Richard Nuccitelli

A Practical Guide to the Preparation of Ca2+ Buffers

Calcium (Ca(2+)) is a critical regulator of an immense array of biological processes, and the intracellular [Ca(2+)] that regulates these processes is ~ 10,000 lower than the extracellular [Ca(2+)]. To study and understand these myriad Ca(2+)-dependent functions requires control and measurement of [Ca(2+)] in the nano- to micromolar range (where contaminating Ca(2+) is a significant problem). As with pH, it is often essential to use Ca(2+) buffers to control free [Ca(2+)] at the desired biologically relevant concentrations. Fortunately, there are numerous available Ca(2+) buffers with different affinities that make this practical. However, there are numerous caveats with respect to making these solutions appropriately with known Ca(2+) buffers. These include pH dependence, selectivity for Ca(2+) (e.g., vs. Mg(2+)), ionic strength and temperature dependence, and complex multiple equilibria that occur in physiologically relevant solutions. Here we discuss some basic principles of Ca(2+) buffering with respect to some of these caveats and provide practical tools (including freely downloadable computer programs) to help in the making and calibration of Ca(2+)-buffered solutions for a wide array of biological applications.

Chapter 1 - A Practical Guide to the Preparation of Ca2 + Buffers

Calcium (Ca2+) is a critical regulator of an immense array of biological processes, and the intracellular [Ca2+] that regulates these processes is ~ 10,000 lower than the extracellular [Ca2+]. To study and understand these myriad Ca2+-dependent functions requires control and measurement of [Ca2+] in the nano- to micromolar range (where contaminating Ca2+ is a significant problem). As with pH, it is often essential to use Ca2+ buffers to control free [Ca2+] at the desired biologically relevant concentrations. Fortunately, there are numerous available Ca2+ buffers with different affinities that make this practical. However, there are numerous caveats with respect to making these solutions appropriately with known Ca2+ buffers. These include pH dependence, selectivity for Ca2+ (e.g., vs. Mg2+), ionic strength and temperature dependence, and complex multiple equilibria that occur in physiologically relevant solutions. Here we discuss some basic principles of Ca2+ buffering with respect to some of these caveats and provide practical tools (including freely downloadable computer programs) to help in the making and calibration of Ca2+-buffered solutions for a wide array of biological applications.

A Role for Endogenous Electric Fields in Wound Healing

This review focuses on the experimental evidence supporting a role for endogenous electric fields in wound healing in vertebrates. Most wounds involve the disruption of epithelial layers composing the epidermis or surrounding organs in the body. These epithelia generate a steady voltage across themselves that will drive an injury current out of the wounded region, generating a lateral electric field that has been measured in four different cases to be 40-200 mV/mm. Many epithelial cells, including human keratinocytes, have the ability to detect electric fields of this magnitude and respond with directed migration. Their response typically requires Ca2+ influx, the presence of specific growth factors and intracellular kinase activity. Protein kinase C is required by neural crest cells and cAMP-dependent protein kinase is used in keratinocytes while mitogen-activated protein kinase is required by corneal epithelial cells. Several recent experiments support a role for electric fields in the stimulation of wound healing in the developing frog neurula, adult newt skin and adult mammalian cornea. Some experiments indicate that when the electric field is removed the wound healing rate is 25% slower. In addition, nearly every clinical trial using electric fields to stimulate healing in mammalian wounds reports a significant increase in the rate of healing from 13 to 50%. However, these trials have utilized many different field strengths and polarities, so much work is needed to optimize this approach for the treatment of mammalian wounds.

Activation of protein kinase C triggers premature compaction in the four-cell stage mouse embryo.

During mouse preimplantation development, the cells of the mouse embryo undergo a progressive subcellular reorganization at compaction, which eventually results in the formation of two distinct cell types. We have investigated the effect that activators of the Ca2(+)-phospholipid-dependent protein kinase (PKC) have on mouse compaction. Phorbol ester activation of PKC caused premature compaction of four-cell embryos within a few minutes of addition followed by a prolonged decompaction phase after 1 hr. This response was dose-dependent to concentrations as low as 250 pg/ml. Diacylglycerides also caused compaction; however, it was more sustained than with phorbol esters and was not followed by a phase of decompaction. Inhibition of PKC with sphingosine blocks induced compaction in a dose-dependent manner and also blocks normal compaction of eight-cell embryos. A monoclonal antibody to the cell adhesion molecule, E-cadherin, which mediates mouse embryo compaction, completely blocks compaction induced by these activators of PKC. Indirect immunofluorescence with a monoclonal antibody to E-cadherin indicates that PKC activation causes a rapid shift in the localization of this cell adhesion molecule, which coincides with the observed compaction. These results suggest that PKC plays a role in the initiation of compaction through its effect either directly or indirectly on E-cadherin.

LOCAL CATION ENTRY AND SELF‐ELECTROPHORESIS AS AN INTRACELLULAR LOCALIZATION MECHANISM*

We are concerned with the mechanisms of intracellular localization that contribute to development. How, for example, does an ameboid cell form a protrusion at one point and not a t another? How does a neuron initiate an outgrowth a t one point and not a t another? How is a neurite’s continued growth oriented? How does a plant egg or spore initiate an outgrowth at one point and not a t another? How are “vegetal” materials localized in one end of an animal egg so that it develops into gut, not skin? Genetic mechanisms have proven to have considerable generality; much of what is true of the genetics of bacteria is likewise true of man. Similarly, we expect morphogenetic mechanisms, in particular those of intracellular localization, to have much generality. Therefore, we have focused our study upon the early development of the fucoid egg. Unlike animal eggs, this common seaweed egg has no preformed animal-vegetal axis. The fucoid zygote is essentially apolar. Then, in the course of a day or less, it initiates growth at one pole, visibly polarizes, and divides into two quite different cells: a rhizoid, or attachment, cell a t the growth pole and a thallus cell a t its antipode (FIGURE 1). This first day of the fucoid egg’s development is a prototype of the localization process. We are further focusing our study upon an essentially electrical hypothesis of localization. According to this hypothesis, the plasma membrane in a growth region, or presumptive growth region, becomes relatively leaky to certain cations that are normally a t a much higher electrochemical potential outside of the cell than within it. These cations could include Caz+, MgZ+, Na+, and H+. The resultant movement of these cations into this region constitutes entry of an electrical current. Movement of this cation flux or current through the resistance of the cytoplasm under the leak will necessarily generate a cytoplasmicjeld that is relatively positive under the leaky portion of the membrane. This field will generate movement. It will tend to pull vesicles and other cytoplasmic constituents with a negative surface charge toward the leaky membrane region. This movement, in turn, may act to make the local membrane leakier, which thus provides the last link in apositive feedback loop. This loop would serve to establish and maintain localized growth, expansion, segregation. and other functions. Specifically, this movement could give such feedback by causing fusion of certain vesicles with the plasma membrane if these vesicles were themselves relatively leaky to particular cations or if they thus released substances that made preexisting parts of the membrane leaky. In our view, the mature nerve synapse may serve as a model of this hypothesis,

Oöplasmic segregation and secretion in the Pelvetia egg is accompanied by a membrane-generated electrical current☆

Abstract Using an ultrasensitive extracellular vibrating electrode, I have studied the membrane-generated electrical currents around the egg of the brown alga, Pelvetia, between fertilization and germination. During this period, the egg chooses an elongation axis and moves wall-precursor vesicles to the prospective growth region where they are secreted. This results in visible ooplasmic segregation which appears under the light microscope as a 1- to 2-μm-thick clear band at the cortex of the growth region. A steady electrical current enters a small region of the membrane and leaves the remainder of the eggs surface as early as 30 min after fertilization. This early spatial current pattern is unstable and shifts position, often with more than one inward current region. However, current enters mainly on the side where germination will occur and is usually largest at the prospective cortical clearing region. The average measured early current density is 0.06 μA/cm2 at 50 μm from the eggs surface, implying a surface current density of between 0.2 and 1 μA/cm2 due to the extrapolation uncertainty. At germination the current increases about twofold, resulting in a total transcellular current on the order of 100 pA. Unilateral growth-orienting light reversal stimulates inward current on the new dark side, and subsequent morphological polarity reversal is preceded by electrical polarity reversal. The steady current tends to increase when the external Ca2+ concentration is increased or the external Na+ concentration is decreased, suggesting that the current is carried in part by Ca2+. This current will generate a transcellular electrical field which may be the force driving the observed ooplasmic segregation.

Characterization of the Sperm-Induced Calcium Wave in Xenopus Eggs Using Confocal Microscopy

We have used confocal microscopy to examine the [Ca2+]i increase in the albino eggs of the frog Xenopus laevis after fertilization. Eggs were placed in agar wells with their animal poles downward so that fertilization occurred preferentially in the equatorial plane, and confocal microscopy was used to provide a two-dimensional optical section through the three-dimensional Ca2+ wave. These data indicate that the wave of increased [Ca2+]i traverses the entire egg and converges uniformly on the antipode. We show that ratioing two different fluorescent dyes to correct for variations in cell thickness is not a reliable technique for this very thick cell due to differential absorption with depth. Indo-1-dextran proves to be a more reliable Ca2+ indicator in this respect. Indo-1-dextran measurements indicate that the resting [Ca2+]i is not uniform throughout the egg but exhibits a 15% higher [Ca2+]i in the cortex than deep in the cytoplasm. This difference is accentuated during wave propagation and is not dependent on extracellular Ca2+. The average peak [Ca2+]i in the center of the egg as the wave propagates through it is 0.7 microM, approximately 60% of the peak cortical [Ca2+]i. The wave velocity through the center of the egg (5.7 micron/s) is slower than that in the cortex (8.9 micron/s), and both velocities vary slightly during transit. The cortical wave speed is particularly high at the beginning (15.7 micron/s) and end (17.2 micron/s) of the wave. Eggs injected with 30-80 microM of 3 kD heparin to compete with inositol-1,4,5,-trisphosphate for binding to its receptor exhibited multiple localized spots of elevated [Ca2+]i, and many of these did not initiate a wave. For those that did lead to a wave, it was usually slow moving and exhibited a reduced (60% reduction) amplitude compared with controls.

Optimized nanosecond pulsed electric field therapy can cause murine malignant melanomas to self-destruct with a single treatment.

We have identified a new, nanosecond pulsed electric field (nsPEF) therapy capable of eliminating murine melanomas located in the skin with a single treatment. When these optimized parameters are used, nsPEFs initiate apoptosis without hyperthermia. We have developed new suction electrodes that are compatible with human skin and have applied them to a xenograft nude mouse melanoma model system to identify the optimal field strength, pulse frequency and pulse number for the treatment of murine melanomas. A single treatment using the optimal pulse parameters (2,000 pulses, 100 ns in duration, 30 kV/cm in amplitude at a pulse frequency of 5–7 pulses/sec) eliminated all 17 melanomas treated with those parameters in 4 mice. This was the highest pulse frequency that we could use without raising the treated skin tumor temperature above 40°C. We also demonstrate that the effects of nsPEF therapy are highly localized to only cells located between electrodes and results in very little scarring of the nsPEF‐treated skin.

Embryonic cell motility can be guided by physiological electric fields

Migratory embryonic quail somitic fibroblasts display a striking sensitivity to small, steady electric fields. There are three components to their response. They begin to orient their long axes perpendicular to the field lines within 5 min of current application at the optimal field strength of 600 mV/mm. The threshold field for significant orientation in 90 min is 150 mV/mm (only 3 mV/cell width). The cells migrate toward the cathode with a similar low threshold. At field strengths greater than 400 mV/mm, the cells also elongate beginning about 1 h after field application. The importance of this embryonic cell galvanotaxis and orientation by electric fields lies in the possible utilization of this behavior both by the embryo in the guidance of embryonic cell migration in vivo and by the investigator to control cell morphology and directionality of movement in vitro in order to study mechanisms of motility.

How do sperm activate eggs

Publisher Summary This chapter provides an overview of the recent evidence for involvement of the inositol cycle and G-proteins in egg activation and also thoughts and hypotheses on egg activation mechanisms. This question of how sperm activate eggs has occupied many developmental biologists over the years and has recently received much attention, stimulated by new information on the signal transduction mechanisms in other cell types. A transient intracellular calcium ion concentration increase and a permanent intracellular pH increase accompany egg activation in many species, and the treatments that activate eggs usually generate these ionic changes. Further evidence for the stimulation of phosphatidylinositol monophosphate (PIP), turnover, and Ins(1,4,5)P 3 and diacylglycerol (DAG) production in sea urchin eggs has appeared. These observations suggest that inositol lipid hydrolysis occurs naturally during egg activation; however, the direct evidence for endogenous Ins( 1,4,5)P, release is underwhelming. Another approach that supported the involvement of PIP hydrolysis is a study of the isolated egg cortices measured in vitro . Yet another line of evidence that suggests the involvement of inositol lipid hydrolysis in egg activation is the activating ability of agents that stimulate or activate guanosine triphosphate (GTP)-binding proteins (G-proteins).