Peter F. Blackmore

Ultrashort electrical pulses open a new gateway into biological cells

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.

Diverse Effects of Nanosecond Pulsed Electric Fields on Cells and Tissues

The application of pulsed electric fields to cells is extended to include nonthermal pulses with shorter durations (10-300 ns), higher electric fields (< or =350 kV/cm), higher power (gigawatts), and distinct effects (nsPEF) compared to classical electroporation. Here we define effects and explore potential application for nsPEF in biology and medicine. As the pulse duration is decreased below the plasma membrane charging time constant, plasma membrane effects decrease and intracellular effects predominate. NsPEFs induced apoptosis and caspase activation that was calcium-dependent (Jurkat cells) and calcium-independent (HL-60 and Jurkat cells). In mouse B10-2 fibrosarcoma tumors, nsPEFs induced caspase activation and DNA fragmentation ex vivo, and reduced tumor size in vivo. With conditions below thresholds for classical electroporation and apoptosis, nsPEF induced calcium release from intracellular stores and subsequent calcium influx through store-operated channels in the plasma membrane that mimicked purinergic receptor-mediated calcium mobilization. When nsPEF were applied after classical electroporation pulses, GFP reporter gene expression was enhanced above that observed for classical electroporation. These findings indicate that nsPEF extend classical electroporation to include events that primarily affect intracellular structures and functions. Potential applications for nsPEF include inducing apoptosis in cells and tumors, probing signal transduction mechanisms that determine cell fate, and enhancing gene expression.

Nanosecond pulsed electric fields modulate cell function through intracellular signal transduction mechanisms

These studies describe the effects of nanosecond (10-300 ns) pulsed electric fields (nsPEF) on mammalian cell structure and function. As the pulse durations decrease, effects on the plasma membrane (PM) decrease and effects on intracellular signal transduction mechanisms increase. When nsPEF-induced PM electroporation effects occur, they are distinct from classical PM electroporation effects, suggesting unique, nsPEF-induced PM modulations. In HL-60 cells, nsPEF that are well below the threshold for PM electroporation and apoptosis induction induce effects that are similar to purinergic agonistmediated calcium release from intracellular stores, which secondarily initiate capacitive calcium influx through store-operated calcium channels in the PM. NsPEF with durations and electric field intensities that do or do not cause PM electroporation, induce apoptosis in mammalian cells with a well-characterized phenotype typified by externalization of phosphatidylserine on the outer PM and activation of caspase proteases. Treatment of mouse fibrosarcoma tumors with nsPEF also results in apoptosis induction. When Jurkat cells were transfected by electroporation and then treated with nsPEF, green fluorescent protein expression was enhanced compared to electroporation alone. The results indicate that nsPEF activate intracellular mechanisms that can determine cell function and fate, providing an important new tool for probing signal transduction mechanisms that modulate cell structure and function and for potential therapeutic applications for cancer and gene therapy.

Cell surface localization of a novel non-genomic progesterone receptor on the head of human sperm

Cell surface receptors for progesterone were visualized in human sperm using fluorescein isothiocyanate-progesterone 3-(O-carboxymethyl) oxime-bovine serum albumin (FITC prog CMO BSA). The receptors were confined to the head and not the midpiece or tail. FITC prog CMO BSA was also an effective stimulus to elevate intracellular free calcium in human sperm as detected by fura-2 fluorescence. The elevation of intracellular free calcium is a stimulus for the acrosome reaction, a process which is necessary to occur for sperm to fertilize the egg. It is proposed that progesterone, which is present in the female reproductive tract, can bind to progesterone receptors located in the plasma membrane of the sperm head and elicit an influx of Ca2+ into the underlying cytoplasm and or acrosome and induce the acrosome reaction and facilitate fertilization.

Nanosecond pulse electric field (nanopulse): A novel non-ligand agonist for platelet activation

Nanosecond pulse stimulation of a variety of cells produces a wide range of physiological responses (e.g., apoptosis, stimulation of calcium (Ca2+) fluxes, changes in membrane potential). In this study, we investigated the effect of nanosecond pulses, which generate intense electric fields (nsPEFs), on human platelet aggregation, intracellular free Ca2+ ion concentration ([Ca2+]i) and platelet-derived growth factor release. When platelet rich plasma was pulsed with one 300ns pulse with an electric field of 30kV/cm, platelets aggregated and a platelet gel was produced. Platelet aggregation was observed with pulses as low as 7kV/cm with maximum effects seen with approximately 30kV/cm. The increases in intracellular Ca2+ release and Ca2+ influx were dose dependent on the electrical energy density and were maximally stimulated with approximately 30kV/cm. The increases in [Ca2+]i induced by nsPEF were similar to those seen with thapsigargin but not thrombin. We postulate that nsPEF caused Ca2+ to leak out of intracellular Ca2+ stores by a process involving the formation of nanopores in organelle membranes and also caused Ca2+ influx through plasma membrane nanopores. We conclude that nsPEFs dose-dependently cause platelets to rapidly aggregate, like other platelet agonists, and this is most likely initiated by the nsPEFs increasing [Ca2+]i, however by a different mechanism.

trans-Resveratrol inhibits calcium influx in thrombin-stimulated human platelets

The phytoestrogenic compound trans‐resveratrol (trans‐3,5,4′‐trihydroxystilbene) is found in appreciable quantities in grape skins and wine. It has been shown that both products rich in trans‐resveratrol and pure trans‐resveratrol inhibit platelet aggregation both in vivo and in vitro. However the mechanism of this action still remains unknown. An essential component of the aggregation process in platelets is an increase in intracellular free Ca2+ ([Ca2+]i). Ca2+ must enter the cell from the external media through specific and tightly regulated Ca2+ channels in the plasma membrane. The objective of this study was to characterize what effect trans‐resveratrol had on the Ca2+ channels in thrombin stimulated platelets. In this study we showed that trans‐resveratrol immediately inhibited Ca2+ influx in thrombin‐stimulated platelets with an IC50 of 0.5 μM. trans‐Resveratrol at 0.1, 1.0 and 10.0 μM produced 20±6, 37±6 and 57±4% inhibition respectively of the effect of thrombin (0.01 u  ml−1) to increase [Ca2+]i. trans‐Resveratrol also inhibited spontaneous Ba2+ entry into Fura‐2 loaded platelets, with 0.1, 1.0 and 10.0 μM trans‐resveratrol producing 10±5, 30±5 and 50±7% inhibition respectively. This indicated that trans‐resveratrol directly inhibited Ca2+ channel activity in the platelets in the absence of agonist stimulation. trans‐Resveratrol also inhibited thapsigargin‐mediated Ca2+ influx into platelets. This suggests that the store‐operated Ca2+ channels are one of the possible targets of trans‐resveratrol. These channels rely on the emptying of the internal Ca2+ stores to initiate influx of Ca2+ into the cell. The phytoestrogens genistein, daidzein, apigenin and genistein‐glucoside (genistin) produced inhibitory effects against thrombin similar to those seen with trans‐resveratrol. We conclude that trans‐resveratrol is an inhibitor of store‐operated Ca2+ channels in human platelets. This accounts for the ability of trans‐resveratrol to inhibit platelet aggregation induced by thrombin.

Effects of hydrogen peroxide on human spermatozoa

PurposeReactive oxygen species (ROS) have been reported widely to cause deleterious effects on sperm viability and function due to peroxidation of membrane lipids. However, their action appears more selective at low concentrations; recent evidence indicates that the superoxide anion can promote capacitation and induce hyperactivated motility (HA) in human spermatozoa and that hydrogen peroxide (H2O2)may participate in capacitation of hamster spermatozoa. The objective of these studies was to investigate the direct effects of H2O2on functions crucial to fertilization in human spermatozoa.MethodsIn these prospective studies, we examined the dose-and time-dependent effects of H2O2on sperm membrane-mediated events (binding to the zona pellucida and changes in intracelłular calcium concentration [Ca2+]i,motility patterns, and acrosome reaction). Sperm from fertile donors were used in the experiments under capacitating conditions after separation of the motile fraction by wash/swim-up. [Ca2+]iwas measured by the fluorescent fura-2 indicator, and sperm-zona pellucida binding was assessed with the hemizona assay (HZA). Hyperactivated motility was evaluated by computerized analysis, and the percentage of acrosomereacted sperm was detected by FITC-Pisum sativumlectin and indirect immunofluorescence.ResultsIn the HZA, H2O2did not influence sperm-zona pellucida binding at low concentrations (0.05 mMand 0.1 mM),but significantly reduced binding at 0.2 mM (P<0.004 vs controls). H2O2significantly decreased HA in a dose-dependent manner (P<0.0001) and had a significant effect (P<0.01) on acrosome reaction (stimulatory effect at 0.01 mM).H2O2did not affect basal[Ca2+]i;however, H2O2 (0.1mMthrough 10 mM)decreased the initial phase of progesterone-induced (P4: 1 μM)enhancement of [Ca2+]iin a dose-and time-dependent fashion. Preincubation of sperm with catalase (20 μg/ml) potentiated the P4-induced increase of [Ca2+]i.H2O2did not significantly modify [Ca2+]iincrease in response to inomycin (10 μM ).ConclusionsThese experiments show that H2O2directly affects sperm functions crucial to fertilization in a dose-and time-dependent fashion. Low concentrations maintain capacitation, whereas higher concentrations have deleterious effects, as determined by the end points of the capacitation process. The latter effects are probably dependent on modifications of plasma membrane and intraceliular homeostasis by the oxidative process.

Rapid non-genomic actions of progesterone stimulate Ca2+ influx and the acrosome reaction in human sperm

This review summarizes some recent findings in human sperm which show that progesterone and 17 alpha hydroxyprogesterone are able rapidly (within seconds) to elevate [Ca2+]i and elicit the acrosome reaction (AR) via a non-genomic cell surface receptor. Progesterone promotes a transient elevation in [Ca2+]i which is blocked by extracellular La3+ and Ni2+ and removal of extracellular Ca2+ following chelation with EGTA. Some studies suggest that polyamines, trypsin-like proteases, and progesterone receptor aggregation are involved in progesterone-induced Ca2+ influx and AR. The receptor is not stimulated by the potent synthetic progestigins (e.g. promegestone, norethynodrel, megestrol acetate, cyproterone acetate) and is weakly antagonized by the genomic anti-progestins RU 486 and ZK 98.299. The sedative-hypnotic 3 alpha hydroxyl A-ring reduced pregnane steroids, which are powerful activators of the GABAA Cl- channel, are weak activators of Ca2+ influx and the AR. These data suggest that human sperm have a cell surface steroid receptor which is unlike the genomic progesterone receptor and the GABAA Cl- channel steroid receptor.

Regulation of intracellular calcium concentration by nanosecond pulsed electric fields

Changes in [Ca(2+)](i) response of individual Jurkat cells to nanosecond pulsed electric fields (nsPEFs) of 60 ns and field strengths of 25, 50, and 100 kV/cm were investigated. The magnitude of the nsPEF-induced rise in [Ca(2+)](i) was dependent on the electric field strength. With 25 and 50 kV/cm, the [Ca(2+)](i) response was due to the release of Ca(2+) from intracellular stores and occurred in less than 18 ms. With 100 kV/cm, the increase in [Ca(2+)](i) was due to both internal release and to influx across the plasma membrane. Spontaneous changes in [Ca(2+)](i) exhibited a more gradual increase over several seconds. The initial, pulse-induced [Ca(2+)](i) response initiates at the poles of the cell with respect to electrode placement and co-localizes with the endoplasmic reticulum. The results suggest that nsPEFs target both the plasma membrane and subcellular membranes and that one of the mechanisms for Ca(2+) release may be due to nanopore formation in the endoplasmic reticulum.

Blockade of hepatic α-adrenergic receptors and responses by chlorpromazine and trifluoperazine

There are many physiological processes in which the calcium-dependent regulator protein calmodulin seems to play an important role [l]. Indeed the protein has been proposed to be ‘a universal receptor of the Ca*+ signal’ in cells. According to this scheme, Ca*+ first binds to calmodulin causing a conformational change. The active conformer of calmodulin then binds to certain enzymes with resultant changes in conformation and activity [ 11. One experimental approach which has been used to show if calmodulin is involved in a particular metabolic process is to examine the effects of antipsychotic phenothiazine drugs such as chlorpromazine and trifluoperazine, which bind to calmodulin and prevent its activation by Ca*+ [2]. However,it has been known for a long time that these drugs also have local anesthetic (>lO-’ M), cholinergic blocking, and a-adrenergic blocking activities [3]. In particular, radioligand binding studies have shown that chlorpromazine and trifluoperazine displace [3H]epinephrine, [3H]norepinephrine, [3H]clonidine and [3H]WB-4101 from brain a-adrenergic receptors [4-61. Hence, studies of the inhibitory effects of these phenothiazines on the actions of ar-adrenergic agonists in tissues should not be interpreted as indicating the involvement of calmodulin in ol-adrenergic action without additional information such as that presented below. However, three examples of such interpretation have appeared [7-91. Another illustration of the need for caution in the use of pharmacological agents to elucidate hormone