Relationship between proteolysis and water-holding of myofibrils.

The purpose of this study was to increase the knowledge on the relationship between proteolysis of myofibrillar proteins and the water-holding of meat. Myofibrils isolated from porcine longissimus thoracis et lumborum muscle were used as a model system. Myofibrils were incubated with either calpain-2, the proteasome or a lysosomal extract at 25°C for 2h. All three proteolytic systems improved the relative water-holding and generally there was a larger effect with increasing amount of enzymes in the incubation. The improved water-holding occurred in parallel to degradation of myofibrillar proteins. Desmin was degraded by calpain-2 as well as by lysosomal enzymes and α-actinin was released by the proteasome. We here propose a model in which degradation of proteins in and around the Z-disk allows overall swelling of the filament lattice and more specifically in the I-band area. In conclusion, proteolytic degradation of myofibrillar proteins by calpain-2, the proteasome or lysosomal enzymes improves the water-holding of myofibrils.

Antioxidant treatment protects diabetic rats from cardiac dysfunction by preserving contractile protein targets of oxidative stress.

BACKGROUND: Animal studies suggest that reactive oxygen species (ROS) play an important role in the development of diabetic cardiomyopathy. HYPOTHESIS: Matrix metalloproteinase-2 (MMP-2) is activated by ROS and contributes to the acute loss of myocardial contractile function by targeting and cleaving susceptible proteins including troponin I (TnI) and alpha-actinin. METHODS: Using the streptozotocin-induced diabetic rat model, we evaluated the effect of daily in vivo administration of sodium selenate (0.3 mg/kg; DMS group), or a pure omega-3 fish oil with antioxidant vitamin E (omega-3E; 50 mg/kg; DMFA group), which has antioxidant-like effects, for 4 weeks on heart function and on several biochemical parameters related to oxidant stress and MMP-2. RESULTS: Although both treatments prevented the diabetes-induced depression in left ventricular developed pressure (LVDP) as well as the rates of changes in developed pressure (+/-dP/dt) (P<.001), the improvement in LVDP of the DMS group was greater compared to that of the DMFA group (P<.001). Moreover, these treatments reduced the diabetes-induced increase in myocardial oxidized protein sulfhydryl and nitrite concentrations (P<.001). Gelatin zymography and Western blot data indicated that the diabetes-induced changes in myocardial levels of MMP-2 and tissue inhibitor of matrix metalloproteinase-4 (TIMP-4) and the reduction in TnI and alpha-actinin protein levels were improved in both the DMS and DMFA groups (P<.001). CONCLUSIONS: These results suggest that diabetes-induced alterations in MMP-2 and TIMP-4 contribute to myocardial contractile dysfunction by targeting TnI and alpha-actinin and that sodium selenate or omega-3E could have therapeutic benefits in diabetic cardiomyopathy.

Adverse effects of cyclosporine A on HSP25, alpha B-crystallin and myofibrillar cytoskeleton in rat heart.

Cyclosporine (CsA) is a universally used immunosuppressive drug which induces adverse side effects in several organs, but its impact on the heart is still controversial. Small heat shock proteins (sHSPs), such as HSP25 and alpha B-crystallin, are cytoprotective stress proteins exceptionally represented in the heart. They act as myofibrillar chaperones that help actin and desmin to maintain their optimum configuration and stability, thereby antagonizing oxidative damage. The present study examined: (1) the cardiac distribution and abundance of HSP25 and alpha B-crystallin in rats receiving CsA at a therapeutic dosage (15 mg/kg/day) for 42 days and 63 days; (2) the presence of myofibrillar proteins, such as actin, alpha-actinin and desmin following the CsA treatments; (3) the subcellular effects of prolonged CsA exposure on the cardiomyocytes by histopathology and transmission electron microscopy. After 63 days CsA intake, sHSPs translocated from a regular sarcomeric pattern to peripheral sarcolemma and intercalated discs, together with actin and desmin. In contrast, the sarcomeric alpha-actinin pattern did not change in all experimental groups. The abundance of actin and HSP25 was unchanged in every time point of treatment while after 63 days CsA, alpha B-crystallin and desmin levels significantly decreased. Furthermore CsA induced fibrosis, irregular sarcomeric alignment and damaged desmosomes. These findings indicate that following prolonged CsA exposure, the cardiac muscle network was affected. In particular, the translocation of sHSPs to intercalated discs merits special consideration as a direct compensatory mechanism to limit CsA cardiotoxicity.

Melatonin protects against piroxicam-induced gastric ulceration.

The antiulcer effect of melatonin on gastric lesions caused by piroxicam was studied with the intent of determining the mechanism of action of this agent. Melatonin dose-dependently lowered piroxicam and indomethacin-induced gastric damage with more than 90% inhibition at a dose of 60 mg/kg BW. Increased lipid peroxidation, augmented protein oxidation and decreased glutathione content of the gastric tissue following piroxicam treatment indicated a possible involvement of oxidative stress in this nonsteroidal anti-inflammatory drug (NSAID)-induced gastropathy. Pretreatment of rats with melatonin prevented these changes. Oral administration of piroxicam to rats caused a threefold increase in the tissue levels of hydroxyl radical generation, a change significantly attenuated by melatonin. Furthermore, a decrease in the activity of gastric peroxidase and an increase in the activity of gastric superoxide dismutase(s) (SOD) because of piroxicam treatment was attenuated by melatonin pretreatment indicating that the indole possibly exerts its gastroprotective effects through its direct as well as indirect antioxidant activities. The results of the present studies also reveal that melatonin may influence the expression of Cu-Zn SOD, catalase, cyclooxygenase as well as alpha-actinin whose levels were found to be altered, following piroxicam treatment. The current studies, therefore, document melatonin's gastroprotective ability against piroxicam-induced gastric damage and the findings raise the possibility of melatonin being considered as a co-therapy with piroxicam or other NSAIDs in reducing the gastropathy when long-term use of these nonsteroidal agents are unavoidable.

Photodamage induced by Zinc(II)-phthalocyanine to microtubules, actin, alpha-actinin and keratin of HeLa cells.

We have studied the photosensitizing effects of zinc(II)-phthalocyanine (ZnPc) on the cytoskeleton of HeLa cells using sublethal (10(-7) M, followed by 1 or 3 min of red light to induce 20%, LD20, or 60%, LD60, cell death, respectively) or lethal (5 x 10(-6) M and 15 min of irradiation, LD100) experimental conditions. The immunofluorescent analysis of the cytoskeleton showed a variable photodamage to microtubules (MT), actin microfilaments (AF) and intermediate filaments of keratin (KF), as well as on alpha-actinin, which was dependent on treatment conditions. Both sublethal treatments induced deep alterations on interphase and mitotic MT. The mitotic index increased with time with the maximum at 18 h (12%) or 24 h (14%) after LD20 or LD60, respectively. The alterations on AF and alpha-actinin were much more severe than those observed on KF at any evaluated time. With the exception of the KF, which remained partially organized, the MT and AF network was severely damaged by the lethal treatment. Western blot analysis for alpha-tubulin, G-actin and alpha-actinin from soluble and insoluble fractions confirmed the results observed by immunofluorescence, thus indicating that these cytoskeletal components are involved in cell damage and death by ZnPc photosensitization.

Fibrillar collagen specifically regulates human vascular smooth muscle cell genes involved in cellular responses and the pericellular matrix environment.

Proliferation and alpha(v)beta(3) integrin-dependent migration of vascular smooth muscle cells are suppressed on polymerized type I collagen. To identify genes specifically regulated in human smooth muscle cells by polymerized collagen, we used the suppressive subtraction hybridization technique. Compared with smooth muscle cells cultured on monomer collagen, polymerized collagen suppresses the following: (1) a number of other extracellular matrix proteins, including fibronectin, thrombospondin-1, tenascin-C, and cysteine-rich protein 61; (2) actin binding proteins including alpha-actinin; (3) signaling molecules; (4) protein synthesis-associated proteins; and (5) genes with unknown functions. Some of the identified genes, including cysteine-rich protein 61, show unique kinetics of mRNA regulation by monomer or polymerized collagen distinct from growth factors, suggesting extracellular matrix-specific gene modulation. Moreover, in vivo balloon catheter-mediated injury to the rat carotid artery induces many of the genes that are suppressed by polymerized collagen. Protein levels of thrombospondin-1 and fibronectin are also suppressed by polymerized collagen. Thrombospondin-1-mediated smooth muscle cell migration on vitronectin is significantly inhibited after culture on polymerized collagen for 24 hours, which is associated with decreased alpha-actinin accumulation at focal adhesions. Thus, polymerized type I collagen dynamically regulates gene expression, pericellular accumulation of extracellular matrix molecules, and the response to a given matrix molecule.

Giardia lamblia rearranges F-actin and alpha-actinin in human colonic and duodenal monolayers and reduces transepithelial electrical resistance.

The mechanisms of epithelial injury in giardiasis remain unknown. The effects of live Giardia lamblia on cellular G-actin, F-actin, alpha-actinin, and electrical resistance of human intestinal epithelial monolayers were investigated using SCBN and Caco2 cell lines grown on chamber slides or Transwell filter membranes. In separate experiments, some monolayers were also exposed to sonicated trophozoites, some to supernatant from live G. lamblia cultures, and some with or without the Ca2+ channel blocker verapamil. After 2, 24, or 48 hr of coincubation with G. lamblia, monolayers were assessed for cytoskeletal arrangement under fluorescence and confocal laser microscopy, and transepithelial electrical resistance was measured. Exposure to live G. lamblia trophozoites induced localized condensation of F-actin and loss of perijunctional alpha-actinin while G-actin remained unchanged. Confocal laser microscopy indicated that F-actin rearrangement was not affected by verapamil and was localized within the terminal web area. Coincubation of monolayers with G. lamblia lysates or with spent medium alone similarly rearranged F-actin. Verapamil alone did not alter F-actin. Electrical resistance of SCBN and Caco2 monolayers exposed to G. lamblia was significantly decreased versus controls regardless of whether live or lysed trophozoite samples were used. The results indicate that G. lamblia-induced epithelial injury is associated with F-actin and alpha-actinin rearrangements in the terminal web area via mechanisms independent of extracellular Ca2+. These alterations are associated with reduced transepithelial electrical resistance and are due at least in part to trophozoite products.

A multi-mutant herpes simplex virus vector has minimal cytotoxic effects on the distribution of filamentous actin, alpha-actinin 2 and a glutamate receptor in differentiated PC12 cells.

To develop effective gene therapy techniques that target populations of neurons in the spinal cord, suitable vectors must be developed that will undergo efficient, retrograde transport from an appropriate peripheral site and will not be cytotoxic. Our previous work (LeVatte et al, 1998a) has demonstrated that a replication defective herpes simplex virus vector 14Hdelta3vhsZ, that has been substantially detoxified, is retrogradely transported from peripheral sites and can infect large numbers of the targeted spinal neurons. We plan to develop targeted gene therapy approaches designed to modulate the excitatory glutamatergic methyl-D-aspartate (NMDA) receptor in spinal cord neurons as a means of ameliorating a form of episodic high blood pressure that occurs after spinal cord injury. In this report, we demonstrate that, in differentiated PC12 cells, a neuronal-like cell line, the virus vector does not appear to alter aspects of the cytoskeletal architecture important to the proper distribution of the NMDA receptor. In turn, the distribution of endogenous NMDA receptor 1 subunit protein (NMDAR1) or a transfected NMDAR1-green fluorescent fusion protein was also found to be unaltered after vector infection. However, whereas endogenous NMDAR1 distribution was maintained, vector infection did tend to reduce the level of its expression. This drop in endogenous NMDAR1 expression coincided with the expression of the HSV immediate early genes ICP0 and ICP27 over the first 24-48 h. These results indicate that the 14Hdelta3vhsZ herpes simplex virus vector is suitable to use in future strategies to alter the level of gene expression in targeted populations of spinal cord neurons.

Cytoskeletal organization and incorporation of beta 3 integrin in thrombin-stimulated platelets: effect of acetylsalicylic acid.

Platelet stimulation by agonists is followed by changes in cytoskeletal organization that includes actin polymerization and association of the membrane skeleton (which is connected with the integrin alpha IIb beta 3) with the underlying cytoplasmic actin filaments. The effect of orally administered acetylsalicylic acid to healthy volunteers on incorporation of contractile protein and beta 3 integrin into the cytoskeletal core of thrombin-stimulated platelets was studied. Stimulation was followed by increased contractile protein and beta 3 incorporation into the cytoskeleton. Acetylsalicylic acid intake resulted in decreased incorporation of myosin and actin (32% and 20%, respectively), and a decrease (36%) in the association of beta 3 integrin with the cytoskeletal elements was evident. In conclusion, we have shown that acetylsalicylic acid, besides the known inhibitory effect on thromboxane synthesis, promotes changes in the cytoskeletal organization of thrombin-stimulated platelets that could limit thrombus formation.

Deuterium oxide (heavy water) accelerates actin assembly in vitro and changes microfilament distribution in cultured cells.

While deuterium oxide (D2O) is known to produce various biological effects in living animals and cultured cells, the detailed mechanisms by which it does so remain unclear. The present study was designed to assess the effects of D2O on microfilaments (MFs) via fluorescence staining of BALB 3T3 cells and in vitro actin polymerization studies. After BALB 3T3 cells had been exposed to a concentration of more than 30% D2O for several hours, stress fibers in the peripheral region became thick and distinct, while the quantity of perinuclear MFs was drastically reduced. This effect was transient and returned to the original distribution within 12 h. Cytoplasmic F-actin (FA) also increased transiently coincident with the enhancement of stress fibers. The pattern of cell locomotion became simpler, and total locomotor activity was suppressed in a D2O concentration-dependent manner. Analysis of in vitro studies demonstrated that, when purified G-actin was polymerized in D2O at a concentration greater than 10%, the rate of actin polymerization was accelerated, whereas the total amount of polymerized actin at the steady state in D2O was the same as that in H2O controls. A gelation assay and transmission electron microscopy (TEM) showed that the network of crosslinked FA with alpha-actinin became denser in 30% D2O than in H2O. These findings concerning actin polymerization and FA gelation suggest that the alteration of stress fibers in cultured cells is caused by a direct effect of D2O on cellular MF dynamics.

Fish muscle cytoskeleton integrity is not dependent on intact thin filaments.

Striated muscle cytoskeleton was studied by ultrastructure and electrophoresis. Treatment of sea bass white muscle myofibrils and glycerinated fibres with calpain caused disruption of costameres, intermediate filaments, and Z-line, without altering sarcomeres. V8 protease also caused loss of costameres and Z-line, and disrupted sarcomeres without affecting the intermediate filaments. Recombinant lipase caused loss of Z-lines and also sarcolemma detachment, without changing sarcomeres or intermediate filaments. DNase-1 removed thin filaments and partially removed Z-lines while leaving intact the sarcolemma attachments and intermediate filaments. Calpain, V8 protease, lipase and DNase-1 treatments induced extensive loss of alpha-actinin from the Z-line, which could be related to titin cleavage (calpain, V8), phosphoinositide hydrolysis (lipase), and actin depolymerisation (DNase-1). These results show that the cytoskeletal components are independent of intact thin filaments.

Cation effects on the conformations of muscle and non-muscle alpha-actinins.

We examined the effects of changing KCl concentration on the secondary structures of alpha-actinins using circular dichroism (CD), 1,1'-bis(4-anilino) naphthalene-5,5'-disulfonic acid (bisANS) fluorescence and proteolysis experiments. Under near-physiological conditions, divalent cations also were added and changes in conformation were investigated. In 25 mM KH2PO4, pH 7.5, increasing KCl from 0 to 120 mM led to decreases in alpha-helix conformation for brain, platelet and heart alpha-actinins (40.5-30.2%, 65.5-37.8% and 37.5-27.8%, respectively). In buffered 120 mM KCl, 0.65 mM calcium produced small changes in the CD spectra of both brain and platelet alpha-actinin, but had no effect on heart alpha-actinin. bisANS fluorescence of all three alpha-actinins also showed significant changes in conformation with increasing KCl. However, in buffered 120 mM KCl increasing concentrations of Ca2+ or Mg2+ did not have significant effects on the bisANS fluorescence of any alpha-actinin. Digestion of brain, platelet and heart alpha-actinins with alpha-chymotrypsin showed an increase of proteolytic susceptibility in 120 mM KCl. These experiments also showed that increasing the concentration of Ca2+ or Mg2+ led to greater changes in digestion fragment patterns in the absence of KCl than in the presence of 120 mM KCl. The results suggest that alpha-actinins exist in different conformations depending on the ionic strength of the medium, which could explain the differences in calcium and F-actin binding results obtained from different alpha-actinins.

Proteolytic cleavage of alpha-actinin by calpain in T cells stimulated with anti-CD3 monoclonal antibody.

Stimulation of the TCR/CD3 complex on T cells initiates rearrangement of the actin cytoskeleton. The results presented show that a temporal increase in the appearance of filamentous actin begins immediately after stimulation of T cells with immobilized anti-CD3 mAb. The formation of filamentous actin in these stimulated cells reaches a steady state within 30 min after anti-CD3 mAb stimulation. At this time, pseudopod formation is observed and becomes progressively more evident over the next several hours. Experiments were done to investigate the role of the actin cytoskeletal associated proteins, alpha-actinin, vinculin, and talin, in the assembly of the actin cytoskeleton in anti-CD3 mAb-stimulated T cells. Using immunofluorescence, these three proteins are detected throughout the cytosol in resting T cells. However, after anti-CD3 mAb stimulation of the T cells, these proteins move to one pole of the cell. Electrophoresis followed by immunoblotting of T cell lysates prepared from resting as well as anti-CD3 mAb-stimulated cells revealed that alpha-actinin, but not vinculin or talin, was modified as a consequence of cell activation. Results show that alpha-actinin exists as a 105-kDa subunit in resting T cells, but that anti-CD3 mAb stimulation of T cells leads to the appearance of an 80-kDa lower molecular form of alpha-actinin. Experiments show that this occurs as a consequence of the 105-kDa subunit being proteolytically cleaved by calpain.

Effects of cytochalasin D on shape and fluid pinocytosis in human neutrophils as related to cytoskeletal changes (actin, alpha-actinin and microtubules).

We report that cytochalasin D (CD) is not a reliable tool to inhibit all forms of cell motility and actin polymerization in neutrophil granulocytes. In addition to the well-established effects of CD such as altered localization of F-actin, inhibition of surface ruffling, fluid pinocytosis and actin polymerization in agonist-stimulated cells, we find that in human neutrophils CD can 1) induce another type of continuous shape changes (10(-6) M and 10(-5) M CD), 2) stimulate fluid pinocytosis (10(-5) M CD), 3) increase actin polymerization (10(-5) to 10(-4) M CD) and alter the localization of F-actin and alpha-actinin (10(-6) to 10(-4) M CD). At 10(-5) M CD F-actin and alpha-actinin are preferentially located in different areas of the cell. At 10(-4) M CD actin and alpha-actinin may colocalize at the membrane but not in cytoplasmic foci. Thus, stimulation of shape changes, pinocytosis, actin polymerization and differential reorganization of the cytoskeleton occur at CD concentrations which are widely used to inhibit cell motility. The results show that CD is not a reliable tool to inhibit all movements of cells and actin polymerization in general. Shape changes, but not fluid pinocytosis and the relative redistribution of F-actin and alpha-actinin induced by 10(-5) M cytochalasin D are suppressed by 10(-5) colchicine. This indicates that also microtubules can play a role in determining neutrophil shape and movements.

Cation binding to chicken gizzard alpha-actinin.

Gizzard alpha-actinin binds 45Ca2+ as shown by the calcium overlay method. Flow dialysis measurements in 20 mM Hepes (pH 7.5) reveal 3.5 +/- 1.8 (S.D.) high affinity calcium binding sites per dimer, with Kd1 = 6.36 +/- 0.34 x 10(-6) M, and 87.3 +/- 7.2 sites with Kd2 = 1.66 +/- 0.44 x 10(-4) M. Chymotrypsin and thermolysin digestion yielded peptides of gizzard alpha-actinin which, if they included the putative EF-hands, bound 45Ca2+ in 10 mM imidazole-HCl (pH 7.4) or 60 mM KCl, 10 mM imidazole-HCl (pH 7.4). In addition, peptides which include a region of the molecule more than 27 kDa from the N-terminal also bind calcium. In contrast, when KCl in the binding buffer was increased to 120 mM, calcium binding was eliminated. Flow dialysis data revealed no high-affinity binding and 82.5 +/- 3.3 calcium binding sites with calculated affinities in the millimolar range. These are divalent cation binding sites, not Ca(2+)-specific sites, because they are eliminated by the addition of up to 5 mM Mg2+. Structural changes produced upon cation binding to alpha-actinin measured by circular dichroism, proteolysis and bisANS fluorescence are substantial when binding K+ with only small changes upon binding of Ca2+ or Mg2+ in the presence of 120 mM KCl. These results suggest that monovalent and divalent cations have different effects on different parts of the molecule with a complete elimination of 45Ca2+ binding to the EF-hands being produced by 120 mM KCl.

Colchicine-induced stimulation of PMN motility related to cytoskeletal changes in actin, alpha-actinin, and myosin.

Colchicine-induced stimulation of polymorphonuclear leukocyte (PMN) locomotion is an interesting model because extension of blebs at the front occurs at a rate (about 2.4 microns/s) which is far above that reported for growth of actin filaments. The following cytoskeletal changes were observed in colchicine-treated PMNs: (1) a small increase in cytoskeleton-associated actin was noted, as well as a somewhat more pronounced increase in cytoskeleton-associated alpha-actinin, as compared with untreated or DMSO-treated controls. There was, however, no measurable increase in F-actin as determined by NBD-phallacidin binding; (2) the values for the ratio (alpha-actinin/actin) are lower in PMNs treated with colchicine for 30 min, as compared with PMNs stimulated with fNLPNTL for 1 minute (non-polar ruffling cells) or 30 min (polarized locomoting cells); thus, this ratio may depend on the type of PMN motility; (3) in polarized PMNs F-actin was mainly located linearly all along the cell membrane; there was more intense staining at the front of the cells; (4) alpha-actinin appeared to colocalize with F-actin at the leading front, but not with F-actin at the tail of polarized cells; (5) myosin was preferentially found at the rear part of polarized cells but not or only to a small extent at the front. Our data indicate a close functional correlation between microtubules and microfilaments. We speculate that F-actin in combination with alpha-actinin promotes expansion of pseudopods, whereas myosin combined with F-actin promotes contraction.(ABSTRACT TRUNCATED AT 250 WORDS)

Rheological measurements of the influence of 1,2-propanediol on actin/alpha-actinin gel structure: the effects of temperature and protein concentrations.

In previous studies, we demonstrated that 1,2-propanediol induces shortening and bundling of actin filaments, both in vitro and in vivo, and that it enhances actin/alpha-actinin interaction, especially at low temperature. 1,2-Propanediol also promotes homogeneous microporous networks which can be vitrified by rapid cooling. In the present study, dynamical rheological measurements were performed under various sets of experimental conditions including temperature (4 or 20 degrees C), protein concentrations (actin and alpha-actinin), and 1,2-propanediol presence or absence. Gelation kinetics were monitored, and the resulting actin mechanical properties investigated, in order to untangle the respective effects of the experimental parameters. Whether in the presence or absence of solvent, low temperature brings about a rigidification of the sample, as does high protein concentration, as expected. However, 1,2-propanediol itself involves either softening of the sample (at high temperature and low protein concentration or at low temperature and high protein concentration) or rigidification in the case of low temperature and low protein concentration. These effects result from the competition between actin/alpha-actinin affinity (enhanced by both low temperature and 1,2-propanediol), bundling of filaments (fostered by alpha-actinin for alpha-actinin/actin ratios used), rate of actin polymerization (higher at high temperature), shortening effect of 1,2-propanediol on actin filaments, and chain mobility (lower at high protein concentration). As discussed, only the combination of low temperature and low protein concentration induces full crosslinking of the system into a viscoelastic solid under the influence of 1,2-propanediol.

TPA has no influence on the expression of myosin heavy chain isoforms in cultured adult cardiac muscle cells.

The effect of a tumor promoter, 12-O-tetradecanoyl phorbol-13-acetate (TPA), on the expression of myosin heavy chain isoforms in cultured rat cardiac ventricular muscle cells was studied. The previous preliminary report [Claycomb WC (1988): "Biology of Isolated Adult Cardiac Myocytes." In Clark WA, Decker RS, Borg TK (eds): New York: Elsevier, pp 284-287] indicated that TPA turns off the expression of myosin heavy chain genes in cultured adult cardiac myocytes. Electrophoretic and immunocytochemical analyses were carried out in the present studies. The myosin heavy chain isoform profiles of cardiac myocytes exposed to TPA at concentrations of 50-250 ng/ml culture medium for varying periods were similar to those of controls that were grown in the absence of TPA, showing predominant isoform V1. Immunofluorescence microscopy with monoclonal antibodies to cardiac ventricular isomyosin revealed the structural organization of myosin in TPA-treated cells. The organization of myosin was variable among different myocytes and within a single myocyte. Immunofluorescence microscopy was extended to the examination of the organization of alpha-actinin which did not differ from that of myosin in some myocytes. In contrast to the previous report [Claycomb, 1988], this study has demonstrated that TPA has no influence on the expression of myosin heavy chain isoforms in cultured adult ventricular cardiac muscle cells.

Calcium channel blocker influences the density of alpha-actinin labeling at the rat neuromuscular junction.

Alpha-actinin is a muscle protein located along the Z-disc. Incubation of frog muscle with the calcium ionophore, A23187, can decrease the immunogold labelling of alpha-actinin. Pyridostigmine (PYR) is an inhibitor of acetylcholinesterase, which causes disruption of Z-discs only in the region of the motor endplate. This is probably due to excess influx of calcium ions, leading to activation of proteases. Pretreating animals with the calcium channel blocker diltiazem can significantly reduce damage to the Z-discs at the motor endplate caused by PYR. It was of interest to determine whether the distribution of alpha-actinin had been altered following PYR administration and whether diltiazem could prevent those changes. There was less alpha-actinin labelling at the motor endplate compared to away from this region for all treatment groups. Animals administered diltiazem showed less labelling compared to PYR, but with no disruption of Z-discs at the motor endplate following diltiazem. Pretreatment with diltiazem reduced the incidence of Z-disc damage, but the degree of alpha-actinin labeling at the endplate was less than that seen with diltiazem alone. The greater effect seen at the endplate implies that neuromuscular activity is an important factor. The drugs may be causing a reduction in alpha-actinin labelling by different mechanisms.