Previous work has shown a number of biochemical changes which accompany atrophy or reduced muscle growth in hindlimb of tail-casted, suspended rats. These results clearly show that altered muscle growth was due to changes in protein turnover. Accordingly, the rise in soleus tyrosine following unloading reflects the more negative protein balance. Other major changes we found included slower synthesis of glutamine as indicated by lower ratios of glutamine/glutamate and reduced levels of aspartate which coincide with slower aspartate and ammonia metabolism in vitro. In conjunction with the study of SL-3 rats, which were subjected to 12 h of post-flight gravity, a study of the effects of 12 h eight bearing on metabolism of 6-day unloaded hindlimb muscles was carried out

Henriksen, E. J.

Jacob, S.

Jaspers, S. R.

Tischler, M. E.

English

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OF POOR QUALITY
RESPONSE OF RAT HINDLIMB MUSCLES TO 12 HOURS
RECOVERY FROM TAIL—CAST SUSPENSION
	
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Marc E. Tischler, Erik J. Henriksen, Stephan Jacob
Paul Cook and Stephen Jaspers
bearing and suspended are reported elsewhere
(6,7).	 Muscles were excised rapidly, weighed and
frozen in liquid nitrogen. Subsequently, pieces
of muscle weret sliced4'off and homogenized in 0.2 N
perchloric acid (15-20 mg muscle/ml). After cen-
trifugation, the supernatant solution was neutral-
ized with KOH.
	
All subsequent analyses were com
pleted within 5 days. Certain metabolites (i.e.,
glutamine, glutamate, and malate) were assayed
within the first 48 h by fluorometric or spectro-
photometric methods (8-10). Other measurements
included: tyrosine (11), aspartate (10), alanine
(12) and protein (13).
i?
Previous work from this laboratory has shown a
number of biochemical changes which accompany
atrophy or reduced muscle growth in hindlimb of
tail—carted, suspended rats (1-5). These results
clearly show that altered muscle growth was due to
changes in protein turnover. Accordingly, the
rise in soleus tyrosine following unloading re-
flects the more negative protein balance (5,6).
Other major changes we found included slower syn-
thesis of glutamine as indicated by lower ratios
of glutamine/glutamate and reduced levels of asp-
artate which coincide with slower aspartate and
ammonia metabolism in vitro (5,7). In conjunction
with our study of SL-3 rats (6,7), which were
subjected to 12 h of post—flight gravity, we car-
ried out a study of the effects of 12 h weight
bearing on metabolism of 6—day unloaded hindlimb
muscles.
RESULTS AND DISCUSSION
	
Protein. As a result of unloading (Table 1)
	 Ln
	
the soleus lost protein while the gastrocnemius,
	 u'1
	
plantaris and extensor digitorum longus muscles
	 ^'1
showed slower accumulation of protein than control Ln
	
muscles (+22a) and a smaller average increase than
	 Co
of the animals' body weights (+18 p ).	 Subjecting	 Z
the animals to 12 h weight bearing after 6 days
unloading did not change the difference in soleus
protein loss but did produce a trend towards accu-
mulation of protein more rapidly in the other
hindlimb muscles.
Tyrosine. The loss of protein by the soleus
muscle was associated with the accumulation of
tyrosine which returned to control values during
recovery. This 15% decrease of tyrosine in the
soleus following unloading (Table 2) may reflect
initial events of the muscle returning to a posi-
tive (growth) protein balance. The plantaris
which showed the largest apparent accumulation of
protein in recovery (+6% during 6 days of unload-
ing to +18% during unloading + 12 h loading) also
showed a significant decline in tyrosine (Table 2)
even relative to the weight bearing muscle.
Alanine.	 Except for the plantaris, the other
muscles showed increased alanine, especially the
soleus.	 Since unloading does not decrease ala-
nine, this change is not specifically one of re-
covery.	 Instead it may reflect some general
change in whole body alanine metabolism. 	 Further
work is needed to understand this response.
Glutamine Synthesis. Both the soleus and
gastrocnemius showed increased glutamine content
during reoovery. Glutamate content was unchanged.
Only in the soleus was a significantly increased
ratio of glutamine/glutamate measured. Based on
our other studies of glutamine metabolism (3,5,7)
these results reflect: a) a restored availability
of intracellular ammonia for synthesis of gluta-
mine and b) the elevated levels of glutamine syn-
thetase. Increased ammonia availability probably
results from renewed flux through the purine
nucleotide cycle which generates ammonia via deam-
ination of AMP.	 Glutamine synthetase activity is
elevated as a result of glucocorticoid action.
Department of Biochemistry
University of Arizona
Tucson, AZ 85724
Hindlimbs of Female Sprague—Dawley rats were
subjected to._p days suspension followed by 12 h of
weight bearing. The soleus (SOL), which atrophied
with suspension, showed increased mass in recovery
but without greater protein content. The extensor
digitorum longus (EDL) and gastrocnemius showed
reduced growth in suspension with no change of
mass or protein in recovery. In contrast, the
plantaris (PLN) recovered mass and protein after
showing reduced growth in suspension.	 In the SOL
during recovery, tyrosine decreased, and malate
and aspartate,
	
and	 the ratio of gluta-
mine/glutamate increased.	 -These changes were
opposite to those occurring in suspension. 	 The
rise in glutamine/glutamate indicates greater
ability of the mus_le to produce glutamine.	 In
the PLN during recovery, tyrosine decreased but no
other significant changes occurred. The fall of
tyrosine in SOL and PLN it probably indicative of
improved protein balance. In the SOL, GAS and EDL
during recovery, muscle alanine increased. These
results show that the SOL is most responsive to
resumption of loading presumably because 'of its
marked atrophy during unloading.
Aspartate and Malate. Changes in aspartate
coincide with the level of muscle activity (5,7)
and are associated generally with parallel changes
in malate.	 Coincident changes of malate and asp-
artate are not surprising since these compounds
METHODS	 have the same carbon backbone structure. That the
soleus muscle is beginning to recover with 12 h of
'.	 Female Sprague—Dawley rats were subjected to 6	 loading is reflected in the rise of aspartate and
days of tail-cast, hindlimb suspension with or 	 malate levels (Table 2) to near normal. Aspartate
without a subsequent 12 h (overnight) period of 	 levels are linked to muscle activity through the
normal weight bearing. Weight bearing controls 	 purine nucleotide cycle, in which aspartate pro
,.	 were also studied but differences between weight 	 vides the nitrogen for amination of IMP.
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Table I. Protein and Tyrosine Content of Muscles
With or Without Recovery
Percent Difference from Weight Bearing
M	
Soleus	 Gastroc- Plantaris . EDL
nemius
ORIGINAL piqcE EfS
Protein
Unloaded -19
	
+6	 +6	 +9
Recovered =22	 +12	 +18	 +15
Tyrosine
Unloaded +34	 NS	 NS	 NS
Recovered NS	 NS	 -20	 NS
Table 2. Effect of 12 h Recovery on Tissue
Metabolites
i0ercent Difference from Unloaded
Soleus Gastroc- Plantaris	 EDL
nemius
Tyrosine	 -15	 NS	 -21	 NS
Alanine	 +120	 +33	 NS	 +22
Glutamine	 +103	 +24	 NS	 NS
Gln/Glu	 +91	 NS	 NS	 NS
Aspartat	 +116	 NS	 NS	 NS
Malate	 +74	 NS	 NS	 NS
Concluding Remarks. While measurement of the
reaccumulation of muscle protein and mass provides
absolute evidence of recovery, such changes in the
atrophied soleus are not 2ikely.to be significant
for several days.	 On the other hand, amino acids
seem to return to their normal metabolic pattern
in a matter of hours. Indeed, we have found
normal glutamine/glutamate after only 4 h. Hence,
amino acid measurements may provide sensitive
indications of variations in muscle activity.
This may be especially true in the case of aspar-
tate and glutamine whose metabolism seems to be
linked to flux through the purine nucleotide cycle
as donor and acceptor, respectively, of nitrogen.
With decreased muscle activity the use of ATP and
hence the formation of AMP declines. The mainten-
ance of a greater energy charge is likely respons-
ible for slower deamination of AMP and hence the
formation of nucleosides, the availability of
ammonia for synthesis of glutamine and the utili-
zation of aspartate for reamination.
ACKNOWLEDGEMENTS
This work was carried out during the tenure of
an Established Investigatorship from the American
Heart Association to M.E. Tischler and a Cusanus-
werk Fellowship from the Federal Republic of Ger-
many to S. Jacob. National Aeronautics and Space
Administration Grant NAGW-227 supported this work.
REFERENCES	 OF POOR QUALITY
1. Jaspers, S.R., and M.E. Tischler. Atrophy and
growth failure of rat hindlimb muscles in tail-
cast suspension. J. Appl. Physiol. 57:1472-1479,
1984.
2. Jaspers, S.R., J.M. Fagan, and M.E. Tischler.
Biochemical response to chronic shortening in
unloaded soleus muscles. J. Appl. Physiol. 59:in
press, 1985.
3. Tischler, M.E., and S.R. Jaspers. Synthesis
of amino acids in weight bearing and non-weight
bearing leg muscles of suspended rats. The Physi-
ologist 25 (suppl.):5155-5156, 1982.
4. Tischler, M.E., S.R. Jaspers, and J.M. Fagan.
Prevention of metabolic alterations caused by
suspension hypokinesia in rats. The Physiologist
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5. Jaspers, S.R.: Metabolic responses of skeletal
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6. Henriksen, E.J., M.E. Tischler, S. Jacob, and
P.H. Cook. Muscle protein and glycogen responses
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7. Tischler, M.E., E.J. Henriksen, S. Jacob, and
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8. Lund, P.	 L-Glutamine. Determination with
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10. Williamson, J.R., and B.E. Corkey. Assays of
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mol. 13:435-513, 1969.
11. Waalkes, T.P., and S. Udenfriend. A fluoro-
metric method for the estimation of tyrosine in
plasma and tissues. J. Lab. Clin. Med. 50:733- 736,
1957.
12. Williamson, D.H. L-Alanine. Determination
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13. Lowry, O.H., N.J. Rosebrough, A.L. Farr, and
R.J. Randall. Protein measurement with folin rea-
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Response of rat hindlimb muscles to 12 hours recovery from tail-cast suspension

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