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.

Thrombin causes neurite retraction in neuronal cells through activation of cell surface receptors.

The mechanism by which thrombin induces neurite retraction was studied in NB2a mouse neuroblastoma cells. The rapid effect of thrombin (completed within minutes) appears to involve an interaction between its anion-binding exosite and the thrombin receptor. Structural alterations of this site increase the EC50 for thrombin-mediated retraction, and a hirudin C-terminal peptide that blocks this site inhibits the response. The thrombin effect was mimicked by a 14 amino acid peptide starting with Ser-42, at the proposed cleavage site of the human thrombin receptor. The protein kinase inhibitors staurosporine and H-7 blocked thrombin-induced retraction. It is therefore proposed that thrombin-mediated neurite retraction is caused by cleavage-induced activation of the thrombin receptor and involves stimulation of a protein kinase(s).

Adenosine 5'-triphosphate synthesis and metabolism localized in neurites of cultured sympathetic neurons.

Adenosine triphosphate synthesis and metabolism in cultured sympathetic neurons was studied after the incorporation of [2-3H]adenine into intact or microdissected neurites to determine whether ATP is provided locally during neurite outgrowth, when and where it is synthesized and how its levels are regulated at rest and following depolarization. Neurites maintained an independent capability for synthesis of ATP at any stage of growth: [3H]ATP levels increased in neurites in direct proportion to neurite length and equivalent amounts of [3H]ATP were synthesized by intact neurites, by neurites separated from cell bodies or by neurites further segmented into sections. Thus, metabolic labelling of cultured neurons with [3H]adenine provides a simple method to measure relative neurite outgrowth. Neurite ATP was maintained mainly by respiration but also by glycolysis and [3H]ATP levels were stable for at least 14 h after adenine withdrawal when cells were at rest. Depolarization overcame respiratory control, causing a quantitative conversion of ATP to adenosine monophosphate (AMP) and inosine monophosphate (IMP) and the release of nucleosides (adenosine and inosine) and nucleotides [adenosine diphosphate (ADP) and adenosine monophosphate (AMP)]. Release of nucleosides, but not of nucleotides or [3H]noradrenaline, was enhanced by NaN3 or 2-deoxyglucose under nondepolarizing conditions and was prevented by the adenosine transport inhibitor p-nitrobenzyl-6-thioinosine. It is concluded that neurites can use local mechanisms for ATP synthesis that do not depend on a functional connection to the cell body. Any metabolic stress which causes ATP breakdown causes these cells to express a transient purinergic phenotype involving release of adenosine and inosine by facilitated diffusion. To promote the release of purine nucleotides, however, more specific stimuli are required.

Simultaneous analysis of adenosine 3',5'-cyclic monophosphate accumulation and adenosine 5'-triphosphate metabolism in cultured cells preincubated with [2-3H]adenine.

A simple and sensitive method based on metabolic labeling was developed for the simultaneous analysis of cyclic AMP accumulation and ATP metabolism in small numbers of cultured cells. Cells are preincubated overnight with [2-3H]adenine to label the ATP pool to a high specific activity. After cell stimulation the metabolites are extracted in a small volume of aqueous acetic acid and chloroform and separated without further manipulation by one-dimensional thin-layer chromatography and the radioactivity incorporated is determined by liquid scintillation counting. With ATP labeled to about 6 Ci/mmol, the lower limit of cyclic AMP detection is 2 fmol, a sensitivity that is comparable to the radioimmunoassay of acetylated cyclic AMP. In primary neurons and a neural cell line, for example, levels of ATP and its metabolites change when large amounts of cyclic AMP are generated, each with its unique pattern. ATP is also depleted when metabolic energy is consumed concomitantly with stimulation of cyclic AMP production by agonists, probably as a result of an increase in ion pump activity following cation influx. As ATP is utilized for cyclic AMP production and simultaneously for many other processes, an assessment of its metabolism in parallel with that of cyclic AMP is critical. We suggest that the method described here is particularly advantageous over other methods for this purpose.

Sympathetic neurons expressing cholinergic properties are poised to allocate choline symmetrically between acetylcholine and the phosphatidylcholine-generating pathway in growing neurites.

We have examined the question of how regenerating sympathetic neurons that are concomitantly induced to become cholinergic regulate choline allocation between ACh and the phospholipid synthetic pathway. The allocation of choline into ACh increased parabolically with time in culture, and by 3 weeks, cultures with neurites of approximately 6 mm length were incorporating over 85% of the choline locally in the neurites into four major metabolites: ACh, phosphorylcholine, cytidine diphosphocholine, and phosphatidylcholine. The near-equivalent distribution of labeled choline between intracellular choline, ACh, and phosphorylcholine was independent of time (5 min to 6 hr) and choline concentration (0.125-30 microM), phosphatidylcholine being the sole metabolite whose level in the neurites increased steadily with incubation time. Relative choline distribution into ACh and phosphorylcholine was unaltered even after a brief depolarizing prepulse, which caused a two- to fourfold enhancement in the total choline incorporated. These observations, allied with the similar half-saturation constants and Vmax values of CAT and choline kinase for intracellular choline, suggest that growing sympathetic neurons are poised to allocate choline symmetrically between the synthesis of ACh and phosphatidylcholine in the neurites. When, however, the supply of choline was limited either by replacement of Na+ in the medium with N-methyl-D-glucamine, or by vesamicol, a 90-97% reduction in intracellular choline caused a similar decline in ACh levels but synthesis of metabolites of the phosphatidylcholine pathway was maintained unperturbed, as if no drug was present. We suggest that this can be accounted for by a 10-fold increase in choline kinase activity. Thus, growing sympathetic neurons that express cholinergic properties not only maintain their chief cellular phosphatidylcholine-synthesizing activity concomitantly with ACh synthesis in the neurites, but may also preserve phosphatidylcholine synthesis more effectively than ACh synthesis when the supply of choline is perturbed. Relinquishing ACh synthesis during growth may be one way of conserving and encouraging neurite regeneration.

Carbachol and bradykinin elevate cyclic AMP and rapidly deplete ATP in cultured rat sympathetic neurons.

The agonists carbachol (CCh) and bradykinin (BK) and 54 mM KCl (high K+) were among the most potent stimulants of cyclic AMP (cAMP) production in cultured rat sympathetic neurons, measured with the use of a high-fidelity assay developed for small samples. The rise in cAMP evoked by CCh (through muscarinic receptors), BK, and high K+ was inhibited in Ca2(+)-depleted medium (1.3 mM Ca2+ and 2 mM BAPTA or EGTA), which also prevented the sustained rise in [Ca2+]i evoked by each of these stimuli, showing that elevation of cAMP requires extracellular Ca2+ and, possibly, Ca2+ influx. Preliminary results obtained with the novel calmodulin inhibitor CGS 9343B, which blocked the elevation of cAMP, and with the cyclogenase inhibitor indomethacin, which partially blocked the actions of the agonists but not those of high K+, suggest that calmodulin and arachidonate metabolites may be two components of the signaling pathway. In addition to their effects on cAMP metabolism, CCh, muscarine, and BK, but not nicotine, caused a 30-40% decrease in ATP levels. This effect was much greater than that evoked by high K+ and was largely inhibited by CGS 9343B but slightly enhanced in the Ca(+)-depleted medium, showing that agonists are still active in the absence of [Ca2+]o. Thus, agonists that activate phosphoinositide metabolism can also increase cAMP production and substantially deplete cells of ATP. These novel actions may have to be taken into account when the mechanisms by which such agonists regulate cell function are being considered.

The thrombin receptor in the nervous system.

Neurite retraction and reversal of astrocyte stellation triggered by the serine protease thrombin are receptor-mediated events. This article summarizes the current knowledge about the cellular effects that are induced by thrombin and its receptor in neural cells. The data presented show that the thrombin receptor messenger RNA is expressed in cultured astrocytes and that the reversal of stellation caused by thrombin in these cells is prevented by the protein kinase inhibitor staurosporine. Peptides based in sequence on the tethered ligand domain of the thrombin receptor were shown to mimic the effect of thrombin in most systems investigated. Platelets of some species, however, aggregate only in response to thrombin but not to the peptides. This observation is confirmed here. Rodent receptor-activating peptides did not cause aggregation of rat or mouse platelets. In contrast, all peptides triggered reversal of stellation in rat astrocytes and neurite retraction in mouse neuroblastoma cells, supporting the proposed mechanism of cleavage-induced receptor activation in neural cells. Finally, evidence is presented that serum withdrawal causes a decrease in the amount of the thrombin receptor mRNA in different types of neuronal cells. The possible role played by the thrombin receptor in the nervous system is discussed.

Glia-derived nexin/protease nexin-1 is expressed by a subset of neurons in the rat brain.

Glia-derived nexin/protease nexin-1 (GDN/PN-1) is a serine protease inhibitor that is secreted by glial cells and fibroblasts in culture. In the adult mammalian nervous system it has been shown to be expressed in the olfactory system and by some glial cells in response to neuronal injury. In situ hybridization and immunocytochemical studies were performed to identify the structures expressing GDN/PN-1 in the developing and adult rat brain. In contrast to a transient widespread expression during pre- and postnatal development, some brain structures constitutively express GDN/PN-1. These include the olfactory nerve layer of the olfactory bulb, basal forebrain, striatum, pyramidal neurons of layer V in the cortex, thalamic nuclei, pars compacta of the substantia nigra, inferior and superior colliculi, and deep cerebellar nuclei. All of these parts, excluding the olfactory nerve layer, are characterized by a high neuronal cell density. Neurons in these regions were immunoreactive for GDN/PN-1. Furthermore GDN/PN-1 expression in cell lines showed that the active protein was synthesized and secreted from B104 but not from NB2a neuroblastoma cells. Although GDN/PN-1 has only been reported to be synthesized by glia, the results presented here demonstrate that in addition, a subset of neurons express this protease inhibitor.

Expression of the thrombin receptor mRNA in rat brain.

Thrombin activates its receptor in a number of cultured cells of neural origin, but the functional significance of this activation in the nervous system is unknown. It is also not known which cells in brain express the thrombin receptor and whether the level of its expression is developmentally regulated. In the present study, Northern blot analysis showed that thrombin receptor mRNA was expressed at higher levels in brain compared to some other tissues, such as skeletal muscle, liver or kidney. The level of expression is substantially higher in the brain of newborn rats compared to that of postnatal day 28 (P28). At embryonic day 18, thrombin receptor mRNA is present throughout in the brain and in dorsal root ganglia as detected by in situ hybridization. The regions of the P28 brain in which the thrombin receptor mRNA was present include the substantia nigra and the ventral tegmental area, the pretectal area, some hypothalamic nuclei and some cells of the cerebral cortex. These results represent one of the first steps needed to understand the role played by the thrombin receptor in the development and function of the nervous system.

Granzyme A released upon stimulation of cytotoxic T lymphocytes activates the thrombin receptor on neuronal cells and astrocytes.

Granzymes are a family of serine proteases that are harbored in cytoplasmic granules of activated T lymphocytes and are released upon target cell interaction. Immediate and complete neurite retraction was induced in a mouse neuronal cell line when total extracts of granule proteins were added. This activity was isolated and identified as granzyme A. This protease not only induced neurite retraction at nanomolar concentrations but also reversed the stellation of astrocytes. Both effects were critically dependent on the esterolytic activity of granzyme A. As neurite retraction is known to be induced by thrombin, possible cleavage and activation of the thrombin receptor were investigated. A synthetic peptide spanning the N-terminal thrombin receptor activation sequence was cleaved by granzyme A at the authentic thrombin cleavage site Leu-Asp-Pro-Arg-Ser. Antibodies to the thrombin receptor inhibited both thrombin and granzyme A-mediated neurite retraction. Thus, T-cell-released granzyme A induces cellular responses by activation of the thrombin receptor. As brain-infiltrating CD4+ lymphocytes are the effector cells in experimental allergic encephalomyelitis, granzyme A released in the brain may contribute to the etiology of autoimmune disorders in the nervous system.

The thrombin receptor is present in myoblasts and its expression is repressed upon fusion.

Cultured myoblasts derived from limb muscle of newborn rats express thrombin receptor immunoreactivity on their surface. Receptor expression is repressed upon myoblast fusion. This is due at least in part to a decrease in the amount of the thrombin receptor mRNA. Addition of thrombin triggers calcium transients only in mono- but not multinucleated muscle cells. Furthermore, thrombin increases the rate of myoblast proliferation that coincides with an activation of mitogen-activated protein kinase. Northern analysis of thrombin receptor mRNA expression in skeletal muscle showed that the transcript is present at a relatively high level at birth, but is almost undetectable in the adult. By in situ hybridization, the mRNA at birth appeared to be present mostly in mononucleated cells grouped in clusters, but not in muscle fibers. Very few nuclei surrounded by a mRNA signal were present on muscle sections of rats 24 days postnatally. These results suggest that the thrombin receptor plays a role in muscle development.

Astrocyte spreading in response to thrombin and lysophosphatidic acid is dependent on the Rho GTPase.

Astrocytes are typically star shaped cells playing diverse roles in the function of the nervous system. In astrocyte cultures established from the cerebral hemispheres of newborn rats, the cells have generally a polygonal fibroblast-like morphology, but acquire a stellate shape upon serum removal. When the serine protease thrombin or the bioactive lipid lysophosphatidic acid is added, the stellate cells revert to the flat morphology. Here we show that the effect of these agents is mediated via activation of the small GTP-binding protein Rho. Neither thrombin nor lysophosphatidic acid induced spreading of astrocytes microinjected with C3 transferase, an exoenzyme which ADP-ribosylates and thereby inactivates Rho. In contrast, the response of cells injected with a dominant negative form of Rac was unaffected. In addition, the injection of active Rho into stellate astrocytes mimicked the effect of thrombin and lysophosphatidic acid and an injection of C3 into flat cells grown in serum induced stellation. The conversion from a stellate to a spread morphology upon activation of Rho resulted in the formation of stress fibers and focal adhesions which most probably are key events in establishing and stabilizing the altered cytoarchitecture. These results suggest that Rho plays a crucial role in determining the shape of astrocytes and thereby may modulate their interaction with neurons in vivo.

Differential effects of sphingomyelin hydrolysis and resynthesis on the activation of NF-kappa B in normal and SV40-transformed human fibroblasts.

The precise role of ceramide in NF-kappaB signaling remains unclear. The recent observation of differential sphingomyelin synthase (SMS) activity in normal (low SMS) versus SV40-transformed (high SMS) WI38 human lung fibroblasts provides an opportunity to assess the involvement of ceramide and SMS in NF-kappaB activation. Treatment of normal WI38 fibroblasts with bacterial sphingomyelinase resulted in a 4-fold elevation of ceramide and blocked NF-kappaB activation by serum stimulation. Such inhibition was not observed in SV40-transformed fibroblasts. Under regular growth conditions, after sphingomyelinase was washed out, normal WI38 did not show SM re-synthesis nor NF-kappaB activation. In SV40-WI38, on the other hand, sphingomyelinase washout induced resynthesis of SM due to the action of SMS on ceramide generated at the plasma membrane. NF-kappaB activation correlated with SM resynthesis. This activation was abrogated by D609, which inhibited SM resynthesis but not the initial formation of ceramide. The differential activity of SMS may explain the effects of ceramide in NF-kappaB signaling: in the absence of significant SMS activity, ceramide inhibits NF-kappaB, whereas with high SMS, the conversion of the ceramide signal to a diacylglycerol signal by the action of SMS stimulates NF-kappaB. These results also suggest a role for SMS in regulating NF-kappaB.

Changes in the expression of protease-activated receptor 1 and protease nexin-1 mRNA during rat nervous system development and after nerve lesion.

HDs racI Thrombin causes profound metabolic and morphological changes in cultured neural cells via activation of the thrombin receptor, also called protease-activated receptor 1 (PAR1). PAR1 mRNA is present in the rat brain, but the role of this receptor in the nervous system remains elusive. The expression of PAR1 and the potent thrombin inhibitor protease nexin-1 (PN-1) was investigated in the developing rat brain and spinal cord and after peripheral nerve lesion. As seen by in situ hybridization, the PAR1 mRNA signal in the late embryonic and early postnatal nervous system was widespread, but generally of low intensity whereas in the adult it was more pronounced and confined to particular neuronal cells. These include the mesencephalic dopaminergic neurons, several thalamic and brainstem nuclei, the mitral cells in the olfactory bulb and the Purkinje cells in the cerebellum. In the spinal cord, PAR1 mRNA was abundant in motoneurons and a particularly high expression was detected in the preganglionic neurons of the autonomic nervous system. High PAR1 mRNA expression was also found in the dorsal root ganglia. Interestingly, strong immunoreactivity for the protease inhibitor PN-1 was present in spinal motoneuron cell bodies, although its transcript was undetectable there. In response to sciatic nerve transection, the signal intensity of PAR1 mRNA as seen by Northern analysis increased in the proximal and the distal part of the lesioned nerve and in the denervated muscle, whereas the PN-1 mRNA signal strongly increased only in the distal part of the nerve but remained unchanged in the proximal part and in the muscle. After facial nerve transection, PAR1 mRNA expression substantially decreased in facial motoneurons. No PAR1 transcript was detected in reactive astrocytes. Similar to PAR1, PN-1 mRNA which was expressed in interneurons within the facial nucleus was also decreased following facial nerve transection.

Nerve growth factor promotes activation of the alpha, beta and gamma isoforms of protein kinase B in PC12 pheochromocytoma cells.

The activation of phosphatidylinositol (PtdIns) 3-kinase is considered to be a key event occurring after stimulation of cells with growth factors. The proto-oncogenic protein kinase B (PKB; also known as RAC protein kinase or Akt) has recently been shown to be a downstream target of PtdIns 3-kinase and may be involved in cell survival. We therefore asked whether stimulation of neuronal cells with nerve growth factor (NGF), on which certain types of neurons are dependent for survival, causes activation of PKB. Stimulation of serum-starved PC12 rat pheochromocytoma cells with NGF caused an increase of up to 14-fold in PKB activity. This activation was detected within 1 min of stimulation and occurred at NGF concentrations that are consistent with TrkA-mediated signaling. PKB activation was accompanied by a decrease in electrophoretic mobility of the kinase, which is characteristic of phosphorylation. Both PKB activation and mobility changes were prevented by wortmannin, indicating the upstream involvement of PtdIns 3-kinase in these events. Analyses employing isoform-specific antibodies for immunoprecipitation suggested that all three isoforms of PKB (alpha, beta and gamma) are activated in response to NGF. G-protein-coupled-receptor agonists, lysophosphatidic acid (lyso-PtdH) and thrombin, which induce rapid neurite retraction, neither stimulated PKB activity, nor affected NGF-induced or insulin-induced kinase activation. Wortmannin treatment did not prevent neurite retraction induced by lyso-PtdH or thrombin. These data suggest that PtdIns 3-kinase and PKB are not involved in cytoskeletal changes mediated by the small GTPase Rho.

The serine protease granzyme A does not induce platelet aggregation but inhibits responses triggered by thrombin.

Granzyme A is a serine protease stored in cytoplasmic granules of cytotoxic and helper T lymphocytes. This protease seems to elicit thrombin receptor-mediated responses in neural cells, thereby triggering neurite retraction and reversal of astrocyte stellation. Here we report that granzyme A does not cause platelet aggregation even at concentrations that are more than two orders of magnitude higher than the EC50 for granzyme A in causing morphological changes in neural cells. However, granzyme A blocks thrombin-induced platelet aggregation in a dose-dependent manner without affecting the response to either ADP or to the peptide agonist of the thrombin receptor SFLLRN that corresponds in sequence to the tethered ligand domain. The inability of granzyme A to cause aggregation and its inhibition of thrombin-induced aggregation were seen in platelets from man, rat and mouse. Granzyme A does not affect the catalytic activity of thrombin in cleaving a chromogenic substrate or the macromolecular substrate fibrinogen. However, granzyme A does seem to cleave the thrombin receptor on platelets to produce a weak Ca2+ signal and reduce the response to subsequent challenge with thrombin, but does not induce a signal in thrombin-stimulated platelets. It is proposed that granzyme A interacts with the thrombin receptor found on platelets in a manner that is insufficient to cause aggregation, but sufficient to compete with thrombin for the receptor. These results suggest that granzyme A cleaves the thrombin receptor at a rate that is insufficient to cause platelet aggregation but is sufficient to cause morphological changes in neural cells. Furthermore, these observations demonstrate that granzyme A release occurring during immune responses within blood vessels would not directly cause platelet aggregation.