Joan M. Lyles

Fibre types in chicken skeletal muscles and their changes in muscular dystrophy

1. Five major fibre types in chicken skeletal muscles are recognized, based upon their histochemical and morphological characteristics. A classification of these which is readily related to a commonly used classification of mammalian muscle fibre types is given.

Developmental changes in levels and forms of cholinesterases in muscles of normal and dystrophic chickens.

Abstract— Acetylcholinesterase (AChE) and pseudocholinesterase (°ChE) were studied in vivo and during the first several months of development of pectoral and posterior latissimi dorsi (PLD) muscles in normal and dystrophic chickens. Muscle extracts were prepared in a high ionic strength‐nonionic detergent medium in the presence of protease inhibitors, in order to obtain complete solubilization and to prevent degradation of intrinsic molecular forms of both enzymes.

Intrinsic forms of acetylcholinesterase in skeletal muscle

Acetylcholinesterase (AChE; EC 3.1.1.7) occurs in mammalian and avian skeletal muscle in a number of molecular forms as characterised by their sedimentation coefficients on sucrose gradients. In rat diaphragm, three principal forms were observed by Hall ]I] , who showed that the heaviest (H) form, 16 S AChE, was specifically associated with the endplate regions of the muscle and was greatly reduced in amount upon denervation. Vigny et al. [2,3] provided additional evidence suggesting that the H form was endplatespecific in various muscles of the rat and the chicken. In all 3 studies, however, the H form accounted for only a minor part of the total AChE activity, the greater part of the enzyme occurring in peaks of medium (

Parallel regulation of acetylcholinesterase and pseudocholinesterase in normal, denervated and dystrophic chicken skeletal muscle

4) or low (L) sedimentation coefficient. Treatment with collagenase or proteases can modify the molecular forms of AChE in skeletal muscle [4-6] , and it has been suggested that endogenous proteases may affect the distribution of AChE species observed in skeletal muscle extracts [ 5,7] . In the present study, therefore, we have examined the effect of certain protease inhibitors on the sedimentation profile of AChE activity in extracts of various chicken muscles and of rat diaphragm. We will demonstrate that use of protease inhibitors in the extraction medium prevents degradation of the intrinsic molecular forms of AChE and.greatly simplifies the sedimentation profiles obtained, Using an appropriate mature of ~hibitors, it is shown that AChE in normal pectoral muscle is predominantly of the H (19.5 S) type. In the pectoral

Changes in the Levels and Forms of Cholinesterases in the Blood Plasma of Normal and Dystrophic Chickens

IN vertebrate skeletal muscle acetylcholinesterase (AChE, EC 3.1.1.7) is usually considered to be involved in the termination of impulse transmission by hydrolysis of acetylcholine1. AChE is often accompanied by a second enzyme2, pseudocholinesterase (ψ/ChE, EC 3.1.1.8), which differs from AChE in substrate specificity and in susceptibility to anticholinesterases3,4. No role comparable with that of AChE in synaptic transmission is known for ψChE, despite its abundance at some sites and its occurrence (as shown histochemically) with ψChE in various muscle endplates1,5. It has been suggested that ψChE is a precursor of AChE in the superior cervical ganglion6,7. Moreover, in ganglia and skeletal muscle of rats, forms of 0ChE have been demonstrated8 homologous with forms of ψChE observed previously9–11. Elevated levels of AChE have been demonstrated in aqueous extracts of fast-twitch muscles of dystrophic as compared with normal chickens12,13; ψChE levels increased similarly13, and also in such extracts of denervated muscles of normal chickens14. We show here that the total amounts and forms of AChE change in parallel with those of ψChE during development of skeletal muscles of both normal and dystrophic chickens, as well as on denervation of normal muscle. Our data strongly indicate that in these tissues the two enzymes are controlled by a common regulatory mechanism.

Comparison of the Molecular Forms of the Cholinesterases in Tissues of Normal and Dystrophic Chickens

Abstract: Acetylcholinesterase (AChE) and pseudocholinesterase (°ChE) were analysed in the blood plasma of developing chickens, both normal and those with inherited muscular dystrophy. The amounts and the molecular forms of each were examined. °ChE concentration rises in the plasma of normal and dystrophic chicks at the end of embryonic development and is maintained after hatching at a constant, relatively high level, accounting for 90‐95% of total cholinesterase activity in normal plasma. This level is maintained in normal and dystrophic chickens. In embryonic plasma of both normal and dystrophic chicks, on the other hand, the levels of AChE are higher than those of °ChE. Immediately after hatching the AChE level decreases rapidly in normal plasma, reaching a very low level by 2‐3 weeks ex ovo. The AChE level in plasma from dystrophic birds, although less than normal from day 19 in ovo to 2 weeks ex ovo, subsequently increases to peak around 4 months at levels 15‐20‐fold of those in normal birds. There is virtually no enzyme of either type in the erythrocytes of normal or dystrophic chickens. The changes of AChE in plasma were correlated with the alterations of AChE in dystrophic fast‐twitch muscles, suggesting that the latter pool is a precursor of the plasma AChE. Both the AChE and the °ChE in plasma exist in multiple molecular forms, which are similar to certain of those found previously in the muscles of these birds. The major form (60‐80%) of both enzymes in the plasma is the M form (sedimentation coefficient ≥11 S) in all cases, but it is accompanied by certain other forms. In no case is there any of the heaviest form (H2, 19‐20 S) of AChE or of °ChE found in normal and dystrophic muscle, which is attached at the synapses in normal muscle. The pattern of forms of plasma °ChE is constant at all ages, and in normal and dystrophic chickens. The pattern of forms of AChE in the plasma, in contrast, varies with age and with dystrophy in a characteristic manner. The sedimentation coefficients and the amounts of the enzymes in fast‐twitch muscle of dystrophic animals are compared with those of the plasma forms, and an interpretation is given of the characteristic patterns of AChE and of χE in their blood.

Disappearance of the 'endplate' form of acetylcholinesterase from a slow tonic muscle.

Abstract: The levels and molecular forms of acetylcholinesterase (AChE, EC 3.1.1.7) and pseudocholinesterase (ΦChE, EC 3.1.1.8) were examined in various skeletal muscles, cardiac muscles, and neural tissues from normal and dystrophic chickens. The relative amount of the heavy (Hc) form of AChE in mixed‐fibre‐type twitch muscles varies in proportion to the percentage of glycolytic fast‐twitch fibres. Conversely, muscles with higher levels of oxidative fibres (i.e., slow‐tonic, oxidative‐glycolytic fast‐twitch, or oxidative slow‐twitch) have higher proportions of the light (L) form of AChE. The effects of dystrophy on AChE and ΦChE are more severe in muscles richer in glycolytic fast‐twitch fibres (e.g., pectoral or posterior latissimus dorsi, PLD); there is no alteration of AChE or ΦChE in a slow‐tonic muscle. In the pectoral or PLD muscles from older dystrophic chickens, however, the AChE forms revert to a normal distribution while the ΦChE pattern remains abnormal. Muscle ΦChE is sensitive to collagenase in a similar way as is AChE, thus apparently having a similar tailed structure. Unlike skeletal muscle, cardiac muscle has very high levels of ΦChE, present mainly as the L form; AChE is present mainly as the medium (M) form, with smaller amounts of L and Hc. The latter pattern of AChE forms resembles that seen in several neural tissues examined. No alterations in AChE or ΦChE were found in cardiac or neural tissues from dystrophic chickens.

Molecular forms of the cholinesterases inside and outside muscle endplates.

Acetylcholinesterase (AChE, EC 3.1 .1.7) and pseudocholinesterase (+ChE, EC 3.1 .1.8) have been shown to exist in muscles and in other tissues in multiple molecular forms [l-5]. In chicken fasttwitch muscles, for example, the occurrence of two light forms (L1 and LZ, s20,w = 4.7 S and 6.6 S), a medium (M, 11 S) and two heavy forms (H, and Hz, 14.7 S and 20 S) of AChE have been followed through development to maturity [6,7]. In mammalian muscles, similar AChE forms (-4 S, 6 S, 10 S, 13 S and 16 S) were described [ l-4,8]. The 16 S mammalian and the 20 S avian H2 forms are widely accepted as constituting the AChE at the motor endplates, since they are not found in non-endplate zones of muscles and since they disappear on denervation [ 1-3 ,8,9] (and a similar conclusion has recently been drawn for their H1 forms [4]); however, in human muscle [lo] some H AChE has been described as being also outside the endplates. The mammalian 16 S, avian 20 S and fish electric organ 16.5 S forms of AChE have been characterised as comprising a collageneous tail to which 12 catalytic monomers are attached in clusters [4,5]. The tail is thought to anchor this structure to the basal lamina [I I], the whole being designed for function external to the synaptic membrane.

Acetylcholinesterase Forms in Dystrophic Chickens. Normal Axonal Transport in Peripheral Nerves, Abnormal Concentration in Fast Twitch Muscles and Plasma

ChE (which differs from AChE in some specificity properties) has recently been shown to exist in a similar set of forms in fast-twitch muscles and ganglia, and the two enzymes appear to be regulated in parallel there [4,7,9]. Again, Hz tiChE, alone, disappears on denervation [9], indicating an endplate location. The slow (tonic) muscles, e.g., the avian anterior

AChE IN CHEMOTHERAPY OF AVIAN MUSCULAR DYSTROPHY

Individual endplates were micro-dissected from chicken fast-twitch muscle, and the molecular forms of acetylcholinesterase and of pseudocholinesterase therein, identified by their sedimentation coefficients, were analysed directly. The forms actually present at the endplate, and those that are non-synaptic9 were established. This analysis was also extended to muscle of the chicken with inherited muscular dystrophy, showing altered distributions of these forms.