II. Beta-adrenergic receptors and catecholamine sensitive adenylate cyclase in the developing rat lung

β-Adrenergic receptors were identified in membrane fractions of fetal and postnatal rat lung with the β-adrenergic antagonist (?)?[³H] dihydroalprenolol, (?)?[³H] DHA. β-Receptor number (Bmax) increased 11-fold from day 18 of gestation to day 28 of postnatal life, 46±7 to 491±69 femtomole·mg^?1 protein. Neither the K_D, approximately 0.8nM for [³H]DHA, nor the β-adrenergic subtype changed with age. Classical agonists competed for the β-receptor with properties characteristic of β₂-adrenergic binding. Analysis of the inhibition of receptor binding by selective β-adrenergic agents demonstrated approximately 75% β₂ and 25% β₁ β-adrenergic subtypes in fetal rat lung membranes. The increase in β-adrenergic receptor during development was associated with adenylate cyclase activity which was sensitive to catecholamines at all ages studied, supporting the possible role of the β-adrenergic receptor system in the postnatal regulation of pulmonary function.     

Calcium binding protein changes of sarcoplasmic reticulum from rat denervated skeletal muscle

Two Ca²⁺ sequestering proteins were studied in fast-twitch (EDL) and slow-twitch (soleus) muscle sarcoplasmic reticulum (SR) as a function of denervation time. Ca²⁺-ATPase activity measured in SR fractions of normal soleus represented 5% of that measure in SR fractions of normal EDL. Denervation caused a severe decrease in activity only in fast-twich muscle. Ca²⁺-ATPase and calsequestrin contents were affected differently by denervation. In EDL SR, Ca²⁺-ATPase content decreased progressively, whereas in soleus SR, no variation was observed. Calsequestrin showed a slight increase in both muscles as a function of denervation time correlated with increased⁴⁵Ca-binding.These results indicate first that Ca²⁺-ATPase activity in EDL was under neural control, and that because of low Ca²⁺-ATPase activity and content in slow-twitch muscle no variation could be detected, and secondly that greater calsequestrin content might represent a relative increasing of heavy vesicles or decreasing of light vesicles as a function of denervation time in the whole SR fraction isolated in both types of muscles.     

Lpaatδ/Agpat4 deficiency impairs maximal force contractility in soleus and alters fibre type in extensor digitorum longus muscle

Lysophosphatidic acid acyltransferase (LPAAT) δ/acylglycerophosphate acyltransferase 4 is a mitochondrial enzyme and one of five homologues that catalyze the acyl-CoA-dependent synthesis of phosphatidic acid (PA) from lysophosphatidic acid. We studied skeletal muscle LPAATδ and found highest levels in soleus, a red oxidative fibre-type that is rich in mitochondria, and lower levels in extensor digitorum longus (EDL) (white glycolytic) and gastrocnemius (mixed fibre-type). Using Lpaatδ-deficient mice, we found no change in soleus or EDL mass, or in treadmill time-to-exhaustion compared to wildtype littermates. There was, however, a significant reduction in the proportion of type I and type IIA fibres in EDL but, surprisingly, not soleus, where these fibre-types predominate. Also unexpectedly, there was no impairment in force generation by EDL, but a significant reduction by soleus. Oxidative phosphorylation and activity of complexes I, I?+?II, III, and IV in soleus mitochondria was unchanged and therefore could not explain this effect. However, pyruvate dehydrogenase activity was significantly reduced in Lpaatδ^?/? soleus and EDL. Analysis of cellular lipids indicated no difference in soleus triacylglycerol, but specific elevations in soleus PA and phosphatidylethanolamine levels, likely due to a compensatory upregulation of Lpaatβ and Lpaatε in Lpaatδ^?/? mice. An anabolic effect for PA as an activator of skeletal muscle mTOR has been reported, but we found no change in serine 2448 phosphorylation, indicating reduced soleus force generation is unlikely due to the loss of mTOR activation by a specific pool of LPAATδ-derived PA. Our results identify an important role for LPAATδ in soleus and EDL.     

Adenylate cyclase system of human skeletal muscle: Subcellular distribution and general properties

The subcellular localization of adenylate cyclase was examined in human skeletal muscle. Three major subcellular membrane fractions, plasmalemma, sarcoplasmic reticulum and mitochondria, were characterized by membrane-marker biochemical studies, by dodecyl sulfate polycrylamide gel electrophoresis and by electron microscopy. About 60% of the adenylate cyclase of the homogenate was found in the plasmalemmal fraction and 10–14% in the sarcoplasmic reticulum and mitochondria. When the plasmalemmal preparation was subjected to discontinuous sucrose gradients, the distribution of adenylate cyclase in different subfractions closely paralleled that of (Na⁺ + K⁺)-ATPase. The highest specific activity was found in a fraction which setteled at the 0.6–0.8 M sucrose interface. The electron microscopic study of this fraction revealed the presence of flattened sacs of variable sizes and was devoid of mitochondrial and myofibrillar material. The electron microscopy of each fraction supported the biochemical studies with enzyme markers. The three major membrane fractions also contained a low K_m phosphodiesterase activity, the highest specific activity being associated with sarcoplasmic reticulum.The plasmalemmal adenylate cyclase was more sensitive to catecholamine stimulation than that associated with sarcoplasmic reticulum or mitochondria. The catecholamine-sensitive, but not the basal, enzyme was further stimulated by GTP. The plasmalemmal adenylate cyclase had typical Michaelis-Menten kinetics with respect to ATP and the apparent K_m for ATP was approx. 0.3. mM. The pH optimum for that enzyme was 7.5. The enzyme required Mg²⁺, and the concentration to achieve half-maximal stimulation was approx. 3 mM. Higher concentrations of Mg²⁺ (about 10 mM) were inhibitory. Solubilization of the plasmalemmal membrane fraction with Lubrol-PX resulted in preferential extraction of 106 000- and 40 000-dalton protein components. The solubilized adenylate cyclase lost its sensitivity for catecholamine stimulation, and the extent of fluoride stimulation was reduced to one-sixth of that of the intact membranes. It is concluded that the catalytically active and hormone-sensitive adenylate cyclase is predominantly localized in the surface membranes of the cells within skeletal muscle. (That “plasmalemmal” fraction is considered likely to contain, in addition to plasmalemma of muscle cells, plasmalemma of bloodvessel cells (endothelium, and perhaps smooth muscle) which may be responsible for a certain amount of the adenylate cyclase activity and other propertiesobserved in that fraction.)The method of preparation used in this study provides a convenient material for evaluating the catecholamine-adenylate cyclase interactions in human skeletal muscle.     

Time course in calpain activity and autolysis in slow and fast skeletal muscle during clenbuterol treatment

Calpains are Ca2+ cysteine proteases that have been proposed to be involved in the cytoskeletal remodeling and wasting of skeletal muscle. Cumulative evidence also suggests that β2-agonists can lead to skeletal muscle hypertrophy through a mechanism probably related to calcium-dependent proteolytic enzyme. The aim of our study was to monitor calpain activity as a function of clenbuterol treatment in both slow and fast phenotype rat muscles. For this purpose, for 21?days we followed the time course of the calpain activity and of the ubiquitous calpain 1 and 2 autolysis, as well as muscle remodeling in the extensor digitorum longus (EDL) and soleus muscles of male Wistar rats treated daily with clenbuterol (4?mg·kg-1). A slow to fast fiber shift was observed in both the EDL and soleus muscles after 9?days of treatment, while hypertrophy was observed only in EDL after 9?days of treatment. Soleus muscle but not EDL muscle underwent an early apoptonecrosis phase characterized by hematoxylin and eosin staining. Total calpain activity was increased in both the EDL and soleus muscles of rats treated with clenbuterol. Moreover, calpain 1 autolysis increased significantly after 14?days in the EDL, but not in the soleus. Calpain 2 autolysis increased significantly in both muscles 6 hours after the first clenbuterol injection, indicating that clenbuterol-induced calpain 2 autolysis occurred earlier than calpain 1 autolysis. Together, these data suggest a preferential involvement of calpain 2 autolysis compared with calpain 1 autolysis in the mechanisms underlying the clenbuterol-induced skeletal muscle remodeling.     

Tissue inhibitor of metalloproteinase‐2 (TIMP‐2) regulates neuromuscular junction development via a β1 integrin‐mediated mechanism

Extracellular matrix (ECM) molecules play critical roles in muscle function by participating in neuromuscular junction (NMJ) development and the establishment of stable, cytoskeleton‐associated adhesions required for muscle contraction. Matrix metalloproteinases (MMPs) are neutral endopeptidases that degrade all ECM components. While the role of MMPs and their inhibitors, the tissue inhibitor of metalloproteinases (TIMPs), has been investigated in many tissues, little is known about their role in muscle development and mature function. TIMP‐2 ^?/? mice display signs of muscle weakness. Here, we report that TIMP‐2 is expressed at the NMJ and its expression is greater in fast‐twitch (extensor digitorum longus, EDL) than slow‐twitch (soleus) muscle. EDL muscle mass is reduced in TIMP‐2^?/? mice without a concomitant change in fiber diameter or number. The TIMP‐2^?/? phenotype is not likely due to increased ECM proteolysis because net MMP activity is actually reduced in TIMP‐2^?/? muscle. Most strikingly, TIMP‐2 colocalizes with β1 integrin at costameres in the wild‐type EDL and β1 integrin expression is significantly reduced in TIMP‐2^?/? EDL. We propose that reduced β1 integrin in fast‐twitch muscle may be associated with destabilized ECM‐cytoskeletal interactions required for muscle contraction in TIMP‐2^?/? muscle; thus, explaining the muscle weakness. Given that fast‐twitch fibers are lost in muscular dystrophies and age‐related sarcopenia, if TIMP‐2 regulates mechanotransduction in an MMP‐independent manner it opens new potential therapeutic avenues. © 2006 Wiley Periodicals, Inc. J Neurobiol, 2006     

Effects of Pleiotrophin Overexpression on Mouse Skeletal Muscles in Normal Loading and in Actual and Simulated Microgravity

Pleiotrophin (PTN) is a widespread cytokine involved in bone formation, neurite outgrowth, and angiogenesis. In skeletal muscle, PTN is upregulated during myogenesis, post-synaptic induction, and regeneration after crushing, but little is known regarding its effects on muscle function. Here, we describe the effects of PTN on the slow-twitch soleus and fast-twitch extensor digitorum longus (EDL) muscles in mice over-expressing PTN under the control of a bone promoter. The mice were maintained in normal loading or disuse condition, induced by hindlimb unloading (HU) for 14 days. Effects of exposition to near-zero gravity during a 3-months spaceflight (SF) into the Mice Drawer System are also reported. In normal loading, PTN overexpression had no effect on muscle fiber cross-sectional area, but shifted soleus muscle toward a slower phenotype, as shown by an increased number of oxidative type 1 fibers, and increased gene expression of cytochrome c oxidase subunit IV and citrate synthase. The cytokine increased soleus and EDL capillary-to-fiber ratio. PTN overexpression did not prevent soleus muscle atrophy, slow-to-fast transition, and capillary regression induced by SF and HU. Nevertheless, PTN exerted various effects on sarcolemma ion channel expression/function and resting cytosolic Ca²⁺ concentration in soleus and EDL muscles, in normal loading and after HU. In conclusion, the results show very similar effects of HU and SF on mouse soleus muscle, including activation of specific gene programs. The EDL muscle is able to counterbalance this latter, probably by activating compensatory mechanisms. The numerous effects of PTN on muscle gene expression and functional parameters demonstrate the sensitivity of muscle fibers to the cytokine. Although little benefit was found in HU muscle disuse, PTN may emerge useful in various muscle diseases, because it exerts synergetic actions on muscle fibers and vessels, which could enforce oxidative metabolism and ameliorate muscle performance.     

Adenylate cyclase system of human skeletal muscle: Characteristics of catecholamine stimulation and nucleotide regulation

The subcellular localization of adenylate cyclase was examined in human skeletal muscle. Three major subcellular membrane fractions, plasmalemma, sarcoplasmic reticulum and mitochondria, were characterized by membrane-marker biochemical studies, by dodecyl sulfate polycrylamide gel electrophoresis and by electron microscopy. About 60% of the adenylate cyclase of the homogenate was found in the plasmalemmal fraction and 10–14% in the sarcoplasmic reticulum and mitochondria. When the plasmalemmal preparation was subjected to discontinuous sucrose gradients, the distribution of adenylate cyclase in different subfractions closely paralleled that of (Na⁺ + K⁺)-ATPase. The highest specific activity was found in a fraction which setteled at the 0.6–0.8 M sucrose interface. The electron microscopic study of this fraction revealed the presence of flattened sacs of variable sizes and was devoid of mitochondrial and myofibrillar material. The electron microscopy of each fraction supported the biochemical studies with enzyme markers. The three major membrane fractions also contained a low K_m phosphodiesterase activity, the highest specific activity being associated with sarcoplasmic reticulum.The plasmalemmal adenylate cyclase was more sensitive to catecholamine stimulation than that associated with sarcoplasmic reticulum or mitochondria. The catecholamine-sensitive, but not the basal, enzyme was further stimulated by GTP. The plasmalemmal adenylate cyclase had typical Michaelis-Menten kinetics with respect to ATP and the apparent K_m for ATP was approx. 0.3. mM. The pH optimum for that enzyme was 7.5. The enzyme required Mg²⁺, and the concentration to achieve half-maximal stimulation was approx. 3 mM. Higher concentrations of Mg²⁺ (about 10 mM) were inhibitory. Solubilization of the plasmalemmal membrane fraction with Lubrol-PX resulted in preferential extraction of 106 000- and 40 000-dalton protein components. The solubilized adenylate cyclase lost its sensitivity for catecholamine stimulation, and the extent of fluoride stimulation was reduced to one-sixth of that of the intact membranes. It is concluded that the catalytically active and hormone-sensitive adenylate cyclase is predominantly localized in the surface membranes of the cells within skeletal muscle. (That “plasmalemmal” fraction is considered likely to contain, in addition to plasmalemma of muscle cells, plasmalemma of bloodvessel cells (endothelium, and perhaps smooth muscle) which may be responsible for a certain amount of the adenylate cyclase activity and other propertiesobserved in that fraction.)The method of preparation used in this study provides a convenient material for evaluating the catecholamine-adenylate cyclase interactions in human skeletal muscle.     

Cholesterol modulation of β-adrenergic receptor characteristics

Cholesterol, a major structural component of plasma membranes, has a profound influence on cell surface receptor characteristics and on adenylate cyclase activity. β-Adrenergic receptor number, adenylate cyclase activity, and receptor-cyclase coupling were assessed in rat lung membranes following preincubation with cholesteryl hemisuccinate. β-Adrenergic receptor number increased by 50% without a change in antagonist affinity. However, β-adrenergic receptor affinity for isoproterenol increased 2-fold as a result of an increase in the affinity of the isoproterenol high-affinity binding site. This increase in agonist affinity did not potentiate hormone-stimulated adenylate cyclase activity, which decreased 3-fold following cholesterol incorporation. However, the ratio of isoproterenol to GTP-stimulated activity was unchanged with cholesterol. Stimulation distal to the receptor by GTP, NaF, GppNHp, Mn²⁺ and forskolin also demonstrated 50–80% reduced enzyme activity following cholesterol incorporation. These data suggest that membrane cholesterol incorporation decreases catalytic unit activity without affecting transduction of the hormone signal.     

Biochemical and histochemical changes in energy supply enzyme pattern of muscles of the rat during old age.

Senile muscles of the rat (28-36 months) show loss of overall activity of glycolytic and aerobic enzymes. However, there is a differential loss and shift of enzyme activity pattern in the three types of muscles. The extensor digitorum longus (EDL) and diaphragm show a decrease of ratios of glycolytic to aerobic-oxidative enzymes. This shift to a more oxidative type of metabolism is not observed in the soleus muscle. Decrease of enzyme activities is least marked in the diaphragm muscle. Biochemical analysis shows a trend to levelling out of metabolic differences between the different muscle types. This trend of 'dedifferentiation' is most marked when comparing EDL and soleus, least marked when comparing EDL and diaphragm muscle. The histochemical analysis shows a shift from the original mixed to a more uniform pattern of muscle fibres in the EDL and soleus muscle; this levelling-out of differences between enzymatic activities of different muscle fibres is not observed in the diaphragm muscle. Preferential atrophy and loss of ATPase activity in II muscle fibres in the soleus muscle and the occurrence of 'type grouping' are further characteristic features of senile muscle change. The findings show general (i.e. loss of enzyme activities) and differential trends of biochemical and histochemical enzyme changes in different types of muscles.     

Excitation-induced Ca(2+) influx in rat soleus and EDL muscle: mechanisms and effects on cellular integrity

In rat skeletal muscle, electrical stimulation increases Ca(2+) influx leading to progressive accumulation of calcium. Excitation-induced Ca(2+) influx in extensor digitorum longus (EDL; fast-twitch fibers) and soleus muscle (slow-twitch fibers) is compared. In EDL and soleus, stimulation at 40 Hz increased (45)Ca uptake 34- and 21-fold and (22)Na uptake 17- and 7-fold, respectively. These differences may be related to the measured 70% higher concentration of Na(+) channels in EDL. Repeated stimulation at 40 Hz elicited a delayed release of lactic acid dehydrogenase (LDH) from EDL (11-fold increase) and soleus (5-fold increase). Continuous stimulation at 1 Hz increased LDH release only from EDL (18-fold). This was associated with increased Ca(2+) content and was augmented at high extracellular Ca(2+) concentration ([Ca(2+)](o)) and suppressed at low [Ca(2+)](o). The data support the hypothesis that excitation-induced Ca(2+) influx is mediated in part by Na(+) channels and that the ensuing increase in intracellular Ca(2+) induces cellular damage. This is most pronounced in EDL, which may account for the repeated observation that prolonged exercise leads to preferential damage to fast-twitch fibers.     

In vitro characterization of skeletal muscle β-adrenergic receptors coupled to adenylate cyclase

[³H]Dihydroalprenolol, a potent ß-adrenergic antagonist, was used to identify the adenylate cyclase-coupled ß-adrenoceptors in isolated membranes of rat skeletal muscle. The receptor sites, as revealed [³H]dihydroalprenolol binding, were predominantly localized in plasmalemmal fraction. That skeletal muscle fraction may also contain the plasmalemma of other intramuscular cells, especially that of blood vessels. Hence, the [³H]dihydroalprenolol binding observed in that fraction may be due partly to its binding to the plasmalemma of blood vessels. Small but consistent binding was also observed in sarcoplasmic reticulum and mitochondria. The level of [³H]dihydroalprenolol binding in different subcellular fractions closely correlated with the level of adenylate cyclase present in those fractions.The binding of [³H]dihydroalprenolol to plasmalemma exhibited saturation kinetics. The binding was rapid, reaching equilibrium within 5 min, and it was readily dissociable. From the kinetics of binding, association (K₁) and dissociation (K₂) rate constants of 2.21 · M^? · min^?1 and 3.21 · 10^?1, respectively, were obtained. The dissociation constant (K_d) of 15 nM for [³H]dihydroalprenolol obtained from saturation binding data closely agreed with the (K_d) derived from the ratio of dissociation and association rate constants (K₂/K₁).Several β-adrenergic agents known to be active on intact skeletal muscle also competed for [³H]dihydroalprenolol binding sites in isolated plasmalemma with essentially similar selectivity and stereospecificity. Catecholamines competed for [³H]dihydroalprenolol binding sites with a potency of isoproterenol > epinephrine > norepinephrine. A similar order of potency was noted for catecholamines in the activation of adenylate cyclase. Effects of catecholamines were stereospecific, (?)-isomers being more than potent than (+)-isomers. Phenylephrine, an α-adrenergic agonist, showed no effect either on [³H]dihydroalprenolol binding or on adenylate cyclase. Known ß-adrenergic antagonists, propranolol and alprenolol, stereospecifically inhibited the [³H]dihydroalprenolol binding and the isoproterenol-stimulated adenylate cyclase. The (K_i) values for the antagonists determined from inhibition of [³H]dihydroalprenolol binding agreed closely with the (K_i) values obtained from the inhibition of adenylate cyclase. The data suggest that the binding of [³H]dihydroalprenolol in skeletal muscle membranes possess the characteristics of a substance binding to the ß-adrenergic receptor.     

Transgenic and tissue culture analyses of the muscle creatine kinase enhancer Trex control element in skeletal and cardiac muscle indicate differences in gene expression between muscle types

The muscle creatine kinase (MCK) gene is expressed at high levels only in differentiated skeletal and cardiac muscle. The activity of the cloned enhancer–promoter has previously been shown to be dependent on the Trex element which is specifically bound by a yet unidentified nuclear factor, TrexBF. We have further characterized the function of the Trex site by comparing wild-type and Trex-mutated MCK transgenes in five mouse skeletal muscles: quadriceps, extensor digitorum longus (EDL), soleus, diaphragm, and distal tongue, as well as in heart ventricular muscle. Several types of statistical analysis including analysis of variance (ANOVA) and rank sum tests were used to compare expression between muscle types and between constructs. Upon mutation of the Trex site, median transgene expression levels decreased 3- to 120-fold in the muscles examined, with statistically significant differences in all muscles except the EDL. Expression in the largely slow soleus muscle was more affected than in the EDL, and expression in the distal tongue and diaphragm muscles was affected more than in soleus. Median expression of the transgene in ventricle decreased about 18-fold upon Trex mutation. Transfections into neonatal rat myocardiocytes confirmed the importance of the Trex site for MCK enhancer activity in heart muscle, but the effect is larger in transgenic mice than in cultured cells.     

Cholinergic and adrenergic receptors on mouse cardiocytes in vitro

The effects of adrenergic and cholinergic receptor agonists and antagonists on single and clustered mouse cardiocytes in culture have been studied. Cardiocytes were obtained from mice, ranging in ages from 9 days in utero to 1 day postpartum, and were grown in culture for 2–14 days. Single isolated cells of every age tested possessed the ability to respond both via a muscarinic cholinergic receptor to the cholinergic agonist, carbamylcholine, and via α- and β-adrenergic receptors to norepinephrine and epinephrine. Thus, cholinergic and adrenergic receptors are simultaneously present on the same cell. Cardiocyte clusters had considerably higher sensitivity to both autonomic agents, but, because of the extensive functional specializations between cells, the localization of functional receptors to specific cells could not be made. [³H]Alprenolol, a potent β-adrenergic receptor antagonist, and [³H]quinuclidinyl benzilate ([³H]QNB), a potent muscarinic cholinergic receptor antagonist, were used to localize β-adrenergic and muscarinic cholinergic receptors by autoradiography. Quantitation of the muscarinic ACh receptor gave ～800 sites/μm², a value comparable to that for the nicotinic ACh receptor on primary skeletal muscle in culture. Electrophysiological and fine-structural studies confirmed the myocardial nature of these cells.     

Simultaneous assay of muscarinic and β-adrenergic receptors using a double isotope technique

Muscarinic receptor and β-adrenergic receptor binding were measured simultaneously in a membrane fraction of bovine tracheal smooth muscle using [³H]-L-quinuclidinyl benzilate and [¹²⁵I]-(?)iodocyanopindolol. The binding characteristics, affinity and receptor density, obtained in the double receptor assay and in the control experiments were the same within experimental error. Moreover, there appears to be neither a significant influence of an excess of d,l-propranolol on [³H]-L-quinuclidinyl benzilate binding nor a significant influence of an excess of l-quinuclidinyl benzilate on [¹²⁵I]-(?)iodocyanopindolol binding. The method is advantageous where both receptors have to be assayed and where limited amounts of biological material, like in biopsy specimen, are available.     

Isolation and characterization of the surface membranes of fast and slow mammalian skeletal muscle

Fast (extensor digitorum longus) and slow (soleus) rat skeletal muscles served as the source for isolation and biochemical comparison of two distinct surface membrane fractions with properties of the sarcolemma and transverse tubular system. Enriched sarcolemmal membrane from soleus demonstrated a lighter density after sucrose density centrifugation. Sialic acid content was 1.5-fold higher in soleus (62 nmol/mg) than extensor (40 nmol/mg). The specific activity of (Na⁺ + K⁺ + Mg²⁺)-ATPase was similar (1.40 and 1.65 μmol P_i/mg per 5 min) with the soleus enzyme displaying a (1) greater resistance to inhibition by ouabain, and (2) broader ionic ratio ($\text{Na}{}^{\mathrm{+}}\text{K}{}^{\mathrm{+}}$) requirement than extensor enzyme. The polypeptide and phospholipid composition showed no major differences between the two muscle types.The second surface membrane fraction, tentatively identified as transverse tubule, differed in membrane composition. The major polypeptide of extensor was of 95 000 molecular weight whereas for soleus a M_r = 28 000 species was dominant. Total phospholipid content of soleus was 1.5-fold greater than extensor due mostly to increased levels of phosphatidylcholine and phosphatidylethanolamine. Endogenous membrane protein kinase for the 28 000 molecular weight polypeptide was found exclusively in this membrane. The reaction conditions were identical for extensor and soleus since both required divalent cations (Ca²⁺ and Mg²⁺) and neither was affected by cyclic AMP. Soleus showed a 2-fold higher capacity for phosphate incorporation than extensor.These studies show that surface membrane fractions derived from fast and slow muscles differ in terms of functional and compositional properties. These differences are specific not only for the surface membrane but for the muscle type and may relate to the known physiological differences observed between fast and slow mammalian muscle.     

Thyroid hormone differentially affects mRNA levels of Ca-ATPase isozymes of sarcoplasmic reticulum in fast and slow skeletal muscle

mRNA levels for the type I and type II isoforms of sarcoplasmic reticulum (SR) Ca-ATPase were determined in soleus (SOL) and extensor digitorum longus (EDL) muscle of euthyroid (normal), hypothyroid, and hyperthyroid rats. Total Ca-ATPase mRNA content of hyperthyroid muscle was 1.5-fold (EDL) and 6-fold (SOL) higher compared to hypothyroid muscle, with corresponding increases in total SR Ca-ATPase activity. EDL contained only type II Ca-ATPase mRNA. In SOL type I mRNA was the major form in hypothyroidism (98%), but the type II mRNA content was stimulated 150-fold by T3, accounting for 50% of the Ca-ATPase mRNA in hyperthyroidism.     

Influence of hindlimb unloading on the modulation of neurotransmitter secretion through the autoreceptor system

In experiments on neuromuscular junctions of fast (m. extensor digitorum longus, EDL) and slow (m. soleus) muscles of rats under hindlimb unloading of varied duration, we compared the intensity of spontaneous quantal secretion of neurotransmitter in response to potassium depolarization and activation of presynaptic cholinoreceptors with a nonhydrolyzable acetylcholine analog. Secretion was assessed by the mean frequency of miniature endplate potentials. In the controls, carbachol raised this index by 363% in EDL and by 62% in soleus. Secretion in the fast muscle was also more sensitive to [K⁺]. Hindlimb unloading abolished the sensitivity to carbachol in EDL while in soleus it did not change. Preservation of the sensitivity of the fast muscle to potassium depolarization suggested that unloading reduced the number of functional presynaptic receptors.     

Demonstration of adrenergic catecholamine receptors in rat myometrium and their regulation by sex steroid hormones.

The molecular basis of sex steroid hormone-modulation of catecholamine-regulated smooth muscle cell contraction in the uterus was investigated at the level of the catecholamine receptor in rat myometrium. Myometrial membrane binding sites for ³H)-dihydroergocryptine bound α-but not β-adrenergic antagonists and stereospecifically bound the α-agonists (?)-norepinephrine > (?)-epinephrine > phenylephrine. Binding sites for (?) (³H)-dihydroalprenolol were specific for β-adrenergic antagonists and stereospecifically bound (?)-isoproterenol > epinephrine ? norepinephrine. These results were consistent with the expected properties of the myometrial α- and β-adrenergic catecholamine receptors. Myometrial content of β- but not α-adrenergic catecholamine receptors was significantly elevated during proestrus and estrus, suggesting a role for sex steroid hormones in the regulation of these receptors. This posibility was substantiated in ovariectomized rats where castration resulted in a reduction in myometrial β-receptor content which was restored in a dose-dependent manner by estrodiol administration. We conclude: 1) rat uterus contains a substantial concentration of α- and β-adrenergic catecholamine receptors, 2) sex steroid hormones may modulated uterine contractility by regulation of these cell surface receptors; 3) modulation of cell responses to surface active hormones and agents by regulation of their cell surface receptors may be a major way in which sex steroids regulate target organ function.     

Effect of anabolic steroids on mitochondria and sarcotubular system of skeletal muscle.

Soleus and extensor digitorum longus (EDL) mitochondria and sarcotubular system were examined in sedentary and trained (treadmill for 12 wk) male rats that were treated with fluoxymesterone or methandrostanolone (2 mg/kg, 5 days/wk, for 8 wk). Neither physical exercise nor anabolic/androgenic steroid administration resulted in a significant change in muscle wet weight. Treatment with the anabolizing androgens increased succinate dehydrogenase activity in fast-twitch muscle mitochondria; this effect was not enhanced by training and was not observed in soleus mitochondria. On the other hand, the content of the slow-twitch muscle in sarcotubular fraction was increased in sedentary rats by fluoxymesterone or methandrostanolone treatment, whereas no significant changes were found in EDL. The training program affected adenosinetriphosphatase (ATPase) activities in the sarcotubular fraction; Mg2(+)-ATPase was increased in both soleus and EDL, but Ca2(+)-ATPase was decreased only in soleus. However, in sedentary animals only the Mg2(+)-dependent activity of EDL was increased by anabolizing androgen treatment, and this change was not potentiated by additional training. The present data indicate that anabolic/androgenic steroids can affect mitochondrial and sarcotubular enzymes in skeletal muscle. The effects are muscle-type specific.