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Calyculin-A-induced fast neurite retraction in nerve growth factor-differentiated rat pheochromocytoma (PC12) cells
ELSEVIER
Neuroscience Letters 183 (1995) 198-201
fiEUROSCHC[ LETTERS
Calyculin-A-induced fast neurite retraction in nerve growth factordifferentiated rat pheochromocytoma (PC 12) cells Bernhard F.X. Reber*, Alexandre Bouron Department of Pharmacology, University of Bern, Friedbiihlstrasse49, CH-3010 Bern, Switzerland Received 2 September 1994; revised version received 11 November 1994; accepted 11 November 1994
Abstract
Rat pheochromocytoma (PC12) cells were treated with nerve growth factor (NGF) for 3--4 days. They formed growth cones and extended neurites. Addition of the phosphatase inhibitor calyculin A (CL-A) caused a concentration-dependent complete retraction of neurites within 15 min. Retraction of growth cones started with the filopodia still present. The cell bodies acquired a grape-like shape opposite to the cell nucleus. These morphological changes were reversible. After washout of the inhibitor, the cell bodies recovered to normal shape within about 30-60 min while neurites started to grow again within 1 day. Okadaic acid (OA) which, compared to CL-A, is less potent as a PP-1 and equally potent as a PP-2A class inhibitor, caused neurite retraction only when added at more than a thousand-fold higher concentration than CL-A. Ca 2÷ levels within neurites and cell bodies remained stable and low during neurite retraction as measured with fura-2. However, cells treated with CL-A showed reduced activity of voltage-gated Ca 2+ channels. The results suggest that the observed reversible changes in cell morphology occur at a constant low Ca 2+ level and are most likely due to the inhibition of PP- 1 class phosphatases. Keywords: Pheochromocytoma; Fura-2; Calycutin-A; Okadaic acid; Neurite retraction
The formation of growth cones and neurites belongs to the most obvious morphological changes of nerve growth factor (NGF)-induced differentiation of rat pheochromocyioma (PC12) cells [8]. In addition, growth cone collapse and neurite retraction belong to the repertoire by which growing neurons can respond to their environment [7]. Much effort has been put into the elucidation of possible signal transduction mechanisms for these phenomena. It has been proposed that the level of Ca 2÷ is an important intracellular regulator of neurite elongation and growth cone motility [ 14]. Furthermore, neurotransmitters may activate other intracellular second messenger systems and, thereby, influence neuronal outgrowth [4,15]. The structure of the neurites and growth cones depends largely on various cytoskeletal elements. The filopodia are formed predominantly by actin-containing structures, whereas the neurites are supported by a microtubular cytoskeleton. Since phosphoproteins are thought to be key intermediates in the regulation of cytoskeletal elements, * Corresponding author, Tel.: +41 31 632 32 81; Fax: +41 31 302 72 30; e.mail: [email protected].
we compared the effects of the phosphatase inhibitors calyculin A (CL-A) and okadaic acid (OA) to obtain information about possible involvement of phosphatase activity in the regulatory events of neurite formation and/or retraction. CL-A, a tumor promoting substance isolated from the marine sponge Discodermia calyx [13], is an inhibitor of protein phosphatase 1 (PP-1) (IC50 = 0.5-1 nM) and protein phosphatase 2A (PP-2A) (IC50 = 2 nM) in rabbit skeletal muscle [10,11]. The inhibitor OA is much more potent for PP-2A (IC50 = 0.51.0 nM) than for PP-1 (IC50 = 60-200 nM). Here we report that CL-A causes a fast, but reversible change in cell morphology of differentiated P C I 2 cells which consists of an initial retraction of neurites followed by a shape change of the cell bodies. OA is about a thousand-fold less potent in causing this effect. The morphological changes occur without obvious changes in the intracellular Ca 2÷ concentration. The rat PC12 cells were grown in DMEM with 10% horse serum (Gibco) in a 5% CO2 atmosphere at 37°C and differentiated by addition of NGF (7S fraction, 100 ng m1-1) as described [18]. Cells were viewed with an in-
0304-3940/95/$09.50 © 1995 Elsevier Science Ireland Ltd. All rights reserved SSDI 0304-3940(94)1 1 150-E
B.F.X. Reber, A. Bouron / Neuroscience Letters 183 (1995) 198-201
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Fig. 1. (A) CL-A-induced retraction of neurites and shape change of NGF-differentiated PC12 cells in culture. (a-c) morphologyof a NGFdifferentiated cell after 0, 4 and 10 min exposure to 20 nM CL-A. (d) Three cells 15 min after addition of CL-A. Note the grape-likestructure (arrow) opposite to the rest of the cell including the cell nucleus. Bar, 20/~m. (B) Time-lapse video images of a single growth cone during CL-A induced retraction (a-d) 3, 4, 7, 8 min. Filopodia remain connected to the retracting growth cone. Black arrow, non-moving reference point. (C) Dose-response relationship of CL-A-induced neurite retraction. The length of individual neurites after CL-A addition (50 nM (O), 20 nM (O), 5 nM (11), 2 nM ([]), 0.5 nM (A), 0.2 nM (/X))wa.s measured on digitized pictures. The mean neurite lengths (n = 5) of 2 or 3 cells is plotted against time. verted microscope (Axiovert 30, Carl Zeiss AG, Zurich, Switzerland). The standard bath solution consisted of (in mM) 150 NaCI, 5 KC1, 1.8 CaC12, 1 MgCI2, 10 HEPES, pH adjusted to 7.4 with NaOH. Photographs were taken using Ilford Pan F film. Time-lapse studies of growth cone movements and cell body rearrangements using high resolution differential interference contrast (DIC) were obtained on a laser scanning microscope (LSM 410, Carl Zeiss AG, Zurich, Switzerland). Data analysis was performed on a Macintosh ® II computer using self-written software programs and the public domain NIH Image software program (written by Wayne Rasband at the US National Institutes of Health). Intracellular free Ca 2÷ concentration ([Ca2+]i) from individual cells was measured using fura-2 [9]. The experimental protocols and data acquisition routines have been described in detail elsewhere [17]. The membrane capacitance of individual cells and membrane currents through Ca 2+ channels were measured using Ba 2÷ as charge carrier in standard bath solution [18]. Culturing of PC12 cells in the presence of NGF led to enlarged cell bodies and simultaneous formation of neurites within 3-4 days [18]. The neurites extended from a few micrometers to several tenths of micrometers after 4 days in culture. Addition of the phosphatase inhibitor CL-A (20 nM, ANAWA Trading SA, Wangen, Switzerland) caused retraction of these neurites within 10 min (Fig. 1A). Growth cone collapse and withdrawal began about 2-3 min after drug addition. Many filopodia were extending from the growth cone tip (Fig. 1B). Not all of
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them collapsed, when the withdrawal of the growth cone began. Some of them adhered firmly to the surface of the coverslip, became extended like rubber bands and remained connected to the retracting neurite tip, while others tore apart. These filopodia are supported internally by actin filaments [6] and seem to be affected little by CL-A treatment. The complete retraction of neurites argues for a breakdown of microtubules. Tension was presumably generated in the neurite at a time when the growth cone adhered firmly to the coverslip. This caused stretching of the cell body before the onset of retraction. A shape change of the cell body accompanied the neurite retraction. The cell bodies showed a characteristic grape-like domain opposite to the cell nucleus (Fig. 1A,d). No obvious budding of cell membrane was observed. We assume that most of the membrane surface of the neurites was taken up into the cell body. Evidence for a reduction of surface membrane area was obtained by means of measurements of the membrane capacitance which decreased from 32 _+ 1.4 pF (n = 13) in control cells to 25.1 _+ 1.1 pF ( n = 20, P < 0.01) in cells after neurite retraction. The dose-dependency of CL-A-induced neurite retraction is shown in Fig. 1C. The elapsed time between the addition of CL-A (>5 nM) and the onset of the retraction was 3 rain. Maximal retraction rates (0.8-1.0/~m/s) of single neurites occurred at different times after drug addition. The lag-time increased and the retraction speed was slower at lower concentrations (0.2-2.0 nM) of the drug. OA is known to inhibit PP-2A activity at concentrations similar to those of CL-A, whereas it is much less effective against PP-1 activity [10]. Therefore, effects of OA on the cell morphology were compared with those observed with CL-A. OA inhibits the NGF-dependent initial outgrowth in PC12 cells at nanomolar concentration [3]. In our study, 20 nM OA induced no changes similar to those of CL-A in NGF-differentiated PC12 cells during exposure times up to 24 h. However, OA at 2/zM concentration was about equally potent as 0.2 nM CL-A. Partial retraction of neurites occurred within 3 h. On the other hand, a down-modulation of the voltage-activated Ba 2+ currents similar to CL-A occurred with OA already at lower concentration in NGF-differentiated PC12 cells [1]. This argues for PP-1 as the main site of action inducing these morphological changes. The shape change of the cell bodies was reversible within 30-60 min when CL-A was washed out after 10 min by means of two rinses with a 1% bovine serum albumin solution. The grape-like structures disappeared and the cell body became larger and round (Fig. 2a,b). When PC12 cells were treated again with NGF (100 ng ml-l), they underwent a second morphological differentiation into neuron-like cells (Fig. 2c,d). Within 2 days the cells formed neurites again. Their number and length increased during the following days. This clearly shows reversibility of the drastic morphological changes induced by CL-A.
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B.F.X. Reber, A. Bouron / Neuroscience Letters 183 (1995) 198-201
Fig. 2. Change in morphology of CL-A-treated cells after washout of the drag. Cells were incubated with 20 nM CL-A for 15 rain and then washed twice with 1% BSA-containing medium. (a,b) 0 rain (end of washout), 60 min. Bar, 10#m. (c,d) Cells regrown in NGF-containing medium for 1 day and 2 days, respectively. Bar, 20/zm.
Morphological changes in cells are probably associated with contractile elements in the cytoskeleton. Changes in [Ca2÷]i may regulate these alterations. However, addition of CL-A did not affect the basal [Ca2+]i. The CL-A-induced retraction of neurites in differentiated PCI2 cells occurs at constant low [Ca2+]i as measured with fura-2 (Fig. 3B). It remained at constant low levels for at least up to 2 h. Retraction occurred in the presence
II l l calcium tre~
B
KCI
C
Ihe~l~in
of external Ca 2+ (Fig. 1A), in nominally Ca 2÷ free solution (Fig. 3A, left), or when [Ca2+]i was transiently increased by K+-depolarization (75 mM [K+]o) (Fig. 3A, middle). Furthermore, the cells underwent CL-A induced neurite retraction after pretreatment with thapsigargin (2/tM, 5 min), arguing for a lack of dependence on intact Ca 2+ stores (Fig. 3A, right) [17]. Similarly, contact-mediated collapse of chick dorsal root ganglion (DRG) growth cone structures has also been described to occur at constant intracellular Ca 2+ levels [12]. Our finding is different from that by Dyer et al. [5], who found a Ca2+-depen dence for the neurite retraction of PC12 cells induced by a factor isolated from serum albumin. It also differs from the results of Bandtlow et al. [2], who described that Ca 2+ release from an internal caffeine-sensitive pool is crucial for the NI-35 induced growth cone collapse in cultured rat DRG neurons. To further analyze the viability of CL-A-treated PC12 cells and to look for other effects of CL-A, we measured the activity of voltage-gated Ca 2+ channels. Fig. 3C shows that short application of 20 nM CL-A (30 s) reduces whole-cell Ba 2+ currents through voltage-activated Ca 2+ channels. The average peak current density of whole-cell Ba 2÷ currents was 19.4 _+0.9 pA/pF (n = 6). Superfusion of the cells with acutely added CL-A (20 nM) reduced the current amplitude by 20 _+9% (n = 4, P < 0.05). A 30-min pre-incubation of the cells with the inhibitor (20 nM) reduced the whole-cell Ba 2+ current by 30% from 19.4 _+0.9 pA/pF (n = 6) to 13.5 _+ 1.1 pA/pF (n = 6, P < 0.05). This result correlates with the reduction in K÷depolarization-induced transient Ca 2+ rises of 50% from 446 _+22 nM (n = 40) to 261 + 12 nM (n = 40; P < 0.01) (Fig. 3D). Half maximal inhibition by CL-A was reached after 5--6 min (n = 3). Therefore, our data show that voltage-activated Ca 2+ channels in PC12 cells are under a modulatory control by phosphorylation/dephosphorylation reactions. In conclusion, CL-A causes retraction of neurites at constant low Ca 2+ levels. The effects are attributed to the inhibition by CL-A of PP-I class phosphatases because OA showed similar effects only when added at much higher concentration.
D
lo0
Fig. 3. (A) CL-A-induced neurite retraction in nominally free Ca 2+ solution, in 75 mM [K+]o, and in presence of 2/~M thapsigargin (2/.tM). Time (min) is indicated at the top. Bar, 20/~m. (B) [Ca2÷]i within retracting neurites. The [Ca2+] i (90 +_5 nM; n = 6) remained constant (94 _+4 nM after 15 min) after CL-A addition (20 nM, arrow). (C) Reversible inhibition of whole-cell Ba 2+ currents by CL-A. Shown are the current traces recorded before (1), in the presence (2, 15 s) and after wash-out of the drug (3, 60 s). (D) Effect of CL-A on the K +depolarization-induced rise in [Ca2+]i. Shown is the pre-incubation time with 20 nM CL-A and net increases in [Ca2+] i (n = 10).
We are very grateful for the encouraging support of H. Reuter and for his suggestions in writing the manuscript. The excellent technical work with the cell cultures by Ms. Ch. Becker and Mrs. Beccioli is gratefully acknowledged. This work was supported by the Swiss National Science Foundation (grant 31-29862.90 to H. Reuter and 4140483.94 to B. Reber). [1] Bouron, A. and Reber, B.F.-X., Differential modulation of Ca 2+ currents by protein kinase C activators and phosphatase inhibitors in nerve-growth-factor-differentiated rat pheochromocytoma (PC12) cells, Pfliigers Arch., 427 (1994) 510-516. [2] Bandtlow, C.E., Schmidt, M.F., Hassinger, T.D., Schwab, M.E.
B.F.X. Reber, A. Bouron / Neuroscience Letters 183 (1995) 198-201
[3]
[4]
[5]
[6]
[7] [8]
[9]
[10]
and Kater, S.B., Role of calcium in NI-35-evoked collapse of neuronal growth cones, Science, 259 (1993) 80-83. Chiou, J.-Y. and Westhead, E.W., Okadalc acid, a protein phosphatase inhibitor, inhibits nerve growth factor-directed neurite outgrowth in PC12 cells, J, Neurochem., 59 (1992) 1963-1966. Cohan, C.S., Connor, J.A. and Kater, S.B., Electrically and chemically mediated increases in intracellular calcium in neuronal growth cones, J. Neurosci., 7 (1987) 3588-3599. Dyer, D., Tigyi, G. and Miledi, R., The effect of active serum albumin on PC12 cells: II lntracellular Ca 2+ transients and their role in neurite retraction, Mol. Brain Res., 14 (1992) 302-309. Fan, J., Mansfield, S.G., Redmont, T., Gordon-Weeks, P.R. and Raper, J.A., The organization of F-actin and microtubules in growth cones exposed to a brain-derived collapsing factor, J. Cell Biol., 121 (1993)867-878. Fawcett, J.W., Growth-cone collapse: too much of a good thing?, Trends Neurosci., 16 (1993) 165-167. Greene, L.A. and Tischler, A.S., PC12 pheochromocytoma cultures in neurobiologicai research, Adv. Cell. Neurobiol., 3 (1982) 373-414. Grynkiewicz, G., Poenie, M. and Tsien, R.Y., A new generation of Ca 2+ indicators with greatly improved fluorescence properties, J. Biol. Chem., 260 (1985) 3340-3450. Ishihara, H., Martin, B.L., Brantigan, D.L., Karak, H., Ozaki, H, Kato, Y., Fusetani, N., Watabe, S., Hashimoto, K., Uemura, D. and Hartshorne, D.J., Calyculin A and okadaic acid: Inhibitors of protein phosphatase activity, Biochem. Biophys. Res. Commun., 159 (1989) 871-877.
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[11] Ishihara, H., Ozaki, H., Sato, K., Hod, M., Karaki, H., Watabe, S., Kato, Y., Fusetani, N., Hashimoto, K., Uemura, D. and Hartshome, D.J., Calcium-independent activation of contractile apparatus in smooth muscle by calyculin-A, J. Pharmacol. Exp. Ther., 250 (1989) 388-396. [12] Ivins, J.K., Raper, J.A. and Pittman, R.N., intracellular calcium levels do not change during contact-mediated collapse of chick DRG growth cone structure, J. Neurosci., 11 (1991) 1597-1608. [13] Kato, Y., Fusetani, N., Matsunaga, S. and Hashimoto, K., Calyculin A a novel antitumor metabolite from the marine sponge Discodermia calyx, J. Am. Chem. Soc., 108 (1986) 2780-2781. [14] Kater, S.B., Mattson, M.P., Cohan, C. and Connor, J., Calcium regulation of the neuronal growth cone, Trends Neurosci., 7 (1988) 315-321. [15] Mattson, M.M., Neurotransmitters in the regulation on neuronal cytoarchitecture, Brain Res. Rev., 13 (1988) 179-212. [16] Neuhaus, R., Reber, B.F.-X. and Reuter, H., Regulation of bradykinin- and ATP-activated Ca2+-permeable channels in rat pheochromocytoma (PCI2) cells, J. Neurosci., 11 (1991) 39843990. [17] Reber, B.F.-X. and Reuter, H., Dependence of cylosolic calcium in differentiating rat pheochromocytoma cells on calcium channels and intracellular stores, J. Physiol. (London), 435 (1991) 145-162. [18] Usowicz, M.M., Porzig, H., Becket, C. and Reuter, H., Differential expression by nerve growth factor of two types of Ca 2+ channels in rat pheochromocytoma cell lines, J. Physiol. (London), 426 (1990) 95-116.
Neuroscience Letters 183 (1995) 198-201
fiEUROSCHC[ LETTERS
Calyculin-A-induced fast neurite retraction in nerve growth factordifferentiated rat pheochromocytoma (PC 12) cells Bernhard F.X. Reber*, Alexandre Bouron Department of Pharmacology, University of Bern, Friedbiihlstrasse49, CH-3010 Bern, Switzerland Received 2 September 1994; revised version received 11 November 1994; accepted 11 November 1994
Abstract
Rat pheochromocytoma (PC12) cells were treated with nerve growth factor (NGF) for 3--4 days. They formed growth cones and extended neurites. Addition of the phosphatase inhibitor calyculin A (CL-A) caused a concentration-dependent complete retraction of neurites within 15 min. Retraction of growth cones started with the filopodia still present. The cell bodies acquired a grape-like shape opposite to the cell nucleus. These morphological changes were reversible. After washout of the inhibitor, the cell bodies recovered to normal shape within about 30-60 min while neurites started to grow again within 1 day. Okadaic acid (OA) which, compared to CL-A, is less potent as a PP-1 and equally potent as a PP-2A class inhibitor, caused neurite retraction only when added at more than a thousand-fold higher concentration than CL-A. Ca 2÷ levels within neurites and cell bodies remained stable and low during neurite retraction as measured with fura-2. However, cells treated with CL-A showed reduced activity of voltage-gated Ca 2+ channels. The results suggest that the observed reversible changes in cell morphology occur at a constant low Ca 2+ level and are most likely due to the inhibition of PP- 1 class phosphatases. Keywords: Pheochromocytoma; Fura-2; Calycutin-A; Okadaic acid; Neurite retraction
The formation of growth cones and neurites belongs to the most obvious morphological changes of nerve growth factor (NGF)-induced differentiation of rat pheochromocyioma (PC12) cells [8]. In addition, growth cone collapse and neurite retraction belong to the repertoire by which growing neurons can respond to their environment [7]. Much effort has been put into the elucidation of possible signal transduction mechanisms for these phenomena. It has been proposed that the level of Ca 2÷ is an important intracellular regulator of neurite elongation and growth cone motility [ 14]. Furthermore, neurotransmitters may activate other intracellular second messenger systems and, thereby, influence neuronal outgrowth [4,15]. The structure of the neurites and growth cones depends largely on various cytoskeletal elements. The filopodia are formed predominantly by actin-containing structures, whereas the neurites are supported by a microtubular cytoskeleton. Since phosphoproteins are thought to be key intermediates in the regulation of cytoskeletal elements, * Corresponding author, Tel.: +41 31 632 32 81; Fax: +41 31 302 72 30; e.mail: [email protected].
we compared the effects of the phosphatase inhibitors calyculin A (CL-A) and okadaic acid (OA) to obtain information about possible involvement of phosphatase activity in the regulatory events of neurite formation and/or retraction. CL-A, a tumor promoting substance isolated from the marine sponge Discodermia calyx [13], is an inhibitor of protein phosphatase 1 (PP-1) (IC50 = 0.5-1 nM) and protein phosphatase 2A (PP-2A) (IC50 = 2 nM) in rabbit skeletal muscle [10,11]. The inhibitor OA is much more potent for PP-2A (IC50 = 0.51.0 nM) than for PP-1 (IC50 = 60-200 nM). Here we report that CL-A causes a fast, but reversible change in cell morphology of differentiated P C I 2 cells which consists of an initial retraction of neurites followed by a shape change of the cell bodies. OA is about a thousand-fold less potent in causing this effect. The morphological changes occur without obvious changes in the intracellular Ca 2÷ concentration. The rat PC12 cells were grown in DMEM with 10% horse serum (Gibco) in a 5% CO2 atmosphere at 37°C and differentiated by addition of NGF (7S fraction, 100 ng m1-1) as described [18]. Cells were viewed with an in-
0304-3940/95/$09.50 © 1995 Elsevier Science Ireland Ltd. All rights reserved SSDI 0304-3940(94)1 1 150-E
B.F.X. Reber, A. Bouron / Neuroscience Letters 183 (1995) 198-201
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Fig. 1. (A) CL-A-induced retraction of neurites and shape change of NGF-differentiated PC12 cells in culture. (a-c) morphologyof a NGFdifferentiated cell after 0, 4 and 10 min exposure to 20 nM CL-A. (d) Three cells 15 min after addition of CL-A. Note the grape-likestructure (arrow) opposite to the rest of the cell including the cell nucleus. Bar, 20/~m. (B) Time-lapse video images of a single growth cone during CL-A induced retraction (a-d) 3, 4, 7, 8 min. Filopodia remain connected to the retracting growth cone. Black arrow, non-moving reference point. (C) Dose-response relationship of CL-A-induced neurite retraction. The length of individual neurites after CL-A addition (50 nM (O), 20 nM (O), 5 nM (11), 2 nM ([]), 0.5 nM (A), 0.2 nM (/X))wa.s measured on digitized pictures. The mean neurite lengths (n = 5) of 2 or 3 cells is plotted against time. verted microscope (Axiovert 30, Carl Zeiss AG, Zurich, Switzerland). The standard bath solution consisted of (in mM) 150 NaCI, 5 KC1, 1.8 CaC12, 1 MgCI2, 10 HEPES, pH adjusted to 7.4 with NaOH. Photographs were taken using Ilford Pan F film. Time-lapse studies of growth cone movements and cell body rearrangements using high resolution differential interference contrast (DIC) were obtained on a laser scanning microscope (LSM 410, Carl Zeiss AG, Zurich, Switzerland). Data analysis was performed on a Macintosh ® II computer using self-written software programs and the public domain NIH Image software program (written by Wayne Rasband at the US National Institutes of Health). Intracellular free Ca 2÷ concentration ([Ca2+]i) from individual cells was measured using fura-2 [9]. The experimental protocols and data acquisition routines have been described in detail elsewhere [17]. The membrane capacitance of individual cells and membrane currents through Ca 2+ channels were measured using Ba 2÷ as charge carrier in standard bath solution [18]. Culturing of PC12 cells in the presence of NGF led to enlarged cell bodies and simultaneous formation of neurites within 3-4 days [18]. The neurites extended from a few micrometers to several tenths of micrometers after 4 days in culture. Addition of the phosphatase inhibitor CL-A (20 nM, ANAWA Trading SA, Wangen, Switzerland) caused retraction of these neurites within 10 min (Fig. 1A). Growth cone collapse and withdrawal began about 2-3 min after drug addition. Many filopodia were extending from the growth cone tip (Fig. 1B). Not all of
199
them collapsed, when the withdrawal of the growth cone began. Some of them adhered firmly to the surface of the coverslip, became extended like rubber bands and remained connected to the retracting neurite tip, while others tore apart. These filopodia are supported internally by actin filaments [6] and seem to be affected little by CL-A treatment. The complete retraction of neurites argues for a breakdown of microtubules. Tension was presumably generated in the neurite at a time when the growth cone adhered firmly to the coverslip. This caused stretching of the cell body before the onset of retraction. A shape change of the cell body accompanied the neurite retraction. The cell bodies showed a characteristic grape-like domain opposite to the cell nucleus (Fig. 1A,d). No obvious budding of cell membrane was observed. We assume that most of the membrane surface of the neurites was taken up into the cell body. Evidence for a reduction of surface membrane area was obtained by means of measurements of the membrane capacitance which decreased from 32 _+ 1.4 pF (n = 13) in control cells to 25.1 _+ 1.1 pF ( n = 20, P < 0.01) in cells after neurite retraction. The dose-dependency of CL-A-induced neurite retraction is shown in Fig. 1C. The elapsed time between the addition of CL-A (>5 nM) and the onset of the retraction was 3 rain. Maximal retraction rates (0.8-1.0/~m/s) of single neurites occurred at different times after drug addition. The lag-time increased and the retraction speed was slower at lower concentrations (0.2-2.0 nM) of the drug. OA is known to inhibit PP-2A activity at concentrations similar to those of CL-A, whereas it is much less effective against PP-1 activity [10]. Therefore, effects of OA on the cell morphology were compared with those observed with CL-A. OA inhibits the NGF-dependent initial outgrowth in PC12 cells at nanomolar concentration [3]. In our study, 20 nM OA induced no changes similar to those of CL-A in NGF-differentiated PC12 cells during exposure times up to 24 h. However, OA at 2/zM concentration was about equally potent as 0.2 nM CL-A. Partial retraction of neurites occurred within 3 h. On the other hand, a down-modulation of the voltage-activated Ba 2+ currents similar to CL-A occurred with OA already at lower concentration in NGF-differentiated PC12 cells [1]. This argues for PP-1 as the main site of action inducing these morphological changes. The shape change of the cell bodies was reversible within 30-60 min when CL-A was washed out after 10 min by means of two rinses with a 1% bovine serum albumin solution. The grape-like structures disappeared and the cell body became larger and round (Fig. 2a,b). When PC12 cells were treated again with NGF (100 ng ml-l), they underwent a second morphological differentiation into neuron-like cells (Fig. 2c,d). Within 2 days the cells formed neurites again. Their number and length increased during the following days. This clearly shows reversibility of the drastic morphological changes induced by CL-A.
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B.F.X. Reber, A. Bouron / Neuroscience Letters 183 (1995) 198-201
Fig. 2. Change in morphology of CL-A-treated cells after washout of the drag. Cells were incubated with 20 nM CL-A for 15 rain and then washed twice with 1% BSA-containing medium. (a,b) 0 rain (end of washout), 60 min. Bar, 10#m. (c,d) Cells regrown in NGF-containing medium for 1 day and 2 days, respectively. Bar, 20/zm.
Morphological changes in cells are probably associated with contractile elements in the cytoskeleton. Changes in [Ca2÷]i may regulate these alterations. However, addition of CL-A did not affect the basal [Ca2+]i. The CL-A-induced retraction of neurites in differentiated PCI2 cells occurs at constant low [Ca2+]i as measured with fura-2 (Fig. 3B). It remained at constant low levels for at least up to 2 h. Retraction occurred in the presence
II l l calcium tre~
B
KCI
C
Ihe~l~in
of external Ca 2+ (Fig. 1A), in nominally Ca 2÷ free solution (Fig. 3A, left), or when [Ca2+]i was transiently increased by K+-depolarization (75 mM [K+]o) (Fig. 3A, middle). Furthermore, the cells underwent CL-A induced neurite retraction after pretreatment with thapsigargin (2/tM, 5 min), arguing for a lack of dependence on intact Ca 2+ stores (Fig. 3A, right) [17]. Similarly, contact-mediated collapse of chick dorsal root ganglion (DRG) growth cone structures has also been described to occur at constant intracellular Ca 2+ levels [12]. Our finding is different from that by Dyer et al. [5], who found a Ca2+-depen dence for the neurite retraction of PC12 cells induced by a factor isolated from serum albumin. It also differs from the results of Bandtlow et al. [2], who described that Ca 2+ release from an internal caffeine-sensitive pool is crucial for the NI-35 induced growth cone collapse in cultured rat DRG neurons. To further analyze the viability of CL-A-treated PC12 cells and to look for other effects of CL-A, we measured the activity of voltage-gated Ca 2+ channels. Fig. 3C shows that short application of 20 nM CL-A (30 s) reduces whole-cell Ba 2+ currents through voltage-activated Ca 2+ channels. The average peak current density of whole-cell Ba 2÷ currents was 19.4 _+0.9 pA/pF (n = 6). Superfusion of the cells with acutely added CL-A (20 nM) reduced the current amplitude by 20 _+9% (n = 4, P < 0.05). A 30-min pre-incubation of the cells with the inhibitor (20 nM) reduced the whole-cell Ba 2+ current by 30% from 19.4 _+0.9 pA/pF (n = 6) to 13.5 _+ 1.1 pA/pF (n = 6, P < 0.05). This result correlates with the reduction in K÷depolarization-induced transient Ca 2+ rises of 50% from 446 _+22 nM (n = 40) to 261 + 12 nM (n = 40; P < 0.01) (Fig. 3D). Half maximal inhibition by CL-A was reached after 5--6 min (n = 3). Therefore, our data show that voltage-activated Ca 2+ channels in PC12 cells are under a modulatory control by phosphorylation/dephosphorylation reactions. In conclusion, CL-A causes retraction of neurites at constant low Ca 2+ levels. The effects are attributed to the inhibition by CL-A of PP-I class phosphatases because OA showed similar effects only when added at much higher concentration.
D
lo0
Fig. 3. (A) CL-A-induced neurite retraction in nominally free Ca 2+ solution, in 75 mM [K+]o, and in presence of 2/~M thapsigargin (2/.tM). Time (min) is indicated at the top. Bar, 20/~m. (B) [Ca2÷]i within retracting neurites. The [Ca2+] i (90 +_5 nM; n = 6) remained constant (94 _+4 nM after 15 min) after CL-A addition (20 nM, arrow). (C) Reversible inhibition of whole-cell Ba 2+ currents by CL-A. Shown are the current traces recorded before (1), in the presence (2, 15 s) and after wash-out of the drug (3, 60 s). (D) Effect of CL-A on the K +depolarization-induced rise in [Ca2+]i. Shown is the pre-incubation time with 20 nM CL-A and net increases in [Ca2+] i (n = 10).
We are very grateful for the encouraging support of H. Reuter and for his suggestions in writing the manuscript. The excellent technical work with the cell cultures by Ms. Ch. Becker and Mrs. Beccioli is gratefully acknowledged. This work was supported by the Swiss National Science Foundation (grant 31-29862.90 to H. Reuter and 4140483.94 to B. Reber). [1] Bouron, A. and Reber, B.F.-X., Differential modulation of Ca 2+ currents by protein kinase C activators and phosphatase inhibitors in nerve-growth-factor-differentiated rat pheochromocytoma (PC12) cells, Pfliigers Arch., 427 (1994) 510-516. [2] Bandtlow, C.E., Schmidt, M.F., Hassinger, T.D., Schwab, M.E.
B.F.X. Reber, A. Bouron / Neuroscience Letters 183 (1995) 198-201
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