Target ß-catenin/CD44/Nanog axis in colon cancer cells by certain N'-(2-oxoindolin-3-ylidene)-2-(benzyloxy)benzohydrazides.

Cell surface molecule CD44 plays a major role in regulation of cancer stem cells CSCs on both phenotypic and functional level, however chemical inhibition approach of CD44 to targets CSCs is poorly studied. Herein, we report the discovery of certain N'-(2-oxoindolin-3-ylidene)-2-(benzyloxy)benzohydrazides as a novel inhibitor of CD44. Molecular docking study showed interference of the scaffold of these compounds with ß-catenin/TCF-4 complex, building a direct relationship between CD44 inhibition and observed well-fitted binding domain. Compound 11a, most potent member elicits inhibition effect on TCF/LEF reporter activity conformed the involvement of Wnt pathway inhibition as a mechanism of action. Furthermore, the treatment by the mentioned compound leads to inhibition of embryonic transcriptional factor Nanog but not Sox2 or Oct-4 suggested specific targeted effect. Moreover, the cytotoxicity and cell cycle effect of this series seems to be dependent on CD44 expression.

Fascin is involved in the chemotherapeutic resistance of breast cancer cells predominantly via the PI3K/Akt pathway.

BACKGROUND: A major therapeutic challenge for breast cancer is the ability of cancer cells to evade killing of conventional chemotherapeutic agents. We have recently reported the actin-bundling protein (fascin) as a major regulator of breast cancer metastasis and survival. METHODS: Survival of breast cancer patients that received chemotherapy and xenograft tumour model was used to assess the effect of chemotherapy on fascin-positive and -negative breast cancer cells. Molecular and cellular assays were used to gain in-depth understanding of the relationship between fascin and chemoresistance. RESULTS: We showed a significant correlation between fascin expression and shorter survival in breast cancer patients who received chemotherapy. In xenograft experiments, fascin-positive cancer cells displayed significantly more resistance to chemotherapy-mediated apoptotic cell death than fascin-negative counterparts. This increased chemoresistance was at least partially mediated through PI3K/Akt signalling, and was paralleled by increased FAK phosphorylation, enhanced expression of the inhibitor of apoptosis proteins (XIAP and Livin) and suppression of the proapoptotic markers (caspase 9, caspase 3 and PARP). CONCLUSIONS: This is the first report to demonstrate fascin involvement in breast cancer chemotherapeutic resistance, supporting the development of fascin-targeting drugs for better treatment of chemoresistance breast cancer.

Pleiotropic effects of YC-1 selectively inhibit pathological retinal neovascularization and promote physiological revascularization in a mouse model of oxygen-induced retinopathy.

Vascular endothelial growth factor (VEGF) and inducible nitric-oxide synthase (iNOS) have been implicated in ischemia-induced retinal neovascularization. Retinal ischemia has been shown to induce VEGF and iNOS expression. It has been postulated that one of the crucial consequences of iNOS expression in the ischemic retina is the inhibition of angiogenesis. Furthermore, iNOS was shown to be overexpressed in Müller cells from patients with diabetic retinopathy. YC-1, a small molecule inhibitor of hypoxia-inducible factor (HIF)-1 alpha, has been shown to inhibit iNOS expression in various tissue models. Our aim was to assess the pleiotropic effects of YC-1 in an oxygen-induced retinopathy (OIR) mouse model and evaluate its therapeutic potential in HIF-1- and iNOS-mediated retinal pathologies. Dual-injections of YC-1 into the neovascular retinas decreased the total retinopathy score, inhibited vaso-obliteration and pathologic tuft formation, and concomitantly promoted physiological retinal revascularization, compared with dimethyl sulfoxide (DMSO)-treated group. Furthermore, YC-1-treated retinas exhibited a marked increase in immunoreactivities for CD31 and von Willebrand factor and displayed significant inhibition in HIF-1alpha protein expression. Furthermore, YC-1 down-regulated VEGF, erythropoietin, endothelin-1, matrix metalloproteinase-9, and iNOS message and protein levels. When hypoxic Müller and neuoroglial cells were treated with YC-1, iNOS mRNA and protein levels were reduced in a dose-dependent fashion. We demonstrate that YC-1 inhibits pathological retinal neovascularization by exhibiting antineovascular activities, which impaired ischemia-induced expression of HIF-1 and its downstream angiogenic molecules. Furthermore, YC-1 enhanced physiological revascularization of the retinal vascular plexuses via the inhibition of iNOS mRNA and protein expressions. The pleiotropic effects of YC-1 allude to its possible use as a promising therapeutic iNOS inhibitor candidate for the treatment of retinal neovascularization.

Modulating the hypoxia-inducible factor signaling pathway as a therapeutic modality to regulate retinal angiogenesis.

Hypoxia-inducible factor (HIF) signaling cascade plays a critical role in angiogenesis by activating the transcription of genes encoding angiogenic growth factors. This study evaluated the effects of YC-1, a HIF-1 inhibitor, on the morphological, biochemical and molecular changes in human retinal microvascular endothelial cells. We found that YC-1 suppressed vascular endothelial cell proliferation, migration and tube formation, while it significantly increased the proteasome activity. Moreover, YC-1 induced a G(0)/G(1) cell-cycle arrest, whereas it exerted only an insignificant proapoptotic effects. Under normoxia or hypoxia, YC-1 did not alter the morphology or the cell viability. Additionally, under hypoxic conditions, YC-1 downregulated HIF-2alpha, VEGF, EPO, ET-1, and MMP-9 mRNA and protein levels, this was accompanied by a significant decrease in the MMP-9 activity. YC-1 decreased the basal expression of HIF-1alpha protein under normoxia, whereas it inhibited HIF-1alpha protein synthesis, stability, and nuclear translocation mechanisms under hypoxia. Furthermore, in a 3D collagen matrix model using mouse retinal explants cultured under normoxic and hypoxic conditions, YC-1; (1) inhibited outgrowth of new vessel sprouts; (2) reduced VEGF expression; (3) dramatically decreased the vessels immunoreactivities for CD31 and von Willebrand Factor (vWF); and (4) was highly effective in reducing the vascular density within the retina, compared to controls. These findings indicate that YC-1 possesses several antiangiogenic properties, both in vitro and ex vivo, which could be exploited as valuable therapeutic potentials to inhibit formation and the growth of new retinal vessels in the hypoxic retina.

Native low density lipoprotein-induced calcium transients trigger VCAM-1 and E-selectin expression in cultured human vascular endothelial cells.

Low density lipoprotein (LDL) interactions with the endothelium are thought to play a major role in the development of atherosclerosis. The mechanism(s) involved are not fully understood, although several lines of evidence support the idea that oxidation of LDL increases its atherogenicity. In this study we report for the first time that native LDL (n-LDL) binding to the LDL receptor (100-700 mug/ml) triggers a rise in intracellular calcium which acts as a second messenger to induce vascular cell adhesion molecule-1 (VCAM-1) expression in human coronary artery (HCAEC) and pig aortic endothelial cells (PAEC) and VCAM-1 and E-selectin expression in human aortic (HAEC) endothelial cells. Preincubation of HCAEC with a monoclonal antibody (IgGC7) to the classical LDL receptor or pretreatment with pertussis toxin blocked the n-LDL-induced calcium transients. Preincubation of each of the endothelial cell lines with the calcium chelator 1,-2-bis(o-aminophenoxy)ethane-N,N,N', N'-tetraacetic acetomethyl ester (BAPTA/AM) prevented the expression of VCAM-1 and E-selectin. The increase in VCAM-1 by n-LDL results in increased monocyte binding to HCAEC which can be attenuated by inhibiting the intracellular calcium rise or by blocking the VCAM-1 binding sites. These studies in human and pig endothelial cells link calcium signaling conferred by n-LDL to mechanisms controlling the expression of endothelial cell adhesion molecules involved in atherogenesis.

Human neutrophil gene expression profiling following xenogeneic encounter with porcine aortic endothelial cells: the occult role of neutrophils in xenograft rejection revealed.

The role of innate immune cells in the recognition and activation of xenogeneic endothelium has always been considered secondary to the initial insult of xenoreactive natural antibodies (XNA) and complement. It was argued, however, that innate immune cells are capable of recognizing and activating xenogeneic endothelium in the absence XNA and complement. Here, we show that porcine aortic endothelial cells (PAECs) activate human neutrophils directly. This contact-dependent activation causes a transient calcium rise leading to increased reactive oxygen metabolite (ROM) production. Neutrophil gene-expression profiling using an adenylate uridylate-rich element-based microarray revealed a dramatic change in the neutrophil gene profiles upon exposure to PAECs. The PAEC-dependent neutrophil transcriptional activity was further confirmed by real-time polymerase chain reaction, which revealed a rapid increase in the mRNA message of a number of inflammatory cytokines. The activation of human neutrophils by PAECs was independent of galactose alpha1,3-galactose (Galalpha1,3-gal) structures, as inclusion of saturating concentrations of anti-Galalpha1,3-gal l antibodies had no significant effect. Furthermore, this activation was inhibited in the presence of the calcium chelator 1,2-bis(O-aminophenyl-ethane-ethane)-N,N,N',N'-tetraacetic acid-acetoxymethyl ester and the ROM inhibitor diphelylene iodonium. Our data illustrate the direct activation of innate immune cells by PAECs in the absence of XNA and complement and suggest alternative recognition sites between PAECs and human innate immune cells.

TSH overcomes Braf(V600E)-induced senescence to promote tumor progression via downregulation of p53 expression in papillary thyroid cancer.

The BRAF(V600E) mutation is found in approximately 40% of papillary thyroid cancers (PTC). Mice with thyroid-specific expression of Braf(V600E) (TPO-Braf(V600E)) develop PTC rapidly with high levels of serum thyroid-stimulating hormone (TSH). It is unclear to what extent the elevated TSH contributes to tumor progression. To investigate the progression of Braf(V600E)-induced PTC (BVE-PTC) under normal TSH, we transplanted BVE-PTC tumors subcutaneously into nude and TPO-Braf(WT) mice. Regression of the transplanted tumors was observed in both nude and TPO-Braf(WT) mice. They were surrounded by heavy lymphocyte infiltration and oncogene-induced senescence (OIS) was demonstrated by strong ß-gal staining and absence of Ki-67 expression. In contrast, BVE-PTC transplants continued to grow when transplanted into TPO-Braf(V600E) mice. The expression of Trp53 was increased in tumor transplants undergoing OIS. Trp53 inactivation reversed OIS and enabled tumor transplants to grow in nude mice with characteristic cell morphology of anaplastic thyroid cancer (ATC). PTC-to-ATC transformation was also observed in primary BVE-PTC tumors. ATC cells derived from Trp53 knockout tumors had increased PI3K/AKT signaling and became resistant to Braf(V600E) inhibitor PLX4720, which could be overcome by combined treatment of PI3K inhibitor LY294002 and PLX4720. In conclusion, BVE-PTC progression could be contained via p53-dependent OIS and TSH is a major disruptor of this balance. Simultaneous targeting of both MAPK and PI3K/AKT pathways offer a better therapeutic outcome against ATC. The current study reinforces the importance of rigorous control of serum TSH in PTC patients.

Actin polymerization modifies stimulus-oxidase coupling in rat neutrophils.

Oxidase activity in rat neutrophils was monitored by oxygen consumption rate and luminol-dependent chemiluminescence. Two agents which inhibit actin polymerization, cytochalasin B and dihydrocytochalasin B, produced a marked enhancement (up to 10-fold) of oxidase activation induced by two Ca2+-dependent stimuli, chemotactic peptide and ionophore A23187. In contrast, activation by the calcium-independent stimulus, phorbol myristate acetate, was unaffected by these agents. Other agents that interact with the cytoskeleton, phalloidin and colchicine have no effect on activation by any stimulus tested. The effect of cytochalasin B, when added after stimulation by chemotactic peptide, was transient with t0.5 approx. 10 s. Similarly, the degree of actin polymerization following stimulation by chemotactic peptide was transient, decaying with a t0.5 of approx. 10 s. The half-maximal concentration of cytochalasin B for inhibition of actin polymerization was similar to that for enhancement of oxidase activation. It was concluded, therefore, that the intracellular Ca2+ rise in rat neutrophils that accompanies stimulation by chemotactic peptide affects actin polymerization in a manner that modifies oxidase activation.

Inflammatory, hemostatic, and clinical changes in a baboon experimental model for heatstroke.

The mortality and neurological morbidity in heatstroke have been attributed to the host's inflammatory and hemostatic responses to heat stress, suggesting that immunomodulation may improve outcome. We postulated that an experimental baboon model of heatstroke will reproduce human responses and clinical outcome to allow testing of new therapeutic strategies. Eight anesthetized juvenile baboons (Papio hamadryas) were subjected to heat stress in an incubator maintained at 44-47 degrees C until rectal temperature attained 42.5 degrees C (moderate heatstroke; n = 4) or systolic arterial pressure fell to <90 mmHg (severe heatstroke; n = 4) and were allowed to recover at room temperature. Four sham-heated animals served as a control group. Rectal temperature at the end of heat stress was 42.5 +/- 0.0 and 43.3 +/- 0.1 degrees C, respectively. All heat-stressed animals had systemic inflammation and activated coagulation, indicated by increased plasma IL-6, prothrombin time, activated partial thromboplastin time, and D-dimer levels, and decreased platelet count. Biochemical markers and/or histology evidenced cellular injury/dysfunction: plasma levels of thrombomodulin, creatinine, creatine kinase, lactic dehydrogenase, and alanine aminotransferase were increased, and varying degrees of tissue damage were present in liver, brain, and gut. No baboon with severe heatstroke survived. Neurological morbidity but no mortality was observed in baboons with moderate heatstroke. Nonsurvivors displayed significantly greater coagulopathy, inflammatory activity, and tissue injury than survivors. Sham-heated animals had an uneventful course. Heat stress elicited distinct patterns of inflammatory and hemostatic responses associated with outcome. The baboon model of heatstroke appears suitable for testing whether immunomodulation of the host's responses can improve outcome.

Correlation of antigenic expression with progress in antibiotic therapy of acute human brucellosis.

Human brucellosis is a zoonotic disease which is endemic in Saudi Arabia. The aim of this study was to investigate the humoral immune responses and identify the target antigens that persist at different stages in human brucellosis during antibiotic therapy. To do this, an acute case of accidental nosocomial infection was studied experimentally. Blood was collected from the patient at the time of diagnosis, and at weekly intervals during therapy until remission. IgG and IgM immunoblotting was used to characterize specific antigenic determinants, and ELISA antibody titration was performed to quantify the circulating antibodies. Results indicated that protein bands of 12-13.5 kDa bound IgG in the patient's sera but did not bind IgM on immunoblots and are probably not specific for, or important in, early stage infections. However, an 18 kDa band persisted during infection through remission. The pivotal and most important findings were that the number of protein bands seen on immunoblots, the magnitude of ELISA antibody titres and the concomitant changes in the intensity of the polypeptide bands of 42-43 kDa were positively correlated with the stage of infection. High numbers of anti-IgG and -IgM immunoblot bands coupled with high ELISA antibody titres and a concomitant increase in intensity of the 42-43 kDa bands were positively correlated with acute and severe infection. Conversely, a reduction in the number of polypeptide bands as well as a decrease in the intensity, until the complete disappearance of the 42-43 kDa bands, coupled with low (baseline) ELISA antibody titration values indicated successful treatment and remission. The routine use of the methods described here to ascertain the stage of the disease, assess the progress of antimicrobial therapy and monitor cases of relapse in human brucellosis is suggested.

Immobilization of rolling NK cells on platelet-borne P-selectin under flow by proinflammatory stimuli, interleukin-12, and leukotriene B4.

Recruitment of leukocytes from bloodstream to extrahematic sites is tightly regulated by a variety of adhesion molecules that are expressed on the leukocytes and the vessel walls. In this manuscript, we describe the interactions between natural killer (NK) cells and activated, autologous platelets under physiologic flow. We found that surface-adherent human platelets are capable of recruiting human NK cells from flow and that this recruitment is characterized by an initial tethering followed by a rolling phase. Both phases were dependent on the adhesion molecule P-selectin and its counter-ligand on the NK cells (P-selectin glycoprotein ligand 1). Activation of rolling NK cells with inflammatory mediators commonly found in atherosclerotic plaques (interleukin-12 and leukotriene B4) causes immediate cessation of the rolling process and conversion to stationary adhesion. Blocking antibodies to the adhesion molecules membrane-activated complex-1 and leukocyte function antigen-1 inhibited this conversion. Our data suggest that platelets deposited at sites of vascular injury may provide an alternative substrate to endothelial cells for initial recruitment of NK cells to the vessel wall. This may result in extravasation of the NK cells if the appropriate chemotactic signal is applied. These data implicate the P-selectin and integrin family of adhesion molecules in the recruitment of NK cells to atherosclerotic sites.

Monomeric human IgE evokes a transient calcium rise in individual human neutrophils.

Digital fluorescence calcium imaging was used to investigate and identify the primary biological responses of human neutrophils to monomeric immunoglobulin E (IgE). Treatment of neutrophils with IgE caused a transient rise in the level of intracellular calcium that was inhibited by pertussis toxin. The calcium rise was due mainly to release from an intracellular membrane-enclosed store that is also sensitive to the chemotactic peptide formyl-Met-Leu-Phe. The IgE-induced calcium transient was independent of Fc gamma receptors and of Fc epsilon receptor ligation. Our data suggest that the mere binding of IgE to neutrophils is sufficient to evoke a biological response without the need for IgE/receptor cross-linking.

The use of fura-2 to determine the relationship between cytoplasmic free Ca2+ and oxidase activation in rat neutrophils.

Incubation of rat neutrophils with fura-2-acetoxy-methyl ester (fura-2/AM) resulted in the loading of fura-2 almost exclusively into the cytoplasm. Despite the additional presence of fura-2/AM esterase activity in the granules, only 1.5% of cell-associated fura-2 was located within these organelles. Fura-2 leaked from neutrophils at an acceptably low rate 0.16 +/- 0.05% min-1 at 37 degrees C. At intracellular concentrations of fura-2 up to 500 microM, there was no effect on oxidase activation; although the cellular ATP content was reduced to approximately 50%. The peptide, f-met-leu-phe (fmlp), 1 microM, produced intensity changes of fluorescence excited at 340nm and 380nm which were consistent with a cytoplasmic Ca2+ rise from the resting level of 94 +/- 13nM to 768 +/- 173nM (n = 6). Intracellular concentrations of fura-2 greater than 1mM were required to buffer effectively this rise, and it was estimated that an intracellular fura-2 concentration required for a high signal:autofluorescence ratio (100 microM) the cytoplasmic Ca2+ buffering capacity of the cells was increased by only 10%. The rise in cytoplasmic free Ca2+ induced by the peptide preceded activation of the oxidase by several seconds, and the magnitude of the response was dependent on the extent of the Ca2+ rise, half-maximal activation being achieved at approx. 600nM. These data were therefore consistent with a secondary messenger role for cytoplasmic Ca2+ in triggering neutrophil oxidase activation.

Botulinum C2 toxin potentiates activation of the neutrophil oxidase. Further evidence of a role for actin polymerization.

Botulinum C2 toxin was employed as a specific inhibitor of actin polymerization in rat neutrophils to determine its role in oxidase activation. This toxin was shown to inhibit actin polymerization and the microfilament-dependent function, phagocytosis. Oxidase activation in response to the chemotactic peptide, f-Met-Leu-Phe (FMLP) was enhanced approx. 3-fold. The enhancement by C2 toxin did not occur in cells pretreated with cytochalasin B. C2 toxin had no significant effect on the FMLP-induced intracellular Ca2+ rise. These data are consistent with an inhibitory role for actin polymerization in oxidase activation.

Vaccinia virus complement control protein is capable of protecting xenoendothelial cells from antibody binding and killing by human complement and cytotoxic cells.

BACKGROUND: Vaccinia virus complement control protein (VCP) was the first secretory microbial protein shown to have structural similarity to the family of complement control proteins. VCP can block both the classical and alternate complement pathways. Recently, VCP has been shown to bind to heparin, and this property contributes to separate functions, making the molecule a multifunctional protein. METHODS: VCP prepared from a natural infection of RK-13 cells with vaccinia virus was purified to homogeneity. Cultured pig aortic endothelial cells (PAECs) were mixed with human serum, anti-Gal alpha1,3 Gal antibody, neutrophils, or natural killer (NK) cells in the presence or absence of VCP and either direct binding of FITC-labeled antibody or killing by cytotoxic cells was estimated. RESULTS: Our cell culture studies demonstrate that VCP blocks complement-mediated killing of PAECs by human serum in a dose-dependent manner. We also demonstrate that VCP is capable of blocking Gal alpha1,3 Gal binding sites on PAECS. Surprisingly, VCP effectively blocked interactions between PAECs and cytotoxic cells such as human naive neutrophils and NK cells. CONCLUSION: VCP is a novel protein amongst the complement control protein family and can, not only block xenorejection by inhibiting complement but also by blocking killing by cytotoxic cells.

Alpha-gal-independent dual recognition and activation of xenogeneic endothelial cells and human naïve natural killer cells.

BACKGROUND: Interaction between vascularized xenograft and host immune system is thought to occur via Galactose alpha (1,3) Galactose (Gala 1,3 gal) structures decorating the xenograft. METHODS: We raised anti-Gala 1,3 gal-BSA polyclonal antibodies in baboons and investigated effect(s) of these antibodies as well as soluble Gala 1,3 gal-BSA on human naive natural killer (NK) cell interactions with porcine aortic endothelial cells. RESULTS: We demonstrate that human naive (unstimulated) NK cells recognize xenogeneic endothelial cells under conditions where binding to the Gala 1,3 gal structures is minimized by the presence of blocking anti-Gala 1,3 gal IgG or soluble Gala 1-3 gal and in the absence of xenoreactive natural antibodies and complement. After xenogeneic encounter both endothelial cells and human NK cells are activated. Endothelial cell activation is rapid and is manifested initially by an intraendothelial calcium transient and subsequently by expression of P-selectin and vascular endothelial cell adhesion molecule-1 on the xenoendothelium surface. NK cell activation is manifested by increased expression of perforin and increased cytotoxicity towards the xenoendothelium. Neither recognition nor activation of the xenoendothelium was affected by the introduction of either anti-Gala 1,3 gal IgG or soluble Gala 1-3 gal. CONCLUSION: Our data provide evidence that innate immune cells, such as NK cells, recognize and activate xenoendothelial cells independently of Gala 1-3 gal structures and raise the possibility of novel interactive sites on both human naive NK cells and discordant xenogeneic endothelium.

Diacylglycerol kinase inhibitor, R59022, potentiates neutrophil oxidase activation by Ca2+-dependent stimuli. Evidence for two separate but convergent pathways.

An inhibitor of diacylglycerol kinase, R59022, enhanced activation of the neutrophil oxidase stimulated by the Ca2+-ionophore, A23187 (1 microM), and by N-formyl-methionyl leucyl-phenylalanine (1 microM). The enhancement was reversed by two inhibitors of c-kinase, retinal (10 microM), and gossypol (20 microM). Activation by phorbol-myristyl-acetate and unopsonised latex beads were not enhanced. It was concluded that the chemotactic peptide generated diacylglycerol, but that maximum activation of c-kinase by this route was not achievable. The role of diacylglycerol in activation by beads remained unclear.

Ca(2+)-dependent production of reactive oxygen metabolites by human neutrophils in response to fluorinated propranolol analogues.

Fluorinated analogues of propranolol, namely trifluoroethyl propranolol (F3), pentafluoropropyl propranolol (F5), and heptafluorobutyl propranolol (F7), were found to induce reactive oxygen metabolite (ROM) production in human neutrophils in a dose-dependent manner. Preincubation of neutrophils with the calcium chelator BAPTA-AM or the tyrosine kinase inhibitor genistein inhibited this ROM production. Direct measurements of intracellular calcium revealed that these analogues caused a transient increase in intracellular calcium. In addition, these fluorinated analogues of propranolol caused a transient increase in actin polymerization. The effects of these compounds were found to be dependent upon the degree of fluorination of the parent compound. Propranolol, on the other hand, had no direct effect on ROM, calcium, or actin polymerization when added alone to neutrophils, although it did modify responses of cells to various stimuli. Whereas ROM production induced by the chemotactic peptide formyl-methionyl-leucyl-phenylalanine was enhanced in a dose-dependent manner, the response to the particulate stimulus, latex beads, was abolished.

Spatial organization of calcium dynamics in growth cones of sensory neurones.

The concentration of calcium ions in the cytosol ([Ca2+]i) has a dominant influence on neuronal development. A [Ca2+]i rise can, depending on the amplitude and location, promote outgrowth or dramatically inhibit it. We have used the fluorescent calcium indicators Fura-2 and Fura-2 dextran to measure [Ca2+]i dynamics in sensory neurones from the adult rat. [Ca2+]i was low and uniform in advancing growth cones, even during specific behaviours such as protrusion, filling and consolidation. A brief train of action potentials caused [Ca2+]i to rise at the extreme leading edge of the growth cone. [Ca2+]i changes in more proximal regions of the growth cone were much smaller. This spatially organized [Ca2+]i change, which may result from a concentration of calcium channels at the growth cone leading edge, is likely to function in spontaneously active regenerating axons in vivo to specifically activate calcium-dependent processes at the growth cone tip.

A narrow window of intracellular calcium concentration is optimal for neurite outgrowth in rat sensory neurones.

We have examined neurite outgrowth in rat sensory neurones when cytosolic free calcium concentration ([Ca2+]i) was varied in the range 0-60 nM. Neurite outgrowth was maximal at 35 nM [Ca2+]i and was reduced at higher and lower values of [Ca2+]i. These results provide direct evidence for Mattson and Kater's suggestion of an optimal calcium range for growth cone function.