Importance of bicarbonate ion for intracellular pH regulation in antigen- and ionomycin-stimulated RBL-2H3 mast cells.

In RBL-2H3 rat basophilic leukemia cells, Ca2+ influx and secretion are activated by antigens that crosslink IgE-receptor complexes and by the Ca2+ ionophore, ionomycin. Here we report that antigen-stimulated Ca2+ influx and secretion are impaired and ionomycin-induced responses are strongly inhibited following the removal of HCO3- from the medium. These results raised the possibility that HCO3(-)-dependent pH regulation mechanisms play a role in the cascade of events leading to mast cell activation. To test this hypothesis, intracellular pH (pHi) was measured by ratio imaging microscopy in individual RBL-2H3 cells labeled with 2',7'-bis-(2-carboxyethyl)-5-(6) carboxyfluorescein (BCECF). In unstimulated cells, it was found that basal pHi in the presence of HCO3- is 7.26, significantly greater than pHi in its absence, 7.09 (P less than 10(-6]. These results, as well as evidence that pHi increases rapidly when HCO3- is added to cells initially incubated in HCO3(-)-free medium, indicate that unstimulated cells use a HCO3(-)-dependent mechanism to maintain cytoplasmic pH. Further analyses comparing unstimulated with stimulated cells showed that antigen causes a small transient acidification in medium containing HCO3- and a larger sustained acidification in HCO3(-)-depleted medium. Ionomycin is a more potent acidifying agent, stimulating a sustained acidification in complete medium and causing further acidification in HCO3(-)-free medium. These results support the hypothesis that the inhibition of antigen- and ionomycin-induced 45Ca2+ influx and secretion in cells incubated in HCO3(-)-free medium is at least partially due to the inactivation of HCO3(-)-dependent mechanisms required to maintain pH in unstimulated cells and to permit pH recovery from stimulus-induced acidification.

Identification of a polypeptide sequence that mediates nuclear localization of the acute phase protein C-reactive protein.

C-reactive protein (CRP) is the prototypic human acute phase serum protein. CRP binds to several nuclear Ag including chromatin, histones, and small nuclear ribonucleoproteins. Binding to sites of tissue inflammation and the nuclei of inflammatory cells has been demonstrated in vivo. We also noticed significant similarity between CRP and nucleoplasmin, a molecule with nuclear localization activity. We therefore decided to test whether CRP was capable of nuclear localization. CRP and the control protein human serum albumin were FITC-conjugated and microinjected into living VERO cells. The cells were incubated at 37 degrees C for 15 min and then examined by fluorescence microscopy. Nuclear localization of CRP but not albumin was rapid and a high nuclear to cytoplasmic ratio was seen, consistent with active nuclear transport. Incubation at reduced temperature inhibited nuclear uptake by CRP. A synthetic peptide, RKSLKK, from the CRP sequence, when coupled to FITC-albumin, also mediated nuclear localization. Nuclear localization of the related protein, serum amyloid P component, was also seen and a homologous nuclear localization signal was identified. Because CRP was previously demonstrated to inhibit RNA transcription and enhance chromatin degradation it is proposed that CRP may play a unique role in injured cells to alter processing of damaged nuclei. Biochemical, structural and sequence comparisons between the CRP/serum amyloid P component family of proteins (pentraxins) and the nucleoplasmin/B23 family of proteins showed regions of sequence homology that may be related to their shared cyclic pentameric structure.

Real-time imaging of single DNA molecules with fluorescence microscopy.

A fluorescence microscopy technique was used to image the dynamics of individual DNA molecules. Lambda, calf thymus, cosmid (circular), and T4 DNA were studied with the fluorescent dye acridine orange. Experiments with DNAase I were conducted, and the results indicate that these observations correspond to DNA molecules. The results of experiments with circular DNA provide strong evidence that these were single DNA molecules. Molecules were observed free in solution or attached to a glass or copper surface at one or several points. The Brownian motion of these molecules was observed, indicating that DNA in solution exists in a partially supercoiled state. Some molecules appeared stretched and were attached to the surface by their termini; the lengths of these molecules were measured. Such molecules also exhibited elastic behavior upon breaking. The power of this technique is demonstrated in images of cosmid DNA molecules, catenanes, and DNA extending from T4 phage particles. These results suggest immediate applications to molecular biology, such as examining the dynamics of protein-DNA interactions. Areas of ongoing research are discussed.

Mapping gold-labeled receptors on cell surfaces by backscattered electron imaging and digital image analysis: studies of the IgE receptor on mast cells.

The response of cells to signaling molecules such as hormones, growth factors, and immune mediators that bind to cell-surface receptors depends in part on the density and distribution of the relevant receptors. We have developed methods to map the distribution of IgE receptors on RBL-2H3 mast cells at high resolution in the scanning electron microscope (SEM). The key elements of our procedure are a new fixative that preserves receptor binding activity; a family of colloidal gold-conjugated probes that bind directly or indirectly to the IgE-receptor complex; an SEM with detectors for both secondary and backscattered electrons (to observe surface topography and gold particles, respectively); and an image processor that can average, digitize, and store these images. Topographical maps are generated by processing and superimposing the digitized images. The methods we describe can be applied to study the density and distribution of any membrane receptor that can be labeled with colloidal gold particles.

An endogenous sodium current may mediate wound healing in Xenopus neurulae.

We have studied healing of Xenopus neurulae in order to investigate the role of endogenous sodium-carried electric currents in wound closure. Transected embryos healed completely within 7 hr in an artificial pond water medium. Wound closure was biphasic, with a rapid purse string-like contraction that was independent of sodium concentration followed by a slower sodium-dependent phase. The initial contraction was reversibly inhibited by cytochalasin B. Healing was prevented when neurulae were wounded and left to heal in sodium-free medium. Healing was also prevented in the presence of amiloride, benzamil, or ouabain, drugs that inhibit sodium flux through the epithelium. The transepithelial potential measured in intact neurulae fell rapidly and reversibly by 70% in response to 10 microM amiloride. Currents measured leaving the wound also decreased by 70% following amiloride addition. Our results indicate that at least one phase of wound healing in Xenopus neurulae is dependent upon an endogenous sodium-carried electric current. We hypothesize that the current may act by guiding epithelial cells to the wound site.

Mapping gold-labeled IgE receptors on mast cells by scanning electron microscopy: receptor distributions revealed by silver enhancement, backscattered electron imaging, and digital image analysis.

Immunogold labeling and silver enhancement techniques are widely used to determine density and distribution of cell membrane receptors by light and transmission electron microscopy. However, these techniques have not been widely used for receptor detection by scanning electron microscopy. We used antigen- or protein A-conjugated colloidal gold particles, together with silver enhancement, sequential secondary and back-scattered electron imaging (SEI and BEI), and digital image processing, to explore cell surface distribution of IgE-receptor complexes on RBL-2H3 cells, a rat leukemia line that provides a model for the study of mucosal mast cells. Cells were first incubated with a monoclonal antidinitrophenol IgE (anti-DNP-IgE) that binds with high affinity to cell surface IgE receptors. The resulting IgE-receptor complexes were cross-linked either with the multivalent antigen, DNP-BSA-gold, or with a polyclonal anti-IgE antibody. Antibody-treated cells were labeled after fixation with protein A-gold. Fixed, gold-labeled cell monolayers were silver enhanced (or not), dehydrated, critical point-dried, and coated with gold-palladium (for SEI analysis) or carbon (for combined SEI/BEI analysis). They were observed in an Hitachi S800 SEM equipped with a field emission tip and a Robinson backscattered electron detector. An image processor (MegaVision 1024XM) digitized images directly from the S800 microscope at 500-1000 line resolution. Silver enhancement significantly improves detection of gold particles in both SEI and BEI modes of SEM. On gold-palladium-coated samples, 20-nm particles are resolved by SEI after enhancement. BEI resolves 15-nm particles without enhancement and 5- or 10-nm particles are resolved by BEI on silver-enhanced, carbon-coated samples. Neither BEI nor SEI alone can yield high resolution topographical maps of receptor distribution (BEI forms images on the basis of atomic number contrast which reveals gold but not surface features). Image analysis techniques were therefore introduced to digitize, enhance, and process BEI and SEI images of the same field of view. The resulting high-contrast, high-resolution images were superimposed, yielding well-resolved maps of the distribution of antigen-IgE-receptor complexes on the surface of RBL-2H3 mast cells. The maps are stored in digital form, as required for computer-based quantitative morphometric analyses. These techniques of silver enhancement, combined BEI/SEI imaging, and digital image analysis can be applied to analyze density and distribution of any gold-labeled ligand on its target cell.

The control of mediator release from RBL-2H3 cells: roles for Ca2+, Na+, and protein kinase C1.

Antigen-stimulated rat basophilic leukemia (RBL-2H3) cells release serotonin and other inflammatory mediators by a process that requires Ca2+ influx and increased cytoplasmic Ca2+ levels, and is mimicked by Ca2+ ionophores. We report here that the Ca2+ response to antigen and to ionomycin has two components, a Ca2+ spike and a Ca2+ plateau. In nominally Ca2+-free medium, both components of the Ca2+ response are inhibited and secretion does not occur. In Na+-free medium, the initial Ca2+ spike induced by antigen or ionomycin occurs, but the plateau is again absent and secretion is inhibited by 30 to 50%. Secretion is also reduced by 10(-4) M amiloride, an inhibitor of Na+ transport pathways, and by 10(-5) M concentrations of two amiloride analogs with greater activity than amiloride, respectively, against Na+ channels and Na+/Ca2+ exchange. Phorbol esters, which stimulate protein kinase C, enhance the Ca2+ plateau and secretion caused by suboptimal amounts of both antigen and ionomycin; this enhancement depends on extracellular Na+. The Na+ ionophore, monensin, mimics the Ca2+ plateau. From these data, we infer that the Ca2+ spike and plateau reflect separate responses of RBL-2H3 cells to antigen or ionomycin. We propose that the Ca2+ plateau results at least in part from the activation of a Na+-dependent Ca2+ influx pathway. One possible mechanism is that antigen binding stimulates a protein kinase C-regulated Na+ transport system. The resulting influx of Na+ may activate a Na+/Ca2+ antiporter that supports the Ca2+ plateau and mediator release.

Heme-linked ionizations of myeloperoxidase detected by Raman difference spectroscopy. A comparison with plant and yeast peroxidases.

The pH-dependence of the oxidation state marker line v4 of human leucocyte myeloperoxidase is determined in the absence of chloride using Raman difference spectroscopy (RDS). A transition in the frequency of v4 with pK of 4.2 +/- 0.3 is found. The pK compares favorably with that previously determined by spectrophotometric titration and kinetic studies. The shift in v4 across the transition is -1.3 cm-1. The shift in v4 and other Raman marker lines indicates enhanced pi charge in the chlorin ring below the transition. The low frequencies of the oxidation state marker lines indicate that a structural change occurs near the chromophore, which results in the formation of a more pi-charge donating protein environment for the chlorin ring at low pH. The Raman results are discussed in terms of a proposed catalytic control mechanism based on charge stabilization of the energy of ring charge-depleted ferryl intermediates of the reaction with peroxide. The myeloperoxidase findings are compared with similar RDS results for ferrous horseradish peroxidase and ferric cytochrome c peroxidase.

Self-generated electrical currents through Xenopus neurulae.

An extracellular vibrating electrode has been used to investigate epithelial current production by Xenopus neurulae. Xenopus neurulae, in 5% DeBoer (DB) solution, stages 14-22, generated an endogenous current which left the blastopores of the embryos, and entered all other locations. The inward current declined near the mid line of the neural groove in stage 15 embryos. The inward current was abolished or drastically reduced by exchange of 5% DB with Na+-free (choline- or bis(2-hydroxyethyl)dimethylammonium (BDAC)-substituted) 5% DB, 5.5 mM-KCl, 1 mM-verapamil in 5% DB, 50 or 500 microM-ouabain in 5% DB, or 1 microM-amiloride in 5% DB. Verapamil (10 microM) in 5% DB, 5.5 mM-NaCl and 1 mM-CoCl2 in 5% DB had little or no effect on the magnitude observed. The current also was present in 5% DB containing Li+ instead of Na+; current carried by Li+ was abolished by amiloride. This is consistent with a Na+ current, similar to that of short-circuited adult frog skin.

Xenopus neural crest cell migration in an applied electrical field.

Xenopus neural crest cells migrated toward the cathode in an applied electrical field of 10 mV/mm or greater. This behavior was observed in relatively isolated cells, as well as in groups of neural crest cells; however, the velocity of directed migration usually declined when a cell made close contact with other cells. Melanocytes with a full complement of evenly distributed melanosomes did not migrate of their own accord, but could be distorted and pulled by unpigmented neural crest cells. Incompletely differentiated melanocytes and melanocytes with aggregated melanosomes displayed the same behavior as undifferentiated neural crest cells, that is, migration toward the cathode. An electrical field of 10 mV/mm corresponded to a voltage drop of less than 1 mV across the diameter of each cell; the outer epithelium of Xenopus embryos drives an endogenous transembryonic current that may produce voltage gradients of nearly this magnitude within high-resistance regions of the embryo. We, therefore, propose that electrical current produced by the skin battery present in these embryos may act as a vector to guide neural crest migration.

Endogenous electrical currents in the water mold Blastocladiella emersonii during growth and sporulation.

We have explored the pattern of electrical currents generated by single cells of the water mold Blastocladiella emersonii at several stages of its life cycle. Extracellular currents were measured with a vibrating probe constructed after the design of Jaffe and Nuccitelli [Jaffe, L. F. & Nuccitelli, R. (1974) J. Cell Biol. 63, 614-628]. In growing cells positive current, of the order of 1 microA/cm2, enters the rhizoid and leaves from the thallus; circumstantial evidence suggests that protons carry much of the current. Sporulation is associated with reversal of the current pattern, such that positive current enters the thallus and leaves from the rhizoidal region; the ions that carry the current have not been identified. These current patterns appear to play a role in the spatial localization of fungal growth and development.

Endogenous electric field around muscle fibres depends on the Na+-K+ pump.

We describe here experiments which reveal a new physiological specialization in the endplate (synaptic) region of skeletal muscle fibres. Using a vibrating microelectrode which can detect small currents flowing in extracellular fluid, we have found that the membrane in the endplate region behaves as though a steady positive current is generated in this location. Current re-enters the fibre in the extrajunctional region. Further experiments show that this current is dependent on the activity of the sodium pump. The electric field created by this current may be important for long-term interactions between muscle and nerve.