scholarly journals Protein kinase A regulates the disposition of Ca2+ which enters the cytoplasmic space through store-activated Ca2+ channels in rat hepatocytes by diverting inflowing Ca2+ to mitochondria

1998 ◽  
Vol 330 (3) ◽  
pp. 1179-1187 ◽  
Author(s):  
C. Kekulu FERNANDO ◽  
B. Roland GREGORY ◽  
J. Greg BARRITT
Keyword(s):  
Protein Kinase ◽  
Protein Kinase A ◽  
Regulatory Protein ◽  
Rat Hepatocytes ◽  
Ruthenium Red ◽  
Maximal Effect ◽  
Isolated Rat Hepatocytes ◽  
Carbonyl Cyanide ◽  
Ca2 Channels ◽  
Kinase A

The roles of a trimeric GTP-binding regulatory protein, protein kinase A and mitochondria in the regulation of store-activated (thapsigargin-stimulated) Ca2+ inflow in freshly-isolated rat hepatocytes were investigated. Rates of Ca2+ inflow were estimated by measuring the increase in the fluorescence of intracellular fura-2 following the addition of extracellular Ca2+ (Ca2+o) to cells incubated in the absence of added Ca2+o. Guanosine 5ʹ-[γ-thio]-triphosphate (GTP[S]) and AlF4- inhibited the thapsigargin-stimulated Ca2+o-induced increase in cytoplasmic free Ca2+ concentration ([Ca2+]c) and this inhibition was prevented by the Rp diastereoisomer of adenosine 3ʹ,5ʹ-(cyclic)phosphoro[thioate]. cAMP, forskolin and glucagon (half-maximal effect at 10 nM) mimicked inhibition of the thapsigargin-stimulated Ca2+o-induced increase in [Ca2+]c by GTP[S], but had little effect on thapsigargin-induced release of Ca2+ from intracellular stores. Azide and carbonyl cyanide p-trifluoromethoxyphenylhydrazone inhibited the thapsigargin-stimulated Ca2+o-induced increase in [Ca2+]c in the presence of increased cAMP (induced by glucagon). In contrast, Ruthenium Red markedly enhanced the thapsigargin-stimulated Ca2+o-induced increase in [Ca2+]c in both the presence and absence of increased cAMP (induced by forskolin and dibutyryl cAMP). It is concluded that, in hepatocytes, protein kinase A regulates the disposition of Ca2+, which enters the cytoplasmic space through store-activated Ca2+ channels, by directing some of this Ca2+ to the mitochondria. The idea that caution should be exercised in using observed values of Ca2+o-induced increase in [Ca2+]c as estimates of rates of agonist-stimulated Ca2+ inflow is briefly discussed.

scholarly journals Spatial targeting of type II protein kinase A to filopodia mediates the regulation of growth cone guidance by cAMP

2007 ◽  
Vol 176 (1) ◽  
pp. 101-111 ◽  
Author(s):  
Jianzhong Han ◽  
Liang Han ◽  
Priyanka Tiwari ◽  
Zhexing Wen ◽  
James Q. Zheng
Keyword(s):  
Protein Kinase ◽  
Protein Kinase A ◽  
Growth Cone ◽  
Regulatory Protein ◽  
Cyclic Adenosine Monophosphate ◽  
Spatial Localization ◽  
Adenosine Monophosphate ◽  
Type Ii ◽  
Regulation Of Growth ◽  
Kinase A

The second messenger cyclic adenosine monophosphate (cAMP) plays a pivotal role in axonal growth and guidance, but its downstream mechanisms remain elusive. In this study, we report that type II protein kinase A (PKA) is highly enriched in growth cone filopodia, and this spatial localization enables the coupling of cAMP signaling to its specific effectors to regulate guidance responses. Disrupting the localization of PKA to filopodia impairs cAMP-mediated growth cone attraction and prevents the switching of repulsive responses to attraction by elevated cAMP. Our data further show that PKA targets protein phosphatase-1 (PP1) through the phosphorylation of a regulatory protein inhibitor-1 (I-1) to promote growth cone attraction. Finally, we find that I-1 and PP1 mediate growth cone repulsion induced by myelin-associated glycoprotein. These findings demonstrate that the spatial localization of type II PKA to growth cone filopodia plays an important role in the regulation of growth cone motility and guidance by cAMP.

cAMP increases liver Na+-taurocholate cotransport by translocating transporter to plasma membranes

1997 ◽  
Vol 273 (4) ◽  
pp. G842-G848 ◽  
Author(s):  
Sunil Mukhopadhayay ◽  
M. Ananthanarayanan ◽  
Bruno Stieger ◽  
Peter J. Meier ◽  
Frederick J. Suchy ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Protein Kinase ◽  
Fusion Protein ◽  
Protein Kinase A ◽  
Plasma Membranes ◽  
Membrane Vesicles ◽  
Rat Hepatocytes ◽  
Plasma Membrane Vesicles ◽  
Short Term ◽  
Kinase A

Adenosine 3′,5′-cyclic monophosphate (cAMP), acting via protein kinase A, increases transport maximum of Na+-taurocholate cotransport within 15 min in hepatocytes (S. Grüne, L. R. Engelking, and M. S. Anwer. J. Biol. Chem. 268: 17734–17741, 1993); the mechanism of this short-term stimulation was investigated. Cycloheximide inhibited neither basal nor cAMP-induced increases in taurocholate uptake in rat hepatocytes, indicating that cAMP does not stimulate transporter synthesis. Studies in plasma membrane vesicles showed that taurocholate uptake was not stimulated by the catalytic subunit of protein kinase A but was higher when hepatocytes were pretreated with cAMP. Immunoblot studies with anti-fusion protein antibodies to the cloned Na+-taurocholate cotransport polypeptide (Ntcp) showed that pretreatment of hepatocytes with cAMP increased Ntcp content in plasma membranes but not in homogenates. Ntcp was detected in microsomes, endosomes, and Golgi fractions, and cAMP pretreatment resulted in a decrease only in endosomal Ntcp content. It is proposed that cAMP increases transport maximum of Na+-taurocholate cotransport, at least in part, by translocating Ntcp from endosomes to plasma membranes.

scholarly journals Tauroursodeoxycholate Protects Rat Hepatocytes from Bile Acid-Induced Apoptosis via β1-Integrin- and Protein Kinase A-Dependent Mechanisms

2015 ◽  
Vol 36 (3) ◽  
pp. 866-883 ◽  
Author(s):  
Annika Sommerfeld ◽  
Roland Reinehr ◽  
Dieter Häussinger
Keyword(s):  
Bile Acid ◽  
Protein Kinase ◽  
Protein Kinase A ◽  
Rat Hepatocytes ◽  
Β1 Integrin ◽  
Dependent Manner ◽  
Mitogen Activated Protein ◽  
Induced Apoptosis ◽  
Jnk Activation ◽  
Kinase A

Background/Aims: Ursodeoxycholic acid, which in vivo is rapidly converted into its taurine conjugate, is frequently used for the treatment of cholestatic liver disease. Apart from its choleretic effects, tauroursodeoxycholate (TUDC) can protect hepatocytes from bile acid-induced apoptosis, but the mechanisms underlying its anti-apoptotic effects are poorly understood. Methods: These mechanisms were investigated in perfused rat liver and isolated rat hepatocytes. Results: It was found that TUDC inhibited the glycochenodeoxycholate (GCDC)-induced activation of the CD95 death receptor at the level of association between CD95 and the epidermal growth factor receptor. This was due to a rapid TUDC-induced β1-integrin-dependent cyclic AMP (cAMP) signal with induction of the dual specificity mitogen-activated protein (MAP) kinase phosphatase 1 (MKP-1), which prevented GCDC-induced phosphorylation of mitogen-activated protein kinase kinase 4 (MKK4) and c-jun-NH2-terminal kinase (JNK) activation. Furthermore, TUDC induced a protein kinase A (PKA)-mediated serine/threonine phosphorylation of the CD95, which was recently identified as an internalization signal for CD95. Furthermore, TUDC inhibited GCDC-induced CD95 targeting to the plasma membrane in a β1-integrin-and PKA-dependent manner. In line with this, the β1-integrin siRNA knockdown in sodium taurocholate cotransporting polypeptide (Ntcp)-transfected HepG2 cells abolished the protective effect of TUDC against GCDC-induced apoptosis. Conclusion: TUDC exerts its anti-apoptotic effect via a β1-integrin-mediated formation of cAMP, which prevents CD95 activation by hydrophobic bile acids at the levels of JNK activation and CD95 serine/threonine phosphorylation.

scholarly journals Convergence of 3′,5′-Cyclic Adenosine 5′-Monophosphate/Protein Kinase A and Glycogen Synthase Kinase-3β/β-Catenin Signaling in Corpus Luteum Progesterone Synthesis

Endocrinology ◽  
2009 ◽  
Vol 150 (11) ◽  
pp. 5036-5045 ◽  
Author(s):  
Lynn Roy ◽  
Claudia A. McDonald ◽  
Chao Jiang ◽  
Dulce Maroni ◽  
Anthony J. Zeleznik ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Protein Kinase A ◽  
Corpus Luteum ◽  
Glycogen Synthase ◽  
Regulatory Protein ◽  
Glycogen Synthase Kinase ◽  
Proximal Promoter ◽  
Progesterone Secretion ◽  
Progesterone Synthesis ◽  
Kinase A

Progesterone secretion by the steroidogenic cells of the corpus luteum (CL) is essential for reproduction. Progesterone synthesis is under the control of LH, but the exact mechanism of this regulation is unknown. It is established that LH stimulates the LH receptor/choriogonadotropin receptor, a G-protein coupled receptor, to increase cAMP and activate cAMP-dependent protein kinase A (PKA). In the present study, we tested the hypothesis that cAMP/PKA-dependent regulation of the Wnt pathway components glycogen synthase kinase (GSK)-3β and β-catenin contributes to LH-dependent steroidogenesis in luteal cells. We observed that LH via a cAMP/PKA-dependent mechanism stimulated the phosphorylation of GSK3β at N-terminal Ser9 causing its inactivation and resulted in the accumulation of β-catenin. Overexpression of N-terminal truncated β-catenin (Δ90 β-catenin), which lacks the phosphorylation sites responsible for its destruction, significantly augmented LH-stimulated progesterone secretion. In contrast, overexpression of a constitutively active mutant of GSK3β (GSK-S9A) reduced β-catenin levels and inhibited LH-stimulated steroidogenesis. Chromatin immunoprecipitation assays demonstrated the association of β-catenin with the proximal promoter of the StAR gene, a gene that expresses the steroidogenic acute regulatory protein, which is a cholesterol transport protein that controls a rate-limiting step in steroidogenesis. Collectively these data suggest that cAMP/PKA regulation of GSK3β/β-catenin signaling may contribute to the acute increase in progesterone production in response to LH.

scholarly journals AKAP79 Orchestrates a Cyclic AMP Signalosome Adjacent to Orai1 Ca2+ Channels

Function ◽  
2021 ◽  
Author(s):  
Pulak Kar ◽  
Pradeep Barak ◽  
Anna Zerio ◽  
Yu-Ping Lin ◽  
Amy J Parekh ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Protein Kinase A ◽  
Adenylyl Cyclase ◽  
Second Messengers ◽  
Neuronal Cell ◽  
Hek293 Cells ◽  
Scaffolding Protein ◽  
Adenylyl Cyclase 8 ◽  
Ca2 Channels ◽  
Kinase A

Abstract To avoid conflicting and deleterious outcomes, eukaryotic cells often confine signalling molecules to spatially restricted sub-compartments. The smallest signalling unit is the Ca2+ nanodomain, forming near open Ca2+ channels. Ca2+ nanodomains near store-operated Orai1 channels stimulate calcineurin, which activates the transcription factor NFAT1, and both enzyme and target are initially attached to the plasma membrane through the scaffolding protein AKAP79. Here we show that a cAMP signalling nexus also forms adjacent to Orai1. Protein kinase A and phosphodiesterase 4, an enzyme that rapidly breaks down cAMP, both associate with AKAP79 and realign close to Orai1 after stimulation. PCR and mass spectrometry failed to show expression of Ca2+-activated adenylyl cyclase 8 in HEK293 cells, whereas the enzyme was observed in neuronal cell lines. FRET and biochemical measurements of bulk cAMP and protein kinase A activity consistently failed to show an increase in adenylyl cyclase activity following even a large rise in cytosolic Ca2+. Furthermore, expression of AKAP79-CUTie, a cAMP FRET sensor tethered to AKAP79, did not report a rise in cAMP after stimulation, despite AKAP79 association with Orai1. Hence HEK293 cells do not express functionally active Ca2+-activated adenylyl cyclases including adenylyl cyclase 8. Our results show that two ancient second messengers are independently generated in nanodomains close to Orai1 Ca2+ channels.

scholarly journals Estradiol Modulates Translocator Protein (TSPO) and Steroid Acute Regulatory Protein (StAR) via Protein Kinase A (PKA) Signaling in Hypothalamic Astrocytes

Endocrinology ◽  
2014 ◽  
Vol 155 (8) ◽  
pp. 2976-2985 ◽  
Author(s):  
Claire Chen ◽  
John Kuo ◽  
Angela Wong ◽  
Paul Micevych
Keyword(s):  
Protein Kinase ◽  
Protein Kinase A ◽  
Primary Cultures ◽  
Regulatory Protein ◽  
Estrogen Receptor Α ◽  
Translocator Protein ◽  
Calcium Stores ◽  
Female Rat ◽  
Transporter Protein ◽  
Kinase A

The ability of the central nervous system to synthesize steroid hormones has wide-ranging implications for physiology and pathology. Among the proposed roles of neurosteroids is the regulation of the LH surge. This involvement in the estrogen-positive feedback demonstrates the integration of peripheral steroids with neurosteroids. Within the female hypothalamus, estradiol from developing follicles stimulates progesterone synthesis in astrocytes, which activate neural circuits regulating gonadotropin (GnRH) neurons. Estradiol acts at membrane estrogen receptor-α to activate cellular signaling that results in the release of inositol trisphosphate-sensitive calcium stores that are sufficient to induce neuroprogesterone synthesis. The purpose of the present studies was to characterize the estradiol-induced signaling leading to activation of steroid acute regulatory protein (StAR) and transporter protein (TSPO), which mediate the rate-limiting step in steroidogenesis, ie, the transport of cholesterol into the mitochondrion. Treatment of primary cultures of adult female rat hypothalamic astrocytes with estradiol induced a cascade of phosphorylation that resulted in the activation of a calcium-dependent adenylyl cyclase, AC1, elevation of cAMP, and activation of both StAR and TSPO. Blocking protein kinase A activation with H-89 abrogated the estradiol-induced neuroprogesterone synthesis. Thus, together with previous results, these experiments completed the characterization of how estradiol action at the membrane leads to the augmentation of neuroprogesterone synthesis through increasing cAMP, activation of protein kinase A, and the phosphorylation of TSPO and StAR in hypothalamic astrocytes.

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