A Note of Caution: Gramicidin Affects Signaling Pathways Independently of Its Effects on Plasma Membrane Conductance.

Gramicidin is a thoroughly studied cation ionophore widely used to experimentally manipulate the plasma membrane potential (PMP). In addition, it has been established that the drug, due to its hydrophobic nature, is capable of affecting the organization of membrane lipids. We have previously shown that modifications in the plasma membrane potential of epithelial cells in culture determine reorganizations of the cytoskeleton. To elucidate the molecular mechanisms involved, we explored the effects of PMP depolarization on some putative signaling intermediates. In the course of these studies, we came across some results that could not be interpreted in terms of the properties of gramicidin as an ionic channel. The purpose of the present work is to communicate these results and, in general, to draw attention to the fact that gramicidin effects can be misleadingly attributed to its ionic or electrical properties. In addition, this work also contributes with some novel findings of the modifications provoked on the signaling intermediates by PMP depolarization and hyperpolarization.

Nature of the neurotoxic membrane actions of amyloid-ß on hippocampal neurons in Alzheimer's disease.

The mechanism by which amyloid-ß (Aß) produces brain dysfunction in patients with Alzheimer's disease is largely unknown. According to previous studies, Aß might share perforating properties with gramicidin, a well-accepted membrane-disrupting peptide. Therefore, we hypothesize that the key steps leading to synaptotoxicity by Aß and gramicidin involve peptide aggregation, pore formation, and calcium dysregulation. Here, we show that Aß and gramicidin form aggregates enriched in ß-sheet structures using electron microscopy, and Thioflavin and Congo Red staining techniques. Also, we found that Aß and gramicidin display fairly similar actions in hippocampal cell membranes, i.e. inducing Ca(2+) entry and synaptoxicity characterized by the loss of synaptic proteins and a decrease in neuronal viability. These effects were not observed in a Ca(2+) free solution, indicating that both Aß and gramicidin induce neurotoxicity by a Ca(2+)-dependent mechanism. Using combined perforated patch clamp and imaging recordings, we found that only Aß produced a perforation that progressed from a small (Cl(-)-selective pore) to a larger perforation that allowed the entry of fluorescent molecules. Therefore, based on these results, we propose that the perforation at the plasma membrane by Aß is a dynamic process that is critical in producing neurotoxicity similar to that found in the brains of AD patients.

Interaction of gramicidin with DPPC/DODAB bilayer fragments.

The interaction between the antimicrobial peptide gramicidin (Gr) and dipalmitoylphosphatidylcholine (DPPC)/dioctadecyldimethylammonium bromide (DODAB) 1:1 large unilamellar vesicles (LVs) or bilayer fragments (BFs) was evaluated by means of several techniques. The major methods were: 1) Gr intrinsic fluorescence and circular dichroism (CD) spectroscopy; 2) dynamic light scattering for sizing and zeta-potential analysis; 3) determination of the bilayer phase transition from extrinsic fluorescence of bilayer probes; 4) pictures of the dispersions for evaluation of coloidal stability over a range of time and NaCl concentration. For Gr in LVs, the Gr dimeric channel conformation is suggested from: 1) CD and intrinsic fluorescence spectra similar to those in trifluoroethanol (TFE); 2) KCl or glucose permeation through the LVs/Gr bilayer. For Gr in BFs, the intertwined dimeric, non-channel Gr conformation is evidenced by CD and intrinsic fluorescence spectra similar to those in ethanol. Both LVs and BFs shield Gr tryptophans against quenching by acrylamide but the Stern-Volmer quenching constant was slightly higher for Gr in BFs confirming that the peptide is more exposed to the water phase in BFs than in LVs. The DPPC/DODAB/Gr supramolecular assemblies may predict the behavior of other antimicrobial peptides in assemblies with lipids.

Water permeability properties of the ovarian oocytes from Bufo arenarum and Xenopus laevis: a comparative study.

The water permeability properties of ovarian oocytes from Xenopus laevis and Bufo arenarum, a toad species found in the Buenos Aires region, were studied. We report that: (i) the water osmotic permeability (Pf, cm/sec x 10(-4)) was significantly higher in Bufo (6 degrees C = 12.3 +/- 2.4; 18 degrees C = 20.8 +/- 4.8) than in Xenopus oocytes (6 degrees C = 5.3 +/- 0.3; 18 degrees C = 6.2 +/- 1.6). The corresponding water diffusion permeability values (Pd, cm/sec x 10(-4)) were: Xenopus = 2.3 +/- 0.3 (6 degrees C) and 4.8 +/- 0.7 (18 degrees C); Bufo = 2.7 +/- 0.4 (6 degrees C) and 6.0 +/- 0.5 (18 degrees C). (ii) Amphotericin B increased the Pf and Pd values. The observed delta Pf/delta Pd ratio was not significantly different from the expected results (n = 3), after amphotericin B incorporation in both species. This means that the influence of unstirred layers and other potential artifactual compounds did not significantly affect our experimental results. (iii) Preincubation with gramicidin during 12 hr induced a clear increase in the oocyte volume. After that, a hypotonic shock only slightly increased the oocyte volume. Conversely, a hypertonic challenge induced a volume change significantly higher than the one observed in control conditions. (iv) Mercury ions did not affect the osmotic permeability in Xenopus oocytes but clearly inhibited, in a reversible way, the osmotic permeability in oocytes from B. arenarum. (v) Mercury ions did not reduce Pd values in either species.(ABSTRACT TRUNCATED AT 250 WORDS)