Journal of Neuroscience Research 26:447454 (1990)
Involvement of Dihydropyridine-Sensitive Calcium Channels in Nerve Growth Factor-Dependent Neurite Outgrowth by Sympathetic Neurons M. Rogers and I. Hendry Division of Neurowcncc. John Curtin School of Medical Recearch. Australian National University,
Canberra, Australia
We have used a number of pharmacological manipulations of calcium influx to alter the nerve growth factor (NCF)-elicited neurite outgrowth response of SCG neurons. Our results indicate that influx of extracellular calcium is critical to sympathetic SCG neurite outgrowth. Effective blockade of this process was produced by the inorganic calcium channel blockers Cd2+ (with an IC,, of 48 pM), Co2+ (129 pM), and Ni2+ (180 pM). More specifically, there is a significant contribution from dihydropyridine-sensitive L-type calcium channels to NGF-activated neurite outgrowth, as evidenced by the significant inhibition of neurite outgrowth by diltiazem (ICso of 17pM) and nifedipine (3 pM). Further, increases in calcium influx can elicit an enhanced neurite outgrowth response, as shown by the calcium channel agonist Bay K 8644 which potentiated neurite outgrowth by up to 40%. Key words: NGF, superior cervical ganglion, cell culture INTRODUCTION The binding of nerve growth factor (NGF) to its receptor elicits numerous effects on target cells over the course of minutes to days. However, the immediate consequences of receptor occupation leading to the generation of key intracellular messengers, and the specific pathways this signalling affects, are still not fully understood. Possible mechanisms of NGF action have either been discounted, as is the case for cyclic AMP (Frazier et al., 1973), or still remain uncertain, as for examplc with the role of inositol trisphosphate (IP,) production and calcium mobilization (Pandiella-Alonso et a1, , 1986). Enhanced turnover of phosphatidylinositides has been demonstrated in response to NGF in superior cervical ganglion (SCG, Lakshmanan: 1978), as well as PC 0 1990 Wiley-lhs, Inc.
12 (Contreras and Guroff, 1987; but see van Caulker et al.. 1989) and adrenal chromaffin cells (PandiellaAlonso et al., 1986). NGF appears lo have a novel action on the hydrylosis of glycophosphatidylinositol (Chan et al., 1989) to produce a second species of diacylglycerol capablc of activating protein kinase C (PK-C). The effects of NGF on neurite extension and protein phosphorylation can be mimicked by activators of PK-C (Hsu et al., 1984; but see Reinhold and Neet, 1989) and inhibited by various kinase blockers (Hall et al., 1988: Koizumi et al., 1988), and NGF itself elevates PK-C activity (Hama et al., 1986). A possible role for calcium ions in NGF action is indicated by the irnportancc of transmembrane calcium flux in supporting neuronal cell survival and modulating neurite outgrowth. Experiments with calcium indicator dyes (Anglister et al., 1982) and agents known to interfere with calcium channel function (Mattson and Kater, 1987) have indicated a correlation between calcium channel activity, intracellular calcium transients, and neurite outgrowth (Connor, 1986; Katcr et al., 1988). The activity of calcium channels is also pronounced in the membranes of growing neurites (Fukuda and Kameyama, 1979) and growth cones (Grinvaltl and Farber, 198 1; Marorn and Dagan, 1987) o f cxplanted and regenReceived November 30. 1989; revised February 5 , 1990; accepted February 6 . 1990. Address reprint requests to Dr. 1. Ilendry, Division of Neuroscience, John Curtin School of .Medical Kesearch, Australian National Univcraity, GPO Box 334, Canberra ACT 2601, Australia. Abbreviations used: Bay K 8644, (methyl- I,4-dihydro-2.6-dimcthyl3-nitro-4-(2-trifluoromethylphenyl)-pyridine-5-carboxylatc~; CAMP. cyclic adenosine monophosphate; DHP. dihydropyridine; DKG. dorsal root ganglion sensory neuron; G-protein, guanosine triphosphatebinding protein; HEPES, (N-(2-hydroxycthyl) piperazine-N'-(2ethanesulphonic acid)); IP,, inositol trisphosphate: NGF, nerve growth factor; PC 12 cells, pheochrornocytoma tumour cell line derived from adrenal chromaffin cell.;; PK-C, protein kinase C; SCC, superior cervical ganglion.
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erating neurons (MacVicar and Llinas, 1985). Similarly, calcium channels have recently been shown to be involved in the depolarization-induced survival of sympathetic SCG neurons (Koike et al., 1989) and chick ciliary, DRG and sympathetic neurons (Collins and Lile, 1989), and to underlie the inhibition of neurite outgrowth by high K t and bradykinin in DRG neurons (Robson and Burgoyne, 1989). The action of NGF on neurite outgrowth from sympathetic neuronc ic cimilarly sensitive to pharmacological manipulation of calcium channels, but evidence for a direct effect or NGF on transmembrane calcium flux is still equivocal (Schubert et al., 1978; Landreth et a]., 1980; Streit and Lux, 1987). With this in mind, we set out to quantify the role of extracellular calcium influx in NGF-mediated neurite outgrowth by using specific ligands to identify the type(s) of calcium channel($) involved. Preliminary reports of this work have been published in abstract form (Rogers et al., 1988; Rogers and Hendry, 1989).
METHODS SCG Culture Dissociated sympathetic cell cultures were prepared as described elsewhere (Mains and Patterson, 1973), with slight modifications. Briefly, superior cervical ganglia were removed from 2-4 day-old Wistar rat pups, cleaned in Dulbecco’s minimum essential medium (DMEM-HEPES buffered), and then incubated with 0.1% collagenase for 30 min, followed by two 10 niin incubations in a mixture of 0.1% collagenase and 0.08% trypsin. The cells were then fully dispersed by gentle trituration through flamed pipettes of decreasing bore. Neurons were suspended in 90% DMEMi 10% foetal calf serum and aliquoted into 96 multiwell plates (Nunc, Denmark) coated with rat tail collagen (prepared according to the method of Bornstem, 1958) after these had been filled with the appropriate test solutions. To half of the wells was added 0.7 pgiml p NGF (prepared from mouse submaxillary glands according to the method of Mobley et a]., 1976) while the other half served as controls for NGF-independent neurite outgrowth. Plates were incubated at 37°C in a 100% air atmosphere to reduce proliferation of non-neuronal cells (Mains and Patterson, 1973). All solutions added to the cells were sterilized by being passed through 0.22 p m filters (Millipore, MA, USA). Dose response curves for drugs were obtained by serial dilution5 of filtered stock solution. Control wells contained drug vehicle alone. Nifedipine and Bay K 8644 were dissolved in ethanol with a maximum concentration in the first well of 0.4%. As both of the dihydropyridines are light sensitive they were kept in shielded
bottles and dispensed in subdued lighting. Flunarizine, diltiazem, and nifedipine were from Sigma Chemical Co., St Louis, MO, USA. Bay K 8644 (Bayer) was a gift from Dr. Graham Lamb.
Neurite Outgrowth Assay Neurite outgrowth was quantified after 24 hr in culture by using phase contrast microscopy to count the total number of healthy neurons (phase-bright, spherical cell body), and the number of such neurons bearing neurites of greater than 1 cell diameter in length. Four fields covering 9% of the entire well were counted and the results expressed as the proportion of neurite-bearing cells per field. Individual data points are an average of at least 4 replicatc wells.
RESULTS General Observations After 24 hr in culture, SCG neurons have large spherical cell bodies about 25-30 p m in diameter. Neurites begin to appear with NGF treatment after 18 hr and continue to form extensive networks for up to several weeks. The presence of NGF is essential to both the survival of postnatal SCG neurons and for their extension of neurites. The average proportion of healthy neurons bearing neurites on collagen after 24 hr in a culture solution of l .8 mM [Ca2+l0and 0.7 yg/rnl NGF was 17.3 k 1.1% (average of means from 161 wells in 17 experiments 2 s.e.m.). SCG neurons grown without NGF express neurites at a much lower rate of 3.0 k 0.7% (n = 161 wells ? s.e.m.), and are clearly deteriorating after 24 hr.
NGF and Calcium Channels Action of inorganic blockers. Cadmium proved to be the most potent blocker of neurite outgrowth, effective at micromolar concentrations with a calculated half-maximal inhibition (ICs0) at 48 p M (Fig. 1A). Cobalt ions were the next most effective, giving an IC,,, of 129 p M (Fig. lB), and nickel gave a slightly higher IC,, of 188 p M (Fig. 1C). The least effective inorganic blocker was manganese, which first significantly affected neurite outgrowth at a concentration of 250 pM, but only completely blocked neurite outgrowth in the millimolar range, to yield an apparent IC,, value of 830 pM (Fig. ID). An intercsting observation with manganese was the appearance of large flattened growth cones at the tips of growing neurites. The relative potencies of these blockers are therefore Cd > Co > Ni > Mn, qualitatively similar to that found in many other neuronal preparations. At high concentrations cadmium, cobalt, and manganese had some effect on neuronal survival (Table I).
Calcium Channels in NGF Action Cadmium
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Cobalt V
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Fig. 1, Action of inorganic calcium channel blockers on NGFactivated neurite uutgrowth. Dose-response curves for cadir ium (n = 8)>cobalt (n = 8), nickel ( n = 7). and manganese (n = 7 - 14) plotted as the mean number (? s.c.m., vertical bars) of neurite-bearing cells against the log concentration for each treatment. Control point represents the mean number of
However, this always occurred at a higher concentration than that having a significant effect on neurite outgrowth. Effect of organic blockers. There are several classes of organic calcium channcl blockers (Table IT) useful in determining a role for various calcium channel types in neuronal systems, and we screened representative samples from three classes for their cffect on NGFmediated neurite outgrowth. Flunarizine was the least effective of all agents tested in this assay, yielding poor inhibitory action (rnaxirnal inhibition of 36% at 22 pM, Fig. 2A) and lacking any statistically significant blocking effects. Stronger inhibition of ncurite outgrowth was shown by diltiazem, which produced statistically significant blockade at all concentrations tested with a maximal inhibition of 60% at higher concentrations, to give an apparent 1C5,, of 17 FM (Fig. 2B). Contrasting results were obtained with the dihydro-
D
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neurite bearing cells in wells containing NGF with addition of vehicle solution. The asterisks inark the first conccntration at which each agent significantly inhibited neurite outgrowth, with statistical significance calculated by two-tailed Students' t test: P valucs * 50.05,
***< 0.001.
TABLE 1. Comparison of Caz+ Channel Ligand Effects on SCG Cell Survival and Neurite Outerowth* Lowest conccntration to significantly affect Treatment
Neurite outgrowth
Cell survival
Cadmium Cobalt Nickel
42 p M 230 p,M 216 p,M 256 pM NS 1 . 1 FM 0.1 p M 1.9 nhl
230 pM 540 pM ?IS 3.0 mM NS NS NS NS
.Manganese
Elunarizinc Diltiazcm Nifedipine Bay K 8644
*Values are the concentration at which survival or neurite outgrowth
was significantly lower than that observed in control wells in the absence of any treatment. Significance was assessed by using twotailed Students' t-tcst, P values 5 0.01, NS = not significanl.
pyridine (DHP) ligands nifedipine and Bay K 8644. The former is a potent calcium channel antagonist and in this assay system produced strong, statistically significant in-
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Rogers and Hendry TABLE 11. Classification of Calcium Channel Blockers* Class of blocker
Ca antagonist
Ca agonist
Ca channel specificity
Inorganic Cadmium Cobalt Nickel Manganese Organic Dihydropyridine Benzothiazepine Diphenyalkylamine Phenylalk ylarnine
Nifedipine ( + ) Bay K 8644 Diltia/.em Flunarizine Verapamil, D600
(-) Bay K 8644
L only L only‘ L onlyd L and Te L onlyd.f
”Calcium channel nomenclature is according to Fox et al. (1987), while the organic blocker types are derived from Spedding and Berg (1984). “Cadtniurn’s specificity for L- and N- over ‘1-type calcium channels is at concentrations of around 20-SO p,M. At 200 p,M cadmium blocks all channel types equivalently (Fox et al., 1987). bNickel is a more specific blocker of T-typc calcium channcls at low (100 pM) concentrations (Fox et al., 1987). ‘Bay K 8644 is listed as an antagonist and agonixt according to its stereoisomeric form; LI racemic mixture of the two having an agonistic effect was used in these experiments. dDiltiazem and the verapamil series of blockers have bcen shown to modulate calcium channels insensitive to dihyropyridines in some tissues (Carboni and Wojcik, 1988). ‘Flunarizine is a potent blocker of smooth muscle L-type currents but also affects T-type calcium channels more effectively in cardiac muscle (Tytgart et al., 1988). ‘Members of the phenylalkylamine group were not tested.
hibition of NGF-mediated neurite outgrowth: the IC,,, calculated from Figurc 2C was 3 pM, and at the highest concentration tested it completely blocked neurite outgrowth. In contrast, the DHP calcium channel agonist Bay K 8644 augmented neurite outgrowth between 10-” and M, producing a statistically significant potentiation of 40% above control at 2 nM (in Fig. 2D). At all concentrations tested the organic calcium channel ligands had no significant effect on the survival of sympathetic neurons (Table I).
DISCUSSION NGF is commonly regarded as the archetypal neurotrophic growth factor, but its mode of action in producing such typical effects as neurite outgrowth is still unclear. It is quite likcly that neurite initiation and elongation are separately regulated events, and so we have used the term neurite outgrowth to indicate the result of both initiation and elongation. Our results indicate that an influx of extracellular calcium is necessary for NGFactivated neurite outgrowth, and that dihydropyridinesensitive L-type calcium channels are specifically implicated in the effects of NGF on neuritogenesis from sympathetic neurons. There may be differing effects of calcium channel modulation upon neurite initiation and/ or elongation, but these cannot be distinguished from the present study.
Factors Affecting Basal Outgrowth Several factors appear to affect the basal level of neurite outgrowth, which in SCG neurons grown on a collagen Substrate in 1.8 mM external calcium averages 17% with an NGF concentration of 0.7 pgiml. Higher concentrations of NGF elicit enhanced rates of neurite outgrowth of up to 30% on collagen (data not shown), and this level is further influenced by the nature of the substrate used. For example, up to 100% of sympathetic neurons grown on poly-l-lysine and laminin produce neurites (Edgar, 1985, and data not shown). Secondly, alterations in the extracellular calcium concentration can influence the production of neurites by rat neurons (Koikc, 1983), 50 comparison of the concentration-dependent effects of NGF should be considered in conjunction with the level of calcium ions in the culture media. Action of Inorganic Blockers Often referred to as universal calcium channel blockers, inorganic divalent cations such as cadmium, cobalt, nickel, and manganese inhibit calcium flux through calcium channels (Hagiwara and Byerly, 1981), and so it was with these that we first assessed the possible involvement of calcium channels in the mode of NGF action and attempted to define the type of channel active in neurite outgrowth. The pharmacological profile of neurite outgrowth blockade by these inorganic ligands was Cd > Co > Ni
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Calcium Channels in NGF Action FI u no rizi ne 25] E cn
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-10.0
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Fig. 2 . Action of organic calcium channel ligands on NGFdependent neurite outgrowth. Dose-response curves for flunarizine (n = 8). diltiazem (n - 4 - 1 I ) , nifcdipinc (n = lo), and Bay K 8644 (n = 4) are expressed as thc mcan (2s.e.m., vertical bars) number of' neurite-bearing cells against ligand concentration. Control point represents the mean number of
neurite bearing cells in wells containing NGF with addition of vehicle solution. Thc asterisks mark the first concentration at which each agent statistically affected neurite outgrowth, with statistical significance calculated by a two-tailed Students' t test: P values *s0.05, **s0.01, ***< 0.001.
> Mn, not dissimilar in either series or effective concentrations from studies of such calcium-dependent process~ as~neurotransmitter release (Gandia et al., 1987), 4 5 ~ I~ 2 uptake (Carboni and Wojcik, 1988), and neurite extension from retinal ganglion cells (Suarez-Isla et al., 1984). If NGF acts upon neuritc outgrowth through changes in calcium channel activity, the potency of cadmium is not unexpected given its potent effect on calcium-dependent processes. Cadmium has a high affinity for Ca2' binding sites associated with the calcium channel pore (Hagiwara and Byerly, 1981; Lansmann et al., 1986) and various intracellular domains involved with neurotransmitter release (Gum et al., 1987). Conversely, the weak effect of Mn2+ upon NGF-activated neurite outgrowth can be explained by its weak competition with Ca2- ions for transport through calcium channels (Lansmann et al., 1986), and in fact its significant conduction through the calcium channel pore may ac-
count for the occurrance of large growth cones seen with manganese, where this ion could substitute for calcium at sites involved with growth cone motility. adherence (Edwards et al., 1988), and neurite extension. Nickel ions at low micromolar concentrations selectively block (i.e., more potently than Cd2+ or C o 2 + ) a low-voltage-activated, transient or T-type calcium channcl in sensory DRG neurons (Fox et al., 1987), and we tested nickel to determine if such a calcium channel was involved in neurite outgrowth from sympathetic neurons. However, the appearance of cadmium and cobalt ahead of nickel in the IC,,, series for inhibition of neurite outgrowth, coupled with the slightly higher IC,, value of 188 p M for nickel, is consistent with electrophysiological cxpcriments which have also failed to find transient T-type calcium channels in SCG neurons (Thayer et a]., 1987; A. Field, personal communication). These inorganic calcium channel blockers are somewhat non-spccific and may be having side effects
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unrelated to calcium channel blockade. For example, cadmium also affects the calmodulin-dependent Ca2 Mg2+ ATPase in some non-neuronal cells (Akerman et al., 1985) and the sodium-calcium exchanger in cardiac muscle (Kimura et al., 1987) and thus may interfere with the overall calcium homeostasis in SCG neurons. accounting for its potent effect upon neurite outgrowth. Nickel at concentrations of 2-5 mM has been claimed to selectively block the electrogenic Na -Ca2 exchanger (Kimura et al., 1987). However, these concentrations are well above those shown here to effectively inhibit NGFactivated neurite outgrowth, and argue against a nonspecific action of these cations on the calcium exchanger in SCG neurons. The results with these inorganic ligands may therefore occur through a combination of effects on calcium channels and intracellular calcium binding sites, upsetting the calcium-dependent processes underlying neurite outgrowth. Further, the effects of these blockers upon cell survival, which can be taken as a measure of non-specific actions, revealed that cadmium, cobalt, and manganese (but not nickel) only affected viability at concentrations above those significantly inhibiting neurite outgrowth (Table I). This effect may be due to their chelation of the high molecular weight 7 s NGF molecule, which normally forms a complex with zinc ions (Greene and Shooter, 1980). Other divalent cations at high enough concentrations might alter this interaction and reduce the availability of NGF to the neurons. A further possibility is that the binding of NGF to the substrate may be altered by these cations, and thus affect the neurite outgrowth response. +
+
+
Effects of Organic Blockers In an effort to further determine whether voltagedependent calcium channels are specifically involved in neurite outgrowth and to idcntify the possible type(s) of calcium channel influenced by NGF, we employed representative agents from three major classes (Spedding and Bcrg, 1984) of organic calcium channel ligands (Table IT). The effective order of potency of these agents on NGF-activated neurite outgrowth by SCG neurons was nifedipine > diltiazem > flunarizine. Additionally, there was a statistically significant potentiation of neurite outgrowth by the calcium channel agonist Bay K 8644. More specifically, these ligands can be used to identify the type of calcium channel involved in neuritogcnesis, as they bind to and modulate specific types of calcium channel. Flunarizine is primarily a blocker of muscle-type calcium channels, being a more selective inhibitor of T-type compared to L-type channels (Tytgart et al., 1988). In this neuronal preparation it proved to be
the least effective organic blocker of neurite outgrowth, perhaps due to the lack of T-type calcium channels in this cell type (Thayer et al., 1987). The benzothiazepine-type, diltiazem-binding organic ligand site is allosterically (Spedding and Berg, 1984) and structurally (Striessnig, 1989) associated with the DHP receptoril-type calcium channel. In several neuronal preparations diltiazem in micromolar concentrations is an effective blocker of processes involving calcium influx, e.g., K+-evoked catecholamine release (Gandia et al., 1987), cerebellar granule cell 45Ca*+ uptake (Carboni and Wojcik, 1988), and smooth muscle calcium currents (Terada et al., 1987) are all inhibited at half-maximal concentrations of 1 to 12 pM. Diltiazem completely inhibited SCG neurite outgrowth in this study at similar concentrations to that required to block cerebellar granule cell calcium uptake, indicating that L-type calcium channels may be involved in both processes. The involvement of L-type calcium channels is further supported by the potent block of NGF-activated neurite outgrowth by the dihydropyridine type antagonist, nifedipine. In our in vitro assay system, concentrations of nifedipine above 10 FM could completely inhibit neuritogenesis, with the calculated IC,, of 3 pM being quite similar to that of 5 pM found to inhibit neurotransmitter release from sensory DRG neurons (Rane et al., 1987). In contrast, the calcium channel agonist Bay K 8644 (Garcia et al., 1984) produced up to 40% potentiation of neurite outgrowth between 0.1 nM and 0.1 pM, concentrations at which Bay K 8644 can be expected to be acting on the DHP receptoril-type calcium channel to prolong channel open times (Brown et al., 1984) and promote the influx of extracellular calcium to support neurite outgrowth. These organic calcium channel ligands are more specific than the inorganic ions also tested in this study. SCG cell viability is not affected at all by concentrations of these ligands that significantly affect Eeurite oulgrowth (Table I), suggesting that calcium channels are indeed specifically implicated in NGF-activated neurite outgrowth. It is therefore unlikely that changes in NGF substrate binding or availability are responsible for the effects seen with the inorganic calcium channel blockers. In neuronal tissue, however, demonstrable DHP sensitivity appears to depend on the stimulating conditions: K -depolarizations or more positive voltage clamp holding potentials favour activity of DHP-sensitive L-type calcium channels and hence reveal DHPmodifiable sites of calcium influx, while electrically or action-potential evoked responses of short duration from resting potentials stimulate calcium influx through calcium channels insensitive to DHPs (Rane et al., 1987). Thus our results with the organic ligands could be interpreted in two ways: +
Calcium Channels in NGF Action
1. A substantial proportion of the calcium influx underlying NGF-mediated neurite outgrowth occurs through DHP-sensitive calcium channels normally active in SCG neurons, or 2. Intracellular and/or membrane effects of NGF, such as a partial depolarization, favour the functioning of DHP-sensitive calcium channels. These particular effects may enhance the sensitivity of calcium influx (and subsequent neurite outgrowth) to DHP modulation.
Consistent with this latter possibility is thc sensitivity of NGF-deprived sympathetic neuron survival in depolarizing K concentrations to blockade of DHP-type calcium channels (Koike et al., 1989), and similarly in sensory DRG neurons where depolarization (and bradykinin) actions on neurite formation are blocked by nifecdipine (Robson and Burgoyne, 1989). Also, DHP-sensitive Ltype calcium channels have recently been identified as having a key role in K t depolarization effects on rat SCG neurotransmitter enzyme expression (Vidal et al., 1989) and upon chick ciliary, DRG, and sympathetic neuron survival (Collins and Lile, 1989). The profound effect of alterations in calcium influx and intracellular calcium concentration upon neurotransmitter-evoked neurite outgrowth (Kater ct al., 1988) and K t depolarization-dependent neuronal survival (Koike et al., 1989) has led to the proposal of an optimal, or set-point’’ , intracellular calcium concentration necessary to support neurite outgrowth (Connor, 1986). SCG neurons have quite a low resting intracellular calcium concentration of 67 nM, and only a small proportion of the Fura-2 calcium transient is dependent on release from intracellular calcium stores (Thayer et al., 1988). The results presented here sugggest that this cell type depends on calcium channels to provide an influx of extracellular calcium to support NGF-mediated neurite outgrowth. One of the questions remaining to be answered with regard to NGF-mediated neurite outgrowth is whether NGF specifically modulates the activity of calcium channels to bring about enhanced entry of extracellular calcium, or whether NGF produces some other signal, such as activation of a phosphorylation cascade or G-protein signalling pathway (Rogers and Hendry, 1989), which indirectly affects calcium channels. Either way, the continued functioning of calcium channels (involving a significant contribution from DHP-sensitive channels) is critical to the process of neurite outgrowth from sympathetic neurons in response to NGF. +
‘i
ACKNOWLEDGMENTS We would like to thank Ms. Jenny Meancy and Mrs. Mary Preston for their ever-helpful technical assis-
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tance, and Dr. Caryl Hill for advice on culturing techniques. Dr. Michael Crouch provided helpful comments on this manuscript. M.R. is supported by an ANU PhD scholarship and assisted by aerobics instructors throughout Australasia!
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L (1984): Dihydropyridine Bay ti 8644 activates chromaffin cell calcium channels. Nature 309:69-71. Greene LA, Shooter EM (1980): The nerve growth factor: Biochemistry. synthesis, and mechanism of action. Annu Rev Neurosci 3:353-402. Grinvald A, Farber IC ( I 98 I ) : Optical recording of calcium action potentials from growth cones of cultured neurons with a laser microbeam. Science 212:1164-1167. Guan Y-Y, Quastel DMJ, Saint DA (1987): Multiple actions of cad mium on transmitter release at the mouse neuromuscular junction. Can J Physiol Pharmacol 6S:2 I3 1-21 36. Hagiwara S , Byerly L (1981): Mernbrane biophysics of calciuni currents. Fed Proc 40:2220-2225. Hall FL, Fernyhough P, Ishii DN, Vulliet PR (1988): Suppression of nerve growth factor-directed neurite outgrowth in PC 12 cells by sphingosine, an inhibitor of protein kinase C . J Biol Chem 263:4460-4466. Hama T, Huang K P, Guroff G (1986): Protein kinase C as a component of a nerve growth factor-sensitivc phosphorylation system in PC 12 cells. Proc Natl Acad Sci USA 83:2353-2357. Hsu L, Natyzak D , Laskin JD (1984): Effects of the turnour promoter 12-0-tetradecanoylphorbol-13-acetate on neurite outgrowth from chick embryo sensory ganglia. Canccr Rcs 44: 4607-46 14. Kater SB, Mattson MP, Cohan C, Connor J (1988): Calcium regulation of the neuronal growth cone. Trends Neurosci 11:315321. Kirnura J , Miyamae S , Noma A (1987): Identification of sodiumcalcium exchange current in single ventricular cells of guineapig. J Physiol (Lond) 384: 199-222. Koike T (1 983): Nerve growth factor-induced neurite outgrowth of rat pheochromocytoma PC 12 cells: Dependence on extracellular Mg2+ and Ca’+. Brain Res 289293-303. Koike T, Martin DP, Johnson EM (1989): Role of Ca2+ channels in the ability of membrane depolarization to prevent neuronal death induced by- trophic factor deprivation: Evidence that levels of internal Ca’+ determine nerve growth factor dependence of sympathetic ganglion cells. Proc Natl Acad Sci USA 86: 6421-642s. Koizumi S. Contreras ML. Matsuda Y, Hama T, Lazarovici P, Guroff G (1988): K - 2 . 5 2 ~A specific inhibitor o f t h e action of nerve growth factor on PC 12 cells. Trends Neurosci 8:715-721. Lakshnianan J (1978): Nerve growth factor-induced turnover of phosphatidylinositol in rat superior cervical ganglia. Biochem Biophys Res Commun 82:767-775. Landrcth G , Cohen P. Shooter EM (1980): CaZt transmembrane fluxes and nerve growth factor action on a clonal rat phcochromocytoma. Nature 283:202-204. L-ansman JB. Hess P, Tsien RW (1986): Blockade of current through single calcium channels by C d z f , Mg’+ and Ca’+. J Gen Physiol 88:321-347. Mains RE, Patterson PH (1973): Primary cultures of dissociated sympathetic neurons 1. Establishment of long-term growth in culture and studies of differentiated properties. J Cell Biol 59: 329-345. Marom S, Dagan L) (lY87): Calciutn currents in growth balls from isolated Helix aspersa neuronal growth cones. Pflugers Arch 409578-58 1, Mattson MP, Kater SB (,1987):Calcium regulation of neurite elongation and growth cone motility. J Neurosci 7:4034-4043. MacVicar BA, Llinas RR (1985): Barium action potentials in regen-
erating axons of the lamprey spinal cord. J Neurosci Res 13: 323-335. Mobley WC, Schenker A , Shooter EM (1976): Characterization and isolation of proteolytically modified nerve growth factor. BioPandiella-Alonso A, Malgaroli A , Vicentini L, Meldolesi J (1986): Early rise of cytosolic Ca2 ’ induced by NGF in PC12 and chroniaffin cells. FEBS Lett 208:48-5 I . Rane SG. Holz GG, Dunlap ti (1987): Dihydropyridine inhibition of neuronal calcium current and substance P release. Pflugers Arch 409:361-366. Reinhold DS, Neet KE (1989): The lack of a role for protein kinase C in neurite extension and in the induction of ornithine decarboxylase by NGF in PC 12 cells. J Riol Chem 264:3538-3544. Robson SJ, Burgoyne RD (1989): L-type calcium channels in the regulation of neurite outgrowth from rat dorsal root ganglion neurons in culture. Neurosci Lett 104:110-1 14. Rogers M, Gage PWG. Hendry 1A (1988): Effects of calciuni channel antagonists on NGF-mediated neurite outgrowth from sympathetic neurons. Proc Aust Physiol Pharrnacol Soc 19:2P. Rogers M , Hendry IA (1989): Involvement of calcium channels and G proteins in KGF-mediated neurite outgrowth from sympathetic neurons. Proc Aust Physiol Pharmacol Soc 20: 19SP. Schubert D, LaCorbiere M, Whitlock C, Stallcup W (1978): Alterations in the surface properties of cells responsive to nerve growth factor. Nature 273:718-723. Spedding M , Berg C (1984): Interactions betwcen a “calcium channel agonist”, Bay K 8644, and calcium antagonists differentiate calcium antagonist subgroups in K ‘-depolarked smooth muscle. Arch Pharmacol 328:69-75. Streit J. Lux HD (1987): Voltage-dependent calcium currents in PC 12 growth cones and cells during NGF-induced cell growth. Pflugers Arch 408:634-641. Striessnig J (1989): Identification of the purified calcium channel benzothiazepine receptor by photoaffinity labelling and anti-benzothiazepine antibodies. Arch I’harmacol 339:R44. Suarez-Isla BA, Pclto DJ. ‘ThompsonJM, Rapoport SI (1984): Blockers of calcium permeability inhibit neurite extension and formation of neuromuscular synapses in cell culture. Dev Brain Res 14:263-270. Terada K, Kitamura K , Kuriyama H (1987): Blocking actions of calcium antagonists on the calcium channels in the smooth muscle cell membrane of rabbit small intestine. Pflugers Arch 408: 552-557. Thaycr SA, Hirning LD, Miller RJ (1987): Distribution of multiple types of calcium channels in rat sympathetic neurons in vitro. Mol Pharmacol 32579-586. Thayer SA, Hirning LD, Miller RJ (1988): The role of caffeinesensitive calcium stores in the regulation of the intracellular free calcium conccntration in rat sympathetic neurons in vitro. Mol Pharmacol 34:664-673. Tytgart J. Vereecke J, Carmeliet E (1988): Differential sensitivity of verapamil and flunarizine on cardiac L-type and T-type calcium channels. Arch Pharmacol 337:690-692. van Calker D , Takahata K, Heumann R (1989): Nerve growth factor potentiates the hormone-stimulated intracellular accumulation of inositol phosphates and Ca2 ’ in rat PC 12 pheochromocytoma cells: Comparison with the effect of EGF. J Neurochem 52~38-45. Vidal S, Raynard B. Weber MJ (1989): The role of calcium channcls of the L-type in neurotransmitter plasticity of culturcd sympathetic neurons. Mol Brain Kes 6:187-196.
Involvement of Dihydropyridine-Sensitive Calcium Channels in Nerve Growth Factor-Dependent Neurite Outgrowth by Sympathetic Neurons M. Rogers and I. Hendry Division of Neurowcncc. John Curtin School of Medical Recearch. Australian National University,
Canberra, Australia
We have used a number of pharmacological manipulations of calcium influx to alter the nerve growth factor (NCF)-elicited neurite outgrowth response of SCG neurons. Our results indicate that influx of extracellular calcium is critical to sympathetic SCG neurite outgrowth. Effective blockade of this process was produced by the inorganic calcium channel blockers Cd2+ (with an IC,, of 48 pM), Co2+ (129 pM), and Ni2+ (180 pM). More specifically, there is a significant contribution from dihydropyridine-sensitive L-type calcium channels to NGF-activated neurite outgrowth, as evidenced by the significant inhibition of neurite outgrowth by diltiazem (ICso of 17pM) and nifedipine (3 pM). Further, increases in calcium influx can elicit an enhanced neurite outgrowth response, as shown by the calcium channel agonist Bay K 8644 which potentiated neurite outgrowth by up to 40%. Key words: NGF, superior cervical ganglion, cell culture INTRODUCTION The binding of nerve growth factor (NGF) to its receptor elicits numerous effects on target cells over the course of minutes to days. However, the immediate consequences of receptor occupation leading to the generation of key intracellular messengers, and the specific pathways this signalling affects, are still not fully understood. Possible mechanisms of NGF action have either been discounted, as is the case for cyclic AMP (Frazier et al., 1973), or still remain uncertain, as for examplc with the role of inositol trisphosphate (IP,) production and calcium mobilization (Pandiella-Alonso et a1, , 1986). Enhanced turnover of phosphatidylinositides has been demonstrated in response to NGF in superior cervical ganglion (SCG, Lakshmanan: 1978), as well as PC 0 1990 Wiley-lhs, Inc.
12 (Contreras and Guroff, 1987; but see van Caulker et al.. 1989) and adrenal chromaffin cells (PandiellaAlonso et al., 1986). NGF appears lo have a novel action on the hydrylosis of glycophosphatidylinositol (Chan et al., 1989) to produce a second species of diacylglycerol capablc of activating protein kinase C (PK-C). The effects of NGF on neurite extension and protein phosphorylation can be mimicked by activators of PK-C (Hsu et al., 1984; but see Reinhold and Neet, 1989) and inhibited by various kinase blockers (Hall et al., 1988: Koizumi et al., 1988), and NGF itself elevates PK-C activity (Hama et al., 1986). A possible role for calcium ions in NGF action is indicated by the irnportancc of transmembrane calcium flux in supporting neuronal cell survival and modulating neurite outgrowth. Experiments with calcium indicator dyes (Anglister et al., 1982) and agents known to interfere with calcium channel function (Mattson and Kater, 1987) have indicated a correlation between calcium channel activity, intracellular calcium transients, and neurite outgrowth (Connor, 1986; Katcr et al., 1988). The activity of calcium channels is also pronounced in the membranes of growing neurites (Fukuda and Kameyama, 1979) and growth cones (Grinvaltl and Farber, 198 1; Marorn and Dagan, 1987) o f cxplanted and regenReceived November 30. 1989; revised February 5 , 1990; accepted February 6 . 1990. Address reprint requests to Dr. 1. Ilendry, Division of Neuroscience, John Curtin School of .Medical Kesearch, Australian National Univcraity, GPO Box 334, Canberra ACT 2601, Australia. Abbreviations used: Bay K 8644, (methyl- I,4-dihydro-2.6-dimcthyl3-nitro-4-(2-trifluoromethylphenyl)-pyridine-5-carboxylatc~; CAMP. cyclic adenosine monophosphate; DHP. dihydropyridine; DKG. dorsal root ganglion sensory neuron; G-protein, guanosine triphosphatebinding protein; HEPES, (N-(2-hydroxycthyl) piperazine-N'-(2ethanesulphonic acid)); IP,, inositol trisphosphate: NGF, nerve growth factor; PC 12 cells, pheochrornocytoma tumour cell line derived from adrenal chromaffin cell.;; PK-C, protein kinase C; SCC, superior cervical ganglion.
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erating neurons (MacVicar and Llinas, 1985). Similarly, calcium channels have recently been shown to be involved in the depolarization-induced survival of sympathetic SCG neurons (Koike et al., 1989) and chick ciliary, DRG and sympathetic neurons (Collins and Lile, 1989), and to underlie the inhibition of neurite outgrowth by high K t and bradykinin in DRG neurons (Robson and Burgoyne, 1989). The action of NGF on neurite outgrowth from sympathetic neuronc ic cimilarly sensitive to pharmacological manipulation of calcium channels, but evidence for a direct effect or NGF on transmembrane calcium flux is still equivocal (Schubert et al., 1978; Landreth et a]., 1980; Streit and Lux, 1987). With this in mind, we set out to quantify the role of extracellular calcium influx in NGF-mediated neurite outgrowth by using specific ligands to identify the type(s) of calcium channel($) involved. Preliminary reports of this work have been published in abstract form (Rogers et al., 1988; Rogers and Hendry, 1989).
METHODS SCG Culture Dissociated sympathetic cell cultures were prepared as described elsewhere (Mains and Patterson, 1973), with slight modifications. Briefly, superior cervical ganglia were removed from 2-4 day-old Wistar rat pups, cleaned in Dulbecco’s minimum essential medium (DMEM-HEPES buffered), and then incubated with 0.1% collagenase for 30 min, followed by two 10 niin incubations in a mixture of 0.1% collagenase and 0.08% trypsin. The cells were then fully dispersed by gentle trituration through flamed pipettes of decreasing bore. Neurons were suspended in 90% DMEMi 10% foetal calf serum and aliquoted into 96 multiwell plates (Nunc, Denmark) coated with rat tail collagen (prepared according to the method of Bornstem, 1958) after these had been filled with the appropriate test solutions. To half of the wells was added 0.7 pgiml p NGF (prepared from mouse submaxillary glands according to the method of Mobley et a]., 1976) while the other half served as controls for NGF-independent neurite outgrowth. Plates were incubated at 37°C in a 100% air atmosphere to reduce proliferation of non-neuronal cells (Mains and Patterson, 1973). All solutions added to the cells were sterilized by being passed through 0.22 p m filters (Millipore, MA, USA). Dose response curves for drugs were obtained by serial dilution5 of filtered stock solution. Control wells contained drug vehicle alone. Nifedipine and Bay K 8644 were dissolved in ethanol with a maximum concentration in the first well of 0.4%. As both of the dihydropyridines are light sensitive they were kept in shielded
bottles and dispensed in subdued lighting. Flunarizine, diltiazem, and nifedipine were from Sigma Chemical Co., St Louis, MO, USA. Bay K 8644 (Bayer) was a gift from Dr. Graham Lamb.
Neurite Outgrowth Assay Neurite outgrowth was quantified after 24 hr in culture by using phase contrast microscopy to count the total number of healthy neurons (phase-bright, spherical cell body), and the number of such neurons bearing neurites of greater than 1 cell diameter in length. Four fields covering 9% of the entire well were counted and the results expressed as the proportion of neurite-bearing cells per field. Individual data points are an average of at least 4 replicatc wells.
RESULTS General Observations After 24 hr in culture, SCG neurons have large spherical cell bodies about 25-30 p m in diameter. Neurites begin to appear with NGF treatment after 18 hr and continue to form extensive networks for up to several weeks. The presence of NGF is essential to both the survival of postnatal SCG neurons and for their extension of neurites. The average proportion of healthy neurons bearing neurites on collagen after 24 hr in a culture solution of l .8 mM [Ca2+l0and 0.7 yg/rnl NGF was 17.3 k 1.1% (average of means from 161 wells in 17 experiments 2 s.e.m.). SCG neurons grown without NGF express neurites at a much lower rate of 3.0 k 0.7% (n = 161 wells ? s.e.m.), and are clearly deteriorating after 24 hr.
NGF and Calcium Channels Action of inorganic blockers. Cadmium proved to be the most potent blocker of neurite outgrowth, effective at micromolar concentrations with a calculated half-maximal inhibition (ICs0) at 48 p M (Fig. 1A). Cobalt ions were the next most effective, giving an IC,,, of 129 p M (Fig. lB), and nickel gave a slightly higher IC,, of 188 p M (Fig. 1C). The least effective inorganic blocker was manganese, which first significantly affected neurite outgrowth at a concentration of 250 pM, but only completely blocked neurite outgrowth in the millimolar range, to yield an apparent IC,, value of 830 pM (Fig. ID). An intercsting observation with manganese was the appearance of large flattened growth cones at the tips of growing neurites. The relative potencies of these blockers are therefore Cd > Co > Ni > Mn, qualitatively similar to that found in many other neuronal preparations. At high concentrations cadmium, cobalt, and manganese had some effect on neuronal survival (Table I).
Calcium Channels in NGF Action Cadmium
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Fig. 1, Action of inorganic calcium channel blockers on NGFactivated neurite uutgrowth. Dose-response curves for cadir ium (n = 8)>cobalt (n = 8), nickel ( n = 7). and manganese (n = 7 - 14) plotted as the mean number (? s.c.m., vertical bars) of neurite-bearing cells against the log concentration for each treatment. Control point represents the mean number of
However, this always occurred at a higher concentration than that having a significant effect on neurite outgrowth. Effect of organic blockers. There are several classes of organic calcium channcl blockers (Table IT) useful in determining a role for various calcium channel types in neuronal systems, and we screened representative samples from three classes for their cffect on NGFmediated neurite outgrowth. Flunarizine was the least effective of all agents tested in this assay, yielding poor inhibitory action (rnaxirnal inhibition of 36% at 22 pM, Fig. 2A) and lacking any statistically significant blocking effects. Stronger inhibition of ncurite outgrowth was shown by diltiazem, which produced statistically significant blockade at all concentrations tested with a maximal inhibition of 60% at higher concentrations, to give an apparent 1C5,, of 17 FM (Fig. 2B). Contrasting results were obtained with the dihydro-
D
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***< 0.001.
TABLE 1. Comparison of Caz+ Channel Ligand Effects on SCG Cell Survival and Neurite Outerowth* Lowest conccntration to significantly affect Treatment
Neurite outgrowth
Cell survival
Cadmium Cobalt Nickel
42 p M 230 p,M 216 p,M 256 pM NS 1 . 1 FM 0.1 p M 1.9 nhl
230 pM 540 pM ?IS 3.0 mM NS NS NS NS
.Manganese
Elunarizinc Diltiazcm Nifedipine Bay K 8644
*Values are the concentration at which survival or neurite outgrowth
was significantly lower than that observed in control wells in the absence of any treatment. Significance was assessed by using twotailed Students' t-tcst, P values 5 0.01, NS = not significanl.
pyridine (DHP) ligands nifedipine and Bay K 8644. The former is a potent calcium channel antagonist and in this assay system produced strong, statistically significant in-
450
Rogers and Hendry TABLE 11. Classification of Calcium Channel Blockers* Class of blocker
Ca antagonist
Ca agonist
Ca channel specificity
Inorganic Cadmium Cobalt Nickel Manganese Organic Dihydropyridine Benzothiazepine Diphenyalkylamine Phenylalk ylarnine
Nifedipine ( + ) Bay K 8644 Diltia/.em Flunarizine Verapamil, D600
(-) Bay K 8644
L only L only‘ L onlyd L and Te L onlyd.f
”Calcium channel nomenclature is according to Fox et al. (1987), while the organic blocker types are derived from Spedding and Berg (1984). “Cadtniurn’s specificity for L- and N- over ‘1-type calcium channels is at concentrations of around 20-SO p,M. At 200 p,M cadmium blocks all channel types equivalently (Fox et al., 1987). bNickel is a more specific blocker of T-typc calcium channcls at low (100 pM) concentrations (Fox et al., 1987). ‘Bay K 8644 is listed as an antagonist and agonixt according to its stereoisomeric form; LI racemic mixture of the two having an agonistic effect was used in these experiments. dDiltiazem and the verapamil series of blockers have bcen shown to modulate calcium channels insensitive to dihyropyridines in some tissues (Carboni and Wojcik, 1988). ‘Flunarizine is a potent blocker of smooth muscle L-type currents but also affects T-type calcium channels more effectively in cardiac muscle (Tytgart et al., 1988). ‘Members of the phenylalkylamine group were not tested.
hibition of NGF-mediated neurite outgrowth: the IC,,, calculated from Figurc 2C was 3 pM, and at the highest concentration tested it completely blocked neurite outgrowth. In contrast, the DHP calcium channel agonist Bay K 8644 augmented neurite outgrowth between 10-” and M, producing a statistically significant potentiation of 40% above control at 2 nM (in Fig. 2D). At all concentrations tested the organic calcium channel ligands had no significant effect on the survival of sympathetic neurons (Table I).
DISCUSSION NGF is commonly regarded as the archetypal neurotrophic growth factor, but its mode of action in producing such typical effects as neurite outgrowth is still unclear. It is quite likcly that neurite initiation and elongation are separately regulated events, and so we have used the term neurite outgrowth to indicate the result of both initiation and elongation. Our results indicate that an influx of extracellular calcium is necessary for NGFactivated neurite outgrowth, and that dihydropyridinesensitive L-type calcium channels are specifically implicated in the effects of NGF on neuritogenesis from sympathetic neurons. There may be differing effects of calcium channel modulation upon neurite initiation and/ or elongation, but these cannot be distinguished from the present study.
Factors Affecting Basal Outgrowth Several factors appear to affect the basal level of neurite outgrowth, which in SCG neurons grown on a collagen Substrate in 1.8 mM external calcium averages 17% with an NGF concentration of 0.7 pgiml. Higher concentrations of NGF elicit enhanced rates of neurite outgrowth of up to 30% on collagen (data not shown), and this level is further influenced by the nature of the substrate used. For example, up to 100% of sympathetic neurons grown on poly-l-lysine and laminin produce neurites (Edgar, 1985, and data not shown). Secondly, alterations in the extracellular calcium concentration can influence the production of neurites by rat neurons (Koikc, 1983), 50 comparison of the concentration-dependent effects of NGF should be considered in conjunction with the level of calcium ions in the culture media. Action of Inorganic Blockers Often referred to as universal calcium channel blockers, inorganic divalent cations such as cadmium, cobalt, nickel, and manganese inhibit calcium flux through calcium channels (Hagiwara and Byerly, 1981), and so it was with these that we first assessed the possible involvement of calcium channels in the mode of NGF action and attempted to define the type of channel active in neurite outgrowth. The pharmacological profile of neurite outgrowth blockade by these inorganic ligands was Cd > Co > Ni
451
Calcium Channels in NGF Action FI u no rizi ne 25] E cn
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Fig. 2 . Action of organic calcium channel ligands on NGFdependent neurite outgrowth. Dose-response curves for flunarizine (n = 8). diltiazem (n - 4 - 1 I ) , nifcdipinc (n = lo), and Bay K 8644 (n = 4) are expressed as thc mcan (2s.e.m., vertical bars) number of' neurite-bearing cells against ligand concentration. Control point represents the mean number of
neurite bearing cells in wells containing NGF with addition of vehicle solution. Thc asterisks mark the first concentration at which each agent statistically affected neurite outgrowth, with statistical significance calculated by a two-tailed Students' t test: P values *s0.05, **s0.01, ***< 0.001.
> Mn, not dissimilar in either series or effective concentrations from studies of such calcium-dependent process~ as~neurotransmitter release (Gandia et al., 1987), 4 5 ~ I~ 2 uptake (Carboni and Wojcik, 1988), and neurite extension from retinal ganglion cells (Suarez-Isla et al., 1984). If NGF acts upon neuritc outgrowth through changes in calcium channel activity, the potency of cadmium is not unexpected given its potent effect on calcium-dependent processes. Cadmium has a high affinity for Ca2' binding sites associated with the calcium channel pore (Hagiwara and Byerly, 1981; Lansmann et al., 1986) and various intracellular domains involved with neurotransmitter release (Gum et al., 1987). Conversely, the weak effect of Mn2+ upon NGF-activated neurite outgrowth can be explained by its weak competition with Ca2- ions for transport through calcium channels (Lansmann et al., 1986), and in fact its significant conduction through the calcium channel pore may ac-
count for the occurrance of large growth cones seen with manganese, where this ion could substitute for calcium at sites involved with growth cone motility. adherence (Edwards et al., 1988), and neurite extension. Nickel ions at low micromolar concentrations selectively block (i.e., more potently than Cd2+ or C o 2 + ) a low-voltage-activated, transient or T-type calcium channcl in sensory DRG neurons (Fox et al., 1987), and we tested nickel to determine if such a calcium channel was involved in neurite outgrowth from sympathetic neurons. However, the appearance of cadmium and cobalt ahead of nickel in the IC,,, series for inhibition of neurite outgrowth, coupled with the slightly higher IC,, value of 188 p M for nickel, is consistent with electrophysiological cxpcriments which have also failed to find transient T-type calcium channels in SCG neurons (Thayer et a]., 1987; A. Field, personal communication). These inorganic calcium channel blockers are somewhat non-spccific and may be having side effects
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unrelated to calcium channel blockade. For example, cadmium also affects the calmodulin-dependent Ca2 Mg2+ ATPase in some non-neuronal cells (Akerman et al., 1985) and the sodium-calcium exchanger in cardiac muscle (Kimura et al., 1987) and thus may interfere with the overall calcium homeostasis in SCG neurons. accounting for its potent effect upon neurite outgrowth. Nickel at concentrations of 2-5 mM has been claimed to selectively block the electrogenic Na -Ca2 exchanger (Kimura et al., 1987). However, these concentrations are well above those shown here to effectively inhibit NGFactivated neurite outgrowth, and argue against a nonspecific action of these cations on the calcium exchanger in SCG neurons. The results with these inorganic ligands may therefore occur through a combination of effects on calcium channels and intracellular calcium binding sites, upsetting the calcium-dependent processes underlying neurite outgrowth. Further, the effects of these blockers upon cell survival, which can be taken as a measure of non-specific actions, revealed that cadmium, cobalt, and manganese (but not nickel) only affected viability at concentrations above those significantly inhibiting neurite outgrowth (Table I). This effect may be due to their chelation of the high molecular weight 7 s NGF molecule, which normally forms a complex with zinc ions (Greene and Shooter, 1980). Other divalent cations at high enough concentrations might alter this interaction and reduce the availability of NGF to the neurons. A further possibility is that the binding of NGF to the substrate may be altered by these cations, and thus affect the neurite outgrowth response. +
+
+
Effects of Organic Blockers In an effort to further determine whether voltagedependent calcium channels are specifically involved in neurite outgrowth and to idcntify the possible type(s) of calcium channel influenced by NGF, we employed representative agents from three major classes (Spedding and Bcrg, 1984) of organic calcium channel ligands (Table IT). The effective order of potency of these agents on NGF-activated neurite outgrowth by SCG neurons was nifedipine > diltiazem > flunarizine. Additionally, there was a statistically significant potentiation of neurite outgrowth by the calcium channel agonist Bay K 8644. More specifically, these ligands can be used to identify the type of calcium channel involved in neuritogcnesis, as they bind to and modulate specific types of calcium channel. Flunarizine is primarily a blocker of muscle-type calcium channels, being a more selective inhibitor of T-type compared to L-type channels (Tytgart et al., 1988). In this neuronal preparation it proved to be
the least effective organic blocker of neurite outgrowth, perhaps due to the lack of T-type calcium channels in this cell type (Thayer et al., 1987). The benzothiazepine-type, diltiazem-binding organic ligand site is allosterically (Spedding and Berg, 1984) and structurally (Striessnig, 1989) associated with the DHP receptoril-type calcium channel. In several neuronal preparations diltiazem in micromolar concentrations is an effective blocker of processes involving calcium influx, e.g., K+-evoked catecholamine release (Gandia et al., 1987), cerebellar granule cell 45Ca*+ uptake (Carboni and Wojcik, 1988), and smooth muscle calcium currents (Terada et al., 1987) are all inhibited at half-maximal concentrations of 1 to 12 pM. Diltiazem completely inhibited SCG neurite outgrowth in this study at similar concentrations to that required to block cerebellar granule cell calcium uptake, indicating that L-type calcium channels may be involved in both processes. The involvement of L-type calcium channels is further supported by the potent block of NGF-activated neurite outgrowth by the dihydropyridine type antagonist, nifedipine. In our in vitro assay system, concentrations of nifedipine above 10 FM could completely inhibit neuritogenesis, with the calculated IC,, of 3 pM being quite similar to that of 5 pM found to inhibit neurotransmitter release from sensory DRG neurons (Rane et al., 1987). In contrast, the calcium channel agonist Bay K 8644 (Garcia et al., 1984) produced up to 40% potentiation of neurite outgrowth between 0.1 nM and 0.1 pM, concentrations at which Bay K 8644 can be expected to be acting on the DHP receptoril-type calcium channel to prolong channel open times (Brown et al., 1984) and promote the influx of extracellular calcium to support neurite outgrowth. These organic calcium channel ligands are more specific than the inorganic ions also tested in this study. SCG cell viability is not affected at all by concentrations of these ligands that significantly affect Eeurite oulgrowth (Table I), suggesting that calcium channels are indeed specifically implicated in NGF-activated neurite outgrowth. It is therefore unlikely that changes in NGF substrate binding or availability are responsible for the effects seen with the inorganic calcium channel blockers. In neuronal tissue, however, demonstrable DHP sensitivity appears to depend on the stimulating conditions: K -depolarizations or more positive voltage clamp holding potentials favour activity of DHP-sensitive L-type calcium channels and hence reveal DHPmodifiable sites of calcium influx, while electrically or action-potential evoked responses of short duration from resting potentials stimulate calcium influx through calcium channels insensitive to DHPs (Rane et al., 1987). Thus our results with the organic ligands could be interpreted in two ways: +
Calcium Channels in NGF Action
1. A substantial proportion of the calcium influx underlying NGF-mediated neurite outgrowth occurs through DHP-sensitive calcium channels normally active in SCG neurons, or 2. Intracellular and/or membrane effects of NGF, such as a partial depolarization, favour the functioning of DHP-sensitive calcium channels. These particular effects may enhance the sensitivity of calcium influx (and subsequent neurite outgrowth) to DHP modulation.
Consistent with this latter possibility is thc sensitivity of NGF-deprived sympathetic neuron survival in depolarizing K concentrations to blockade of DHP-type calcium channels (Koike et al., 1989), and similarly in sensory DRG neurons where depolarization (and bradykinin) actions on neurite formation are blocked by nifecdipine (Robson and Burgoyne, 1989). Also, DHP-sensitive Ltype calcium channels have recently been identified as having a key role in K t depolarization effects on rat SCG neurotransmitter enzyme expression (Vidal et al., 1989) and upon chick ciliary, DRG, and sympathetic neuron survival (Collins and Lile, 1989). The profound effect of alterations in calcium influx and intracellular calcium concentration upon neurotransmitter-evoked neurite outgrowth (Kater ct al., 1988) and K t depolarization-dependent neuronal survival (Koike et al., 1989) has led to the proposal of an optimal, or set-point’’ , intracellular calcium concentration necessary to support neurite outgrowth (Connor, 1986). SCG neurons have quite a low resting intracellular calcium concentration of 67 nM, and only a small proportion of the Fura-2 calcium transient is dependent on release from intracellular calcium stores (Thayer et al., 1988). The results presented here sugggest that this cell type depends on calcium channels to provide an influx of extracellular calcium to support NGF-mediated neurite outgrowth. One of the questions remaining to be answered with regard to NGF-mediated neurite outgrowth is whether NGF specifically modulates the activity of calcium channels to bring about enhanced entry of extracellular calcium, or whether NGF produces some other signal, such as activation of a phosphorylation cascade or G-protein signalling pathway (Rogers and Hendry, 1989), which indirectly affects calcium channels. Either way, the continued functioning of calcium channels (involving a significant contribution from DHP-sensitive channels) is critical to the process of neurite outgrowth from sympathetic neurons in response to NGF. +
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ACKNOWLEDGMENTS We would like to thank Ms. Jenny Meancy and Mrs. Mary Preston for their ever-helpful technical assis-
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tance, and Dr. Caryl Hill for advice on culturing techniques. Dr. Michael Crouch provided helpful comments on this manuscript. M.R. is supported by an ANU PhD scholarship and assisted by aerobics instructors throughout Australasia!
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