Electroporation by using pulsed electric fields with long durations compared with the charging time of the plasma membrane can induce cell fusion or introduce xenomolecules into cells. Nanosecond pulse power technology generates pulses with high‐intensity electric fields, but with such short durations that the charging time of the plasma membrane is not reached, but intracellular membranes are affected. To determine more specifically their effects on cell structure and function, human cells were exposed to high intensity (up to 300 kV/cm) nanosecond (10–300 ns) pulsed electric fields (nsPEF) and were analyzed at the cellular and molecular levels. As the pulse duration decreased, plasma membrane electroporation decreased and appearances of apoptosis markers were delayed. NsPEF induced apoptosis within tens of minutes, depending on the pulse duration. Annexin‐V binding, caspase activation, decreased forward light scatter, and cytochrome c release into the cytoplasm were coincident. Apoptosis was caspase‐ and mitochondria‐dependent but independent of plasma membrane electroporation and thermal changes. The results suggest that with decreasing pulse durations, nsPEF modulate cell signaling from the plasma membrane to intracellular structures and functions. NsPEF technology provides a unique, high‐power, energy‐independent tool to recruit plasma membrane and/or intracellular signaling mechanisms that can delete aberrant cells by apoptosis.

An electrical model for biological cells predicts that for pulses with durations shorter than the charging time of the outer membrane, there is an increasing probability of electric field interactions with intracellular structures. Experimental studies in which human cells were exposed to pulsed electric fields of up to 300-kV/cm amplitude, with durations as short as 10 ns, have confirmed this hypothesis. The observed effects include the breaching of intracellular granule membranes without permanent damage to the cell membrane, abrupt rises in intracellular free calcium levels, and enhanced expression of genes. At increased electric fields, the application of submicrosecond pulses induces apoptosis (programmed cell death) in biological cells, an effect that has been shown to reduce the growth of tumors. Possible applications of the intracellular electroeffect are enhancing gene delivery to the nucleus, controlling cell functions that depend on calcium release (causing cell immobilization), and treating tumors.

Pulse power technology using high intensity (up to 300 kV/cm) nanosecond pulsed electric fields (nsPEF) has been applied for decontamination and amelioration of biofouling, but until now effects on human cells have not been investigated. To analyze structural and functional changes in human cells and solid tumors following exposure to nsPEF, we utilized flow cytometry and immunofluorescence microscopy. We provide further support for the hypothesis that as the pulse duration is decreased, there is a lower incidence of electric field interactions at the plasma membrane and a higher incidence of interactions with intracellular structures. The nsPEF effects are pulse duration/electric field intensity-dependent and energy density- or temperature-independent. We also show that nsPEF induces programmed cell. death (apoptosis) in cultured cells as indicated by cell shrinkage, Annexin-V-FITC binding to phosphatidylserine on intact cells, and caspase activation. Mouse fibrosarcoma tumors exposed to nsPEF exhibit fragmented DNA and reduced tumor growth in a mouse model. These studies show that nsPEF effects are distinctly different than electroporation pulses and provide the first evidence for the potential application of nsPEF to induce apoptosis and inhibit tumor growth.

Progesterone and 17 alpha-hydroxyprogesterone (but not other steroids such as testosterone, corticosterone, beta-estradiol, estrone, dehydroepiandrosterone, 20 alpha-hydroxypregnen-3-one, androstenedione, and pregnenolone) were shown to cause an immediate increase, in free cytosolic calcium ([Ca2+]i) in both capacitated and noncapacitated human sperm, using the fluorescent indicator fura 2. Significant increases in [Ca2+]i were observed with 10 ng/ml progesterone, while maximum effects were seen with 1 microgram/ml progesterone. Two other steroids 11 beta-hydroxyprogesterone and 5 alpha-pregnane-3,20-dione exhibited significant activity to increase [Ca2+]i. This increase in [Ca2+]i elicited by progesterone was entirely due to Ca2+ influx from the extracellular medium since the increase in [Ca2+]i was blocked by the Ca2+ chelator EGTA (2.5 mM) and the Ca2+ channel antagonist La3+ (0.25 mM) when added to the medium containing 2.5 mM Ca2+. Progesterone also stimulated the uptake of Mn2+ into sperm as measured by the quenching of fura 2 fluorescence. Progesterone has been found in human follicular fluid at levels capable of stimulating increases in [Ca2+]i. The similarities in responses induced by human follicular fluid and progesterone an increase in [Ca2+]i, and hence the acrosome reaction, is progesterone and/or 17 alpha-hydroxyprogesterone. Progesterone (1 microgram/ml) did not increase [Ca2+]i in somatic cells such as adipocytes, hepatocytes, Balb/c 3T3 cells, normal rat kidney, or DDT1 MF-2 cells. The effects of these progestins to increase [Ca2+]i, by activating a receptor-operated calcium channel, is the first report of such an activity in sperm. This phenomena possibly opens up a new field of steroid action in the area of sterility, fertility, and contraception at the level of the sperm.