RESUMEN
The pathogenesis of diabetes-associated motility disorders are multifactorial and attributed to abnormalities in extrinsic and intrinsic innervation, and a decrease in the number of interstitial cells of Cajal, and nNOS expression and activity. Here we studied the effect of hyperglycemia on smooth muscle function. Using smooth muscles from the fundus of ob/ob mice and of wild type (WT) mice treated with 30 mM glucose (HG), we identified the molecular mechanism by which hyperglycemia upregulates RhoA/Rho kinase pathway and muscle contraction. RhoA expression, Rho kinase activity and muscle contraction were increased, while miR-133a expression was decreased in smooth muscle of ob/ob mice and in smooth muscle treated with HG. Intraperitoneal injections of pre-miR-133a decreased RhoA expression in WT mice and reversed the increase in RhoA expression in ob/ob mice. Intraperitoneal injections of antagomiR-133a increased RhoA expression in WT mice and augmented the increase in RhoA expression in ob/ob mice. The effect of pre-miR-133a or antagomiR-133a in vitro in smooth muscle treated with HG was similar to that obtained in vivo, suggesting that the expression of RhoA is negatively regulated by miR-133a and a decrease in miR-133a expression in diabetes causes an increase in RhoA expression. Oxidative stress (levels of reactive oxygen species and hydrogen peroxide, and expression of superoxide dismutase 1 and NADPH oxidase 4) was increased in smooth muscle of ob/ob mice and in HG-treated smooth muscle. Treatment of ob/ob mice with N-acetylcysteine (NAC) in vivo or addition of NAC in vitro to HG-treated smooth muscle reversed the effect of glucose on the expression of miR-133a and RhoA, Rho kinase activity and muscle contraction. NAC treatment also reversed the decrease in gastric emptying in ob/ob mice. We conclude that oxidative stress in diabetes causes a decrease in miR-133a expression leading to an increase in RhoA/Rho kinase pathway and muscle contraction.
Asunto(s)
Contracción Muscular/fisiología , Músculo Liso/fisiología , Estrés Oxidativo , Quinasas Asociadas a rho/metabolismo , Proteína de Unión al GTP rhoA/metabolismo , Acetilcisteína/farmacología , Animales , Western Blotting , Células Cultivadas , Diabetes Mellitus/genética , Diabetes Mellitus/metabolismo , Diabetes Mellitus/fisiopatología , Depuradores de Radicales Libres/farmacología , Mucosa Gástrica/metabolismo , Expresión Génica/efectos de los fármacos , Glucosa/farmacología , Hiperglucemia/genética , Hiperglucemia/metabolismo , Hiperglucemia/fisiopatología , Ratones Endogámicos C57BL , Ratones Obesos , MicroARNs/genética , Músculo Liso/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal/genética , Estómago/fisiología , Regulación hacia Arriba , Quinasas Asociadas a rho/genética , Proteína de Unión al GTP rhoA/genéticaRESUMEN
Antimicrobial peptides (AMPs) are of importance in defense mechanism of many organisms and are potential candidate for treatment of infections in animals and humans. AMPs exhibit a wide range of immunomodulatory activities related to innate immunity, wound healing, and inflammation. AMPs also serve as drug delivery vectors, antitumor agents, and mitogenic agents. Here, we describe the investigation of anticancer and cytotoxic activities of antimicrobial peptides by colorimetric MTT assay using smooth muscle, dental pulp stem cell, human colon cancer cell line (SW620), and human oral squamous carcinoma cell line (HSC4).
Asunto(s)
Péptidos Catiónicos Antimicrobianos/farmacología , Antineoplásicos/farmacología , Recuento de Células/métodos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Neoplasias del Colon , Pulpa Dental/citología , Humanos , Neoplasias de la Boca , Músculo Liso/citología , Células Madre/efectos de los fármacos , Células Madre/metabolismoRESUMEN
We examined whether CB1 receptors in smooth muscle conform to the signaling pattern observed with other Gi-coupled receptors that stimulate contraction via two Gßγ-dependent pathways (PLC-ß3 and phosphatidylinositol 3-kinase/integrin-linked kinase). Here we show that the anticipated Gßγ-dependent signaling was abrogated. Except for inhibition of adenylyl cyclase via Gαi, signaling resulted from Gßγ-independent phosphorylation of CB1 receptors by GRK5, recruitment of ß-arrestin1/2, and activation of ERK1/2 and Src kinase. Neither uncoupling of CB1 receptors from Gi by pertussis toxin (PTx) or Gi minigene nor expression of a Gßγ-scavenging peptide had any effect on ERK1/2 activity. The latter was abolished in muscle cells expressing ß-arrestin1/2 siRNA. CB1 receptor internalization and both ERK1/2 and Src kinase activities were abolished in cells expressing kinase-deficient GRK5(K215R). Activation of ERK1/2 and Src kinase endowed CB1 receptors with the ability to inhibit concurrent contractile activity. We identified a consensus sequence (102KSPSKLSP109) for phosphorylation of RGS4 by ERK1/2 and showed that expression of a RGS4 mutant lacking Ser103/Ser108 blocked the ability of anandamide to inhibit acetylcholine-mediated phosphoinositide hydrolysis or enhance Gαq:RGS4 association and inactivation of Gαq. Activation of Src kinase by anandamide enhanced both myosin phosphatase RhoA-interacting protein (M-RIP):RhoA and M-RIP:MYPT1 association and inhibited Rho kinase activity, leading to increase of myosin light chain (MLC) phosphatase activity and inhibition of sustained muscle contraction. Thus, unlike other Gi-coupled receptors in smooth muscle, CB1 receptors did not engage Gßγ but signaled via GRK5/ß-arrestin activation of ERK1/2 and Src kinase: ERK1/2 accelerated inactivation of Gαq by RGS4, and Src kinase enhanced MLC phosphatase activity, leading to inhibition of ACh-stimulated contraction.
Asunto(s)
Arrestinas/metabolismo , Quinasa 5 del Receptor Acoplado a Proteína-G/metabolismo , Sistema de Señalización de MAP Quinasas/fisiología , Músculo Liso/fisiología , Receptor Cannabinoide CB1/fisiología , Familia-src Quinasas/metabolismo , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Ácidos Araquidónicos/farmacología , Endocannabinoides/farmacología , Subunidades alfa de la Proteína de Unión al GTP Gq-G11/metabolismo , Músculo Liso/efectos de los fármacos , Alcamidas Poliinsaturadas/farmacología , Conejos , Receptor Cannabinoide CB1/biosíntesis , Receptor Cannabinoide CB1/efectos de los fármacos , beta-Arrestinas , Quinasas Asociadas a rho/metabolismoRESUMEN
We examined expression of protease-activated receptors 2 (PAR2) and characterized their signaling pathways in rabbit gastric muscle cells. The PAR2 activating peptide SLIGRL (PAR2-AP) stimulated Gq, G13, Gi1, PI hydrolysis, and Rho kinase activity, and inhibited cAMP formation. Stimulation of PI hydrolysis was partly inhibited in cells expressing PAR2 siRNA, Gaq or Gai minigene and in cells treated with pertussis toxin, and augmented by expression of dominant negative regulator of G protein signaling (RGS4(N88S)). Stimulation of Rho kinase activity was abolished by PAR-2 or Ga13 siRNA, and by Ga13 minigene. PAR2-AP induced a biphasic contraction; initial contraction was selectively blocked by the inhibitor of PI hydrolysis (U73122) or MLC kinase (ML-9), whereas sustained contraction was selectively blocked by the Rho kinase inhibitor (Y27632). PAR2-AP induced phosphorylation of MLC20, MYPT1 but not CPI-17. PAR2-AP also caused a decrease in the association of NF-kB and PKA catalytic subunit: the effect of PAR2-AP was blocked by PAR2 siRNA or phosphorylation-deficient RhoA (RhoA(S188A)). PAR2-AP-induced degradation of IkBa and activation of NF-kB were abolished by the blockade of RhoA activity by Clostridium botulinum C3 exoenzyme suggesting RhoA-dependent activation of NF-kB. PAR2-AP-stimulated Rho kinase activity was significantly augmented by the inhibitors of PKA (myristoylated PKI), IKK2 (IKKIV) or NF-kB (MG132), and in cells expressing dominant negative mutants of IKK (IKK(K44A), IkBa (IkBa (S32A/S36A)) or RhoA(S188A), suggesting feedback inhibition of Rho kinase activity via PKA derived from NF-kB pathway. PAR2-AP induced phosphorylation of RhoA and the phosphorylation was attenuated in cells expressing phosphorylation-deficient RhoA(S188A). Our results identified signaling pathways activated by PAR2 to mediate smooth muscle contraction and a novel pathway for feedback inhibition of PAR2-stimulated RhoA. The pathway involves activation of the NF-kB to release catalytic subunit of PKA from its binding to IkBa and phosphorylation of RhoA at Ser(188).
Asunto(s)
Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Proteínas de Unión al GTP/metabolismo , Músculo Liso/metabolismo , Receptor PAR-2/metabolismo , Transducción de Señal , Adenilil Ciclasas/metabolismo , Animales , Activación Enzimática , Contracción Muscular , Músculo Liso/citología , Músculo Liso/enzimología , FN-kappa B/metabolismo , Fosfoinositido Fosfolipasa C/metabolismo , ConejosRESUMEN
Previous studies have identified differences in the expression of proteins that regulate myosin light chain phosphorylation and contraction in tonic and phasic smooth muscle. cGMP plays a critical role in smooth muscle relaxation and is important for optimal function of phasic and tonic smooth muscle. The intracellular cGMP levels are regulated by its hydrolysis via phosphodiesterase 5 (PDE5) and efflux via novel multidrug resistance protein 5 (MRP5). In the present study we tested the hypothesis that the differences in the phasic and tonic behavior of smooth muscles may be related to differences in mechanisms that terminate cGMP signaling. Expression of PDE5 and MRP5 was significantly (more than 2-fold) higher in fundus compared with antrum. The NO donor S-nitrosoglutathione (GSNO) caused an increase in PDE5 activity and intra- and extracellular cGMP levels in both fundus and antrum. Stimulation of PDE5 activity and increase in extracellular cGMP were significantly higher in fundus, whereas increase in intracellular cGMP was significantly higher in antrum. GSNO-induced increase in extracellular cGMP was blocked in dispersed cells by the cyclic nucleotide export blocker probenecid and in cultured muscle cells by depletion of ATP or suppression of MRP5 by siRNA, providing evidence that cGMP efflux was mediated by ATP-dependent export via MRP5. Consistent with the higher expression and activity levels of PDE5 and MRP5, GSNO-induced PKG activity and muscle relaxation were significantly lower in muscle cells from fundus compared with antrum. Thus higher expression of PDE5 and MRP5 in muscle cells from fundus correlates with tonic phenotype of muscle.
Asunto(s)
GMP Cíclico/metabolismo , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 5/metabolismo , Fundus Gástrico/enzimología , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/metabolismo , Contracción Muscular , Músculo Liso/enzimología , Miocitos del Músculo Liso/enzimología , Antro Pilórico/enzimología , Adenosina Trifosfato/metabolismo , Animales , Células Cultivadas , Proteínas Quinasas Dependientes de GMP Cíclico/metabolismo , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 5/genética , Relación Dosis-Respuesta a Droga , Fundus Gástrico/citología , Fundus Gástrico/efectos de los fármacos , Hidrólisis , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/genética , Contracción Muscular/efectos de los fármacos , Relajación Muscular , Músculo Liso/citología , Músculo Liso/efectos de los fármacos , Miocitos del Músculo Liso/efectos de los fármacos , Óxido Nítrico/metabolismo , Donantes de Óxido Nítrico/farmacología , Fenotipo , Antro Pilórico/citología , Antro Pilórico/efectos de los fármacos , Interferencia de ARN , ARN Mensajero/metabolismo , Conejos , Sistemas de Mensajero Secundario , Factores de Tiempo , TransfecciónRESUMEN
BACKGROUND: The Jk(a-b-) phenotype is rare in most populations and often detected after transfusion or pregnancy. After immunisation, anti-Jk3 forms and it can be difficult to find compatible Jk(a-b-) donors. Using anti-Jk(a) and anti-Jk(b) in a conventional tube method is unsuitable for identifying Jk(a-b-) in mass screening of blood donors. Jk(a-b-) phenotypes are associated with the absence of urea transporters on erythrocytes, making red blood cells (RBC) resistant to lysis by 2M urea, while Jk(a+b-), Jk(a-b+) and Jk(a+b+) phenotypes are susceptible to lysis. MATERIALS AND METHODS: We screened for Jk(a-b-) phenotypes in blood donors by the urea lysis test using a 96-well microplate. The Jk(a-b-) phenotypes were confirmed by the indirect antiglobulin test (IAT). RESULTS: Altogether, 20,163 blood samples from Thai blood donors were tested and only RBC from five samples were resistant to lysis by 2M urea, while 20,158 samples were completely lysed within 5 min. In an IAT, both anti-Jk(a) and anti-Jk(b) failed to agglutinate RBC from all five samples. Using a micro-titre plate, the direct urea lysis test, costs * 0.01, about 480 times less than IAT. Moreover, the test time for each plate (94 samples) is about 18 times less than that for IAT. CONCLUSION: Jk(a-b-) phenotype screening by the direct urea lysis test on samples in a micro-titre plate is simple, cost-effective and practical for mass screening of blood donors.
Asunto(s)
Donantes de Sangre , Tipificación y Pruebas Cruzadas Sanguíneas/métodos , Sistema del Grupo Sanguíneo de Kidd , Tamizaje Masivo , Urea/química , Femenino , Humanos , Masculino , Fenotipo , Embarazo , TailandiaRESUMEN
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.
Asunto(s)
Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Subunidades alfa de la Proteína de Unión al GTP Gq-G11/metabolismo , Músculo Liso/metabolismo , Fosfatasa de Miosina de Cadena Ligera/metabolismo , Receptores del Ácido Lisofosfatídico/metabolismo , Transducción de Señal , Quinasas Asociadas a rho/metabolismo , Animales , Células Cultivadas , Activación Enzimática , Humanos , Lipopolisacáridos/farmacología , Ratones , Contracción Muscular/efectos de los fármacos , Músculo Liso/efectos de los fármacos , Miosinas/metabolismo , FN-kappa B/metabolismo , Fosfolipasa C beta/metabolismo , Fosforilación/efectos de los fármacos , Fosfotreonina/metabolismo , Conejos , Quinasas Asociadas a rho/genéticaRESUMEN
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.
Asunto(s)
Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Endocitosis/fisiología , Quinasa 2 del Receptor Acoplado a Proteína-G/metabolismo , Miocitos del Músculo Liso/metabolismo , Receptores de Tipo II del Péptido Intestinal Vasoactivo/metabolismo , Animales , Unión Competitiva/fisiología , Células Cultivadas , AMP Cíclico/metabolismo , Relación Dosis-Respuesta a Droga , Endocitosis/efectos de los fármacos , Retroalimentación Fisiológica/efectos de los fármacos , Retroalimentación Fisiológica/fisiología , Quinasa 2 del Receptor Acoplado a Proteína-G/química , Quinasa 2 del Receptor Acoplado a Proteína-G/genética , Mucosa Gástrica/metabolismo , Mutación/genética , Miocitos del Músculo Liso/efectos de los fármacos , Fosforilación/efectos de los fármacos , Conejos , Ensayo de Unión Radioligante , Receptores de Tipo II del Péptido Intestinal Vasoactivo/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiología , Estómago/citología , Péptido Intestinal Vasoactivo/metabolismo , Péptido Intestinal Vasoactivo/farmacologíaRESUMEN
Smooth muscle of the gut undergoes rhythmic cycles of contraction and relaxation. Various constituents in the pathways that mediate muscle contraction could act to cross-regulate cAMP or cGMP levels and terminate subsequent relaxation. We have previously shown that cAMP levels are regulated by PKA-mediated phosphorylation of cAMP-specific phosphodiesterase 3A (PDE3A) and PDE4D5; the latter is the only PDE4D isoform expressed in smooth muscle. In the present study we have elucidated a mechanism whereby cholecystokinin (CCK) and, presumably, other contractile agonists capable of activating PKC can cross-regulate cAMP levels. Forskolin stimulated PDE4D5 phosphorylation and PDE4D5 activity. CCK significantly increased forskolin-stimulated PDE4D5 phosphorylation and activity and attenuated forskolin-stimulated cAMP levels. The effect of CCK on forskolin-induced PDE4D5 phosphorylation and activity and on cAMP levels was blocked by the inhibitors of PLC or PKC and in cultured muscle cells by the expression of Galpha(q) minigene. The effects of CCK on PDE4D5 phosphorylation, PDE4D5 activity, and cAMP levels were mimicked by low (1 nM) concentrations of okadaic acid, but not by a low (10 nM) concentration of tautomycin, suggesting involvement of PP2A. Purified catalytic subunit of PP2A but not PP1 dephosphorylated PDE4D5 in vitro. Coimmunoprecipitation studies demonstrated association of PDE4D5 with PP2A and the association was decreased by the activation of PKC. In conclusion, cAMP levels are cross-regulated by contractile agonists via a mechanism that involves PLC-beta-dependent, PKC-mediated inhibition of PP2A activity that leads to increase in PDE4D5 phosphorylation and activity and inhibition of cAMP levels.
Asunto(s)
Colecistoquinina/metabolismo , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 4/metabolismo , Mucosa Gástrica/metabolismo , Contracción Muscular , Miocitos del Músculo Liso/metabolismo , Proteína Quinasa C/metabolismo , Proteína Fosfatasa 2/metabolismo , Transducción de Señal , Inhibidores de Adenilato Ciclasa , Adenilil Ciclasas/metabolismo , Animales , Células Cultivadas , Colforsina/farmacología , AMP Cíclico/metabolismo , Activación Enzimática , Activadores de Enzimas/farmacología , Inhibidores Enzimáticos/farmacología , Subunidades alfa de la Proteína de Unión al GTP Gq-G11/genética , Subunidades alfa de la Proteína de Unión al GTP Gq-G11/metabolismo , Contracción Muscular/efectos de los fármacos , Miocitos del Músculo Liso/efectos de los fármacos , Miocitos del Músculo Liso/enzimología , Ácido Ocadaico/farmacología , Fosfolipasa C beta/antagonistas & inhibidores , Fosfolipasa C beta/metabolismo , Fosforilación , Proteína Quinasa C/antagonistas & inhibidores , Proteína Fosfatasa 2/antagonistas & inhibidores , Conejos , Transducción de Señal/efectos de los fármacos , Estómago/citología , Estómago/efectos de los fármacos , Estómago/enzimología , TransfecciónRESUMEN
The aim of the study was to examine the mechanisms by which ACh, acting via m2 receptors, regulates GRK2-mediated VPAC(2) receptor desensitization in gastric smooth muscle cells. VIP induced VPAC(2) receptor phosphorylation and internalization in freshly dispersed smooth muscle cells. Co-stimulation with acetylcholine (ACh), in the presence of m3 receptor antagonist, 4-DAMP, augmented VPAC(2) receptor phosphorylation and internalization. The m2 receptor antagonist methoctramine or the c-Src inhibitor PP2 blocked the effect of ACh, suggesting that the augmentation was mediated by c-Src, derived from m2 receptor activation. ACh induced activation of c-Src and phosphorylation of GRK2 and the effects of ACh were blocked by methoctramine, PP2, or by uncoupling of m2 receptors from G(i3) with pertussis toxin. In conclusion, we identified a novel mechanism of cross-regulation of GRK2-mediated phosphorylation and internalization of G(s)-coupled VPAC(2) receptors by G(i)-coupled m2 receptors via tyrosine phosphorylation of GRK2 and stimulation of GRK2 activity.
Asunto(s)
Proteínas Proto-Oncogénicas pp60(c-src)/metabolismo , Receptor Muscarínico M2/fisiología , Receptores de Tipo II del Péptido Intestinal Vasoactivo/metabolismo , Quinasas de Receptores Adrenérgicos beta/metabolismo , Acetilcolina/farmacología , Animales , Proteína Quinasa Tipo II Dependiente de AMP Cíclico , Relación Dosis-Respuesta a Droga , Endocitosis/fisiología , Músculo Liso/citología , Músculo Liso/efectos de los fármacos , Músculo Liso/metabolismo , Fosforilación/efectos de los fármacos , Conejos , Receptor Muscarínico M2/metabolismo , Péptido Intestinal Vasoactivo/farmacologíaRESUMEN
In gastrointestinal smooth muscle cells, VPAC(2) receptor desensitization is exclusively mediated by G protein-coupled receptor kinase 2 (GRK2). The present study examined the mechanisms by which acetylcholine (ACh) acting via M(3) receptors regulates GRK2-mediated VPAC(2) receptor desensitization in gastric smooth muscle cells. Vasoactive intestinal peptide induced VPAC(2) receptor phosphorylation, internalization, and desensitization in both freshly dispersed and cultured smooth muscle cells. Costimulation with ACh in the presence of M(2) receptor antagonist (i.e., activation of M(3) receptors) inhibited VPAC(2) receptor phosphorylation, internalization, and desensitization. Inhibition was blocked by the selective protein kinase C (PKC) inhibitor bisindolylmaleimide, suggesting that the inhibition was mediated by PKC, derived from M(3) receptor activation. Similar results were obtained by direct activation of PKC with phorbol myristate acetate. In the presence of the M(2) receptor antagonist, ACh induced phosphorylation of Raf kinase inhibitory protein (RKIP), increased RKIP-GRK2 association, decreased RKIP-Raf-1 association, and stimulated ERK1/2 activity, suggesting that, upon phosphorylation by PKC, RKIP dissociates from its known target Raf to associate with, and block the activity of, GRK2. In muscle cells expressing RKIP(S153A), which lacks the PKC phosphorylation site, RKIP phosphorylation was blocked and the inhibitory effect of ACh on VPAC(2) receptor phosphorylation, internalization, and desensitization and the stimulatory effect on ERK1/2 activation were abolished. This study identified a novel mechanism of cross-regulation of G(s)-coupled receptor phosphorylation and internalization by G(q)-coupled receptors. The mechanism involved phosphorylation of RKIP by PKC, switching RKIP from association with Raf-1 to association with, and inhibition of, GRK2.
Asunto(s)
Proteínas de Unión a Fosfatidiletanolamina/metabolismo , Proteína Quinasa C/metabolismo , Receptor Muscarínico M3/fisiología , Receptores de Tipo II del Péptido Intestinal Vasoactivo/fisiología , Quinasas de Receptores Adrenérgicos beta/metabolismo , Acetilcolina/farmacología , Adenilil Ciclasas/metabolismo , Animales , AMP Cíclico/metabolismo , Proteína Quinasa Tipo II Dependiente de AMP Cíclico , Diaminas/farmacología , Endocitosis/efectos de los fármacos , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Indoles/farmacología , Maleimidas/farmacología , Modelos Biológicos , Antagonistas Muscarínicos/farmacología , Mutación , Miocitos del Músculo Liso/efectos de los fármacos , Miocitos del Músculo Liso/metabolismo , Miocitos del Músculo Liso/fisiología , Proteínas de Unión a Fosfatidiletanolamina/genética , Fosforilación/efectos de los fármacos , Proteína Quinasa C/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/farmacología , Conejos , Receptor Cross-Talk , Receptor Muscarínico M2/antagonistas & inhibidores , Receptor Muscarínico M3/antagonistas & inhibidores , Receptor Muscarínico M3/metabolismo , Receptores de Tipo II del Péptido Intestinal Vasoactivo/metabolismo , Acetato de Tetradecanoilforbol/farmacología , Transfección , Péptido Intestinal Vasoactivo/farmacologíaRESUMEN
In smooth muscle of the gut, G(q)-coupled receptor agonists activate preferentially PLC-beta1 to stimulate phosphoinositide (PI) hydrolysis and inositol 1,4,5-trisphosphate (IP(3)) generation and induce IP(3)-dependent Ca(2+) release. Inhibition of Ca(2+) mobilization by cAMP- (PKA) and cGMP-dependent (PKG) protein kinases reflects inhibition of PI hydrolysis by both kinases and PKG-specific inhibitory phosphorylation of IP(3) receptor type I. The mechanism of inhibition of PLC-beta1-dependent PI hydrolysis has not been established. Neither G(q) nor PLC-beta1 was directly phosphorylated by PKA or PKG in gastric smooth muscle cells. However, both kinases 1) phosphorylated regulator of G protein signaling 4 (RGS4) and induced its translocation from cytosol to plasma membrane, 2) enhanced ACh-stimulated association of RGS4 and Galpha(q).GTP and intrinsic Galpha(q).GTPase activity, and 3) inhibited ACh-stimulated PI hydrolysis. RGS4 phosphorylation and inhibition of PI hydrolysis were blocked by selective PKA and PKG inhibitors. Expression of RGS4(S52A), which lacks a PKA/PKG phosphorylation site, blocked the increase in GTPase activity and the decrease in PI hydrolysis induced by PKA and PKG. Blockade of PKA-dependent effects was only partial. Selective phosphorylation of G protein-coupled receptor kinase 2 (GRK2), which contains a RGS domain, by PKA augmented ACh-stimulated GRK2:Galpha(q).GTP association; both effects were blocked in cells expressing GRK2(S685A), which lacks a PKA phosphorylation site. Inhibition of PI hydrolysis induced by PKA was partly blocked in cells expressing GRK2(S685A) and completely blocked in cells coexpressing GRK2(S685A) and RGS4(S52A) or Galpha(q)(G188S), a Galpha(q) mutant that binds GRK2 but not RGS4. The results demonstrate that inhibition of PLC-beta1-dependent PI hydrolysis by PKA is mediated via stimulatory phosphorylation of RGS4 and GRK2, leading to rapid inactivation of Galpha(q).GTP. PKG acts only via phosphorylation of RGS4.
Asunto(s)
Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Proteínas Quinasas Dependientes de GMP Cíclico/metabolismo , Subunidades alfa de la Proteína de Unión al GTP Gq-G11/metabolismo , Isoenzimas/antagonistas & inhibidores , Proteínas RGS/metabolismo , Fosfolipasas de Tipo C/antagonistas & inhibidores , Quinasas de Receptores Adrenérgicos beta/metabolismo , Acetilcolina/farmacología , Animales , Células Cultivadas , GTP Fosfohidrolasas/metabolismo , Subunidades alfa de la Proteína de Unión al GTP Gq-G11/genética , Hidrólisis/efectos de los fármacos , Isoenzimas/metabolismo , Isoproterenol/farmacología , Contracción Muscular/efectos de los fármacos , Mutación , Miocitos del Músculo Liso/metabolismo , Nitroprusiato/farmacología , Fosfatidilinositoles/metabolismo , Fosfolipasa C beta , Fosforilación , Conejos , Estómago , Fosfolipasas de Tipo C/metabolismoRESUMEN
Sustained smooth-muscle contraction or its experimental counterpart, Ca2+ sensitization, by G(q/13)-coupled receptor agonists is mediated via RhoA-dependent inhibition of MLC (myosin light chain) phosphatase and MLC20 (20 kDa regulatory light chain of myosin II) phosphorylation by a Ca2+-independent MLCK (MLC kinase). The present study identified the corresponding pathways initiated by G(i)-coupled receptors. Somatostatin acting via G(i)1-coupled sstr3 receptor, DPDPE ([D-Pen2,D-Pen5]enkephalin; where Pen is penicillamine) acting via G(i)2-coupled delta-opioid receptors, and cyclopentyl adenosine acting via G(i)3-coupled adenosine A1 receptors preferentially activated PI3K (phosphoinositide 3-kinase) and ILK (integrin-linked kinase), whereas ACh (acetylcholine) acting via G(i)3-coupled M2 receptors preferentially activated PI3K, Cdc42 (cell division cycle 42)/Rac1, PAK1 (p21-activated kinase 1) and p38 MAPK (mitogen-activated protein kinase). Only agonists that activated ILK induced sustained CPI-17 (protein kinase C potentiated inhibitor 17 kDa protein) phosphorylation at Thr38, MLC20 phosphorylation at Ser19, and contraction, consistent with recent evidence that ILK can act as a Ca2+-independent MLCK capable of phosphorylating the MLC phosphatase inhibitor, CPI-17, at Thr38. ILK activity, and CPI-17 and MLC20 phosphorylation were inhibited by LY294002 and in muscle cells expressing ILK(R211A) or treated with siRNA (small interfering RNA) for ILK. ACh acting via M2 receptors activated ILK, and induced CPI-17 and MLC20 phosphorylation and muscle contraction, but only after inhibition of p38 MAPK; all these responses were inhibited in cells expressing ILK(R211A). Conversely, ACh activated PAK1, a step upstream of p38 MAPK, whereas the three other agonists did so only in cells transfected with ILK(R211A) or siRNA for ILK. The results demonstrate reciprocal inhibition between two pathways downstream of PI3K, with ILK inhibiting PAK1, and p38 MAPK inhibiting ILK. Sustained contraction via G(i)-coupled receptors is dependent on CPI-17 and MLC20 phosphorylation by ILK.
Asunto(s)
Subunidades alfa de la Proteína de Unión al GTP Gi-Go/fisiología , Proteínas Musculares/metabolismo , Miocitos del Músculo Liso/metabolismo , Cadenas Ligeras de Miosina/metabolismo , Fosfatidilinositol 3-Quinasas/fisiología , Fosfoproteínas/metabolismo , Procesamiento Proteico-Postraduccional/fisiología , Proteínas Serina-Treonina Quinasas/fisiología , Receptores Acoplados a Proteínas G/fisiología , Transducción de Señal/fisiología , Adenosina/análogos & derivados , Adenosina/farmacología , Agonistas del Receptor de Adenosina A1 , Animales , Azepinas/farmacología , Calcio/fisiología , Células Cultivadas/efectos de los fármacos , Células Cultivadas/metabolismo , Cromonas/farmacología , Encefalina D-Penicilamina (2,5)/farmacología , Estrenos/farmacología , Imidazoles/farmacología , Morfolinas/farmacología , Contracción Muscular/efectos de los fármacos , Contracción Muscular/fisiología , Fosfatasa de Miosina de Cadena Ligera/metabolismo , Fosforilación/efectos de los fármacos , Procesamiento Proteico-Postraduccional/efectos de los fármacos , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Serina-Treonina Quinasas/genética , Proteínas Proto-Oncogénicas c-akt/genética , Proteínas Proto-Oncogénicas c-akt/fisiología , Piridinas/farmacología , Pirrolidinonas/farmacología , ARN Interferente Pequeño/farmacología , Conejos , Receptor de Adenosina A1/fisiología , Receptor Muscarínico M2/agonistas , Receptor Muscarínico M2/fisiología , Receptores Opioides delta/agonistas , Receptores Opioides delta/fisiología , Receptores de Somatostatina/agonistas , Receptores de Somatostatina/fisiología , Proteínas Recombinantes de Fusión/fisiología , Somatostatina/farmacología , Transfección , Quinasas p21 Activadas , Proteínas Quinasas p38 Activadas por Mitógenos/antagonistas & inhibidores , Proteínas Quinasas p38 Activadas por Mitógenos/fisiologíaRESUMEN
The signaling cascades initiated by motilin receptors in gastric and intestinal smooth muscle cells were characterized. Motilin bound with high affinity (IC(50) 0.7 +/- 0.2 nM) to receptors on smooth muscle cells; the receptors were rapidly internalized via G protein-coupled receptor kinase 2 (GRK2). Motilin selectively activated G(q) and G(13), stimulated G alpha(q)-dependent phosphoinositide (PI) hydrolysis and 1,4,5-trisphosphate (IP(3))-dependent Ca(2+) release, and increased cytosolic free Ca(2+). PI hydrolysis was blocked by expression of G alpha(q) minigene and augmented by overexpression of dominant negative RGS4(N88S) or GRK2(K220R). Motilin induced a biphasic, concentration-dependent contraction (EC(50) = 1.0 +/- 0.2 nM), consisting of an initial peak followed by a sustained contraction. The initial Ca(2+)-dependent contraction and myosin light-chain (MLC)(20) phosphorylation were inhibited by the PLC inhibitor U-73122 and the MLC kinase inhibitor ML-9 but were not affected by the Rho kinase inhibitor Y27632 or the PKC inhibitor bisindolylmaleimide. Sustained contraction and MLC(20) phosphorylation were RhoA dependent and mediated by two downstream messengers: PKC and Rho kinase. The latter was partly inhibited by expression of G alpha(q) or G alpha(13) minigene and abolished by coexpression of both minigenes. Sustained contraction and MLC(20) phosphorylation were partly inhibited by Y27632 and bisindolylmaleimide and abolished by a combination of both inhibitors. The inhibition reflected phosphorylation of two MLC phosphatase inhibitors: CPI-17 via PKC and MYPT1 via Rho kinase. We conclude that motilin initiates a G alpha(q)-mediated cascade involving Ca(2+)/calmodulin activation of MLC kinase and transient MLC(20) phosphorylation and contraction as well as a sustained G alpha(q)- and G alpha(13)-mediated, RhoA-dependent cascade involving phosphorylation of CPI-17 by PKC and MYPT1 by Rho kinase, leading to inhibition of MLC phosphatase and sustained MLC(20) phosphorylation and contraction.