Nerve Growth Factor influences potassium movements in chick embryo dorsal root ganglionic cells

Copyright 0 1981 by Academic Press. Inc. All right5 of reproduction in any form reserved 0014-4827/81/020353-09$02.00/0

Experimental

Cell Research 131 (1981) 353-361

NERVE GROWTH FACTOR INFLUENCES

POTASSIUM MOVEMENTS

CHICK EMBRYO DORSAL ROOT GANGLIONIC

IN

CELLS

STEPHEN D. SKAPER and SILVIO VARON Department

of Biology, School of Medicine, University San Diego, La Jolla, CA 92093, USA

of California,

SUMMARY Recently we have shown that Nerve Growth Factor (NGF) influences the movement of Na+ across the membrane of chick embryo dorsal root anglion (DRG) ceils. When cell dissociates from 8-day embryonic chick DRG, equilibrated with f 6Rb+ (a K+ analog) in the presence of NGF, were transferred to NGF-free medium a marked loss of intracellular K+ occurred over several hours. The time course of K+ loss was similar to the time course of Na+ accumulation which occurs in the absence of NGF. NGF-denrived. K+-depleted DRG cells reaccumulated K’ within minutes of delayed NGF presentationi”ust as delayed NGF administration results in the rapid ext.rusion of Nat from Na+-loaded ccl r’s. Restoratton of K+ competence was dependent upon NGF concentration. The occurrence of this K+ response to exogenous NGF in other ganglionic preparations correlated with traditional responses to NGF in culture and previously observed Na+ responses. Neither the development nor the expression of the ionic defect (K+ depletion, Na+ filhng) during NGF deprivation required the presence of both cations in the medium. NGFdependent restoration of intracellular K+ in NGF-deprived chick DRG cells required the resence of intracellular Naf, and NGF-dependent extrusion of Naf required extracellular K+. T&s NGF appears to influence the coupled (active) movemet$s o
Nerve Growth Factor (NGF) is essential for the normal growth, development and maintenance of sympathetic ganglionic and sensory neurons [ 1, 21, The mechanism whereby NGF accomplishes this trophic role remains to be elucidated. Such a trophic role of NGF would imply, however, that the factor acts to control some crucial function of its target neurons, in turn regulating quantitative or qualitative expression of cellular machineries [3,4]. The traditional responses to NGF are only observed a number of hours following presentation of the factor. We believe that short-latency responses, occurring within minutes of NGF presentation are more likely to reflect

primary events involved in the mode of action of NGF. Recent experiments in this laboratory have demonstrated that cell dissociates from embryonic chick dorsal root ganglia (DRG) lose their competence to maintain a low intracellular Na+ when incubated without NGF for 6 h, and promptly (within minutes) recover it on delayed presentation of NGF [5]. Additional studies [6, 71 have shown that (i) NGF causes an overall efflux of Na+ from *‘Na+-loaded cells rather than an accelerated equilibration; (ii) Na+ extrusion is dependent upon NGF concentration; (iii) in all ganglionic preparations examined, the occurrence of Na+ responses Exp Ce//Res 131 (1981)

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acid) to exogenous NGF correlates with tradi- (N-2-hydroxypiperazine-N’-2-ethanesulfonic Sigma Co., St Louis, MO; bovine serum albumin tional responses to NGF in culture; and (iv) from (fraction V) from Armour Pharmaceutical Co., Chicaserum proteins and several other defined go, Ill.; fiberglass filter discs (Whatman GF/C, 2.4 cm from Reeve Angel Co., Clifton, NJ. Nerve growth agents do not prevent the effects of NGF 0) factor, 7s form, was prepared from the submaxillary deprivation or mimic the NGF induction of glands of adult male mice as previously described @I. Concentrations of NGF were expressed in Biological Na+ extrusion. Units (BU)/ml, where 1 BU/ml is the concentration reThese fi&lings encouraged the view that quired to elicit an optimal biological response in the traditional bioassay [8]. All chemicals were reagent ionic control may be an important element grade. in the mode of action of NGF. It was thus of interest to investigate whether potassium regulation might also be controlled by Measurement of K+ movements in NGF. In the studies to be presented here, @@ionic dissociates 86Rb+ was used as a K+ analog to show Dorsal root ganglia (DRG) from S-day-old White Leghorn chick embryos were dissociated as described that chick DRG cells become unable to previously [5], in a Tris-Hepes albumin medium maintain their intracellular K+ level when (THAM) consisting of 40 mM Tris-Hepes, pH 7.4, 140 mM NaCI, 5 mM KCI, 1 mM MgC&, 0.1 mM CaC12, deprived of exogenous NGF over 4-6 h. and 1% (w/v) bovine serum albumin. DRG from neoThe NGF-deprived and K+ depleted cells natal Swiss ICR mice and sympathetic ganglia (SG) from 1l-day chick embryos were dissociated by simireaccumulated K+ within minutes upon de- 1ar procedures [7]. For studies on K+ retention,ganlayed NGF presentation, and do so in an glionic cell suspensions of 1x lo6 cells/ml for chick DRG and SG and 0.25~ lo6 cells/ml for mouse DRG NGF concentration-dependent manner. In were allowed to first equilibrate with %Rb+ by incuwith *6RbCl at 0.5 &i/ml for 2 h at 37”C, in the other ganglionic preparations examined, bation presence of 10 BU/ml of NGF. The cell suspension the occurrence of K+ responses to exoge- was then divided into two parts, the cells pelleted by a brief centrifugation (300 g, 3 min), washed once with nous NGF correlated with previously ob- fresh THAM, centrifuged as above and resuspended in served Na+ responses and with traditional the initial volume of fresh THAM with %RbCI (0.5 NGF (10 BU/ml) was present in the resuspenresponses to NGF in culture. Loss of K+ &i/ml). sion medium for only one of the two samples. Incubaand accumulation of Na+ under NGF depri- tion was carried out for an additional 6 h. At different during the course of the incubations, aliquots vation appeared to be passive phenomena, times (0.14.2 ml) of cell suspension were transferred to occurring independently from each other. moist GF/C fiberglass filters under suction. The filters were washed with 10 ml of ice-cold THAM, dried, and In contrast, when NGF was administered counted by liquid scintillation techniques [9]. Filter to restore ionic control, intracellular Na+ blanks consisted of 0.1-0.2 ml of THAM with %RbCI no cells, carried through as the cell samples were. was required for K+ reaccumulation, and but Values expressed as cpm per cell number were in conversely, Na+ extrusion required extra- terms of initial cell numbers. Duplicate samples generagreed to within 15%. Variations of this basic cellular K+. NGF may thus influence cou- ally procedure with regard to incubation times or alteration pled movements of Na+ and K+ across the of the standard ionic composition of THAM medium will be presented in the pertinent portions of the text membrane of its target cells, possibly in- or figure captions. volving the classical Na+, K+-ATPase To measure K+ reaccumulation by the cells, cell pump.

MATERIALS

AND METHODS

Materials Sources of materials were the following: rubidium86 (carrier-free %RbCl) from Amersham, Arlington Heights, III.; sodium-22 (carrier-free “NaCI) from New England Nuclear, Boston, Mass.; Tris and Hepes Exp Cell RPS I31 (1981)

dissociates were allowed to lose their K+ by incubation for 6 h in the absence of NGF (see Results). NGF (10 BU/ml) was then added to some samples, while controls received an equal volume of THAM. In some cases, %RbCI (0.5&ilml) was also added at this time, while in other instances the %Rb’ was also continuously present during the preceding period of NGF deprivation. At different times aliquots of cell suspension were transferred to GF/C filters, washed and counted for radioactivity as described above. Variations of this procedure with regard to NGF concentration or replacement of the basic THAM medium with

Nerve growth factor action on K+ flux in ganglionic cells

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Hours of incubation

Fig.

1. Equilibration and retention of 86RbCby chick DRG cells in the presence and absence of NGF. (A) Dissociated DRG cells were incubated in THAM medium with %RbCI (0.5 &i/ml) with 10 BU/ml of NGF for 2 h at 37°C. then transferred to fresh THAM containing 0.5 PCcrnl s6RbCI with 0, 10 BU/ml NGF or 0, no NGF. as described under Methods. At different times, 0.1 ml of cell suspension was taken for determi-

nation of radioactivity. (B) Dissociated DRG cells were incubated in THAM medium with %RbCl (0.5 @/ml) in 0, the continuous presence or 0, absence of 10 BU/ml NGF. At different times, aliquots (0.1 ml) were taken for determination of radioactivity as described under Methods. Each point represents the average of duplicate measurements from two separate experiments.

other ionic compositions will be presented in the pertinent portions of the text or figure captions.

studied by the use of the K+ analog 86Rb+. Cell dissociates of chick DRG were first allowed to equilibrate with 86Rb+ for 2 h in the presence of NGF (fig. 1A). At this time the cells were divided into two parts, with one part now having the NGF removed via centrifugation and washing of the cells (in all other respects both samples were treated the same). Incubation was continued for another 6 h, with 86Rb+ still present in the incubation medium. As fig. 1A shows, equilibration with “Rb+ was completed in 90-120 min. In the subsequent 6 h incubation, the presence of NGF permitted only a small (15 %) decrease in the intracellular K+ content over the first 2 h, with a stable value maintained after this time. Removal of the NGF, however, resulted in a progressive and drastic loss of K+ from the DRG cells, reaching a final value only onesixth that of the NGF-maintained cells. A similar experiment was carried out in which both equilibration and subsequent behavior of “Rb+ were compared in the continuous presence or absence of NGF

Measurement of K+ movements in intact ganglia The following tissues were used: I-day chick embryo DRG; SG from 1l-day-old chick embryos; DRG from neonatal Swiss ICR mice. Dissected ganglia were placed in glass tubes containing 125 ~1 of THAM. Typically, each tube contained 5 DRG or one SG chain (about 10 ganglia). WRbCl was added to the samples at 0.5 @/ml, with some samples also receiving NGF at 10 BU/ml (controls received an equal volume of THAM). After 6 h at 37°C or 30 min following presentation of NGF to some NGF-deprived samples after 6 h, samples were processed for determination of radioactivity. After diluting the incubation medium with 4 ml of ice-cold THAM, the ganglia were collected by centrifugation (300 g, 3 min), followed by washing with 3 ml of THAM and centrifugation again. The ganglia were transferred in about 0.2 ml of THAM using siliconized Pasteur pipets to scintillation counting vials containing the previously described scintillation cocktail [9] and counted. Zero-time blanks consisted of ganglia processed immediately after receiving the %RbCl. Duplicate samples generally agreed to within 20 %.

RESULTS Effect of NGF on the maintenance of intracellular K’ of chick DRG cells In the experiments illustrated in fig. 1, the intracellular movements of K ions were

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Table 1. Effect of NGF on K+ movements in various dissociated and intact ganglia Chick SG

Chick DRG

Mouse DRG

Condition

10’ cells

5 ganglia

lo5 cells

10 ganglia

IO5cells

15 ganglia

+NGF -NGF -NGF -NGF

1 270 190 1 340 200

950 140 1 070 120

1 180 210 1 230 190

1600

1 240

240

270

1 430 170

1 340 210

1 320 1590 1 620 1 470

(6 h) (6 h) (6 h), +NGF (30 min) (6 h), -NGF (30 min)

All values are given in cpm. The ganglionic dissociates prepared in THAM were incubated with s6RbCI (0.5 &i/ml) for 6 h with 10 BU/ml NGF or no NGF. Aliquots (0.1 ml) were sampled for %Rb retained as described under Methods. NGF was also added to some NGF-deprived samples after 6 h, and additional measurements made after 30 min. Intact ganglia were incubated in 125 ,LLIof THAM with 86RbC1+10 BU/ml NGF. 86Rb+ retention and reaccumulation were measured as described under Methods. Each value represents the average of duplicate measurements from two separate experiments.

(fig. 1B). In the presence of NGF the DRG cells again reached equilibration with the 86Rb+ after about 2 h, followed by a small decrease over the next 2 h and a leveling off. In the absence of NGF, the cells began to equilibrate with 86Rb+ but failed to

1400 1200

j 1000 “0 2- 600 8 600 P p

400 200 I5 Minutes

10 after

15 NGF

20 presentation

25

L 30

Fig. 2. Effect of NGF and NGF concentration on the

time course of K+ reaccumulation by NGF-deprived chick DRG cells. A DRG cell dissociate at 1x10” cells/ml in THAM was incubated for 6 h at 37°C with %RbCl (0.5 &i/ml) in the absence of NGF. At this time NGF was added at the following concentrations: n , 0.1 BU/ml; q ,l BU/ml; 0,100 BU/ml. At different times, 0.1 ml aliquots of cell suspension were taken for determination of radioactivity. The upper and lower broken lines represent the values for cells maintained 6 h in the continuous presence of 1 BU/ml NGF or no NGF, respectively. Values represent the average of duplicate measurements from two separate experiments. Exp Cell Res 131 (1981)

reach the NGF-supported level, possibly reflecting the incipient development during these first 2 h of a defect in the ability to retain K+. The cells continued to lose K+ during the remainder of the 7 h incubation period, with a time course and a final low level that were identical with that of the NGF-deprived cells described in fig. 1A. Thus, NGF appears necessary for the cell to maintain its normal intracellular K+ content, in this system. The reason for the small decrease in radioactivity observed with NGF-maintained cells over the first few hours is not known. Effect of NGF concentration on K+ reaccumulation by NGF-deprived, KCdepleted chick DRG cells To examine the influence of NGF concentration on the ability of chick DRG cells to reaccumulate the K+ lost during NGF deprivation, cells incubated for 6 h without NGF but with 86Rb+ were presented with NGF at 0.1-100 BU/ml and sampled for accumulation of radioactivity at different times. As fig. 2 shows, both 1 and 100 BU/ml of NGF resulted in approximately the same final accumulation of K+, to a level equal to that of 6 h NGF-maintained

Nerve growth factor

cells (upper broken line). NGF at 100 BU/ml, however, resulted in a more rapid rate of K+ accumulation than did 1 BU/ml. An NGF concentration of 0.1 BU/ml produced the slowest rate of K+ accumulation, and the steady-state value achieved by 30 min was only about one-half that of the 6 h NGF-maintained level. The lower broken line in fig. 2 represents the radioactivity content of chick DRG cells maintained for 6 h with “Rb+ in the absence of NGF. Effect of NGF on K+ levels in other ganglionic preparations

Previous studies [7] had shown that in all ganglionic preparations examined, the occurrence of Na+ responses to exogenous NGF correlated with traditional responses to NGF in culture. The present investigation of K+ responses to NGF were therefore extended to these same tissues, namely chick DRG and SG and mouse DRG. The data are summarized in table 1. Dissociates prepared from g-day chick embryo DRG, II-day chick embryo SG, and neonatal mouse DRG when incubated for 6 h with 86Rb+ maintained an intracellular level of radioactivity approx. six times greater in the presence than in the absence of NGF. Presentation of NGF to factor-deprived ganglionic cells after 6 h resulted in the reaccumulation of radioactivity at 30 min to values not substantially different from those of cells maintained for 6 h with NGF. In contrast, addition of medium only to 6 h NGF-deprived cells yielded low levels of intracellular radioactivity after 30 min, similar to values for NGF-deprived cells incubated for 6 h with 86Rb. Taken together, the data indicate that all these ganglionic cells are able to (i) maintain their intracellular K+ content for 6 h in the presence of exogenous NGF, but lose it in the absence of NGF; and (ii) reacquire the ini-

action on K’ flux in ganglionic

cells

357

tial intracellular K+ level when NGF is presented after 6 h of NGF deprivation. One must caution against a direct comparison of the data among ganglionic materials from different sources, since the various ganglia used yielded different numbers of cells per ganglion upon dissociation, and neurons occurred in different proportions in the various ganglionic dissociates. The same caveat applies to comparisons between dissociated and intact ganglia of the same source (see next). Intact chick embryo DRG and SG exhibited behaviors similar to those of the dissociated tissues (table 1). Ganglionic radioactivity levels after 6 h were some 6-fold greater in the presence of NGF than in its absence. When ganglia deprived of NGF for 6 h were presented with NGF, they reaccumulated radioactivity after 30 min to values comparable to those seen for 6 h NGF-maintained ganglia. NGF-deprived ganglia which received only medium failed to accumulate substantial radioactivity in the same 30 min period. The mouse DRG, however, retained the same radioactivity level regardless of the absence or presence of NGF. These observations are consistent with NGF-related behaviors of intact and dissociated ganglia in culture [7]. Unlike chick embryo DRG and SG explants, neonatal mouse DRG explants do not require the addition of NGF in order to elicit a fiber outgrowth halo. Mouse DRG neurons in monolayer culture, like chick embryo DRG and SG neurons, however, require exogenous NGF. Mutual independence of Na’ and K’ movements in NGF-deprived chick DRG cells

As fig. 1 has illustrated, chick DRG cells pre-equilibrated with 86Rb+ lose most of Exp Cd Res 131 (1981)

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Skaper and Varon 1200

1000 v) j

BOO

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Fig. 3. Movement of K+ and Na* in NGF-deprived cluck DRG cells in the absence of one or the other ion. (A) A DRG cell dissociate at 1x lo6 cells/ml in THAM was incubated for 90 min at 37°C with 86RbCI (0.5 @/ml) and 10 BU/ml NGF. The cell suspension was sampled for radioactivity (time 0) and then divided into five parts. These were centrifuged at 300 g for 4 min, washed once in their respective new medium containing no NGF, and resuspended in the starting volume of A, standard THAM with 10 BU/ml NGF (one portion); Cl, NGF-free THAM modified to contain 140 mM ChCl in place of NaCl (three portions); H, NGF-free standard THAM (one portion). All media At different time contained %RbCI (0.5 &i/ml). points over 6 h, aliquots (0.1 ml) of cell suspension were taken for determination of radioactivity. (B) After 6 h, the remaining two portions that contained ChCl in place of NaCl were transferred (via centrifugation and washing as above) to fresh NGF-free THAM medium containing 140 mM NaCl and either the standard 5 mM KC1 (0). or 5 mM ChCl(0) in its stead. 22NaCI (7.5 @.X/ml) was now added, and 0.1 ml aliquots of each cell suspension were removed at different short time intervals to measure accumulation of 22Na+ radioactivity. Each value represents the average of duplicate measurements from two different experiments.

their radioactivity (K+ content) when incubated for 54 h in the absence of NGF. Under the same circumstances, the cells have been repeatedly shown to also accumulate Na+ IS], presumably until intracellular Na+ concentrations have matched the extracellular ones [6]. These two ionic consequences of NGF deprivation do not appear to depend on each other for their occurrence, as illustrated in fig. 3. When DRG cells, first equilibrated with 86Rb+ in the presence of NGF, are transferred to medium lacking both Na+ and NGF, intracellular K+ is lost over the usual 6 h period Exp Cell Res 131 (1981)

10

5 Minutes

after

NGF

15

presentation

4. Influence of Na+ on the K’ accumulation response of chick DRG cells. A DRG cell dissociate at 1x106 cells/ml prepared in THAM was divided into four equal volumes, centrifuged at 300 g for 4 min, and the cell pellets were resuspended in equal volumes of standard THAM (0, n ) or THAM modiied to contain 140 mM ChCl in place of NaCl (0, Cl). NGF was omitted from all samples. After 6 h of incubation at 37”C, =RbCI was added (0.5 &i/ml) to all samples. Some samples also received 10 BU/ml NGF (0, 0); the remainder received medium only (m, 0). At different times, aliquots (0.1 ml) of cell suspension were taken for determination of radioactivity. The lower broken line corresponds to the average of the three lower sets of values. Values represent the average of duplicate measurements from two separate experiments. Fig.

to the same extent as when the cells are deprived of NGF in the p!esence of Na+ (fig. 3A). Thus, the K+ depletion of chick DRG cells under NGF deprivation does not require the concurrent filling of the cells with Na+. The converse independence is equally true, namely Na+ accumulation in NGF-deprived cells does not require the intracellular or extracellular presence of K+. DRG cells, depleted of K+ in the absence of both Na+ and NGF, were transferred to NGF-free medium containing 140 mM NaCl (and **Na as tracer) but with the 5 mM KC1 replaced by choline chloride (ChCl). As seen in fig. 3 B, the cells rapidly fill with Na+ to the same extent as when K+ is also present [cf 51. Taken together, these data indicate that the K+ depletion

Nerve growth factor

and the Na+ accumulation which occur in chick DRG cells when they are deprived of exogenous NGF over a period of hours are strictly passive processes, operating down their initial concentration gradients without concurrent (coupled) movement of the opposite cation. Coupled behavior of Na and K movements under delayed NGF administration

The recovery of ionic control by chick DRG cells re-supplied with NGF also appears to occur in a complementary fashion, i.e., the cells extrude the excess Na+ acquired under NGF deprivation while simultaneously re-acquiring the K+ that had been concurrently lost. One needs, therefore, to ask with regard to the NGF-induced recovery the same question that was previously raised with regard to the development of ionic control under NGF deprivation, namely whether the two ionic events are independent from or coupled to each other. Chick DRG cells were incubated for 6 h in standard THAM or in THAM containing ChCl instead of NaCl, in both cases without NGF. In either case, the cells were expected to lose their K+ content (cf fig. 3A), but only the cells incubated in Na+containing medium will have accumulated Na+ [5]. After the 6 h incubation, both suspensions received 86Rb+ and some portions also were given 10 BU/ml of NGF. As fig. 4 demonstrates, when NGF was presented to NGF-deprived cells only those cells incubated in Na-containing medium (and, thus, having a high intracellular Naf) were able to recover K+. The level of 86Rb radioactivity reached was comparable to those of the earlier experiments (figs 1, 2, table 1). Thus, the K+ reaccumulation response to delayed NGF administration requires the presence of intracellular Na+.

action on Kf jlux in ganglionic

/--‘~ 1000 1200

L

z E 600 % c ; 600 a M=

400

-

200

-

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l

cells

359

_,.....5 0

.

L

1 2 Minutes

4 after

6 NGF

a presentation

10

Fig. 5. Effect of K+ on the Naf extrusion response of

chtck DRG cells. A DRG cell dissociate at 1~10~ cells/ml in THAM was incubated for 6 h at 37°C with **NaCI (7.5 &i/ml) in the absence of NGF. to achieve **Na+ loading [5, 61. The cell suspension &as divided into four equal volumes, followed by centrifugation at 300 g for 4 min. The cell pellets were resuspended in equal volumes of standard THAM (0, n ) or THAM modified to contain 5 mM ChCl in place of KC1 (0, 0). Both media contained 7.5 ,&i/ml **NaCI. One portion from each set also received 10 BU/ml NGF (0, 0), while the others received medium only (m, 0). At different times, aliquots (0.1 ml) of cell suspension were taken for determination of radioactivity. The upper broken line corresponds to the average of the three upper sets of values. Each value represents the average of duplicate measurements from two separate experiments.

In order to ask the converse question, does the NGF-induced Na+ extrusion require the presence of extracellular K+, chick DRG cells were first loaded with **Na+ by incubation in THAM with **Na+ and no NGF for 6 h [5, 61. The cells were then transferred to fresh THAM containing the standard 5 mM KCl, or with the KC1 replaced by 5 mM ChCl (both media contained **Na+). One portion in each set also received NGF at 10 BU/ml. The cell suspensions were sampled at different times for radioactivity retained. As fig. 5 clearly illustrates, the delayed administration of NGF to **Na-loaded cells resulted in the expected rapid loss of radioactivity only in the presence of extracellular K+. Substitution of ChCl for KC1 in the incubation meExp Cell Res I31 (1981)

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presentation results in the rapid extrusion of Na+ from the intracellular milieu. While the beta subunit of NGF was not tested in these K+ studies, beta NGF has been shown to elicit the corresponding Na+ responses in a cell dissociate of chick DRG [5]. The Na+ response to 7s NGF occurred equally well with a nutrient-rich medium, serum, and certain hormones and other proteins present [6]. Since the present data indicate a coupled movement of Naf and K+ in these cells, it would not be unreasonable to expect that beta NGF or a glucosecontaining medium could be substituted for 7S NGF or glucose-free THAM medium, respectively, in these K+ experiments. DISCUSSION As with the Na+ response, restoration of Previous studies from this laboratory [5, 61 K+ competence by NGF was seen to vary have shown that NGF influences the move- in both speed and magnitude with the conment of Na+ across the membrane of chick centration of NGF presented (fig. 2). We DRG cells. Intact as well as dissociated have stated before [6, 101that such obserchick embryo DRG, intact and dissociated vations are consistent with a binding of chick embryo sympathetic ganglia, and dis- NGF to a specific cell surface receptor sociated neonatal mouse DRG-all tradi- [l l-131 and the formation of an NGF-retional target tissues for NGF-showed an ceptor complex as the first step for an ionic ‘Na response’ to NGF, namely a require- response. Receptor saturation would give ment for NGF to sustain or recover their rise to a complete ionic response. Subsatuability to control intracellular Na+ levels rating NGF concentrations (e.g., 0.1 [7]. In the present studies, we have ex- BU/ml) would in turn result in only a subtended our investigation to show that K+ optimal response. Supersaturating concenmovements are also controlled by NGF in trations of NGF (e.g., 100 BU/ml) would these ‘Na responsive’ tissues. When chick lead to faster saturation of available reDRG cells were first allowed to equilibrate ceptors, with a correspondingly greater with 86Rb+ (a K+ analog) in the presence of speed, but not magnitude, of Na+ extrusion NGF, subsequent removal of the NGF led and K+ accumulation by the cells. to a marked loss of intracellular K+ over a Following the initial observation [S] that period of several hours. The time course of NGF regulates intraganglionic Na+ levels, K+ loss was very similar to the time course it was subsequently shown [6] that NGF of Na+ accumulation observed in these action is consistent with a model in which cells as a consequence of NGF deprivation. NGF acts through an Na+ pump rather Delayed administration of NGF to NGF- than by restricting Na+ intluxes. The nadeprived DRG cells resulted in a rapid ture of such an Na+ pump is unknown at (minutes) reacquisition of intracellular K+, this time. The data on K+ fluxes presented in much the same way that delayed NGF here may, however, shed some light on this

dium at this time completely abolished the Na+ response elicited by NGF. Together, the data of figs 4 and 5 show that the Na+ and K+ movements following delayed NGF administration, unlike those developing during NGF deprivation, require each the concurrent occurrence of the other. In other words, recovery of ionic control under re-administration of NGF results in the restoration of high K+ and low Naf intracellular levels by coupled active (uphill) transports of the two cations in opposite directions.

Exp Cell Res 131 (1981)

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question. During NGF deprivation, neither will have to be addressed by future investithe development nor the expression of the gations. One area concerns the events that ionic defect (K+ depletion, Na+ tilling) re- must link the restoration of Na and K conquired the presence of both cations in the trol to the binding reaction between NGF medium (fig. 3). In contrast, NGF-de- and cell surface receptors, and the question pendent restoration of intracellular K+ in whether intracellular components are also NGF-deprived chick DRG cells required involved beside membrane ones. The other the presence of intracellular Na+ (fig. 4), area concerns the consequences of ionic and NGF-dependent extrusion of excess control by NGF with regard to the later, Na+ required extracellular K+ (fig. 5). In traditional effects of NGF on neuronal surother words, in the latter case the fluxes of vival, neurite stimulation and, possibly, sethese two cations seem to be coupled. The lective regulation of transmitter enzyme ionic pump which NGF appears to infhi- synthesis. ence, then, is one that requires, for the active movement of Na+ or K+ across the This work was supported by USPHS grant NS-07606 the National Institute of Neurological and Complasma membrane, the presence of the oth- from municative Disorders and Stroke. er cation on the opposite side of the membrane. The classical Na+, K+-ATPase pump REFERENCES would fit the description of just such an 1. Levi-Montalcini, R 8~Angeletti, P U, Physiol rev ionic pump. If NGF were to control in 48 (1968) 534. some manner the operation of the NaK2. Varon, S, Exp neurol48/3 (part 2) (1975) 75. ATPase, lack of NGF would result in a 3. Varon, S &. Bunge, P, Ann rev neurosci 1 (1978) progressive decline in its function aimed at 4. 327. Varon, S & Adler, R, Curr top dev neurobiol. In press. offsetting ongoing ‘leakages’ of K+ out5. Skaper, S D & Varon, S, Biochem biophys res ward and Na+ inward. The passive loss of commun 88 (1979) 563. intracellular K+ and rise of intracellular 6. - J neurochem 34 (1980) 1654. - Brain res (1980). In press. Na+ occurring under NGF deprivation are 7. 8. Varon, S, Nomura, J, Perez-Polo, J R & Shooter, E M, Methods and techniques of neurosciences consistent with such an interpretation, and (ed R Fried) p. 203. Dekker, New York (1972). its relatively slow time scale would reflect a 9. Skater. S D 81 Varon, S, Brain res 163 (1979) 89. slow rate of pump inactivation. Converse- 10. Vat&t, S & Skaper, S D, Tissue culture in neurobiology (ed E Giacobini, A Shaher & A Vemadaly, delayed presentation of NGF would kis) p. 333. Raven, New York (1980). derepress-the 11. Banejee, S H, Cuatrecasas, P & Snyder, S H, J activate-or promptly them 251 (1976) 5680. pump and, thus, the coupled correction of 12. biol Costrini. N V & Bradshaw, R A, Proc natl acad sci US 76 (1976) 3242. the aberrant intracellular levels of the two A, Riopelle, R J, Harris-Warrick, R M & ions. More direct evidence is now being 13. Sutter, Shooter, E M, J biol them 254 (1979) 5972. sought to substantiate such a hypothesis. Received May 22, 1980 Regardless of a validation of the Na+, Revised version received July 28, 1980 K+-ATPase hypothesis, two main areas Accepted August 28, 1980

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Exp Cell Res 131 (1981)