Source: LOUISIANA STATE UNIVERSITY submitted to
REAL-TIME MEASUREMENT OF ETHANOL'S EFFECT ON CYCLIC AMP METABOLISM IN LIVE CELLS
Sponsoring Institution
Cooperating Schools of Veterinary Medicine
Project Status
TERMINATED
Funding Source
Reporting Frequency
Annual
Accession No.
0217394
Grant No.
(N/A)
Project No.
LAV-2807
Proposal No.
(N/A)
Multistate No.
(N/A)
Program Code
(N/A)
Project Start Date
Jul 20, 2008
Project End Date
Jun 30, 2011
Grant Year
(N/A)
Project Director
yoshimura, M.
Recipient Organization
LOUISIANA STATE UNIVERSITY
(N/A)
BATON ROUGE,LA 70893
Performing Department
Comparative Biomedical Sciences
Non Technical Summary
cAMP signal transduction has been postulated to play a critical role in the physiological and behavioral responses to ethanol in animals and in the development of and predisposition to alcoholism in humans. The cAMP signaling system can be modulated by both acute and chronic ethanol exposure. The effect of ethanol on the levels of cAMP and on the activity of adenylyl cyclase was measured by immunological and radiochemical methods in past studies, which had poor temporal resolution and no spatial resolution. Recently, several groups have developed single polypeptide chain cAMP sensor molecules, which can monitor cAMP levels by fluorescence resonance energy transfer (FRET). Using these sensor molecules it is possible to study real-time dynamic changes in the concentration of cAMP at a subcellular level with high spacio-temporal resolution. In this project we will utilize this technology for studying the effects of ethanol on the cAMP signaling system in real-time at a subcellular level with the hypothesis that ethanol influences cAMP metabolism in a subcellular compartment specific manner. The knowledge we will obtain is crucial for understanding the effects of ethanol on cAMP metabolism at the single cell and subcellular level. The technical expertise we will gain during the course of this study is indispensable for future studies of ethanol??s effects on cAMP metabolism in the brain.
Animal Health Component
(N/A)
Research Effort Categories
Basic
100%
Applied
(N/A)
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
7237010100020%
7237010104040%
7237010118040%
Goals / Objectives
Using a single polypeptide chain cAMP sensor molecule, which can monitor cAMP levels by fluorescence resonance energy transfer (FRET), we proposed 1) to determine the effects of ethanol on cAMP metabolism in subcellular compartments of HeLa cells, 2) to determine the contributions of phosphodiesterase (PDE), protein kinase A (PKA), and A-kinase anchoring proteins (AKAPs) on the observed effects of ethanol, and 3) to determine the effects of ethanol on cAMP metabolism in subcellular compartments of neuronal cells in primary culture.
Project Methods
Cells will be cultured on glass coverslips and transfected with genes encoding a FRET-based cAMP sensor molecule. The coverslips with cells will be mounted in a perfusion chamber on the microscope stage, through which drugs will be applied to the cells. Fluorescent images of the cells will be captured at preset intervals for the duration of time-lapse experiments using a fluorescent imaging workstation which consists of an inverted microscope, a cooled charge-coupled device camera, dual filter wheels, and a Xenon light source, all controlled by a computer with image analysis software. The intensity of the FRET signal that is free from contamination will be calculated on a pixel-by pixel basis for the entire image. The statistical significance of the drug treatment will be examined by two-way RM ANOVA.

Progress 07/20/08 to 06/30/11

Outputs
OUTPUTS: Activities: Using original cAMP sensor, Epac1-camp, effect of different concentrations of ethanol on cAMP level in Hela cells transfected with type 7 adenylyl cyclase (AC7) and dopamine D1A receptor was examined by FRET analysis of individual cells. This was compared to the effect of ethanol determined by cAMP accumulation assay using radioactive reagents. Epac1-camp targeted to cytoplasm, Nucleus, and plasma membrane, respectively, were created and used to examine effect of ethanol on cAMP in each compartments. This was also carried out using cells transfected with AC3. Using a perfusion chamber effect of acute exposure (5 sec and 2 min) was examined. In addition, longer exposure (4 hr and 24 hr) to ethanol was examined. All planned experiments were carried out. Effect of acute exposure to ethanol on cAMP level was examined in the presence of PDE inhibitor, IBMX. Currently, effects of isoform specific PDE inhibitors and PKA inhibitors are being studied. Primary culture of rat cortical neurons was established using commercially available preparations of cells. Expression of epac1-camp (original and subcellular targeted forms) was carried out. Initial FRET analysis indicated detectable cAMP response in these cells. However, cell density and transfection efficiency were too low to obtain enough number of cell recordings. Events: Presented poster at 2009, 2010, 2011 RSA annual meeting and 2010 ISBRA World Congress Services: N/A Products: N/A PARTICIPANTS: Individuals: Masami Yoshimura (PI), Usa Dokphrom, Ratna Gupta Partner Organizations: N/A Collaborators and contacts: N/A Training or professional development: N/A TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
1. Real-time detection of forskolin-stimulated activity of AC6 in HEK293 cells. Addition of forskolin triggered a rapid decrease in FRET signals indicating a rapid increase in intracellular cAMP. The intracellular cAMP level reached a plateau in 150 sec and stayed at the same level during the recorded period up to 500 sec. 2. Real-time detection of ethanol's effect on cAMP in HEK293 cells. Addition of 200 mM ethanol caused a slight but steady decrease in the FRET signals over 600 sec. The results are comparable to the data obtained by a radiochemical method in our lab. 3. Measurement of cAMP change in cortical neurons. Addition of forskolin triggered a rapid decrease in FRET signals indicating a rapid increase in intracellular cAMP. 4. Staurosporine-induced apoptosis detected with FRET. A FRET-based apoptosis sensor was created by joining mTagBFP and mTagGFP with the 20 amino acid linker. The large FRET efficiency of the mTagBFP-mTagGFP pair enabled the detection of even weak proteolitic activity in each cell at the beginning of apoptosis, when only a fraction of the substrate was cleaved. 5. Real-time detection of ethanol's effect on cAMP in HeLa cells. Addition of ethanol together with dopamine further increased cytosolic cAMP in a concentration dependent manner. The change of cAMP concentration followed a similar time course as stimulation with dopamine alone but time to reach to the peak concentration and plateau was longer in the presence of ethanol. Overall, the results are consistent with our previous observations. 6. Effect of long preexposure to ethanol. Cytosolic cAMP level of the cells expressing AC7was examined after 24 hr preexposure to 50 mM ethanol. The results indicated that preexposure to ethanol had little effects on cAMP generation system and on the enhancing effect of ethanol 7. Effect of ethanol on very short stimulation with dopamine. Cytosolic cAMP level of the cells expressing AC7 was examined when dopamine was added for 5 sec. The results indicated that addition of ethanol during 5 sec stimulation with dopamine decreased the activity of AC7 to the contrary to the observation with 2 min stimulation mentioned above and previous studies using radiochemical assay with population of cells.

Publications

  • Subach, O.M., Gundorov, I.S., Yoshimura, M., Subach, F.V., Zhang, J., Gruenwald, D., Souslova, E.A., Chudakov, D.M. and Verkhusha, V.V. (2008) Conversion of red fluorescent protein into a bright blue probe. Chem. Biol. 15, 1116-1124. (PMC2585067).
  • Dokphrom, U., Qualls-Creekmore, E. and Yoshimura, M. (2011) Effects of alcohols on recombinant adenylyl cyclase type 7 expressed in bacteria. Alcohol. Clin. Exp. Res. 35, 1915-1922.
  • Dokphrom, U. and Yoshimura, M. (2009) Effect of alcohols on a recombinant adenylyl cyclase expressed in bacteria. Alcohol. Clin. Exp. Res. 33 (6), 16A.
  • Yoshimura, M. (2009) Real-time measurement of ethanol effects upon cyclic AMP in living cells. Alcohol. Clin. Exp. Res. 33 (6), 17A.
  • Dokphrom, U. and Yoshimura, M. (2010) Effect of alcohols on a soluble form of adenylyl cyclase type 7 expressed in bacteria. FASEB J. 24, 962.1.
  • Dokphrom, U. and Yoshimura, M. (2010) Effect of alcohols on a soluble form of adenylyl cyclase type 7 expressed in bacteria. Alcohol. Clin. Exp. Res. 34 (6), 14A.
  • Yoshimura, M., Castillo, M.I., Hasanuzzaman, M. and Dokphrom, U. (2010) Isoform specific effects of alcohols on adenylyl cyclase activity. Alcohol. Clin. Exp. Res. 34 (8), 150A.
  • Gupta, R and Yoshimura, M. (2011) Temporal dynamics of effect of short ethanol exposure on cyclic AMP in cells expressing adenylyl cyclase type 7. Alcohol. Clin. Exp. Res. 35 (6), 256A.


Progress 01/01/10 to 12/31/10

Outputs
OUTPUTS: Activities: Change in intracellular cAMP was measured by using FRET based cAMP sensor, Epac1-camp. The following experiments were conducted: 1) Real-time detection of dopamine D1A receptor effect on AC7 in HELA cells 2) Real-time detection of ethanol effect on cAMP in HeLa cells expressing AC7. 3) Real-time detection of effect of different duration of stimulation by ethanol on AC7. 4) Real-time detection of ethanol effect on AC7 in the presence of phosphodiesterease inhibitor. Events: Presented one poster at 2010 ISBRA World Congress Services: N/A Products: N/A PARTICIPANTS: Individuals: Masami Yoshimura (PI), Usa Dokphrom, Ratna Gupta Partner Organizations: N/A Collaborators and contacts: N/A Training or professional development: N/A TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
Our results indicate that using current microscope setting it is possible to carry out FRET based cAMP measurement in single cells in real-time and that data obtained with FRET based measurement are consistent with those obtained with radiochemical measurement. Our results indicate that in the presence of ethanol the activation of cAMP generating system is slower comparing to in the absence of ethanol. This type of observation in time course of cAMP change is possible using the FRET sensor.

Publications

  • Yoshimura, M., Castillo, M.I., Hasanuzzaman, M. and Dokphrom, U. (2010) Isoform specific effects of alcohols on adenylyl cyclase activity. (abstract) Alcohol. Clin. Exp. Res. 34 (s3), 150A.


Progress 01/01/09 to 12/31/09

Outputs
OUTPUTS: Activities: Change in intracellular cAMP was measured by using FRET based cAMP sensor, Epac1-camp. The following experiments were conducted: 1) Real-time detection of forskolin–stimulated activity of AC6 in HEK293 cells. 2) Real-time detection of ethanol effect on cAMP in HEK293 cells. 3) Real-time detection of ethanol effect on cAMP in HeLa cells. 4) Expression of Epac1-camp in subcellular compartments. 5) Measurement of cAMP change in cortical neurons. FRET based sensor for apoptosis was constructed and used to monitor staurosporine induced apoptosis in HeLa cells. Events: Presented three posters at Annual meeting for Research Society on Alcoholism. Services: N/A Products: Modified Epac1-camp targeted to either plasma membrane, nucleus, or cytoplasm were created. PARTICIPANTS: Individuals: Masami Yoshimura (PI), Maria I. Castillo, Usa Dokphrom TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
Our results indicate that using current microscope setting it is possible to carry out FRET based cAMP measurement in single cells in real-time and that data obtained with FRET based measurement are consistent with those obtained with radiochemical measurement. Our results indicate that initiation of cAMP synthesis after addition of G protein coupled receptor agonists is very fast and that there is no detectable delay in the enhancing effect of ethanol on cAMP accumulation. The addition of signaling peptides to the cAMP sensor protein proved to be successful and provided a new set of tools for subcellular compartment specific cAMP measurement. We also demonstrated that newly developed fluorescent proteins, mTagBFP and mTagGFP, can be very useful as FRET based sensors.

Publications

  • Yoshimura, M. (2009) Real-time measurement of ethanol effects upon cyclic AMP in living cells. (poster) Alcohol. Clin. Exp. Res. 33 (s1), 17A.


Progress 01/01/08 to 12/31/08

Outputs
OUTPUTS: Effects of alcohols on the activity of different isoforms of mammalian adenylyl cyclases were examined by using transfected HeLa cells. Real-time measurement of cyclic AMP change in single cells was measured by fluorescent resonance energy transfer. PARTICIPANTS: Individuals: Masami Yoshimura (PI), Usa Dokphrom TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Not relevant to this project.

Impacts
We were establishing hardware system and cell culture condition to measure cyclic AMP change in single cells in real-time.

Publications

  • Subach, O.M., Gundorov, I.S., Yoshimura, M., Subach, F.V., Zhang, J., Gruenwald, D., Souslova, E.A., Chudakov, D.M. and Verkhusha, V.V. (2008) Conversion of red fluorescent protein into a bright blue probe. Chem. Biol. 15, 1116-1124.