Ca2+ transients are not required as signals for long-term neurite outgrowth from cultured sympathetic neurons

A method for clamping cytosolic free Ca2+ ([Ca2+]i) in cultures of rat sympathetic neurons at or below resting levels for several days was devised to determine whether Ca2+ signals are required for neurite outgrowth from neurons that depend on Nerve Growth Factor (NGF) for their growth and survival. To control [Ca2+]i, normal Ca2+ influx was eliminated by titration of extracellular Ca2+ with EGTA and reinstated through voltage-sensitive Ca2+ channels. The rate of neurite outgrowth and the number of neurites thus became dependent on the extent of depolarization by KCl, and withdrawal of KCl caused an immediate cessation of growth. Neurite outgrowth was completely blocked by the L type Ca2+ channel antagonists nifedipine, nitrendipine, D600, or diltiazem at sub- or micromolar concentrations. Measurement of [Ca2+]i in cell bodies using the fluorescent Ca2+ indicator fura-2 established that optimal growth, similar to that seen in normal medium, was obtained when [Ca2+]i was clamped at resting levels. These levels of [Ca2+]i were set by serum, which elevated [Ca2+]i by integral of 30 nM, whereas the addition of NGF had no effect on [Ca2+]i. The reduction of [Ca2+]o prevented neurite fasciculation but this had no effect on the rate of neurite elongation or on the number of extending neurites. These results show that neurite outgrowth from NGF-dependent neurons occurs over long periods in the complete absence of Ca2+ signals, suggesting that Ca2+ signals are not necessary for operating the basic machinery of neurite outgrowth.     

Evidence that Protein Kinase C Activities Involved in Regulating Neurite Growth Are Localized to Distal Neurites

Abstract: Previously, we observed that long-term treatment of distal nerve fibers of rat sympathetic neurons in compartmented cultures with phorbol 12-myristate 13-acetate (PMA) caused a reduction in the rate of neurite elongation by >50%. In the present report we show that protein kinase C (PKC) activity could be measured in extracts of distal neurites by an assay of the Ca²⁺-dependent phosphorylation of a PKC-specific octapeptide substrate. We found that local application of 1 µ M PMA for 24 h to distal neurites caused nearly complete down-regulation of Ca²⁺-dependent PKC activity measured in this manner. We determined that the inhibition of neurite elongation by PMA was mediated by local mechanisms in the neurites because local application of PMA to center compartments containing cell bodies and proximal neurites did not inhibit the rate of elongation of distal neurites. We then investigated the effects of the recently available PKC inhibitors, calphostin C and chelerythrine, finding that, like PMA, these inhibited the growth of distal neurites when applied locally to them, and had no effect when applied to cell bodies and proximal neurites. However, the inhibition of neurite growth by calphostin C occurred at a concentration far below its IC₅₀ value for protein kinase inhibition, and both calphostin C and chelerythrine inhibited distal neurite growth even in neurons pretreated with PMA. Thus, it appears that these agents do not all inhibit neurite growth through the same mechanisms. Although the PKC activities involved in neurite elongation in sympathetic neurons have not been precisely defined, these data presented in this study indicate that protein kinases localized to growth cones play a complex and important role in regulating axonal growth.     

Ca2+ influx and neurite growth in response to purified N-cadherin and laminin

The signaling mechanisms underlying neurite growth induced by cadherins and integrins are incompletely understood. In our experiments, we have examined these mechanisms using purified N-cadherin and laminin (LN). We find that unlike the neurite growth induced by fibroblastic cells expressing transfected N-cadherin (Doherty, P., and F.S. Walsh. 1992. Curr. Opin. Neurobiol. 2:595-601), growth induced by purified N- cadherin in chick ciliary ganglion (CG), sensory, or forebrain neurons is not sensitive to inhibition by pertussis toxin. Using fura-2 imaging of single cells, we show that soluble N-cadherin induces Ca2+ increases in CG neuron cell bodies, and, importantly, in growth cones. In contrast, N-cadherin can induce Ca2+ decreases in glial cells. N- cadherin-induced neuronal Ca2+ responses are sensitive to Ni2+, but are relatively insensitive to diltiazem and omega-conotoxin. Similarly, neurite growth induced by purified N-cadherin is inhibited by Ni2+, but is unaffected by diltiazem and conotoxin. Soluble LN also induced small Ca2+ responses in CG neurons. LN-induced neurite growth, like that induced by N-cadherin, is insensitive to diltiazem and conotoxin, but is highly sensitive to Ni2+ inhibition. K+ depolarization experiments suggest that voltage-dependent Ca2+ influx pathways in CG neurons (cell bodies and growth cones) are largely blocked by the combination of diltiazem and Ni2+. Our results demonstrate that cadherin signaling involves cell type-specific Ca2+ changes in responding cells, and in particular, that N-cadherin can cause Ca2+ increases in neuronal growth cones. Our findings are consistent with the current idea that distinct neuronal transduction pathways exist for cell adhesion molecules compared with integrins, but suggest that the involvement of Ca2+ signals in both of these pathways is more complex than previously appreciated.     

Mouse semaphorin H induces PC12 cell neurite outgrowth activating Ras-mitogen-activated protein kinase signaling pathway via Ca(2+) influx.

We recently showed that mouse semaphorin H (MSH), a secreted semaphorin molecule, acts as a chemorepulsive factor on sensory neurites. In this study, we found for the first time that MSH induces neurite outgrowth in PC12 cells in a dose-dependent manner. Comparison of Ras-mitogen-activated protein kinase (MAPK) signaling pathways between MSH and nerve growth factor (NGF) revealed that these pathways are crucial for MSH action as well as NGF. K-252a, an inhibitor of tyrosine autophosphorylation of tyrosine kinase receptors (Trks), did not inhibit the action of MSH, suggesting that MSH action occurs via a different receptor than NGF. L- and N-types of voltage-dependent Ca(2+) channel blockers, diltiazem and omega-conotoxin, inhibited MSH-induced neurite outgrowth and MAPK phosphorylation in a Ca(2+)-dependent manner. A transient elevation in intracellular Ca(2+) level was observed upon MSH stimulation. These findings suggest that extracellular Ca(2+) influx, followed by activation of the Ras-MAPK signaling pathway, is required for MSH induced PC12 cell neurite outgrowth.     

Contribution of Ca2+ calmodulin-dependent protein kinase II and mitogen-activated protein kinase kinase to neural activity-induced neurite outgrowth and survival of cerebellar granule cells

In this report we describe our studies on intracellular signals that mediate neurite outgrowth and long-term survival of cerebellar granule cells. The effect of voltage-gated calcium channel activation on neurite complexity was evaluated in cultured cerebellar granule cells grown for 48 h at low density; the parameter measured was the fractal dimension of the cell. We explored the contribution of two intracellular pathways, Ca2+ calmodulin-dependent protein kinase II and mitogen-activated protein kinase kinase (MEK1), to the effects of high [K+ ]e under serum-free conditions. We found that 25 mm KCl (25K) induced an increase in calcium influx through L subtype channels. In neurones grown for 24-48 h under low-density conditions, the activation of these channels induced neurite outgrowth through the activation of Ca2+ calmodulin-dependent protein kinase II. This also produced an increase in long-term neuronal survival with a partial contribution from the MEK1 pathway. We also found that the addition of 25K increased the levels of the phosphorylated forms of Ca2+ calmodulin-dependent protein kinase II and of the extracellular signal-regulated kinases 1 and 2. Neuronal survival under resting conditions is supported by the MEK1 pathway. We conclude that intracellular calcium oscillations can triggered different biological effects depending on the stage of maturation of the neuronal phenotype. Ca2+ calmodulin-dependent protein kinase II activation determines the growth of neurites and the development of neuronal complexity.     

Glutamate regulates neurite outgrowth of cultured descending brain neurons from larval lamprey

In larval lamprey, descending brain neurons, which regenerate their axons following spinal cord injury, were isolated and examined in cell culture to identify some of the factors that regulate neurite outgrowth. Focal application of 5 mM or 25 mM L-glutamate to single growth cones inhibited outgrowth of the treated neurite, but other neurites from the same neuron were not inhibited, an effect that has not been well studied for neurons in other systems. Glutamate-induced inhibition of neurite outgrowth was abolished by 10 mM kynurenic acid. Application of high potassium media to growth cones inhibited neurite outgrowth, an effect that was blocked by 2 mM cobalt or 100 microM cadmium, suggesting that calcium influx via voltage-gated channels contributes to glutamate-induced regulation of neurite outgrowth. Application of glutamate to growth cones in the presence of 2 microM omega-conotoxin MVIIC (CTX) still inhibited neurite outgrowth, while CTX blocked high potassium-induced inhibition of neurite outgrowth. Thus, CTX blocked virtually all of the calcium influx resulting from depolarization. To our knowledge, this is the first direct demonstration that calcium influx via ligand-gated ion channels can contribute to regulation of neurite outgrowth. Finally, focal application of glutamate to the cell bodies of descending brain neurons inhibited outgrowth of multiple neurites from the same neuron, and this is the first demonstration that multiple neurites can be regulated in this fashion. Signaling mechanisms involving intracellular calcium, similar to those shown here, may be important for regulating axonal regeneration following spinal cord injury in the lamprey.     

Extracellular calcium-dependent regulation of transmembrane calcium fluxes in murine keratinocytes.

Because the level of extracellular Ca2+ is an important stimulus for differentiation of epidermal cells in vitro, we characterized the extracellular Ca(2+)-dependent transmembrane Ca2+ fluxes in BALB/MK mouse keratinocytes. Increasing levels of extracellular Ca2+, ranging from 0.07 to 1.87 mM, stimulated the rate of 45Ca2+ uptake into these cells 10- to 70-fold and doubled the rate of 45Ca2+ efflux. The divalent cations, Ni2+ and Co2+, were able to block the influx of Ca2+, but dihydropyridines and verapamil were not. Furthermore, 10 to 100 microM of the trivalent cation La3+ induced a dose-dependent 2- to 100-fold increase of Ca2+ uptake, independently of the level of extracellular Ca2+. These observations suggest that keratinocytes possess a cell-surface "Ca(2+)-receptor," activation of which stimulates the influx of 45Ca2+ through a type of voltage-independent, receptor-operated Ca2+ channels. Epidermal growth factor induced an accumulation of 45Ca2+ of a much smaller magnitude than elevations of the level of extracellular Ca2+, without a detectable increase of Ca2+ efflux. Thus, the divergent cellular responses of keratinocytes to EGF and extracellular Ca2+ may be due, in part, to the distinct changes in transmembrane Ca2+ fluxes that these two stimuli generate. Treatment of cells with type beta transforming growth factor led to a gradual 6-fold increase of the Ca(2+)-activated rate of Ca2+ uptake over a period of 4 hours, but reduced the Ca2+ efflux by approximately 50% within 10 minutes. Thus, type beta transforming growth factor apparently stimulates Ca2+ influx indirectly, but may control the differentiation of keratinocytes by direct inhibition of Ca2+ efflux pumps.     

Neuronal calcium sensor-1 modulation of optimal calcium level for neurite outgrowth

Neurite extension and branching are affected by activity-dependent modulation of intracellular Ca2+, such that an optimal window of [Ca2+] is required for outgrowth. Our understanding of the molecular mechanisms regulating this optimal [Ca2+]i remains unclear. Taking advantage of the large growth cone size of cultured primary neurons from pond snail Lymnaea stagnalis combined with dsRNA knockdown, we show that neuronal calcium sensor-1 (NCS-1) regulates neurite extension and branching, and activity-dependent Ca2+ signals in growth cones. An NCS-1 C-terminal peptide enhances only neurite branching and moderately reduces the Ca2+ signal in growth cones compared with dsRNA knockdown. Our findings suggest that at least two separate structural domains in NCS-1 independently regulate Ca2+ influx and neurite outgrowth, with the C-terminus specifically affecting branching. We describe a model in which NCS-1 regulates cytosolic Ca2+ around the optimal window level to differentially control neurite extension and branching.     

Effects of soluble laminin on organelle transport and neurite growth in cultured mouse dorsal root ganglion neurons: difference between primary neurites and branches

Laminin, an extracellular matrix molecule, is known to promote neurite growth. In the present study, the effects of soluble laminin on organelle transport and their relation to neurite growth were investigated in cultured dissociated mouse dorsal root ganglion (DRG) neurons. Laminin added into the extracellular medium was deposited on the surface of DRG neurons. DRG neurons incubated with soluble laminin exhibited branched, long, and thin neurites. Time-lapse study demonstrated that many small-diameter branches were newly formed after the addition of laminin. Thus, the growths of large-diameter primary neuritis, arising from cell bodies and branches extended from growth cones of primary neuritis, were analyzed separately. Laminin decreased the growth rate of primary neurites but increased that of branches. In primary neurites, acute addition of laminin rapidly decreased organelle movement in the neurite shaft and growth cone, accompanied by slowing of the growth cone advance. Branching of primary neurites occurred in response to laminin in some growth cones. In these growth cones, organelles protruded into nascent branches. In branches, soluble laminin increased organelle movement in the growth cone and the distal portion of the shaft. These results suggest that laminin inhibits the elongation of primary neurites but promotes branching and elongation of branches, all of which seem to be closely related to organelle transport.     

Ca2+ waves in PC12 neurites: a bidirectional, receptor-oriented form of Ca2+ signaling

Spatial and temporal aspects of Ca2+ signaling were investigated in PC12 cells differentiated with nerve growth factor, the well known nerve cell model. Activation of receptors coupled to polyphosphoinositide hydrolysis gave rise in a high proportion of the cells to Ca2+ waves propagating non decrementally and at constant speed (2-4 microns/s at 18 degrees C and approximately 10-fold faster at 37 degrees C) along the neurites. These waves relied entirely on the release of Ca2+ from intracellular stores since they could be generated even when the cells were incubated in Ca(2+)-free medium. In contrast, when the cells were depolarized with high K+ in Ca(2+)-containing medium, increases of cytosolic Ca2+ occurred in the neurites but failed to evolve into waves. Depending on the receptor agonist employed (bradykinin and carbachol versus ATP) the orientation of the waves could be opposite, from the neurite tip to the cell body or vice versa, suggesting different and specific distribution of the responsible surface receptors. Cytosolic Ca2+ imaging results, together with studies of inositol 1,4,5-trisphosphate generation in intact cells and inositol 1,4,5-trisphosphate-induced Ca2+ release from microsomes, revealed the sustaining process of the waves to be discharge of Ca2+ from the inositol 1,4,5-trisphosphate- (and not the ryanodine-) sensitive stores distributed along the neurites. The activation of the cognate receptor appears to result from the coordinate action of the second messenger and Ca2+. Because of their properties and orientation, the waves could participate in the control of not only conventional cell activities, but also excitability and differential processing of inputs, and thus of electrochemical computation in nerve cells.     

Characterization of the bradykinin-stimulated calcium influx pathway of cultured vascular endothelial cells. Saturability, selectivity, and kinetics

Measurement of fura-2 fluorescence and 45Ca2+ uptake was used to evaluate Ca2+ influx in cultured bovine aortic endothelial cells (BAECs) stimulated by bradykinin (BK). The BK-stimulated influx pathway was characterized with respect to its 1) sensitivity to extracellular Ca2+, 2) inhibition by membrane depolarization, and 3) permeability to Ba2+ and Sr2+. The results indicate that the activity of the influx pathway is a saturable function of extracellular Ca2+ and that membrane depolarization inhibits Ca2+ influx by changing the apparent affinity and maximal capacity of the pathway for Ca2+. Fura-2 fluorescence was used to compare the profiles for BK-stimulated changes in cytosolic Ca2+, Sr2+, and Ba2+ (Ca2+i, Ba2+i, and Sr2+i). Addition of Ca2+ and Sr2+ to Ca2+-depleted cells in the presence of BK produced a transient increase in Ca2+i and Sr2+i. Following the peak of the response, Ca2+i and Sr2+i declined within 2 min to a steady elevated level. Blockade of influx by the addition of La3+ at the peak of the response to Ca2+ and Sr2+ immediately reduced Ca2+i and Sr2+i to basal levels. Addition of Ba2+ to Ca2+-depleted cells in the presence of BK produced an increase in Ba2+i which continued to rise with time to a steady level. Addition of La3+ after Ba2+, however, did not reduce Ba2+i. These results suggest that 1) Ca2+ and Sr2+ (but not Ba2+) are sequestered by intracellular mechanisms and that the declining phase of the Ca2+ and Sr2+ response reflects a time and divalent cation-dependent inactivation of the influx pathway. The inactivation of the influx pathway was further demonstrated by measuring the kinetics of BK-stimulated 45Ca2+ uptake into BAECs. The results of these experiments demonstrate that BK stimulates a 100- to 150-fold increase in Ca2+ permeability of the BAEC but that the influx pathway turns off or inactivates within 2 min. The magnitude of the flux, the voltage sensitivity, and the ability to conduct Ca2+, Sr2+, and Ba2+ are suggestive of a channel mechanism.     

Axonal synthesis of phosphatidylcholine is required for normal axonal growth in rat sympathetic neurons

The goal of this study was to assess the relative importance of the axonal synthesis of phosphatidylcholine for neurite growth using rat sympathetic neurons maintained in compartmented culture dishes. In a double-labeling experiment [14C]choline was added to compartments that contained only distal axons and [3H]choline was added to compartments that contained cell bodies and proximal axons. The specific radioactivity of labeled choline was equalized in all compartments. The results show that approximately 50% of phosphatidylcholine in distal axons is locally synthesized by axons. The requirement of axonal phosphatidylcholine synthesis for neurite growth was investigated. The neurons were supplied with medium lacking choline, an essential substrate for phosphatidylcholine synthesis. In the cells grown in choline-deficient medium for 5 d, the incorporation of [3H]palmitate into phosphatidylcholine was reduced by 54% compared to that in cells cultured in choline-containing medium. When phosphatidylcholine synthesis was reduced in this manner in distal axons alone, growth of distal neurites was inhibited by approximately 50%. In contrast, when phosphatidylcholine synthesis was inhibited only in the compartment containing cell bodies with proximal axons, growth of distal neurites continued normally. These experiments imply that the synthesis of phosphatidylcholine in cell bodies is neither necessary nor sufficient for growth of distal neurites. Rather, the local synthesis of phosphatidylcholine in distal axons is required for normal growth.     

Ionic responses and growth stimulation induced by nerve growth factor and epidermal growth factor in rat pheochromocytoma (PC12) cells

Rat pheochromocytoma cells (clone PC12) respond to nerve growth factor (NGF) by the acquirement of a phenotype resembling neuronal cells. In an earlier study we showed that NGF causes an increase in Na+,K+ pump activity, as monitored by ouabain-sensitive Rb+ influx. Here we show that addition of epidermal growth factor (EGF) to PC12 cells resulted in a stimulation of Na+,K+ pump activity as well. The increase of Na+,K+ pump activity by NGF or EGF was due to increased Na+ influx. This increased Na+ influx was sensitive to amiloride, an inhibitor of Na+,H+ exchange. Furthermore, no changes in membrane potential were observed upon addition of NGF or EGF. Amiloride-sensitive Na+,H+ exchange in PC12 cells was demonstrated by H+ efflux measurements and the effects of weak acids on Na+ influx. These observations suggest that both NGF and EGF activate an amiloride-sensitive, electroneutral Na+,H+ exchange mechanism in PC12 cells. These findings were surprising in view of the opposite ultimate biological effects of NGF and EGF, e.g., growth arrest vs. growth stimulation. However, within 24 h after addition, NGF was found to stimulate growth of PC12 cells, comparable to EGF. In the presence of amiloride, this stimulated growth by NGF and EGF was abolished. In contrast, amiloride did not affect NGF-induced neurite outgrowth of PC12 cells. From these observations it is concluded that in PC12 cells: (a) NGF has an initial growth stimulating effect; (b) neurite outgrowth is independent of increased amiloride-sensitive Na+ influx; and (c) growth stimulation by NGF and EGF is associated with increased amiloride-sensitive Na+ influx.     

Characterization of the Ca2+ influx into embryonic cells after stimulation of the embryonic muscarinic receptor.

Embryonic cells transiently express an embryonic muscarinic system during morphogenesis. Stimulation of the embryonic muscarinic receptor results in biphasic intracellular Ca2+ mobilization: an initial "peak" due to Ca2+ release from intracellular stores is followed by a sustained "plateau" of enhanced cytoplasmic Ca2+ due to influx of extracellular Ca2+. In the present investigation, we characterized the Ca2+ influx by measuring the cytoplasmic free Ca2+ concentration [Ca2+]i using the Ca2+ indicator fura-2: 1. The increase of [Ca2+]i during the plateau depended linearly on the logarithm of the extracellular calcium concentration whereas the initial peak was almost independent from extracellular calcium. 2. The organic Ca2+ entry blockers verapamil, gallopamil, nifedipine, nitrendipine and the inorganic blockers Mn2+, Mg2+ and La3+ were without effect on both phases of Ca2+ mobilization. Only Ni2+ at concentrations above 1 mM was able to reduce the influx without affecting the intracellular Ca2+ release. 3. Substitution of extracellular Na+ by guanidine+, choline+ or tris+ and membrane depolarisation by increasing the extracellular K+ concentration had no effect on either phase of Ca2+ mobilization. We conclude that a non-voltage dependent, receptor-operated influx mechanism, probably a "second messenger operated Ca2+ channel", is responsible for the Ca2+ influx after stimulation of the embryonic muscarinic receptor.     

Survival of mammalian B104 cells following neurite transection at different locations depends on somal Ca2+ concentration

We report that cell survival after neurite transection in a mammalian neuronal model (cultured B104 cells) critically depends on somal [Ca2+]i, a novel result that reconciles separate long-standing observations that somal survival decreases with more-proximal axonal transections and that increased somal Ca2+ is cytotoxic. Using fluorescence microscopy, we demonstrate that extracellular Ca2+ at the site of plasmalemmal transection is necessary to form a plasmalemmal barrier, and that other divalent ions (Ba2+, Mg2+) do not play a major role. We also show that extracellular Ca2+, rather than injury per se, initiates the formation of a plasmalemmal barrier and that a transient increase in somal [Ca2+]i significantly decreases the percentage of cells that survive neurite transection. Furthermore, we show that the increased somal [Ca2+]i and decreased cell survival following proximal transections are not due to less frequent or slower plasmalemmal sealing or Ca2+ entry through plasmalemmal Na+ and Ca2+ channels. Rather, the increased somal [Ca2+]i and lethality of proximal neurite injuries may be due to the decreased path length/increased diameter for Ca2+ entering the transection site to reach the soma. A ryanodine block of Ca2+ release from internal stores before transection has no effect on cell survival; however, a ryanodine- or thapsigargin-induced buildup of somal [Ca2+]i before transection markedly reduces cell survival, suggesting a minor involvement of Ca2+-induced release from internal stores. Finally, we show that cell survival following proximal injuries can be enhanced by increasing intracellular Ca2+ buffering capacity with BAPTA to prevent the increase in somal [Ca2+]i.     

Sensitization of rat hepatocytes to hyperthermia by calcium

The viability of isolated rat hepatocytes, as assayed by trypan blue exclusion, decreases in a dose-dependent fashion during exposure to hyperthermia (D0 [43 degrees C] = 105 +/- 10 min, D0 [45 degrees C] = 24 +/- 4 min). Hyperthermic sensitivity varies as a function of extracellular Ca2+ concentration in a biphasic manner; optimum survival occurs at 1-5 mM Ca2+, with sensitization in the absence of Ca+ and increasing sensitization at Ca2+ concentrations greater than 10 mM. Ca influx does not correlate well with loss of viability for hepatocytes in 4 mM extracellular Ca2+; influx does not occur until viability decreases to less than 1%. Under sensitizing conditions, Ca2+ influx proceeds loss of viability. Influx begins within 15 min at 45 degrees C in 15 mM Ca2+, and the ionophore A23187 is a potent hyperthermic sensitizer in the presence of extracellular Ca2+. Thus, Ca2+ influx, whether caused by high extracellular Ca2+ or A23187, increases cellular damage caused by supraoptimal temperatures, although some Ca2+ is necessary for maximum resistance, probably because of stabilization of Ca2+ binding proteins against thermal denaturation or possibly to Ca2+-induced decrease in lipid fluidity.     

Evidence for extensive methylation of ribosomal RNA genes in a rat XC cell line

The effects of extracellular Na+, K+ and Cl- on neurite outgrowth of PC12 pheochromocytoma cells were studied. Nerve growth factor (NGF)-induced neurite formation was inhibited upon substitution of choline chloride for NaCl under normal culture conditions. It was found that neurite formation increased proportionately with the concentration of Na+ in medium up to 150 mM. When PC12 cells were exposed to NGF in suspension culture followed by transfer to new dishes, they showed neurite extention in response to NGF in an RNA- and protein synthesis-independent manner. Under these conditions, neurite outgrowth occurred normally in 60-150 mM Na+, whereas it decreased significantly at lower concentrations of Na+. Na+ dependency was also observed for cyclic AMP-mediated neurite formation of PC12 cells. In contrast neurite outgrowth was independent of K+ in the range 5-106 mM, suggesting that membrane potential did not play a role in this process. No alterations were observed in neurite outgrowth with Cl- replaced by NO3-, SO2-4, or 2-hydroxyethanesulfonate. Thus, extracellular Na+ plays a role in controlling neurite formation of these cells. An attempt was made to relate this effect to a decrease in cytoplasmic Ca2+ concentration monitored by a fluorescent dye sensitive to Ca2+.     

Microtubule dynamics in nerve cells: analysis using microinjection of biotinylated tubulin into PC12 cells

To study microtubule (MT) dynamics in nerve cells, we microinjected biotin-labeled tubulin into the cell body of chemically fused and differentiated PC12 cells and performed the immunofluorescence or immunogold procedure using an anti-biotin antibody followed by secondary antibodies coupled to fluorescent dye or colloidal gold. Incorporation of labeled subunits into the cytoskeleton of neurites was observed within minutes after microinjection. Serial electron microscopic reconstruction revealed that existing MTs in PC12 neurites incorporated labeled subunits mainly at their distal ends and the elongation rate of labeled segments was estimated to be less than 0.3 micron/min. Overall organization of MTs in the nerve cells was different from that in undifferentiated cells such as fibroblasts. Namely, we have not identified any MT-organizing centers from which labeled MTs are emanating in the cell bodies of the injected cells. Stereo electron microscopy revealed that some fully labeled segments seemed to start in the close vicinity of electron dense material within the neurites. This suggests new nucleation off some structures in the neurites. We have also studied the overall pattern of the incorporation of labeled subunits which extended progressively from the proximal part of the neurites toward their tips. To characterize the mechanism of tubulin incorporation, we have measured mean density of gold labeling per unit length of labeled segments at different parts of the neurites. The results indicate access of free tubulin subunits into the neurites and local incorporation into the neurite cytoskeleton. Our results lead to the conclusion that MTs are not static polymers but dynamic structures that continue to elongate even within the differentiated nerve cell processes.     

Alpha-actinins, calspectin (brain spectrin or fodrin), and actin participate in adhesion and movement of growth cones

We have used biochemical and immunocytochemical techniques to investigate the possible involvement of membrane cytoskeletal elements such as alpha-actinin, calspectin (brain spectrin or fodrin), and actin in growth cone activities. During NGF-induced differentiation of PC12 cells, alpha-actinin increased in association with neurite outgrowth and was predominantly distributed throughout the entire growth cone and the distal portion of neurites. Filopodial movements were sensitive to Ca2+ flux. Two types of alpha-actinin, with Ca2(+)-sensitive and -insensitive actin binding abilities, were identified in the differentiated cells. Ca2(+)-sensitive alpha-actinin and actin filaments were concentrated in filopodia. The Ca2(+)-insensitive protein was distributed from the body of the growth cone to the distal portion of neurites, corresponding to the substratum-adhesive sites. The location of calspectin in growth cones was similar to that of the Ca2(+)-insensitive alpha-actinin. These results are consistent with the hypothesis that Ca2(+)-sensitive alpha-actinin and actin filaments are involved in Ca2(+)-dependent filopodial movement and Ca2(+)-insensitive alpha-actinin and calspectin are associated with adhesion of growth cones.