Neurotrophic regulation of rat muscle glucose-6-phosphate dehydrogenase in vitro

Brain Research, 225 (1981) 387-399

387

Elsevier/North-Holland Biomedical Press

N E U R O T R O P H I C R E G U L A T I O N OF RAT MUSCLE GLUCOSE-6-PHOSP H A T E D E H Y D R O G E N A S E IN VITRO

NORMAN ROBBINS Department of Anatomy, Case Western Reserve School of Medicine, Cleveland, Ohio 44106 (U.S.A.)

(Accepted April 9th, 1981) Key words: trophic function - - neuromuscular junction - - denervation - - glucose-6-phosphate

dehydrogenase - - neurotrophic regulation

SUMMARY In organ culture of rat diaphragm, the presence of a 2-2.5 cm phrenic nerve stump delays the time of failure of miniature endplate potentials and eliminates the increase in glucose-6-phosphate dehydrogenase (G6PD) activity which otherwise occurs at 16.5 h in muscles cultured without nerve stumps. The nerve stump effect persists in the presence of blocking doses of o-tubocurarine but is eliminated by nerve crush. As shown by studies of amino acid incorporation into protein, the effect does not involve an overall change in protein synthesis. Effluents collected over 1-2 h from unstimulated or stimulated phrenic nerve-muscle preparations hadno effect on G6PD activity when applied to muscles cultured without nerve stumps. However, medium conditioned by use in organ cultures with long nerve stumps partially countered the denervation-like effect in host cultures. Thus, the nerve maintains muscle G6PD by a humoral mechanism probably unrelated to impulse activity or nicotinic receptor activation.

INTRODUCTION Considerable attention has been directed to the means by which innervation controis membrane properties and membrane proteins of muscle. For these parameters both electrical activity and neurotrophic factors appear to play a role (see reviews 5, 7, 9, 16). However, denervation affects a wide variety of cellular parameters and the possibility that intracellular or cytoplasmic proteins have different mechanisms of neuronalregulation is comparatively unexplored. The mechanism of neuronal influence on a cytoplasmic enzyme, glucose-6-phosphate dehydrogenase (G6PD), was investigated because it responds more rapidly to denervation than other enzymes so far

388 examined (e.g. refs. 12, 20), possibly because it plays a rate-limiting regulatory role. In addition, histochemical analysis has revealed that most of the increased G6PD activity after denervation was located within muscle cells rather than in satellite or other 'extrinsic' cells within muscle tissue 19 whereas such information is lacking for many other enzymes rapidly affected by denervation. The relative rapidity of the change in G6PD also suggests that there is a shorter chain of events between loss of innervation and muscle response, compared to most denervation phenomena. This feature makes short-term in vitro experiments feasible and thus provides a good opportunity to analyze and uncover neuronal factors regulating muscle G6PD. Recently it was shown that the time of increase in G6PD activity is delayed if a long nerve stump is left attached to the denervated muscle 19,21. Since denervated muscles with or without a long nerve stump are both presumably electrically silent, these experiments suggest that a neuronal factor independent of activity regulates muscle G6PD. Despite the fact that analogous nerve stump effects have long been known (cf. reviews cited above and ref. 4), there has been little attempt to analyze the underlying mechanism. Therefore an organ culture system was devised in order to examine this and other modes of regulation more thoroughly. In this system, as reported here, the relative regulatory roles of synaptic transmission, acetylcholine, and soluble neurotrophic factors could be evaluated. METHODS

Organ culture technique All operations were carried out with sterile technique in uv-sterilized tissue culture hoods equipped with Zeiss dissection microscopes. Female rats, 150-190 g, were killed by concussion, the previously shaved skin of the chest was sterilized with acetone, and the left hemi-diaphragm along with a 2-2.5 cm length of phrenic nerve was rapidly removed to a dish of oxygenated Krebs solution. The diaphragm was temporarily pinned at rest length while extraneous tissue was removed, and the indirect twitch was observed to ensure that the nerve was functional. Two strips of diaphragm were cut parallel to the muscle fibers, each about 6-7 mm wide. One strip (M + N) retained a 2-2.5 cm length of phrenic nerve while the other (M) had no exterior nerve stump. Thus, in M, the length of nerve stump ranged from 0 at one edge to 5-7 mm at the other. Each strip was then attached by stainless steel pins to Silastic mounts on a rectangular glass platform. After the costal margin was attached, the tendinous end was pinned while the central tendon was pulled by a hook lever which transmitted horizontally 1 g tension (the optimal twitch tension found in separate experiments). The attached portion of the rib cage was pinned almost vertically against one of the Silastic mounts so that almost the entire length of the muscle fibers was exposed to the culture medium (Fig. 1A). In M + N cultures, the proximal end of the phrenic nerve was pinned to the Silastic support through an attached piece of connective tissue in order to prevent movement and mechanical jarring during the culture. Finally, the platform holding the muscle strip was placed in a 66 × 20 mm rectangular compartment containing 7.5 ml of'199' medium (GIBCO) supplemented

389

A

B

J Fig. 1. Side (A) and top (B) views of rat diaphragm muscle prepared for organ culture. (per ml) with 50 units penicillin, 50/~g streptomycin, 10 units mycostatin, 50 /~g insulin and 20/~g glutamine. The dishes were covered with an overhanging top containing a conduit for 95 % 02-5 ~ CO2 to bubble the medium at the opposite end from the muscle (Fig. 1B). The gas flowed through a water trap and a sterilizing (0.2 #m) Millipore filter before reaching the culture medium. Flow was adjusted to 60 ml/min by inserting a meter in series at the beginning of the culture. Finally, the culture dishes were placed on a platform which sat on a motor-driven cam, such that the muscle platform was lifted out of the solution at an angle of 30 ° for 1.75 min out of every 2 min 1. The whole culture system was placed in a water-jacketed temperature-controlled incubator at 36 °C. The medium was changed at 1 and at 11 h - - a procedure which was empirically adopted since it yielded optimal denervation-like effects.

G6PD activity After organ culture, extraneous tissue and injured muscle on the sides of the diaphragm strips were removed and the blotted muscle fragment was weighed and homogenized in cold 10 mM KH2PO4 (pH 7.0), 5 mM EDTA, 1 mg/ml albumin, and 0.1 mM dithiothreitol. The homogenate was centrifuged at low speed (1000 g), and the supernatant further centrifuged at 15,000 × g, both done at 4 °C. The final supernarant was used in a reaction mixture at 35 °C containing 60 ~1 muscle supernatant, 8.4 mM glucose-6-phosphate, 0.1 mM dithiothreitol, 1 mg/ml bovine serum albumin, 2 mM MgC12, 2.1 mM N A D P H , and 3 units 6-phosphogluconate dehydrogenase (Sigma) in 0.01 M phosphate buffer, pH 7.06. Duplicates with and without substrate were run for exactly 20 min, then read at 340 nm in a Beckman spectrophotometer. Previous work with a continuous recording spectrophotometer showed that the enzyme reaction was linear with time and amount of supernatant. After subtraction of the no-substrate sample average, the reaction rate (nmol N A D H formed/min) was

390 calculated. Since wet weight and protein were highly correlated, the final results were all normalized to blotted muscle wet weight.

Amino acid uptake and incorporation Radio-labeled amino acids were either L-[aSS]methionine (sp. act. 500 Ci/mmol, New Eng. Nuclear) or L-4-[SH]glutamic acid (sp. act. 19 Ci/mmol) kindly supplied by Dr. David Love. In the course of organ culture and at times indicated, muscles were incubated for 2 h in 3 #Ci/ml [85S]methionine or 10 #Ci/ml [3H]glutamic acid. At the end of the incubation, muscles were washed in 2-3 changes of Krebs solution at room temperature (22-24 °C) over 20 min to wash out most extracellular label, and were then cut and frozen as for G6PD analysis. Next, they were homogenized in distilled water, brought to a final concentration of 10 ~ trichloroacetic acid (TCA), placed at 4 °C for 90 min, and centrifuged at 1000 × g for 2 min. The supernatant was pooled with that resulting from two additional washings of the pellet by resuspension in 5 TCA and centrifugation, and an aliquot was counted. The pellet was dissolved in 1 N N a O H at 40 °C overnight, then neutralized with HC1 and counted. Scintillation counts were converted to dpm by appropriate quench curves and external standardization.

Miniature end-plate potentials (m.e.p.p.s) At selected intervals in vitro, the muscle strip still pinned to the glass platform was transferred briefly to a chamber at room temperature (22-24 °C) containing flowing Krebs solution pre-oxygenated with 9 5 ~ O 2 - 5 ~ CO2. M.e.p.p.s were recorded with conventional 2.5 M KCl-filled 15 Mf~ resistance glass electrodes via a WP MP 207 or a Bak ELSA amplifier with oscilloscopic display. Over a 15 min period, about 15 fibers were impaled at points near visible nerve branches and the presence or absence of m.e.p.p.s, was noted. The muscle was then returned for further incubation at 36 °C until the next test period. The data were expressed as percentage of impaled fibers with m.e.p.p.s visible in about 1 min of recorded time per fiber. In reconstructing the time sequence of m.e.p.p.s failure from sequential test impalements at 30-45 min intervals, only the time in culture at 36 °C (and not the time for physiological study at room temperature) was included. The assumption that nerve terminal degeneration at room temperature was negligible was validated by comparing the percentage of fibers with m.e.p.p.s in the last of several tests (with deduction of time at room temperature) to that in muscle first tested after the same time in organ culture.

Potassium contractures The isotonic potassium contracture provided a simple, rapid and non-destructive measure of muscle viability of organ-cultured muscles. At the end of the culture, the costal end of the muscles was pinned to a rubber mount which fitted into a 5 ml vertical chamber filled with Krebs solution bubbled with 95 ~o 02-5 ~ COz at room temperature (22-24 °C). The Krebs solution consisted of: NaC1 135 mM, KC1 5 mM, MgSO4 1 mM, Na2HPO4 1 mM, NaHCOs 15 mM, calcium gluconate 2.5 mM and glucose 11 mM. The tendinous end was attached to the balance lever of a Brush

391 isotonic transducer and the output was displayed on a Brush M a r k 220 Recorder. After a baseline was established, the fluid was changed rapidly to 100 m M potassium sulphate and the contracture was followed to the peak value. Lastly, the chamber was evacuated and re-filled with Krebs solution to determine baseline stability after 5 minutes. Contractures of freshly removed muscles were 5 - 6 m m ; after 16.5 h in vitro, values were generally 3.5-5.5 mm, with exceptions as noted. Muscles with contractures less than 3.0 m m were not included in the data to be presented. The potassium contracture served as an overall index of culture conditions but was insensitive to mechanical injury of up to 50 ~o of the fibers. The latter, however, was easily detectable when the muscles were transilluminated either at the time of initial dissection or at the end of the culture. Muscle strips with more than 25 ~ visible damage were rejected and in all cases reported, only regions of muscle with uninjured fibers were taken for biochemical analysis.

Collection of effluents Phrenic nerve-muscle preparations obtained under sterile conditions from 150-250 g rats were pre-incubated 30-60 min at 22-24 °C in oxygenated 199 medium containing 2/zg/ml D-tubocurarine to block transmission. After a change of medium, the nerve was stimulated via a suction electrode during 4 sequential cycles of 15 min stimulation at 7 Hz and 5 min rest. The medium was removed 1-2 h after the last stimulation and was stored at 4 °C for not longer than 1.5 days before use in culture. Variations in the procedure included stimulation without D-tubocurarine, or for 1 h either continuously or with trains of 350 msec at 40 Hz interrupted by 500 msec rest periods. The results were pooled because the negative outcomes were the same. Effluents from unstimulated or denervated preparations were collected in like manner. Denervations were performed as described previously 19. Treatment of data The entire data from any rat were excluded if either of the paired cultured muscle strips yielded a potassium contracture less than 3 m m (see above) or if G 6 P D activity were less than that of the fresh-dissected control from the same rat by 50 nmol N A D P H / m i n / g or more. The latter criterion was chosen because the largest decrease in G 6 P D activity found in vivo, 12 h after long-stump denervation, was 23 nmol N A D P H / m i n / g (data from same experiments presented in ref. 19). Depressions more than twice that value after organ culture were considered to be products of aberrant culture conditions. Even with both criteria for excluding data, less than one quarter of all cultures were rejected. For statistical evaluation, data were analyzed with Student's t-test (two-tailed) either as paired differences or paired ratios. In the case of ratios, logarithmic transformation of data was employed to eliminate distortion. RESULTS

Functional survival of nerve terminals in vitro In vivo, a long phrenic nerve stump delays functional failure of nerve termi-

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Fig. 2. Persistence of m.e.p.p.s after nerve section as a function of nerve stump length in vivo and in vitro. For in vitro data (solid lines), the presence of m.e.p.p.s was determined in muscle strips cultured with no external stump(O, short nerve stump) or with a 2-2.5 cm nerve stump (O, long nerve stump). In these data, cultures were initiated and nerves were sectioned at the same time. Each point is the mean of 2~42 muscle fibers from 2 to 3 cultures. For comparison, the in vivo data of Miledi and Slaterzz are plotted on identical axes. In their experiments (interrupted lines), nerves were sectioned at a distance of a few mm (A, short nerve stump) or 4 cm (A) from the point of nerve entry and the animals were allowed to recover. M.e.p.p.s were then investigated in muscles removed at the later times indicated. nals la. I f in vitro c o n d i t i o n s were c o m p a r a b l e , the survival a n d d e g e n e r a t i o n o f nerve terminals should foUow a similar pattern. M.e.p.p.s were r e c o r d e d at intervals f r o m o r g a n - c u l t u r e d d i a p h r a g m muscle strips with a n d w i t h o u t a 2 cm nerve s t u m p ( ' M + N ' a n d ' M ' respectively). I n freshly r e m o v e d d i a p h r a g m , m.e.p.p.s were easily r e c o r d e d in 80-95 ~ fibers i m p a l e d : a 100 ~ yield was n o t expected because some endplates were far r e m o v e d f r o m the location o f the visible nerve twigs. Both M + N a n d M showed a b o u t an 85-95 ~ yield o f m.e.p.p.s until a b o u t 10.5 h in vitro when the yield b e g a n to decline in M strips (Fig. 2). This decline was delayed by 5-6 h in the M q- N strip, in qualitative agreement with in vivo results (Fig. 2): quantitative differences will be discussed below.

lncrease of G6PD in freshly denervated muscles in vitro: the effect of a long nerve stump and comparison to in vivo results In a suitable o r g a n culture system, G 6 P D activity should increase rapidly in M strips a n d should be affected by the presence o f a l o n g nerve stump, as f o u n d in vivo 19. G 6 P D activity o f M strips h a d n o t changed after 12 h in vitro, b u t b y a b o u t 16 h, the increased enzyme activity was c o m p a r a b l e to t h a t observed in vivo at 12 h after d e n e r v a t i o n (Fig. 3). Similarly, M + N strips showed no significant increase in enzyme activity at 16 h (Fig. 3) as h a d been f o u n d in vivo at 12 h in muscles supplied b y a long nerve, where even a small decrease was observed 19. Thus, the in vitro increase o f G 6 P D activity in M c o m p a r e d to p a i r e d M ÷ N strips resulted entirely f r o m the increased activity in M strips a n d m a i n t e n a n c e o f n o r m a l G 6 P D activity in M

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-20 Fig. 3. Comparison of changes in G6PD activity after denervation in vivo or in vitro. Muscles were denervated 12 h in vivo (data from ref. 19) or cultured 15.5-16.5 h, in both cases with or without a phrenic nerve stump (designated M + N and M, respectively). The contralateral innervated hemidiaphragm (in vivo) or a strip of muscle from the same hemidiaphragm as that used for culture is designated C. All data are expressed as mean paired differences (± S.E.M.) in G6PD activity between muscles of the same animal given different treatments. Thus, 'M-C' is the difference in enzyme activity between 'M' muscles, denervated in vivo or cultured with a short nerve stump, and controls. C: analogous differences between M ÷ N and C muscles are labeled (M ÷ N)--C, and between M and (M ÷ N) muscles, M-(M ÷ N). In vivo data labeled M--(M + N) were from a set of experiments in which parts of the same hemidiaphragm were denervated with short or long nerve stumps. Numbers of paired data are in parentheses. * Indicates difference significantly different from 0 (P < 0.05). q- N strips. These events occurred somewhat later in vitro t h a n in vivo, as will be discussed. F o r the m o m e n t , the i m p o r t a n t p o i n t is that both the effect of d e n e r v a t i o n a n d that o f the nerve s t u m p o n G 6 P D , previously f o u n d in vivo, were reproduced in vitro. Because o f the i n t e r a n i m a l v a r i a t i o n in control values, m e a n enzyme activities were n o t as sensitive as paired differences. F o r instance, in the in vitro data of Fig. 3, absolute values ( n m o l N A D P H / m i n / g wet wt. 4- S.E.) were 179 4- 10, 185 4- 18 a n d 252 4- 20, for c o n t r o l strips a n d for muscles cultured with a n d w i t h o u t a long nerve stump, respectively. I n c o m p a r i n g M a n d M ÷ N, the probability of the null hypothesis was P < 0.02 for group differences of absolute values, a n d P < 0.001 for b o t h the paired ratio, M / ( M ÷ N), or the paired difference, M - - (M 4- N). Except as noted, statistical validity was similar whether paired differences or paired ratios were used.

Leakage from nerve stump I f the nerve s t u m p effects in vitro were due to leakage of some factor from extraneous tissue or from the cut nerve end or nerve trunks, the effect should persist

394 even if the nerve were crushed at the point of entry into the muscle throughout the culture period. In fact, when M strips were paired with M ÷ N strips with crushed nerve, the paired difference of enzyme activities after 16.5 h in vitro was not significantly different from zero (Fig. 4). That is, the inclusion of a crushed nerve stump had no effect: the nerve stump effect required intact nerve connection to the nerve terminals.

Effect of D-tubocurarine Terminals supplied by a long nerve stump released acetylcholine for a longer time than those with a short stump (Fig. 2). Therefore, the differential effects of nerve stump length on G6PD could result from a postsynaptic action of acetylcholine, e.g. receptor binding, depolarization, or muscle activity (if there was spontaneous firing of axons). All of these actions of acetylcholine were eliminated in a series of experiments in which both M and M + N strips were cultured in the presence of 2-8/~g/ml D-tubocurarine. The results (Fig. 4) clearly demonstrated that the nerve stump effect persisted unimpaired despite blockage of nicotinic postsynaptic acetylcholine action.

Relation to protein synthesis The nerve stump effect in vitro could simply represent a manifestation of a generalized reduction of protein synthesis in M + N strips. To test this possibility, amino acid uptake into muscle and incorporation into protein was studied in paired M and M q- N strips (see Methods). Because of its high specific activity, [35S]methionine was used to measure incorporation into rapidly synthesized proteins during the last 2 h of culture. In a separate series, [4-3H]glutamic acid, which is degraded and therefore not reutilized 14, was present in the culture medium from I to 16.5 h in order to detect incorporation into proteins turning over more slowly. In the [35S]methionine experiments, G6PD activity was assayed in the same muscles, and a significantly increased M/(M q- N) ratio of G6PD activity (1.19) was found. With both isotopes and durations of exposure, paired M and M q- N strips showed essentially the same free amino acid uptake and incorporation into protein (Table I). Thus, the nerve stump dependent difference in G6PD activity in vitro does not reflect an overall influence of innervation on protein synthesis.

Soluble factors mediating neurotrophic regulation of G6PD Experiments were done to test whether soluble factors from nerve or muscle mediated muscle G6PD activity. First, some negative results are given because the outcomes for G6PD appeared to differ from those reported for other muscle parameters studied in vitro. In most cases (with exceptions noted), the G6PD activities of paired M strips were compared after 16.5 h in vitro, one strip treated with the factor to be tested, the other with suitable control medium (Table II). Also, in order to minimize and compensate for possible destruction of trophic factors by lytic enzymes leaking into the medium from the cultured muscle, both fresh medium and test substances were resupplied at the 11 h medium change. No differential effect on the rise of G6PD activity in paired M strips was observed with effluents from fresh-dissected unstimulat-

395 80

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Fig. 4. Elimination and persistence of the nerve stump effect on G6PD activity with nerve crush and Dtubocurarine, respectively. In the former group, nerve in M ÷ N was crushed at site of entry into muscle but left attached. In the D-tubocurarine experiments, drug concentration was 2-8 /zg/ml. Culture duration 15.5-16.5 h. All results are expressed as the mean i S.E.M. of paired differences between muscles of the same animal given different treatments. These differences M - - C , (M 4- N)--C, etc. are labeled as in Fig. 3. Numbers of paired data are in parentheses. Single asterisk indicates significant difference P < 0.05. Double asterisk indicates significant difference 0.1 > P > 0.05.

ed (innervated) vs that from pre-denervated muscles (case 1, Table II). Effluents from stimulated fresh nerve-muscle preparations also had no effect, whether added to the usual M strips or to muscles denervated 11-16 h in vivo before culture (cases 2 and 3, Table II). In the denervated host muscles, G6PD activity was already rapidly increasing 19. One explanation for the negative results in cases 1-3 (Table II) is that a putative neurotrophic factor might leak only very slowly from nerve terminals so that it would take several hrs of incubation of an M 4- N strip to accumulate sufficient factor in the culture medium to influence an M strip. Therefore, an experimental scheme was devised in which the medium conditioned by M or M -t- N strips from 11 to 16.5 h in vitro was saved and transferred to a different pair of M strips for the same 11-16.5 h

TABLE I

Labeled amino acid uptake and incorporation into protein: comparison o f paired M and M 4- N strips cultured l 6.5 h Amino acid

No. o f M / ( M + N) pairs

[zsS]methionine 7 [SH]glutamic acid 6

Duration o f exposure to label (h) *

2 15.5

Ratio, M / ( M + N) Uptake free amino acid per mg wet wt.

Incorporation into protein per mg wet wt.

0.97 1.00

1.02 1.05

* Time in vitro of exposure to label was either from 14.5 to 16.5 h ([zsS]methionine) or from I to 16.5 h ([SH]glutamic acid). Ratios are means (antilog of mean after logarithmic transformation, see Methods), none of which were significantly different from 1.0.

396 TABLE I1 Effects of effluents and conditioned media on G6 PD activity of organ-cultured M strips

Data are means ± S.E.M. of paired comparisons. C = paired fresh-dissected muscle. Case Treatment A

1

Difference in G6PD activity (nmol NADPH/min/g tissue) B

Effluent, stimulated phrenic nerve-muscle preparation*** Same as above applied to cultured denervated muscle**** Effluent from innervated muscle

Effluent, unstimulated phrenic nervemuscle preparation Same as above applied to cultured denervated muscle**** Effluent from 3-day denervated muscle Medium from M ± N Medium from M culculture (conditioned ture (conditioned 11 to 16.5 hin vitro) as in A)

2

3 4

A-C

B-C

N

B-.4

36.5 ± 16.0" 63.5 -- 14.6" 28.5 d_ 17.3 13

52.2 ± 18.9" 37.7 ± 15.5"

14.7 ± 22.8

9

66.2 ± 26.1" 54.5 ± 22.3* --11.3 ± 31.7 10 27.9 ± 16.2 58.4 ± 17.8" 30.6 ± 13.4"* 9

* P < 0.05 for null hypothesis (no difference). P = 0.05 with paired differences but P < 0.05 with log transformation (see Methods). * * * Stimulated at 7 Hz for 10 min with 5 min rest. Cycle repeated 4 times. D-Tubocurarine (2/~g/ml) present to block contractile response. **** Diaphragm denervated 16 h in vivo at time cultures were made. This was the only experiment in which recipient cultured M strips were denervated prior to culture. * *

period. I n both the d o n o r a n d host M strips, it was reasoned that with failure of nerve terminals b e g i n n i n g at 9-10 h (Fig. 2), putative trophic factors would be lacking thereafter. The result was significant a n d as predicted. Strips treated with a m e d i u m c o n d i t i o n e d by M strips showed a significantly greater increase in G 6 P D activity t h a n strips treated with M + N conditioned m e d i u m (case 4, Table II), in which G 6 P D activity was n o t significantly greater t h a n control. These results are consistent with those of the nerve stump experiments presented earlier in p o i n t i n g towards a soluble factor leaking slowly a n d s p o n t a n e o u s l y from nerve terminals a n d influencing muscle G 6 P D activity. DISCUSSION Organ culture vs in vivo events

O r g a n culture provided a suitable model system to analyze postdenervation events because both the n a t u r e a n d schedule of these events were qualitatively similar in vivo a n d in vitro. I n both situations, there was a rapid rise in G 6 P D activity a n d functional failure of the nerve terminal d e p e n d e n t on nerve s t u m p length. The overall delay of a few hours in vitro could be due to the lower temperature (36 °C rather t h a n 37 °C) a n d a Q10 of 2. However, the time shifts with respect to in vivo data were n o t

397 uniform: m.e.p.p, failure in 50~o of fibers was delayed 1.5 h in preparations with short nerve stumps, and almost 5 h in those with long nerves (compare ref. 13). Also, an increase in G6PD activity was detected at 15-16.5 h in vitro vs 12 h in vivo 19. Part of this delay could be a consequence of the longer period for nerve degeneration. The remainder is attributable to the temperature difference, depletion of medium ingredients, persistence of agents arising from the preparation which would normally be removed by the circulation, or absence of hormonal factors of known importance in the G6PD response to denervationll, is. Finally, comparable to results in vivo 17, actinomycin D had no effect on the G6PD activity of muscle cultured with a long nerve stump, but did prevent the increase in activity in muscles cultured without a nerve stump (Robbins, unpublished). In sum, with respect to G6PD activity, the number of resemblances between in vivo and in vitro events after denervation suggest that the phenomena in the two systems are similar if not identical.

Mechanisms of the nerve stump effect Some possible mechanisms of the nerve stump effect on G6PD can be considered in the light of the present results. First, the repetition of such effects in organ culture demonstrates that they do not involve or require changes of blood flow or continuous participation of circulating cells or factors. Second, curarization did not alter the nerve stump effect, and hence the possibility of indirect stimulation by spontaneous firing of axons in the long nerve stump was excluded. The same experiments serve to rule out any role of acetylcholine, released in quantal or nonquantal form, via a classical action on nicotinic receptors. These results parallel those reported by Politoff and Blitz 15, in which an in vitro nerve stump effect on postdenervation increase in RNA synthesis was unimpaired by gentamicin-induced neuromuscular blockade. Third, effluents from acute (2 h)unstimulated or stimulated nerve-muscle preparations, when added to the culture medium, did not prevent the rise in muscle G6PD activity. On the other hand, 11-16 h medium conditioned by muscle with a long nerve stump, was significantly although not completely effective. Hence, at least part of the nerve stump effect may be due to slow leakage of water-soluble trophic agent(s). Further, only experiments in which there was steady leakage from viable nerve terminals for 5.5 h led to sufficient accumulation of trophic factor(s) for these to be detectable. It is unlikely that the trophic agent in the conditioned medium experiment is acetylcholine, since the latter would be destroyed by cholinesterases present in the preparation and the medium a. Whatever the agent, the possibility that it derived from muscle under regulation by the nerve terminals rather than from nerve terminals themselves, was not excluded. Furthermore, soluble factors from M q- N cultures did not completely reproduce the effect of the long nerve stump. Therefore, additional events may be important, such as the altered synaptic structure which may occur within hours of denervation 1°. Nerve muscle effluents such as used here partially reverse denervation changes in acetylcholinesterase in mature muscle denervated prior to culture 3. However, similar additives were ineffective in the present case even though a soluble trophic factor was

398 indicated by c o n d i t i o n e d m e d i u m experiments. The inference is that a factor with different characteristics is involved in m a i n t e n a n c e of n o r m a l level of muscle G 6 P D . ACKNOWLEDGEMENTS This work was supported by grants from the M u s c u l a r Dystrophy Association a n d N a t i o n a l Institute of Health (AG-00795). A critical review of the m a n u s c r i p t by Dr. Stephen Y o u n k i n a n d the able technical assistance of Ms. C y n t h i a T o m c k o a n d Mrs. M a r i l y n Meigs were m u c h appreciated.

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