Ultrafast Ca2+ wave in cultured vascular smooth muscle cells aligned on a micropatterned surface.

Communication between vascular smooth muscle cells (SMCs) allows control of their contraction and so regulation of blood flow. The contractile state of SMCs is regulated by cytosolic Ca2+ concentration ([Ca2+]i) which propagates as Ca2+ waves over a significant distance along the vessel. We have characterized an intercellular ultrafast Ca2+ wave observed in cultured A7r5 cell line and in primary cultured SMCs (pSMCs) from rat mesenteric arteries. This wave, induced by local mechanical or local KCl stimulation, had a velocity around 15 mm/s. Combining of precise alignment of cells with fast Ca2+ imaging and intracellular membrane potential recording, allowed us to analyze rapid [Ca2+]i dynamics and membrane potential events along the network of cells. The rate of [Ca2+]i increase along the network decreased with distance from the stimulation site. Gap junctions or voltage-operated Ca2+ channels (VOCCs) inhibition suppressed the ultrafast Ca2+ wave. Mechanical stimulation induced a membrane depolarization that propagated and that decayed exponentially with distance. Our results demonstrate that an electrotonic spread of membrane depolarization drives a rapid Ca2+ entry from the external medium through VOCCs, modeled as an ultrafast Ca2+ wave. This wave may trigger and drive slower Ca2+ waves observed ex vivo and in vivo.

Red blood cell permeabilization by hypotonic treatments, saponin, and anticancer avicins.

Plasma membrane permeabilization by saponin and anticancer avicins was studied using light dispersion measurements, since high correlation between light dispersion changes and hemolysis has been demonstrated. Nevertheless, we observed that rat red blood cell swelling in moderately hypotonic media was accompanied by up to 20% decrease of light dispersion, when hemolysis was not yet detectable. Avicin G and avicin D were significantly more efficient than saponin in inducing cytotoxicity in PC3 human prostate cancer cells. We found that the preincubation of avicins with the plasma membrane, but not with the cytosolic fraction of previously lysed red blood cells, completely protected fresh cells against permeabilization. The data suggest that the plasma membrane can tightly bind the avicins, but not the saponin. Using the "osmotic protection" method with 100mOsm PEGs of increasing molecular weight in isotonic media, the size of the pores generated by avicin G and avicin D in the plasma membrane was estimated to be higher than the hydrodynamic radius of PEG-8000. The obtained results indicate that the anticancer activity of avicin G and avicin D could be related, at least partially, to their high ability to permeabilize biological membranes. These data might represent interest for possible applications of these anticancer drugs in vivo.

Hemoglobin precipitation by polyethylene glycols leads to underestimation of membrane pore sizes.

The size of pores formed in the plasma membrane by various substances is frequently determined using polyethylene glycols as osmotic protectants. In this work, we have found that the size of pores formed by saponin in the red blood cell membrane determined by hemolysis versus molecular weight of polyethylene glycol was different to that estimated by light dispersion of cell suspensions. After complete swelling of cells induced by saponin in semiisotonic salt media containing 150 mOsm PEG-4000 or PEG-3000, a significant increase in the light absorbance at 640 nm was developed resulting from the formation of hemoglobin precipitates. Easily sedimenting aggregates were also formed when the supernatant of lysed cells was added to the equiosmotic solutions of polyethylene glycols with molecular weight higher than 1000. We suggest that the real size of large pores could be underestimated due to the phenomenon of hemoglobin precipitation by polyethylene glycols.