Pig Skeletal Muscle Research Articles

Ontogeny of epidermal growth factor (EGF), EGF receptor (EGFR) and basic fibroblast growth factor (bFGF) mRNA levels in pancreas, liver, kidney, and skeletal muscle of pig

Epidermal growth factor (EGF), EGF receptor (EGFR), and basic fibroblast growth factor (bFGF) messenger RNA (mRNA) levels were examined by Northern blot analysis in four tissues (pancreas, liver, kidney, and skeletal muscle) of pig from fetal 90 d to postnatal 180 d of age. The present study shows for the first time that EGF mRNA increased with advancing age in the kidney and skeletal muscle of pig. A high level of EGF mRNA was observed in the kidney compared with the liver and skeletal muscle. In the pancreas, high levels of EGF mRNA were found in fetuses and newborns and were low in older pigs. Pancreatic EGFR mRNA level parallelled its EGF mRNA, whereas in the kidney and skeletal muscle, patterns of EGFR mRNA were reversed to their EGF mRNA levels. In the liver, EGFR mRNA was abundant but EGF mRNA was undetected. In the pancreas and skeletal muscle, the highest levels of bFGF mRNA were found in fetuses of 90 d of age and then decreased with advancing age. In the liver and kidney, there were no major changes in bFGF mRNA levels during the examined developmental periods. These results show that EGF, EGFR, and bFGF mRNA levels are developmentally and tissue specifically regulated in pig. In the pancreas, mRNA levels of EGF, EGFR and bFGF were high in fetal and neonatal life and low thereafter. In the kidney and skeletal muscle, EGF mRNA increased with advancing age. EGF may play a role in muscle growth and maintenance in growing pigs during the later stage of development.

Expressional downregulation of neuronal-type NO synthase I in guinea pig skeletal muscle in response to bacterial lipopolysaccharide

We have investigated the expression of neuronal-type NO synthase I (NOS I) and inducible-type NOS II in guinea pig skeletal muscle (diaphragm). Expression of NOS I mRNA and protein was highest in muscle of specific pathogen-free animals, lower in normally bred animals, and lowest in lipopolysaccharide (LPS)-treated animals. NOS II mRNA and protein levels were highest in muscle of LPS-treated animals. Elevated NOS activity in muscle from LPS-treated animals was less susceptible to the NOS I-selective inhibitor N G-nitro- l-arginine. Expressional downregulation of NOS I in sepsis may have implications for contractile function of skeletal muscle.

Inducible NO synthase II and neuronal NO synthase I are constitutively expressed in different structures of guinea pig skeletal muscle: implications for contractile function

The expression of NOS isoforms was studied in guinea pig skeletal muscle at the mRNA and protein level, and the effect of NO on contractile response was examined. Ribonuclease protection analyses demonstrated NOS I and NOS II mRNAs in diaphragm and gastrocnemius muscle. In Western blots, NOS I and NOS II immunoreactivities were found in the particulate but not the soluble fraction of skeletal muscle. NOS activity was found almost exclusively in the particulate fraction. About 50% of this activity was Ca2+ independent. In immunohistochemistry, the anti-NOS I antibody stained distinct membrane regions of muscle fibers. The most intense staining was seen in neuromuscular endplates identified by labeling with alpha-bungarotoxin. The anti-NOS II antibody labeled muscle fibers that contained alkali-labile myosin ATPase (type I fibers). NOS II was located to intracellular structures and was also seen in "specific pathogen-free" animals. Pretreatment of guinea pigs with bacterial lipopolysaccharide (LPS) markedly intensified NOS II staining. Significant NOS III immunoreactivity was detected only in vascular endothelium. In functional experiments, tetanic muscle contractions were induced in diaphragm and gastrocnemius muscle by electrical stimulation of the innervating nerves. Pretreatment of guinea pigs with LPS or addition of S-nitroso-N-acetyl-D,L-penicillamine to the organ bath markedly decreased tetanic contractions. N(G)-nitro-L-arginine, on the other hand, increased contractile force and reversed the effect of LPS. Our data indicate that NOS II and NOS I are expressed in different structures of skeletal muscle and are involved in the regulation of contractile response.

Calmodulin sensitivity of the sarcoplasmic reticulum ryanodine receptor from normal and malignant-hyperthermia-susceptible muscle.

Ca2+ release from sarcoplasmic reticulum (SR) of malignant-hyperthermia-susceptible (MHS) muscle is hypersensitive to Ca2+ and caffeine. To determine if an abnormal calmodulin (CaM) regulation of the SR Ca(2+)-release-channel-ryanodine-receptor complex (RYR1) contributes to this hypersensitivity, we investigated the effect of CaM on high-affinity [3H]ryanodine binding to isolated SR vesicles from normal and MHS pig skeletal muscle. CaM modulated [3H]ryanodine binding in a Ca(2+)-dependent manner. In the presence of maximally activating Ca2+ concentrations, CaM inhibited [3H]ryanodine binding with no differences between normal and MHS vesicles. In the absence of Ca2+, however, CaM activated [3H]ryanodine binding with a 2-fold-higher potency in MHS vesicles. Significant differences between normal and MHS tissue were observed for CaM concentrations between 50 nM and 10 microM. A polyclonal antibody raised against the central region of RYR1 specifically inhibited this activating effect of CaM without affecting the inhibition by CaM. This indicates that the central region of RYR1 is a potential binding domain for CaM in the absence of Ca2+. It is suggested that in vivo an enhanced CaM sensitivity of RYR1 might contribute to the abnormal high release of Ca2+ from the SR of MHS muscle.

Bradykinin B2 receptors on skeletal muscle are coupled to inositol 1,4,5-trisphosphate formation.

To determine the presence of bradykinin receptors in skeletal muscle, we examined in both displacement and saturation studies the binding of [125I-Tyr8]bradykinin or [3H]bradykinin in three types of skeletal muscle preparations: membrane fractions from guinea pig hindlimb quadriceps, dog semimembranosus and semitendinosus muscles, and L8 rat skeletal muscle myoblasts. Scatchard analysis of [125I-Tyr8]bradykinin x bradykinin competition binding demonstrated specific bradykinin binding of 4.9 and 3.2 fmol/mg protein in dog and guinea pig skeletal muscle preparations, respectively. Unlabeled bradykinin specifically displaced [125I-Tyr8]bradykinin with IC50 values of 36.5 +/- 6 and 118.0 +/- 16.0 pmol/l from dog and guinea pig muscle membranes, respectively. The B2 bradykinin receptor antagonist HOE 140 and the B1 bradykinin receptor antagonist des-Arg9[Leu8]bradykinin displaced the binding of [3H]bradykinin from dog membranes with IC50 values of 0.38 and 217.3 nmol/l, respectively, suggesting that bradykinin binds to a B2-type receptor. In addition, unlabeled bradykinin competed with [3H]bradykinin for binding to dog skeletal muscle membrane preparations in a biphasic manner. To assess whether this represents multiple bradykinin receptor subtypes present in skeletal muscle homogenates or several affinity states of a single binding site, we examined bradykinin receptors on a pure skeletal muscle system, the L8 neonatal rat skeletal muscle myoblast cell line. These myoblasts also contain specific [3H]bradykinin-binding sites with a Bmax of 271 fmol/mg protein and a Kd of 0.83 nmol/l. Competitive agonist binding curves were biphasic (high-affinity IC50 = 3.9 pmol/l, low-affinity IC50 = 22.6 nmol/l) in the absence of guanosine 5'-O-(3-thio-trisphosphate) (GTP gamma S); they shifted to a model of one affinity (8.1 nmol/l) in the presence of GTP gamma S. Because the enzyme neutral endopeptidase 24.11 is an important kininase in skeletal muscle, we examined the effect of the neutral endopeptidase inhibitor phosphoramidon on the binding of bradykinin to dog skeletal muscle membranes. We found that phosphoramidon decreased the apparent Bmax from 7.3 to 5.8 fmol/mg protein. In addition, in this cell line we investigated the action of bradykinin on phosphoinositide hydrolysis. Inositol 1,4,5-trisphosphate (IP3) was measured with a radioreceptor assay. Bradykinin (0.1 nmol/l to 1 mumol/l) induced IP3 formation in a dose-dependent manner (EC50 = 1.42 nmol/l) from a basal level of 72.8 +/- 16 pmol/mg protein to 433 +/- 35.5 at the highest (1 mumol/l) concentration. We conclude that bradykinin B2 receptors are expressed in skeletal muscle. Phosphoinositide hydrolysis upon stimulation of this receptor is an indicator of intracellular signal transduction. Part of the bradykinin binding in skeletal muscle is due to interaction with the enzyme neutral endopeptidase.

Acute ischaemic preconditioning protects against skeletal muscle infarction in the pig

The aims were to investigate the efficacy of acute ischaemic preconditioning for protection of skeletal muscles against infarction and its effect on muscle blood flow and ischaemic muscle metabolism. The efficacy of preconditioning was tested by subjecting pig latissimus dorsi and gracilis muscles to different numbers and durations of ischaemia/reperfusion cycles before 4 h of global ischaemia. Infarction was assessed at 48 h of reperfusion, using nitroblue tetrazolium dye. Blood flow in the latissimus dorsi was measured at the end of preconditioning and 1.5 and 3.0 h of reperfusion, using the radioactive microsphere (15 microns) technique. Muscle biopsies were taken from the latissimus dorsi before ischaemia, at the end of 2 and 4 h of ischaemia, and 1.5 h of reperfusion. At least three cycles of 10 min ischaemia and 10 min reperfusion were required for preconditioning of latissimus dorsi and gracilis muscles for protection against infarction. Preconditioning reduced the total infarct size by 44% and 62% in latissimus dorsi and gracilis muscles, respectively. Preconditioning did not affect preischaemia muscle blood flow but it reduced the muscle content (preischaemia reserve) of phosphocreatine and ATP and the muscle energy charge potential (ECP) by 13.5%*, 27.5%*, and 8%* (*P < 0.05), respectively. In spite of a lower preischaemia reserve of phosphocreatine and ATP, the muscle contents of phosphocreatine and ATP and muscle ECP were maintained higher and the lactate lower (*P < or = 0.05) in the preconditioned than in the non-preconditioned (control) muscles at the end of 4 h of ischaemia [phosphocreatine 8.0(SEM 0.4) v 3.2(0.3)*; ATP 9.8(0.7) v 7.8(0.3); ECP 0.72(0.02) v 0.66(0.01)*; lactate 115.4(8.6) v 160.5(11.8)* mumol.g-1 dry muscle]. The level of ATP and ECP also remained significantly higher and the level of lactate significantly lower in the preconditioned than in the non-preconditioned latissimus dorsi muscles at 1.5 h of reperfusion. Hyperaemia was seen in the preconditioned latissimus dorsi muscles at 1.5 h of reperfusion and it subsided by the end of 3h of reperfusion. The protective effect of preconditioning can be induced in pig skeletal muscle but at a higher threshold than reported previously in pig cardiac muscle (one cycle). Preconditioning of pig skeletal muscle is associated with a lower energy metabolism during sustained ischaemia. At the present time, it is not known if this energy sparing effect is a major mechanism of ischaemic preconditioning against infarction in skeletal muscles.

Pattern of muscle fiber type formation in the pig.

The aim of this study was to analyze the temporal sequence of expression of the myosin isoforms in the populations of muscle fibers in the pig and to bring more information on the origin of the strikingly different pattern of fiber composition and distribution between the deep medial red (oxido-glycolytic) and superficial white (glycolytic) portions of semitendinosus (ST) muscle. Muscle samples were taken from 49-, 55-, 75-, 90-, 103-, and 113- (birth) day-old fetuses, from 6-, 11-, 21-, 35-, 50-, and 80-day-old piglets, and from a 3-year-old pig. Our results confirm the sequential formation of primary and secondary generation fibers. The use of immunohistochemistry and heterologous monoclonal antibodies (mAb) directed against specific myosin heavy chain (MHC) isoforms revealed a different pattern of gene expression between the two portions of the ST muscle for both generations of fibers. By 75 days of gestation (dg), primary myotubes from the deep medial portion stained positively for the anti-slow MHC mAb and negatively for the adult anti-fast MHC, whereas the opposite was observed in the superficial portion. Secondary fibers never expressed slow MHC until late gestation. Instead, they expressed an adult fast MHC isoform as soon as they formed in the deep medial portion and later on in the superficial portion. From late gestation to the first 3 postnatal weeks, slow MHC began to be expressed in a subpopulation of secondary fibers. These fibers were in the direct vicinity of primary myotubes in the deep medial portion, whereas their location could not be established in the superficial portion. The remaining secondary fibers matured to type IIA in the direct vicinity of these type I fibers and to type IIB at the periphery of the islets. In both portions of the muscle, a subpopulation of secondary fibers, the first ones to express slow MHC, also transitorily expressed a MHC that was identical or closely related to the alpha-cardiac MHC during the early postnatal period. A third generation of small diameter fibers was observed shortly after birth and reacted with the anti-fetal MHC mAb; their destiny remains to be established.(ABSTRACT TRUNCATED AT 400 WORDS)

Acute pharmacologic preconditioning as a new concept and alternative approach for prevention of skeletal muscle ischemic necrosis

The phenomenon of ischemic preconditioning for augmentation of ischemic tolerance has been well documented in the myocardium of common laboratory animals and human cardiomyocytes. The cellular mechanism of ischemic preconditioning is unclear, but adenosine is most likely the mediator in the rabbit, dog, pig and human. We have demonstrated recently that the protective effect of ischemic preconditioning and adenosine against ischemic injury can also be induced in pig skeletal muscles [116]. We speculate that adenosine is a potential treatment modality for prevention of skeletal muscle ischemic injury in vascular and musculoskeletal reconstructive surgery and in muscle and limb procurement for transplantation in the future. It is hoped that this review will stimulate workers at other laboratories to join the adventure in exploring the cellular mechanism and clinical application of adenosine for augmentation of skeletal muscle ischemic tolerance.

Fibre type differentiation during postnatal development of miniature pig skeletal muscles

Histochemical differentiation of 12 skeletal muscles with a different fibre type composition was studied in miniature pigs from 80 d of gestation to 1 year of age. Two fetal myofibre types were distinguished at 100 d of gestation by the mATPase reaction after acid preincubation. The staining for oxidative enzyme activities showed no conspicuous differences between fibres up to the 6th day after birth. Starting from this age it was possible to distinguish 3 fibre categories: SO (slow-twitch oxidative); FOG (fast-twitch oxidative-glycolytic); and FG (fast-twitch glycolytic). A characteristic cluster distribution of the 3 fibre types was observed in all studied muscles with the exception of the masseter muscle which consisted only of the type SO and FOG fibres with a mosaic arrangement. The frequencies of both SO and FG fibre types increased and the proportion of type FOG fibres decreased during the postnatal period. These changes could be explained by developmental transformations among the individual fibre types. The type FOG fibres converted preferably to the fibre type (SO or FG) that prevailed in the muscles of adult animals.

Ion selectivity of porcine skeletal muscle Ca2+ release channels is unaffected by the Arg615 to Cys615 mutation

The Arg615 to Cys615 mutation of the sarcoplasmic reticulum (SR) Ca2+ release channel of malignant hyperthermia susceptible (MHS) pigs results in a decreased sensitivity of the channel to inhibitory Ca2+ concentrations. To investigate whether this mutation also affects the ion selectivity filter of the channel, the monovalent cation conductances and ion permeability ratios of single Ca2+ release channels incorporated into planar lipid bilayers were compared. Monovalent cation conductances in symmetrical solutions were: Li+, 183 pS +/- 3 (n = 21); Na+, 474 pS +/- 6 (n = 29); K+, 771 pS +/- 7 (n = 29); Rb+, 502 pS +/- 10 (n = 22); and Cs+, 527 pS +/- 5 (n = 16). The single-channel conductances of MHS and normal Ca2+ release channel were not significantly different for any of the monovalent cations tested. Permeability ratios measured under biionic conditions had the permeability sequence Ca2+ >> Li+ > Na+ > K+ > or Rb+ > Cs+, with no significant difference noted between MHS and normal channels. This systematic examination of the conduction properties of the pig skeletal muscle Ca2+ release channel indicated a higher Ca2+ selectivity (PCa2+:Pk+ approximately 15.5) than the sixfold Ca2+ selectivity previously reported for rabbit skeletal (Smith et al., 1988) or sheep cardiac muscle (Tinker et al., 1992) Ca2+ release channels. These results also indicate that although Ca2+ regulation of Ca2+ release channel activity is altered, the Arg615 to Cys615 mutation of the porcine Ca2+ release channel does not affect the conductance or ion selectivity properties of the channel.

The Superfamily of Receptors for Growth Hormone, Prolactin, Erythropoietin, and Cytokines

Postnatal developmental pretranslational regulation of skeletal muscle alpha-actin gene expression was investigated. Northern blot analysis of skeletal muscle alpha-actin and beta-tubulin mRNA from 1- and 28-day-old pigs indicated that there are developmental increases in alpha-actin mRNA abundance (P < 0.03) and no significant changes in beta-tubulin mRNA (P > 0.1). A system for isolation of nuclei from porcine skeletal muscle and for transcriptional "run-on" analysis was established in order to investigate the regulatory mechanism of developmental changes in porcine skeletal muscle protein. Skeletal muscle nuclei were isolated from longissimus dorsi (LD) muscle of 1- and 28-day-old pigs by adapting a method to isolate nuclei from cardiac muscle. Results from a [3H]-UTP incorporation assay indicate that these nuclei preparations have the capacity to synthesize RNA and attain maximum incorporation after 40-45 min at 26 degrees C. Messenger RNA syntheses from skeletal muscle nuclei from 1- and 28-day-old pigs were not significantly different (P > 0.25). All nascent tRNA, rRNA, and mRNA in the nuclei were elongated since [3H]-UTP incorporation was reduced after addition of 0.05 micrograms/ml alpha-amanitin to the transcription mixture. Transcription "run-on" assay results indicated that more (P < 0.02) skeletal muscle alpha-actin pre-mRNA was synthesized in the 28-day-old pig skeletal muscle nuclei than in the 1-day-old pig skeletal muscle nuclei. These results indicate that the relative increase in skeletal muscle alpha-actin mRNA observed in the older animals was due, at least in part, to an increase in the transcriptional activity of the skeletal muscle alpha-actin gene.

Calcium-dependent block of ryanodine receptor channel of swine skeletal muscle by direct binding of calmodulin

The interaction of the Ca 2+ binding protein calmodulin (CaM) with the ryanodine receptor of the sarcoplasmic reticulum (SR) of pig skeletal muscle was investigated by [ 3H]-ryanodine binding, planar bilayer recordings, and rapid filtration of 45Ca 2+-loaded SR. Inhibition of [ 3H]-ryanodine binding by CaM was phosphorylation-independent, had an IC 50 of approximately 0.1 μM and was optimal at 10 μM Ca 2+. CaM also inhibited [ 3H]-ryanodine binding to CHAPS-solubilized and purified ryanodine receptors, suggesting a direct CaM-ryanodine receptor interaction. In single channel recordings, CaM blocked Ca 2+ release channels in a Ca 2+-dependent manner by decreasing the number of open events per unit time without affecting the mean open time or unitary channel conductance. Rapid filtration of 45Ca 2+ passively loaded into SR vesicles showed that CaM blocked Ca 2+ release within milliseconds of exposure of SR to a Ca 2+ release medium containing 10 μM CaM. In controls, an increase in extravesicular Ca 2+ from 7 nM to 10 μM resulted in a release of 47 ± 10% of the 45Ca 2+ in 20 ms. CaM reduced the release to 23 ± 12% in the same period. These results are compatible with a direct mechanism of Ca 2+ release channel blockade by CaM and suggest that CaM could play a significant role in the inactivation of SR Ca 2+ release during excitation-contraction coupling.

Cloning and characterization of pig muscle cDNAs by an expressed sequence tag approach

Abstract To provide additional unique marker sequences for genome mapping, we have cloned and partially sequenced 14 pig skeletal muscle cDNAS, representing 11 independent genes. Random selection from an adult skeletal muscle cDNA library, coupled with dot blot hybridization of the cDNA clones with complex probes representing muscle and non‐muscle gene expression, was used to identify putative muscle‐specific cDNAs. These cDNAs were then partially sequenced and the resulting primary structural information was used to screen the Genbank/European Molecular Biology Laboratory (EMBL) and Protein Information Resource (PIR) databases. Pig cDNAs with significant similarity to α‐actin, α7‐integrin, α‐actinin2, myosin binding protein H, and myosin light chain kinase were identified. Northern analysis of α‐actinin2 showed the expression pattern of this pig gene closely matched that reported for human α‐actinin2. Six cDNAs had no significant database match indicating that these genes have not been sequenced in other...

An indirect immunofluorescence procedure for staining the same cryosection with two mouse monoclonal primary antibodies.

Indirect immunofluorescence procedures reported thus far are not effective at localizing two antigens in the same preparation when both primary antibodies are raised in the same species. In this case, the secondary antibodies can crossreact with both primary antibodies. We report here a protocol in which mouse monoclonal antibodies (MAb) specific for actin and myosin were used sequentially to stain the same frozen section of guinea pig skeletal muscle. The myosin-specific mAb was applied first and was localized with a rabbit anti-mouse IgG-rhodamine secondary antibody. The sections were then "blocked" with a non-binding mouse MAb and unconjugated goat anti-mouse IgG F(ab) fragments. The actin-specific mAb was then applied and localized with a rabbit anti-mouse IgG-fluorescein secondary antibody. Laser scanning confocal microscopy and image analysis demonstrated that the I-bands, the A-bands, and the H-bands of each sarcomere were clearly identifiable by this approach. This protocol is not limited to use with mouse MAb but can be easily modified to permit indirect immunolocalization of two antigens in the same sample using any pair of same-species primary antibodies.

Activation of the Ca2+ release channel of skeletal muscle sarcoplasmic reticulum by palmitoyl carnitine

Studies of [3H]ryanodine binding, 45Ca2+ efflux, and single channel recordings in planar bilayers indicated that the fatty acid metabolite palmitoyl carnitine produced a direct stimulation of the Ca2+ release channel (ryanodine receptor) of rabbit and pig skeletal muscle junctional sarcoplasmic reticulum. At a concentration of 50 microM, palmitoyl carnitine (a) stimulated [3H]ryanodine binding 1.6-fold in a competitive manner at all pCa in the range 6 to 3; (b) released approximately 65% (30 nmol) of passively loaded 45Ca2+/mg protein; and (c) increased 7-fold the open probability of Ca2+ release channels incorporated into planar bilayers. Neither carnitine nor palmitic acid could reproduce the effect of palmitoyl carnitine on [3H]ryanodine binding, 45Ca2+ release, or channel open probability. 45Ca2+ release was induced by several long-chain acyl carnitines (C14, C16, C18) and acyl coenzyme A derivatives (C12, C14, C16), but not by the short-chain derivative C8 or by free saturated fatty acids of chain length C8 to C18, at room temperature or 36 degrees C. This newly identified interaction of esterified fatty acids and ryanodine receptors may represent a pathway by which metabolism of skeletal muscle could influence intracellular Ca2+ and may be responsible for the pathophysiology of disorders of beta-oxidation such as carnitine palmitoyl transferase II deficiency.

Analysis of lipid composition of sarcoplasmic reticulum membranes from normal and malignant hyperthermic pig skeletal muscle.

In search of a general membrane defect hypothesis for malignant hyperthermia syndrome, we analysed the lipid profiles of heavy sarcoplasmic reticulum membranes isolated from normal and malignant hyperthermia longissimus dorsi pig muscle. Malignant hyperthermia susceptibility was assessed by halothane challenge of pigs. Sarcoplasmic reticulum membranes from malignant hyperthermia susceptible pigs differed significantly from control ones in the cholesterol content and phosphatidylethanolamine/phosphatidylcholine ratio; both were higher in former membranes. These latter lipid modifications were in agreement with the significant increase of their bulk lipid viscosity, as evidenced by an increase of diphenyl hexatriene fluorescence anisotropy. The increased level of phosphatidylethanolamine associated with the decreased content of phosphatidylcholine in malignant hyperthermic membranes was shown to be a potential consequence of depressed activities of both phospholipid N-methyltransferase I and II activities. Finally, the distribution of fatty acids in these particular phospholipids showed no change in phosphatidylcholine molecules, whereas the percentage of arachidonate and stearate in the phosphatidylethanolamine species were respectively higher and lower in malignant hyperthermic membranes. These differences in major phospholipids content and the enrichment of a metabolically important fatty acyl chains in malignant hyperthermia sarcoplasmic reticulum membranes strongly suggest that the lipid metabolism may contribute to the molecular mechanism of malignant hyperthermia syndrome.

Ontogenic study of Expression of IGF-I and Gh-Receptor (GHR) mRNA in pig Liver and Skeletal Muscle.

Many of the effects of growth hormone are now thought to be mediated via the stimulation of insulin-like growth factor-I (IGF-I) production by many tissues, especially the liver, with this stimulation being dependent upon the presence of the GH-receptor (GHR). IGF-I gene expression occurs via alternative promoters giving rise to class 1 and 2 transcripts, of which class 2 is thought to be preferentially responsive to GH (Saunders, Dickson, Pell &amp; Gilmour, 1991).The effects of IGF-I include the stimulation of DNA synthesis (mitogenesis) and protein synthesis in most cell types, together with the differentiation of many cell types into mature tissue, including the differentiation of muscle cells into muscle fibres. Thus the GH/IGF-I axis has been found to play a major part in the control of animal growth. For this reason, we studied the age related changes in IGF-I and GHR mRNA expression in pig liver and skeletal muscle.

Determination of the relative abundance of skeletal muscle alpha actin mRNA in muscle of livestock species

Three market-weight animals of meat-producing livestock species were slaughtered to obtain porcine (barrows), bovine (steers), ovine (wethers), and avian (cockerels) tissue samples. The four tissues of interest were skeletal muscle, heart, smooth muscle (stomach or gizzard), and liver. Total RNA was isolated from each tissue and then hybridized to a human skeletal (sk)-alpha-actin [32P]cDNA probe using both dot blot and Northern blot hybridization. No hybridization was observed with RNA from liver or smooth muscle from any of the species, suggesting little or no hybridization to nonmuscle and smooth muscle beta- and gamma-actin isoforms. The human sk-alpha-actin probe hybridized to RNA from skeletal muscle of pigs, cattle, sheep, and chickens, although relative hybridization was 75% less with chicken RNA. The hybridization was limited specifically to a band at 1.6 kb (kilobases), the known length of sk-alpha-actin mRNA. Hybridization was observed with RNA from pig heart (1.6 kb) and the relative abundance was consistently 7 to 10% of that observed with porcine skeletal muscle, even as stringency conditions were increased. These results indicate that the human sk-alpha-actin probe can be used to determine alpha-actin mRNA expression in skeletal muscle for pigs, cattle, and sheep.

Confocal laser microscopy of dystrophin localization in guinea pig skeletal muscle fibers.

A confocal laser microscope was used to analyze the localization pattern of dystrophin along the sarcolemma in guinea pig skeletal muscle fibers. Hind leg muscles of the normal animals were freshly dissected and frozen for cryostat sections, which were then stained with a monoclonal antidystrophin antibody. In confocal laser microscopy, immunofluorescence staining in relatively thick sections could be sharply imaged in thin optical sections. When longitudinal and transverse sections of muscle fibers were examined, the immunostaining of dystrophin was seen as linearly aligned fluorescent dots or intermittent lines along the sarcolemma. In longitudinally cut muscle fibers, many fluorescent dots, but not all, corresponded to the sarcomere pattern, especially the I band. Sections cut tangential to the sarcolemma also showed a lattice-like pattern of longitudinal and transverse striations of fluorescent dots. Double staining for dystrophin and vinculin showed that the two proteins were not exactly colocalized. The end portions of muscle fibers were much more intensely stained with antidystrophin antibody than the central portions, following the contour of elaborate surface specializations at the myo-tendon junction. The staining pattern at the myo-tendon junction was also discontinuous. These confocal microscopic observations suggest that dystrophin may be localized in a nonuniform, discontinuous pattern along the sarcolemma and in some relationship with the underlying myofibrils.

Stimulation of protein synthesis in pig skeletal muscle by infusion of amino acids during constant insulin availability.

Incorporation of L-[1-13C]leucine into muscle protein and leg exchange of L-[15N]phenylalanine were used to assess the effects over 240 min of amino acid supply on leg protein turnover in anesthetized, overnight-fasted (Landrace x Great White) female pigs. In all pigs, plasma insulin and glucagon stability was ensured by infusion of somatostatin (8 micrograms.kg-1.h-1), insulin (6 mU.kg-1.h-1), and glucagon (72 ng.kg-1.h-1). Mixed amino acid infusion (260 mg.kg-1.h-1) caused a 2- to 2.5-fold elevation of arterial plasma phenylalanine and leucine; in a control group (no amino acid infusion), an increase in phenylalanine and leucine concentration was observed as a result of the hormone clamp. Plasma insulin and glucagon concentrations were steady and not significantly different between control and amino acid-infused groups during the final 240 min, but plasma glucose fell (P less than 0.05) in both groups (4.57 +/- 0.17 to 3.15 +/- 0.73 mM). Muscle protein synthetic rate (estimated from the change in L-[1-13C]leucine incorporation compared with labeling of [13C]leucyl-tRNA) was greater in amino acid-infused (0.076%/h) than in control (0.053%/h) pigs. In the control group, leg amino acid balance was negative (Phe alone, -10.2 +/- 9.4 nmol Phe.100 g-1.min-1; total amino acids, -0.27 +/- 1.04 micrograms amino N.100 g-1.min-1), but during amino acid infusion, balance was positive (Phe alone, +33.6 +/- 8.8 nmol Phe.100 g-1.min-1; total amino acids, +58.2 +/- 4.9 micrograms amino N.100 g-1.min-1).(ABSTRACT TRUNCATED AT 250 WORDS)