Laminin polymerization induces a receptor-cytoskeleton network.

The transition of laminin from a monomeric to a polymerized state is thought to be a crucial step in the development of basement membranes and in the case of skeletal muscle, mutations in laminin can result in severe muscular dystrophies with basement membrane defects. We have evaluated laminin polymer and receptor interactions to determine the requirements for laminin assembly on a cell surface and investigated what cellular responses might be mediated by this transition. We found that on muscle cell surfaces, laminins preferentially polymerize while bound to receptors that included dystroglycan and alpha7beta1 integrin. These receptor interactions are mediated through laminin COOH-terminal domains that are spatially and functionally distinct from NH2-terminal polymer binding sites. This receptor-facilitated self-assembly drives rearrangement of laminin into a cell-associated polygonal network, a process that also requires actin reorganization and tyrosine phosphorylation. As a result, dystroglycan and integrin redistribute into a reciprocal network as do cortical cytoskeleton components vinculin and dystrophin. Cytoskeletal and receptor reorganization is dependent on laminin polymerization and fails in response to receptor occupancy alone (nonpolymerizing laminin). Preferential polymerization of laminin on cell surfaces, and the resulting induction of cortical architecture, is a cooperative process requiring laminin- receptor ligation, receptor-facilitated self-assembly, actin reorganization, and signaling events.

The actin-driven movement and formation of acetylcholine receptor clusters.

A new method was devised to visualize actin polymerization induced by postsynaptic differentiation signals in cultured muscle cells. This entails masking myofibrillar filamentous (F)-actin with jasplakinolide, a cell-permeant F-actin-binding toxin, before synaptogenic stimulation, and then probing new actin assembly with fluorescent phalloidin. With this procedure, actin polymerization associated with newly induced acetylcholine receptor (AChR) clustering by heparin-binding growth-associated molecule-coated beads and by agrin was observed. The beads induced local F-actin assembly that colocalized with AChR clusters at bead-muscle contacts, whereas both the actin cytoskeleton and AChR clusters induced by bath agrin application were diffuse. By expressing a green fluorescent protein-coupled version of cortactin, a protein that binds to active F-actin, the dynamic nature of the actin cytoskeleton associated with new AChR clusters was revealed. In fact, the motive force generated by actin polymerization propelled the entire bead-induced AChR cluster with its attached bead to move in the plane of the membrane. In addition, actin polymerization is also necessary for the formation of both bead and agrin-induced AChR clusters as well as phosphotyrosine accumulation, as shown by their blockage by latrunculin A, a toxin that sequesters globular (G)-actin and prevents F-actin assembly. These results show that actin polymerization induced by synaptogenic signals is necessary for the movement and formation of AChR clusters and implicate a role of F-actin as a postsynaptic scaffold for the assembly of structural and signaling molecules in neuromuscular junction formation.

Poloxamer 188 failed to prevent exercise-induced membrane breakdown in mdx skeletal muscle fibers.

We sought to determine the effectiveness of poloxamer 188 (P188) in protecting dystrophin-deficient, mdx skeletal muscle fiber membrane against exercise-induced breaches. mdx mice were treated with either P188 or placebo via intraperitoneal injections and run on a treadmill for 60-90 min. Membrane breakdown was quantified in cross-sections of rectus femoris muscle pretreated with Evans blue dye (in vivo). The mean % dye-penetrated muscle in the P188 and placebo groups was not significantly different in each of three trials. These results contrast with a recent report of P188 being highly effective in protecting the stretch- and dobutamine-stressed mdx heart muscle. The most likely explanations for the disparity are: (1) the exercise stress we used was beyond the protective range of P188, (2) P188 delivery and serum concentration were sub-optimal, or (3) the mdx skeletal myopathy and cardiomyopathy have fundamentally different responses to treatment.

Morphofunctional compensation of masseter muscles in unilateral posterior crossbite patients.

Unilateral posterior crossbite is a widespread, asymmetric malocclusion characterized by an inverse relationship of the upper and lower buccal dental cusps, in the molar and premolar regions, on one side only of the dental arch. Patients with unilateral posterior crossbite exhibit an altered chewing cycles and the crossbite side masseter results to be less active with respect to the contralateral one. Few studies about morphological features of masticatory muscle in malocclusion disorders exist and most of these have been performed on animal models. The aim of the present study was to evaluate morphological and protein expression characteristics of masseter muscles in patients affected by unilateral posterior crossbite, by histological and immunofluorescence techniques. We have used antibody against PAX-7, marker of satellite cells, and against α-, ß-, γ-, δ-, ε- and ζ-sarcoglycans which are transmembrane glycoproteins involved in sarcolemma stabilization. By statistical analysis we have evaluated differences in amount of myonucley between contralateral and ipsilateral side. Results have shown: i) altered fibers morphology and atrophy of ipsilateral muscle if compared to the contralateral one; ii) higher number of myonuclei and PAX-7 positive cells in contralateral side than ipsilateral one; iii) higher pattern of fluorescence for all tested sarcoglycans in contralateral side than ipsilateral one. Results show that in unilateral posterior crossbite hypertrophic response of contralateral masseter and atrophic events in ipsilateral masseter take place; by that, in unilateral posterior crossbite malocclusion masticatory muscles modify their morphology depending on the function. That could be relevant in understanding and healing of malocclusion disorders; in fact, the altered balance about structure and function between ipsilateral and contralateral muscles could, long-term, lead and/ or worsen skeletal asymmetries.

Low affinity binding of taurine to phospholiposomes and cardiac sarcolemma.

A sarcolemma-enriched membrane fraction was prepared from the hearts of Sprague-Dawley rats and its ability to bind taurine (0.5-150 mM) was measured. In the absence of cations, the sarcolemma bound a maximum of 661 nmol taurine/mg protein, with a dissociation constant of 19.2 mM and a Hill coefficient of 1.9, indicating positive cooperativity. Scatchard analysis of taurine binding to sarcolemma gave a bell-shaped curve. Neither beta-alanine nor guanidinoethane sulfonate, inhibitors of taurine transport, affected the degree of taurine binding to sarcolemma. However, hypotaurine was an effective antagonist. Equimolar concentrations of Ca2+, Na+ or K+ also reduced taurine binding. Heterogeneous phospholipid vesicles of phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, and phosphatidylserine (18:19:2:1) also bound taurine with positive cooperativity, yielding a bell-shaped Scatchard curve. The affinity of taurine for these mixed phospholipid vesicles was enhanced by the inclusion of cholesterol (50%). Taurine associated in a maximum ratio of 1:1 with homogeneous vesicles of phosphatidylcholine or phosphatidylserine. Vesicles of phosphatidylethanolamine bound taurine in a maximum ratio of 2:1, whereas those of phosphatidylinositol bound insignificant amounts of taurine. These studies demonstrate a low affinity binding to sarcolemma of taurine at concentrations normally present in rat heart. Similar levels of binding were observed in phospholipid vesicles, suggesting that the interaction of taurine with biological membranes involves phospholipids.

Protein methylation inhibits Na+-Ca2+ exchange activity in cardiac sarcolemmal vesicles.

We have examined the effect of membrane methylation on the Na+-Ca2+ exchange activity of canine cardiac sarcolemmal vesicles using S-adenosyl-L-methionine as methyl donor. Methylation leads to approximately 40% inhibition of the initial rate of Nai+-dependent Ca2+ uptake. The inhibition is due to a lowering of the Vmax for the reaction. The inhibition is not due to an effect on membrane permeability and is blocked by S-adenosyl-L-homocysteine, an inhibitor of methylation reactions. The following experiments indicated that inhibition of Na+-Ca2+ exchange was due to methylation of membrane protein and not due to methylated phosphatidylethanolamine (PE) compounds (i.e., phosphatidyl-N-monomethylethanolamine (PMME) or phosphatidyl-N,N'-dimethylethanolamine (PDME]: (1) We solubilized sarcolemma and reconstituted activity into vesicles containing no PE. The inhibition by S-adenosyl-L-methionine was not diminished in this environment. (2) We reconstituted sarcolemma into vesicles containing PMME or PDME. These methylated lipid components had no effect on Na+-Ca2+ exchange activity. (3) We verified that many membrane proteins, probably including the exchanger, become methylated.

Phospholipid composition modulates the Na+-Ca2+ exchange activity of cardiac sarcolemma in reconstituted vesicles.

Na+-Ca2+ exchange activity in cardiac sarcolemmal vesicles is known to be sensitive to charged, membrane lipid components. To examine the interactions between membrane components and the exchanger in more detail, we have solubilized and reconstituted the Na+-Ca2+ exchanger into membranes of defined lipid composition. Our results indicate that optimal Na+-Ca2+ exchange activity requires the presence of certain anionic phospholipids. In particular, phosphatidylserine (PS), cardiolipin, or phosphatidic acid at 50% by weight results in high Na+-Ca2+ exchange activity, whereas phosphatidylinositol and phosphatidylglycerol provide a poor environment for exchange. In addition, incorporation of cholesterol at 20% by weight greatly facilitates Na+-Ca2+ exchange activity. Thus, for example, an optimal lipid environment for Na+-Ca2+ exchange is phosphatidylcholine (PC, 30%)/PS (50%)/cholesterol (20%). Na+-Ca2+ exchange activity is also high when cardiac sarcolemma is solubilized and then reconstituted into asolectin liposomes. We fractionated the lipids of asolectin into subclasses for further reconstitution studies. When sarcolemma is reconstituted into vesicles formed from the phospholipid component of asolectin, Na+-Ca2+ exchange activity is low. When the neutral lipid fraction of asolectin (including sterols) is also included in the reconstitution medium, Na+-Ca2+ exchange activity is greatly stimulated. This result is consistent with the requirement for cholesterol described above. Proteinase treatment, high pH, intravesicular Ca2+ and dodecyl sulfate all stimulate Na+-Ca2+ exchange in native sarcolemmal vesicles. We examined the effects of these interventions on exchange activity in reconstituted vesicles of varying lipid composition. In general, Na+-Ca2+ exchange could be stimulated only when reconstituted into vesicles of a suboptimal lipid composition. That is, when reconstituted into asolectin or PC/PS/cholesterol (30:50:20), the exchanger is already in an activated state and can no longer be stimulated. The one exception was that the Na+-Ca2+ exchanger responded to altered pH in an identical manner, independent of vesicle lipid composition. The mechanism of action of altered pH on the exchanger thus appears to be different from other interventions.

Solubilization and reconstitution of the sarcolemmal Na+-Ca2+ exchange system of vascular smooth muscle.

The Na+-Ca2+ exchange system of the sarcolemma of rat mesenteric artery was solubilized and reconstituted in soybean phospholipid vesicles. In the reconstituted system, the exchange process showed about 4-fold higher specific activity compared to that of native vesicles. The inhibitory effect of monensin and the stimulatory effect of valinomycin in the presence of K+ on Na+ gradient-dependent Ca2+ uptake were preserved and were pronounced in the reconstituted system. The stimulation by valinomycin indicates that the exchange process is electrogenic. Thus, the stoichiometry, the characteristics and the mechanism of action which were difficult to study in the native vesicles can now be determined conveniently using the reconstituted system. Also, solubilization and reconstitution of the exchange system confirms its existence in vascular smooth muscle.

Defective sarcolemmal phosphorylation associated with noninsulin-dependent diabetes.

Noninsulin-dependent diabetes is associated with a decrease in the activity of sarcolemmal phosphatase 1, but no change in the activities of phosphatase 2A, 2B, or 2C. Also unaffected by diabetes were the activities of protein kinase C, cAMP-dependent protein kinase and calcium-calmodulin protein kinase. Because of the decrease in phosphatase 1 activity, 32P incorporation into sarcolemmal phosphoproteins catalyzed by either intrinsic protein kinases or extrinsic cAMP-dependent protein kinase was elevated in the diabetic. Among the proteins whose phosphorylation was elevated in diabetes was the phospholamban-like protein, which has been implicated in the regulation of ATP-dependent calcium transport. The phosphate-linked increase could be prevented by exposing the membranes to a phosphatase inhibitor and either extrinsic cAMP-dependent protein kinase or alamethicin. In addition to the phosphatase-linked effects, analysis of individual sarcolemmal phosphoproteins by SDS-polyacrylamide gel electrophoresis indicated that diabetes caused a specific elevation in membrane phosphorylation of some proteins (43 kDa and 78 kDa), but a decrease in the phosphorylation state of other phosphoproteins (31 kDa and 49 kDa). The data indicate that membrane phosphorylation is dramatically altered by diabetes. The possibility that this contributes to altered myocardial function is discussed.

Biochemical and structural evidence for pig myocardium adherens junction disruption by cardiopulmonary bypass.

BACKGROUND: Given that cardiopulmonary bypass (CPB) is associated with edema and heart dysfunction and that adherens junctions may regulate vascular permeability barrier integrity and cardiomyocyte function, we investigated adherens junction protein steady-state levels in a pig model of CPB. METHODS AND RESULTS: Pigs were subjected to normothermic CPB for 90 minutes, followed by post-CPB perfusion for 90 minutes. Atrial and ventricular myocardium tissue samples were harvested before institution of bypass (basal levels) and at the end of post-CPB perfusion. Adherens junctions were analyzed by either total lysate or cadherin immunoprecipitates that were immunoblotted for pan-cadherin, VE-cadherin, beta-catenin, and gamma-catenin. Adherens junction solubility was addressed with Triton X-100 extraction. Frozen tissue sections were labeled with the same antibodies, and adherens junctions were visualized by confocal microscopy. Immunoblotting of total lysates revealed an increase in smaller-molecular-weight fragments of VE-cadherin, beta-catenin, and gamma -catenin after post-CPB perfusion, indicating partial protein degradation. Smaller-molecular-weight fragments recognized by VE-cadherin and beta-catenin antibodies were also obtained from VE-cadherin immunoprecipitation, indicating degradation of endothelial cell adherens junctions. A prominent increase in adherens junction complex solubility was observed in post-CPB perfusion samples. Confocal microscopy of hearts obtained before CPB showed a continuous, homogeneous pattern of cell-cell labeling that contrasted with an irregular, discontinuous, punctuate, or zigzag pattern observed in post-CPB perfusion samples, corroborating biochemical data. CONCLUSIONS: These results indicate that CPB is associated with signs of degradation of endothelial and cardiomyocytes adherens junctions, pointing to a molecular mechanism leading to increased vascular permeability and cardiomyocyte dysfunction.

The selective activation of the cardiac sarcolemmal sodium-calcium exchanger by plasmalogenic phosphatidic acid produced by phospholipase D.

Since plasmalogens are the predominant phospholipid of cardiac sarcolemma, the activation of the sodium-calcium exchanger by either plasmenylethanolamine or plasmalogenic phosphatidic acid generated by phospholipase D was explored. Sodium-calcium exchange activity was 7-fold greater in proteoliposomes comprised of plasmenylethanolamine compared to proteoliposomes comprised of only plasmenylcholine. Phospholipase D treatment of proteoliposomes resulted in 1 mol % conversion of plasmenylcholine or phosphatidylcholine to their respective phosphatidic acid molecular species with a concomitant 8-fold or 2-fold activation of sodium-calcium exchange activity, respectfully. Thus, phospholipase D-mediated hydrolysis of plasmalogens to phosphatidic acid may be an important mechanism for the regulation of the sodium-calcium exchanger.

Plasmalogen and anionic phospholipid dependence of the cardiac sarcolemmal sodium-calcium exchanger.

Although plasmalogens are the predominant phospholipids of cardiac sarcolemma, their physiological role has not been forthcoming. Since the cardiac sarcolemmal sodium-calcium exchanger has been proposed to be regulated by anionic phospholipids, the roles of plasmalogens and anionic phospholipids as regulators of the sodium-calcium exchanger were explored. Reconstituted sodium-calcium exchange activity in plasmalogen-containing proteoliposomes was 10-fold higher than that in control proteoliposomes comprised of only diacyl phospholipids. Additionally, exchange activity in plasmalogen-containing proteoliposomes was regulated by anionic phospholipids. Thus, plasmalogens provide a critical lipid environment in which anionic phospholipids serve as boundary lipids for the regulation of the trans-sarcolemmal sodium-calcium exchanger.

Glycosphingolipids of skeletal muscle: II. Modulation of Ca2(+)-flux in triad membranes by gangliosides.

Membrane vesicles of rabbit skeletal muscle were prepared and separated by sucrose density gradient centrifugation. The fractions obtained (in the order of increasing density) were sarcolemma (SL), T-tubules (TT), sarcoplasmic reticulum (SR1 and SR2) and triads/mitochondria (Tr/M) as characterized by their specific marker enzymes, ligand binding, and ion flux activities. The distribution of neutral glycosphingolipids and gangliosides in these membrane preparations has been documented in the preceding paper (J. Müthing, U. Maurer, U. Neumann, B. Kniep, and S. Weber-Schürholz, Carbohydr, Res., (1988) 135-145). GM3(Neu5Ac) is the dominant ganglioside, neolacto-series gangliosides are moderately expressed and ganglio-series gangliosides were found in minor quantities, however, all showing different qualitative and quantitative membrane-type specific patterns. The voltage dependent Ca(2+)-channels of skeletal muscle reside prevalently in the triad enriched membrane fractions deduced from highest binding capacity of 1,4-dihydropyridines. Calcium channel complexes of triads were reconstituted into unilamellar phospholipid vesicles of 400 nm defined size and the active 45Ca(2+)-uptake into intravesicular space was measured after incorporation of muscle specific gangliosides into the outer vesicle lipid bilayer in parallel to control liposomes without gangliosides. GM3(Neu5Ac) strongly increased the uptake of 45Ca2+ (+285%) whereas GM3(Neu5Gc) severely inhibited the ion flux (-61%). Neolacto-series gangliosides evoked miscellaneous effects upon 45Ca(2+)-flux depending on isomeric sialic acid configuration, oligosaccharide size and fatty acid chain length of the ceramide portion. VI3Neu5Ac-nLcOse6Cer (C24-fatty acid), IV3Neu5Ac-nLcOse4Cer (C16-fatty acid) and IV6Neu5Ac-nLcOse4Cer (C16-fatty acid) strongly enhanced the 45Ca(2+)-flux (+208, +162, and +120%, respectively, whereas IV3Neu5Ac-nLcOse4Cer (C24-fatty acid), VI3Neu5Ac-nLcOse6Cer (C16-fatty acid) and IV6Neu5Ac-nLcOse4Cer (C24-fatty acid) slightly reduced 45Ca(2+)-flux (-3, -6, and -17%, respectively). Out of all gangliosides tested in this study, GM1 showed the strongest stimulatory effect (+327%). GD1a and GT1b gave rise to remarkable flux-stimulation of +283 and +255%, respectively, whereas GD1b exhibited only a slightly positive effect (+38%). This data suggest a functional role of gangliosides in subcellular muscle membranes giving strong evidence that gangliosides are capable of modulating the cytosolic calcium level of muscle, which regulates muscle contraction.

[Mechanism of changes in the calcium permeability of the sarcolemma of vascular smooth muscle cells during hypoxia]. / Mekhanizmy izmeneniia kal'tsievoi pronitsaemosti sarkolemmy gladkomyshechnykh kletok sosudov pri gipoksii.

In isolated preparations of smooth muscle of the rat portal vein, removal of Ca2+ from perfusate or addition of Ca2+--antagonists as well as hypoxia entailed inhibition of vascular smooth muscle contractile activity. Uptake of 45Ca by vascular smooth muscle cells was diminished in hypoxia. Liposomes filled with Ca2+ or Cr but not ATP prevented the hypoxic relaxation of vascular smooth muscle. One of the main reasons of the decrease of vascular smooth muscle contractility in hypoxia seems to be the disturbance of calcium channel phosphorylation and the decrease of sarcolemma calcium permeability.

[Binding of GTP and GTPase activity of rabbit myocardial sarcolemma]. / Zv'iazuvannia GTP sarkolemoiu miokardu krolia ta ïï GTPazna aktyvnist'.

Binding [3H] GTP and GTP-ase activity were studied on the isolated preparation of myocardium sarcolemma. The Muscarinic acetylcholine receptors against carbocholine (10(-4) M) was shown to stimulate both the GTP binding by sarcolemma and its GTP-ase activity. The activating effect of carbocholine is blocked by the Muscarinic acetylcholine receptors antagonist atropine (10(-6) M). The GTP-ase activity of sarcolemma is reduced in presence of sodium fluoride which points out the enzyme activity of G-proteins. Thus, functioning of G-proteins and their Muscarinic acetylcholine receptors regulation were showed during in vitro experiments on preparation of myocardium sarcolemma.

[Effect of N-palmitoylethanolamine on energy-dependent transport of Ca2+ in vesicles of myometrium sarcolemma and their phospholipid composition]. / Vliianie n-pal'mitoilétanolamina na énergozavisimyi transport Ca2+ v vezikulakh sarkolemmy miometriia i ikh fosfolipidnyi sostav.

N-palmitoylethanolamine (NPE) was studied for their effect on calcium pump of pig myometrium sarcolemma. NPE in concentration of 10 microM, stimulated by 28-46% Mg2+, ATP-dependent accumulation of Ca2+ in vesicles of plasmatic membrane of uterus myocytes taking absolutely no effect on passive release of this cation from them. NPE modified phospholipid composition of sarcolemma, causing the increase of percentage content of phosphatidylinositol (by 20.2%) and lysophosphatidylcholine (2.7 times). While NPE effects transport Ca2+, Mg(2+)-ATPase solubilized from plasmatic membrane and purified due to the method of affinity chromatography on calmodulin-sepharose 4B, no activating effect of NPE on the calcium pump was observed. And what is more, a weakly expressed tendency to inhibition (by 14-15%, respectively) of the rate of Ca2+, Mg(2+)-dependent enzymic hydrolysis of ATP has been revealed. It is supposed that the effect of NPE on active transmembrane transport of Ca2+ is an important link in the general mechanism of contraction-relax of the myometrium and is, apparently, connected with its modifying effect on the lipid composition of the sarcolemma.

Tubulin polymerization modifies cardiac sodium channel expression and gating.

AIMS: Treatment with the anticancer drug taxol (TXL), which polymerizes the cytoskeleton protein tubulin, may evoke cardiac arrhythmias based on reduced human cardiac sodium channel (Na(v)1.5) function. Therefore, we investigated whether enhanced tubulin polymerization by TXL affects Na(v)1.5 function and expression and whether these effects are beta1-subunit-mediated. METHODS AND RESULTS: Human embryonic kidney (HEK293) cells, transfected with SCN5A cDNA alone (Na(v)1.5) or together with SCN1B cDNA (Na(v)1.5 + beta1), and neonatal rat cardiomyocytes (NRCs) were incubated in the presence and in the absence of 100 microM TXL. Sodium current (I(Na)) characteristics were studied using patch-clamp techniques. Na(v)1.5 membrane expression was determined by immunocytochemistry and confocal microscopy. Pre-treatment with TXL reduced peak I(Na) amplitude nearly two-fold in both Na(v)1.5 and Na(v)1.5 + beta1, as well as in NRCs, compared with untreated cells. Accordingly, HEK293 cells and NRCs stained with anti-Na(v)1.5 antibody revealed a reduced membrane-labelling intensity in the TXL-treated groups. In addition, TXL accelerated I(Na) decay of Na(v)1.5 + beta1, whereas I(Na) decay of Na(v)1.5 remained unaltered. Finally, TXL reduced the fraction of channels that slow inactivated from 31% to 18%, and increased the time constant of slow inactivation by two-fold in Na(v)1.5. Conversely, slow inactivation properties of Na(v)1.5 + beta1 were unchanged by TXL. CONCLUSION: Enhanced tubulin polymerization reduces sarcolemmal Na(v)1.5 expression and I(Na) amplitude in a beta1-subunit-independent fashion and causes I(Na) fast and slow inactivation impairment in a beta1-subunit-dependent way. These changes may underlie conduction-slowing-dependent cardiac arrhythmias under conditions of enhanced tubulin polymerization, e.g. TXL treatment and heart failure.

Calcium efflux through cardiac calcium channels reconstituted into liposomes--flux measurement with fura-2.

Cardiac Ca2+ channels were solubilized and reconstituted into liposomes, and Ca2+ efflux from the proteoliposomes was measured with the fluorescent dye fura-2. The Ca2+ efflux, induced by K+ depolarization, was sensitive to Ca2+ channel modulators such as nifedipine, D-600 and Bay K 8644, and was dependent on the membrane potential. Furthermore, the efflux was increased by phosphorylation of proteoliposomes with cAMP-dependent protein kinase. These results suggest that the reconstituted cardiac Ca2+ channels retain the voltage-dependent gating properties, pharmacological sensitivities and modulation by phosphorylation.