The situation, however, was complicated because pirenzepine was indicated to show similar affinity for the two receptor preparations solubilized with digitonin
from either rat brain or heart.
The cells were lysed with 50[micro]M digitonin
followed by addition of 1mM Ca[Cl.
Permeabiliszation and cell viability of PFFs treated with digitonin
were assessed by 0.
3-[(3-Cholamidopropyl)-dimethylammonio]-1-propanesulphonate (CHAPS) and 3-[(3-cholamidopropyl)-dimethylammonio]-2-hydroxy-1-propanesulphonate (CHAPSO) were from Calbiochem (Darmstadt, Germany); N-(2-hydroxyethyl)-piperazine-N'-(2-ethanesulphonic acid) (Hepes), tris(hydroxymethyl)aminomethane (Tris), ethylene glycol-bis([beta]-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA), 1,6-diphenyl-1,3,5-hexatriene (DPH), sodium dodecyl sulphate (SDS), phenylmethylsulphonyl fluoride (PMSF), aprotinin, benzamidine, digitonin
, cholic acid, deoxycholic acid, n-dodecyl-[beta]-D-maltoside (DBM), and dodecylpoly[(ethylene-glycolether).
Collagenase, ethylenediaminetetraacetic acid (EDTA), ethylene glycol tetra-acetic acid (EGTA), sodium pyrophosphate, sodium orthovanadate, sodium fluoride, okadaic acid, 1% protease inhibitor cocktail, dithiothreitol, magnesium chloride, K-lactobionate, taurine, potassium phosphate, HEPES, digitonin
, pyruvate, malic acid, glutamic acid, adenosine diphosphate (ADP), succinic acid, oligomycin, carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP), antibody to [beta]-actin (mouse), phenylephrine and acetylcholine, trypsin inhibitor, and cytochrome c were procured from SigmaAldrich (MO, USA).
Additionally, in threedrug combinations of lignans with the saponin digitonin
and doxorubicin MDR reversal activity was even stronger enhanced.
The results were expressed as the percentage of ATP released relative to the total ATP present in cells lysed by means of digitonin
(50 [micro]M) (16).
94] described that, in digitonin
permeabilized cardiomyocytes from young hearts, induction of SR [Ca.
14]C]-radio-labeled VLCFA substrates and one of two available methods: either in intact human skin fibroblasts cultured in monolayer (5); or in isolated fibroblasts permeabilized with digitonin
2])GMP, isobutylmethylxanthine (IBMX), and digitonin
were from Fluka (Ronkonkoma, NY).
min] values were determined by addition of digitonin
(50 [micro]M) and [Mn.
The first colorimetric assay for cholesterol in serum, reported by Grigaut in 1910, combined purification by digitonin
with an acid reagent.