Tyrosine kinase inhibitors can differentially inhibit integrin- dependent and CAM-stimulated neurite outgrowth

We have used monolayers of parental 3T3 cells and 3T3 cells expressing one of three transfected cell adhesion molecules (CAMs) (NCAM, N- cadherin, and L1) as a culture substrate for rat cerebellar neurons. A number of tyrosine kinase inhibitors have been tested for their ability to inhibit neurite outgrowth over parental 3T3 monolayers which we show to be partly dependent on neuronal integrin receptor function, as compared with neurite outgrowth stimulated by the above three CAMs. Whereas genistein (100 microM), lavendustin A (20 microM), and tyrphostins 34 and 47 (both at 150 microM) had no effect on integrin dependent or CAM stimulated neurite outgrowth, the erbstatin analogue (10-15 micrograms/ml) and tyrphostins 23 and 25 (both at 150 microM) specifically inhibited the response stimulated by all three CAMs. CAM stimulated neurite outgrowth can be accounted for by a G-protein- dependent activation of neuronal calcium channels; experiments with agents that directly activate this pathway localized the erbstatin analogue site of action upstream of the G-protein and calcium channels, whereas tyrphostins have sites of action downstream from calcium channel activation. These data suggest that activation of an erbstatin sensitive tyrosine kinase is an important step upstream of calcium channel activation in the second messenger pathway underlying the neurite outgrowth response stimulated by a variety of CAMs, and that this kinase is not required for integrin-dependent neurite outgrowth.     

A developmentally regulated switch in neuronal responsiveness to NCAM and N-cadherin in the rat hippocampus.

Monolayers of control 3T3 fibroblasts and 3T3 cells expressing transfected NCAM or N-cadherin have been used as a culture substratum for rat hippocampal neurons. Both NCAM and N-cadherin are expressed in the hippocampus through embryonic day 17 (E17) to postnatal day 4 (PND4); however, whereas E17 neurons responded to transfected NCAM by extending considerably longer neurites, PND4 neurons responded very poorly. The converse was true for responsiveness to N-cadherin. These data demonstrate a switch in neuronal responsiveness to NCAM and N-cadherin in the developing hippocampus. NCAM-dependent neurite outgrowth from E17 neurons was largely dependent on the presence of alpha 2-8-linked polysialic acid (PSA) on neuronal NCAM. NCAM-dependent neurite outgrowth could be fully inhibited by pertussis toxin or a combination of L- and N-type calcium channel antagonists thus providing direct evidence concerning the nature of the second messenger pathway activated in primary neurons by cell adhesion molecules (CAMs).     

Neurite Outgrowth Stimulated by the Tyrosine Kinase Inhibitor Herbimycin A Requires Activation of Tyrosine Kinases and Protein Kinase C

Abstract: Activation of tyrosine kinases is established as an important mechanism for controlling growth cone motility and neurite outgrowth. We have tested the effects of a range of tyrosine kinase inhibitors on neurite outgrowth from postnatal day 4 cerebellar granule cells cultured over confluent monolayers of 3T3 fibroblasts. The only agent that had any effect was herbimycin A, which stimulated neurite outgrowth. The response is shown to be attributable to a direct effect of this tyrosine kinase inhibitor on neurones. The neurite outgrowth response to herbimycin A was inhibited by two other tyrosine kinase inhibitors, which on their own did not affect neurite outgrowth. The data suggest that the response to herbimycin A reflects either a direct or indirect activation of one or more protein tyrosine kinases. Independent signalling events downstream from tyrosine kinase activation underlying the neurite outgrowth response to herbimycin A include increased activity of protein kinase C and calcium influx into neurones through both N-and L-type calcium channels.     

Direct activation of second messenger pathways mimics cell adhesion molecule-dependent neurite outgrowth

We present evidence that direct activation of neuronal second messenger pathways in PC12 cells by opening voltage-dependent calcium channels mimics cell adhesion molecule (CAM)-induced differentiation of these cells. PC12 cells were cultured on monolayers of control 3T3 cells or 3T3 cells expressing transfected N-cadherin in the presence of KCl or a calcium channel agonist Bay K 8644. Both potassium depolarization and agonist-induced activation of calcium channels promoted substantial neurite outgrowth from PC12 cells cultured on control 3T3 monolayers and increased neurite outgrowth from those cultured on N-cadherin-expressing 3T3 monolayers. The potassium-induced response could be inhibited by L- and N-type calcium channel antagonists and by kinase inhibitor K-252b but was unaffected by pertussis toxin. In contrast activators of protein kinase C did not stimulate neurite outgrowth, and the neurite outgrowth response induced by activation of protein kinase A was not inhibited by calcium channel antagonists or pertussis toxin. These studies support the postulate that CAM-induced neuronal differentiation involves a specific transmembrane signaling pathway and suggest that activation of this pathway after CAM binding may be more important for the neurite outgrowth response than CAM-dependent adhesion per se.     

Calcium influx into neurons can solely account for cell contact- dependent neurite outgrowth stimulated by transfected L1

We have used monolayers of control 3T3 cells and 3T3 cells expressing transfected human L1 as a culture substrate for rat PC12 cells and rat cerebellar neurons. PC12 cells and cerebellar neurons extended longer neurites on human L1 expressing cells. Neurons isolated from the cerebellum at postnatal day 9 responded equally as well as those isolated at postnatal day 1-4, and this contrasts with the failure of these older neurons to respond to the transfected human neural cell adhesion molecule (NCAM). Human L1-dependent neurite outgrowth could be blocked by antibodies that bound to rat L1 and, additionally, the response could be fully inhibited by pertussis toxin and substantially inhibited by antagonists of L- and N-type calcium channels. Calcium influx into neurons induced by K+ depolarization fully mimics the L1 response. Furthermore, we show that L1- and K+(-)dependent neurite outgrowth can be specifically inhibited by a reduction in extracellular calcium to 0.25 microM, and by pretreatment of cerebellar neurons with the intracellular calcium chelator BAPTA/AM. In contrast, the response was not inhibited by heparin or by removal of polysialic acid from neuronal NCAM both of which substantially inhibit NCAM-dependent neurite outgrowth. These data demonstrate that whereas NCAM and L1 promote neurite outgrowth via activation of a common CAM-specific second messenger pathway in neurons, neuronal responsiveness to NCAM and L1 is not coordinately regulated via posttranslational processing of NCAM. The fact that NCAM- and L1-dependent neurite outgrowth, but not adhesion, are calcium dependent provides further evidence that adhesion per se does not directly contribute to neurite outgrowth.     

Thy-1 antibody-triggered neurite outgrowth requires an influx of calcium into neurons via N- and L-type calcium channels

We present evidence that the neurite out-growth stimulated by the binding of Thy-1 antibodies to PC12 cells is mediated by calcium influx through both N- and L-type calcium channels. PC12 cells cultured on a noncellular substratum in the presence of NGF, or on a cellular substratum in the absence of NGF, responded to soluble Thy-1 antibody by extending longer neurites. The response required bivalent antibody and could be blocked by removing Thy-1 from the surface of PC12 cells with phosphatidylinositol specific phospholipase C. The response could also be blocked by reducing extracellular calcium to 0.25 mM, or by antagonists of L- and N-type calcium channels. Additionally, the response could be fully inhibited by preloading PC12 cells with BAPTA/AM which buffers changes in intracellular calcium. A heterotrimeric G-protein is also implicated in the pathway as the response could be fully inhibited by pertussis toxin. These data suggest that antibody-induced clustering of Thy-1 stimulates neurite outgrowth by activating a second messenger pathway that has previously been shown to underlie cell adhesion molecule (NCAM, N-cadherin, and L1), but not integrin or NGF-dependent neurite outgrowth.     

Phospholipase C and diacylglycerol lipase activities associated with plasma membranes of chromaffin cells isolated from bovine adrenal medulla

The plasma membranes of bovine adrenal chromaffin cells were isolated and the activities of enzymes involved in arachidonic acid liberation were investigated. Only a minute activity of phospholipase A2 (phosphatide 2-acylhydrolase, EC 3.1.1.4) could be detected using externally added phosphatidylcholine (PC) and phosphatidylethanolamine (PE) as substrate. When membranes were treated with exogenous phospholipase C (orthophosphoric acid diester phosphohydrolase, EC 3.1.4.1) there was a liberation of free fatty acids from the sn-2 position of PC. The enzyme responsible for this effect could be demonstrated to be a diacylglycerol lipase (glycerol ester hydrolase, EC 3.1.1.3) localized in the plasma membrane. Using phosphatidylinositol (PI) as a substrate, it was found that an endogenous phospholipase C exists which co-purifies with the membrane preparation. The produced diacylglycerol is subsequently hydrolyzed by diacylglycerol lipase liberating arachidonic acid. The two enzymes, phospholipase C and diacylglycerol lipase were characterized. Phospholipase C was found to be calcium dependent and PI specific, showing an activity of 60 pmol/micrograms protein per h (1.2 mM Ca2+), whereas the diacylglycerol lipase was calcium independent hydrolyzing diacylglycerol at a rate of 7.2 pmol/micrograms protein per h. The lipase but not the phospholipase C was inhibited 50% by 1.7 mM para-bromophenacylbromide.     

Ca2+-dependent and Ca2+-independent pathways for release of arachidonic acid from phosphatidylinositol in endothelial cells

The pathways for degradation of phosphatidylinositol (PI) were investigated in sonicated suspensions prepared from confluent cultures of bovine pulmonary artery endothelial cells. The time courses of formation of 3H-labeled and 14C-labeled metabolites of phosphatidyl-[3H]inositol ([3H]Ins-PI) and 1-stearoyl-2-[14C] arachidonoyl-PI were determined at 37 degrees C and pH 7.5 in the presence of 2 mM EDTA with or without a 2 mM excess of Ca2+. The rates of formation of lysophosphatidyl-[3H]inositol ([3H]Ins-lyso-PI) and 1-lyso-2-[14C] arachidonoyl-PI were similar in the presence and absence of Ca2+, and the absolute amounts of the two radiolabeled lyso-PI products formed were nearly identical. This indicated that lyso-PI was formed by phospholipase A1, and phospholipase A2 was not measurable. In the presence of EDTA, [14C]arachidonic acid release from 1-stearoyl-2-[14C]arachidonoyl-PI paralleled release of glycerophospho-[3H]inositol ([3H]GPI) from [3H]Ins-PI. Formation of [3H]GPI was inhibited by treatment with the specific sulfhydryl reagent, 2,2'-dithiodipyridine, and this was accompanied by an increase in [3H]Ins-lyso-PI. In the presence of Ca2+, [14C] arachidonic acid release from 1-stearoyl-2-[14C]arachidonoyl-PI was increased 2-fold and was associated with Ca2+-dependent phospholipase C activity. Under these conditions, [3H]inositol monophosphate production exceeded formation of [14C]arachidonic acid-labeled phospholipase C products, diacylglycerol plus monoacylglycerol, by an amount that was equal to the amount of [14C]arachidonic acid formed in excess of [3H]GPI. Low concentrations of phenylmethanesulfonyl fluoride (15-125 microM) inhibited Ca2+-dependent [14C]arachidonic acid release, and the decrease in [14C] arachidonic acid formed was matched by an equivalent increase in 14C label in diacylglycerol plus monoacyclglycerol. These data supported the existence of two pathways for arachidonic acid release from PI in endothelial cells; a phospholipase A1-lysophospholipase pathway that was Ca2+-independent and a phospholipase C-diacylglycerol lipase pathway that was Ca2+-dependent. The mean percentage of arachidonic acid released from PI via the phospholipase C-diacylglycerol lipase pathway in the presence of Ca2+ was 65 +/- 8%. The mean percentage of nonpolar phospholipase C products of PI metabolized via the diacylglycerol lipase pathway to free arachidonic acid was 28 +/- 3%.     

A major role for phospholipase A2 in antigen-induced arachidonic acid release in rat mast cells.

Cross-linking of IgE receptors by antigen stimulation leads to histamine release and arachidonic acid release in rat peritoneal mast cells. Investigators have reported a diverse distribution of [3H]arachidonate that is dependent on labelling conditions. Mast cells from rat peritoneal cavity were labelled with [3H]arachidonic acid for different periods of time at either 30 or 37 degrees C. Optimum labelling was found to be after 4 h incubation with [3H]arachidonate at 30 degrees C, as judged by cell viability (Trypan Blue uptake), responsiveness (histamine release) and distribution of radioactivity. Alterations in 3H-radioactivity distribution in mast cells labelled to equilibrium were examined on stimulation with antigen (2,4-dinitrophenyl-conjugated Ascaris suum extract). The results indicated that [3H]arachidonic acid was lost mainly from phosphatidylcholine and, to a lesser extent, from phosphatidylinositol. A transient appearance of radiolabelled phosphatidic acid and diacylglycerol indicated phosphatidylinositol hydrolysis by phospholipase C. Pretreatment with a phospholipase A2 inhibitor, mepacrine, substantially prevented the antigen-induced liberation of [3H]arachidonic acid from phosphatidylcholine. It can be thus concluded that, in the release of arachidonic acid by antigen-stimulated mast cells, the phospholipase A2 pathway, in which phosphatidylcholine is hydrolysed, serves as the major one, the phospholipase C/diacylglycerol lipase pathway playing only a minor role.     

Guanine nucleotides stimulate arachidonic acid release by phospholipase A2 in saponin-permeabilized human platelets

GTP or GTP gamma S alone caused low but significant liberation of arachidonic acid in saponin-permeabilized human platelets but not in intact platelets. GTP or GTP gamma S also enhanced thrombin-induced [3H]arachidonic acid release in permeabilized platelets. Inhibitors of the phospholipase C (neomycin)/diacylglycerol lipase (RHC 80267) pathway for arachidonate liberation did not reduce the [3H]arachidonic acid release. The loss of [3H]arachidonate radioactivity from phosphatidylcholine was almost equivalent to the increase in released [3H]arachidonic acid, suggesting the hydrolysis of phosphatidylcholine by phospholipase A2. The effect of GTP gamma S was greater at lower Ca2+ concentrations. These data indicate that the release of arachidonic acid by phospholipase A2 in saponin-treated platelets may be linked to a GTP-binding protein.     

The inhibition of arachidonic acid metabolism in human platelets by RHC 80267, a diacylglycerol lipase inhibitor

The diacylglycerol lipase inhibitor, RHC 80267, 1,6-di(O-(carbamoyl)cyclohexanone oxime)hexane, was tested for its ability to block the release of arachidonic acid from human platelets. At a concentration (10 microM) reported to completely inhibit diacylglycerol lipase in fractions of broken platelets, RHC 80267 had no effect on diacylglycerol lipase activity or the release of arachidonic acid from washed human platelets stimulated with collagen. At a high concentration (250 microM), the compound inhibited the formation of arachidonyl-monoacylglycerol by 70% and the release of arachidonate by 60%. However, at this concentration RHC 80267 was found to inhibit cyclooxygenase activity, phospholipase C activity and the hydrolysis of phosphatidylcholine (PC) (presumably by inhibiting phospholipase A2). The phospholipase C inhibition was attributed to the inhibition of prostaglandin H2 formation, as it was alleviated by the addition of the endoperoxide analog, U-46619. PC hydrolysis was only partially restored with U-46619, suggesting that RHC 80267 directly alters phospholipase A2 activity. The inhibition of arachidonate release observed was accounted for by the inhibition of PC hydrolysis. We conclude that RHC 80267, because of its lack of specificity at concentrations needed to inhibit diacylglycerol lipase, is an unsuitable inhibitor for studying the release of arachidonic acid in intact human platelets.     

Stimulation of arachidonic acid release by guanine nucleotide in saponin-permeabilized neutrophils: evidence for involvement of GTP-binding protein in phospholipase A2 activation

Addition of a guanine nucleotide analog, guanosine 5'-O-(thiotriphosphate) (GTP gamma S)(1-100 microM) induced release of [3H]arachidonic acid from [3H]arachidonate-prelabeled rabbit neutrophils permeabilized with saponin. The chemotactic peptide N-formyl-methionyl-leucyl-phenylalanine (fMLP)-induced arachidonate release was enhanced by GTP gamma S, Ca2+, or their combination. Ca2+ alone (up to 100 microM) did not effectively stimulate lipid turnover. However, the combination of fMLP plus GTP gamma S elicited greater than additional effects in the presence of resting level of free Ca2+. The addition of 100 microM of GTP gamma S reduced the Ca2+ requirement for arachidonic acid liberation induced by fMLP. Pretreatment of neutrophils with pertussis toxin resulted in the abolition of arachidonate release and diacylglycerol formation. Neomycin (1 mM) caused no significant reduction of arachidonate release. In contrast, about 40% of GTP gamma S-induced arachidonate release was inhibited by a diacylglycerol lipase inhibitor, RHC 80267 (30 microM). These observations indicate that liberation of arachidonic acid is mediated by phospholipase A2 and also by phospholipase C/diacylglycerol lipase pathways. Fluoride, which bypasses the receptor and directly activates G proteins, induced arachidonic acid release and diacylglycerol formation. The fluoride-induced arachidonate release also appeared to be mediated by these two pathways. The loss of [3H]arachidonate was seen in phosphatidylinositol, phosphatidylcholine, and phosphatidylethanolamine. These data indicate that a G protein is involved between the binding of fMLP to its receptor and activation of phospholipase A2, and also that the arachidonic acid release is mediated by both phospholipase A2 and phospholipase C/diacylglycerol lipase.     

The alpha 1-adrenergic transduction system in hamster brown adipocytes. Release of arachidonic acid accompanies activation of phospholipase C.

Previous studies of brown adipocytes identified an increased breakdown of phosphoinositides after selective alpha 1-adrenergic-receptor activation. The present paper reports that this response, elicited with phenylephrine in the presence of propranolol and measured as the accumulation of [3H]inositol phosphates, is accompanied by increased release of [3H]arachidonic acid from cells prelabelled with [3H]arachidonic acid. Differences between stimulated arachidonic acid release and formation of inositol phosphates included a requirement for extracellular Ca2+ for stimulated release of arachidonic acid but not for the formation of inositol phosphates and the preferential inhibition of inositol phosphate formation by phorbol 12-myristate 13-acetate. The release of arachidonic acid in response to phenylephrine was associated with an accumulation of [3H]arachidonic acid-labelled diacylglycerol, and this response was not dependent on extracellular Ca2+ but was partially prevented by treatment with the phorbol ester. The release of arachidonic acid was also stimulated by melittin, which increases the activity of phospholipase A2, by ionophore A23187, by lipolytic stimulation with forskolin and by exogenous phospholipase C. The arachidonic acid response to phospholipase C was completely blocked by RHC 80267, an inhibitor of diacylglycerol lipase, but this inhibitor had no effect on release stimulated with melittin or A23187 and inhibited phenylephrine-stimulated release by only 40%. The arachidonate response to forskolin was additive with the responses to either phenylephrine or exogenous phospholipase C. These data indicate that brown adipocytes are capable of releasing arachidonic acid from neutral lipids via triacylglycerol lipolysis, and from phospholipids via phospholipase A2 or by the sequential activities of phospholipase C and diacylglycerol lipase. Our findings also suggest that the action of phenylephrine to promote the liberation of arachidonic acid utilizes both of these reactions.     

Arachidonic Acid Liberated by Diacylglycerol Lipase Is Essential for the Release Mechanism in Chromaffin Cells from Bovine Adrenal Medulla

Chromaffin cells from bovine adrenal medulla secrete catecholamines on stimulation with acetylcholine. In addition to the activation of the phosphatidylinositol cycle, arachidonic acid is generated, which was thought to be the result of phospholipase A2 activation. We have demonstrated in isolated plasma membranes of these cells that arachidonic acid is generated by a two-step reaction of diacylglycerol and monoacylglycerol lipase splitting diacylglycerol, which originates from the action of phospholipase C on phosphatidylinositols. No phospholipase A2 activity could be detected in plasma membranes so far. External addition of arachidonic acid increases the release in the absence and in the presence of agonist. Inhibition of the diacylglycerol lipase by RHC 80267 suppresses the catecholamine release, which is restored on addition of arachidonic acid. This effect, however, is reversed by lipoxygenase inhibitors, indicating that it is not arachidonic acid itself, but one of its lipoxygenase products, that is essential for inducing exocytosis.     

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.     

Stimulation of inositol phosphate and diacylglycerol production by RHC 80267, a diacylglycerol-lipase inhibitor, in rat gastric parietal cells: effects on hydrogen ion secretion

RHC 80267, on inhibitor of diacylglycerol lipase, was used to investigate the role of diacylglycerol in acid secretion by isolated rat gastric parietal cells. Unexpectedly, RHC 80267 stimulated the production of inositol phosphates in [3H]inositol-prelabeled cells and increased levels of 32P-labeled phosphatidic acid to the same degree as did carbachol. RHC 80267 increased diacylglycerol to a greater extent than did carbachol, and additionally decreased levels of [3H]arachidonic acid. This suggests that RHC 80267 stimulated phospholipase C and inhibited diacylglycerol lipase in parietal cells. RHC inhibited [14C]aminopyrine uptake, a measure of acid secretion, stimulated by carbachol or by simultaneous addition of carbachol and dibutyryl-cAMP. These data support the model that the diacylglycerol/protein kinase C branch of the phosphoinositide system is inhibitory to acid secretion.     

Involvement of phospholipase D activation in endothelin-1-induced release of arachidonic acid in osteoblast-like cells

In a previous study, we have shown that endothelin-1 (ET-1) activates phospholipase D independently from protein kinase C in osteoblast-like MC3T3-E1 cells. It is well recognized that phosphatidylycholine hydrolysis by phospholipase D generates phosphatidic acid, which can be further degraded by phosphatidic acid phosphohydrolase to diacylglycerol. In the present study, we investigated the role of phospholipase D activation in ET-1-induced arachidonic acid release and prostaglandin E₂ (PGE₂) synthesis in osteoblast-like MC3T3-E1 cells. ET-1 stimulated arachidonic acid release dose-dependently in the range between 0.1 nM and 0.1 μM. Propranolol, an inhibitor of phosphatidic acid phosphohydrolase, significantly inhibited the ET-1-induced arachidonic acid release in a dose-dependent manner as well as the ET-1-induced diacylglycerol formation. 1,6-bis-(cyclohexyloxyminocarbonylamino)-hexane (RHC-80267), an inhibitor of diacylglycerol lipase, significantly suppressed the ET-1-induced arachidonic acid release. The pretreatment with propranolol and RHC-80267 also inhibited the ET-1-induced PGE₂ synthesis. These results strongly suggest that phosphatidylcholine hydrolysis by phospholipase D is involved in the arachidonic acid release induced by ET-1 in osteoblast-like cells. J. Cell. Biochem. 64:376–381. © 1997 Wiley-Liss, Inc.     

Neurite Outgrowth Stimulated by L1 Requires Calcium Influx into Neurons but is Not Associated with Changes in Steady State Levels of Calcium in Growth Cones

L1, NCAM and N-cadherin are cell adhesion molecules (CAMs), present on neuronal growth cones, which promote cell-contact dependent axonal growth by activating a second messenger pathway in neurons that requires calcium influx through L- and N- type calcium channels. In the present study we show that two of these CAMs, (L1 and N-cadherin) can stimulate neurite regeneration from axotomised adult dorsal root ganglion (DRG) neurons cultured in vitro and that this response can be fully inhibited by agents that block or negate the effect of calcium influx into the neurons. However although the response required calcium influx into neurons, it was not associated with an increase in the steady state levels of calcium in neuronal growth cones. These results suggest that small localised changes, or increases in the rate of calcium cycling, in growth cones and/or filopodia, are more important for regulating axonal growth than changes in the steady-state level of calcium.     

Neurite Outgrowth Stimulated by L1 Requires Calcium Influx into Neurons but is Not Associated with Changes in Steady State Levels of Calcium in Growth Cones

L1, NCAM and N-cadherin are cell adhesion molecules (CAMs), present on neuronal growth cones, which promote cell-contact dependent axonal growth by activating a second messenger pathway in neurons that requires calcium influx through L- and N- type calcium channels. In the present study we show that two of these CAMs, (L1 and N-cadherin) can stimulate neurite regeneration from axotomised adult dorsal root ganglion (DRG) neurons cultured in vitro and that this response can be fully inhibited by agents that block or negate the effect of calcium influx into the neurons. However although the response required calcium influx into neurons, it was not associated with an increase in the steady state levels of calcium in neuronal growth cones. These results suggest that small localised changes, or increases in the rate of calcium cycling, in growth cones and/or filopodia, are more important for regulating axonal growth than changes in the steady-state level of calcium.