RESUMEN
Spatiotemporal regulation of protein kinase A (PKA) activity involves the manipulation of compartmentalized cAMP pools. Now we demonstrate that the muscle-selective A-kinase anchoring protein, mAKAP, maintains a cAMP signaling module, including PKA and the rolipram-inhibited cAMP-specific phosphodiesterase (PDE4D3) in heart tissues. Functional analyses indicate that tonic PDE4D3 activity reduces the activity of the anchored PKA holoenzyme, whereas kinase activation stimulates mAKAP-associated phosphodiesterase activity. Disruption of PKA- mAKAP interaction prevents this enhancement of PDE4D3 activity, suggesting that the proximity of both enzymes in the mAKAP signaling complex forms a negative feedback loop to restore basal cAMP levels.
Asunto(s)
3',5'-AMP Cíclico Fosfodiesterasas/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , AMP Cíclico/metabolismo , Proteínas Musculares/metabolismo , Miocardio/metabolismo , Animales , Células Cultivadas , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 4 , Retroalimentación , Ventrículos Cardíacos/citología , Ventrículos Cardíacos/metabolismo , Modelos Biológicos , Miocardio/citología , Unión Proteica , Ratas , Transducción de SeñalRESUMEN
During pregnancy in the rat, there is a change in the ability of chlorophenylthio (CPT)-cAMP to inhibit myometrial phosphatidylinositide turnover. This is accompanied by a change in the association of proteins with a plasma membrane A kinase anchoring protein (AKAP). Both CPT-cAMP and isoproterenol inhibited oxytocin-stimulated phosphatidylinositide turnover on days 12 through 20 of gestation, whereas neither agent had an effect on day 21. Accompanying this change was a dramatic decrease in the concentration and activity of cAMP-dependent protein kinase [protein kinase A (PKA)] and an increase in the concentration of protein phosphatase 2B (PP2B) in plasma membranes from day 21 compared with day 19 pregnant rats. In contrast, both PKA and PP2B concentrations and activities increased in total myometrial homogenates. Both PKA and PP2B coimmunoprecipitated with an antibody against the 150-kDa AKAP found in rat myometrial plasma membranes. More PKA was associated with AKAP150 on day 19 than on day 21, while the reverse was true for PP2B. Disruption of PKA/AKAP association in day 19 pregnant rat myometrial cells with the specific interaction inhibitor peptide S-Ht31 resulted in the loss of the cAMP-inhibitory effect on phosphatidylinositide turnover. PP2B activity in myometrial homogenates dephosphorylated PLCbeta3, a PKA substrate targeted in the inhibition of Galphaq-stimulated phosphatidylinositide turnover. The dramatic loss of the cAMP-inhibitory effect on day 21 of pregnancy may alter the balance between uterine contraction and relaxation near parturition. The changes in the relative concentrations of PKA and PP2B associated with AKAP150 are consistent with a functional role for AKAP150 scaffolding in the alteration of cellular signaling.
Asunto(s)
Proteínas Portadoras/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , AMP Cíclico/farmacología , Miometrio/metabolismo , Preñez/metabolismo , Animales , Calcineurina/metabolismo , Membrana Celular/metabolismo , Femenino , Edad Gestacional , Miometrio/efectos de los fármacos , Fosfatidilinositoles/metabolismo , Embarazo , Ratas , Ratas Sprague-DawleyRESUMEN
The importance of the localization of protein kinase A (PKA) to the plasma membrane for cAMP-mediated inhibition of phosphatidylinositide turnover was tested in an immortalized pregnant human myometrial (PHM1-41) cell line, and the putative A kinase anchoring protein (AKAP) involved was identified. Preincubation in PHM1-41 cells with chlorophenylthio-cAMP (CPT-cAMP), forskolin, or relaxin inhibited the ability of oxytocin to stimulate phosphatidylinositide turnover. The addition of a peptide that specifically disrupts interactions of PKA RII subunits with AKAPs (S-Ht31) reversed the effects of these agents, whereas a control peptide was ineffective. The pharmacology of S-Ht31 on this particular membrane event was further characterized. A 10-min incubation with S-Ht31 at a concentration of 1 microM completely reversed the inhibitory effect of relaxin on phosphatidylinositide turnover. S-Ht31 inhibited cAMP-stimulated PKA activity in PHM1-41 cell plasma membranes and decreased the concentration of PKA. Overlay analysis detected a single AKAP of approximately 86 kDa associated with the plasma membrane of PHM1-41 cells, suggesting that the association of PKA with this AKAP is important for the cAMP inhibitory mechanism. The mol wt of this AKAP was similar to that of an AKAP associated with the plasma membrane in the human brain, AKAP79. Antibodies against AKAP79 recognized a band at 86 kDa in purified plasma membranes from the PHM1-41 cells, indicating similar determinants in these proteins. These data suggest that PKA is anchored to the myometrial plasma membrane through association with an AKAP similar to AKAP79, and that this anchoring is required for the cAMP-mediated inhibition of phosphatidylinositide turnover in PHM1-41 cells.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales , Proteínas Portadoras/metabolismo , Membrana Celular/enzimología , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , AMP Cíclico/farmacología , Miometrio/enzimología , Fosfatidilinositoles/metabolismo , Proteínas de Anclaje a la Quinasa A , Animales , Western Blotting , Línea Celular Transformada , AMP Cíclico/análogos & derivados , Proteínas Quinasas Dependientes de AMP Cíclico/antagonistas & inhibidores , Femenino , Humanos , Péptidos y Proteínas de Señalización Intercelular , Peso Molecular , Oxitocina/farmacología , Péptidos/farmacología , Embarazo , Ratas , Tionucleótidos/farmacologíaRESUMEN
Abstract The primary goal of this investigation was to explore how the quality of parent-student relationships relates to coping style by examining multiple aspects of this relationship-including support and conflict-and by examining the contribution that both parents' and students' perceptions of their relationship make to students' coping reports. We found that perceptions of conflict and depth in the parent-child relationship were each associated with different styles of coping. Moreover, parents' and students' perceptions each accounted for unique variance in students' coping reports. Greater perceived depth (both parent and student reports) predicted higher problem-focused scores, while students' perceptions of conflict predicted higher emotion-focused coping scores. Specific support provisions reported by students and parents also related differentially to the specific coping styles. Finally, the extent to which parents and students reported coping in a similar fashion was predicted by the quality of their relationship.
RESUMEN
Heterotrimeric G proteins are actively involved in intracellular signalling in the myometrium and play important roles in regulating myometrial contraction and relaxation. Increases in intracellular calcium can be induced by agents that stimulate uterine contractions. In a number of instances, these increases in intracellular calcium are attributed to stimulation of phospholipase C by either G alpha or G betagamma subunits as a result of activation of G protein-coupled plasma membrane receptors. This mechanism also stimulates calcium entry through calcium release-activated channels, either directly or indirectly. Thus, while phospholipase C can be activated by other pathways and calcium can enter myometrial cells through other channels, G proteins play a major role in these processes. Similarly, activation of protein kinase A and protein kinase C are consequences of G protein activation. Protein kinase A and protein kinase C exert a number of regulatory influences on phospholipase C, ion channel activity and other processes in the myometrium. The mitogen-activated protein kinase pathway can also be activated directly or indirectly by the action of G proteins in myometrium. Responsiveness to G proteins can be altered during pregnancy and depends on the relative expression of all of the components of the signalling pathways involved. The balance between G protein-mediated stimulatory and inhibitory signalling pathways has important consequences for the control of myometrial contractile activity.
Asunto(s)
Proteínas de Unión al GTP/fisiología , Relajación Muscular/fisiología , Miometrio/metabolismo , Transducción de Señal , Contracción Uterina/fisiología , Adenilil Ciclasas/metabolismo , Animales , Femenino , Humanos , Canales Iónicos/metabolismo , Embarazo , Fosfolipasas de Tipo C/metabolismoRESUMEN
The mechanism by which protein kinase A (PKA) inhibits Galphaq -stimulated phospholipase C activity of the beta subclass (PLCbeta ) is unknown. We present evidence that phosphorylation of PLCbeta3 by PKA results in inhibition of Galphaq -stimulated PLCbeta3 activity, and we identify the site of phosphorylation. Two-dimensional phosphoamino acid analysis of in vitro phosphorylated PLCbeta3 revealed a single phosphoserine as the putative PKA site, and peptide mapping yielded one phosphopeptide. The residue was identified as Ser1105 by direct sequencing of reverse-phase high pressure liquid chromatography-isolated phosphopeptide and by site-directed mutagenesis. Overexpression of Galphaq with PLCbeta3 or PLCbeta (Ser1105--> Ala) mutant in COSM6 cells resulted in a 5-fold increase in [3H]phosphatidylinositol 1,4,5-trisphosphate formation compared with expression of Galphaq, PLCbeta3, or PLCbeta3 (Ser1105 --> Ala mutant alone. Whereas Galpha1-stimulated PLCbeta3, activity was inhibited by 58-71% by overexpression of PKA catalytic subunit, Galphaq-stimulated PLCbeta3 (Ser1105 --> Ala) mutant activity was not affected. Furthermore, phosphatidylinositide turnover stimulated by presumably Galpha1-coupled M1 muscarinic and oxytocin receptors was completely inhibited by pretreating cells with 8-[4-chlorophenythio]-cAMP in RBL-2H3 cells expressing only PLCbeta3. These data establish that direct phosphorylation by PKA of Ser1105 in the putative G-box of PLCbeta3 inhibits Galphaq-stimulated PLCbeta3 activity. This can at least partially explain the inhibitory effect of PKA on Galphaq-stimulated phosphatidylinositide turnover observed in a variety of cells and tissues.
Asunto(s)
Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Proteínas de Unión al GTP/metabolismo , Isoenzimas/antagonistas & inhibidores , Serina/metabolismo , Fosfolipasas de Tipo C/antagonistas & inhibidores , Animales , Sitios de Unión , Células Cultivadas , Clonación Molecular , Activación Enzimática , Isoenzimas/genética , Mutagénesis Sitio-Dirigida , Fosfolipasa C beta , Fosforilación , Porcinos , Fosfolipasas de Tipo C/genéticaRESUMEN
The effects of cAMP on the oxytocin-stimulated increase in phosphatidylinositide turnover and the possible pathways involved were investigated in a human myometrial cell line (PHM1-41) and in COS-M6 cells overexpressing the oxytocin receptor. Preincubation with chlorophenylthio-cAMP (CPT-cAMP), forskolin, or relaxin inhibited oxytocin-stimulated phosphatidylinositide turnover in PHM1-41 cells, and the inhibition was reversed by H-89, a relatively specific protein kinase A inhibitor. Both CPT-cAMP and transiently expressed protein kinase A catalytic subunit inhibited stimulation by oxytocin and carbachol of [3H]inositol 1,3,4-trisphosphate formation in COS-M6 cells expressing oxytocin or muscarinic M1 receptors, respectively. CPT-cAMP also inhibited phosphatidylinositide turnover stimulation by endothelin-1 in PHM1-41 cells, further demonstrating the generality of the cAMP-inhibitory mechanism. Since G betagamma activation of phospholipase Cbeta2 (PLCbeta2) is a suggested target of protein kinase A, the possibility that the oxytocin receptor couples to PLCbeta2 via G alpha(i)G betagamma activation was explored. Western blot analysis of PHM1-41 cells and COS-M6 cells detected PLCbeta1 and PLCbeta3, but not PLCbeta2. In PHM1-41 cells, pertussis toxin reduced the oxytocin-stimulated increase in [3H]inositol 1,3,4-trisphosphate by 53%, and this was reversed completely by H-89. Thus, the inhibitory effect of pertussis toxin may result from an indirect effect of cAMP elevation. These data suggest that receptor/G alpha(q)-coupled stimulation of PLCbeta1 or PLCbeta3 can be inhibited by cAMP through a phosphorylation mechanism involving protein kinase A that does not involve PLCbeta2. In smooth muscle, this mechanism could constitute potentially important cross-talk between pathways regulating contraction and relaxation.
Asunto(s)
Proteínas Quinasas Dependientes de AMP Cíclico/farmacología , Proteínas de Unión al GTP/metabolismo , Receptores Muscarínicos/metabolismo , Receptores de Oxitocina/metabolismo , Fosfolipasas de Tipo C/metabolismo , Animales , Western Blotting , Células COS , Carbacol/farmacología , Línea Celular , AMP Cíclico/farmacología , Femenino , Humanos , Fosfatos de Inositol/metabolismo , Miometrio/metabolismo , Oxitocina/farmacología , Toxina del Pertussis , Fosforilación , Embarazo , Receptor Muscarínico M1 , Receptores Muscarínicos/genética , Receptores de Oxitocina/genética , Transfección , Factores de Virulencia de Bordetella/farmacologíaRESUMEN
Peroxynitrite (ONOO-) inhibits energy metabolism in isolated cells and mitochondria and may be involved in the depression of cardiac mechanical function during pathophysiological states. We determined the actions of ONOO- on cardiac function and energy metabolism in isolated working rat hearts and compared them with the NO donor S-nitroso-DL-acetylpenicillamine (SNAP). After a 15-min baseline aerobic perfusion, ONOO- (4 or 40 microM), SNAP (40 microM), or their vehicles were infused over a 60-min period. ONOO- or SNAP (40 microM each) caused a rapid and sustained rise in coronary flow. Infusion of 40 microM (but not 4 microM) ONOO- caused a marked depression in cardiac work with a delayed onset but no change in O2 consumption, resulting in a marked loss of cardiac efficiency. Cardiac work, O2 consumption, and cardiac efficiency remained constant in vehicle- and SNAP-treated hearts. ONOO- (40 microM) enhanced glycolysis and glucose oxidation but did not change pyruvate oxidation compared with its vehicle control, whereas SNAP was without effect. ONOO(-)-mediated depression in cardiac efficiency may be due to reduced coupling between ATP production and mechanical work.