[Show abstract][Hide abstract] ABSTRACT: Formation of ATP from ADP on the external surface of vascular endothelial cells has been attributed to plasma membrane ATP
synthase, ectoadenylate kinase (ecto-AK), and/or ectonucleoside diphosphokinase. These enzymes or their catalytic products
have been causatively linked to the elaboration of vascular networks and the regulation of capillary function. The amount
of ATP generated extracellularly is small, requiring sensitive analytical methods for quantification. Human umbilical vein
endothelial cells were used to revisit extracellular ATP synthesis using a reliable tetrazolium reduction assay and multiwell
plate cultures. Test conditions compatible with AK stability were established. Extracellular AK activity was found to be <1%
of the total (intracellular and extracellular), raising the possibility that the external enzyme could have leaked from living
cells and/or a few dying cells. To determine whether AK inadvertently leaked from the cells, the activity of another cytoplasmic
enzyme, glucose-6-phosphate dehydrogenase (G6PD), was also measured. G6PD is present in the cytoplasm in similar abundance
to AK. The activity ratio of G6PD (extracellular/total) was found to be similar to that of AK. Because G6PD in the medium
was probably due to leakage, other cytoplasmic macromolecules, including AK, should be released proportionately from the cells.
The role of plasma membrane ATP synthase in extracellular ATP formation was examined using Hanks' balanced salt solution with
and without selective inhibitors of AK and ATP synthase activities. With P1,P5-di(adenosine 5′)-pentaphosphate (inhibitor of AK activity), no extracellular ATP synthesis was detected, whereas with oligomycin,
piceatannol, and aurovertin (inhibitors of F1F0-ATP synthase and F1-ATPase activities), no inhibition of extracellular ATP synthesis was observed. AK activity alone could account for the observed
extracellular ATP synthesis. The possible impact of ADP impurity in the assays is discussed.
[Show abstract][Hide abstract] ABSTRACT: A reliable, indirect method (GPD/INT assay) for estimating the number of live animal cells in multiwell culture has been devised. It is based on the glucose-6-phosphate dehydrogenase (Gpdh) and 6-phosphogluconate dehydrogenase activities present in the cytoplasm of viable eukaryotic cells but not in their bathing medium nor in nonviable cells. A single reagent mixture, buffered at pH 7.8 and containing Tris, Triton X-100, glucose-6-phosphate, nicotinamide adenine dinucleotide phosphate (NADP), phenazine methosulfate, and iodonitrotetrazolium violet, is added to the cultures. The Triton X-100 releases the cytoplasmic contents into the medium, facilitating enzyme-catalyzed oxidation of the glucose-6-phosphate and 6-phosphogluconate by NADP. The resulting reduced nicotinamide adenine dinucleotide phosphate, NADPH, reduces tetrazolium violet to its formazan, the color of which reflects the number of living cells that were in the culture. The assay was tested on recombinant Gpdh and the several types of animal and insect cell lines to verify the premise that there is proportionality between the amount of GPdh and number of viable cells in the cultures. The method has been used to quantitate the effects of growth inhibitors on cells in 96-well cultures.
[Show abstract][Hide abstract] ABSTRACT: A method is described for estimating the numbers ofanimal cells in multi-well culture by simultaneouslymeasuring the lactate dehydrogenase activity of thetotal culture and the medium. The difference betweenthe two reflects the dehydrogenase content of thecells and correlates with cell number. This LDH/INTmethod was tested using several lines of normal andtransformed suspension and adherent cells. Thelactate dehydrogenase activities of duplicate cultureswere determined colourimetrically using reactioncocktails containing lactate, NAD(+), diaphorase,and p-iodonitrotetrazolium violet, with and withoutTriton X-100. The difference in absorbance at 490 nm(DeltaA(490) = A(490, test) - A(490, control)) was used to calculate the lactatedehydrogenase activity of the total culture (+ Triton)and the medium (- Triton). The cellular lactatedehydrogenase activity (difference between totaland medium dehydrogenaseactivities) was proportional to viable cell number. The effects on cell growth of four metabolicinhibitors, sodium azide, actinomycin D,cycloheximide, and taxol, were determined using theLDH/INT assay and direct cell counting. The inhibitorconcentrations that caused decreases in the LDHactivity and cell number by 50% were similar. TheLDH/INT assay is quick and sensitive, works equallywell for adherent and suspension cells, and providesinformation about LDH activities of both the mediumand cells. It is particularly useful for screeningpotential cell-growth inhibitors.