Membrane action of polyhexamethylene guanidine hydrochloride revealed on smooth muscle cells, nerve tissue and rat blood platelets: A biocide driven pore-formation in phospholipid bilayers.

A well-known cationic biocide of guanidine polymer family, polyhexamethylene guanidine hydrochloride (PHMG) has been tested against smooth muscle cells isolated from swine myometrium, synaptosomes of rat brain nerve terminals and rat blood platelets for the membrane action. It was established that PHMG blocked the activity of Na+,K+-ATPase of smooth muscle cells plasma membrane by 82.2 ±â€¯0.9% at a concentration of 7 ppm, whilst a dose-dependent depolarization of synaptosomes and platelets became appreciable at 100-500 ppm. Comparative studies by the methods of mass spectrometry (MALDI-TOF and PDMS-TOF), viscosimetry, dynamic light scattering and model phospholipid membranes revealed PHMG oligomers with various number of repeat units (8-16) that formed K+-selective potential-dependent pores in sterol-free phosphatidylethanolamine-containing phospholipid bilayers at a concentration of 1 ppm. Obtained results suggest that besides acidic lipids and membrane proteins phosphatidylethanolamine and cholesterol are the other major factors responsible for the differences between PHMG-induced plasma membrane depolarization of microbial and eukaryotic cells and thus, diverse modes of PHMG membrane action.

The phenomenon of synaptic vesicle clustering as the prefusion state in the model system of exocytosis.

Our findings concern to the synaptic vesicle interactions that were reconstructed in the cell-free system and are thought to represent the different states of exocytosis pathway. The combination of different technical approaches allowed to study the features of aggregation and calcium-dependent homotypic fusion of synaptic vesicles. Electron microscopy observations of synaptic vesicle fraction purified from the rat brain showed the appearance of large particles formed by aggregated synaptic vesicles in the presence of the nerve terminal cytosolic proteins only. This data were confirmed by dynamic light scattering measurements indicating an importance of the cytosolic proteins for the formation of synaptic vesicle clusters. The scanning confocal microscopy and imaginative exploitation of fluorescence probe R18 allowed to distinguish the process of synaptic vesicle clustering from the synaptic vesicle fusion. The stimulating effect of antiepileptic drug, ethosuximide and sodium valproate on the formation of synaptic vesicle aggregates has been revealed. Experiments with the removal of cholesterol showed that such modification of synaptic vesicle membranes did not change the ability of synaptic vesicles to form the clusters, reducing their Ca2+-triggered membrane fusion. Thus, our data have shown that aggregated state of synaptic vesicles represent an intermediate stage of the fusion pathway, where aggregation of synaptic vesicles is preceded by Ca2+-dependent membrane fusion.