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Journal of Neurophysiology Dec 2006Neurotransmitter release is a steep function of the intracellular calcium ion concentration ([Ca(2+)](i)) at the release sites. Both the Ca(2+) amplitude and the time...
Neurotransmitter release is a steep function of the intracellular calcium ion concentration ([Ca(2+)](i)) at the release sites. Both the Ca(2+) amplitude and the time course appear to be important for specifying neurotransmitter release. Ca(2+) influx regulates the number of vesicles exocytosed as well as the amount of neurotransmitter each individual vesicle releases. In our study we stimulated mouse chromaffin cells in two different ways to alter Ca(2+) presentation at the release sites. One method, digitonin permeabilization followed by exposure to Ca(2+), allows for a large uniform global elevation of [Ca(2+)](i), whereas the second method, application of nicotine, depolarizes chromaffin cells and activates voltage-dependent Ca(2+) channels, thereby producing more phasic and localized changes in [Ca(2+)](i). Using amperometry to monitor catecholamine release, we show that both kinds of stimuli elicit the exocytosis of similar quantities of neurotransmitter per large dense core vesicles (LDCVs) released. Even so, the release process was quite different for each stimulus; nicotine-elicited events were small and slow, whereas digitonin events were, in comparison, large and fast. In addition, the transient opening of the fusion pore, called the "foot," was essentially absent in digitonin-stimulated cells, but was quite common in nicotine-stimulated cells. Thus even though both strong stimuli used in this study elicited the release of many vesicles it appears that the differences in the Ca(2+) levels at the release sites were key determinants for the fusion and release of individual vesicles.
Topics: Animals; Calcium; Catecholamines; Chromaffin Cells; Digitonin; Electric Stimulation; Electrophysiology; Exocytosis; In Vitro Techniques; Mice; Neurotransmitter Agents; Nicotine; Nicotinic Agonists; Stimulation, Chemical; Synaptic Vesicles
PubMed: 16956996
DOI: 10.1152/jn.00017.2006 -
Vision Research Oct 1994The visual pigment of the main rhabdom of the crayfish (P533) is unstable in digitonin. While slowly hydrolyzing to N-retinylidene opsin, a portion passes through a...
The visual pigment of the main rhabdom of the crayfish (P533) is unstable in digitonin. While slowly hydrolyzing to N-retinylidene opsin, a portion passes through a long-lived intermediate (P'505) with absorption similar to metarhodopsin but with the retinal still in the cis configuration. Crayfish metarhodopsin (M515) is similarly unstable in digitonin, and a portion converts to M'508 while bleaching slowly in the dark. Both P'505 and M'508 are light sensitive and bleach through an intermediate absorbing at still shorter wavelengths, M'460. The photobleaching of M'508 is likely a two-photon process, possibly involving P'505 as an intermediate. The persistence of these altered forms of the pigment with lambda max near 510 nm has compromised earlier efforts to analyze extracts of crayfish rhodopsin by partial bleaching. First, because of the incomplete decay of M515 (a portion of which liners as M'508), the difference spectrum for a red light exposure followed by dark decay has lambda max at 562 nm, but this difference spectrum does not describe a pigment. Because of the photosensitivity of M'508, a second bleaching exposure reveals the presence of a pigment with lambda max near 510 nm, but it is not a visual pigment and it is not present in the extract initially.
Topics: Animals; Astacoidea; Chromatography, High Pressure Liquid; Digitonin; Hot Temperature; In Vitro Techniques; Light; Photoreceptor Cells, Invertebrate; Rhodopsin; Spectrophotometry
PubMed: 7975305
DOI: 10.1016/0042-6989(94)90224-0 -
Biochimica Et Biophysica Acta Jan 2002A single shock wave generated by a shock tube is able to effectively deliver macromolecules such as fluorescein isothiocyanate-dextran into the cytoplasm of living cells...
A single shock wave generated by a shock tube is able to effectively deliver macromolecules such as fluorescein isothiocyanate-dextran into the cytoplasm of living cells without causing cytotoxicity. We report on the effect of varying the molecular weight of the dextran and the number of shock waves on the efficiency of delivery into a cancer cell line. The fraction of cells permeabilized and the total fluorescence delivered were measured by flow cytometry, and the cellular viability by a tetrazolium assay on adherent cells and these values were compared to cell permeabilization using digitonin. Shock waves can deliver molecules of up to 2000000 molecular weight into the cytoplasm of cells without toxicity and may have applications in gene therapy.
Topics: Cell Membrane Permeability; Cell Survival; Cytoplasm; Dextrans; Digitonin; Flow Cytometry; Fluorescein-5-isothiocyanate; Genetic Therapy; High-Energy Shock Waves; Humans; Microscopy, Confocal; Microscopy, Phase-Contrast; Molecular Weight; Tumor Cells, Cultured
PubMed: 11853891
DOI: 10.1016/s0167-4889(01)00177-x -
The Journal of Biological Chemistry Nov 1986PC12 cells, a cloned rat pheochromocytoma cell line, were treated with digitonin to render the plasma membrane permeable to ions and proteins. At a cell density of 2-6 X...
PC12 cells, a cloned rat pheochromocytoma cell line, were treated with digitonin to render the plasma membrane permeable to ions and proteins. At a cell density of 2-6 X 10(5) cells/cm2, incubation with 7.5 microM digitonin permitted a Ca2+-dependent release of 25-40% of the catecholamine within 18 min in the presence of 10 microM Ca2+. Half-maximal secretion occurred at 0.5-1 microM Ca2+. PC12 cultures at lower cell densities were more sensitive to digitonin and gave more variable results. Secretion in the presence of digitonin and Ca2+ began after a 2-min lag and continued for up to 30 min. When cells were treated for 3 min in digitonin and then stimulated with Ca2+ in the absence of digitonin, secretion occurred in the same manner but without the initial lag. Optimal secretion from PC12 cells was also dependent upon the presence of Mg2+ and ATP. Permeabilized PC12 cells exhibited a slow time-dependent loss of secretory responsiveness which was correlated with the release of a cytosolic marker, lactate dehydrogenase (134 kDa). This suggests that digitonin permeabilization allows soluble constituents necessary for secretion to leave the cell in addition to allowing Ca2+ and ATP access into the cell interior. Ca2+-dependent secretion was completely inhibited by exposure of digitonin-permeabilized cells to 100 micrograms/ml trypsin (27 kDa), whereas secretion was only slightly inhibited by trypsin exposure prior to digitonin treatment. Thus, an intracellular, trypsin-sensitive protein is probably involved in secretion. The data also indicate that the same population of digitonin-treated cells which responded to Ca2+ was permeable to a 27-kDa protein. 1,2-Dioctanoylglycerol and phorbol esters which activate protein kinase C enhanced the Ca2+-dependent and Ca2+-independent secretion in digitonin-permeabilized PC12 cells. Thus, protein kinase C appears to be involved in the regulation of catecholamine secretion from permeabilized PC12 cells.
Topics: Adenosine Triphosphate; Adrenal Gland Neoplasms; Animals; Calcium; Catecholamines; Cell Line; Clone Cells; Digitonin; Kinetics; Pheochromocytoma; Phorbol Esters; Protein Kinase C; Rats
PubMed: 3490477
DOI: No ID Found -
Proceedings of the National Academy of... Oct 2019Cytochrome oxidase (CcO), a membrane enzyme in the respiratory chain, catalyzes oxygen reduction by coupling electron and proton transfer through the enzyme with a...
Cytochrome oxidase (CcO), a membrane enzyme in the respiratory chain, catalyzes oxygen reduction by coupling electron and proton transfer through the enzyme with a proton pump across the membrane. In all crystals reported to date, bovine CcO exists as a dimer with the same intermonomer contacts, whereas CcOs and related enzymes from prokaryotes exist as monomers. Recent structural analyses of the mitochondrial respiratory supercomplex revealed that CcO monomer associates with complex I and complex III, indicating that the monomeric state is functionally important. In this study, we prepared monomeric and dimeric bovine CcO, stabilized using amphipol, and showed that the monomer had high activity. In addition, using a newly synthesized detergent, we determined the oxidized and reduced structures of monomer with resolutions of 1.85 and 1.95 Å, respectively. Structural comparison of the monomer and dimer revealed that a hydrogen bond network of water molecules is formed at the entry surface of the proton transfer pathway, termed the K-pathway, in monomeric CcO, whereas this network is altered in dimeric CcO. Based on these results, we propose that the monomer is the activated form, whereas the dimer can be regarded as a physiological standby form in the mitochondrial membrane. We also determined phospholipid structures based on electron density together with the anomalous scattering effect of phosphorus atoms. Two cardiolipins are found at the interface region of the supercomplex. We discuss formation of the monomeric CcO, dimeric CcO, and supercomplex, as well as their role in regulation of CcO activity.
Topics: Animals; Cardiolipins; Cattle; Crystallography, X-Ray; Digitonin; Electron Transport; Electron Transport Complex I; Electron Transport Complex IV; Hydrogen Bonding; Hydrogen-Ion Concentration; Mitochondria, Heart; Mitochondrial Membranes; Molecular Conformation; Oxidation-Reduction; Oxygen; Phospholipids; Phosphorus; Protein Binding; Protein Conformation; Protein Multimerization
PubMed: 31533957
DOI: 10.1073/pnas.1907183116 -
Identification of Novel Natural Products as Effective and Broad-Spectrum Anti-Zika Virus Inhibitors.Viruses Nov 2019Zika virus (ZIKV) infection during pregnancy leads to severe congenital Zika syndrome, which includes microcephaly and other neurological malformations. No therapeutic...
Zika virus (ZIKV) infection during pregnancy leads to severe congenital Zika syndrome, which includes microcephaly and other neurological malformations. No therapeutic agents have, so far, been approved for the treatment of ZIKV infection in humans; as such, there is a need for a continuous effort to develop effective and safe antiviral drugs to treat ZIKV-caused diseases. After screening a natural product library, we have herein identified four natural products with anti-ZIKV activity in Vero E6 cells, including gossypol, curcumin, digitonin, and conessine. Except for curcumin, the other three natural products have not been reported before to have anti-ZIKV activity. Among them, gossypol exhibited the strongest inhibitory activity against almost all 10 ZIKV strains tested, including six recent epidemic human strains. The mechanistic study indicated that gossypol could neutralize ZIKV infection by targeting the envelope protein domain III (EDIII) of ZIKV. In contrast, the other natural products inhibited ZIKV infection by targeting the host cell or cell-associated entry and replication stages of ZIKV. A combination of gossypol with any of the three natural products identified in this study, as well as with bortezomib, a previously reported anti-ZIKV compound, exhibited significant combinatorial inhibitory effects against three ZIKV human strains tested. Importantly, gossypol also demonstrated marked potency against all four serotypes of dengue virus (DENV) human strains in vitro. Taken together, this study indicates the potential for further development of these natural products, particularly gossypol, as the lead compound or broad-spectrum inhibitors against ZIKV and other flaviviruses, such as DENV.
Topics: Alkaloids; Animals; Antiviral Agents; Biological Products; Cell Survival; Chlorocebus aethiops; Curcumin; Dengue Virus; Digitonin; Drug Synergism; Gossypol; Humans; Molecular Structure; Vero Cells; Zika Virus; Zika Virus Infection
PubMed: 31684080
DOI: 10.3390/v11111019 -
Journal of Biochemistry Aug 1980The effects of the presence of digitonin in the reaction mixture on photophosphorylation, light-induced H+ uptake, and the size of isolated spinach chloroplasts were...
The effects of the presence of digitonin in the reaction mixture on photophosphorylation, light-induced H+ uptake, and the size of isolated spinach chloroplasts were studied. Digitonin inactivated photosystem II and dissipated light-induced pH increase without affecting the photophosphorylation driven by photosystem I. Digitonin increased the concentration of NH4Cl required for uncoupling. This effect of digitonin was diminished by valinomycin which barely affects phosphorylation by itself. Although such properties have all been observed with digitonin subchloroplast particles, digitonin at the concentration required for acquiring such properties had little effect on the size of chloroplasts. These results suggest that the characteristic effects of digitonin subchloroplast particles are caused by thylakoid membranes penetrated by digitonin but not by the size of membrane vesicles. Both in chloroplasts with digitonin in the reaction mixture and in digitonin subchloroplast particles, N,N'-dicyclohexylcarbodiimide, purine nucleoside di- and tri-phosphates, and organic bases with low pKa values (pyridine and aniline) made the light-induced pH increase detectable, and this was lost upon the addition of proton ionophores. These results suggest that the lack of the light-induced pH increase by chloroplasts with digitonin in the reaction mixture and by digitonin subchloroplast particles is caused both by a membrane leaky to protons and by the loss of internal buffering capacity of thylakoids. Furthermore, the phosphorylation of chloroplasts with digitonin in the reaction mixture and of digitonin subchloroplast particles is considered to be driven by the proton motive force as is that of chloroplasts without digitonin.
Topics: Chloroplasts; Dicyclohexylcarbodiimide; Digitonin; Hydrogen-Ion Concentration; Kinetics; Light; Methylphenazonium Methosulfate; Photophosphorylation; Plants
PubMed: 7419505
DOI: 10.1093/oxfordjournals.jbchem.a132992 -
Biochemistry May 2006Nitroxide sensors were placed in rhodopsin at sites 140, 227, 250, and 316 to monitor the dynamics and conformation of the receptor at the cytoplasmic surface in... (Comparative Study)
Comparative Study
Nitroxide sensors were placed in rhodopsin at sites 140, 227, 250, and 316 to monitor the dynamics and conformation of the receptor at the cytoplasmic surface in solutions of dodecyl maltoside (DM), digitonin, and phospholipid bilayers of two compositions. The EPR spectra reveal a remarkable similarity of rhodopsin structure and the activating conformational change in DM and bilayers, the hallmark of which is an outward tilt of transmembrane helix VI. This conformational change is blocked in solutions of digitonin, although changes in optical absorbance accompany activation, showing that absorbance and structural changes are not necessarily coupled. In DM and bilayers, the receptor is apparently in equilibrium between conformational substates whose populations are modulated by activation. Despite the general similarity in the two environments, the receptor conformations have increased flexibility in DM relative to bilayers. For the activated receptor in DM and bilayers, a pH-dependent conformational equilibrium is identified that may correspond to the optically characterized MII(a)()-MII(b)() equilibrium. No specific effects of headgroup composition on receptor conformation in lipid bilayers were found.
Topics: Amino Acid Substitution; Animals; Cyclic N-Oxides; Digitonin; Electron Spin Resonance Spectroscopy; Glucosides; Lipid Bilayers; Membranes, Artificial; Mesylates; Micelles; Protein Conformation; Rhodopsin; Spin Labels
PubMed: 16634635
DOI: 10.1021/bi060101v -
The Journal of Biological Chemistry Dec 1986The effects of phorbol esters, dioctanoylglycerol (DiC8), and micromolar Ca2+ on protein phosphorylation and catecholamine secretion in digitonin-treated chromaffin...
The effects of phorbol esters, dioctanoylglycerol (DiC8), and micromolar Ca2+ on protein phosphorylation and catecholamine secretion in digitonin-treated chromaffin cells were investigated. [gamma-32P]ATP was used as a substrate for phosphorylation in the permeabilized cells. 12-O-Tetradecanoylphorbol-13-acetate (TPA) enhanced Ca2+-dependent catecholamine secretion from digitonin-permeabilized cells. The enhancement required MgATP. Only those phorbol esters which activate protein kinase C in vitro enhanced both catecholamine secretion and protein phosphorylation. DiC8, which activates protein kinase C in vitro and mimics phorbol ester effects in situ, also enhanced both catecholamine secretion and protein phosphorylation. Preincubation of intact cells with TPA or DiC8 was necessary for maximal effects on both catecholamine secretion and protein phosphorylation in subsequently digitonin-treated chromaffin cells. The TPA-induced enhancement of protein phosphorylation was almost entirely Ca2+-independent, whereas DiC8-induced enhancement of protein phosphorylation was mainly Ca2+-dependent. Micromolar Ca2+ alone also enhanced the phosphorylation of a large number of proteins. Most of the proteins phosphorylated in response to TPA or potentiated by DiC8 in combination with Ca2+ were also phosphorylated by micromolar Ca2+ in the absence of exogenous protein kinase C activators. In intact cells, 1,1-dimethyl-4-phenylpiperazinium (DMPP) induced Ca2+-dependent phosphorylation of at least 17 proteins which were detected by two-dimensional gel electrophoresis. All of the proteins phosphorylated upon incubation with 1,1-dimethyl-4-phenylpiperazinium were phosphorylated upon incubation with micromolar Ca2+ in digitonin-treated cells. These results demonstrate that TPA- or DiC8-enhanced Ca2+-dependent catecholamine secretion is associated with enhanced protein phosphorylation which is probably mediated by protein kinase C and that activation of protein kinase C modulates catecholamine secretion from digitonin-treated chromaffin cells.
Topics: Adenosine Triphosphate; Adrenal Medulla; Animals; Calcium; Cattle; Cell Membrane Permeability; Cells, Cultured; Digitonin; Diglycerides; Glycerides; Kinetics; Norepinephrine; Phosphoproteins; Phosphorylation; Proteins; Tetradecanoylphorbol Acetate
PubMed: 3491075
DOI: No ID Found -
Journal of Neurochemistry Jan 1985The relationship between catecholamine secretion and arachidonic acid release from digitonin-treated chromaffin cells was investigated. Digitonin renders permeable the...
The relationship between catecholamine secretion and arachidonic acid release from digitonin-treated chromaffin cells was investigated. Digitonin renders permeable the plasma membranes of bovine adrenal chromaffin cells to Ca2+, ATP, and proteins. Digitonin-treated cells undergo exocytosis of catecholamine in response to micromolar Ca2+ in the medium. The addition of micromolar Ca2+ to digitonin-treated chromaffin cells that had been prelabeled with [3H]arachidonic acid caused a marked increase in the release of [3H]arachidonic acid. The time course of [3H]arachidonic acid release paralleled catecholamine secretion. Although [3H]arachidonic acid release and exocytosis were both activated by free Ca2+ in the micromolar range, the activation of [3H]arachidonic acid release occurred at Ca2+ concentrations slightly lower than those required to activate exocytosis. Pretreatment of the chromaffin cells with N-ethylmaleimide (NEM) or p-bromophenacyl bromide (BPB) resulted in dose-dependent inhibition of 10 microM Ca2+-stimulated [3H]arachidonic acid release and exocytosis. The IC50 of NEM for both [3H]arachidonic acid release and exocytosis was 40 microM. The IC50 of BPB for both events was 25 microM. High concentrations (5-20 mM) of Mg2+ caused inhibition of catecholamine secretion without altering [3H]arachidonic acid release. A phorbol ester that activates protein kinase C, 12-O-tetradecanoylphorbol-13-acetate (TPA), caused enhancement of both [3H]arachidonic acid release and exocytosis. The findings demonstrate that [3H]arachidonic acid release is stimulated during catecholamine secretion from digitonin-treated chromaffin cells and they are consistent with a role for phospholipase A2 in exocytosis from chromaffin cells. Furthermore the data suggest that protein kinase C can modulate both arachidonic acid release and exocytosis.
Topics: Acetophenones; Animals; Arachidonic Acid; Arachidonic Acids; Calcium; Catecholamines; Cattle; Chromaffin Granules; Chromaffin System; Digitonin; Ethylmaleimide; Exocytosis; Lipid Metabolism; Magnesium; Norepinephrine; Phorbols; Tetradecanoylphorbol Acetate
PubMed: 3917291
DOI: 10.1111/j.1471-4159.1985.tb07140.x