Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.7.11.1 (protein kinase)
81,284 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Decidualization of human endometrial stromal (ES) cells in vitro is induced by cAMP analogs and ligands that elevate cellular cAMP levels. A marker of this differentiation process is the activation of the decidual PRL (dPRL) promoter. In a primary ES cell culture system we show that relaxin not only acutely but permanently elevates cellular cAMP levels and leads to induction of PRL secretion after 6 days Northern and Western blot analyses revealed that all regulatory subunit isoforms (RI alpha, RI beta, RII alpha, and RII beta) and catalytic subunits C alpha and C beta of protein kinase A (PKA) are expressed in ES cells. Transcript levels of PKA subunit isoforms are not altered during decidualization but in decidualized ES cells, exposed to relaxin for more than 6 days a significant reduction of RI alpha protein level occurs, whereas levels of all other forms remain unchanged. Reduction of R subunits might result in a net increase in free C subunit activity. This alteration is not due to a change in the mitotic state of the cells, as proliferating cell nuclear antigen is evenly expressed in undifferentiated and differentiated ES cell cultures. In transient transfections of undifferentiated ES cells, the dPRL promoter is activated by 8-bromo-cAMP and the C subunit (C beta) of PKA. This induction as well as the differentiation-dependent activity of the dPRL promoter in transfected decidualized cells are effectively abolished by the coexpression of protein kinase inhibitor. We demonstrate that 332 bp of the dPRL promoter are sufficient to mediate full inducibility by cAMP. Activation of the dPRL promoter by cAMP in ES cells occurs in two steps: an initial weak induction within 12 h and a subsequent, much more pronounced induction after 12 h. The secondary induction is not seen with a control construct driven by a consensus cAMP response element (CRE) linked to a minimal promoter and is absent from a uterine cell line that does not express the endogenous dPRL gene. The early response of the dPRL promoter depends upon a noncanonical CRE at position -12, as mutation of this sequence leads to abolition of the early, but not the delayed, induction. The major activation depends upon a different region within 332 bp of the dPRL promoter; is probably indirect, as it follows different kinetics compared to a classical CRE-mediated response; and is specific to ES cells.
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PMID:Activated protein kinase A is required for differentiation-dependent transcription of the decidual prolactin gene in human endometrial stromal cells. 904 92

The peptide hormone relaxin has direct, positive inotropic and chronotropic effects on rat hearts in vivo and in vitro. Relaxin's effects on the electrophysiological properties of single quiescent atrial cells from normal rats were investigated with a whole cell patch clamp. Relaxin had a significant inhibitory effect on outward potassium currents. The outward potassium current consisted of a transient component (I(to)) and a sustained component (I(S)). The addition of 100 ng/ml of relaxin inhibited the peak I(to) in a voltage-dependent manner (74% inhibition at a membrane potential of -10 mV to 30% inhibition at +70 mV). The time to reach peak I(to) and the apparent time constant of inactivation of I(to) were increased by relaxin. Dialysis with the protein kinase A inhibitor 5-24 amide (2 microM) prevented relaxin's effects, suggesting an obligatory role for this kinase in the relaxin-dependent regulation of the potassium current.
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PMID:Effects of relaxin on rat atrial myocytes. I. Inhibition of I(to) via PKA-dependent phosphorylation. 913 64

Relaxin produces positive inotropic and chronotropic effects in rat hearts. The effect of relaxin on the action potential duration (APD) of single quiescent rat atrial cells was investigated with a whole cell patch clamp. Relaxin induced a significant, dose-dependent prolongation of the APD. This effect was maximal at 200 ng/ml (nominal concentration of 33.6 nM), which caused, on average, a 57% increase in the time taken to reach 90% repolarization. The effect of relaxin was blocked by the protein kinase A inhibitor 5-24 amide, indicating that its effect is mediated by an adenosine 3',5'-cyclic monophosphate-dependent mechanism. The increased APD induced by relaxin caused an enhanced entrance of calcium, with the charge carried through voltage-activated calcium channels increased by approximately 25%. This increase was not due to a direct modulation of calcium currents (20); rather, it was a consequence of the longer period of cellular depolarization. Our findings that relaxin increased the APD and therefore increased the calcium influx in atrial myocytes could explain the positive inotropic effects induced by relaxin in atrial preparations.
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PMID:Effects of relaxin on rat atrial myocytes. II. Increased calcium influx derived from action potential prolongation. 913 65

The aim of this study was to investigate the role of the adenylyl cyclase pathway, and in particular cyclic AMP-dependent protein kinase A, in the relaxant action of relaxin in the isolated uterus of the nonpregnant rat. The purportedly selective inhibitor of cAMP-dependent protein kinase A N-[2-(methylamino) ethyl]-5-isoquinolinesulfonamide hydrochloride (H-8) (at 100 mumol l-7) antagonized relaxin, salbutamol (an agonist at beta-adrenoceptors) and levcromakalim (a K+ channel opener) to a similar extent (by factors of 3.1, 1.9 and 2.8, respectively), demonstrating that it is not a selective inhibitor. Relaxin and levcromakalim were less potent and had smaller, maximal, relaxant effects in longitudinal myometrium than in intact uterus cut in the longitudinal plane. By contrast, nifedipine (a Ca2+ channel blocker) was equipotent in the two preparations and salbutamol only slightly less potent in the longitudinal myometrium. Relaxin did not alter the cyclic AMP-dependent protein kinase A activity ratio in longitudinal myometrium, but did increase the activity ratio by a factor of 2.0 +/- 0.2 in the intact uterus. Salbutamol, the positive control, increased this activity ratio in both longitudinal myometrium (by 1.9 +/- 0.3 times) and in the intact uterus (by 3.8 +/- 0.3 times), whereas the negative control levcromakalim had no effect. Relaxin seems to act as a relaxant of longitudinal myometrium by a cyclic AMP-independent mechanism but possibly interacts with the circular myometrium or endometrium to release a relaxant factor via a cyclic-AMP-dependent mechanism.
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PMID:The role of a cAMP-dependent pathway in the uterine relaxant action of relaxin in rats. 915 39

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.
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PMID:Evidence for inhibition by protein kinase A of receptor/G alpha(q)/phospholipase C (PLC) coupling by a mechanism not involving PLCbeta2. 956 32

The process of decidualization involves the morphological and functional transformation of stromal fibroblasts to decidual cells. The objective of this study was to define appropriate in vitro culture conditions required for decidualization of baboon stromal cells. Parallel studies were also done with human endometrial stromal cells for comparative analysis. Human stromal cells produced prolactin and insulin-like growth factor-binding protein (IGFBP)-1 in response to hormones (estradiol-17beta [36 nM], medroxyprogesterone acetate [1 microM], and relaxin [100 ng/ml]), and production was enhanced in the presence of 0.1 mM dibutyryl cAMP (dbcAMP). By contrast, baboon cells did not produce any detectable levels of prolactin, even in the presence of hormones and dbcAMP. IGFBP-1 expression in baboon stromal cells was detectable by Day 6 of hormone and dbcAMP treatment and increased exponentially thereafter. In both human and baboon stromal cells, alpha smooth muscle actin (alphaSMA) expression, an early marker for decidualization in the baboon in vivo, was induced spontaneously under normal culture conditions. Furthermore, a decrease in alphaSMA expression was observed in cells producing high levels of IGFBP-1. Human cells produced significant levels of IGFBP-1 (p < or = 0.01) in response to short-term dbcAMP treatment (48 h) after 2 and 12 days of hormone treatment. However, baboon stromal cells required 17 days of hormonal treatment before cells became responsive to short-term dbcAMP treatment (p < or = 0.01). Finally, human endometrial stromal cells expressed the protein kinase A regulatory subunits RIalpha, RIbeta, RIIalpha, and RIIbeta whereas baboon stromal cells expressed RIalpha, RIIalpha, and RIIbeta. No difference in the mRNA expression of these isoforms was observed in decidualized or nondecidualized cells of either human or baboon endometrium. Our observations indicate that baboon stromal cells can be induced to decidualize in vitro and that this requires dbcAMP in addition to hormones. This is the first report demonstrating in vitro decidualization in a nonhuman primate.
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PMID:Comparative studies on the in vitro decidualization process in the baboon (Papio anubis) and human. 967 7

Decidualization of human endometrial stromal (ES) cells in vitro is induced by cAMP analogues and ligands that elevate cellular cAMP levels in a manner resembling the gonadotrophins, prostaglandin E2 and relaxin (RLX). This differentiation process is marked by the onset of decidual prolactin (PRL) production in the late luteal phase of the cycle. Using transfection assays and a primary ES cell culture system, we have demonstrated that decidual PRL gene transcription is driven by an alternative upstream promoter (dPRL), approximately 6 kb upstream of the pituitary transcription start site. In primary cell cultures, RLX not only acutely but also permanently elevated cellular cAMP levels and induced PRL secretion after 6 days. Northern and Western blot analyses revealed all regulatory subunit isoforms (RIalpha, RIbeta, RIIalpha, RIIbeta) and catalytic subunits Calpha and Cbeta of protein kinase A (PKA) in ES cells. Transcript levels of PKA subunit isoforms are not altered during decidualization, but in decidualized ES cells exposed to elevated cellular cAMP levels by stimulation with RLX for >6 days, RIalpha protein levels were significantly reduced, whereas levels of all other forms remained unchanged. Reducing the availability of R subunits changed the R:C subunit ratio in favour of C and increased kinase activity. In transient transfections of undifferentiated ES cells, the dPRL promoter was activated by 8-Br-cAMP and by C subunit (Cbeta) of PKA. This induction, and the differentiation-dependent activity of the dPRL promoter in transfected decidualized cells, was effectively abolished by the co-expression of protein kinase inhibitor (PKI). A fragment of 332 bp of 5'-flanking region of the dPRL transcription start site was sufficient to mediate full inducibility by cAMP. cAMP activation of the dPRL promoter in ES cells was biphasic as an initial weak induction within 12 hours was followed by a subsequent, much more intense induction after 12 hours. The secondary induction was not seen with a control construct driven by a consensus cAMP response element (CRE) linked to a minimal promoter. The early response of the dPRL promoter depended upon a non-palindromic CRE at position -12 and mutation of this sequence led to omission of the early, but not of the delayed, induction. The major activation of the dPRL promoter depended upon a different region between position -332 and -270 since its deletion significantly reduced inducibility by cAMP. Its action was probably indirect as its kinetics differed from classic CRE-mediated responses, and it was specific to ES cells.
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PMID:Marker genes of decidualization: activation of the decidual prolactin gene. 1002 98

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.
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PMID:Protein kinase A anchoring to the myometrial plasma membrane is required for cyclic adenosine 3',5'-monophosphate regulation of phosphatidylinositide turnover. 1053 45

Relaxin is an insulin-like serum protein secreted during pregnancy and found in many tissues, including the lung. Relaxin is reported to stimulate epithelial cell proliferation, but the effects of relaxin on airway epithelium are unknown. We tested the hypothesis that relaxin would stimulate the increased migration of bronchial epithelial cells (BEC) in response to wounding. Using monolayers of BEC in a wound-healing model, relaxin augmented wound closure with maximal closure occurring at 12 hr (1 micro M). Unlike cytokines, relaxin did not stimulate increased BEC interleukin-8 (IL-8) release. Relaxin caused a significant stimulation of ciliary beat frequency (CBF) in BEC. Because protein kinase (PKA) activation increases CBF and relaxin can elevate intracellular cAMP levels, we measured PKA activity in BEC treated with relaxin. Relaxin increased PKA activity 3-4 fold by approximately 4 hr, with a return to baseline levels by 8-10 hr. Relaxin-stimulated PKA activity differs temporally from the rapid (1 hr) beta-adrenergic activation of PKA in BEC. These data suggest that relaxin augments epithelial repair by increasing airway cell migration and CBF via PKA-dependent mechanisms.
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PMID:Relaxin stimulates bronchial epithelial cell PKA activation, migration, and ciliary beating. 1248 16

We have recently demonstrated that relaxin (RLX) acts as compensatory mediator in human heart failure. RLX inhibits the stimulation of endothelin-1, the most potent vasoconstrictor in heart failure. Upregulation of the endothelin type-B receptor (ET(B)), which mediates endothelin-1 clearance and endothelial release of NO, represents a pivotal mode of RLX action. However, signal transduction and abundance of this phenomenon are unknown. Therefore, we investigated RLX-induced regulation of ET(B) in human umbilical vein endothelial, epithelial (HeLa), and vascular smooth muscle cells. In human umbilical vein endothelial cells and HeLa cells, but not in human vascular smooth muscle cells, RLX upregulated ET(B) expression and activated extracellular signal-regulated kinase-1/2 (ERK-1/2) and nuclear factor-kappaB (NF-kappaB), a transcription factor. PD-98059, a selective inhibitor of the mitogen-activated protein kinase kinase-1 (MEK-1)-ERK-1/2 pathway, abolished ERK-1/2 and NF-kappaB activation and ET(B) upregulation. NF-kappaB inhibition also prevented RLX-mediated ET(B) stimulation. In NF-kappaB-luciferase reporter assays, we demonstrated complete inhibition of RLX-induced NF-kappaB activation in cells transfected with dominant-negative Raf-1, MEK-1, or ERK-1/2 constructs, whereas dominant-negative Ras had no effect. In rat aorta and mesenteric artery, RLX pretreatment, in an ET(B)-dependent fashion, mitigated the maximum contractile response to endothelin-1, by 38+/-4% and 43+/-6%, and the endothelin-1 sensitivity (-log[EC(50)]: aorta, 8.2+/-0.2 for vehicle versus 7.2+/-0.2 for RLX; mesenteric artery, 8.0+/-0.2 for vehicle versus 7.1+/-0.1 for RLX). RLX pretreatment augmented the dilator effect of the ET(B) agonist endothelin-3 by 100+/-8% and 133+/-13%. In conclusion, RLX stimulates endothelial and epithelial ET(B) via a Ras-independent Raf-1-MEK-1-ERK-1/2 pathway that activates NF-kappaB. On vascular smooth muscle cells, ET(B), a contributor to endothelin-mediated vasoconstriction, remains unaffected. This renders RLX a functional endothelin-1 antagonist.
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PMID:Relaxin, a pregnancy hormone, is a functional endothelin-1 antagonist: attenuation of endothelin-1-mediated vasoconstriction by stimulation of endothelin type-B receptor expression via ERK-1/2 and nuclear factor-kappaB. 1252 18


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