University of Wisconsin - Green Bay


Research Council

Research
Success

Craig Hanke

Craig Hanke

Spring 2010

"Development of a fluorescence method for quantification of intracellular calcium concentration"

Grant in Aid of Research

Final Report: "Thanks to funding from the Research Council, we were able to purchase necessary FLUO-3 dye and cell culture media to continue our studies on the H295R cell line. FLUO-3 is a small, membrane permeable, calcium sensitive dye that can be loaded into cultured cells to allow measurement of intracellular calcium release. Intracellular calcium is a universal trigger for many cells, resulting in various cellular reactions such as muscle contraction, neurotransmitter release and hormone release. FLUO 3 dye will fluoresce at 525 nm when it is bound to calcium and fluorescence intensity can be measured with digital imaging. The intensity of fluorescence is directly related to calcium concentration within the cell. To date these studies have indicated a strong fluorescence response from the H295R cells in response to typical stimuli. H295R cells are an immortalized cell line capable of producing the hormone aldosterone. Aldosterone production is typically triggered by angiotensin II, high potassium concentrations and adrenocorticotropic hormone release in healthy animals. Both angiotensin II and potassium ions will trigger intracellular calcium release and the corresponding fluorescence of the FLUO-3 dye in our H295R cell studies. Thus, the first step in our experimental system is working properly and cellular responses to normal stimuli can be measured.During our most recent studies we have observed an unexpected FLUO -3 response to ethanol. This ethanol–stimulated fluorescence has not previously been reported and is a concern due to the common use of ethanol as a solvent for many calcium-stimulating drugs. If our findings are correct, many FLUO 3 users could potentially measure false positive effects when they dissolve their test compounds in ethanol. We have continued to investigate whether the effect could be due to contaminated ethanol stocks or a direct effect of ethanol on the cell membranes of the H295R cells. The data indicate that the ethanol stocks have not been contaminated. The data also indicate that the mechanism and rate of ethanol administration is critical to the cellular response. Slow administration of ethanol shows a substantially lower fluorescent response than rapid injection of even small volumes (10 µl) of ethanol in a cell culture well containing 1 ml of buffer. We now intend to characterize this ethanol response and publish our findings as a short communication alerting other FLUO-3 users of this potential problem. Once we have resolved the ethanol problem, we intend to finish our study of the use of FLUO 3 for quantitative measurements of intracellular calcium."


Spring 2007

"The role of cyclic ADP ribose-stimulated calcium release in the adrenal H295R cell line"

Grant in Aid of Research


Spring 2006

"Control of Adrenal Zonal Glomerulosa cell intracellular calcium and aldosterone synthesis by cyclic adenosine diphosphate ribose"

Grant in Aid of Research


Fall 2004

"Study the Effects of Probenecid Treatment on Aldeosterone Release from Adrenal Zona glomerulosa cells"

Grant in Aid of Research


Fall 2003

"The Release of Aldosterone from Cultured Adrenal cells before and after treatment with Organic Anion transporter inhibitors such as Probenecid"

Grant in Aid of Research