| 1. |
- Hugonin, Loïc, et al.
(författare)
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Calcium influx into phospholipid vesicles caused by dynorphin neuropeptides
- 2008
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Ingår i: Biochimica et Biophysica Acta - Biomembranes. - : Elsevier BV. - 0005-2736 .- 1879-2642 .- 0006-3002. ; 1778:5, s. 1267-1273
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Tidskriftsartikel (refereegranskat)abstract
- Dynorphins, endogeneous opioid peptides, function as ligands to the opioid kappa receptors but also induce non-opioid excitotoxic effects. Dynorphin A can increase the intra-neuronal calcium concentration through a non-opioid and non-NMDA mechanism. In this investigation, we show that big dynorphin, dynorphin A and to some extent dynorphin A (1-13), but not dynorphin B, allow calcium to enter into large unilamellar phospholipid vesicles with partly negative headgroups. The effects parallel the previously studied potency of dynorphins to translocate through biological membranes and to cause calcein leakage from large unilamellar phospholipid vesicles. There is no calcium ion influx into vesicles with zwitterionic headgroups. We have also investigated if the dynorphins can translocate through the vesicle membranes and estimated the relative strength of interaction of the peptides with the vesicles by fluorescence resonance energy transfer. The results show that dynorphins do not translocate in this membrane model system. There is a strong electrostatic contribution to the interaction of the peptides with the membrane model system.
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| 2. |
- Marinova, Zoya, et al.
(författare)
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Translocation of dynorphin neuropeptides across the plasma membrane. A putative mechanism of signal transmission.
- 2005
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Ingår i: Journal of Biological Chemistry. - 0021-9258 .- 1083-351X. ; 280:28
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Tidskriftsartikel (refereegranskat)abstract
- Several peptides, including penetratin and Tat, are known to translocate across the plasma membrane. Dynorphin opioid peptides are similar to cell-penetrating peptides in a high content of basic and hydrophobic amino acid residues. We demonstrate that dynorphin A and big dynorphin, consisting of dynorphins A and B, can penetrate into neurons and non-neuronal cells using confocal fluorescence microscopy/immunolabeling. The peptide distribution was characterized by cytoplasmic labeling with minimal signal in the cell nucleus and on the plasma membrane. Translocated peptides were associated with the endoplasmic reticulum but not with the Golgi apparatus or clathrin-coated endocytotic vesicles. Rapid entry of dynorphin A into the cytoplasm of live cells was revealed by fluorescence correlation spectroscopy. The translocation potential of dynorphin A was comparable with that of transportan-10, a prototypical cell-penetrating peptide. A central big dynorphin fragment, which retains all basic amino acids, and dynorphin B did not enter the cells. The latter two peptides interacted with negatively charged phospholipid vesicles similarly to big dynorphin and dynorphin A, suggesting that interactions of these peptides with phospholipids in the plasma membrane are not impaired. Translocation was not mediated via opioid receptors. The potential of dynorphins to penetrate into cells correlates with their ability to induce non-opioid effects in animals. Translocation across the plasma membrane may represent a previously unknown mechanism by which dynorphins can signal information to the cell interior.
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| 3. |
- Vukojević, Vladana, et al.
(författare)
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Mu-opioid receptor activation in live cells
- 2008
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Ingår i: The FASEB Journal. - : Wiley. - 0892-6638 .- 1530-6860. ; 22:10, s. 3537-3548
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Tidskriftsartikel (refereegranskat)abstract
- Interaction of the mu-opioid receptor (MOP) with selected ligands was investigated in live cells using advanced imaging by confocal laser scanning microscopy integrated with fluorescence correlation spectroscopy and fluorescence cross-correlation spectroscopy. In PC12 cells stably transformed to express the fluorescently labeled MOP-enhanced green fluorescent protein construct, two pools of MOP were identified that could be discriminated by differences in their lateral mobility in the cell membrane. The majority of MOP receptors (80 +/- 10%) were characterized by a diffusion coefficient D-MOP,D-1 = (4 +/- 2) X 10(-11) m(2) s(-1), compared with the slowly moving fraction, D-MOP,D- 2 = (4 +/- 2) x 10(-12) m(2) s(-1). On stimulation with selected agonists ([D-Ala(2),N-MePhe(4),Gly-ol(5)]enkephalin, enkephalin-heptapeptide Tyr-Gly-Gly-Phe-Met-Arg-Phe, morphine, and methadone), surface density of the MOP decreased, whereas the lateral mobility increased. In contrast, antagonists (naloxone and naltrexone) "froze" the receptor in the membrane, i.e., increased MOP surface density and decreased lateral mobility. Agonist activation was also accompanied by pronounced changes in the dynamics of plasma membrane lipids, as revealed by the general lipid marker 1,1'-dioctadecyl-3,3,3',3'-tetramethylindo-carbocyanine perchlorate dye. The results provide new information about MOP activation in live cells at the molecular level, with a special focus on the dynamics of the intricate interplay between this receptor and the surrounding lipids.
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