Despite earlier notions that intracellular pH (pHi) was invariant with time, recent studies have documented pHi changes of from 0.1 to 1.6 U during metabolic and developmental transitions in a variety of cells. Here we review the evidence for pHi-mediated regulation of gamete activation, cellular dormancy, the cell cycle, and stimulus-response coupling. Intracellular Ca2+ level changes also accompany many of these same transitions, and mounting evidence suggests that pHi and Ca2+ changes can be interdependent, both in their mechanisms and their effects. Although the significance of such interactions is still largely unclear, one example--the pronounced pH dependence of Ca2+ binding by calmodulin--suggests their potential importance in metabolic regulation. Similar evidence suggests that pHi changes also influence intracellular adenosine 3',5'-cyclic monophosphate levels, and vice versa. Finally we show that changes in adenylate energy charge can significantly alter pHi. In light of these interactions--and because pHi, unlike most other effectors, does not require specialized receptors--we suggest that pHi functions as a synergistic messenger, providing a metabolic context within and through which the actions of other effectors are integrated.

We have discovered a new, drug-free therapy for treating solid skin tumors. Pulsed electric fields greater than 20 kV/cm with rise times of 30 ns and durations of 300 ns penetrate into the interior of tumor cells and cause tumor cell nuclei to rapidly shrink and tumor blood flow to stop. Melanomas shrink by 90% within two weeks following a cumulative field exposure time of 120 μs. A second treatment at this time can result in complete remission. This new technique provides a highly localized targeting of tumor cells with only minor effects on overlying skin. Each pulse deposits 0.2 J and 100 pulses increase the temperature of the treated region by only 3 °C, ten degrees lower than the minimum temperature for hyperthermia effects. KeywordsSkin cancer; Cancer therapy; Tumor; Pulsed electric fields; Pyknosis; Inhibiting angiogenesis; DNA; Nucleus Electric fields have been employed in several different types of cancer therapy. Some of these involve radiofrequency or microwave devices that heat the tumor to greater than 43 °C to kill the cells via hyperthermia [1,2]. Others use pulsed electric fields to permeabilize the tumor cells to allow the introduction of toxic drugs or DNA [3][4][5]. We have discovered that ultrashort electrical pulses can be used as a purely electrical cancer therapy that kills tumors without hyperthermia or drugs. Previous work from this laboratory found that fibrosarcoma tumors treated in vivo with ten 300 ns pulses exhibited a reduced growth rate compared to control tumors in the same animal [6]. Here, we report that when melanoma tumors are treated with four hundred of these pulses, tumors shrink by 90% within two weeks and a subsequent treatment can result in complete remission.The main characteristics of these nanosecond pulsed electric fields (nsPEF) are their low energy that leads to very little heat production and their ability to penetrate into the cell to permeabilize intracellular organelles [7,8] and release calcium [9][10][11] from the endoplasmic reticulum [11]. They provide a new approach for physically targeting intracellular organelles with many applications, including the initiation of apoptosis in cultured cells [12][13][14] and tumors [6], enhancement of gene transfection efficiency [13,14], and inhibiting tumor growth [6]. During the past year, we have treated over 300 murine melanomas in 120 mice with 40 kV/cm electric field pulses 300 ns in duration with dramatic results. Every tumor exposed to 400 such pulses exhibits rapid pyknosis and reduced blood flow and shrinks by an average of 90% within two The efficacy of this nsPEF treatment depends on two separate electric field parameters: pulse duration and amplitude. The effect of pulse duration can be understood by considering the process of membrane charging when the cell is placed in an electric field. Ions in the cell interior will respond to the electric field by moving in the field direction and charging the highly resistive membrane until they experience no further force. By definition this will only occur ...

We describe a vibrating probe system for measuring relatively steady electrical current densities near individual living cells. It has a signal-to-noise ratio at least 100 times greater than previously available techniques. Thus it can be used to detect current densities as small as 10 n A / c m ~ in serum when a 30-/~m diameter probe is vibrated at 200 Hz between two points 30 #m apart, and the amplifier's time constant is set at 10 s. Moreover, it should be generally insensitive to interference by concentration gradients. It has been first used to reveal and study 100-s long current pulses which developing fucoid embryos drive through themselves.

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Electrical models for biological cells predict that reducing the duration of applied electrical pulses to values below the charging time of the outer cell membrane (which is on the order of 100 ns for mammalian cells) causes a strong increase in the probability of electric field interactions with intracellular structures due to displacement currents. For electric field amplitudes exceeding MV/m, such pulses are also expected to allow access to the cell interior through conduction currents flowing through the permeabilized plasma membrane. In both cases, limiting the duration of the electrical pulses to nanoseconds ensures only nonthermal interactions of the electric field with subcellular structures. This intracellular access allows the manipulation of cell functions. Experimental studies, in which human cells were exposed to pulsed electric fields of up to 30 MV/m amplitude with durations as short as 3 ns, have confirmed this hypothesis and have shown that it is possible to selectively alter the behavior and/or survival of cells. Observed nanosecond pulsed effects at moderate electric fields include intracellular release of calcium and enhanced gene expression, which could have long term implications on cell behavior and function. At increased electric fields, the application of nanosecond pulses induces a type of programmed cell death, apoptosis, in biological cells. Cell survival studies with 10 ns pulses have shown that the viability of the cells scales inversely with the electrical energy density, which is similar to the "dose" effect caused by ionizing radiation. On the other hand, there is experimental evidence that, for pulses of varying durations, the onset of a range of observed biological effects is determined by the electrical charge that is transferred to the cell membrane during pulsing. This leads to a similarity law for nanosecond pulse effects, with the product of electric field intensity, pulse duration, and the square root of the number of pulses as the similarity parameter. The similarity law allows one not only to predict cell viability based on pulse parameters, but has also been shown to be applicable for inducing platelet aggregation, an effect which is triggered by internal calcium release. Applications for nanosecond pulse effects cover a wide range: from a rather simple use as preventing biofouling in cooling water systems, to advanced medical applications, such as gene therapy and tumor treatment. Results of this continuing research are leading to the development of wound healing and skin cancer treatments, which are discussed in some detail.

Epithelial layers in developing embryos are known to drive ion currents through themselves that will, in turn, generate small electric fields within the embryo . We hypothesized that the movement of migratory embryonic cells might be guided by such fields, and report here that embryonic quail somite fibroblast motility can be strongly influenced by small DC electric fields . These cells responded to such fields in three ways: (a) The cells migrated towards the cathodal end of the field by extending lamellipodia in that direction . The threshold field strength for this galvanotaxis was between 1 and 10 mV/mm when the cells were cultured in plasma. (b) The cells oriented their long axes perpendicular to the field lines. The threshold field strength for this response for a 90-min interval in the field was 150 mV/mm in F12 medium and between 50 and 100 mV/mm in plasma. (c) The cells elongated under the influence of field strengths of 400 mV/mm and greater . These fibroblasts were therefore able to detect a voltage gradient at least as low as 0.2 mV across their width. Electric fields of at least 10-fold larger in magnitude than this threshold field have been detected in vivo in at least one vertebrate thus far, so we believe that these field effects encompass a physiological range .