A model for the modulation of microvessel permeability by junction strands

To investigate the effect of junction strands on microvessel permeability, we extend the previous analytical model developed by Fu et al. (1994, J. Biomech. Eng., 116, pp. 502-513), for the interendothelial cleft to include multiple junction strands in the cleft and an interface between the surface glycocalyx layer and the cleft entrance. Based on the electron microscopic observations by Adamson et al. (1998, Am. J. Physiol., 274(43), pp. H1885-H1894), that elevation of intracellular cAMP levels would increase number of tight junction strands, this two-junction-strand and two-pore model can successfully account for the experimental data for the decreased permeability to water, small and intermediate-sized solutes by cAMP.     

Biochemical localisation of the 5-HT2A (serotonin) receptor in rat skeletal muscle

The 5-HT2A receptor was recently shown to localise morphologically to the transverse tubules (TT) in rat foetal myoblasts. Receptor activation enhanced the expression of genes involved in myogenesis, and its TT localisation has led to the suggestion that it may participate in excitation-contraction coupling. In order to gain further insights into 5-HT2A receptor function in muscle we have (i) investigated its biochemical localisation in adult rat skeletal muscle and (ii) determined whether receptor expression is dependent upon muscle type. Immunoblot analysis of muscle membranes, isolated by subcellular fractionation, revealed that adult muscle expresses the 5-HT2A receptor and that it resides exclusively in plasma membranes and not in TT. No differences in 5-HT2A abundance were observed between red and white muscle, suggesting that receptor expression does not correlate with the metabolic or contractile properties of the muscle fibre. Our data indicate that 5-HT2A expression in skeletal muscle is maintained into adulthood and that its absence from TT make it an unlikely participant in the excitation-contraction coupling process.     

Vanilloid receptor expressed in the sarcoplasmic reticulum of rat skeletal muscle

Vanilloid receptor subtype 1 (VR1) was cloned as a capsaicin receptor from neuronal cells of dorsal root ganglia. VR1 was subsequently found in a few non-neuronal tissues, including skeletal muscle [Onozawa et al., Tissue distribution of capsaicin receptor in the various organs of rats, Proc. Jpn. Acad. Ser. B 76 (2000) 68-72]. We confirmed the expression of VR1 in muscle cells using the RT-PCR method and Western blot analysis. Immunostaining studies with a confocal microscope and an electron microscope indicated that VR1 was present in the sarcoplasmic reticulum (SR), a store of Ca2+. The SR releases Ca2+ to cause a contraction when a muscle is excited. However, SR still releases a small amount of Ca2+ under relaxed conditions. We found that this leakage was enhanced by capsaicin and was antagonized by capsazepine, a capsaicin blocker, indicating that leakage of Ca2+ occurs through a channel composed of VR1.     

Perturbed skeletal muscle insulin signaling in the adult female intrauterine growth-restricted rat

To determine the molecular mechanism(s) linking fetal adaptations in intrauterine growth restriction (IUGR) to adult maladaptations of type 2 diabetes mellitus, we investigated the effect of prenatal seminutrient restriction, modified by early postnatal ad libitum access to nutrients (CM/SP) or seminutrient restriction (SM/SP), vs. early postnatal seminutrient restriction alone (SM/CP) or control nutrition (CM/CP) on the skeletal muscle postreceptor insulin-signaling pathway in the adult offspring. The altered in utero hormonal/metabolic milieu was associated with no change in basal total IRS-1, p85, and p110beta subunits of PI 3-kinase, PKCtheta, and PKCzeta concentrations but an increase in basal IRS-2 (P < 0.05) only in the CM/SP group and an increase in basal phospho (p)-PDK-1 (P < 0.05), p-Akt (P < 0.05), and p-PKCzeta (P < 0.05) concentrations in the CM/SP and SM/SP groups. Insulin-stimulated increases in p-PDK-1 (P < 0.05) and p-Akt (P < 0.0007), with no increase in p-PKCzeta, were seen in both CM/SP and SM/SP groups. SHP2 (P < 0.03) and PTP1B (P < 0.03) increased only in SM/SP with no change in PTEN in CM/SP and SM/SP groups. Aberrations in kinase and phosphatase moieties in the adult IUGR offspring were initiated in utero but further sculpted by the early postnatal nutritional state. Although the CM/SP group demonstrated enhanced kinase activation, the SM/SP group revealed an added increase in phosphatase concentrations with the net result of heightened basal insulin sensitivity in both groups. The inability to further respond to exogenous insulin was due to the key molecular distal roadblock consisting of resistance to phosphorylate and activate PKCzeta necessary for GLUT4 translocation. This protective adaptation may become maladaptive and serve as a forerunner for gestational and type 2 diabetes mellitus.     

Nutritional modulation of training-induced skeletal muscle adaptations

Skeletal muscle displays remarkable plasticity, enabling substantial adaptive modifications in its metabolic potential and functional characteristics in response to external stimuli such as mechanical loading and nutrient availability. Contraction-induced adaptations are determined largely by the mode of exercise and the volume, intensity, and frequency of the training stimulus. However, evidence is accumulating that nutrient availability serves as a potent modulator of many acute responses and chronic adaptations to both endurance and resistance exercise. Changes in macronutrient intake rapidly alter the concentration of blood-borne substrates and hormones, causing marked perturbations in the storage profile of skeletal muscle and other insulin-sensitive tissues. In turn, muscle energy status exerts profound effects on resting fuel metabolism and patterns of fuel utilization during exercise as well as acute regulatory processes underlying gene expression and cell signaling. As such, these nutrient-exercise interactions have the potential to activate or inhibit many biochemical pathways with putative roles in training adaptation. This review provides a contemporary perspective of our understanding of the molecular and cellular events that take place in skeletal muscle in response to both endurance and resistance exercise commenced after acute and/or chronic alterations in nutrient availability (carbohydrate, fat, protein, and several antioxidants). Emphasis is on the results of human studies and how nutrient provision (or lack thereof) interacts with specific contractile stimulus to modulate many of the acute responses to exercise, thereby potentially promoting or inhibiting subsequent training adaptation.     

Chronic alcohol ingestion alters the calcium permeability of sarcoplasmic reticulum of rat skeletal muscle

Heavy sarcoplasmic reticulum (SR) was prepared from skeletal muscle of control and chronic alcoholic rats, and the effect of in vitro addition of ethanol on the passive Ca2+ permeability was studied. The SR was loaded with Ca2+ in the absence of ATP. Then efflux was initiated by adding an EGTA solution to decrease the extravesicular Ca2+ concentration. The decrease of Ca2+ content of the SR was measured by an optical method using an encapsulated metallochromic indicator (calcein). The Ca2+ permeability of alcoholic rat SR was higher than that of control rats, especially at low external Ca2+ concentrations (below 1 microM). An in vitro (acute) exposure of SR to ethanol increased the Ca2+ permeability of the SR. However, the degree of increase in alcoholic rat SR was smaller than that in control rat SR.     

Levonorgestrel and norethindrone alter insulin action on skeletal muscle of the female rat

Prospective studies of women receiving oral contraceptives suggest that the progestin component may induce insulin resistance and variable deterioration of glucose tolerance. Because the tissue sites and nature of this insulin antagonism are not well-defined, we studied the effects of two parenterally administered progestins, levonorgestrel (NG) and norethindrone (NE), on insulin-regulated glucose uptake and phenylalanine release by the perfused rat hindquarter. Female rats were injected sc for 14 days with NG or NE (10 or 30 micrograms/kg/day). Low-dose NG and high-dose NE approximate the per kg dose received by women taking a high-dose progestin oral contraceptive. Phenylalanine release and glucose uptake (nmole/min/g) by the perfused hindquarters were calculated from the A-V difference for each. Progestin treatment (30 micrograms/kg/d) significantly reduced phenylalanine release from hindquarters perfused without exogenous insulin. Hindquarters from the high dose NG and low and high dose NE rats perfused with insulin (100 microU/ml) released 22% less phenylalanine than control rats perfused with the same insulin concentration (P less than 0.01) but the net suppression below baseline was similar in the control and steroid-treated groups. High-dose progestin treatment did not alter glucose uptake by hindquarters perfused without exogenous insulin. Insulin (100 microU/ml) increased glucose uptake by hindquarters of control and progestin-treated rats as compared to animals in the same treatment group perfused without exogenous insulin (P less than 0.01). High dose NE impaired insulin-stimulated glucose uptake 24% below values of the control group (P less than 0.01). The other NE and NG doses had no effect.(ABSTRACT TRUNCATED AT 250 WORDS)     

Adenosine A2A receptors inhibit the conductance of NMDA receptor channels in rat neostriatal neurons

Summary Whole-cell patch clamp experiments were carried out in rat striatal brain slices. In a subset of striatal neurons (70–80%), NMDA-induced inward currents were inhibited by the adenosine AZA receptor selective agonist CGS 21680. The non-selective adenosine receptor antagonist 8-(p-sulphophenyl)-theophylline and the AZA receptor selective antagonist 8-(3chlorostyryl) caffeine abolished the inhibitory action of CGS 21680. Intracellular GDP--S, which is known to prevent G protein-mediated reactions, also eliminated the effect of CGS 21680. Extracellular dibutyryl cAMP, a membrane permeable analogue of cAMP, and intracellular Sp-cAMPS, an activator of cAMP-dependent protein kinases (PKA), both abolished the CGS 21680-induced inhibition. By contrast, Rp-cAMPS and PKI 14–24 amide, two inhibitors of PKA had no effect. Intracellular U-73122 (a phospholipase C inhibitor) and heparin (an inositoltriphosphate antagonist) prevented the effect of CGS 21680. Finally, a more efficient buffering of intracellular Ca²⁺ by a substitution of EGTA (11 mM) by BAPTA (5.5 mM) acted like U-73122 or heparin. Hence, AZA receptors appear to negatively modulate NMDA receptor channel conductance via the phospholipase C/inositoltriphosphate/Ca²⁺ pathway rather than the adenylate cyclase/PKA pathway.     

Adenosine A 2B receptors modulate cAMP levels and induce CREB but not ERK1/2 and p38 phosphorylation in rat skeletal muscle cells

The present study examined the existence of the adenosine A(1),A(2A), and A(2B) receptors and the effect of receptor activation on cAMP accumulation and protein phosphorylation in primary rat skeletal muscle cells. Presence of mRNA and protein for all three receptors was demonstrated in both cultured and adult rat skeletal muscle. NECA (10(-9)-10(-4)M) increased the cAMP concentration in cultured muscle cells with an EC(50) of (95% confidence interval)=15 (5.9-25.1) micro M, whereas CGS 21680 (10(-9)-10(-4)M) had no effect on cAMP accumulation. Concentrations of [R]-PIA below 10(-6)M had no effect on cAMP accumulation induced by either isoproterenol or forskolin. NECA resulted in phosphorylation of CREB with an EC(50) of (95% confidence interval)=1.7 (0.40-7.02) micro M, whereas ERK1/2 and p38 phosphorylation was unchanged. The results show that, although the A(1),A(2A), and A(2B) receptors are all present in skeletal muscle cells, the effect of adenosine on adenylyl cyclase activation and phosphorylation of CREB is mainly mediated via the adenosine A(2B) receptor.     

A high-molecular-weight cysteine endopeptidase from rat skeletal muscle

A cytosolic enzyme of high molecular weight (about 500 000), which attacks native or denatured proteins (inter alia, casein, globin and hexokinase) was purified about 1000-fold from mixed rat skeletal muscles, including muscles freed of mast cells by prior treatment of the animals with the degranulator, compound 48/80. Peptides of varying size were generated from radioactively labelled globin, but no free amino acids were formed; free tyrosine was also not released from azocasein. The pH optimum was 7.5 and the presence of an essential cysteine group was suggested because dithiothreitol (1 mM) stimulated the activity and N-ethylmaleimide (5 mM) and p-chloromercuriphenylsulphonic acid (1 mM) were inhibitors. The activity was markedly inhibited by Zn2+ but not by leupeptin, chymostatin or pepstatin. The enzyme was stabilized by ATP, at concentrations as low as 0.1 mM, against inactivation at 42 degrees C. The endopeptidase was clearly separated on gel chromatography from another large protease, also sensitive to Zn2+, but with marked aminopeptidase activity and the properties of hydrolase H. The activity levels of the protease, assayed after chromatography on Sepharose 6B of high-speed supernatant fractions, did not vary significantly in skeletal muscle samples which were derived from denervated, starved, diabetic or hyperthyroid animals, in all of which the abnormal physiological states expressed themselves as enhanced rates of tyrosine released by incubated soleus and extensor digitorum longus muscles. Nevertheless, the enzyme described here may be part of an ATP-dependent, multi-component proteolytic system similar to that already known to be present in reticulocytes.     

A skeletal muscle ryanodine receptor interaction domain in triadin

Excitation-contraction coupling in skeletal muscle depends, in part, on a functional interaction between the ligand-gated ryanodine receptor (RyR1) and integral membrane protein Trisk 95, localized to the sarcoplasmic reticulum membrane. Various domains on Trisk 95 can associate with RyR1, yet the domain responsible for regulating RyR1 activity has remained elusive. We explored the hypothesis that a luminal Trisk 95 KEKE motif (residues 200-232), known to promote RyR1 binding, may also form the RyR1 activation domain. Peptides corresponding to Trisk 95 residues 200-232 or 200-231 bound to RyR1 and increased the single channel activity of RyR1 by 1.49±0.11-fold and 1.8±0.15-fold respectively, when added to its luminal side. A similar increase in [(3)H]ryanodine binding, which reflects open probability of the channels, was also observed. This RyR1 activation is similar to activation induced by full length Trisk 95. Circular dichroism showed that both peptides were intrinsically disordered, suggesting a defined secondary structure is not necessary to mediate RyR1 activation. These data for the first time demonstrate that Trisk 95's 200-231 region is responsible for RyR1 activation. Furthermore, it shows that no secondary structure is required to achieve this activation, the Trisk 95 residues themselves are critical for the Trisk 95-RyR1 interaction.     

Characterization of skeletal muscle insulin receptor

The insulin receptor from rat skeletal muscle was characterized. Treatment of muscle membranes with the photoactive insulin analog, 125I[N-epsilonB29-monoazidobenzoyl]-insulin revealed a single protein band of 135,000 Da, the alpha subunit. Iodination of total membrane protein followed by Triton X-100 solubilization and immunoprecipitation demonstrated the presence of a protein band of 90,000 Da, the beta subunit, together with a protein band of 190,000 Da, which may be the receptor precursor. In partially purified receptor preparations, the beta subunit exhibited dose-dependent, insulin-stimulated phosphorylation with incorporation of phosphate solely into tyrosine residues, which was also observed in the 190,000-Da receptor precursor. Purified plasma membranes contained a large amount of insulin-degrading activity which had to be inactivated prior to performing insulin-binding studies. If degradation of insulin was not prevented, apparent enhanced binding in the presence of unlabeled insulin was observed.     

Adenosine A2A receptor is involved in cell surface expression of A2B receptor

The A2A and A2B adenosine receptors (A2AR and A2BR) are implicated in many physiological processes. However, the mechanisms of their intracellular maturation and trafficking are poorly understood. In comparative studies of A2AR versus A2BR expression in transfected cells, we noticed that the levels of cell surface expression of A2BR were significantly lower than those of A2AR. A large portion of the A2BR was degraded by the proteasome. Studies of cell surface expression of A2BR chimeric molecules in transfectants suggested that A2BR does not have the dominant forward transport signal for export from the endoplasmic reticulum to the cell surface. A2BR surface expression was increased in A2BR chimeras where the A2BR carboxyl terminus (CT) was replaced or fused with the A2AR CT. Co-transfection of A2AR with A2BR enhanced surface expression of A2BR though the F(X)(6)LL motif in the A2AR CT. The requirements of A2AR expression for better A2BR cell surface expression was not only established in transfectants but also confirmed by observations of much lower levels of A2BR-induced intracellular cAMP accumulation in response to A2BR-activating ligand in splenocytes from A2AR(-/-) mice than in wild type mice. The results of mechanistic studies suggested that poor A2BR expression at the cell surface might be accounted for mainly by the lack of a dominant forward transport signal from the endoplasmic reticulum to the plasma membrane; it is likely that A2BR forms a hetero-oligomer complex for better function.     

Effects of adenosine receptor antagonism on protein tyrosine phosphatase in rat skeletal muscle

Earlier studies have shown that whole body adenosine receptor antagonism increases skeletal muscle insulin sensitivity in insulin-resistant Zucker rats. To find which steps in the insulin signaling pathway are influenced by adenosine receptors, muscle from lean and obese Zucker rats, treated for 1 week with the adenosine receptor antagonist, 1,3-dipropyl-8-(4-acrylate)-phenylxanthine (BWA1433), were analyzed. All rats were first anesthetized and injected intravenously (i.v.) with 1 IU of insulin. About 3 min later the gastrocnemius was freeze clamped. Insulin receptors were partially purified on wheat germ agglutinin (WGA) columns and insulin receptor kinase activity measured in control and BWA1433-treated lean and obese Zucker rats. Protein tyrosine phosphatase (PTPase) activity was also analyzed in subcellular fractions, including the cytosolic fraction, a high-speed particulate fraction and the insulin receptor fraction eluted from WGA columns. Administration of BWA1433 increased insulin receptor kinase activity in obese but not lean Zucker rats. PTPase activities were higher in the untreated obese rat muscle particulate fractions than in the lean rat particulate fractions. The BWA1433 administration lowered the PTPase activity of the obese rats but not the lean rats. Although the PTPase activity in WGA eluate fractions containing crude insulin receptors were similar in lean and obese animals, BWA1433 administration was found to lower the PTPase activities in the fractions obtained from obese but not from the lean rats. PTPases may be upregulated in muscles from obese rats due to activated adenosine receptors. Adenosine receptor blockade, by reducing PTPase activity, may thereby increase insulin signaling.     

Proton modulation of a Ca(2+)-activated K+ channel from rat skeletal muscle incorporated into planar bilayers

The effect of pH on the activation of a Ca-activated K+ [K(Ca)] channel from rat skeletal muscle incorporated into planar lipid bilayers was studied. Experiments were done at different intracellular Ca2+ and proton concentrations. Changes in pH modified channel kinetics only from the Ca-sensitive face of the channel. At constant Ca2+ concentration, intracellular acidification induced a decrease in the open probability (Po) and a shift of the channel activation curves toward the right along the voltage axis. The displacement was 23.5 mV per pH unit. This displacement was due to a change in the half saturation voltage (Vo) and not to a change in channel voltage dependence. The shifts in Vo induced by protons appeared to be independent of Ca2+ concentration. The slope of the Hill plot of the open-closed equilibrium vs. pH was close to one, suggesting that a minimum of one proton is involved in the proton-driven channel closing reaction. The change in Po with variations in pH was due to both a decrease in the mean open time (To) and an increase in the mean closed time (Tc). At constant voltage, the mean open time of the channel was a linear function of [Ca2+] and the mean closed time was a linear function of 1/[Ca2+]2. Changes in the internal pH modified the slope, but not the intercept of the linear relations To vs. [Ca2+] and Tc vs. 1/[Ca2+]2. On the basis of these results an economical kinetic model of the effect of pH on this channel is proposed. It is concluded that protons do not affect the open-closed reaction, but rather weaken Ca2+ binding to all the conformational states of the channel. Moreover, competitive models in which Ca2+ and H+ cannot bind to the same open or closed state are inconsistent with the data.     

Adenosine receptor on human basophils: modulation of histamine release.

Adenosine, at physiologic concentrations, inhibits in vitro IgE-mediated human basophil histamine release in a dose-dependent fashion. The inhibition dose-response curve is paralleled by an adenosine-induced increase in cAMP levels of human leukocyte preparations. Further evidence that the adenosine effect is related to changes in cAMP levels is that the nucleoside inhibits only in the first stage of antigen-induced histamine release and fails to inhibit the release caused by ionophore A23187. A poorly metabolized derivative of adenosine, 2-chloroadenosine inhibits as effectively as adenosine; dipyridamole, which blocks adenosine uptake, does not impair the inhibition caused by adenosine. Finally, theophylline, which is a competitive antagonist of adenosine in human lymphocytes also blocks the inhibition of release caused by adenosine. These data suggest that adenosine acts via a specific cell-surface receptor linked to adenylate cyclase. It appears that the human basophil has a specific receptor for adenosine and that this nucleoside may modulate the in vivo release of the mediators of immediate hypersensitivity reactions.     

Identification of different receptor types for toxic phospholipases A2 in rabbit skeletal muscle.

Oxyuranus scutellatus scutellatus toxins 1 (OS1) and 2 (OS2) are two phospholipase A2S (PLA2) isolated from the venom of the Australian Taipan snake. Their iodinated derivatives have been used to characterize PLA2 binding sites on rabbit skeletal muscle. Competition and cross-linking experiments indicate that 125I-labelled OS2 binding sites in rabbit skeletal muscle in vivo are distributed into two classes of receptors. One class binds OS2 and OS1 and is insensitive to the bee venom PLA2. It is composed of a 180 kDa binding protein. This class of PLA2 receptor is expressed at a high level in rabbit myotube membranes. The other class of PLA2 receptor identified with 125I-OS2 also binds with high affinity the bee venom PLA2 but not OS1 and is composed of major polypeptides of 34, 48 and 82 kDa. This second class of receptor is similar to the one found in brain membranes. The density of the two classes of receptors varies during muscle development.