scholarly journals Monitoring of cAMP Synthesis and Degradation in Living Cells

Physiology ◽  
2006 ◽  
Vol 21 (2) ◽  
pp. 86-92 ◽  
Author(s):  
Viacheslav O. Nikolaev ◽  
Martin J. Lohse
Keyword(s):  
Protein Kinase ◽  
Protein Kinase A ◽  
Cyclic Nucleotide ◽  
Living Cells ◽  
Cellular Effects ◽  
Cyclic Nucleotide Gated Channels ◽  
Camp Synthesis ◽  
Spatio Temporal ◽  
Synthesis And Degradation ◽  
Kinase A

cAMP is an important second messenger with a plethora of cellular effects and biological roles. To monitor and visualize cAMP in intact living cells, electrophysiological and fluorescent methods have been developed based on activation of all three types of cAMP effectors: protein kinase A, cyclic nucleotide-gated channels, and exchange protein directly activated by cAMP. In this review, we describe and compare these techniques in terms of their robustness, sensitivity and spatio-temporal resolution.

scholarly journals A Membrane Permeable Prodrug of S223 for Selective Epac2 Activation in Living Cells

Cells ◽  
2019 ◽  
Vol 8 (12) ◽  
pp. 1589 ◽  
Author(s):  
Yunjian Xu ◽  
Frank Schwede ◽  
Hans Wienk ◽  
Anders Tengholm ◽  
Holger Rehmann
Keyword(s):  
Protein Kinase ◽  
Protein Kinase A ◽  
Cyclic Nucleotide ◽  
Adenosine Monophosphate ◽  
Living Cells ◽  
Effector Proteins ◽  
Nucleotide Exchange ◽  
Acetoxymethyl Ester ◽  
Different Types ◽  
Kinase A

Signalling by cyclic adenosine monophosphate (cAMP) occurs via various effector proteins, notably protein kinase A and the guanine nucleotide exchange factors Epac1 and Epac2. These proteins are activated by cAMP binding to conserved cyclic nucleotide binding domains. The specific roles of the effector proteins in various processes in different types of cells are still not well defined, but investigations have been facilitated by the development of cyclic nucleotide analogues with distinct selectivity profiles towards a single effector protein. A remaining challenge in the development of such analogues is the poor membrane permeability of nucleotides, which limits their applicability in intact living cells. Here, we report the synthesis and characterisation of S223-AM, a cAMP analogue designed as an acetoxymethyl ester prodrug to overcome limitations of permeability. Using total internal reflection imaging with various fluorescent reporters, we show that S223-AM selectively activates Epac2, but not Epac1 or protein kinase A, in intact insulin-secreting β-cells, and that this effect was associated with pronounced activation of the small G-protein Rap. A comparison of the effects of different cAMP analogues in pancreatic islet cells deficient in Epac1 and Epac2 demonstrates that cAMP-dependent Rap activity at the β-cell plasma membrane is exclusively dependent on Epac2. With its excellent selectivity and permeability properties, S223-AM should get broad utility in investigations of cAMP effector involvement in many different types of cells.

scholarly journals Dependence of Electrical Activity and Calcium Influx-Controlled Prolactin Release on Adenylyl Cyclase Signaling Pathway in Pituitary Lactotrophs

2006 ◽  
Vol 20 (9) ◽  
pp. 2231-2246 ◽  
Author(s):  
Arturo E. Gonzalez-Iglesias ◽  
Yonghua Jiang ◽  
Melanija Tomić ◽  
Karla Kretschmannova ◽  
Silvana A. Andric ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Protein Kinase A ◽  
Adenylyl Cyclase ◽  
Guanylyl Cyclase ◽  
Cyclic Nucleotide ◽  
Action Potentials ◽  
Soluble Guanylyl Cyclase ◽  
Cgmp Production ◽  
Camp And Cgmp ◽  
Kinase A

Abstract Pituitary lactotrophs in vitro fire extracellular Ca2+-dependent action potentials spontaneously through still unidentified pacemaking channels, and the associated voltage-gated Ca2+ influx (VGCI) is sufficient to maintain basal prolactin (PRL) secretion high and steady. Numerous plasma membrane channels have been characterized in these cells, but the mechanism underlying their pacemaking activity is still not known. Here we studied the relevance of cyclic nucleotide signaling pathways in control of pacemaking, VGCI, and PRL release. In mixed anterior pituitary cells, both VGCI-inhibitable and -insensitive adenylyl cyclase (AC) subtypes contributed to the basal cAMP production, and soluble guanylyl cyclase was exclusively responsible for basal cGMP production. Inhibition of basal AC activity, but not soluble guanylyl cyclase activity, reduced PRL release. In contrast, forskolin stimulated cAMP and cGMP production as well as pacemaking, VGCI, and PRL secretion. Elevation in cAMP and cGMP levels by inhibition of phosphodiesterase activity was also accompanied with increased PRL release. The AC inhibitors attenuated forskolin-stimulated cyclic nucleotide production, VGCI, and PRL release. The cell-permeable 8-bromo-cAMP stimulated firing of action potentials and PRL release and rescued hormone secretion in cells with inhibited ACs in an extracellular Ca2+-dependent manner, whereas 8-bromo-cGMP and 8-(4-chlorophenyltio)-2′-O-methyl-cAMP were ineffective. Protein kinase A inhibitors did not stop spontaneous and forskolin-stimulated pacemaking, VGCI, and PRL release. These results indicate that cAMP facilitates pacemaking, VGCI, and PRL release in lactotrophs predominantly in a protein kinase A- and Epac cAMP receptor-independent manner.

scholarly journals Role for YakA, cAMP, and Protein Kinase A in Regulation of Stress Responses ofDictyostelium discoideumCells

2002 ◽  
Vol 13 (7) ◽  
pp. 2266-2275 ◽  
Author(s):  
Alexandre Taminato ◽  
Raquel Bagattini ◽  
Renata Gorjão ◽  
Guokai Chen ◽  
Adam Kuspa ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Protein Kinase ◽  
Protein Kinase A ◽  
Stress Responses ◽  
Site Mutation ◽  
Camp Synthesis ◽  
Different Types ◽  
Arrest Growth ◽  
Pka Pathway ◽  
Kinase A

The Dictyostelium protein kinase YakA is required for the growth-to-development transition. During growth YakA controls the cell cycle, regulating the intervals between cell divisions. When starved for nutrients Dictyosteliumcells arrest growth and undergo changes in gene expression, decreasing vegetative mRNAs and inducing the expression of pkaC. YakA is an effector of these changes, being necessary for the decrease of vegetative mRNA expression and the increase of protein kinase A (PKA) activity that will ultimately regulate expression of adenylyl cyclase, cAMP synthesis, and the induction of development. We report a role for this kinase in the response to nitrosoative or oxidative stress of Dictyostelium cells. Hydrogen peroxide and sodium nitroprusside arrest the growth of cells and trigger cAMP synthesis and activation of PKA in a manner similar to the well-established response to nutrient starvation. We have found thatyakA null cells are hypersensitive to nitrosoative/oxidative stress and that a second-site mutation inpkaC suppresses this sensitivity. The response to different stresses has been investigated and YakA, cAMP, and PKA have been identified as components of the pathway that regulate the growth arrest that follows treatment with compounds that generate reactive oxygen species. The effect of different types of stress was evaluated in Dictyostelium and the YakA/PKA pathway was also implicated in the response to heat stress.

scholarly journals Quantitation of protein kinase A-mediated trafficking of cardiac sodium channels in living cells

2006 ◽  
Vol 72 (2) ◽  
pp. 250-261 ◽  
Author(s):  
H HALLAQ ◽  
Z YANG ◽  
P VISWANATHAN ◽  
K FUKUDA ◽  
W SHEN ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Protein Kinase A ◽  
Sodium Channels ◽  
Living Cells ◽  
Cardiac Sodium Channels ◽  
Kinase A

The Role of ABC Transporter Abcc4 in Platelets Physiologic Function and Its Impact On Collagen Meditated Platelet Aggregation

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 1063-1063
Author(s):  
Satish B. Cheepala ◽  
Kazumasa Takenaka ◽  
Tamara I. Pestina ◽  
Carl W. Jackson ◽  
John D. Schuetz
Keyword(s):  
Plasma Membrane ◽  
Platelet Aggregation ◽  
Protein Kinase ◽  
Protein Kinase A ◽  
Platelet Activation ◽  
Cyclic Nucleotide ◽  
Dense Granules ◽  
Collagen Receptor ◽  
Kinase A

Abstract Abstract 1063 Platelet activation is a highly regulated process, and cyclic nucleotide mediated signaling pathways are crucial to effective platelet activation. Vascular injury produces, exposed collagen which binds circulating platelets through the platelet's “collagen” receptor, GPVI, resulting in the activation of guanyly/adenlyl cyclases. These interactions result in the rapid alterations in the cyclic nucleotide concentration inside the platelets leading to activation of protein kinase A and G signaling pathways to modulate platelet function. While, ABCC4 functions as a plasma membrane transporter for cyclic nucleotides its contribution to platelet activation has been obscured because it was reportedly as primarily intracellular in the platelets dense granules. This original report (Jedlitschky, Tirschmann et al. 2004) evaluated ABCC4 localization by immune-fluorescence of platelets attached to collagen coated coverslips. However, attachment via collagen produces platelet activation leading to mobilization and fusion of alpha and dense granules to the plasma membrane, thus under these conditions distinguishing between plasma membrane and dense granules is not possible. We resolved this problem by labeling quiescent platelets with a cell impermeable biotinylating agent (EZ-Link Sulfo-NHS-LC-LC Biotin). Isolation of membrane and internal fraction demonstrated that of over ninety percent of Abcc4 localizes to the plasma membrane. Furthermore, confocal microscopy of platelets stained with specific antibodies against Abcc4 confirmed Abcc4 localization to the plasma membrane. We extended these studies to the Abcc4- knockout (KO) mouse model. The Abcc4- KO mouse does not have any change in the number of platelet or dense granules compared to the wild type mouse. Platelet activation in vivo can be initiated by interaction with collagen through the GPVI receptor that is expressed at the plasma membrane of the platelets. At the molecular level, the initiation of platelet activation by collagen results in an increase in the cyclic nucleotide concentration leading to activation of signaling cascade through protein kinase A or G. Expose of Abcc4-KO platelets to collagen and revealed impaired activation in response to collagen. However, Abcc4-KO platelets activated by either thrombin or ADP (which activate either G-coupled PAR receptors or P2Y12 receptor respectively) shows an aggregation profile almost identical to wildtype platelets, thus indicating the defect in Abcc4 -KO platelet aggregation is specific to the collagen pathway. To understand the basis for the impaired collagen aggregation of Abcc4-KO platelets, we investigated the collagen receptor (GPVI) signaling pathway in Abcc4-KO platelets. Interestingly, in the Abcc4-KO platelets after the platelet activation with collagen, cyclic nucleotide dependent phosphorylation of VASP through protein kinase A or G at Ser-157 or Ser-239 respectively is reduced compared to the wildtype. Notably, Abcc4-KO platelets had reduced GPVI surface expression that correlated with the reduced phosphorylation of VASP after collagen stimulation. The similar, protein levels of Syk and Plcg2, (downstream signaling molecules of GPVI signaling pathway), in the Abcc4 wildtype and KO platelets implies that GPVI expression is the primary defect in Abcc4 deficiency. These results suggest that Abcc4 plays a crucial role in regulating cyclic nucleotides in response to GPVI activation by collagen. These findings suggest ABCC4/Mrp4 loss of function or inhibition (by drugs) may disrupt platelet aggregation under conditions of vascular injury. As, many antiplatelet drugs are potent inhibitors of Abcc4 (e.g., Dipyridamole and Sildenafil) these conclusions have strong implications for not just the development of antiplatelet drugs, but also for further exploring the role of Abcc4 in regulating intracellular nucleotide levels and platelet biology. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Comparative thermodynamic analysis of cyclic nucleotide binding to protein kinase A

2007 ◽  
Vol 388 (2) ◽  
Author(s):  
Daniela Moll ◽  
Sonja Schweinsberg ◽  
Christian Hammann ◽  
Friedrich W. Herberg
Keyword(s):  
Protein Kinase ◽  
Protein Kinase A ◽  
Thermodynamic Analysis ◽  
Cyclic Nucleotide ◽  
Nucleotide Binding ◽  
Cyclic Nucleotide Binding ◽  
Kinase A
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