VASP phosphorylation was inhibited by small interfering RNA-mediated knockdown of RhoA, whereas Galpha13-dependent VASP phosphorylation was inhibited by a specific RhoA inhibitor botulinum toxin C3 and by a dominant negative mutant of MEKK1. We determined that Galpha13-dependent VASP phosphorylation was also inhibited by specific PKA inhibitors, PKI and H-89. In addition, the expression of phosphorylation-deficient IkappaB and pretreatment with the proteasome inhibitor MG-132 abolished Galpha13- and alpha-thrombin-induced VASP phosphorylation. In summary, we have described a novel pathway of Galpha13-induced VASP phosphorylation that involves activation of RhoA and MEKK1, phosphorylation and degradation of IkappaB, release of PKA catalytic subunit from the complex with IkappaB and NF-kappaB, and subsequent phosphorylation of VASP.     

A novel PAR-1-type thrombin receptor signaling pathway: cyclic AMP-independent activation of PKA in SNB-19 glioblastoma cells

Cellular effects of thrombin are mediated by members of a new subfamily of G protein-coupled receptors designated proteinase-activated receptors (PARs) with the prototype PAR-1. Investigation of PAR-1-induced signaling has been shown to be very important in clarifying thrombin's role in cell metabolism, differentiation, and growth. We evaluated connection of PAR-1 with the cAMP/PKA pathway in SNB-19 glioblastoma cells. Alpha-thrombin and the synthetic PAR-1 agonist SFLLRN stimulated PKA as shown by increased PKA activity and translocation of the catalytic PKA alpha subunits (PKA(cat)alpha) into the nucleus. However, no effect on cAMP could be observed. PKA(cat)alpha was found to be associated with nuclear factor-kappa B (NF-kappaB) p65 and its inhibitor protein IkappaB in SNB-19 cells. After PAR-1 stimulation, this association was markedly diminished. We conclude that PAR-1 mediates PKA activation without altering cAMP levels but includes NF-kappaB-associated PKA(cat)alpha in SNB-19 glioblastoma cells. This is the first evidence for a cAMP-independent PKA signaling by a G protein-coupled receptor.     

G(q)-dependent signalling by the lysophosphatidic acid receptor LPA(3) in gastric smooth muscle: reciprocal regulation of MYPT1 phosphorylation by Rho kinase and cAMP-independent PKA

The present study characterized the signalling pathways initiated by the bioactive lipid, LPA (lysophosphatidic acid) in smooth muscle. Expression of LPA(3) receptors, but not LPA(1) and LPA(2), receptors was demonstrated by Western blot analysis. LPA stimulated phosphoinositide hydrolysis, PKC (protein kinase C) and Rho kinase (Rho-associated kinase) activities: stimulation of all three enzymes was inhibited by expression of the G(alphaq), but not the G(alphai), minigene. Initial contraction and MLC(20) (20 kDa regulatory light chain of myosin II) phosphorylation induced by LPA were abolished by inhibitors of PLC (phospholipase C)-beta (U73122) or MLCK (myosin light-chain kinase; ML-9), but were not affected by inhibitors of PKC (bisindolylmaleimide) or Rho kinase (Y27632). In contrast, sustained contraction, and phosphorylation of MLC(20) and CPI-17 (PKC-potentiated inhibitor 17 kDa protein) induced by LPA were abolished selectively by bisindolylmaleimide. LPA-induced activation of IKK2 {IkappaB [inhibitor of NF-kappaB (nuclear factor kappaB)] kinase 2} and PKA (protein kinase A; cAMP-dependent protein kinase), and degradation of IkappaBalpha were blocked by the RhoA inhibitor (C3 exoenzyme) and in cells expressing dominant-negative mutants of IKK2(K44A) or RhoA(N19RhoA). Phosphorylation by Rho kinase of MYPT1 (myosin phosphatase targeting subunit 1) at Thr(696) was masked by phosphorylation of MYPT1 at Ser(695) by PKA derived from IkappaB degradation via RhoA, but unmasked in the presence of PKI (PKA inhibitor) or C3 exoenzyme and in cells expressing IKK2(K44A). We conclude that LPA induces initial contraction which involves activation of PLC-beta and MLCK and phosphorylation of MLC(20), and sustained contraction which involves activation of PKC and phosphorylation of CPI-17 and MLC(20). Although Rho kinase was activated, phosphorylation of MYPT1 at Thr(696) by Rho kinase was masked by phosphorylation of MYPT1 at Ser(695) via cAMP-independent PKA derived from the NF-kappaB pathway.     

Apolipoprotein E binding to low density lipoprotein receptor-related protein-1 inhibits cell migration via activation of cAMP-dependent protein kinase A

Smooth muscle cell migration and proliferation contribute to neointimal hyperplasia and vascular stenosis after endothelial denudation. Previous studies revealed that apolipoprotein E (apoE) is an effective inhibitor of platelet-derived growth factor-directed smooth muscle cell migration and proliferation and that the anti-migratory function is mediated via apoE binding to low density lipoprotein receptor-related protein-1 (LRP-1). This study was undertaken to identify the intracellular pathway by which apoE binding to LRP-1 results in inhibition of smooth muscle cell migration. The results showed that apoE increased intracellular cAMP levels 3-fold after 5 min, and the increase was sustained for more than 1 h. As a consequence, apoE also increased protein kinase A (PKA) activity in smooth muscle cells. Importantly, suppression of PKA activity with a cell-permeable peptide inhibitor of PKA abolished the inhibitory effect of apoE on smooth muscle cell migration. These results indicated that apoE inhibition of smooth muscle cell migration is mediated via the activation of cAMP-dependent PKA. Additional experiments revealed that apoE also inhibited fibroblasts migration toward platelet-derived growth factor by a similar mechanism of cAMP-dependent PKA activation. It is noteworthy that apoE failed to increase cAMP levels or inhibit migration of LRP-1-negative mouse embryonic fibroblasts and LRP-1-deficient smooth muscle cells. Taken together, these findings established the mechanism by which apoE inhibits cell migration, i.e. via cAMP-dependent protein kinase A activation as a consequence of its binding to LRP-1.     

Inflammatory gene expression by human colonic smooth muscle cells

Intestinal mucosal cells and invading leukocytes produce inappropriate levels of cytokines and chemokines in human colitis. However, smooth muscle cells of the airway and vasculature also synthesize cytokines and chemokines. To determine whether human colonic myocytes can synthesize proinflammatory mediators, strips of circular smooth muscle and smooth muscle cells were isolated from human colon. Myocytes and muscle strips were stimulated with 10 ng/ml of IL-1beta, TNF-alpha, and IFN-gamma, respectively. Expression of mRNA for IL-1beta, IL-6, IL-8, and cyclooxygenase-2 (COX-2) was induced within 2 h and continued to increase for 8-12 h. Regulated on activation, normal T cell-expressed and -secreted (RANTES) mRNA expression was slower, appearing at 8 h and increasing linearly through 20 h. Expression of all five mRNAs was inhibited by 0.1 microM MG-132, a proteosome inhibitor that blocks NF-kappaB activation. Expression of IL-1beta, IL-6, IL-8, and COX-2 mRNA was reduced by 30 microM PP1, an Src family tyrosine kinase inhibitor, and by 25 microM SB-203580, a p38 MAPK inhibitor. MAPK/extracellular regulated kinase-1 inhibitor PD-98059 (25 microM) was much less effective. In conclusion, human colonic smooth muscle cells can synthesize and secrete interleukins (IL-1beta and IL-6) and chemokines (IL-8 and RANTES) and upregulate expression of COX-2. Regulation of cytokine, chemokine, and COX-2 mRNA depends on multiple signaling pathways, including Src-family kinases, extracellular regulated kinase, p38 MAPKs, and NF-kappaB. SB-203580 was a consistent, efficacious inhibitor of inflammatory gene expression, suggesting an important role of p38 MAPK in synthetic functions of human colonic smooth muscle.     

Effects of dehydroepiandrosterone on mitogen-activated protein kinase in human aortic smooth muscle cells.

The objective of the present study was to determine whether dehydroepiandrosterone (DHEA) modifies growth factor-induced mitogen-activated protein kinase (MAPK) activation, based on our previous study demonstrating that DHEA attenuates fetal calf serum-induced proliferation in human male aortic smooth muscle cells (human male aortic SMCs). Human male aortic SMCs were used for this study. Platelet-derived growth factor-BB (PDGF-BB), epidermal growth factor (EGF), and basic fibroblast growth factor (bFGF), but not insulin-like growth factor-1 (IGF-1), stimulated MAPK activity. Only MAPK activation induced by PDGF-BB was reduced by pretreatment with DHEA, although DHEA did not affect the MAPK activation induced by EGF or bFGF. The basal and PDGF-stimulated MAPK activity were decreased by two types of cyclic AMP (cAMP) elevating agents and increased by cAMP-dependent protein kinase (PKA) inhibitor in human male aortic SMCs, suggesting that cAMP regulates MAPK negatively. The intracellular cAMP was increased by PDGF-BB. The increase of cAMP by PDGF-BB was augmented by pretreatment with DHEA, although DHEA alone did not affect cAMP. Neither EGF nor bFGF affected cAMP with and without DHEA pretreatment. Secretion of PGE2 induced by PDGF was augmented by pretreatment with DHEA. Stimulatory effects of DHEA on the production of PGE2 and cAMP were partially canceled by aromatase inhibitor and completely canceled by indomethacin or selective inhibitor of cyclooxygenase-2. These results suggest that DHEA inhibited MAPK activation induced by PDGF-BB via PGE2 overproduction and subsequent cAMP-dependent pathway in human male aortic SMCs.     

Functional significance of protein kinase A activation by endothelin-1 and ATP: negative regulation of SRF-dependent gene expression by PKA

Endothelin-1 (ET1) and ATP stimulate contraction and hypertrophy of vascular smooth muscle cells (VSMC) by activating diverse signalling pathways. In this study, we show that in VSMC, ET1 and ATP stimulate transient and sustained activation of protein kinase A (PKA), respectively. Using a dominant negative PKA mutant (PKA-DN), we examined the functional significance of PKA activation in the signalling of ET1 and ATP. Overexpression of PKA-DN did not alter the ET1- or ATP-induced phosphorylation of the extracellular signal-regulated protein kinase, Erk2. ATP stimulated a profound, PKA-dependent activation of cAMP-response element (CRE), whereas the effect of ET1 was negligible. Both ET1 and ATP stimulated serum response factor (SRF)-dependent gene expression. Overexpression of PKA-DN potentiated the effects of ET1 and ATP on SRF activity, whereas stimulation of PKA by isoproterenol, forskolin or by overexpression of the PKA catalytic subunit decreased SRF activity. These data demonstrate that (i) PKA negatively regulates SRF activity and (ii) ET1 and ATP stimulate opposing pathways, whose balance determines the net activity of SRF.     

PKA-dependent activation of PDE3A and PDE4 and inhibition of adenylyl cyclase V/VI in smooth muscle

Regulation of adenylyl cyclase type V/VI and cAMP-specific, cGMP-inhibited phosphodiesterase (PDE) 3 and cAMP-specific PDE4 by cAMP-dependent protein kinase (PKA) and cGMP-dependent protein kinase (PKG) was examined in gastric smooth muscle cells. Expression of PDE3A but not PDE3B was demonstrated by RT-PCR and Western blot. Basal PDE3 and PDE4 activities were present in a ratio of 2:1. Forskolin, isoproterenol, and the PKA activator 5,6-dichloro-1-beta-D-ribofuranosyl benzimidazole 3',5'-cyclic monophosphate, SP-isomer, stimulated PDE3A phosphorylation and both PDE3A and PDE4 activities. Phosphorylation of PDE3A and activation of PDE3A and PDE4 were blocked by the PKA inhibitors [protein kinase inhibitor (PKI) and H-89] but not by the PKG inhibitor (KT-5823). Sodium nitroprusside inhibited PDE3 activity and augmented forskolin- and isoproterenol-stimulated cAMP levels; PDE3 inhibition was reversed by blockade of cGMP synthesis. Forskolin stimulated adenylyl cyclase phosphorylation and activity; PKI blocked phosphorylation and enhanced activity. Stimulation of cAMP and inhibition of inositol 1,4,5-trisphosphate-induced Ca(2+) release and muscle contraction by isoproterenol were augmented additively by PDE3 and PDE4 inhibitors. The results indicate that PKA regulates cAMP levels in smooth muscle via stimulatory phosphorylation of PDE3A and PDE4 and inhibitory phosphorylation of adenylyl cyclase type V/VI. Concurrent generation of cGMP inhibits PDE3 activity and augments cAMP levels.     

The cAMP-responsive Rap1 guanine nucleotide exchange factor, Epac, induces smooth muscle relaxation by down-regulation of RhoA activity

Agonist activation of the small GTPase, RhoA, and its effector Rho kinase leads to down-regulation of smooth muscle (SM) myosin light chain phosphatase activity, an increase in myosin light chain (RLC(20)) phosphorylation and force. Cyclic nucleotides can reverse this process. We report a new mechanism of cAMP-mediated relaxation through Epac, a GTP exchange factor for the small GTPase Rap1 resulting in an increase in Rap1 activity and suppression of RhoA activity. An Epac-selective cAMP analog, 8-pCPT-2'-O-Me-cAMP ("007"), significantly reduced agonist-induced contractile force, RLC(20), and myosin light chain phosphatase phosphorylation in both intact and permeabilized vascular, gut, and airway SMs independently of PKA and PKG. The vasodilator PGI(2) analog, cicaprost, increased Rap1 activity and decreased RhoA activity in intact SMs. Forskolin, phosphodiesterase inhibitor isobutylmethylxanthine, and isoproterenol also significantly increased Rap1-GTP in rat aortic SM cells. The PKA inhibitor H89 was without effect on the 007-induced increase in Rap1-GTP. Lysophosphatidic acid-induced RhoA activity was reduced by treatment with 007 in WT but not Rap1B null fibroblasts, consistent with Epac signaling through Rap1B to down-regulate RhoA activity. Isoproterenol-induced increase in Rap1 activity was inhibited by silencing Epac1 in rat aortic SM cells. Evidence is presented that cooperative cAMP activation of PKA and Epac contribute to relaxation of SM. Our findings demonstrate a cAMP-mediated signaling mechanism whereby activation of Epac results in a PKA-independent, Rap1-dependent Ca(2+) desensitization of force in SM through down-regulation of RhoA activity. Cyclic AMP inhibition of RhoA is mediated through activation of both Epac and PKA.     

Cyclic adenosine 3',5'monophosphate/protein kinase A and mitogen-activated protein kinase 3/1 pathways are involved in adenylate cyclase-activating polypeptide 1-induced common alpha-glycoprotein subunit gene (Cga) expression in mouse pituitary gonadotroph LbetaT2 cells

Adenylate cyclase-activating polypeptide 1 (ADCYAP1) binds both Gs- and Gq-coupled receptors and stimulates adenylate cyclase/cAMP and protein kinase C/mitogen-activated protein kinase 3/1 (MAPK3/1) signaling pathways in pituitary gonadotrophs. In this study, we investigated the cAMP and MAPK3/1 signaling pathways induced by ADCYAP1 stimulation and examined the effects of ADCYAP1 on the expression of gonadotropin subunit genes using a clonal gonadotroph cell line, LbetaT2. ADCYAP1 increased intracellular cAMP accumulation up to 19-fold in LbetaT2 cells. Common alpha-glycoprotein subunit gene (Cga) promoter activity was strongly activated by both ADCYAP1 and the cyclic-AMP analog, 8-(4-chlorophenylthio) adenosine 3',5'-cyclic monophosphate (CPT-cAMP). Both had little effect on luteinizing hormone beta (Lhb) and follicle-stimulating hormone beta (Fshb) promoter activities. Cga promoter activity was significantly increased by transfection with constitutively active cAMP-dependent protein kinase (PKA). Activities of the Lhb and Fshb promoters were only modestly increased. Both ADCYAP1 and CPT-cAMP induced MAPK3/1 activation in LbetaT2 cells. The MEK inhibitor, U0126, and the PKA inhibitors, H89 and cAMP-dependent protein kinase peptide inhibitor (PKI), completely inhibited MAPK3/1 activation by either ADCYAP1 or CPT-cAMP. Using luciferase reporter constructs containing cis-elements, the cAMP response element (Cre) promoter was stimulated about 4-fold by ADCYAP1. ADCYAP1-induced Cre promoter activity was completely inhibited by H89, but not by U0126. ADCYAP1 also increased the activity of the serum response element (Sre) promoter, a target for MAPK3/1, and treatment of the cells with U0126 completely inhibited ADCYAP1-induced Sre promoter activity. ADCYAP1-increased Cga promoter activity was inhibited partially by both H89 and U0126. Although combining the inhibitors showed an additive inhibition effect, it did not result in complete inhibition. These results suggest that in LbetaT2 cells, ADCYAP1 mainly increases Cga through activation of PKA and MAPK3/1, as well as through an additional unknown pathway.     

Adrenomedullin inhibits angiotensin II-induced contraction in human aortic smooth muscle cells

The vasodilating peptide adrenomedullin (AM) has been reported to regulate vascular tone as well as proliferation and differentiation of various cell types in an autocrine/paracrine manner. Our study was designed to investigate the effect of AM on Ang II-induced contraction on human aortic smooth muscle cells (HASMC) in vitro, evaluating the signal pathways involved. Our findings indicate that AM was able to inhibit HASMC Ang II-induced contraction (IC50 19 nM). AM stimulated cAMP production in a dose-dependent fashion as well. SQ 22.536, an adenylate cyclase inhibitor, and KT5720, a PKA inhibitor, blunted the AM effect, suggesting that it was mediated by the activation of the cAMP transduction pathway. Our results suggest that AM plays a role in the regulation of HASMC contraction by antagonizing the Ang II effects and may be involved in conditions of altered regulation of the blood vessels.     

Interaction of heterotrimeric G13 protein with an A-kinase-anchoring protein 110 (AKAP110) mediates cAMP-independent PKA activation.

Heterotrimeric G proteins and protein kinase A (PKA) are two important transmitters that transfer signals from a wide variety of cell surface receptors to generate physiological responses. The established mechanism of PKA activation involves the activation of the Gs-cAMP pathway. Binding of cAMP to the regulatory subunit of PKA (rPKA) leads to a release and subsequent activation of a catalytic subunit of PKA (cPKA). Here, we report a novel mechanism of PKA stimulation that does not require cAMP. Using yeast two-hybrid screening, we found that the alpha subunit of G13 protein interacted with a member of the PKA-anchoring protein family, AKAP110. Using in vitro binding and coimmunoprecipitation assays, we have shown that only activated G alpha 13 binds to AKAP110, suggesting a potential role for AKAP110 as a G alpha subunit effector protein. Importantly, G alpha 13, AKAP110, rPKA, and cPKA can form a complex, as shown by coimmunoprecipitation. By characterizing the functional significance of the G alpha 13-AKAP110 interaction, we have found that G alpha 13 induced release of the cPKA from the AKAP110-rPKA complex, resulting in a cAMP-independent PKA activation. Finally, AKAP110 significantly potentiated G alpha 13-induced activation of PKA. Thus, AKAP110 provides a link between heterotrimeric G proteins and cAMP-independent activation of PKA.     

Suppression of WEE1 and stimulation of CDC25A correlates with endothelin-dependent proliferation of rat aortic smooth muscle cells

Proliferation of vascular smooth muscle cells plays a key role in the pathogenesis of several disorders of the vascular wall. Endothelin (ET), a vasoactive peptide that signals through a G protein-coupled receptor, has been linked to mitogenesis in vascular smooth muscle cells, but the mechanistic details underlying this activity remain incompletely understood. In the present study, we demonstrate that ET-dependent mitogenesis in rat neonatal and adult aortic smooth muscle (RASM) cells is accompanied by an increase (up to 10-fold) in CDK2 activity, but not CDK2 protein levels. This effect is blocked almost entirely by PD98059 and UO126, implying involvement of the MEK/ERK signal transduction cascade in the activation. Extracts of ET-treated cells phosphorylate the N terminus of WEE1, an inhibitory kinase, which negatively regulates CDK2 activity through phosphorylation at Tyr(15), leading to a decrease in WEE1 activity and a reduction in levels of phospho-Tyr(15) in the CDK2 protein. ET also increases expression and activity of CDC25A, the regulatory phosphatase responsible for dephosphorylating Tyr(15). All of these effects are reversible following treatment with the MEK inhibitor PD98059. ET also increases levels of CDC2 activity in these cells in association with a decrease in levels of phospho-Tyr(15) on the CDC2 molecule. Phosphorylation of WEE1 is linked to ERK while phosphorylation of MYT1 (CDC2-selective inhibitory kinase) is tied to the ribosomal S6 kinase (RSK). In summary, ET controls progression through the cell cycle, in part, by increasing CDK2 and CDC2 activity through the MEK/ERK/RSK signal transduction pathway(s). This results from the phosphorylation and subsequent inactivation of two inhibitory kinases (WEE1 and MYT1) that tonically suppress CDK2 and CDC2 activity and activation of a phosphatase (CDC25A) that increases CDK2 activity.     

Autoinhibition of endothelial nitric oxide synthase (eNOS) in gut smooth muscle by nitric oxide

Nitric oxide in the gut is produced by nNOS in enteric neurons and by eNOS in smooth muscle cells. The eNOS in smooth muscle is activated by vasoactive intestinal peptide (VIP) released from enteric neurons. In the present study, we examined the effect of nitric oxide on VIP-induced eNOS activation in smooth muscle cells isolated from human intestine and rabbit stomach. NOS activity was measured as formation of the 1:1 co-product, l-citrulline from l-arginine. VIP caused an increase in l-citrulline production that was inhibited by NO in a concentration dependent manner (IC(50)~25 microM; maximal inhibition 72% at 100 microM NO). Basal l-citrulline production, however, was unaffected by NO. The effect was not mediated by cGMP/PKG since the PKG inhibitor KT5823 had no effect on eNOS autoinhibition. The autoinhibition was selective for NO since the co-product l-citrulline had no effect on VIP-induced NOS activation. Similar effects were obtained in rabbit gastric and human intestinal smooth muscle cells. The results suggest that NO produced in smooth muscle cells as a result of the activation of eNOS by VIP exerts an autoinhibitory restraint on eNOS thereby regulating the balance of the VIP/cAMP/PKA and NO/cGMP/PKG pathways that regulate the relaxation of gut smooth muscle.     

PACAP-38 induces neuronal differentiation of human SH-SY5Y neuroblastoma cells via cAMP-mediated activation of ERK and p38 MAP kinases

The intracellular signaling pathways mediating the neurotrophic actions of pituitary adenylate cyclase-activating polypeptide (PACAP) were investigated in human neuroblastoma SH-SY5Y cells. Previously, we showed that SH-SY5Y cells express the PAC(1) and VIP/PACAP receptor type 2 (VPAC(2)) receptors, and that the robust cAMP production in response to PACAP and vasoactive intestinal peptide (VIP) was mediated by PAC(1) receptors (Lutz et al. 2006). Here, we investigated the ability of PACAP-38 to differentiate SH-SY5Y cells by measuring morphological changes and the expression of neuronal markers. PACAP-38 caused a concentration-dependent increase in the number of neurite-bearing cells and an up-regulation in the expression of the neuronal proteins Bcl-2, growth-associated protein-43 (GAP-43) and choline acetyltransferase: VIP was less effective than PACAP-38 and the VPAC(2) receptor-specific agonist, Ro 25-1553, had no effect. The effects of PACAP-38 and VIP were blocked by the PAC(1) receptor antagonist, PACAP6-38. As observed with PACAP-38, the adenylyl cyclase activator, forskolin, also induced an increase in the number of neurite-bearing cells and an up-regulation in the expression of Bcl-2 and GAP-43. PACAP-induced differentiation was prevented by the adenylyl cyclase inhibitor, 2',5'-dideoxyadenosine (DDA), but not the protein kinase A (PKA) inhibitor, H89, or by siRNA-mediated knock-down of the PKA catalytic subunit. PACAP-38 and forskolin stimulated the activation of extracellular signal-regulated kinase (ERK), mitogen-activated protein kinase (MAP; p38 MAP kinase) and c-Jun N-terminal kinase (JNK). PACAP-induced neuritogenesis was blocked by the MEK1 inhibitor PD98059 and partially by the p38 MAP kinase inhibitor SB203580. Activation of exchange protein directly activated by cAMP (Epac) partially mimicked the effects of PACAP-38, and led to the phosphorylation of ERK but not p38 MAP kinase. These results provide evidence that the neurotrophic effects of PACAP-38 on human SH-SY5Y neuroblastoma cells are mediated by the PAC(1) receptor through a cAMP-dependent but PKA-independent mechanism, and furthermore suggest that this involves Epac-dependent activation of ERK as well as activation of the p38 MAP kinase signaling pathway.     

Phosphorylation of GRK2 by PKA augments GRK2-mediated phosphorylation, internalization, and desensitization of VPAC2 receptors in smooth muscle

The smooth muscle of the gut expresses mainly G(s) protein-coupled vasoactive intestinal peptide (VIP)/pituitary adenylate cyclase-activating peptide receptors (VPAC(2) receptors), which belong to the secretin family of G protein-coupled receptors. The extent to which PKA and G protein-coupled receptor kinases (GRKs) participate in homologous desensitization varies greatly among the secretin family of receptors. The present study identified the novel role of PKA in homologous desensitization of VPAC(2) receptors via the phosphorylation of GRK2 at Ser(685). VIP induced phosphorylation of GRK2 in a concentration-dependent fashion, and the phosphorylation was abolished by blockade of PKA with cell-permeable myristoylated protein kinase inhibitor (PKI) or in cells expressing PKA phosphorylation-site deficient GRK2(S685A). Phosphorylation of GRK2 increased its activity and binding to G betagamma. VIP-induced phosphorylation of VPAC(2) receptors was abolished in muscle cells expressing kinase-deficient GRK2(K220R) and attenuated in cells expressing GRK2(S685A) or by PKI. VPAC(2) receptor internalization (determined from residual (125)I-labeled VIP binding and receptor biotinylation after a 30-min exposure to VIP) was blocked in cells expressing GRK2(K220R) and attenuated in cells expressing GRK2(S685A) or by PKI. Finally, VPAC(2) receptor degradation (determined from residual (125)I-labeled VIP binding and receptor expression after a prolonged exposure to VIP) and functional VPAC(2) receptor desensitization (determined from the decrease in adenylyl cyclase activity and cAMP formation after a 30-min exposure to VIP) were abolished in cells expressing GRK2(K220R) and attenuated in cells expressing GRK2(S685A). These results demonstrate that in gastric smooth muscle VPAC(2) receptor phosphorylation is mediated by GRK2. Phosphorylation of GRK2 by PKA enhances GRK2 activity and its ability to induce VPAC(2) receptor phosphorylation, internalization, desensitization, and degradation.     

肾上腺髓质素和降钙素基因相关肽受体在血管平滑肌细胞的脱敏—受体活性修饰蛋白的作用

新近研究发现,肾上腺髓质素（adrenomedullin,ADM)和降钙素基因相关肽（calcitonin gene-related peptide,CGRP)均能与降钙素受体样受体（calcitoni receptor-like receptor,CRLR)结合,其配体特异性由受体活性修饰蛋白（receptor activity-modifying protein RAMP)调控,本工作在离体培养的大鼠胸主动脉血管平滑肌细胞（vsacular smooth muscle cells,VSMCs)上观察ADM和CGRP受体脱敏现象,以探讨CRLR／RAMP假说在心血管组织方面的意义,用无血清培养基（serum-free medium,SFM)和含有10^-8mol/L ADM,CGRP和肾上腺髓素质前体原N－末端20肽（proadrenomedullin N-terminal 20 peptide PAMP）的SFM培养,再用10^-8mol/L ADM或 CGRP和磷酸二酯酶的抑制剂异丙基次黄苷（isobutyryl methyxanthine,IBMX)与VSMCs进行第二次孵育,然后收集细胞,测定VSMCs cAMP含量。10^-8mol/LADM,CGRP和PAMP单独与VSMCs孵育,VSMCs cAMP含量分别较SFM组高191％（P＜0．01）,385％（P＜0．01）和67％（P＜0．05）,预先用10^-8mol/L ADM ak CGRP与VSMCs孵育可降低随后的CGRP刺激VSMCs产生cAMP,分别较单次CGRP育少44％（P＜0．05）和48％（P＜0．01）,预先用100nmol/L蛋白激酶A（PKA）抑制剂H－89处理VSMCs,可完全阻断ADM和CGRP预处理诱导的第二次CGRP刺激的VSMCs cAMP含量减少,表明VSMCs对CGRP的脱敏过程是通过PKA途径实现的,预先用ADM,CGRP处理VSMCs后,用ADM第二次孵育,细胞内cAMP含量与单次ADM孵育无明显改变,PKA抑制H－89与VSMCs孵育,无论对欠ADM刺激或对ADM和CGRP处理的第二次刺激的cAMP生成均无影响,用PAMP处理VSMCs后,ADM和CGRP的第二次刺激的VSMCs cAMP水平无明显改变（P＞0．05）。结果提示,在离体培养的大鼠VSMCs,ADM epc wsg i euk txgtdmj CGRP受体对预先用ADM和CGRP处理后的激动剂的第二次刺激都脱敏,表明ADM和CGRP的脱敏现象不一致。     

cAMP inhibits IP(3)-dependent Ca(2+) release by preferential activation of cGMP-primed PKG

The singular effects and interplay of cAMP- and cGMP-dependent protein kinase (PKA and PKG) on Ca(2+) mobilization were examined in dispersed smooth muscle cells. In permeabilized muscle cells, exogenous cAMP and cGMP inhibited inositol 1,4,5-trisphosphate (IP(3))-induced Ca(2+) release and muscle contraction via PKA and PKG, respectively. A combination of cAMP and cGMP caused synergistic inhibition that was exclusively mediated by PKG and attenuated by PKA. In intact muscle cells, low concentrations (10 nM) of isoproterenol and sodium nitroprusside (SNP) inhibited agonist-induced, IP(3)-dependent Ca(2+) release and muscle contraction via PKA and PKG, respectively. A combination of isoproterenol and SNP increased PKA and PKG activities: the increase in PKA activity reflected inhibition of phosphodiesterase 3 activity by cGMP, whereas the increase in PKG activity reflected activation of cGMP-primed PKG by cAMP. Inhibition of Ca(2+) release and muscle contraction by the combination of isoproterenol and SNP was preferentially mediated by PKG. In light of studies showing that PKG phosphorylates the IP(3) receptor in intact and permeabilized muscle cells, whereas PKA phosphorylates the receptor in permeabilized cells only, the results imply that inhibition of IP(3)-induced Ca(2+) release is mediated exclusively by PKG. The effect of PKA on agonist-induced Ca(2+) release probably reflects inhibition of IP(3) formation.     

Calcitonin gene-related peptide regulates mitogen-activated protein kinase pathway to decrease transforming growth factor β1-induced hepatic plasminogen activator inhibitor-1 mRNA expression in HepG2 cells

Transforming growth factor (TGF) β1-induced plasminogen activator inhibitor (PAI)-1 is one of factors associated with the development of hepatic fibrosis. Calcitonin gene-related peptide (CGRP) shows hepatoprotective effect during hepatic injuries, including fibrosis. However, the effects of CGRP on PAI-1 expression induced by TGFβ1 are unknown. In this study, we investigated the effect of CGRP on TGFβ1-induced PAI-1 expression and its regulatory mechanisms in HepG2 cells. CGRP inhibited TGFβ1-induced PAI-1 expression. H89, a protein kinase A inhibitor, abolished the inhibition of TGFβ1-induced PAI-1 expression by CGRP. TGFβ1 activated mitogen-activated protein kinase (MAPK), including extracellular signal-regulated kinase, c-jun NH₂-terminal kinase, and p38, and this activation was abolished by CGRP. These results show that the CGRP-induced cAMP/PKA activation suppresses activation of MAPK induced by TGFβ1, leading to decreased PAI-1 expression in HepG2 cells.