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Biomedical subjects

G M Stephenson

Publications and source records attributed to G M Stephenson.

At least 19 recordsLinked to original sources
PubMed2000

Ca2+-activation characteristics of single fibres from chemically skinned rat muscle incubated with glucose-6-phosphate.

In this study we examined the effects of 3-24 h of incubation of chemically skinned rat fast-twitch muscle with the glycolytic metabolite glucose 6-phosphate (G6-P) on the contractile properties and myosin ATPase activity in single muscle fibres, and on the carbohydrate content of myosin heavy chains (MHCs). Exposure of the permeabilised muscle to 10 mM G6-P for 24 h at 22+/-1 degrees C in a rigor solution containing protease inhibitors and a reducing agent (dithiothreitol, DTT) significantly decreased maximum Ca(2+)-activated force output by 31%, lowered the Ca2+ threshold for contraction by 0.1 pCa units and produced shallower force-pCa curves compared with controls. Furthermore, under these conditions, G6-P-treated muscle displayed lower myofibrillar MgATPase activity and a markedly higher carbohydrate content of MHCs, as identified with an immunoblot protocol for glycoprotein detection. Shorter incubations under the same conditions or 24-h incubations with 5 mM G6-P generally resulted in smaller changes in the contractile activation parameters. These findings suggest that reducing sugars acting as metabolic intermediates in the glycolytic pathway can have important non-energy-related effects on the contractile activation characteristics of mammalian skeletal muscle. These effects are consistent with the glycation of muscle proteins, in particular that of the MHC.

Adenosine Triphosphatases↗
PubMed1999

Glycogen content and excitation-contraction coupling in mechanically skinned muscle fibres of the cane toad.

1. Mechanically skinned skeletal muscle fibres from the twitch region of the iliofibularis muscle of cane toads were used to investigate the relationship between fibre glycogen content and fibre capacity to respond to transverse tubular (T-) system depolarization. 2. A large proportion of total fibre glycogen remained in mechanically skinned muscle fibres exposed to aqueous solutions. This glycogen pool (about 80% of total fibre glycogen) was very stable when the preparation was incubated in a rigor solution (pH 7.0) but decreased gradually at a rate of 0.59+/-0.20% min-1 in a relaxing solution (200 nM [Ca2+]). The rate was considerably higher (2.66+/-0.38% min(-1)) when the preparations were exposed to 30 microM [Ca2+]. An even greater rate of glycogen loss was found after T-system depolarization-induced contractions. The Ca2+-dependent loss of fibre glycogen was caused by endogenous glycogenolytic processes. 3. Silver stained SDS gels of components eluted into relaxing solution from single skinned fibres revealed a rapid (2 min) loss of parvalbumin and at least 10 other proteins varying in molecular mass between 10 and 80 kDa but there was essentially no loss of myosin heavy and light chains and actin. Subsequent elution for a further 30 min in either relaxing or maximally Ca2+-activating solution did not result in additional, appreciable detectable loss of fibre protein. 4. Depletion of fibre glycogen was associated with loss of fibre ability to respond to T-system depolarization even though the bathing solutions contained high levels of ATP (8 mM) and creatine phosphate (10 mM). 5. The capacity of mechanically skinned fibres to respond to T-system depolarization was highly positively correlated (P<0.0001) with initial fibre glycogen concentration. 6. In conclusion, the results show that (i) the capacity of skeletal muscle to respond to T-system depolarization is related directly or indirectly to the non-washable glycogen pool in fibres, (ii) this relationship holds for conditions where glycogen is not required as a source of energy and (iii) the mechanically skinned fibre preparation is well suited to study the regulation of endogenous glycogenolytic enzymes.

Adenosine Triphosphate↗
PubMed1999

An electrophoretic study of myosin heavy chain expression in skeletal muscles of the toad Bufo marinus.

In this study we developed an SDS-PAGE protocol which for the first time separates effectively all myosin heavy chain (MHC) isoforms expected to be expressed in iliofibularis (IF), pyriformis (PYR), cruralis (CRU) and sartorius (SAR) muscles of the toad Bufo marinus on the basis of previously reported fibre type composition. The main feature of the method is the use of alanine instead of glycine both in the separating gel and in the running buffer. The correlation between the MHC isoform composition of IF, SAR and PYR muscles determined in this study and the previously reported fibre type composition of IF and SAR muscles in the toad and of PYR muscle in the frog was used to tentatively identify the MHC isoforms expressed by twitch fibre types 1, 2 and 3 and by tonic fibres. The alanine-SDS electrophoretic method was employed to examine changes in the MHC composition of IF, PYR, CRU and SAR muscles with the ontogenetic growth of the toad from post-natal life (body weight < 1 g) to late adulthood (body weight 200-450 g). The developmental changes in the MHC isoform composition of the toad IF muscle observed in this study are in very good agreement with those in the fibre type composition of the developing IF muscle reported in the literature.

Animals↗
PubMed1999

Fiber type populations and Ca2+-activation properties of single fibers in soleus muscles from SHR and WKY rats.

Electrophoretic analyses of muscle proteins in whole muscle homogenates and single muscle fiber segments were used to examine myosin heavy chain (MHC) and myosin light chain 2 (MLC2) isoform composition and fiber type populations in soleus muscles from spontaneously hypertensive rats (SHRs) and their age-matched normotensive controls [Wistar-Kyoto (WKY) rats], at three stages in the development of high blood pressure (4 wk, 16 wk, and 24 wk of age). Demembranated (chemically skinned with 2% Triton X-100), single fiber preparations were used to determine the maximum Ca2+-activated force per cross-sectional area, calcium sensitivity, and degree of cooperativity of the contractile apparatus and Ca2+-regulatory system with respect to Ca2+. The results show that, at all ages examined, 1) SHR soleus contained a lower proportion of MHCI and MLC2 slow (MLC2s) and a higher proportion of MHCIIa, MHCIId/x, and MLC2 fast (MLC2f ) isoforms than the age-matched controls; 2) random dissection of single fibers from SHR and WKY soleus produced four populations of fibers: type I (expressing MHCI), type IIA (expressing MHCIIa), hybrid type I+IIA (coexpressing MHCI and MHCIIa), and hybrid type IIA+IID (coexpressing MHCIIa and MHCIId/x); and 3) single fiber dissection from SHR soleus yielded a lower proportion of type I fibers, a higher proportion of fast-twitch fibers (types IIA and IIA+IID), and a higher proportion of hybrid fibers (types I+IIA and IIA+IID) than the homologous muscles from the age-matched WKY rats. Because the presence of hybrid fibers is viewed as a marker of muscle transformation, these data suggest that SHR soleus undergoes transformation well into adulthood. Our data show also that, for a given fiber type, there are no significant differences between SHR and WKY soleus muscles with respect to any of the Ca2+-activation properties examined. This finding indicates that the lower specific tensions reported in the literature for SHR soleus muscles are not due to strain- or hypertension-related differences in the function of the contractile apparatus or regulatory system.

Animals↗
PubMed1998

Microfluorometric analyses of glycogen in freshly dissected, single skeletal muscle fibres of the cane toad using a mechanically skinned fibre preparation.

The main objective of this study was to analyse glycogen in single muscle fibres, using a recently developed microfluorometric method which detects subpicomol amounts of NADPH, glucose and glycogen (as glucosyl units) (detection limit 0.16-0.17 pmol in a 25 nl sample) without fluorochrome amplification. The fibres were freshly dissected from the twitch region of the iliofibularis muscle of the cane toad (Bufo marinus), and were mechanically skinned under paraffin oil to gain access to the intracellular compartments. The results show that (1) glycogen concentrations in toad skeletal muscle fibres range between 25.8 and 369 mmol glucosyl units/litre fibre volume; (2) there is a large variation in glycogen content between individual fibres from the iliofibularis muscle of one animal; (3) there are seasonal differences in the glycogen content of toad single muscle fibres; (4) the total amount of glycogen in single muscle fibres of the toad does not decrease significantly when storing the tissue, under paraffin oil, at 20-25 degree C for up to 6 h or at 4 degree C for up to 24 h; and (5) 15-26% of fibre glycogen can be washed in an aqueous solution at pH 5-7, within 5 min, while 74-85% of fibre glycogen remains associated with the washed skinned fibre, even after 40 min exposure of the skinned fibre preparation to the aqueous environment. The retention of most glycogen in the fibre preparation after mechanical removal of the plasma membrane and extensive washing indicates that in toad skeletal muscle fibres the largest proportion of glycogen is tightly bound to intracellular structures. The results also show that the skinned muscle fibre preparation is well suited for microfluorometric glycogen determination, since low molecular weight non-glycogen contributors to the fluorescence signal can be removed from the myoplasmic space prior to the glycogen hydrolysis step.

Animals↗
PubMed1998

Events of the excitation-contraction-relaxation (E-C-R) cycle in fast- and slow-twitch mammalian muscle fibres relevant to muscle fatigue.

The excitation-contraction-relaxation cycle (E-C-R) in the mammalian twitch muscle comprises the following major events: (1) initiation and propagation of an action potential along the sarcolemma and transverse (T)-tubular system; (2) detection of the T-system depolarization signal and signal transmission from the T-tubule to the sarcoplasmic reticulum (SR) membrane; (3) Ca2+ release from the SR; (4) transient rise of myoplasmic [Ca2+]; (5) transient activation of the Ca2+-regulatory system and of the contractile apparatus; (6) Ca2+ reuptake by the SR Ca2+ pump and Ca2+ binding to myoplasmic sites. There are many steps in the E-C-R cycle which can be seen as potential sites for muscle fatigue and this review explores how structural and functional differences between the fast- and slow-twitch fibres with respect to the E-C-R cycle events can explain to a great extent differences in their fatiguability profiles.

Animals↗
PubMed1996

Effects of ammonium ions on the depolarization-induced and direct activation of the contractile apparatus in mechanically skinned fast-twitch skeletal muscle fibres of the rat.

Mechanically skinned fibre preparations from the extensor digitorum longus muscle of the rat were used to test whether a rise in myoplasmic [NH+4] in the range 2-10 mM interferes with the mechanism of excitation-contraction coupling in fast-twitch mammalian muscle. Under our conditions (pH 7.10, Mg2+ 1 mM, temperature 23 degrees C), [NH+4] up to 10 mM had little effect on the Ca(+)-activated force and on the peak of the t-system depolarization-induced force response. However, the duration of the depolarization-induced force response was decreased significantly at [NH+4] > or = 2 mM. From these data we conclude that the intracellular accumulation of NH+4 is not likely to play a major role in fatigue. Nevertheless, the build up of NH+4 during fatigue, may have a significant inhibitory effect on the force output by decreasing the duration of the t-system depolarization-induced activation of the contractile apparatus.

Animals↗
PubMed1994

Single-fiber study of contractile and biochemical properties of skeletal muscles in streptozotocin-induced diabetic rats.

Mechanically skinned, single muscle fibers, isometrically activated in pH and Ca2+ (Sr2+) buffered solutions were used to examine the function of the contractile apparatus in slow- and fast-twitch fibers from soleus (SOL, predominantly slow-twitch) and extensor digitorum longus (EDL, predominantly fast-twitch) muscles of streptozotocin (STZ)-induced diabetic rats and age-matched controls. Three and 14 days after STZ administration, the contractile properties of muscle fibers from diabetic rats did not differ significantly from those of controls with respect to several mechanical parameters, such as maximum Ca-activated tension, activation threshold, and sensitivity to Ca2+ and Sr2+. In contrast, 28 days after STZ administration, 37.5% of the fast-twitch EDL fibers developed maximum activated tensions (77.1 +/- 10.4 kN/m2), which were significantly lower than those developed by controls (244.0 +/- 14.3 kN/m2), and the slow-twitch SOL fibers displayed a significantly higher sensitivity to Ca2+ (and Sr2+) than the controls. All fibers from diabetic rats, including the low-tension EDL fibers and higher Ca sensitivity SOL fibers displayed control-like electrophoretic profiles of the major myofibrillar proteins. Taken together with data from earlier studies on the effects of long-term diabetes on whole skeletal muscle contractility, these results strongly suggest that 1) the decrease in tetanic tension output of EDL muscles induced by diabetes is caused mainly by direct effects of the diabetic condition on the contractile/regulatory system of a subpopulation of fast-twitch fibers, which develop little force, and 2) the diabetes-induced slowing of twitch times of SOL muscles is caused in part by the increased sensitivity to Ca2+ of the contractile apparatus in the slow-twitch fibers.

Analysis of Variance↗
PubMed1993

Endogenous MLC2 phosphorylation and Ca(2+)-activated force in mechanically skinned skeletal muscle fibres of the rat.

A method has been developed for measuring the level of phosphorylation of myosin regulatory light chains (MLC2) by the endogenous myosin light chain kinase in mechanically skinned skeletal muscle fibres. The method was used to characterize the endogeous MLC2 phosphorylation capacity of single fast-twitch fibres from the rat and to investigate the relationship between the endogenous MLC2 phosphorylation and the Ca(2+)-activated force. The results show that (1) about 50% of MLC2 were 32P-phosphorylated after activation of the skinned fibre preparation by 30 microM [Ca2+] for longer than 30 s, but that there was variability between fibres; (2) most of the endogenous phosphorylating system diffused out of the skinned fibre preparation after 5 min exposure to an aqueous solution; (3) the MLC2 phosphorylation by the endogenous phosphorylating system followed with a delay of the order of 1-2 s after the sudden rise in [Ca2+] from below 10 nM to 30 microM; and (4) the sensitivity of the contractile apparatus to Ca2+ was markedly increased when the MLC2 were phosphorylated by the endogenous phosphorylating system following a rise in [Ca2+]. The Kd for MgATP of the endogenous MLC2 phosphorylating system was estimated to be less than 300 microM. These results unequivocally demonstrate that prolonged activation of the fast-twitch muscle fibre leads to increased Ca2+ sensitivity of the contractile apparatus and that mechanically skinned fibres can be successfully used to study the regulation of the endogenous MLC2 phosphorylation capacity at single muscle fibre level.

Adenosine Triphosphate↗
PubMed1991

A comparative study of microsomal and cytosolic S6 phosphatase activities in rat liver.

Spontaneous S6 phosphatase activities dephosphorylating Ser(P)-235 and Ser(P)-236 of the ribosomal protein S6 were measured and compared in microsomes and cytosol of rat liver. The substrate used, small (40S) ribosomal subunits 32P-labelled in vitro by protein kinase A, contained phosphorylated S6 (mainly in the diphosphorylated form) and some minor phosphorylated species. The microsomal and cytosolic S6 phosphatase activities displayed a number of distinct properties. The microsomal activity, representing ca 20% of the S6 phosphatase activity in the post-mitochondrial supernatant, was mainly due to a type-1 phosphatase and dephosphorylated only S6. The remaining post-mitochondrial S6 phosphatase activity, which was fully recovered in the cytosol, and appeared to result from a combination of type-1 (43%) and type 2 (57%) phosphatases, acted on S6 as well as on the minor phosphorylated species. The microsomal activity was 50% inhibited by MgCl2 (10 mM) and was stimulated at least 4.3 fold by MnCl2 (1 mM), while the cytosolic activity was inhibited only 18% by Mg2+ (10 mM) and was increased 2.2 fold by Mn2+ (1 mM). The microsomal activity was increased 10% (P less than 0.06) by lower doses of insulin (25 U/Kg) and 14% (P less than 0.05) by vanadate, but was not significantly (P greater than 0.10) affected by larger doses of insulin (100 U/kg), hepatectomy or cycloheximide. By comparison the cytosolic S6 phosphatase activity was unresponsive to insulin and vanadate, but was decreased 14% and 17% (P less than 0.05) by hepatectomy and cycloheximide.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1991

A comparative study of the microsomal S6 phosphatase and phosphorylase phosphatase activities in rat liver.

Rat liver microsomes contain type-1 S6 phosphatase (acting on the serine residues phosphorylated by protein kinase A) and type-1 phosphorylase phosphatase activities. The main aim of this study has been to characterize the microsomal S6 phosphatase activity and to compare its properties with those of the phosphorylase phosphatase activity in the same microsomal preparation. The specific activities of both microsomal S6 phosphatase and phosphorylase phosphatase were 1.6- to 1.7-fold higher in the smooth endoplasmic reticulum than in the rough sarcoplasmic reticulum. Both phosphatase activities were inhibited to a similar extent by MgCl2 (10 mM) and NaF (22 mM), were completely suppressed by glycerophosphate (80 mM) and ZnCl2(10 mM), and were stimulated by MnCl2(1 mM). When analyzed by gel filtration on Sephadex G-100 superfine, both phosphatase activities eluted as broad peaks, stretching from the void volume to 45-60 kDa. The microsomal S6 phosphatase and phosphorylase phosphatase activities also displayed the following distinct characteristics: (a) Mn2+ stimulated the S6 phosphatase activity 2.9-fold more than the phosphorylase phosphatase activity, (b) limited trypsin digestion of microsomal preparations increased the phosphorylase phosphatase activity by 1.5- to 2-fold, but decreased the S6 phosphatase activity by 50%, (c) a synthetic peptide analog of S6 (S6229-239) (200 microM), which did not act as a substrate for the microsomal S6 phosphatase and did not affect its activity, inhibited the microsomal phosphorylase phosphatase activity by about 50%, and (d) the elution profile of the phosphorylase phosphatase activity was markedly broader than that of the S6 phosphatase activity. A series of in vivo studies showed that streptozotocin-diabetes and insulin replacement therapy as well as ip injection of insulin or vanadate, which modified the microsomal S6 phosphatase activity, had no statistically significant effects on the microsomal phosphorylase phosphatase activity. Taken together, these results suggest that the microsomal S6 phosphatase and phosphorylase phosphatase activities are due to two distinct enzyme populations.

Animals↗
PubMed1981

Visual communication and the content and style of conversation.

Previous research suggests that visual communication plays a number of important roles in social interaction. In particular, it appears to influence the content of what people say in discussions, the style of their speech, and the outcomes they reach. However, the findings are based exclusively on comparisons between face-to-face conversations and audio conversations, in which subjects sit in separate rooms and speak over a microphone-headphone intercom which precludes visual communication. Interpretation is difficult, because visual communication is confounded with physical presence, which itself makes available certain cues denied to audio subjects. The purpose of this paper is to report two experiments in which the variables were separated and content and style were re-examined. The first made use of blind subjects, and again compared the face-to-face and audio conditions. The second returned to sighted subjects, and examined four experimental conditions: face-to-face; audio; a curtain condition in which subjects sat in the same room but without visual communication; and a video condition in which they sat in separate rooms and communicated over a television link. Neither visual communication nor physical presence proved to be critical variable. Instead, the two sources of cues combined, such that content and style were influenced by the aggregate of available cues. The more cueless the settings, the more task-oriented, depersonalized and unspontaneous the conversation. The findings also suggested that the primary effect of cuelessness is to influence verbal content, and that its influence on both style and outcome occurs indirectly, through the mediation of content.

Adult↗