Brain Research, 520 (1990) 159-165 Elsevier
159
BRES 15599
Volume regulation in response to hypo-osmotic stress in goldfish retinal ganglion cell axons regenerating in vitro Brian T. Edmonds and Edward Koenig Department of Physiology, State University of New York at Buffalo, Buffalo, N Y 14214 (U.S.A.)
(Accepted 12 December 1989) Key words: Regulatory volume decrease; Immature axon; Potassium channel; Cytoskeleton; ATP-~,S
Goldfish retinal ganglion cell (RGC) axons regenerating in vitro were used to investigate the volume regulatory response to hypo-osmotic stress. Reducing the tonicity of the bathing medium to half strength caused an immediate swelling of axons; however, within 1 rain a progressive volume reduction ensued which stabilized at near control volume over a period of 10 min. This regulatory volume decrease (RVD) was attenuated by elevated [K+]o, Ca2+-activated K + channel antagonists, and calmidazolium, a potent calmodulin inhibitor. Inclusion of ATP-)'S in the hypotonic bathing medium led to a loading of stressed axons which resulted in an excessive volume reduction that reflected an overshooting of the RVD response. The latter suggested the importance of phosphorylation/dephosphorylation reactions in the RVD response pathway. Cytochalasin D and colchicine had no effect on the development of the typical RVD response, providing no evidence of involvement of actin or microtubule cytoskeletons in the volume reduction mechanism of these immature axons. The results are consistent with the hypothesis that hypo-osmotic stress activates a calcium/calmodulin dependent membrane pathway, which probably involves transient phosphorylation, leading to a loss of cellular K + and osmotically obligated water which restorates normal axonal volume.
INTRODUCTION M a n y cell types display the capacity to reduce cell volume in response to hypo-osmotic stress 25 which has b e e n t e r m e d regulatory volume decrease ( R V D ) . In some cases, the R V D response appears to be associated with a loss of cellular KCI through calcium-sensitive m e m b r a n e pathways (for recent reviews, see refs. 12, 17). Little is known, however, about the volume regulatory b e h a v i o r of neurons at the cellular level (for review, see ref. 1). A t t e n t i o n to neural tissue responses to h y p o - o s m o t i c conditions has focussed on either alterations in ion content of whole brain (see ref. 31), or p o p u l a t i o n s of cultured glia or glioma cell lines ls'19. In both cases, hypotonic conditions resulted in a loss of K + and C1- with time and the restoration of near preshock volume. The mechanisms of the ion loss and R V D response were unclear, but were r e g a r d e d to be similar in the case of glia to o t h e r cell types 2°. Recently, work utilizing an in vitro n e u r o b l a s t o m a m o d e l has a p p e a r e d which r e p o r t e d findings consistent with a role for K + channels in R V D response 4. In the latter study, it was suggested that increases in cell soma v o l u m e o p e n mechanosensitive non-selective cation pathways in the p l a s m a m e m b r a n e which results in m e m b r a n e d e p o l a r i z a t i o n and subsequent activation of voltage-
d e p e n d e n t K + and anion channels. A r e p o r t indicating an antagonism of the R V D response by cytochalasin B in isolated m a t u r e axons from the green crab Carcinus maenas 7 suggested a role for the actin-containing cytoskeleton in R V D . The latter observations would a p p e a r to be consistent with involvement of mechanosensitive cation channels (i.e. stretch channels) coupled to the m e m b r a n e skeleton in regulation of cell volume 34-36. In the present study, goldfish retinal ganglion cell ( R G C ) axons regenerating in retinal explant culture have been e m p l o y e d as a m o d e l to investigate the response of n a k e d neuronal processes to anisotonic conditions. These axons are very i m m a t u r e and contain p r o m i n e n t protruding varicosities, whose ultrastructural features and mobile dynamic behavior have been described 3,23. The mobile behavior of varicosities reflects an intrinsic capability of redistributing axoplasm rapidly in an aggregate bulk form, which requires a significant cytoskeletal plasticity that would not be possible in axons containing a fully d e v e l o p e d neurofilament cytoskeleton 3,23. Given the relative paucity of an i m p o r t a n t structurally stabilizing influence of neurofilaments, and the large surfaceto-volume ratio of such axons, the possibility of significant changes in volume in response to ionic and solute fluxes w o u l d a p p e a r to m a k e volume regulation an important intrinsic capability.
Correspondence: E. Koenig, State University of New York at Buffalo, 313 Cary Hall, Buffalo, New York, 14214, U.S.A.
0006-8993/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
160 The
question
of R V D
f u n c t i o n in R G C
axons is
p r o b e d in the p r e s e n t study. F o r this p u r p o s e , t i m e - l a p s e , p h a s e - c o n t r a s t v i d e o m i c r o s c o p y has b e e n used to m o n i tor d y n a m i c structural changes in varicosity v o l u m e in r e s p o n s e to a l t e r a t i o n s in b a t h i n g m e d i u m tonicity. O u r findings s h o w that R G C axons r e c o v e r to control volu m e s within 10 min o f initial e x p o s u r e to h y p o t o n i c stress. This R V D
r e s p o n s e is a t t e n u a t e d
by e l e v a t e d
[K÷]o, s o m e K ÷ c h a n n e l antagonists and a c a l m o d u l i n inhibitor, and o v e r s h o o t s with l o a d i n g of A T P - y S . O n the o t h e r h a n d , c y t o s k e l e t a l p e r t u r b a n t s had no effect on the RVD
response.
The
results
are consistent
with the
Inc.). The phase image was displayed on a video monitor (Sanyo) using a DAGE NC-67M video camera with a Newvicon tube (DAGE-MTI, Inc.) mounted on a trinocular head of the microscope. Experiments were recorded in a time-lapse mode with a video recorder (TLC 2001, GYYR, Inc.), where time was compressed by a factor of 12. Still photographs were taken from the monitor screen using a Polaroid CU-5 Land camera type 665 positive/negative Polaroid film, or directly through the microscope with an attached 35 mm photomicrograph system (Olympus PM10AD). In those experiments employing fluorescence microscopy, axonal fields were viewed under epi-iUumination (Olympus). Photomicrographs were taken on T-Max film (Kodak) at exposure times of 18-25 s and processed to ASA 400-800 with T-Max developer (Kodak).
h y p o t h e s i s that C a Z ÷ / c a l m o d u l i n - a c t i v a t e d K ÷ c h a n n e l s
Determination of varicosity volume
play a p r e d o m i n a n t role in r e g u l a t i o n of v o l u m e reduc-
Axonal varicosities were used in some experiments to monitor changes in axonal volume. These structures are prominent, geometrically symmetrical and sensitive to volume changes. Photomicrographs were taken of axonal fields and enlarged prints were made and used to measure axial and transverse axes of identifiable varicosities before after treatment. As varicosities resemble prolate spheroids in shape, the following equation was used to approximate the volume: V = 4/3az ab2; where V = volume, a = axial length, and b = transverse width. Changes in relative volume (Vr~l) were determined by the ratio: volume after/volume before. Student's t-test was used to test for significance at a level of 0.05. As reported below, one feature of hypo-osmotic treatment of axonal fields is the de novo formation of varicosities along axonal segments previously devoid of such structures. Such varicosities are distinguishable from pre-existing varicosities because they disappear during a typical RVD response. In order to gauge the magnitude of the RVD recovery, a varicosity coefficient ratio (Kvar) was determined, in which the total number of varicosities within a given microscopic field before hypo-osmotic shock was compared to that after the RVD response; i.e., Kvar = number of varicosities after/number of varicosities before treatment. Non-parametric statistical analysis of ratios was performed with a microcomputer software package (Number cruncher Statistical Systems, Kayesville, UT) at a 0.05 level of significance. The two sided sign test was used to determine if a given treatment had an effect on transport activity; i.e. if the median ratio after/before the treatment was or was not equal to 1.0.
tion in i m m a t u r e axons, and that the c y t o s k e l e t o n w o u l d a p p e a r to be of s e c o n d a r y i m p o r t a n c e . I n d i r e c t e v i d e n c e that t r a n s i e n t p h o s p h o r Y l a t i o n m a y also be i m p o r t a n t in the m e c h a n i s m is also a d d u c e d . MATERIALS AND METHODS
Biochemicals The following were purchased from Sigma: cytochalasin D, poly-L-lysine, 5-fluorodeoxyuridine, gentamycin sulfate, uridine, methyl cellulose, N-methyl-D-glucamine, sodium and calcium salts of gluconic acid, sorbitol, valinomycin, digitonin, ionomycin, A23187, tetraethylammonium bromide (TEA) and dibutyryladenosine 3"-5" cyclic monophosphate (dbcAMP). Calmidazolium was purchased from Boehringer Mannheim. Quinine and 8-bromocAMP were gifts of Dr. Michael Duffey, 3,3"-diethylthiadicarbocyanine bromide (diS-C3-(5)) was a gift of Dr. James Goldinger, and Lucifer yellow was a gift of Dr. Dennis Higgins.
Retinal explant preparation Goldfish retinal explants were prepared as described elsewhere 22' 23 and were used for experimental observations after 3-5 days in culture. Briefly, 2-4 weeks after crushing the optic nerve, the retina was isolated, chopped into squares (0.65 x 0.65 mm), plated out onto polylysine-coated no. 1.5 circular coverslips, and cultured in L-15 (Gibco) medium, supplemented with 10% fetal calf serum (Flow), 0.02 M HEPES, 0.l mM 5-fluorodeoxyuridine, 0.1 mg/ml gentamycin sulfate, 0.2 mM uridine and 0.6% methyl cellulose. Explants were cultured in humid air atmosphere at 27 °C.
Video and fluorescence microscopy Details of the videomicroscopy system and the procedures for viewing have been described elsewhere (Edmonds and Koenig; submitted). In general, the circular coverslip with attached explants was inverted over a rectangular no. 2 coverslip (35 x 50 mm) mounted in a holder and supported by 1 mm thick silastic spacers. This chamber allowed the rapid exchange of the bathing medium within 1 rain, using a Pasteur pipette to apply the fluid to one side of the chamber, while fluid was removed with Whatman filter paper from the opposite side. The standard bathing medium was a modified Cortland physiological fish saline 22 composed of (in mM): NaCI 132, KCI 5, MgCI2 1.6, CaCIz 1.8, glucose 5.5, Hepes 20, adjusted to pH 7.2 with Tris. All experiments were conducted at 22-24 °C. Axonal fields were viewed under phase-contrast microscopy (Olympus BHS microscope) with a X100 oil immersion planapochromat objective (Zeiss N.A. = 1.25) combined with an achromat condenser (Olympus, N.A. = 1.4) oiled to the bottom coverslip. The microscope stage was isolated from external vibration by a Vibraplane air-suspension table top platform (Kinetic Systems,
RESULTS A x o n a l fields e x p o s e d to h a l f - s t r e n g t h C o r t l a n d saline, diluted 1:1 with d e i o n i z e d w a t e r (final o s m o l a r i t y , 150 m O s m ) u n d e r g o an i m m e d i a t e swelling (Fig. 1). This swelling is c h a r a c t e r i z e d by a d r a m a t i c i n c r e a s e in the v o l u m e of pre-existing varicosities (see Fig. 2) and the e x t e n s i v e de n o v o f o r m a t i o n o f s w o l l e n , p h a s e - l u c e n t varicosities a l o n g a x o n a l s e g m e n t s (Fig. 1B). W i t h i n 1 - 2 rain of the initial h y p o t o n i c shock, t h e swollen varicosities begin to d e c r e a s e in v o l u m e , and within 10 m i n , the axonal fields h a v e r e t u r n e d to t h e i r p r e - e x i s t i n g m o r p h o l ogy (cf. Fig. l a , c ) . B e c a u s e t h e initial v o l u m e i n c r e a s e of varicosities was highly v a r i a b l e a m o n g varicosities the d a t a c o u l d n o t be p o o l e d . T h e r e f o r e , the c h a n g e s in r e l a t i v e v o l u m e o v e r t i m e of a single p r e - e x i s t i n g varicosity in r e s p o n s e to e x p o s u r e to h a l f - s t r e n g t h tonicity of C o r t l a n d saline in Fig. 2 is s h o w n as a typical e x a m p l e . W i t h i n 1 min after
161 attaining m a x i m u m volume in response to hypotonic stress, a rapid phase of volume reduction occurs, which stabilizes at a v o l u m e very close to control level within 10 min. T h e variability in the initial volume increase and the f o r m a t i o n of transient varicosities a p p a r e n t l y reflect a non-uniformity in the compliance of the i m m a t u r e axon along its extent. While the basis for this non-uniformity is u n k n o w n , it m a y reflect a non-uniformity in the m a k e u p o r organization of the cortical cytoskeleton. N o n e t h e l e s s , as shown in Table I, final varicosity volumes following R V D were only slightly different from prestress
3.0
2.0
"6 1.0
0
2
4
6
8
10
Time (rain.) Fig. 2. Changes in the relative volume of a single varicosity in response to exposure to a hypotonic bathing medium. Cortland saline diluted 1:1 with deionized water (final tonicity, 150 mOsm) was added at to (arrow), and varicosity volumes were calculated at subsequent 1-min intervals (see Materials and Methods). The curve was fitted by inspection.
controls (Vre ~ = 0.84 + 0.38). It is n o t e w o r t h y that this dramatic reversal of axonal swelling occurs while hypotonic conditions are m a i n t a i n e d , which raises the important question of what mechanisms m a y underlie the volume recovery process. A s already n o t e d above, R V D responses to hypoosmotic stress in some o t h e r cell types have been shown to be related to an increased efflux of KCI, possibly through Ca 2÷ regulated m e m b r a n e pathways. It was of interest, therefore, to test for similar mechanisms operating in R G C axons. Two p a r a m e t e r s were m o n i t o r e d as indicators of the R V D response (i.e. recovery of volume during hypotonic shock): (1) reduction in the relative volumes of varicosities (Vrel) ; and (2) d i s a p p e a r a n c e of varicosities f o r m e d de novo with e x p o s u r e to hypotonic conditions (see Fig. l b , c ) . P e r t u r b a t i o n s in the R V D response was evaluated by calculating a 'varicosity coefficient ratio' (Kvar) , which t r a c k e d the total n u m b e r
TABLE I Effects o f variouz, treatments on varicosities 10 rain after hypo-osmotic stress
n, number of varicosities monitored; Vrel, volume after/volume before (mean +_ S.D.); Kvar, varicosity coefficient (see Materials and Methods). Significance tested at a level of 0.05 by two-sided sign test.
Fig. 1. Changes in RGC axonal morphology induced by exposure to a hypertonic bathing medium. Phase-contrast micrographs (a) before, (b) immediately after exposure to hypotonic Cortland saline, and (c) after 10 min under hypotonic conditions. Bar = 10 /~m.
Treatment
n
Conc.
Vret
P
Kvar
Control DiS-Ca-(5 ) Calmidazolium Elevated [K+]o Quinine CSC12 TEA Cytochalasin D Colchicine
11 14 8 13 14 11 4 8 12
1/~M 1#M 70 mM 250/~M 10 mM 50mM 10#M 5 mM
0.84 + 0.38 2.80 + 2.0 3.30 + 1.4 2.63 + 1.2 1.30 + 0.46 1.08 + 0.63 1.30+ 1.19 0.96 + 0.27 1.19 + 0.46
0.03 0.0001 0.004 0.002 0.09 0.38 0.5 0.63 0.19
1.08 6.8 6.2 3.2 3.9 0.99 1.5 1.3 1.2
162
Fig. 3. Evidence of membrane permeability changes in RGC axons during hypotonic swelling. (a) Phase-contrast, and (b) fluorescence micrograph of a small fascicle containing a single varicosity after volume recovery in response to a hypotonic bathing medium (150 mOsm) containing Lucifer yellow (0.1%). Bar = 5/~m.
of varicosities present within a given microscopic field before and after a given treatment (see Materials and Methods). The results of these experiments are summarized in Table I.
2
The typical R V D response is attenuated when axonal fields are exposed to a hypo-osmotic medium containing 70 mM potassium gluconate (150 mOsm, total). For these experiments, gluconate was substituted for chloride as the major anion in order to prevent swelling that accompanies membrane depolarization with potassium 24. In the presence of elevated [K+]o, V r e I is 2.63 + 3.2 and the g v a r is 3.2, compared to control preparations, in which Vre I is 0.84 "1- 0.38 and the Kvar is 1.08. When this bathing medium is exchanged for a bathing medium of identical osmolarity with low [K+]o (i.e. 2.5 mM), R V D occurs as usual. These results suggest that an outwardly directed K + gradient is important for the R V D response. Agents that have been reported to block Ca 2+activated K ÷ channels in other cell types yielded variable results. The carbocyanine dye, diS-C3-(5), a potent inhibitor of the Ca2+-dependent K ÷ channel in erythrocytes 38 at a concentration of 1/~M appeared to be the most effective of the agents tested in preventing volume recovery (Vre I = 2.8 + 2.0) and loss of de novo formed varicosities (Kvar = 6.8). Quinine (0.25-1 mM) 32 was less effective in attenuating volume recovery (Ire ~ = 1.3 + 0.46), but partially inhibited the disappearance of transient varicosities (Kvar = 3.9). CsCI 2 (10 mM; VreI = 1.08 + 0.63; g v a r = 0.99) and T E A (50 mM; VreI = 1.3 + 1 . 1 9 ; g v a r = 1.5) were relatively ineffective in blocking the R V D response. Calmodulin has been implicated in Ca2+-mediated activation of K + channels in other cell types 9'13A5' 16,23,28,29 and experiments were also conducted to test for a calmodulin dependence in the R V D response. Calmidazolium (1 ~M), a potent antagonist of calmodulin 4°, effectively blocked the typical R V D volume recovery ( V r e I = 3.3 + 1.4) and varicosity loss (Kwr = 6.2). Although the foregoing results suggest a role for calmodulin in the R V D response of immature R G C axons, as has been suggested for other cell types, experiments designed to investigate a calcium dependency were inconclusive. R V D occurred in these axons with the usual time course, both in hypotonic Ca 2÷-
>o
• 0
....... . iiiIiiiiiiiii 10
20
30
Time (min.) Fig. 4. Changes in the relative volume of a single Varicosity in response to exposure to a hypotonic bathing medium containing ATP-),S (5 mM) was added at to (arrow), and varicosity volumes were calculated at subsequent 5-min intervals (see Materials and Methods). The curvd was fitted by inspection. Note that endstage volume constitutes -30% of prehypo-osmotic stress volume.
TABLE II Changes in relative varicosity volume 20 min following hypotonic loading o f A TP or A TP-yS
n, number of varicosities monitored; Vre~, volume after/volume before (mean +~ S.D.); (see Materials and Methods). Significance tested at a level of 0~05. Loading
n
VreI
P*
ATP ATP-yS
9 6
1.14 + 0.26 0.42 + 0.2
0.34 0.016
* Comparison is with control (see Table I).
163 free/EGTA-buffered, and Ca2+-containing media. These results appear to make the requirement of a Ca 2+ influx unlikely. Attempts were made to reduce the Ca 2+ content of intracellular storage sites by preincubating some axonal fields for 1 h prior to hypotonic stress with the membrane permeant calcium chelator BAPTA-AM (10 ¢tM) combined with ionomycin (5/~M) in a Ca 2+free/EGTA (1 mM) buffered Cortland medium. These preparations also displayed the normal RVD response; however, it was not possible to verify in these axons that calcium actually had been depleted. It has been reported that cytochalasin B treatment interferes with RVD of gallbladder epithelial cells 5 and of mature axons isolated from C a r c i n u s m a e n a s 7. In order to test for potential cytoskeletal involvement in RVD of these growing axons, axonal fields were treated with either cytochalasin D (10/~M) or colchicine (0.1-5 raM) for 15 min prior to hypotonic stress. As shown in Table I, neither cytochalasin D (Vre I = 0.96 + 0.27; Kvar = 1.3), nor colchicine (Vrel = 1.19 + 0.46; g v a r ~- 1.2) had an appreciable effect on attenuating the typical RVD response. Previous work has shown that both of these agents induce cytoskeletal alterations of R G C axons within this time period (data not shown). These observations provide no evidence that actin or microtubular cytoskeletons play a direct role in volume regulation of immature R G C axons. Our experiments with hypotonic stress showed that it could also provide a means for loading low molecular weight solutes into axons. For example, Lucifer yellow, an impermeant anionic fluorescent dye (MW 457 Da), made up in half-strength Cortland saline (0.1%), was taken up during hypotonic stress and remained in axons after RVD reached a steady state and the medium was exchanged for a dye-free one (Fig. 3b). This technique was subsequently used to load ATPwS, a thiophosphate ATP analog in order to probe the potential effect of thiophosphorylation. In the presence of 5 mM ATP, a typical RVD response occurs after exposure to hypotonic stress (i.e. Vre~= 1.24 + 0.26); however, in the presence of 5 mM ATP-yS, the relative varicosity volume is significantly lower than control (i.e. Vrej = 0.42 + 0.2; P = 0.02 - see Table II). Fig. 4 shows the RVD response of a representative single varicosity exposed to half-strength saline, containing 5 mM ATP-yS. After exposure to the ATP-yS-containing hypotonic medium for 30 min, the Vr~~ ratio was 0.3 in this particular case. ATP-~S can serve as a substrate for some kinases which results in a stable thiophosphorylated product resistant to phosphatase activity s. These observations suggest that one or more phosphorylation events accompany the RVD response, and, if not reversed, leads to an irreversible 'overshoot' of the R V D .
DISCUSSION R G C axons regenerating in vitro are capable of reducing their volume in response to hypo-osmotic stress. The RVD response and its mechanism appear to be similar to those described for other cell types, in that an outwardly directed K + gradient is requisite for the recovery of normal volume. Also, agents reported to block Ca2+-activated K + channels antagonize the typical RVD response (see Table I). An area of controversy relates to the role of intracellular Ca 2÷ as a second messenger in mediating the RVD response. In the case of immature R G C axons, the RVD response was unaffected by the absence of extracellular calcium, and additional attempts to deplete and/or buffer [Ca2+]i . These results would suggest that calcium is not required for the RVD response in R G C axons. Studies, using intracellular fluorescent Ca 2÷ indicators in other cell types, however, have yielded variable and conflicting findings. Similar to the present results, Rink et al. 33, using Quin2 as an indicator, was unable to demonstrate an elevation in [Ca2+]i in lymphocytes suspended in a hypotonic medium, notwithstanding the pharmacological evidence to the contrary 9. Yet, recently, experiments with fura2, as an indicator, have provided evidence that [Ca2+]i increases slightly during RVD in hypotonically swollen osteosarcoma cells, even in the absence of extracellular calcium 42. The principal evidence for a potential role of Ca 2÷ in the RVD response in R G C axons is based on two sets of experimental findings. First, apparent antagonism of calmodulin blocks the RVD response. Recent work has implicated calmodulin as a likely target involved in RVD 29. However, the issue of specificity remains a potential problem with regard to calmodulin antagonists 39. Nonetheless, recent work with sulfoxide derivatives of phenothiazine calmodulin antagonists indicates that they produce the same non-specific effects as the unmodified phenothiazines but do not bind to calmodulin and do not block restoration of volume, while the unmodified agents do block recovery 29. The calmodulin inhibitor used in the present study has a much higher affinity for calmodulin than the phenothiazines4°,, which increases the likelihood for a role of calmodulin in the RVD response of RGC axons. Second,, increasing [Ca2+]i in R G C axons under isotonic conditions with 100/tM [Ca2+]o by treating them with ionomycin, a calcium ionophore, causes a Volume reduction that exceeds 40%; a similar effect is produced with 1/~M [Ca2+]o in a Na+-free bathing medium. The volume reduction is blocked by the same treatments that antagonize the RVD response, including elevated [K+]o, selected K + channel blockers, and calmidazolium (Ed-
164 monds and Koenig; in preparation). Volume reductions stimulated by calcium ionophores in other cell types have been shown to be a result of a KC1 efflux 9'11'12"15"16 and observations in R G C axons are consistent with this interpretation. The calcium-mediated shrinkage in R G C axons, which is also blocked by a p o t e n t calmodulin antagonist, suggests that R G C axons have an intrinsic capability to reduce volume by a calcium-dependent pathway. It appears likely, therefore, that this or a similar pathway is utilized in response to hypo-osmotic stress; however, as already noted, a direct correlation between an increase in [Ca2+]i and R V D remains to be demonstrated. A n interesting aspect of the R V D response is the feedback mechanism which limits the volume reduction to near p r e h y p o - o s m o t i c stress levels. The finding, however, that ATP-yS loading during hypo-osmotic stress leads to an overshoot of the volume reduction, not only suggests that at least one phosphorylation step is a requisite event in the R V D pathway, but that dephosphorylation m a y also be requisite to prevent overshooting the volume 'setpoint'. Recently, reports have a p p e a r e d which provide evidence of increased phosphorylation of m e m b r a n e proteins 1° and phosphoinositides 27 induced by cell shrinkage, and by hypo-osmotic stress 3°. In the latter case, it was suggested that m e m b r a n e protein phosphorylation was due to an increased Ca2+-stimulated calmodulin activity. While cytochalasin B treatment attenuated the typical R V D response in m a t u r e invertebrate axons 7, which could implicate the potential involvement of cytoskeletally coupled m e m b r a n e pathway in triggering the R V D response, there was a lack of an observable effect by either cytochalasin D or colchicine on the R V D response
REFERENCES 1 Ballanyi, K. and Grafe, P., Cell volume regulation in the nervous system, Renal Physiol. Biochem., 3-5 (1988) 142-157. 2 Cala, P.M., Volume regulation by Amphiuma red blood cells, Mol. Physiol., 8 (1985) 199-214. 3 Edmonds, B.T. and Koenig, E., Powering of bulk transport (varicosities) and differential sensitivities of directional transport in growing axons, Brain Research, 406 (1987) 288-293. 4 Falke, L.C. and Misler, S., Activity of ion channels during volume regulation by clonal N1E115 neuroblastoma cells, Proc. Natl. Acad. Sci. U.S.A., 86 (1989) 3919-3923. 5 Foskett, J.K. and Spring, K.R., Involvement of calcium and cytoskeleton in gallbladder epithelial cell volume regulation, Am. J. Physiol., 248 (1985) C27-C36. 6 Gilbert, D.S., Axoplasm architecture and physical properties as seen in the Myxicola giant axon, J. Physiol. (Lond.), 253 (1975) 257-301. 7 Gilles, R., Delpine, E., Duchare, C., Cornet, M. and Pequeux, A., The effect of cytochalasin B on the volume regulation response of isolated axons of the green crab Curcinus maenas submitted to hypo-osmotic media, Comp. Biochem. Physiol., 85A (1986) 523-525.
in R G C axons. The m o d e l put forth by Sachs 35 is that m e m b r a n e tension is coupled to stretch channels through cytoskeletal elements, p r o b a b l y consisting of in-series coupling by spectrin, and in-parallel coupling with actin filaments. A s also pointed out by Sachs, however, while the mechanism of transduction m a y be universal 34, the mechanical coupling p r o b a b l y varies widely and may account for variations in response a m o n g different cell types 35. O n e m a j o r difference b e t w e e n i m m a t u r e and m a t u r e R G C axons is the relative paucity of neurofilaments in the former, which we believe contributes to the capability of immature axons to exhibit m a r k e d structural plasticity associated with the rapid directional bulk redistribution of axoplasm 3'23. Thus, in the m a t u r e axon, the neurofilament cytoskeleton plays m a j o r roles in structural stabilization, shape and d e t e r m i n a t i o n of caliber 634, and may influence the organization and p r o p e r t i e s of the actin-containing cortical cytomatrix. H o w e v e r , as crustacean axons a p p e a r not to have neurofilaments 41, the latter considerations would p r e s u m a b l y not account for the divergent findings in the m a t u r e crab axon 7. The present study extends to growing axons the R V D response that characterizes m a n y n o n - n e u r o n a l cell types. While the data are consistent with activation of K + channels as a basic mechanism for loss of an important axoplasmic osmolyte, the elucidation of the potential roles of calcium, calmodulin, and phosphorylation/dephosphorylation, also implicated in the mechanism, requires further investigation. Acknowledgements. This research was supported in part by NS21843 from the NINCDS. The authors wish to thank Drs. James Goldinger and Michael Duffey for helpful discussions during the course of this investigation.
8 Gratecos, D. and Fischer, E.H., Adenosine 5"-O(3-thiotriphosphate) in the control of phosphorylase activity, Biochem. Biophys. Res. Commun., 58 (1974) 960-967. 9 Grinstein, S., Dupre, A. and Rothstein, A., Volume regulation by human lymphocytes: role of calcium, J. Gen. Physiol., 79 (1982) 849-868. 10 Grinstein, S., Goetz-Smith, J.D., Stewart, D., Bereford, B.J. and Mellors, A., Protein phosphorylation during activation of Na+/K ÷ exchange by phorbol esters and by osmotic shrinkage, J. Biol. Chem., 261 (1986) 8009-8016. 11 Grinstein, S. and Cohen, S., Cytoplasmic [Ca2÷] and intracellular pH in lymphocytes, J. Gen. Physiol., 89 (1987) 185-213. 12 Grinstein, S. and Dixon, S.J., Ion transport, membrane potential and cytoplasmic pH in lymphocytes: changes during activation, Physiol. Rev., 69 (1989) 417-481. 13 Hinrichsen, R.D., Burgess-Cassler, A., Soltveldt, B.C., Hennessey, T. and Kung, C., Restoration by calmodutin of a Ca ++ dependent K ÷ current missing in a mutant Paramedium, Science, 232 (1986) 503-506. 14 Hoffman, P.N., Griffin, J.W. and Price, D.L., Control of axonal caliber by neurofilament transport, J. Cell Biol., 99 (1985) 705-714. 15 Hoffmann, E.K., Role of separate K ÷ and CI channels and of
165
16
17
18
19
20
21 22
23
24
25 26
27
28
Na+/K + cotransport in volume regulation in Ehrlich cells, Fed. Proc., 44 (1985) 2513-2519. Hoffmann, E.K., Lambert, I.H. and Simonsen, L.O., Separate Ca + +-activated K ÷ and CI- transport pathways in Ehrlich acites tumor cells, J. Membr. Biol., 91 (1986) 227-244. Hoffmann, E.K. and Simonsen, L.O., Membrane mechanisms in volume and pH regulation in vertebrate cells, Physiol. Rev., 69 (1989) 315-382. Kempski, O., Chausey, L., Gross, U., Zimmer, M. and Baetmann, A., Volume regulation and metabolism of suspended C6 glioma cells: an in vitro model to study cytotoxic brain edema, Brain Research, 279 (1983) 217-228. Kimelberg, H.K. and Frangakis, M.V., Furosemide- and bumetanide-sensitive ion transport and volume control in primary astrocyte cultures from rat brain, Brain Research, 361 (1985) 125-134. Kimelberg, H.K. and O'Connor, E., Swelling of astrocytes causes membrane potential depolarization, Glia, 1 (1988) 219224. Omitted. Koenig, E. and Adams, P., Local protein synthesizing activity in axonal fields regenerating in vitro, J. Neurochem., 39 (1982) 386-400. Koenig, E., Kinsman, S., Repasky, E. and Sultz, L., Rapid motility of motile varicosities and inclusions containing spectrin, actin and calmodulin in regenerating axons in vitro, J. Neurosci., 5 (1985) 715-729. Lipton, P., Effects of membrane depolarization on light scattering by cerebral cortical slices, J. Physiol. (Lond.), 231 (1973) 365-383. Macknight, A.D.C., Principles of cell volume regulation, Renal Physiol. Biochem., 3-5 (1988) 114-141. McCann, J.D. and Welsh, M.J., Neuroleptics antagonize a calcium-activated potassium channel in airway smooth muscle, J. Gen. Physiol., 89 (1987) 339-352. Orlov, S.N., Pokudin, N.I., Kotelevtsev, Y.V. and Gulak, P.V., Volume-dependent regulation of ion transport and membrane phosphorylation in human and rat erythrocytes, J. Membr. Biol., 89 (1989) 339-352. Pierce, S.K., Politis, A.D., Smith, L.H., Jr. and Rowland, L.M., A Ca 2÷ influx in response to hypo-osmotic stress may alter osmolyte permeability by a phenothiazine-sensitive mech-
anism, Cell Calcium, 9 (1988) 129-140. 29 Pierce, S.K., Politis, A.D., Cronkite, D.H., Rowland, L.M., Smith, L.H., Jr., Evidence of calmodulin involvement in cell volume recovery following hypo-osmotic stress, Cell Calcium, 10 (1989) 159-169. 30 Politis, A.D. and Pierce, S.K., Hypoosmotic stress induces phosphorylation of two specific proteins that may be involved in cell volume recovery, J. Cell Biol., 107 (1988) 286a. 31 Pollock, A. and Arieff, A., Abnormalities of cell volume regulation and their functional consequences, Am. J. Physiol., 239 (1980) F195-F205. 32 Reichstein, E. and Rothstein, A., Effects of quinine on Ca + +-induced K + efflux from human red blood cells, J. Mernbr. Biol., 59 (1981) 57-63. 33 Rink, T.J., Sanchez, A., Grinstein, S. and Rothstein, A., Volume restoration in osmotically swollen lymphocytes does not involve changes in free Ca ++ concentration, Biochem. Biophys. Acta, 762 (1983) 593-596. 34 Sachs, F., Mechanical transduction: unification? News Physiol. Sci., 1 (1986) 98-100. 35 Sachs, F., Baroreceptor mechanisms at the cellular level, Fed. Proc., 46 (1987) 12-16. 36 Sachs, F., Mechanical transduction and stretch activated ion channels, Biophys. J., 53 (1988) 410a. 37 Sachs, F., Mechanical transduction in biological systems, Crit. Rev. Biomed. Eng., 16 (1988) 141-169. 38 Simons, T.J.B., Actions of a carbocyanine dye on calciumdependent potassium transport in human red cell ghosts, J. Physiol. (Lond.), 288 (1979) 481-507. 39 Stoclet, J.-C., Gerard, D., Kilhoffer, M.-C., Lugnier, C., Miller, R. and Schaeffer, P., Calmodulin and its role in intracellular calcium regulation, Prog. Neurobiol., 29 (1987) 483-494. 40 Van Belle, H., R24571: a potent inhibitor of calmodulinactivated enzymes, Cell Calcium, 2 (1981) 483-494. 41 Warren, R.H. and Rubin, R.W., Microtubules and actin in giant nerve fibers of the spiny lobster, Panulirus argus, Tissue & Cell, 10 (1978) 687-697. 42 Yamaguchi, D.T., Green, J., Kleeman, C.R. and Muallem, S., Characterization of volume-sensitive, calcium-permeating pathways in osteosarcoma cell line UMR-106-01, J. Biol. Chem., 264 (1989) 4383-4390.
159
BRES 15599
Volume regulation in response to hypo-osmotic stress in goldfish retinal ganglion cell axons regenerating in vitro Brian T. Edmonds and Edward Koenig Department of Physiology, State University of New York at Buffalo, Buffalo, N Y 14214 (U.S.A.)
(Accepted 12 December 1989) Key words: Regulatory volume decrease; Immature axon; Potassium channel; Cytoskeleton; ATP-~,S
Goldfish retinal ganglion cell (RGC) axons regenerating in vitro were used to investigate the volume regulatory response to hypo-osmotic stress. Reducing the tonicity of the bathing medium to half strength caused an immediate swelling of axons; however, within 1 rain a progressive volume reduction ensued which stabilized at near control volume over a period of 10 min. This regulatory volume decrease (RVD) was attenuated by elevated [K+]o, Ca2+-activated K + channel antagonists, and calmidazolium, a potent calmodulin inhibitor. Inclusion of ATP-)'S in the hypotonic bathing medium led to a loading of stressed axons which resulted in an excessive volume reduction that reflected an overshooting of the RVD response. The latter suggested the importance of phosphorylation/dephosphorylation reactions in the RVD response pathway. Cytochalasin D and colchicine had no effect on the development of the typical RVD response, providing no evidence of involvement of actin or microtubule cytoskeletons in the volume reduction mechanism of these immature axons. The results are consistent with the hypothesis that hypo-osmotic stress activates a calcium/calmodulin dependent membrane pathway, which probably involves transient phosphorylation, leading to a loss of cellular K + and osmotically obligated water which restorates normal axonal volume.
INTRODUCTION M a n y cell types display the capacity to reduce cell volume in response to hypo-osmotic stress 25 which has b e e n t e r m e d regulatory volume decrease ( R V D ) . In some cases, the R V D response appears to be associated with a loss of cellular KCI through calcium-sensitive m e m b r a n e pathways (for recent reviews, see refs. 12, 17). Little is known, however, about the volume regulatory b e h a v i o r of neurons at the cellular level (for review, see ref. 1). A t t e n t i o n to neural tissue responses to h y p o - o s m o t i c conditions has focussed on either alterations in ion content of whole brain (see ref. 31), or p o p u l a t i o n s of cultured glia or glioma cell lines ls'19. In both cases, hypotonic conditions resulted in a loss of K + and C1- with time and the restoration of near preshock volume. The mechanisms of the ion loss and R V D response were unclear, but were r e g a r d e d to be similar in the case of glia to o t h e r cell types 2°. Recently, work utilizing an in vitro n e u r o b l a s t o m a m o d e l has a p p e a r e d which r e p o r t e d findings consistent with a role for K + channels in R V D response 4. In the latter study, it was suggested that increases in cell soma v o l u m e o p e n mechanosensitive non-selective cation pathways in the p l a s m a m e m b r a n e which results in m e m b r a n e d e p o l a r i z a t i o n and subsequent activation of voltage-
d e p e n d e n t K + and anion channels. A r e p o r t indicating an antagonism of the R V D response by cytochalasin B in isolated m a t u r e axons from the green crab Carcinus maenas 7 suggested a role for the actin-containing cytoskeleton in R V D . The latter observations would a p p e a r to be consistent with involvement of mechanosensitive cation channels (i.e. stretch channels) coupled to the m e m b r a n e skeleton in regulation of cell volume 34-36. In the present study, goldfish retinal ganglion cell ( R G C ) axons regenerating in retinal explant culture have been e m p l o y e d as a m o d e l to investigate the response of n a k e d neuronal processes to anisotonic conditions. These axons are very i m m a t u r e and contain p r o m i n e n t protruding varicosities, whose ultrastructural features and mobile dynamic behavior have been described 3,23. The mobile behavior of varicosities reflects an intrinsic capability of redistributing axoplasm rapidly in an aggregate bulk form, which requires a significant cytoskeletal plasticity that would not be possible in axons containing a fully d e v e l o p e d neurofilament cytoskeleton 3,23. Given the relative paucity of an i m p o r t a n t structurally stabilizing influence of neurofilaments, and the large surfaceto-volume ratio of such axons, the possibility of significant changes in volume in response to ionic and solute fluxes w o u l d a p p e a r to m a k e volume regulation an important intrinsic capability.
Correspondence: E. Koenig, State University of New York at Buffalo, 313 Cary Hall, Buffalo, New York, 14214, U.S.A.
0006-8993/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
160 The
question
of R V D
f u n c t i o n in R G C
axons is
p r o b e d in the p r e s e n t study. F o r this p u r p o s e , t i m e - l a p s e , p h a s e - c o n t r a s t v i d e o m i c r o s c o p y has b e e n used to m o n i tor d y n a m i c structural changes in varicosity v o l u m e in r e s p o n s e to a l t e r a t i o n s in b a t h i n g m e d i u m tonicity. O u r findings s h o w that R G C axons r e c o v e r to control volu m e s within 10 min o f initial e x p o s u r e to h y p o t o n i c stress. This R V D
r e s p o n s e is a t t e n u a t e d
by e l e v a t e d
[K÷]o, s o m e K ÷ c h a n n e l antagonists and a c a l m o d u l i n inhibitor, and o v e r s h o o t s with l o a d i n g of A T P - y S . O n the o t h e r h a n d , c y t o s k e l e t a l p e r t u r b a n t s had no effect on the RVD
response.
The
results
are consistent
with the
Inc.). The phase image was displayed on a video monitor (Sanyo) using a DAGE NC-67M video camera with a Newvicon tube (DAGE-MTI, Inc.) mounted on a trinocular head of the microscope. Experiments were recorded in a time-lapse mode with a video recorder (TLC 2001, GYYR, Inc.), where time was compressed by a factor of 12. Still photographs were taken from the monitor screen using a Polaroid CU-5 Land camera type 665 positive/negative Polaroid film, or directly through the microscope with an attached 35 mm photomicrograph system (Olympus PM10AD). In those experiments employing fluorescence microscopy, axonal fields were viewed under epi-iUumination (Olympus). Photomicrographs were taken on T-Max film (Kodak) at exposure times of 18-25 s and processed to ASA 400-800 with T-Max developer (Kodak).
h y p o t h e s i s that C a Z ÷ / c a l m o d u l i n - a c t i v a t e d K ÷ c h a n n e l s
Determination of varicosity volume
play a p r e d o m i n a n t role in r e g u l a t i o n of v o l u m e reduc-
Axonal varicosities were used in some experiments to monitor changes in axonal volume. These structures are prominent, geometrically symmetrical and sensitive to volume changes. Photomicrographs were taken of axonal fields and enlarged prints were made and used to measure axial and transverse axes of identifiable varicosities before after treatment. As varicosities resemble prolate spheroids in shape, the following equation was used to approximate the volume: V = 4/3az ab2; where V = volume, a = axial length, and b = transverse width. Changes in relative volume (Vr~l) were determined by the ratio: volume after/volume before. Student's t-test was used to test for significance at a level of 0.05. As reported below, one feature of hypo-osmotic treatment of axonal fields is the de novo formation of varicosities along axonal segments previously devoid of such structures. Such varicosities are distinguishable from pre-existing varicosities because they disappear during a typical RVD response. In order to gauge the magnitude of the RVD recovery, a varicosity coefficient ratio (Kvar) was determined, in which the total number of varicosities within a given microscopic field before hypo-osmotic shock was compared to that after the RVD response; i.e., Kvar = number of varicosities after/number of varicosities before treatment. Non-parametric statistical analysis of ratios was performed with a microcomputer software package (Number cruncher Statistical Systems, Kayesville, UT) at a 0.05 level of significance. The two sided sign test was used to determine if a given treatment had an effect on transport activity; i.e. if the median ratio after/before the treatment was or was not equal to 1.0.
tion in i m m a t u r e axons, and that the c y t o s k e l e t o n w o u l d a p p e a r to be of s e c o n d a r y i m p o r t a n c e . I n d i r e c t e v i d e n c e that t r a n s i e n t p h o s p h o r Y l a t i o n m a y also be i m p o r t a n t in the m e c h a n i s m is also a d d u c e d . MATERIALS AND METHODS
Biochemicals The following were purchased from Sigma: cytochalasin D, poly-L-lysine, 5-fluorodeoxyuridine, gentamycin sulfate, uridine, methyl cellulose, N-methyl-D-glucamine, sodium and calcium salts of gluconic acid, sorbitol, valinomycin, digitonin, ionomycin, A23187, tetraethylammonium bromide (TEA) and dibutyryladenosine 3"-5" cyclic monophosphate (dbcAMP). Calmidazolium was purchased from Boehringer Mannheim. Quinine and 8-bromocAMP were gifts of Dr. Michael Duffey, 3,3"-diethylthiadicarbocyanine bromide (diS-C3-(5)) was a gift of Dr. James Goldinger, and Lucifer yellow was a gift of Dr. Dennis Higgins.
Retinal explant preparation Goldfish retinal explants were prepared as described elsewhere 22' 23 and were used for experimental observations after 3-5 days in culture. Briefly, 2-4 weeks after crushing the optic nerve, the retina was isolated, chopped into squares (0.65 x 0.65 mm), plated out onto polylysine-coated no. 1.5 circular coverslips, and cultured in L-15 (Gibco) medium, supplemented with 10% fetal calf serum (Flow), 0.02 M HEPES, 0.l mM 5-fluorodeoxyuridine, 0.1 mg/ml gentamycin sulfate, 0.2 mM uridine and 0.6% methyl cellulose. Explants were cultured in humid air atmosphere at 27 °C.
Video and fluorescence microscopy Details of the videomicroscopy system and the procedures for viewing have been described elsewhere (Edmonds and Koenig; submitted). In general, the circular coverslip with attached explants was inverted over a rectangular no. 2 coverslip (35 x 50 mm) mounted in a holder and supported by 1 mm thick silastic spacers. This chamber allowed the rapid exchange of the bathing medium within 1 rain, using a Pasteur pipette to apply the fluid to one side of the chamber, while fluid was removed with Whatman filter paper from the opposite side. The standard bathing medium was a modified Cortland physiological fish saline 22 composed of (in mM): NaCI 132, KCI 5, MgCI2 1.6, CaCIz 1.8, glucose 5.5, Hepes 20, adjusted to pH 7.2 with Tris. All experiments were conducted at 22-24 °C. Axonal fields were viewed under phase-contrast microscopy (Olympus BHS microscope) with a X100 oil immersion planapochromat objective (Zeiss N.A. = 1.25) combined with an achromat condenser (Olympus, N.A. = 1.4) oiled to the bottom coverslip. The microscope stage was isolated from external vibration by a Vibraplane air-suspension table top platform (Kinetic Systems,
RESULTS A x o n a l fields e x p o s e d to h a l f - s t r e n g t h C o r t l a n d saline, diluted 1:1 with d e i o n i z e d w a t e r (final o s m o l a r i t y , 150 m O s m ) u n d e r g o an i m m e d i a t e swelling (Fig. 1). This swelling is c h a r a c t e r i z e d by a d r a m a t i c i n c r e a s e in the v o l u m e of pre-existing varicosities (see Fig. 2) and the e x t e n s i v e de n o v o f o r m a t i o n o f s w o l l e n , p h a s e - l u c e n t varicosities a l o n g a x o n a l s e g m e n t s (Fig. 1B). W i t h i n 1 - 2 rain of the initial h y p o t o n i c shock, t h e swollen varicosities begin to d e c r e a s e in v o l u m e , and within 10 m i n , the axonal fields h a v e r e t u r n e d to t h e i r p r e - e x i s t i n g m o r p h o l ogy (cf. Fig. l a , c ) . B e c a u s e t h e initial v o l u m e i n c r e a s e of varicosities was highly v a r i a b l e a m o n g varicosities the d a t a c o u l d n o t be p o o l e d . T h e r e f o r e , the c h a n g e s in r e l a t i v e v o l u m e o v e r t i m e of a single p r e - e x i s t i n g varicosity in r e s p o n s e to e x p o s u r e to h a l f - s t r e n g t h tonicity of C o r t l a n d saline in Fig. 2 is s h o w n as a typical e x a m p l e . W i t h i n 1 min after
161 attaining m a x i m u m volume in response to hypotonic stress, a rapid phase of volume reduction occurs, which stabilizes at a v o l u m e very close to control level within 10 min. T h e variability in the initial volume increase and the f o r m a t i o n of transient varicosities a p p a r e n t l y reflect a non-uniformity in the compliance of the i m m a t u r e axon along its extent. While the basis for this non-uniformity is u n k n o w n , it m a y reflect a non-uniformity in the m a k e u p o r organization of the cortical cytoskeleton. N o n e t h e l e s s , as shown in Table I, final varicosity volumes following R V D were only slightly different from prestress
3.0
2.0
"6 1.0
0
2
4
6
8
10
Time (rain.) Fig. 2. Changes in the relative volume of a single varicosity in response to exposure to a hypotonic bathing medium. Cortland saline diluted 1:1 with deionized water (final tonicity, 150 mOsm) was added at to (arrow), and varicosity volumes were calculated at subsequent 1-min intervals (see Materials and Methods). The curve was fitted by inspection.
controls (Vre ~ = 0.84 + 0.38). It is n o t e w o r t h y that this dramatic reversal of axonal swelling occurs while hypotonic conditions are m a i n t a i n e d , which raises the important question of what mechanisms m a y underlie the volume recovery process. A s already n o t e d above, R V D responses to hypoosmotic stress in some o t h e r cell types have been shown to be related to an increased efflux of KCI, possibly through Ca 2÷ regulated m e m b r a n e pathways. It was of interest, therefore, to test for similar mechanisms operating in R G C axons. Two p a r a m e t e r s were m o n i t o r e d as indicators of the R V D response (i.e. recovery of volume during hypotonic shock): (1) reduction in the relative volumes of varicosities (Vrel) ; and (2) d i s a p p e a r a n c e of varicosities f o r m e d de novo with e x p o s u r e to hypotonic conditions (see Fig. l b , c ) . P e r t u r b a t i o n s in the R V D response was evaluated by calculating a 'varicosity coefficient ratio' (Kvar) , which t r a c k e d the total n u m b e r
TABLE I Effects o f variouz, treatments on varicosities 10 rain after hypo-osmotic stress
n, number of varicosities monitored; Vrel, volume after/volume before (mean +_ S.D.); Kvar, varicosity coefficient (see Materials and Methods). Significance tested at a level of 0.05 by two-sided sign test.
Fig. 1. Changes in RGC axonal morphology induced by exposure to a hypertonic bathing medium. Phase-contrast micrographs (a) before, (b) immediately after exposure to hypotonic Cortland saline, and (c) after 10 min under hypotonic conditions. Bar = 10 /~m.
Treatment
n
Conc.
Vret
P
Kvar
Control DiS-Ca-(5 ) Calmidazolium Elevated [K+]o Quinine CSC12 TEA Cytochalasin D Colchicine
11 14 8 13 14 11 4 8 12
1/~M 1#M 70 mM 250/~M 10 mM 50mM 10#M 5 mM
0.84 + 0.38 2.80 + 2.0 3.30 + 1.4 2.63 + 1.2 1.30 + 0.46 1.08 + 0.63 1.30+ 1.19 0.96 + 0.27 1.19 + 0.46
0.03 0.0001 0.004 0.002 0.09 0.38 0.5 0.63 0.19
1.08 6.8 6.2 3.2 3.9 0.99 1.5 1.3 1.2
162
Fig. 3. Evidence of membrane permeability changes in RGC axons during hypotonic swelling. (a) Phase-contrast, and (b) fluorescence micrograph of a small fascicle containing a single varicosity after volume recovery in response to a hypotonic bathing medium (150 mOsm) containing Lucifer yellow (0.1%). Bar = 5/~m.
of varicosities present within a given microscopic field before and after a given treatment (see Materials and Methods). The results of these experiments are summarized in Table I.
2
The typical R V D response is attenuated when axonal fields are exposed to a hypo-osmotic medium containing 70 mM potassium gluconate (150 mOsm, total). For these experiments, gluconate was substituted for chloride as the major anion in order to prevent swelling that accompanies membrane depolarization with potassium 24. In the presence of elevated [K+]o, V r e I is 2.63 + 3.2 and the g v a r is 3.2, compared to control preparations, in which Vre I is 0.84 "1- 0.38 and the Kvar is 1.08. When this bathing medium is exchanged for a bathing medium of identical osmolarity with low [K+]o (i.e. 2.5 mM), R V D occurs as usual. These results suggest that an outwardly directed K + gradient is important for the R V D response. Agents that have been reported to block Ca 2+activated K ÷ channels in other cell types yielded variable results. The carbocyanine dye, diS-C3-(5), a potent inhibitor of the Ca2+-dependent K ÷ channel in erythrocytes 38 at a concentration of 1/~M appeared to be the most effective of the agents tested in preventing volume recovery (Vre I = 2.8 + 2.0) and loss of de novo formed varicosities (Kvar = 6.8). Quinine (0.25-1 mM) 32 was less effective in attenuating volume recovery (Ire ~ = 1.3 + 0.46), but partially inhibited the disappearance of transient varicosities (Kvar = 3.9). CsCI 2 (10 mM; VreI = 1.08 + 0.63; g v a r = 0.99) and T E A (50 mM; VreI = 1.3 + 1 . 1 9 ; g v a r = 1.5) were relatively ineffective in blocking the R V D response. Calmodulin has been implicated in Ca2+-mediated activation of K + channels in other cell types 9'13A5' 16,23,28,29 and experiments were also conducted to test for a calmodulin dependence in the R V D response. Calmidazolium (1 ~M), a potent antagonist of calmodulin 4°, effectively blocked the typical R V D volume recovery ( V r e I = 3.3 + 1.4) and varicosity loss (Kwr = 6.2). Although the foregoing results suggest a role for calmodulin in the R V D response of immature R G C axons, as has been suggested for other cell types, experiments designed to investigate a calcium dependency were inconclusive. R V D occurred in these axons with the usual time course, both in hypotonic Ca 2÷-
>o
• 0
....... . iiiIiiiiiiiii 10
20
30
Time (min.) Fig. 4. Changes in the relative volume of a single Varicosity in response to exposure to a hypotonic bathing medium containing ATP-),S (5 mM) was added at to (arrow), and varicosity volumes were calculated at subsequent 5-min intervals (see Materials and Methods). The curvd was fitted by inspection. Note that endstage volume constitutes -30% of prehypo-osmotic stress volume.
TABLE II Changes in relative varicosity volume 20 min following hypotonic loading o f A TP or A TP-yS
n, number of varicosities monitored; Vre~, volume after/volume before (mean +~ S.D.); (see Materials and Methods). Significance tested at a level of 0~05. Loading
n
VreI
P*
ATP ATP-yS
9 6
1.14 + 0.26 0.42 + 0.2
0.34 0.016
* Comparison is with control (see Table I).
163 free/EGTA-buffered, and Ca2+-containing media. These results appear to make the requirement of a Ca 2+ influx unlikely. Attempts were made to reduce the Ca 2+ content of intracellular storage sites by preincubating some axonal fields for 1 h prior to hypotonic stress with the membrane permeant calcium chelator BAPTA-AM (10 ¢tM) combined with ionomycin (5/~M) in a Ca 2+free/EGTA (1 mM) buffered Cortland medium. These preparations also displayed the normal RVD response; however, it was not possible to verify in these axons that calcium actually had been depleted. It has been reported that cytochalasin B treatment interferes with RVD of gallbladder epithelial cells 5 and of mature axons isolated from C a r c i n u s m a e n a s 7. In order to test for potential cytoskeletal involvement in RVD of these growing axons, axonal fields were treated with either cytochalasin D (10/~M) or colchicine (0.1-5 raM) for 15 min prior to hypotonic stress. As shown in Table I, neither cytochalasin D (Vre I = 0.96 + 0.27; Kvar = 1.3), nor colchicine (Vrel = 1.19 + 0.46; g v a r ~- 1.2) had an appreciable effect on attenuating the typical RVD response. Previous work has shown that both of these agents induce cytoskeletal alterations of R G C axons within this time period (data not shown). These observations provide no evidence that actin or microtubular cytoskeletons play a direct role in volume regulation of immature R G C axons. Our experiments with hypotonic stress showed that it could also provide a means for loading low molecular weight solutes into axons. For example, Lucifer yellow, an impermeant anionic fluorescent dye (MW 457 Da), made up in half-strength Cortland saline (0.1%), was taken up during hypotonic stress and remained in axons after RVD reached a steady state and the medium was exchanged for a dye-free one (Fig. 3b). This technique was subsequently used to load ATPwS, a thiophosphate ATP analog in order to probe the potential effect of thiophosphorylation. In the presence of 5 mM ATP, a typical RVD response occurs after exposure to hypotonic stress (i.e. Vre~= 1.24 + 0.26); however, in the presence of 5 mM ATP-yS, the relative varicosity volume is significantly lower than control (i.e. Vrej = 0.42 + 0.2; P = 0.02 - see Table II). Fig. 4 shows the RVD response of a representative single varicosity exposed to half-strength saline, containing 5 mM ATP-yS. After exposure to the ATP-yS-containing hypotonic medium for 30 min, the Vr~~ ratio was 0.3 in this particular case. ATP-~S can serve as a substrate for some kinases which results in a stable thiophosphorylated product resistant to phosphatase activity s. These observations suggest that one or more phosphorylation events accompany the RVD response, and, if not reversed, leads to an irreversible 'overshoot' of the R V D .
DISCUSSION R G C axons regenerating in vitro are capable of reducing their volume in response to hypo-osmotic stress. The RVD response and its mechanism appear to be similar to those described for other cell types, in that an outwardly directed K + gradient is requisite for the recovery of normal volume. Also, agents reported to block Ca2+-activated K + channels antagonize the typical RVD response (see Table I). An area of controversy relates to the role of intracellular Ca 2÷ as a second messenger in mediating the RVD response. In the case of immature R G C axons, the RVD response was unaffected by the absence of extracellular calcium, and additional attempts to deplete and/or buffer [Ca2+]i . These results would suggest that calcium is not required for the RVD response in R G C axons. Studies, using intracellular fluorescent Ca 2÷ indicators in other cell types, however, have yielded variable and conflicting findings. Similar to the present results, Rink et al. 33, using Quin2 as an indicator, was unable to demonstrate an elevation in [Ca2+]i in lymphocytes suspended in a hypotonic medium, notwithstanding the pharmacological evidence to the contrary 9. Yet, recently, experiments with fura2, as an indicator, have provided evidence that [Ca2+]i increases slightly during RVD in hypotonically swollen osteosarcoma cells, even in the absence of extracellular calcium 42. The principal evidence for a potential role of Ca 2÷ in the RVD response in R G C axons is based on two sets of experimental findings. First, apparent antagonism of calmodulin blocks the RVD response. Recent work has implicated calmodulin as a likely target involved in RVD 29. However, the issue of specificity remains a potential problem with regard to calmodulin antagonists 39. Nonetheless, recent work with sulfoxide derivatives of phenothiazine calmodulin antagonists indicates that they produce the same non-specific effects as the unmodified phenothiazines but do not bind to calmodulin and do not block restoration of volume, while the unmodified agents do block recovery 29. The calmodulin inhibitor used in the present study has a much higher affinity for calmodulin than the phenothiazines4°,, which increases the likelihood for a role of calmodulin in the RVD response of RGC axons. Second,, increasing [Ca2+]i in R G C axons under isotonic conditions with 100/tM [Ca2+]o by treating them with ionomycin, a calcium ionophore, causes a Volume reduction that exceeds 40%; a similar effect is produced with 1/~M [Ca2+]o in a Na+-free bathing medium. The volume reduction is blocked by the same treatments that antagonize the RVD response, including elevated [K+]o, selected K + channel blockers, and calmidazolium (Ed-
164 monds and Koenig; in preparation). Volume reductions stimulated by calcium ionophores in other cell types have been shown to be a result of a KC1 efflux 9'11'12"15"16 and observations in R G C axons are consistent with this interpretation. The calcium-mediated shrinkage in R G C axons, which is also blocked by a p o t e n t calmodulin antagonist, suggests that R G C axons have an intrinsic capability to reduce volume by a calcium-dependent pathway. It appears likely, therefore, that this or a similar pathway is utilized in response to hypo-osmotic stress; however, as already noted, a direct correlation between an increase in [Ca2+]i and R V D remains to be demonstrated. A n interesting aspect of the R V D response is the feedback mechanism which limits the volume reduction to near p r e h y p o - o s m o t i c stress levels. The finding, however, that ATP-yS loading during hypo-osmotic stress leads to an overshoot of the volume reduction, not only suggests that at least one phosphorylation step is a requisite event in the R V D pathway, but that dephosphorylation m a y also be requisite to prevent overshooting the volume 'setpoint'. Recently, reports have a p p e a r e d which provide evidence of increased phosphorylation of m e m b r a n e proteins 1° and phosphoinositides 27 induced by cell shrinkage, and by hypo-osmotic stress 3°. In the latter case, it was suggested that m e m b r a n e protein phosphorylation was due to an increased Ca2+-stimulated calmodulin activity. While cytochalasin B treatment attenuated the typical R V D response in m a t u r e invertebrate axons 7, which could implicate the potential involvement of cytoskeletally coupled m e m b r a n e pathway in triggering the R V D response, there was a lack of an observable effect by either cytochalasin D or colchicine on the R V D response
REFERENCES 1 Ballanyi, K. and Grafe, P., Cell volume regulation in the nervous system, Renal Physiol. Biochem., 3-5 (1988) 142-157. 2 Cala, P.M., Volume regulation by Amphiuma red blood cells, Mol. Physiol., 8 (1985) 199-214. 3 Edmonds, B.T. and Koenig, E., Powering of bulk transport (varicosities) and differential sensitivities of directional transport in growing axons, Brain Research, 406 (1987) 288-293. 4 Falke, L.C. and Misler, S., Activity of ion channels during volume regulation by clonal N1E115 neuroblastoma cells, Proc. Natl. Acad. Sci. U.S.A., 86 (1989) 3919-3923. 5 Foskett, J.K. and Spring, K.R., Involvement of calcium and cytoskeleton in gallbladder epithelial cell volume regulation, Am. J. Physiol., 248 (1985) C27-C36. 6 Gilbert, D.S., Axoplasm architecture and physical properties as seen in the Myxicola giant axon, J. Physiol. (Lond.), 253 (1975) 257-301. 7 Gilles, R., Delpine, E., Duchare, C., Cornet, M. and Pequeux, A., The effect of cytochalasin B on the volume regulation response of isolated axons of the green crab Curcinus maenas submitted to hypo-osmotic media, Comp. Biochem. Physiol., 85A (1986) 523-525.
in R G C axons. The m o d e l put forth by Sachs 35 is that m e m b r a n e tension is coupled to stretch channels through cytoskeletal elements, p r o b a b l y consisting of in-series coupling by spectrin, and in-parallel coupling with actin filaments. A s also pointed out by Sachs, however, while the mechanism of transduction m a y be universal 34, the mechanical coupling p r o b a b l y varies widely and may account for variations in response a m o n g different cell types 35. O n e m a j o r difference b e t w e e n i m m a t u r e and m a t u r e R G C axons is the relative paucity of neurofilaments in the former, which we believe contributes to the capability of immature axons to exhibit m a r k e d structural plasticity associated with the rapid directional bulk redistribution of axoplasm 3'23. Thus, in the m a t u r e axon, the neurofilament cytoskeleton plays m a j o r roles in structural stabilization, shape and d e t e r m i n a t i o n of caliber 634, and may influence the organization and p r o p e r t i e s of the actin-containing cortical cytomatrix. H o w e v e r , as crustacean axons a p p e a r not to have neurofilaments 41, the latter considerations would p r e s u m a b l y not account for the divergent findings in the m a t u r e crab axon 7. The present study extends to growing axons the R V D response that characterizes m a n y n o n - n e u r o n a l cell types. While the data are consistent with activation of K + channels as a basic mechanism for loss of an important axoplasmic osmolyte, the elucidation of the potential roles of calcium, calmodulin, and phosphorylation/dephosphorylation, also implicated in the mechanism, requires further investigation. Acknowledgements. This research was supported in part by NS21843 from the NINCDS. The authors wish to thank Drs. James Goldinger and Michael Duffey for helpful discussions during the course of this investigation.
8 Gratecos, D. and Fischer, E.H., Adenosine 5"-O(3-thiotriphosphate) in the control of phosphorylase activity, Biochem. Biophys. Res. Commun., 58 (1974) 960-967. 9 Grinstein, S., Dupre, A. and Rothstein, A., Volume regulation by human lymphocytes: role of calcium, J. Gen. Physiol., 79 (1982) 849-868. 10 Grinstein, S., Goetz-Smith, J.D., Stewart, D., Bereford, B.J. and Mellors, A., Protein phosphorylation during activation of Na+/K ÷ exchange by phorbol esters and by osmotic shrinkage, J. Biol. Chem., 261 (1986) 8009-8016. 11 Grinstein, S. and Cohen, S., Cytoplasmic [Ca2÷] and intracellular pH in lymphocytes, J. Gen. Physiol., 89 (1987) 185-213. 12 Grinstein, S. and Dixon, S.J., Ion transport, membrane potential and cytoplasmic pH in lymphocytes: changes during activation, Physiol. Rev., 69 (1989) 417-481. 13 Hinrichsen, R.D., Burgess-Cassler, A., Soltveldt, B.C., Hennessey, T. and Kung, C., Restoration by calmodutin of a Ca ++ dependent K ÷ current missing in a mutant Paramedium, Science, 232 (1986) 503-506. 14 Hoffman, P.N., Griffin, J.W. and Price, D.L., Control of axonal caliber by neurofilament transport, J. Cell Biol., 99 (1985) 705-714. 15 Hoffmann, E.K., Role of separate K ÷ and CI channels and of
165
16
17
18
19
20
21 22
23
24
25 26
27
28
Na+/K + cotransport in volume regulation in Ehrlich cells, Fed. Proc., 44 (1985) 2513-2519. Hoffmann, E.K., Lambert, I.H. and Simonsen, L.O., Separate Ca + +-activated K ÷ and CI- transport pathways in Ehrlich acites tumor cells, J. Membr. Biol., 91 (1986) 227-244. Hoffmann, E.K. and Simonsen, L.O., Membrane mechanisms in volume and pH regulation in vertebrate cells, Physiol. Rev., 69 (1989) 315-382. Kempski, O., Chausey, L., Gross, U., Zimmer, M. and Baetmann, A., Volume regulation and metabolism of suspended C6 glioma cells: an in vitro model to study cytotoxic brain edema, Brain Research, 279 (1983) 217-228. Kimelberg, H.K. and Frangakis, M.V., Furosemide- and bumetanide-sensitive ion transport and volume control in primary astrocyte cultures from rat brain, Brain Research, 361 (1985) 125-134. Kimelberg, H.K. and O'Connor, E., Swelling of astrocytes causes membrane potential depolarization, Glia, 1 (1988) 219224. Omitted. Koenig, E. and Adams, P., Local protein synthesizing activity in axonal fields regenerating in vitro, J. Neurochem., 39 (1982) 386-400. Koenig, E., Kinsman, S., Repasky, E. and Sultz, L., Rapid motility of motile varicosities and inclusions containing spectrin, actin and calmodulin in regenerating axons in vitro, J. Neurosci., 5 (1985) 715-729. Lipton, P., Effects of membrane depolarization on light scattering by cerebral cortical slices, J. Physiol. (Lond.), 231 (1973) 365-383. Macknight, A.D.C., Principles of cell volume regulation, Renal Physiol. Biochem., 3-5 (1988) 114-141. McCann, J.D. and Welsh, M.J., Neuroleptics antagonize a calcium-activated potassium channel in airway smooth muscle, J. Gen. Physiol., 89 (1987) 339-352. Orlov, S.N., Pokudin, N.I., Kotelevtsev, Y.V. and Gulak, P.V., Volume-dependent regulation of ion transport and membrane phosphorylation in human and rat erythrocytes, J. Membr. Biol., 89 (1989) 339-352. Pierce, S.K., Politis, A.D., Smith, L.H., Jr. and Rowland, L.M., A Ca 2÷ influx in response to hypo-osmotic stress may alter osmolyte permeability by a phenothiazine-sensitive mech-
anism, Cell Calcium, 9 (1988) 129-140. 29 Pierce, S.K., Politis, A.D., Cronkite, D.H., Rowland, L.M., Smith, L.H., Jr., Evidence of calmodulin involvement in cell volume recovery following hypo-osmotic stress, Cell Calcium, 10 (1989) 159-169. 30 Politis, A.D. and Pierce, S.K., Hypoosmotic stress induces phosphorylation of two specific proteins that may be involved in cell volume recovery, J. Cell Biol., 107 (1988) 286a. 31 Pollock, A. and Arieff, A., Abnormalities of cell volume regulation and their functional consequences, Am. J. Physiol., 239 (1980) F195-F205. 32 Reichstein, E. and Rothstein, A., Effects of quinine on Ca + +-induced K + efflux from human red blood cells, J. Mernbr. Biol., 59 (1981) 57-63. 33 Rink, T.J., Sanchez, A., Grinstein, S. and Rothstein, A., Volume restoration in osmotically swollen lymphocytes does not involve changes in free Ca ++ concentration, Biochem. Biophys. Acta, 762 (1983) 593-596. 34 Sachs, F., Mechanical transduction: unification? News Physiol. Sci., 1 (1986) 98-100. 35 Sachs, F., Baroreceptor mechanisms at the cellular level, Fed. Proc., 46 (1987) 12-16. 36 Sachs, F., Mechanical transduction and stretch activated ion channels, Biophys. J., 53 (1988) 410a. 37 Sachs, F., Mechanical transduction in biological systems, Crit. Rev. Biomed. Eng., 16 (1988) 141-169. 38 Simons, T.J.B., Actions of a carbocyanine dye on calciumdependent potassium transport in human red cell ghosts, J. Physiol. (Lond.), 288 (1979) 481-507. 39 Stoclet, J.-C., Gerard, D., Kilhoffer, M.-C., Lugnier, C., Miller, R. and Schaeffer, P., Calmodulin and its role in intracellular calcium regulation, Prog. Neurobiol., 29 (1987) 483-494. 40 Van Belle, H., R24571: a potent inhibitor of calmodulinactivated enzymes, Cell Calcium, 2 (1981) 483-494. 41 Warren, R.H. and Rubin, R.W., Microtubules and actin in giant nerve fibers of the spiny lobster, Panulirus argus, Tissue & Cell, 10 (1978) 687-697. 42 Yamaguchi, D.T., Green, J., Kleeman, C.R. and Muallem, S., Characterization of volume-sensitive, calcium-permeating pathways in osteosarcoma cell line UMR-106-01, J. Biol. Chem., 264 (1989) 4383-4390.
