655
Journal of Muscle Research and Cell Motility 21: 655±662, 2000.
Ó 2000 Kluwer Academic Publishers. Printed in the Netherlands.
Glycogen stability and glycogen phosphorylase activities in isolated skeletal muscles from rat and toad CRAIG A. GOODMAN and GABRIELA M. M. STEPHENSON* Muscle Cell Biochemistry Laboratory, School of Life Sciences and Technology, Victoria University, P.O. Box 14428 MCMC, Melbourne, Victoria, Australia, 8001 Received 29 July 2000; accepted in revised form 18 September 2000
Abstract There is increasing evidence that endogenous glycogen depletion may aect excitation±contraction (E±C) coupling events in vertebrate skeletal muscle. One approach employed in physiological investigations of E±C coupling involves the use of mechanically skinned, single ®bre preparations obtained from tissues stored under paran oil, at room temperature (RT: 20±24°C) and 4°C for several hours. In the present study, we examined the eect of these storage conditions on the glycogen content in three muscles frequently used in research on E±C coupling: rat extensor digitorum longus (EDL) and soleus (SOL) and toad ilio®bularis (IF). Glycogen content was determined ¯uorometrically in homogenates prepared from whole muscles, stored under paran oil for up to 6 h at RT or 4°C. Control muscles and muscles stored for 0.5 and 6 h were also analysed for total phosphorylase (Phostotal) and phosphorylase a (Phos a) activities. No signi®cant change was observed in the glycogen content of EDL and SOL muscles stored at RT for 0.5 h. In rat muscles stored at RT for longer than 0.5 h, the glycogen content decreased to 67.6% (EDL) and 78.7% (SOL) of controls after 3 h and 25.3% (EDL) and 37.4% (SOL) after 6 h. Rat muscles stored at 4°C retained 79.0% (EDL) and 92.5% (SOL) of glycogen after 3 h and 75.2% (EDL) and 61.1% (SOL) after 6 h. The glycogen content of IF muscles stored at RT or 4°C for 6 h was not signi®cantly dierent from controls. Phostotal was unchanged in all muscles over the 6 h period, at both temperatures. Phos a was also unchanged in the toad IF muscles, but in rat muscles it decreased rapidly, particularly in EDL (4.1-fold after 0.5 h at RT). Taken together these results indicate that storage under paran oil for up to 6 h at RT or 4°C is accompanied by minimal glycogen loss in toad IF muscles and by a time- and temperature-dependent glycogen loss in EDL and SOL muscles of the rat. Introduction It is now well established that glycogen plays an important role in skeletal muscle performance (Bergstrom, 1967; Hultman, 1967; Conlee, 1987), but the cellular mechanisms underlying this role are not yet understood (for reviews see Green, 1991; Fitts, 1994). Using ilio®bularis (IF) muscles of the cane toad, Stephenson et al. (1999) have shown that the capacity of skinned twitch ®bre preparations to respond to Tsystem depolarisation is positively correlated with the initial concentration of ®bre glycogen, when the activating solution contains large and constant amounts of ATP and creatine phosphate (8 and 10 mM, respectively). In the same study, these authors found that the depletion of ®bre glycogen, by T-system depolarizationinduced activation of the contractile apparatus, was accompanied by a marked decrease in the capacity of the skinned ®bre preparation to further respond to Tsystem depolarization. Based on these data, Stephenson et al. (1999) suggested that, in cane toad skeletal muscle, *To whom correspondence should be addressed: Fax: 61-3-9688 4995; E-mail:
[email protected]
endogenous glycogen plays a protective role in excitation±contraction (E±C) coupling, which may be related to its proximity to the sarcoplasmic reticulum (SR) (Wanson and Drochmans, 1972; Friden et al., 1989; Cuenda et al., 1995) and to its association with various molecular species (such as kinases and phosphatases) (DiMauro et al., 1971; Entman et al., 1980) that could be implicated in the E±C coupling process. It has yet to be determined whether the relationship between glycogen content and contractile responsiveness to T-system depolarization found in toad muscle ®bres holds also for mammalian muscle. A methodological prerequisite for physiological studies which seek to correlate the glycogen content in mechanically skinned single ®bres with parameters of E± C coupling is that any loss of glycogen and associated enzymes occurring during muscle storage is kept to a minimum. In this type of studies, a muscle is dissected carefully from tendon to tendon, and kept at room temperature (RT), under a thin layer of paran oil, while a single ®bre segment is isolated, mechanically skinned, mounted onto the force measuring system and then placed in an aqueous solution which mimics the myoplasmic environment. The time spent by each single ®bre
656 segment under oil, from the moment it is isolated from the muscle until it is transferred to the aqueous solution, does not exceed 10 min. When not used for ®bre preparation, the muscle is stored, while still under oil, in a refrigerator (4±6°C) or on cold packs (see for example Lamb and Cellini, 1999). The total time spent by the muscle tissue, under oil, at RT and at 4°C depends on the skill of the experimenter and the functional state of the muscle, but does not usually exceed 6 h. According to Nguyen et al. (1998), under these conditions the amount of glycogen in toad single ®bres does not decrease signi®cantly from the post-dissection level. There is evidence to suggest that glycogen in mammalian skeletal muscle is less stable than that in amphibian muscle. For example, Calder and Geddes (1990) found very rapid glycogen loss (60% in the ®rst 5 min after animal death, 82% within the ®rst 2 h) in rat hindlimb muscles left in the body post-mortem. In contrast, Sahyun (1931) reported no decrease in glycogen content in frog (Rana pipiens) skeletal muscle left in situ for over 2 h, at RT. If glycogen in isolated mammalian muscle is less stable than in isolated amphibian muscle, then shorter muscle storage times may need to be used in studies of the correlation between initial glycogen content and E±C coupling in mammalian skinned ®bre preparations. In the present study, which has been prompted by the current lack of available data on the relative stability of glycogen in isolated muscles from amphibians and mammals, we determined and compared the time course of endogenous glycogen depletion in toad IF muscle (IF; predominantly fast-twitch; Hoy et al., 1994; Nguyen and Stephenson, 1999) and in two rat hindlimb muscles that are extensively used in physiological studies, viz extensor digitorum longus (EDL; predominantly fasttwitch glycolytic; Armstrong and Phelps, 1984) and soleus (SOL; predominantly slow-twitch oxidative; Armstrong and Phelps, 1984). The enzyme predominantly responsible for the breakdown of skeletal muscle glycogen is glycogen phosphorylase, which is present in both active (phosphorylase a; Phos a) and inactive (phosphorylase b; Phos b) forms. Phos b can be either activated allosterically, by increased concentrations of adenosine monophosphate (AMP), or can be covalently converted to Phos a by phosphorylase kinase-catalysed phosphorylation (for review see Cohen, 1983b). To date there have been several reports concerned with glycogen phosphorylase activities in EDL and SOL muscles of the rat (Villa Moruzzi et al., 1981; James and Kraegen, 1984; Chasiotis, 1985; Chasiotis et al., 1985). In contrast, no equivalent studies have been performed using the IF muscle of the cane toad and there are no comparative studies on glycogen phosphorylase activities in rat and toad skeletal muscles. Thus, a second aim of the present study was to determine and compare total phosphorylase (Phostotal) and Phos a activities in isolated toad IF and rat EDL and SOL muscles at dierent time points over the period of muscle storage.
Materials and methods Animals The animals used in this study were male rats (DRY and Sprague±Dawley; 11±13 weeks) and male and female cane toads (Bufo marinus; 160±400 g). The rats, supplied by the University of Melbourne, were exposed to a 12:12 h dark:light cycle at a temperature range of 16± 24°C and had access to standard rat chow and water ad libitum. The toads, obtained from Peter Krauss Ltd. (North Queensland, Australia) were kept unfed in a dark, moist environment (16±24°C) for up to 1 week. Muscle preparation Rats were killed by deep halothane inhalation, while toads were double pithed. Both procedures were approved by the Animal Experimentation and Ethics Committee at Victoria University. Previous studies have demonstrated no adverse eect of rat exposure to anaesthetic halothane on skeletal muscle glycogen (Musch et al., 1989; Ferreira et al., 1998) or glycogen phosphorylase activity (Ferreira et al., 1998). To minimise any diurnal variation in muscle glycogen, all rats were killed at the same time of day (Cohn and Joseph, 1971; Conlee et al., 1976). The muscles used in the present study were SOL and EDL from the rat and IF from the cane toad. Muscles from both hindlimbs were dissected rapidly (less than 4 min) and simultaneously by two people, taking care not to cut or activate the muscle. All muscles were blotted dry on ®lter paper (Whatman 1) and weighed. The muscles to be stored, either at RT or at 4°C (see below), were placed in a Petri dish under light mineral oil (Sigma) kept at RT. In all experiments (determination of glycogen content and phosphorylase activity), one muscle was used as a control and processed immediately while the contralateral muscle was stored as described below and processed as appropriate at the required time. In glycogen determination experiments, control muscles were homogenised immediately after weighing, while the contralateral muscles were homogenised after being incubated (see below). For glycogen phosphorylase assays, control muscles were rapidly clamped with aluminium tongs precooled in liquid N2 and freeze-dried for 48 h, while stored muscles were blotted free of oil on ®lter paper, freezeclamped and freeze-dried. Due to technical constraints, glycogen determination and phosphorylase activity assays were performed, for each storage time point, on muscles obtained from dierent animals, and dierent rats were used for supplying the EDL and SOL muscles. Muscle storage Muscles were stored under paran oil at either RT (20± 24°C) or at 4°C. These temperatures were chosen based on the protocol employed in studies using mechanically
657 skinned single ®bre preparations, which involves storage of the muscle tissue at low temperature and isolation of single ®bres, mechanical skinning and ®bre mounting onto the force measuring system at RT (Stephenson and Stephenson, 1996; Lamb and Cellini, 1999). For glycogen stability experiments, muscles were stored at RT for 0.5 (EDL and SOL), 3 h (EDL and SOL) and 6 h (EDL, SOL and IF) and at 4°C for 3 h (EDL and SOL) and 6 h (EDL, SOL and IF). For measurements of phosphorylase activity, rat SOL and EDL muscles were stored for 0.5 h (RT) and 6 h (RT and 4°C), while toad muscles were stored for 6 h (RT and 4°C) only. Glycogen determination Whole muscle glycogen was determined using a two-step ¯uorometric method based on the analytical protocol of Passonneau and Lowry (1993). All enzymes were purchased from Boehringer±Mannheim. Brie¯y, whole muscles were mechanically homogenised (Omni, Warrenton, VA, USA) on ice in 6 volumes of ice cold 0.5 M acetate buer (0.25 M acetic acid and 0.25 M sodium acetate), pH 5.0. Amyloglucosidase (amylo-a1,4-a-1,6glucosidase) was added to the homogenate at a ®nal concentration of 20 lg/ml and samples were incubated at RT for 4 h (step 1). After this period, samples were centrifuged for 5 min at 12,000 ´g. An aliquot of 7.5± 10 ll was taken from the supernatant and added to 3 ml of glucose reagent, which consisted of 0.05 M Tris/HCl, pH 8.1, 1.0 mM MgCl2, 0.5 mM DTT, 0.3 mM ATP, 0.05 mM NADP+ and 1.76 lg/ml glucose-6-phosphate dehydrogenase. To start the reaction, hexokinase was added at a ®nal concentration of 0.4 lg/ ml. The sample±reagent mixture was then incubated for 15 min in the dark (step 2) and subsequently read ¯uorometrically for NADPH content (Perkin-Elmer, LC100). To determine any other endogenous contributors to the ¯uorescence signal (i.e. glucose, G-6-P, NADH & NADPH), a portion of the original homogenate, without the addition of AG, was subjected to the second reaction step and the ¯uorescence signal analysed. The concentration of these non-glycogen ¯uorescence contributors was subtracted from the glycogen concentration. Glycogen concentration was expressed as mmol glucosyl residues/kg wet weight or as mmol glucosyl residues/kg dry weight (mmol glucosyl residues/ kg wet weight ´ conversion factor). The conversion factors used in this study, 3.66 (for EDL and SOL) and 4.34 (for IF), were calculated based on our ®nding that, for the three muscles examined, the dry weight (expressed as percent of wet weight), was 27.5 0.3% (n 14; EDL), 27.5 0.4% (n 15; SOL) and 22.7 0.2% (n 15; IF). Phosphorylase activities Phostotal and Phos a activities were determined at 30°C (the temperature commonly used in phosphorylase
assays) in the direction of glycogen degradation using the method of Ren et al. (1992). All enzymes were purchased from Boehringer±Mannheim. Brie¯y, 10± 25 mg of freeze dried muscle was homogenised on ice in 0.4 ml of homogenisation solution containing 100 mM Tris/HCl, pH 7.5, 60% glycerol (v/v), 50 mM NaF and 10 mM EDTA. The homogenate was then diluted with 1.6 ml of the above solution without glycerol and further homogenised on ice. Fifty microliters of homogenate was then added to 0.5 ml of a phosphorylase stock reagent containing 50 mM Imidazole/HCl at pH 7.0, 1.25 mM MgCl2, 20 mM K2HPO4, 0.25% BSA, 62.5 mM glycogen, 5 lM G-1-6-P, 0.5 mM DTT and 2.5 lg/ml phosphoglucomutase. Phosphorylase activity was determined in the presence (Phostotal) or absence (Phos a) of 3 mM AMP. After 10 min at 30°C, the reaction was stopped with 60 ll of 0.5 N HCl. The glucose-6-phosphate generated was measured by adding 50 ll sample (Phostotal assays) or 100 ll sample (Phos a assays) to 2 ml of G-6-P reagent containing 50 mM Tris/HCl, pH 8.1, 1 mM EDTA, 250 lM NADP+ and 0.5 lg/ml G-6-P dehydrogenase. Samples were subsequently incubated for 15 min in the dark at room temperature, then read ¯uorometrically. Phostotal activity was expressed as lmol/min/g dry weight while Phos a activity was expressed both as lmol/min/g dry weight and as percentage of Phostotal activity. Statistics All results are presented as means and standard error of the mean (M SEM). Student's paired t-test was used to detect dierences between two groups. One way analysis of variance (ANOVA) with Bonferroni's posttest was used for multiple group comparisons. Signi®cance was set at P < 0.05. Results Glycogen content in control muscles As described in the Methods, muscle glycogen content was determined in homogenates prepared from wet (not freeze-dried) muscles. Control experiments showed no signi®cant dierence between the glycogen content of wet muscles and that of the freeze-dried contra-lateral muscles (data not shown). The glycogen content (expressed as mmol glucosyl residues/kg wet weight and in bracket as mmol glucosyl residues/kg dry weight), in freshly dissected EDL, SOL and IF muscles was 41.1 1.5 (150.4 5.5; n 38), 41.7 1.5 (152.6 5.5; n 37) and 51.4 2.8 (223.1 12.1; n 18), respectively. The conversion factors used are given in the Methods. The intracellular glycogen pool in both rat muscles was signi®cantly lower than that in toad IF. There was no signi®cant dierence between EDL and SOL with respect to glycogen content.
658 Glycogen content in muscles stored under oil at RT As seen in Figure 1A, 6 h storage at RT was accompanied by a marked decrease in glycogen content both in EDL (by 74.7 3.4%; n 7) and in SOL (by 62.6 3.9%; n 7). In contrast, under the same conditions, there was only a slight decrease (by 13.5 6.2%; n 6) in IF glycogen and this decrease was statistically non-signi®cant. The proportions of glycogen retained in both EDL and SOL muscles after 6 h storage at RT were not signi®cantly dierent (ANOVA with Bonferroni's post-test; P > 0.05), but both were signi®cantly lower than the proportion of glycogen found in IF muscle stored for the same length of time (ANOVA with Bonferroni's post-test; P < 0.05). To determine the time course of glycogen breakdown in rat EDL and SOL over the 6 h time period, additional samples were stored at RT for 0.5 and 3 h. No change in the glycogen content of EDL (106.5 1.9%; n 4) or SOL (102.8 3.8%; n 4) was observed after 0.5 h, while the 3 h storage period decreased the glycogen content of both muscles by 32.4 8.7% (n 4) and 21.3 4.0% (n 4), respectively (Figure 1A). No signi®cant dierence was observed between the proportions of glycogen retained in EDL and SOL after either 0.5 or 3 h storage (ANOVA with Bonferroni's post-test; P > 0.05). Glycogen content in muscles stored under oil at 4°C As shown in Figure 1B, 6 h storage under oil at 4°C induced a signi®cant loss of glycogen from both EDL (24.8 3.8%, n 7) and SOL (38.9 2.6%, n 7). By comparison, the same storage conditions produced only a small decrease in the glycogen content of IF muscles (14.1 4.4%; n 6). The proportion of glycogen retained at 6 h in the two predominantly fasttwitch muscles (EDL and IF) was signi®cantly higher than that in SOL (Student's t-test; P < 0.05). After 3 h storage at 4°C, the glycogen content in EDL and SOL decreased non-signi®cantly by 21.0 5.1% (n 4) and 7.5 4.8% (n 4), respectively. No signi®cant dier-
ence was found between the two muscles with respect to the proportions of glycogen retained at this time point. Phostotal and Phos a activities in control muscles The activities of Phostotal and Phos a in control, EDL, SOL and IF muscles are presented in Figure 2. The Phostotal (expressed as lmol/g dry weight/min) for EDL (152.9 4.4; n 23) was signi®cantly higher than for IF (66.3 5.7; n 12), which in turn was signi®cantly higher than for SOL (29.9 2.3; n 22) (Figure 2A). By comparison, the Phos a activity (expressed both as lmol/g dry weight/min and, in bracket, as a percentage of Phostotal activity) was 72.4 4.0 (48.1 1.8%; n 23) for EDL, 6.0 0.6 (19.2 1.7%; n 22) for SOL and 3.1 0.7 (4.9 1.1%; n 12) for IF (Figure 2B). The values of Phos a activity in SOL and IF, shown in Figure 2B, were signi®cantly lower than the Phos a activity in EDL. Phostotal and Phos a activity in muscles stored under oil at RT and 4°C. In all three muscles examined in this study (EDL, SOL and IF), Phostotal did not change over the entire 6 h period regardless of the storage temperature (Table 1), indicating no signi®cant storage-induced degradation of glycogen phosphorylase. As shown in Figure 3A, after 6 h of storage at RT, Phos a activity (expressed both as lmol/g dry weight/min and, in bracket, as a percentage of Phostotal activity) in EDL decreased 9-fold from 74.6 4.7 (49.6 2.3%; n 11) to 8.3 1.5 (6.3 1.1%; n 6). Six hour storage at RT of SOL muscles was accompanied by a 4-fold decrease in Phos a activity from 6.3 0.9 (19.6 2.5%; n 12) to 1.6 0.6 (4.9 1.7%; n 6). No decrease in Phos a activity was observed in IF muscles stored for 6 h at RT [4.2 1.7 (6.6 3.2%; n 6) vs. 3.5 1.0 (5.5 1.8%)]. To ®nd out how fast did the Phos a activity decrease in EDL and SOL, the enzyme was assayed also in muscles stored at RT for 0.5 h. As seen in Figure 3A, a marked decrease in Phos a activity was observed relatively early both in EDL (4.1-fold) and in SOL (2.4-fold).
Fig. 1. The time course of glycogen breakdown in rat EDL (j), SOL (h) and IF (n) muscles stored under oil at RT (20±24°C) (A) or 4°C (B). Results are M SEM. *Signi®cantly dierent (P < 0.05; ANOVA with Bonferroni's post-test) from controls (0 h storage).
659
Fig. 2. (A) Total phosphorylase activities (lmol/min/g dry weight) in rat EDL and SOL and toad IF control muscles. Results are M SEM. *Signi®cantly dierent (P < 0.05; ANOVA with Bonferroni's post-test) from EDL; #Signi®cantly dierent (P < 0.05; ANOVA with Bonferroni's post-test) from IF. (B) Phosphorylase a activities (expressed as lmol/min/g dry weight and as percentage of total phosphorylase) in rat EDL and SOL and toad IF control muscles. Results are M SEM. *Signi®cantly dierent from EDL. #Signi®cantly dierent (P < 0.05; ANOVA with Bonferroni's post-test) from IF.
After 6 h storage at 4°C, Phos a activities decreased 7.3-fold in EDL from 72.0 5.3 (46.6 2.8%; n 12) to 9.9 0.6 (7.6 0.6%; n 6) and only 3.3-fold in the SOL muscles [from 6.3 0.7 (18.6 2.3%; n 10) to 2.0 0.4 (6.2 0.8%; n 6)] (Figure 3B). There was no decrease in the Phos a activity of IF muscles stored for 6 h at 4°C. In Figure 3B, the values for Phos a activity in rat muscles stored for 0.5 h at 4°C are also shown. After this period of time, the Phos a activity decreased 2.5-fold to 29.3 2.1 (19.6 1.5%; n 6) in EDL and 1.3-fold to 4.9 1.7 (13.7 4.2%; n 4) in SOL muscles. Discussion The results presented in this study clearly show that storage under oil for up to 6 h at RT or 4°C, is accompanied by minimal glycogen loss in toad IF and by a time- and temperature-dependent glycogen loss in rat EDL and SOL. These data strengthen the evidence that glycogen is more stable in skeletal muscles of the toad (amphibian) than those in the rat (mammal). Glycogen content and phosphorylase activities in freshly dissected (control) muscles The absolute values for EDL and SOL glycogen determined in the present study are similar to those Table 1. Ratio Phostotal 6 h/Phostotal control for EDL, SOL and IF. Number of samples given in brackets Storage temperature
EDL
RT (20±24°C) 4°C
0.97 0.03 (6) 1.34 0.15 (6) 1.06 0.14 (6) 0.92 0.05 (6) 0.92 0.04 (6) 1.41 0.23 (6)
SOL
IF
reported by other investigators who used Sprague± Dawley rats (Witzmann et al., 1983; Chasiotis, 1985). In our study, we found no signi®cant dierence in the glycogen content of these two rat muscles. This result is in agreement with the data of Witzmann et al. (1983) and James and Kraegen (1984), but in disagreement with those of Villa Moruzzi and Bergamini (1983), Chasiotis (1985) and Hutber and Bonen (1989), who reported that the glycogen content in EDL is slightly higher than in SOL. This discrepancy between dierent studies can be explained by the large animal-related variability in muscle glycogen content observed both by us [the coecient of variance (CV) for both EDL and SOL glycogen was about 20%], and by others (Villa Moruzzi and Bergamini, 1983; James and Kraegen, 1984; Chasiotis, 1985). To our knowledge this is the ®rst study in which the glycogen content of rat EDL and SOL is compared with that of the cane toad IF muscle, using the same experiment. Our data show that the toad IF (a predominantly fast-twitch muscle; Nguyen and Stephenson, 1999) contains a signi®cantly higher concentration of glycogen than EDL and SOL of the rat. This somewhat surprising result, given that the toads were kept unfed while the rats had access to food ad libitum, may be explained by species-related dierences in dietary habits, muscular activity patterns, or metabolic adaptations to environmental conditions. The absolute values of Phostotal determined in this study for freshly dissected and rapidly freeze-dried EDL and SOL muscles are similar to those reported by others for Sprague±Dawley rats (Chasiotis, 1985; Chasiotis et al., 1985). Under our experimental conditions, the rat EDL Phostotal activity was 4±5-fold higher than that in the SOL. This result is in agreement with previous reports that rat fast-twitch muscles have a higher Phostotal activity than slow-twitch muscles (Villa Moruzzi et al., 1981; Chasiotis et al., 1985). In this
660
Fig. 3. Phosphorylase a activities (expressed as percentage of total phosphorylase) in rat EDL and SOL and toad IF muscles stored under oil at RT (20±24°C) (A) or 4°C (B) for up to 6 h. Results are M SEM. *Signi®cantly dierent (P < 0.05; ANOVA with Bonferroni's post-test) from controls (0 h).
context, it is interesting to note that the Phostotal activity displayed by toad IF, also a predominantly fast-twitch muscle, was also higher than that in rat SOL, but was lower (less than half) than that found in EDL. According to earlier reports (Conlee et al., 1979; Ren and Hultman, 1988; Ren et al., 1992), the proportion of Phos a activity in resting mammalian muscle (rat and human) represents approximately 5±10% of the total phosphorylase. In one of these studies, Conlee et al. (1979) found that the proportion of Phos a in the `resting' red region (slow-twitch) of rat gastrocnemius was 52%, whereas that in the white region (fast-twitch) was 60% of the total phosphorylase. The authors attributed the high values found for Phos a in gastrocnemius muscle to the activation of glycogen phosphorylase by the cutting and pulling of the tissue prior to freeze-drying. The proportions of Phos a in the freshly dissected and rapidly freeze-dried rat muscles used in the present study were also high (48.1% in EDL and 19.2% in SOL). These values may also be attributed to the Ca2+ release induced by stretching the muscle during freeze clamping (as suggested by Conlee et al., 1979) and/or inadvertent mechanical stimulation during dissection (Cohen, 1983a), because, as it will be discussed later, the proportion of Phos a activity in the rat muscles (particularly in EDL) decreased markedly when the tissues were freeze-clamped after being stored under oil for at least 0.5 h. In contrast to the rat muscles, the freshly dissected toad IF, which was subjected to the same dissecting and freeze-drying protocol, displayed a low Phos a activity (4.9% of Phostotal). This dierence in Phos a activity between the toad and rat muscles suggests that the mechanisms of regulation of phosphorylase kinase and phosphorylase activities in the toad may be dierent from those in the rat. To our knowledge, there is currently no information regarding the proportion of Phos a or the cellular mechanisms involved in the regulation of the glycogenolytic system in `resting' toad IF muscles.
Glycogen loss and phosphorylase activities in muscles stored under oil at RT and at 4°C In this study we found no glycogen breakdown in the rat muscles stored for 0.5 h, under paran oil, at either RT or 4°C. This result strongly suggests that, under these conditions, stored muscles were not strongly hypoxic because in the experiments performed by Ren et al. (1992) on rat epitrochlearis muscles stored for 20 min under strongly hypoxic conditions (Krebs±Henseleit bicarbonate buer bubbled with 95% N2±5% CO2) lost 36% of the initial glycogen, while the controls (muscles stored in oxygenated Krebs±Henseleit bicarbonate buffer) lost only 1%. It is highly likely, however, that all muscles stored under paran oil for 6 h at RT would have become hypoxic. If this was the case, it is interesting to note that the hypoxic conditions were accompanied by a large drop (>60%) in the glycogen content of rat muscles, but only by a small drop (
