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Effects of p-bromophenacyl bromide on neurite growth at different levels of nerve growth factor
Neuroscience Letters, 86 (I g88) 35(~ _:;¢~ti Elsevier Scientific Publishers Ireland Lid
356
NSL 05230
Effects of p-bromophenacyl bromide on neurite growth at different levels of nerve growth factor Claudia Cei de Job and Angela Maria Suburo lnstituto de Neurobiologia, Buenos Aires (Argentina) (Received 16 June 1987; Revised version received 4 November 1987; Accepted 18 December 1987)
Key words." Neurite growth; p-Bromophenacyl bromide; Nerve growth factor; Rat dorsal root ganglion explant We have previously been shown that the phospholipase inhibitor p-bromophenacyl bromide (BPB) modifies neurite morphology and growth rate in rat dorsal root ganglion explants. Neurite extension is stimulated by 2.5 x 10- 7 M BPB but it is prevented by concentrations higher than 10 5 M. Under our experimental conditions, no neurites appeared in the absence of added nerve growth factor (NGF). Therefore, we have tested the effect of a low BPB concentration at different levels of exogenous NGF. Thoracic and lumbar ganglia were obtained from 19.5-day-old fetal rats and were cultured for 24 h on a polyornithine substrate. Neurite growth and development of a dense halo were NGF-dependent in both ganglionic populations; however, each of them showed a characteristic dose--response curve. Incubation with BPB induced neurites in the absence of added NGF and stimulated their growth when low levels of this factor were added. In lumbar ganglia incubated with higher levels of N(3F, BPB had no detectable effects. On the contrary, similarly treated thoracic ganglia showed an inhibition of neurite extension. Our findings suggest a relationship between the effects of BPB and the initial, membrane-activating actions of NGF.
We have recently reported that the extension of neural cell processes can be facilitated or prevented by different concentrations of p-bromophenacyl bromide (BPB) and other phospholipase A2 (PLA2) inhibitors. This effect has been shown both in dorsal root ganglion (DRG) explants [14, 15] and in neural retina monolayers [16]. In the D R G explants, a decrease in the growth rate of neural processes is produced by 2.5 x 10 -5 M or higher BPB concentrations, whereas increases in neurite length are observed around 10 -7 M. It is well known that neuron survival and neurite extension in D R G explants require nerve growth factor (NGF) [8, 18]. Besides, under our culture conditions, fetal rats D R G s do not extend neural processes without added N G F , and their neuritic outgrowth can be regulated by the N G F concentration in the culture medium. Since the stimulant effect of PLA2 inhibitors could be more evident at suboptimal concentrations of N G F , we have assayed the effect of a low concentration of BPB in D R G explants grown at different N G F levels.
Correspondence: A.M. Suburo, Instituto de Neurobiologia, Serrano 661, 1414 Buenos Aires, Argentina. 0304-3940/88/$ 03.50 © 1988 Elsevier Scientific Publishers Ireland Ltd.
357
Ganglia were obtained from 19.5-day-old rat fetuses (Holtzmann inbred rats, day 0 being the day when spermatozoids were found in vaginal smears). Pregnant rats were anesthetized with chloral hydrate and fetuses were removed under aseptic conditions. Thoracic and lumbar ganglia were dissected and explanted onto a 0.1 mg/ml polyornithine substrate made on plastic dishes. Culture medium was Eagle's minimal medium with Hanks' salts (Difco) supplemented with 5% fetal calf serum, 2 mM glutamine, 5 g/liter glucose, antibiotics and various concentrations of N G F . The latter was purified from mouse submaxillary glands [9] and biologically assayed [18]. Explants were incubated at 37°C in a 95:5 air:CO2 atmosphere saturated with water vapor. Dilutions of BPB (Sigma) were prepared as previously reported [15] and added after the first 5 h in vitro. One day-old cultures were examined under phase optics and classified according to the following criteria: (a) presence of neurites; (b) appearance of neuritic halos, defined as outgrowths with processes equal or longer than 225/,m in their 4 quadrants; (c) development of large halos, defined as those showing 330/lm or longer neurites in at least one of their quadrants. Outward migration of non-neuronal cells was not observed in these cultures. In control experiments, the proportion of ganglia bearing neurites increased with N G F concentration in the culture medium. No neurites were observed when this factor was excluded from the medium. The shortest neurites measured about 45 pro, whereas the longest ones extended up to 3 0 0 4 5 0 ,um. As longer neurites appeared, a dense outgrowth was formed around the explants. Halo development required the addition of higher levels of N G F than the appearance of neurites (Table I), and only the highest N G F concentrations induced the formation of large halos. Lumbar ganglia explants appeared more sensitive to added N G F than those from the thoracic region. Thus, every lumbar explant exhibited neurites at 1.2 BU/ml and the thoracic population reached that situation at 4.8 BU/ml. Moreover, very few of the thoracic explants showed halos at 0.48 BU/ml, whereas at the same N G F concentration halos were tbund in 33% of the lumbar explants (Table I). Spinal ganglia develop along a cephalocaudal gradient [5], and differences between the thoracic and lumbar populations might reflect different ontogenetic stages. On the other hand, survival of ganglionic neurons is NGF-dependent during a limited developmental period [8]. Therefore, if N G F sensitivity also develops along a cephalocaudal grandient, thoracic ganglia would lose it before the lumbar ones. However, the peculiar thoracic and lumbar responses to added N G F could also be explained by differences in the endogenous N G F production, the peripheral field innervated in vivo or the volume of each type of ganglion. Lumbar ganglia were larger than the thoracic ones, their mean diameters being 538 _+ 17 and 433 _+8/,m, respectively. Neurite extension patterns in the presence of 2.5 x 10- 7 M BPB were not the same as m controls (Table II). The most remarkable response, shared by the thoracic and lumbar explants, was neurite growth in the absence of added N G F . Albeit short, neurites appeared in a significant proportion of the ganglia cultured without added NGF. For the thoracic explants, BPB increased the proportion of neurite-bearing explants when low N G F concentrations were added, but no differences in this pro-
358 TABLE 1 NEURITE OUTGROWTH AT DIFFERENT NGF CONCENTRATIONS - CONTROL CULTURES NGF (BU/ml)
0.00 0.12 0.48 1.20 4.80 12.00
Thoracic ganglia
Lumbar ganglia
Neurite +
Halo +
(n)
Neurite +
Halo +
(n)
0.00 0.36 0.61 0.85 1.00 1.00
0.00 0.00 0.06 0.18 0.74 0.71
24 14 18 20 19 17
0.00 0.57 0.89 1.00 1.00 1.00
0.00 0.00 0.33 0.60 0.88 1.00
9 7 9 5 8 8
Neurite + and Halo + indicate the proportion of the explants bearing neurites and halos, respectively. The number of explants for each expeirmental condition is shown under (n). After incubation with BPB, a significant proportion (P < 0.01) of neurite-bearing explants appeared in cultures growth without added NGF (*). Besides, a statistically significant decrease in the porportion of halos was detected in thoracic explants grown in media with 4.8 BU/ml of exogenous BPB (**P<0.01). The different effects of BPB at low and high concentrations of added NGF could be further shown when the thoracic explants (treated vs non-treated) were compared as two groups grown at 0-0.48 BU/ml and 1.2-12.0 BU/ml of added NGF. For the low level groups there was a significant difference in the frequency of neurite-bearing explants (Z2=4.14; df= 1; P<0.05). A similar comparison showed no differences between the treated and nontreated explants grown at high levels of exogenous NGF. TABLE II NEURITE OUTGROWTH AT DIFFERENT NGF CONCENTRATIONS - CULTURES INCUBATED WITH 2.5 x 10 -7 M BPB NGF (BU/ml)
0.00 0.12 0.48 ! .20 4.80 12.00
Thoracic ganglia
Lumbar ganglia
Neurite +
Halo +
(n)
Neurite +
Halo +
(n)
0.19" 0.52 0.73 0.86 0.94 1.00
0.00 0.00 0.18 0.30 0.33** 0.55
26 21 22 17 18 20
0.38* 0.75 0.80 1.00 1.00 1.00
0.00 0.00 0.50 1.00 1.00 1.00
8 8 l0 6 5 8
portion were found when NGF was added at concentrations higher than 1.2 BU/ml. Development of neurite halos was apparently enhanced by BPB treatment at low NGF concentrations and decreased under the two highest NGF levels (Table It). This decrease in neurite outgrowth was more evident when large halos were considered, since they were found in 53% of control cultures but only in 15% of the BPBtreated explants grown at 12 BU/ml. Lumbar ganglia exposed to BPB also showed an increase in neurite outgrowth under low concentrations of added NGF. However, differences between BPB-treated
359
and control explants disappeared at higher N G F concentrations, when maximal responses were elicited on both types of culture (Table II). The proportion of explants with large halos was not affected by BPB treatment. These experiments confirmed our previous studies [14, 15] and further demonstrated that the extent of the D R G response to BPB can be regulated by N G F levels. Bromophenacyl bromide selectively binds to a histidine residue in the active site of PLA~ and different concentrations of this reagent produce different degrees of PLA~ inactivation [20]. Since this enzyme is associated both with the deacylation reacylation of several phospholipids and with the synthesis of arachidonate metabolites and other important cellular messengers, different degrees of PLA2 activity could induce selective changes in the turnover of membrane components. Several pieces of evidence support the hypothesis that BPB has a direct effect on neural cell membranes. In DRG explants, changes of growth cone morphology can be observed alter an exposure of just 20 rain and less than 24 h are required for an increase in ncurite length [14]. Besides, in neural retina monolayers, the appearance of cell processes is enhanced in 30 min [16]. Moreover, recent experiments from our laboratory (in preparation) indicate that each concentration of BPB produced characteristic patterns of [-~H]arachidonate incorporation into phospholipids of neural retina monolayers. Therefore, it can be postulated that enhancement of neurite growth induced by low concentrations of BPB would be due to modifications of membrane proportions such as viscosity and adhesivity. Nerve growth factor has several actions on sensitive neurons. Elongation and stabilization of neurite shafts are late responses requiring synthesis and deployment of cytoskeletal proteins [1]. On the other hand, short-latency responses to N G F include the modification of ionic fluxes [20] and the increase of filopodia, lamellipodia and other membranous structures involved in growth cone motility [3, 6, 7]. As suggested by our morphological evidence [14, 16], positive interactions between BPB and N G F would be associated with short-latency responses. Partial inhibition of PLA2 could conceivably induce changes of lipid metabolism similar to those produced by N G F [10, 12, 17]. Besides, it has also been postulated that N G F modulates PLA2 activity [10]. Thus, membrane changes induced by BPB could either mimic the short-latency responses to N G F or modulate cell sensitivity to this and other neurotrophic tactors. An increase of endogenous N G F production, such as that observed alier iris denervation [1 I] cannot be excluded at the present time. However, the fast morphological and biochemical responses to BPB argue against this possibility. Although ganglionic non-neuronal cells normally synthesize N G F [2], under our experimental conditions, neurite growth was not supported by endogenous N G F in the absence of BPB. Furthermore, neurites induced by the latter when N G F was not added to the medium were rather shorL suggesting that they lacked the organized cytoskeletal array induced by this factor. Thus, the increase in neurite length induced by BPB [13, 14] would be a secondary effect, requiring neurotrophic or neuritogenic factors for the elongation and stabilization of the neurite shaft. At high levels of added N G F , when both its immediate and late response are already maximal, BPB would not be able to induce changes upon neurite length.
360 S u c h a p h e n o m e n o n , o b s e r v e d in o u r l u m b a r e x p l a n t s , r e s e m b l e d the s t i m u l a n t effect o f G M I g a n g l i o s i d e o n c h i c k s y m p a t h e t i c g a n g l i a , t h a t c a n o n l y be d e m o n s t r a t e d at low concentrations of added NGF
[13]. tn t h e t h o r a c i c g a n g l i a c u l t u r e d w i t h high
levels o f e x o g e n o u s N G F , B P B a p p a r e n t l y i n h i b i t e d n e u r i t e e l o n g a t i o n a n d h a l o form a t i o n . T h i s p a r a d o x i c a l effect c o u l d be r e l a t e d to t h e o u t g r o w t h i n h i b i t i o n [18] a n d n e u r i t e r e t r a c t i o n [4] p r o d u c e d by high N G F c o n c e n t r a t i o n s . A l t h o u g h f u r t h e r studies a r e r e q u i r e d to u n d e r s t a n d it, this p h e n o m e n o n
w o u l d also suggest t h a t 10
M BPB somehow enhances NGF actions. R e s e a r c h r e p o r t e d in this p a p e r h a s b e e n s u p p o r t e d by C O N I C E T
( P I D 3- 903302/
85), a n d by F u n d a c i 6 n I n s t i t u t o de N e u r o b i o l o g i a . C . C . J . is a f e l l o w f r o m the ' C o n sejo N a c i o n a l d e I n v e s t i g a c i o n e s Cientificas, C O N I C E T ' ,
a n d A M S is a m e m b e r o f
the " C a r r e r a del I n v e s t i g a d o r ' t ¥ o m the s a m e I n s t i t u t i o n . I Black, M.M., Aletta, J. and Greene, L.A., Regulation of microtubule composition and stability during nerve growth factor-promoted neurite outgrowth, J. Cell Biol., 103 (1986) 545--557. 2 Burnham, P.A., Raiborn, C. and Varon, S., Replacement of nerve growth factor by ganglionic nonneuronal cells for the survival in vitro of dissociated ganglionic neurons, Proc. Natl. Acad. Sci. USA, 69 (1972) 3556-3660. 3 Connoly, J.L., Seeley, P.J. and Greene, L.A., Regulation of growth cone morphology by nerve growth factor: a comparative study by scanning electron microscopy, J. Neurosci. Res., 13 (1985) 183 198. 4 Griffin, C.G. and Letorneau, P.C., Rapid retraction of neurites by sensory neurons in response to increased concentrations of nerve growth factor, J. Cell Biol.. 86 (1980) 156-161. 5 Hamilton, H., Lillie's Development of the Chick, 3rd. edn., Holt, New York, 1952. 6 Johnston, R.N. and Wessells, N.K., Regulation of the elongating nerve fiber, Curr. Top. Dev. Biol., 16 (1980) 165-206. 7 Letorneau, P., Axonal growth and guidance, Trends Neurosci., 6 (1983) 451-455. 8 Levi-Montalcini, R., Developmental neurobiology and the natural history of nerve growth factor, Annu. Rev. Neurosci., 5 (1982) 341 362. 9 Mobley, C., Schenker, A. and Shooter, E.M., Characterization and isolation of proteolitically modified nerve growth factor, Biochemistry, 15 (1976) 5543-555 I. 10 Pfenninger, K.H. and Johnston, M.P., Nerve growth factor stimulates phospholipid methylation in growing neurites, Proc. Natl. Acad. Sci. USA, 78 (1981) 7797-7800. 11 Rush, R.A., lmmunohistochemical localization of endogenous nerve growth factor, Nature (Lond.), 312 (1984) 364 367. 12 Skaper, S.D. and Varon, S., Nerve growth factor stimulates phospholipid methylation in target ganglion neurons independently of the cyclic AMP and sodium pump responses, J. Neurochem., 42 (1984) 116 122. 13 Skaper, S.D. and Varon, S., Ganglioside GMI overcomes serum inhibition of neurite outgrowth, Int. J. Dev. Neurosci., 3 (1985) 187 198. 14 Suburo, A.M. and Cei de Job, C., Effect of p-bromophenacyl bromide on axon growth in vitro, Comp. Biol., 3 (1984) 159 164. 15 Suburo, A.M. and Cei de Job, C., The biphasic effect of phospholipase A2 inhibitors on axon elongation, Int. J. Dev. Neurosci., 4 (1986) 363-367. 16 Suburo, A.M. and Cei de Job, C., Early changes of surface morphology in monolayer cultures of neural retina cells: the effect of p-bromophenacyl bromide, Int. J. Dev. Neurosci., in press. 17 Traynor, A.E., Schubert~ D. and Allen, W.R., Alterations of lipid metabolism in response to nerve growth factor, J. Neurochem., 39 (1982) 1677 1683. 19 Varon, S. and Skaper, S.D., The Na ÷, K+-pump may mediate control of nerve cells by nerve growth factor, Trends Biochem. Sci., 8 (I983) 22 ~25. 20 Volwerk, J.J., Pieterson. W.A. and de Haas, G.H.. Histidine at the active site of phospholipasc A2, Biochemistry. 13 (1974l 1446 1454.
356
NSL 05230
Effects of p-bromophenacyl bromide on neurite growth at different levels of nerve growth factor Claudia Cei de Job and Angela Maria Suburo lnstituto de Neurobiologia, Buenos Aires (Argentina) (Received 16 June 1987; Revised version received 4 November 1987; Accepted 18 December 1987)
Key words." Neurite growth; p-Bromophenacyl bromide; Nerve growth factor; Rat dorsal root ganglion explant We have previously been shown that the phospholipase inhibitor p-bromophenacyl bromide (BPB) modifies neurite morphology and growth rate in rat dorsal root ganglion explants. Neurite extension is stimulated by 2.5 x 10- 7 M BPB but it is prevented by concentrations higher than 10 5 M. Under our experimental conditions, no neurites appeared in the absence of added nerve growth factor (NGF). Therefore, we have tested the effect of a low BPB concentration at different levels of exogenous NGF. Thoracic and lumbar ganglia were obtained from 19.5-day-old fetal rats and were cultured for 24 h on a polyornithine substrate. Neurite growth and development of a dense halo were NGF-dependent in both ganglionic populations; however, each of them showed a characteristic dose--response curve. Incubation with BPB induced neurites in the absence of added NGF and stimulated their growth when low levels of this factor were added. In lumbar ganglia incubated with higher levels of N(3F, BPB had no detectable effects. On the contrary, similarly treated thoracic ganglia showed an inhibition of neurite extension. Our findings suggest a relationship between the effects of BPB and the initial, membrane-activating actions of NGF.
We have recently reported that the extension of neural cell processes can be facilitated or prevented by different concentrations of p-bromophenacyl bromide (BPB) and other phospholipase A2 (PLA2) inhibitors. This effect has been shown both in dorsal root ganglion (DRG) explants [14, 15] and in neural retina monolayers [16]. In the D R G explants, a decrease in the growth rate of neural processes is produced by 2.5 x 10 -5 M or higher BPB concentrations, whereas increases in neurite length are observed around 10 -7 M. It is well known that neuron survival and neurite extension in D R G explants require nerve growth factor (NGF) [8, 18]. Besides, under our culture conditions, fetal rats D R G s do not extend neural processes without added N G F , and their neuritic outgrowth can be regulated by the N G F concentration in the culture medium. Since the stimulant effect of PLA2 inhibitors could be more evident at suboptimal concentrations of N G F , we have assayed the effect of a low concentration of BPB in D R G explants grown at different N G F levels.
Correspondence: A.M. Suburo, Instituto de Neurobiologia, Serrano 661, 1414 Buenos Aires, Argentina. 0304-3940/88/$ 03.50 © 1988 Elsevier Scientific Publishers Ireland Ltd.
357
Ganglia were obtained from 19.5-day-old rat fetuses (Holtzmann inbred rats, day 0 being the day when spermatozoids were found in vaginal smears). Pregnant rats were anesthetized with chloral hydrate and fetuses were removed under aseptic conditions. Thoracic and lumbar ganglia were dissected and explanted onto a 0.1 mg/ml polyornithine substrate made on plastic dishes. Culture medium was Eagle's minimal medium with Hanks' salts (Difco) supplemented with 5% fetal calf serum, 2 mM glutamine, 5 g/liter glucose, antibiotics and various concentrations of N G F . The latter was purified from mouse submaxillary glands [9] and biologically assayed [18]. Explants were incubated at 37°C in a 95:5 air:CO2 atmosphere saturated with water vapor. Dilutions of BPB (Sigma) were prepared as previously reported [15] and added after the first 5 h in vitro. One day-old cultures were examined under phase optics and classified according to the following criteria: (a) presence of neurites; (b) appearance of neuritic halos, defined as outgrowths with processes equal or longer than 225/,m in their 4 quadrants; (c) development of large halos, defined as those showing 330/lm or longer neurites in at least one of their quadrants. Outward migration of non-neuronal cells was not observed in these cultures. In control experiments, the proportion of ganglia bearing neurites increased with N G F concentration in the culture medium. No neurites were observed when this factor was excluded from the medium. The shortest neurites measured about 45 pro, whereas the longest ones extended up to 3 0 0 4 5 0 ,um. As longer neurites appeared, a dense outgrowth was formed around the explants. Halo development required the addition of higher levels of N G F than the appearance of neurites (Table I), and only the highest N G F concentrations induced the formation of large halos. Lumbar ganglia explants appeared more sensitive to added N G F than those from the thoracic region. Thus, every lumbar explant exhibited neurites at 1.2 BU/ml and the thoracic population reached that situation at 4.8 BU/ml. Moreover, very few of the thoracic explants showed halos at 0.48 BU/ml, whereas at the same N G F concentration halos were tbund in 33% of the lumbar explants (Table I). Spinal ganglia develop along a cephalocaudal gradient [5], and differences between the thoracic and lumbar populations might reflect different ontogenetic stages. On the other hand, survival of ganglionic neurons is NGF-dependent during a limited developmental period [8]. Therefore, if N G F sensitivity also develops along a cephalocaudal grandient, thoracic ganglia would lose it before the lumbar ones. However, the peculiar thoracic and lumbar responses to added N G F could also be explained by differences in the endogenous N G F production, the peripheral field innervated in vivo or the volume of each type of ganglion. Lumbar ganglia were larger than the thoracic ones, their mean diameters being 538 _+ 17 and 433 _+8/,m, respectively. Neurite extension patterns in the presence of 2.5 x 10- 7 M BPB were not the same as m controls (Table II). The most remarkable response, shared by the thoracic and lumbar explants, was neurite growth in the absence of added N G F . Albeit short, neurites appeared in a significant proportion of the ganglia cultured without added NGF. For the thoracic explants, BPB increased the proportion of neurite-bearing explants when low N G F concentrations were added, but no differences in this pro-
358 TABLE 1 NEURITE OUTGROWTH AT DIFFERENT NGF CONCENTRATIONS - CONTROL CULTURES NGF (BU/ml)
0.00 0.12 0.48 1.20 4.80 12.00
Thoracic ganglia
Lumbar ganglia
Neurite +
Halo +
(n)
Neurite +
Halo +
(n)
0.00 0.36 0.61 0.85 1.00 1.00
0.00 0.00 0.06 0.18 0.74 0.71
24 14 18 20 19 17
0.00 0.57 0.89 1.00 1.00 1.00
0.00 0.00 0.33 0.60 0.88 1.00
9 7 9 5 8 8
Neurite + and Halo + indicate the proportion of the explants bearing neurites and halos, respectively. The number of explants for each expeirmental condition is shown under (n). After incubation with BPB, a significant proportion (P < 0.01) of neurite-bearing explants appeared in cultures growth without added NGF (*). Besides, a statistically significant decrease in the porportion of halos was detected in thoracic explants grown in media with 4.8 BU/ml of exogenous BPB (**P<0.01). The different effects of BPB at low and high concentrations of added NGF could be further shown when the thoracic explants (treated vs non-treated) were compared as two groups grown at 0-0.48 BU/ml and 1.2-12.0 BU/ml of added NGF. For the low level groups there was a significant difference in the frequency of neurite-bearing explants (Z2=4.14; df= 1; P<0.05). A similar comparison showed no differences between the treated and nontreated explants grown at high levels of exogenous NGF. TABLE II NEURITE OUTGROWTH AT DIFFERENT NGF CONCENTRATIONS - CULTURES INCUBATED WITH 2.5 x 10 -7 M BPB NGF (BU/ml)
0.00 0.12 0.48 ! .20 4.80 12.00
Thoracic ganglia
Lumbar ganglia
Neurite +
Halo +
(n)
Neurite +
Halo +
(n)
0.19" 0.52 0.73 0.86 0.94 1.00
0.00 0.00 0.18 0.30 0.33** 0.55
26 21 22 17 18 20
0.38* 0.75 0.80 1.00 1.00 1.00
0.00 0.00 0.50 1.00 1.00 1.00
8 8 l0 6 5 8
portion were found when NGF was added at concentrations higher than 1.2 BU/ml. Development of neurite halos was apparently enhanced by BPB treatment at low NGF concentrations and decreased under the two highest NGF levels (Table It). This decrease in neurite outgrowth was more evident when large halos were considered, since they were found in 53% of control cultures but only in 15% of the BPBtreated explants grown at 12 BU/ml. Lumbar ganglia exposed to BPB also showed an increase in neurite outgrowth under low concentrations of added NGF. However, differences between BPB-treated
359
and control explants disappeared at higher N G F concentrations, when maximal responses were elicited on both types of culture (Table II). The proportion of explants with large halos was not affected by BPB treatment. These experiments confirmed our previous studies [14, 15] and further demonstrated that the extent of the D R G response to BPB can be regulated by N G F levels. Bromophenacyl bromide selectively binds to a histidine residue in the active site of PLA~ and different concentrations of this reagent produce different degrees of PLA~ inactivation [20]. Since this enzyme is associated both with the deacylation reacylation of several phospholipids and with the synthesis of arachidonate metabolites and other important cellular messengers, different degrees of PLA2 activity could induce selective changes in the turnover of membrane components. Several pieces of evidence support the hypothesis that BPB has a direct effect on neural cell membranes. In DRG explants, changes of growth cone morphology can be observed alter an exposure of just 20 rain and less than 24 h are required for an increase in ncurite length [14]. Besides, in neural retina monolayers, the appearance of cell processes is enhanced in 30 min [16]. Moreover, recent experiments from our laboratory (in preparation) indicate that each concentration of BPB produced characteristic patterns of [-~H]arachidonate incorporation into phospholipids of neural retina monolayers. Therefore, it can be postulated that enhancement of neurite growth induced by low concentrations of BPB would be due to modifications of membrane proportions such as viscosity and adhesivity. Nerve growth factor has several actions on sensitive neurons. Elongation and stabilization of neurite shafts are late responses requiring synthesis and deployment of cytoskeletal proteins [1]. On the other hand, short-latency responses to N G F include the modification of ionic fluxes [20] and the increase of filopodia, lamellipodia and other membranous structures involved in growth cone motility [3, 6, 7]. As suggested by our morphological evidence [14, 16], positive interactions between BPB and N G F would be associated with short-latency responses. Partial inhibition of PLA2 could conceivably induce changes of lipid metabolism similar to those produced by N G F [10, 12, 17]. Besides, it has also been postulated that N G F modulates PLA2 activity [10]. Thus, membrane changes induced by BPB could either mimic the short-latency responses to N G F or modulate cell sensitivity to this and other neurotrophic tactors. An increase of endogenous N G F production, such as that observed alier iris denervation [1 I] cannot be excluded at the present time. However, the fast morphological and biochemical responses to BPB argue against this possibility. Although ganglionic non-neuronal cells normally synthesize N G F [2], under our experimental conditions, neurite growth was not supported by endogenous N G F in the absence of BPB. Furthermore, neurites induced by the latter when N G F was not added to the medium were rather shorL suggesting that they lacked the organized cytoskeletal array induced by this factor. Thus, the increase in neurite length induced by BPB [13, 14] would be a secondary effect, requiring neurotrophic or neuritogenic factors for the elongation and stabilization of the neurite shaft. At high levels of added N G F , when both its immediate and late response are already maximal, BPB would not be able to induce changes upon neurite length.
360 S u c h a p h e n o m e n o n , o b s e r v e d in o u r l u m b a r e x p l a n t s , r e s e m b l e d the s t i m u l a n t effect o f G M I g a n g l i o s i d e o n c h i c k s y m p a t h e t i c g a n g l i a , t h a t c a n o n l y be d e m o n s t r a t e d at low concentrations of added NGF
[13]. tn t h e t h o r a c i c g a n g l i a c u l t u r e d w i t h high
levels o f e x o g e n o u s N G F , B P B a p p a r e n t l y i n h i b i t e d n e u r i t e e l o n g a t i o n a n d h a l o form a t i o n . T h i s p a r a d o x i c a l effect c o u l d be r e l a t e d to t h e o u t g r o w t h i n h i b i t i o n [18] a n d n e u r i t e r e t r a c t i o n [4] p r o d u c e d by high N G F c o n c e n t r a t i o n s . A l t h o u g h f u r t h e r studies a r e r e q u i r e d to u n d e r s t a n d it, this p h e n o m e n o n
w o u l d also suggest t h a t 10
M BPB somehow enhances NGF actions. R e s e a r c h r e p o r t e d in this p a p e r h a s b e e n s u p p o r t e d by C O N I C E T
( P I D 3- 903302/
85), a n d by F u n d a c i 6 n I n s t i t u t o de N e u r o b i o l o g i a . C . C . J . is a f e l l o w f r o m the ' C o n sejo N a c i o n a l d e I n v e s t i g a c i o n e s Cientificas, C O N I C E T ' ,
a n d A M S is a m e m b e r o f
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