Biomedical subjects

C D McCaig

Publications and source records attributed to C D McCaig.

At least 19 recordsLinked to original sources

PubMed1999

A small, physiological electric field orients cell division.

We report on an observation that the orientation of cell division is directed by small, applied electric fields (EFs). Cultured human corneal epithelial cells were exposed to a direct-current EF of physiological magnitude. Cells divided while attached to the culture dish, and most did so with a cleavage plane perpendicular to the EF vector. There are many instances in which cell divisions in vivo occur in the presence of direct-current physiological EF, for example, during embryonic morphogenesis, neuronal and epithelial differentiation, wound healing, or tumor formation. Endogenous physiological EFs may play important roles in some or all of these processes by regulating the axis of cell division and, hence, the positioning of daughter cells.

Cell Division↗

PubMed1999

Electric field-directed cell motility involves up-regulated expression and asymmetric redistribution of the epidermal growth factor receptors and is enhanced by fibronectin and laminin.

Wounding corneal epithelium establishes a laterally oriented, DC electric field (EF). Corneal epithelial cells (CECs) cultured in similar physiological EFs migrate cathodally, but this requires serum growth factors. Migration depends also on the substrate. On fibronectin (FN) or laminin (LAM) substrates in EF, cells migrated faster and more directly cathodally. This also was serum dependent. Epidermal growth factor (EGF) restored cathodal-directed migration in serum-free medium. Therefore, the hypothesis that EGF is a serum constituent underlying both field-directed migration and enhanced migration on ECM molecules was tested. We used immunofluorescence, flow cytometry, and confocal microscopy and report that 1) EF exposure up-regulated the EGF receptor (EGFR); so also did growing cells on substrates of FN or LAM; and 2) EGFRs and actin accumulated in the cathodal-directed half of CECs, within 10 min in EF. The cathodal asymmetry of EGFR and actin staining was correlated, being most marked at the cell-substrate interface and showing similar patterns of asymmetry at various levels through a cell. At the cell-substrate interface, EGFRs and actin frequently colocalized as interdigitated, punctate spots resembling tank tracks. Cathodal accumulation of EGFR and actin did not occur in the absence of serum but were restored by adding ligand to serum-free medium. Inhibition of MAPK, one second messenger engaged by EGF, significantly reduced EF-directed cell migration. Transforming growth factor beta and fibroblast growth factor also restored cathodal-directed cell migration in serum-free medium. However, longer EF exposure was needed to show clear asymmetric distribution of the receptors for transforming growth factor beta and fibroblast growth factor. We propose that up-regulated expression and redistribution of EGFRs underlie cathodal-directed migration of CECs and directed migration induced by EF on FN and LAM.

Actins↗

PubMed1998

The direction of neurite growth in a weak DC electric field depends on the substratum: contributions of adhesivity and net surface charge.

We investigated the influence of the growth surface on the direction of Xenopus spinal neurite growth in the presence of a dc electric field of physiological magnitude. The direction of galvanotropism was determined by the substratum; neurites grew toward the negative electrode (cathode) on untreated Falcon tissue culture plastic or on laminin substrata, which are negatively charged, but neurites growing on polylysine, which is positively charged, turned toward the positive electrode (anode). Growth was oriented randomly on all substrata without an electric field. We tested the hypothesis that the charge of the growth surface was responsible for reversed galvanotropism on polylysine by growing neurons on tissue culture dishes with different net surface charges. Although neurites grew cathodally on both Plastek substrata, the frequency of anodal turning was greater on dishes with a net positive charge (Plastek C) than on those with a net negative charge (Plastek M). The charge of the growth surface therefore influenced the frequency of anodal galvanotropism but a reversal in surface charge was insufficient to reverse galvanotropism completely, possibly because of differences in the relative magnitude of the substratum charge densities. The influence of substratum adhesion on galvanotropism was considered by growing neurites on a range of polylysine concentrations. Growth cone to substratum adhesivity was measured using a blasting assay. Adhesivity and the frequency of anodal turning were graded over the range of polylysine concentrations (0 = 0.1 < 1 < 10 = 100 microg/ml). The direction of neurite growth in an electric field is therefore influenced by both substratum charge and growth cone-to-substratum adhesivity. These data are consistent with the idea that spatial or temporal variation in the expression of adhesion molecules in embryos may interact with naturally occurring electric fields to enhance growth cone pathfinding.

Animals↗

PubMed1997

Physiological electrical fields modify cell behaviour.

Steady direct current (dc) electric fields exist in many biological systems over many hours. At these times cells are dividing, differentiating, moving to final locations and extending motile processes. Each of these events may be influenced by physiological electric fields in tissue culture and when electric fields are disrupted in vivo, major developmental abnormalities arise. The likelihood of physiological electric fields playing a role in cell behaviours and some potential mechanisms are outlined.

Animals↗

PubMed1997

Human corneal epithelial cells reorient and migrate cathodally in a small applied electric field.

PURPOSE: To test whether human corneal epithelial cells (HCECs) respond to small applied electric fields (EFs) in a similar manner to bovine corneal epithelial cells (BCECs), the orientation and directed migration in small EFs of both primary cultures and of a human corneal epithelial cell line were quantified. METHODS: Primary cultures of human corneal epithelial cells (PHCECs) and transformed human corneal epithelial cells (THCECs) were exposed to EFs (100 mV/mm-250 mV/mm) in different media. Cell migration was traced using an image analyser. RESULTS: PHCECs and THCECs reoriented and migrated towards the cathode (negative pole) when cultured in small direct current (dc) EFs. Both the reorientation and directional migration were voltage- and serum-dependent, as shown previously for bovine cells. PHCECs and THCECs showed significant perpendicular orientation in EFs at 150 mV/mm in medium with serum, while at the same voltage, no significant orientation was found in serum free medium. PHCECs started to show perpendicular reorientation around 30 min after onset of EF at 150 mV/mm. They showed significant directional migration at 150 mV/mm, with directedness of 0.35 +/- 0.07 and a migration rate of 9.1 +/- 0.7 microns/h (n = 90), both significantly higher than that of cells in serum free medium. Addition of EGF-induced significant reorientation and directional migration of THCECs at 100 mV/mm. Additionally, as for BCECs, which remained viable and responsive to electric fields for at least 75 h at 150 mV/mm, THCECs also remained viable and showed responsiveness during long periods of exposure to EFs (at least 20 h). CONCLUSIONS: Cultured human primary CECs and a human corneal epithelial cell line both responded to small EFs with perpendicular reorientation and cathodally-directed migration. Cell responses were qualitatively similar to those reported previously for bovine CECs. The endogenous EFs generated by wounded cornea may play an important role in promoting cell shape changes and directed migration of CECs during the healing process.

Animals↗

PubMed1997

Integrated interactions between chondroitin sulphate proteoglycans and weak dc electric fields regulate nerve growth cone guidance in vitro.

During development and regenerative growth, neuronal pathways are defined in part by several endogenous cues that collectively determine directed growth. The interactions between such cues largely are unknown. To address potential interactions, we have examined in vitro the combined effect on nerve growth of two endogenous growth cone guidance cues: chondroitin sulphate proteoglycans and weak dc electric fields. Addition to the culture medium of a chondroitin 6-sulphate/keratan sulphate containing PG (BNC-PG) markedly enhanced the cathodal re-orientation of embryonic Xenopus neurites in an electric field, whereas a proteoglycan containing chondroitin 4-sulphate (RC-PG) was inhibitory. These effects of BNC-PG and RC-PG were reproduced by their chondroitin sulphate glycosaminoglycan side chains alone. Chondroitin 6-sulphate or chondroitin 4-sulphate, respectively, enhanced and inhibited cathodally-directed nerve re-orientation. This was dependent on the integrity of the glycosaminoglycan chain structure; when digested into their disaccharide subunits both molecules became inactive. Keratan sulphate, a minor component of BNC-PG, was found to be inhibitory, whereas dermatan sulphate, an epimer of chondroitin 4-sulphate, had no effect. We conclude that in vitro specific interactions between these two nerve guidance cues do occur and that the specificity of the response is critically dependent on the charge pattern of the proteoglycans chondroitin sulphate side chains. The expression of a host of proteoglycans with differing glycosaminoglycan side chains varies in both time and place in the developing nervous system, thus the scope is vast for spatial and temporal modulation of nerve guidance by interacting cues.

Animals↗

PubMed1996

Lectins implicate specific carbohydrate domains in electric field stimulated nerve growth and guidance.

Both endogenous lectins and DC electric fields may control aspects of early nerve growth and nerve guidance. To test whether such endogenous cues interact, lectins of varying sugar affinity and valency were studied for effects on electric field induced growth and reorientation of cultured Xenopus neurites. Concanavalin A (Con A), succinylated concanavalin A (S-Con A), and wheat germ agglutinin all completely inhibited field-induced cathodal reorientation. Lentil and pea lectins, which share the same sugar affinity as Con A/S-Con A, were only partially effective in inhibiting reorientation. Because S-Con A does not alter lateral mobility of membrane receptors, the previously accepted notion that Con A inhibited field-induced reorientation by preventing receptors from translocating and becoming redistributed asymmetrically in the membrane may be oversimplified. There are likely to be additional steric interactions that Con A and S-Con A share that inactivate asymmetrically redistributed receptors and prevent reorientation. Additionally, nerves growing in an applied field branch more commonly toward the cathode. Con A and S-Con A alone prevented this development of asymmetric branching. All the lectins tested prevented the normal field-induced increase in nerve growth rate, while all, except peanut agglutinin, prevented the usual faster growth cathodally than anodally. We suggest that lectin interactions with electric field effects in vitro may involve modulation of neuronal nicotinic acetylcholine receptors, neurotrophin receptors, or voltage-dependent calcium channels. Similar interactions between endogenous lectins and endogenous electric fields are to be expected.

Animals↗

PubMed1996

Orientation and directed migration of cultured corneal epithelial cells in small electric fields are serum dependent.

Reorientation and migration of cultured bovine corneal epithelial cells (CECs) in an electric field were studied. Electric field application was designed to model the laterally directed, steady direct current electric fields which arise in an injured corneal epithelium. Single cells cultured in media containing 10% foetal bovine serum showed significant galvanotropism, reorienting to lie perpendicular to electric field vector with a threshold field strength of less than 100 mV/mm. Cells cultured in serum-free medium showed no reorientation until 250 mV/mm. Addition of EGF, bFGF or TGF-beta 1 singly or in combination to serum free medium significantly restored the reorientation response at low field strengths. Both the mean translocation rate and directedness of cell migration were serum dependent. Cultured in medium with serum or serum plus added EGF, single cells showed obvious cathodal migration at 100 mV/mm. Increasing electric field strength enhanced the cathodal directedness of single cell migration. Supplementing serum free medium with growth factors restored the cathodal directed migration of single cells and highest directedness was found for the combination of EGF and TGF-beta 1. Corneal epithelial sheets also migrated towards the cathode in electric fields. Serum or individual growth factors stimulated CEC motility (randomly directed). Applied fields did not further augment migration rates but added a vector to stimulated migration. Electric fields which are present in wounded cornea interact with other environmental factors and may impinge on CECs migration during wound healing. Therapies which combine the application of growth factors and electric fields may be useful clinically.

Animals↗

PubMed1996

Directed migration of corneal epithelial sheets in physiological electric fields.

PURPOSE: To characterize the effects of small applied electric fields (EFs) (100 to 250 mV/ mm) on cultured bovine corneal epithelial cell (CEC) sheets and to determine how EFs interact with other environmental cues in directing CEC sheet migration. METHODS: Primary cultures of bovine CECs were exposed to EFs in medium with or without serum, epithelial growth factor, basic fibroblast growth factor, or transforming growth factor-beta 1. Cell sheet migration was traced using an image analyzer. RESULTS: Cell sheets migrated toward the cathode (negative pole). The directional migration was voltage dependent, and, at low field strength (up to 200 mV/mm), it required serum in the medium. Sheets showed no migration responses up to 200 mV/mm in serum-free medium, whereas those in medium with serum showed evident migration toward the cathode, at an average rate of approximately 15 microns/h (n = 15 approximately 20) at 150 mV/mm. When serum was present, the threshold was below 100 mV/mm, very close to the measured wound field strength (approximately 42 mV/mm). After supplementing serum-free medium with individual growth factors or with combinations of epithelial growth factor, basic fibroblast growth factor, and transforming growth factor-beta 1, significant restoration of cathode-directed migration occurred at 150 mV/ mm. Lamellipodia were abundant at the leading edges of migrating sheets, extending the area of sheets covered. The extension of cell membranes toward the cathode was more prominent in cell sheets than in single cells. CONCLUSIONS: The endogenous EFs generated by wounded cornea could play an important role by interacting with other environmental factors to promote changes in shape and in directed migration of CEC sheets.

Animals↗

PubMed1995

The effects of lyotropic anions on electric field-induced guidance of cultured frog nerves.

1. Dissociated Xenopus neurites turn cathodally in small applied electric fields. Increasing the external polycation concentration alters the direction and extent of field-induced orientation. A decrease in membrane surface charge may underlie these effects. 2. Lyotropic anions increase membrane surface charge and we have examined the effect of perchlorate (ClO4-), thiocyanate (SCN-) and sulphate (SO4(2-)) on galvanic nerve orientation. 3. Perchlorate and SCN- had no effect on field-induced cathodal turning, whereas incubation with SO4(2-) was inhibitory. In addition to its effects on surface charge, SO4(2-) increases production of the second messengers diacylglycerol and inositol trisphosphate. Interestingly, lithium (Li+), a blocker of polyphosphoinositide metabolism, had a similar effect to SO4(2-) on field-induced neurite orientation. 4. We conclude that increasing surface charge with lyotropic anions neither enhances galvanotropic orientation nor underlies the inhibitory effects of SO4(2-) and suggest that modulation of galvanotropism by SO4(2-) occurs owing to changes in the inositolphospholipid second messenger system.

Animals↗

PubMed1995

Electric field-directed growth and branching of cultured frog nerves: effects of aminoglycosides and polycations.

The direction and rate of earliest nerve growth are critical determinants of neuronal architecture. One extrinsic cue that influences these parameters is a small direct current electric field, although the underlying mechanisms are unclear. We have studied the orientation, rate of growth, and branching behavior of embryonic Xenopus spinal neurites exposed to aminoglycoside antibiotics, to raised external cations, to applied direct current electric fields, and to combinations of these treatments. Field-induced cathodal turning and cathodal branching of neurites were blocked by the aminoglycosides, by raised extracellular calcium ([Ca2+]0) and by raised extracellular magnesium ([Mg2+]0). Neomycin together with high external Ca2+, by contrast, induced a reversal in the polarity of turning and branching, with neurites reorienting and branching more frequently anodally. Aminoglycosides decreased neurite growth rates, and for neomycin this was partially reversed by high external Ca2+. Raised [Ca2+]0 alone but not raised [Mg2+]0 altered growth rates in a field-strength dependent manner. Modulation of membrane surface charge may underlie altered galvanotropic orientation and branching. Such an effect is insufficient to explain the changes in growth rates, which may result from additional perturbations to Ca2+ influx and inositol phospholipid metabolism.

Animals↗

PubMed1995

Growth cone neurotransmitter receptor activation modulates electric field-guided nerve growth.

We have studied the interactions between two nerve guidance cues, which alone induce substantial growth cone turning: endogenous neurotransmitters and small dc electric fields. d-tubocurarine, a nicotinic AChR (acetylcholine receptor) antagonist, inhibited field-induced cathodal orientation of cultured neurites, whereas atropine, a muscarinic AChR blocker, and suramin, a P2-purinoceptor antagonist, markedly enhanced the guidance properties of the applied field. These experiments implicate the activation of growth cone nicotinic AChRs by self-released acetylcholine in the mechanism underpinning electric field-induced neurite orientation and raise the possibility that growth cones release neurotransmitter prior to target interaction in order to assist their own pathfinding. Additionally, they provide the first evidence that coactivation of several neurotransmitter receptors may interact to regulate directed nerve growth. Such interaction in vivo, where guidance signals coexist, would add further levels of control to neurite guidance.

Animals↗

PubMed1995

Calcium channel subtypes and intracellular calcium stores modulate electric field-stimulated and -oriented nerve growth.

In culture, embryonic spinal neurites from Xenopus laevis show striking growth responses to steady dc electric fields, at a time when endogenous electric fields of similar size impinge on the developing nervous system. A high proportion of cultured neurites reorient, with both turning and branching directed cathodally. Neurite growth rates are increased and growth is differential (faster cathodally than anodally). Voltage-dependent calcium channels and calcium release from intracellular stores are shown to control these events. However, the pharmacological sensitivities of these phenomena differ, indicating different control mechanisms.

Animals↗

PubMed1994

Electric fields induce curved growth of Enterobacter cloacae, Escherichia coli, and Bacillus subtilis cells: implications for mechanisms of galvanotropism and bacterial growth.

Directional growth in response to electric fields (galvanotropism) is known for eukaryotic cells as diverse as fibroblasts, neurons, algae, and fungal hyphae. The mechanism is not understood, but all proposals invoke actin either directly or indirectly. We applied electric fields to bacteria (which are inherently free of actin) to determine whether actin was essential for galvanotropism. Field-treated (but not control) Enterobacter cloacae and Escherichia coli cells curved rapidly toward the anode. The response was both field strength and pH dependent. The direction of curvature was reversed upon reversal of field polarity. The directional growth was not due to passive bending of the cells or to field-induced gradients of tropic substances in the medium. Field-treated Bacillus subtilis cells also curved, but the threshold was much higher than for E. cloacae or E. coli. Since the curved morphology must reflect spatial differences in the rates of cell wall synthesis and degradation, we looked for regions of active wall growth. Experiments in which the cells were decorated with latex beads revealed that the anode-facing ends of cells grew faster than the cathode-facing ends of the same cells. Inhibitors of cell wall synthesis caused spheroplasts to form on the convex regions of field-treated cells, suggesting that the initial curvature resulted from enhanced growth of cathode-facing regions. Our results indicate that an electric field modulates wall growth spatially and that the mechanism may involve differential stimulation of wall growth in both anode- and cathode-facing regions. Electric fields may therefore serve as valuable tools for studies of bacterial wall growth. Use of specific E. coli mutants may allow dissection of the galvanotropic mechanism at the molecular level.

Bacillus subtilis↗

PubMed1993

Hippocampal growth cone responses to focally applied electric fields.

A wide variety of cell types respond to electric fields in culture. Despite evidence for electric fields existing in the mammalian embryo, there are few studies testing the effects electric fields exert on neurons from the mammalian central nervous system (CNS). The present study demonstrates orientation responses to focally applied electric fields of embryonic rat hippocampal neurons isolated in culture. The most striking result from this study is that different growth cones of the same neuron can show differential responsiveness to focally applied electric fields: growth cones on the short, straight processes that are destined to become dendrites, oriented toward the cathode, whereas growth cones on the longest process, the presumptive axon, did not orient. The present experiments bring a significant increase in resolution to the study of neuronal growth cone orientation by applied electric fields: a novel examination of the early events leading to orientation. Growth cones on dendrites displayed a spectrum of orientation responses: directed lamellipodial extension, directed filopodial extension and/or reorientation, cytoplasmic swelling of existing filopodia, consolidation of filopodia, and rapid elongation of the entire process. Individual growth cones displayed only one or two of these responses. Additionally, not all growth cones on these short processes sustained their initial orientation response: 35% adapted within 6 min.

Animals↗

PubMed1993

Raised cyclic-AMP and a small applied electric field influence differentiation, shape, and orientation of single myoblasts.

The effects of the adenyl cyclase activator forskolin and of a small applied electric field on the differentiation, morphology, and orientation of processes from single Xenopus myoblasts has been studied. Forskolin promoted myoblast differentiation (elongation from spherical cells) and induced extra process growth, such that normally bipolar muscle cells possessed on average five or six processes. Both events appeared to depend on elevated levels of cyclic AMP, since they were mimicked by another adenyl cyclase activator, cholera toxin, and by two membrane-permeable analogues of cyclic AMP. By contrast, the forskolin analogue dideoxyforskolin, which does not elevate adenyl cyclase levels, was without effect. Forskolin-stimulated differentiation depended on new protein synthesis, while excess process production required both new protein synthesis and intact microfilaments. Both control and forskolin-treated myoblasts developed a long axis perpendicular to an applied electric field. In addition, many processes on forskolin-treated muscle cells turned to grow toward the cathode; untreated processes did not turn to either pole. Since this nerve-like orientation behavior was inhibited by a protein synthesis inhibitor, the expression of an integral component of galvanotropism may be stimulated by forskolin treatment and by raised cyclic AMP levels in myoblasts.

Animals↗

PubMed1992

The effects of melanocortins and electrical fields on neuronal growth.

The effects of the neuropeptide molecules, adrenocorticotrophic hormone or alpha-melanocyte stimulating hormone, on various parameters of in vitro nerve growth, have been studied in the presence and absence of a small applied electric field. Striking effects on rates of nerve growth occurred. Selected concentrations of either substance alone, produced a three- to fourfold increase in nerve growth rate; other concentrations were inhibitory. The applied electric field alone increased rates of growth threefold. Further augmented rates of growth were seen when either neuropeptide was present simultaneously with an applied field. Under these latter conditions, galvanotropic (cathodal) orientation persisted, while nerve branching was suppressed. Given the clinical interest in melanocortin-stimulated nerve regeneration, perhaps a combined electrical and neuropeptide approach would be warranted.

Adrenocorticotropic Hormone↗

PubMed1992

Electric field-induced orientation of rat hippocampal neurones in vitro.

Rat hippocampal neurones in primary culture exhibited directional growth in response to DC electric fields. Neurites lay perpendicular to the field after exposure to 28, 80 or 219 mV/mm but there was no orientation effect at 9 mV/mm or in unexposed control cultures. Fields that evoked perpendicular responses also reduced the number of cathode-facing neurites. Orientation was the same for neurite initiation sites and the direction of overall neurite growth.

Animals↗