Lloyd Guth

Qualitative differences between actomyosin ATPase of slow and fast mammalian muscle

Abstract Cross-reinnervation of slow and fast muscles increases the contraction velocity of slow muscles and decreases that of fast ones. It, therefore, appears that the innervation regulates this physiological property of muscle. If actomyosin ATPase activity is, indeed, rate-limiting in the sequence of chemical events mediating contraction velocity, a comparison of the properties of this enzyme from slow and fast muscles should help elucidate the neural regulatory mechanism. Actomyosin, isolated from fast muscle of the cat, had threefold greater ATPase activity and was relatively more alkali-stabile and acid-labile than was actomyosin from slow muscle. These differences were demonstrable histochemically. Fibers having high ATPase activity predominate in fast muscles and fibers having low ATPase activity predominate in slow ones. By exposing frozen sections to acid or alkali before staining for ATPase, it was shown that the high-ATPase fibers are alkali-stabile and acid-labile, whereas as low-ATPase fibers are acid-stabile and alkali-labile. In addition, the fibers having high-ATPase activity could be further subdivided. The large-diameter fibers that predominate in the superficial parts of fast muscle are inhibited by formaldehyde, whereas the small-diameter fibers found mainly in the deeper parts of fast muscle (and in slow muscle of some species) are not. Preliminary observations indicate that under the influence of a foreign nerve, some acid-stabile fibers are converted to alkali-stabile ones. We conclude, on the basis of pH stability, that there are at least two qualitatively distinct actomyosin ATPases, and suggest that the nerve regulates the type of enzyme found in the muscle fiber.

The dynamic nature of the so-called “fiber types” of mammalian skeletal muscle

Abstract In general, when mammalian muscles are stained by a histochemical procedure, three categories of muscle fibers become evident. However, careful analysis of individual fibers in serial sections stained histochemically for different enzymes reveals muscle fibers with diverse combinations of enzyme activities. Thus, the “metabolic profile” of individual muscle fibers is more varied than might be supposed and, on this basis, there are considerably more than three fiber types in mammalian skeletal muscle. To ascertain whether the histochemical characteristics of individual fibers undergo transformations in response to increased work, we excised several muscles of the posterior compartment of the rats hind leg, leaving only the soleus or plantaris muscles as the primary extensors of the ankle joint. Subsequently, alternate serial sections of each muscle were studied histochemically for actomyosin ATPase, intermyofibrillar ATPase, and succinic dehydrogenase activities. Within 2 weeks, profound changes in these enzymes and in the size of certain muscle fibers was observed; this remodelling of the muscle fibers continued throughout the 21-week duration of the study. We interpret these observations as indicating that muscle cells undergo continual alteration throughout life in adaptation to changing functional demands, and that the histochemically demonstrable “fiber types” merely reflect each muscle fibers constitution at a given moment in time. We submit that a new, more flexible approach to fiber typing is needed to appreciate the dynamic nature of the muscle cell. Furthermore, the physiological factors regulating each enzyme must be evaluated in order to achieve a fuller understanding of the significance of the histochemical attributes of normal, exercised, or diseased muscle fibers.

Quantitative changes in cholinesterase activity of denervated muscle fibers and sole plates

Abstract Histochemical studies have indicated that normal muscle fibers exhibit little cholinesterase activity at regions other than the sole plate and that denervated muscle fibers manifest no decreased activity within 2 to 4 weeks. Because histochemical data may be quantitatively unreliable, it was decided to reinvestigate the distribution of cholinesterase with a microchemical modification of Ellmans quantitative cholinesterase method. Alternate frozen cross section (28 μ) from muscle stretched to constant length were examined histochemically and quantitatively. By counting the number of sole plates in the histochemical preparations it was possible to estimate the relative contributions of the sole-plate and the nonsole-plate tissue to the cholinesterase activity of the adjacent quantitatively analyzed tissue slice. Following denervation the sole-plate and non-sole-plate activity both decreased rapidly, reaching approximately 50 per cent of normal within 1 week. A very small additional loss in activity occurred during the following 7 weeks. The total protein content of the tissue slices declined more gradually, reaching 50 per cent of normal after 3 weeks. It is concluded that there is considerable cholinesterase activity not associated with sole plates and that denervation produces a very rapid decrease in cholinesterase activity. The neural influence on cholinesterase level is fairly specific inasmuch as denervation produces a more rapid decrease in cholinesterase activity than in total protein content whereas tenotomy produces less of a decrease in cholinesterase activity than in total protein content of the muscle.

Selectivity in the re-establishment of synapses in the superior cervical sympathetic ganglion of the cat

Abstract Two experiments were performed to determine whether regenerating preganglionic sympathetic fibers establish synapses randomly with any postganglionic neuron or selectively with specific postganglionic neurons. Experiment I . In normal cats the cervical sympathetic trunk (CST) is composed of nerve fibers deriving from the thoracic sympathetic rami T1 to T7. Of these rami, T1 contributes largely pupillodilator fibers and T4 contributes largely vasoconstrictor fibers (i.e., electrical stimulation of T1 produces pupillary dilation without decrease in ear temperature and stimulation of T4 produces a decrease in ear temperature without pupillary dilation). Following transection and regeneration of the CST, these relationships are re-established, indicating that the sympathetic preganglionics from T1 have established synapses preferentially with pupillary postganglionic cells and that the preganglionics from T4 have established synapses preferentially with vasomotor postganglionic cells. Experiment II . In normal cats pupillary dilation is elicited by electrical stimulation of rami T1 to T3, but not T4 to T7. However, stimulation of T4 to T7 does produce pupillary dilation 1 month after crushing T1 to T3 (i.e., before the crushed fibers have regenerated). This effect results from the formation of terminal collateral connections between the residual nonpupillary (T4 to T7) preganglionic nerve fibers and pupillary postganglionic cells (11). Six months postoperatively, pupillary dilation is elicited by stimulation of T1 to T3, but not T4 to T7. Apparently, after T1 to T3 fibers have regenerated, the nonpupillary collaterals (T4 to T7) become functionally inactive and the regenerated fibers again establish functional connections with the pupillary postganglionic cells. From these two experiments we conclude that there is a strong specific affinity between regenerating sympathetic preganglionic fibers and their appropriate postganglionic nerve cells.

AN ELECTROPHYSIOLOGICAL STUDY OF THE EARLY STAGES OF PERIPHERAL NERVE REGENERATION.

Abstract The sciatic nerve of adult rats was crushed at the sciatic notch and tested for nerve regeneration at intervals up to 56 days postoperatively. At the time of testing, the sciatic nerve was removed and immersed in mineral oil (37 C) in a chamber containing sixty-six parallel electrodes at 1 mm intervals. The proximal nerve end was stimulated with single shocks and the compound action potential recorded millimeter by millimeter down the nerve. Immediately postoperatively action potentials could be recorded no farther than 2 mm proximal to the lesion. By 4 days, potentials were observed up to 2 mm distal to the lesion. The rate of regeneration accelerated exponentially from an initial rate of 0.3 mm per day immediately postoperatively to 3.0 mm per day by 18 days. By the twenty-eighth postoperative day the nerve fibers had regenerated into the leg muscles. Although the amplitude of the action potentials increased steadily it was only 20% of normal by 56 days. The conduction velocity increased steadily to 75% of normal by 28 days and did not change during the ensuing 28 days.

Nonselectivity in establishment of neuromuscular connections following nerve regeneration in the rat.

Abstract Experiments were performed to determine if the fibers of the sciatic or tibial nerves that normally innervate the soleus and plantaris muscles will, after regeneration, selectively reinnervate these muscles. In addition to nine unoperated control rats, thirty-seven operated animals were studied 4 months after one of the following procedures: sciatic nerve transection and resuture in adults (9), sciatic nerve crush in adults (8), tibial nerve transection and resuture in adults (5), tibial nerve crush in adults (5), sciatic nerve transection in neonatal rats (5), sciatic nerve crush in neonatal rats (5). Recordings were made of the isometric tensions (T) developed by the soleus and plantaris muscles during supramaximal tetanic stimulation of lumbar nerves L4 and L5 singly and simultaneously (L4 + 5). In normal rats L4 supplies a proportionately greater functional innervation to the plantaris than to the soleus, the ratio TL4/TL5 being significantly higher in the plantaris than in the soleus. In none of the six operated groups could any such difference in the ratio of the two muscles be demonstrated. Consequently there is no evidence of selectivity in the regeneration of the plantaris and soleus nerve fiber constituents of the sciatic or tibial nerves. Both the plantaris and soleus muscles of each operated group showed greater tension overlap than the corresponding muscles of the unoperated control group. Tension overlap was computed as follows: (TL4 + TL5 − TL4 + 5) TL4 + 5 . Gold chloride impregnations of motor end plates indicate that the increased overlap results only in part from increased numbers of dually innervated (presumably bisegmentally innervated) muscle fibers.

The neural regulation of gene expression in the muscle cell

Abstract Although it has long been known that the myosin adenosine triphosphatase (ATPase) activity of fast muscle is greater than that of slow muscle, qualitative differences between these myosins have only recently been demonstrated. The ATPase of slow myosin is relatively acid-stabile and alkali-labile whereas that of fast muscle is acid-labile and alkali-stabile. Moreover, the myosin from each of these types of muscle possesses specific closely associated proteins of low molecular weight (subunits) that are electrophoretically distinct. Thus, in these respects, the myosins of slow and fast muscles appear to be different proteins. After cross-reinnervation of cat slow and fast muscles, these qualitative properties of myosin become altered. The cross-reinnervated slow muscle does not merely develop increased myosin ATPase activity (as has been been previously reported), but the enzyme becomes more alkali-stabile and acid-labile; furthermore, the myosin loses its characteristic subunits and acquires those of fast-muscle myosin. The converse changes occur in cross-reinnervated fast muscle. Although the specific activity of enzymes is regulated in many ways (inhibition by metabolites, binding to intracellular constituents, changes in rate of protein degradation), the synthesis of proteins is qualitatively specified by gene action. Our observation that a qualitatively different myosin appears in cross-reinnervated muscle indicates that a new species of protein has been synthesized, and we therefore suggest that the nerve influences gene expression in the muscle cell.

A correlated histochemical and quantitative study on cerebral glycogen after brain injury in the rat.

Abstract Cerebral injury results in accumulation of glycogen in astrocytes. A quantitative micromethod for glycogen is needed to permit precise investigations of changes in amount, as well as distribution, of glycogen. In the present investigation a highly specific, microchemical, enzymatic assay was employed. Quantitative recovery of glycogen could be obtained from paraffin sections of brain that had been perfused with 4% phosphate-buffered paraformaldehyde, provided that the fixed brain was not washed in water or dehydrated in low grades of alcohol. The fixation and special dehydration procedures used also gave excellent histochemical demonstration of glycogen. Six hours after making a stab wound lesion in the rat cerebrum, a pericapillary accumulation of glycogen was noted in the vicinity of the lesion. Between 24 and 96 hours postoperatively glycogen deposition was observed in the astrocyte cell bodies and processes. Although these histochemical changes were evident only in the immediate vicinity of the lesion, the quantitative assays revealed increased glycogen throughout the ipsilateral hemisphere. This increase was observed at 6, 24, and 96 hours postoperatively. During the subsequent 2 weeks the glycogen values returned toward normal. These observations emphasize the value of correlated histochemical and quantitative studies on adjacent tissue sections because, if applied independently, the histochemical techniques reveal only gross increases in glycogen and the more sensitive quantitative methods are unable to give any information regarding the distribution of the changes.

Fact and artifact in the histochemical procedure for myofibrillar ATPase

Abstract A recent quantitative biochemical study has shown that skeletal muscle mitochondria have high ATPase activity under the same conditions that are employed in the histochemical procedure for myofibrillar (actomyosin) ATPase. However, mitochondrial staining is not observed when this histochemical procedure is applied to frozen sections of skeletal muscle. The present investigation was performed to resolve this discrepancy. Frozen sections of muscle were incubated in the presence of inorganic phosphate (in place of ATP) and then treated sequentially with calcium chloride, cobalt chloride, and ammonium sulfide to determine whether inorganic phosphate binds to specific sarcoplasmic constituents. Binding of phosphate did occur and, furthermore, it was localized to the myofibrils and not at all to the mitochondria. It is concluded that the hydrolysis of ATP in the histochemical reaction can result from ATPase activity of myofibrils, mitochondria, or both. Since the released inorganic phosphate binds to the myofibril, the localization of the reaction product does not necessarily reveal all sites of enzymatic activity. Thus the histochemical procedure does not reflect myofibrillar ATPase activity exclusively, and one therefore cannot use this technique to infer that individual muscle fibers are physiologically fast or slow according to the intensity of the ATPase staining of their myofibrils.

Effects of cross-reinnervation on some chemical properties of red and white muscles of rat and cat

Abstract It appears that highly specific attributes of the nerve regulate the speed of contraction of slow and fast muscle. The present study was undertaken to determine whether the innervation is also specifically responsible for some of the metabolic differences between slow and fast muscles. Self-reinnervation and cross-reinnervation of slow and fast muscles of adult rats and cats were obtained by nerve anastomosis. Six months postoperatively the muscles were examined histochemically and biochemically (quantitatively) for enzymes representative of the glycolytic, oxidative, and pentose-shunt pathways of carbohydrate metabolism, and electrophoretically for soluble-protein pattern. In comparison with self-reinnervated controls, the cross-reinnervated slow muscles exhibited many of the histochemical, quantitative and electrophoretic characteristics of fast muscle, though the conversion was not complete. The cross-reinnervated white muscles showed very little histochemical or quantitative evidence of conversion. However, the electrophoretic protein pattern of the rostral portion of the cross-reinnervated white medial gastrocnemius became converted to a pattern similar to that of red muscle; thus operative failure alone could not have accounted for the relative lack of change in the cross-reinnervated white muscle. The results are consistent with those of several recent reports and support the conclusion that muscle enzymes are neurally regulated. However, the incompleteness of the conversions observed in all these studies indicates that the degree of conversion is probably determined by an interaction between specific neural factors and other physiological influences.