Naslov
Microcalorymetric measurement of ecto-nucleotidase activity of endothelial cells

Autori
Čulić, Ognjen ; Schrader, Juergen ; Novak Mirčetić, Renata ; Žanić Grubišić, Tihana

Vrsta, podvrsta i kategorija rada
Sažeci sa skupova, sažetak, znanstveni

Izvornik
Ecto-ATPases and Related Ectonucleotidases, Proceedingsof the Second International Workshop on Ecto-ATPases and Related Ectonucleotidases / Vanduffel, Luc ; Leemmens, Raf ; - Maastricht : Shaker, 1999

Skup
Ecto-ATPases and Related Ectonucleotidases

Mjesto i datum
Diepenbeek, Belgija, 14.06.1999. - 18.06.1999

Vrsta sudjelovanja
Poster

Vrsta recenzije
Međunarodna recenzija

Ključne riječi
microcalorymetria; ectonucleotidase; endothelial cells

Sažetak
Heat flux (HF) reflecting the metabolic activity of the perfused pig aortic endothelial cells (PAEC) grown on microcarrier beads was measured microcalorimetrically (LKB thermal monitor) and found to be 231.0 ą65.5 ěW/mg cell protein or 46.34 ą 8.48 pW per single cell. Upon addition of ATP (2 mM) in a perfusion medium a significant increase of the HF could be observed. Since ATP is a charged molecule it is believed that it can not freely cross the plasma membrane. We assume that the observed increase in HF is due to ecto-nucleotidase enzyme activities that are present on the cell surface. The following findings are supportive for this conclusion: 1. Other nucleoside triphosphates (GTP, ITP, UTP, and CTP), as well as, nucleoside diphosphates (ADP, GDP) caused quantitatively similar increase of the HF, thus indicating the non-specific character of the observed (hydrolytic) activity. 2. ATP-induced HF was completely independent of the intracellular ATP concentration. Cells perfused with medium that had no exogenous substrate reduced basal heat flux to less than 5% of the initial value. Still, this drastic intracellular ATP depletion and energy turnover reduction did not influence HF signal triggered by addition of extracellular ATP. 3. Preincubation of cells with thapsigagrin (inhibitor of ER Ca2+ -ATPase and intracellular Ca2+ -releasing compound) did not influence the stimulation of the HF caused by addition of the CTP, as a substrate. Thapsigagrin influenced the subsequent ATP challenge, only to a minor extent, thus indicating that the observed HF effect is not likely to be due to activation of the purinoceptors and involvement of intracellular Ca2+ as a second messenger. Taking into account that enthalpy of ATP hydrolysis is about 60kJ/mol it can be calculated that the released heat corresponds to ATP-hydrolysing activity of approx. 16 nmol/min-1x 10-6 cells which was determined spectrophotometrically, in the phosphate liberation assay. Stimulation of the HF in the case of AMP as a substrate was less than 10% of the ATPase activity, which is close to the reported Vmax value for 5’-nucleotidase of PAEC. Both methods (calorymetry and spectrophotometry revealed some rather atypical properties of the ecto-ATPase in PAEC like: 1. Very poor stimulatory effect of externally added cation (Mg2+ ) 2. Reaction rates of the ATP and ADP hydrolysis were significantly decreasing during the course of enzyme reaction, amounting to only 30% of the initial value after 60 minutes of incubation, indicating inhibition, downregulation or downexpression of the enzyme. In conclusion, our study has indicated that microcalorimetry can be used as a suitable method for continuos measurement of ecto-nucleotidase activity, including studies of its kinetic properties, expression, stability and inducibility.

Izvorni jezik
Engleski

Znanstvena područja
Temeljne medicinske znanosti

POVEZANOST RADA