Identification of triadin and of histidine-rich Ca(2+)-binding protein as substrates of 60 kDa calmodulin-dependent protein kinase in junctional terminal cisternae of sarcoplasmic reticulum of rabbit fast muscle.

The endogenous calmodulin-protein kinase system of sarcoplasmic reticulum terminal cisternae of rabbit fast-twitch muscle was studied. Investigation of a single Ca(2+)-channel in terminal cisternae fused to planar lipid bilayers demonstrated that the endogenous kinase inhibits the channel, although it remained unclear whether the phosphorylation sites are on the channel protein or on other junctional sarcoplasmic reticulum specific proteins [Hain et al., (1994) Biophys. J. 67, 1823-1833]. Our results, which show that two junctional sarcoplasmic reticulum specific proteins, i.e., triadin and histidine-rich Ca(2+)-binding protein, but not the ryanodine receptor/Ca(2+)-channel protein, are phosphorylated by membrane-bound 60 kDa protein kinase, seem to be able to resolve this ambiguity. Furthermore, such a probably specific protein isoform of calmodulin-protein kinase, by its substrate specificity and exposure to the cytoplasmic side of terminal cisternae at the junctional membrane domain and based on protease sensitivity, also seems to possess some of the potential requirements for a regulatory role in the functional state of the Ca(2+)-channel.

Heparin neutralization by platelet-rich thrombi. Role of platelet factor 4.

BACKGROUND: Platelets contain several factors that inhibit heparin. This study was designed to assess the heparin-neutralizing activity present in acute, platelet-rich arterial thrombi formed at sites of arterial injury in animals. METHODS AND RESULTS: Platelet-rich thrombi (n = 3) were induced in pig coronary arteries by balloon catheter-mediated arterial injury. Soluble extracts were prepared from each thrombus and assayed for the capacity to inhibit heparin in an in vitro clotting assay (activated partial thromboplastin time). Mean heparin-neutralizing activity was 28 U of heparin neutralized per milliliter of thrombus, indicating that 1 vol of coronary thrombus completely inhibited the heparin present in 140 vols of therapeutically anticoagulated (0.2 U heparin/mL) plasma. In contrast, thrombus extracts had no effect on the anticoagulant activity of hirudin, a direct-acting thrombin inhibitor. The heparin-neutralizing activity present in coronary thrombi bound to heparin-agarose and was eluted from it by 1.4 mol/L NaCl, suggesting that platelet factor 4 mediated the antiheparin effect of thrombi. Consistent with this hypothesis, a murine monoclonal antibody to rabbit platelet factor 4 nearly completely inhibited the heparin-neutralizing activity present in rabbit thrombi (n = 3) generated by carotid artery injury. CONCLUSIONS: Extracts prepared from platelet-rich arterial thrombi significantly inhibit the in vitro anticoagulant potency of heparin but not of hirudin. This antiheparin effect appears to be mediated by platelet factor 4. These results are consistent with the hypothesis that localized inhibition of heparin at sites of platelet activation may reduce its antithrombotic efficacy. In addition, they suggest an additional mechanism for the apparent superiority of hirudin over heparin as a thrombin inhibitor at sites of arterial injury.

Production of novel monoclonal antibodies against rabbit platelet factor four.

Ten hybridomas producing monoclonal antibodies (Mabs) against rabbit platelet factor 4 (PF4) were obtained from the fusion of splenocytes from mice immunized with purified rabbit PF4 and NSO mouse myeloma cells. When the reactivities of these monoclonal antibodies were determined by enzyme-linked immunosorbent assay and immunoblotting with human and rabbit PF4, they showed a high degree of specificity. Only one Mab recognized an epitope common to the human and rabbit molecules, the other nine reacted only with the rabbit protein. All the antibodies recognized, in crude platelet lysates, a band that comigrates with the purified PF4 protein. None of these antibodies cross-reacted with major rabbit or human platelet-poor plasma proteins. The significance of the Mabs in immunological and physiological studies is discussed.

Neutralization of the antiheparin activity of platelet factor 4 by a monoclonal antibody.

We have produced a panel of monoclonal antibodies (mAbs) against rabbit platelet factor 4 (PF4). Two of these mAbs have been characterized in this study. In particular the antibody called 10B2, which also recognizes the human molecule, is able to block PF4's ability to neutralize heparin in a modified Heparin-Factor Xa chromogenic assay. The inhibition appears to be more than 95% at 1:1 mAb/PF4 molar ratio both for purified rabbit and human PF4. Similar results were obtained using supernatants from stimulated human platelets (90% of inhibition at 1:1 mAb/PF4 molar ratio) or using Fab fragments from 10B2. Studies to determine the antigenic determinant against which 10B2 is directed, show that this is an assembled epitope which involves disulfide bonds of the PF4.

Relationship between plasma leucine concentration and clearance in normal and type 1 diabetic subjects.

In a series of studies in normal and type 1 diabetic subjects, we analysed the relationship between isotope-calculated leucine clearance and plasma leucine concentration. All studies were performed under euglycaemic conditions. Plasma leucine concentrations were either experimentally decreased by means of insulin infusion, or increased by means of exogenous amino acid infusion in the presence of hyperinsulinaemia. Leucine clearance rates were compared in normal and diabetic subjects at similar plasma insulin levels. The effect of hyperinsulinaemia was examined by measuring clearance rates in normal subjects at comparable leucine levels but different insulin concentrations. Our data show that leucine clearance is inversely related to leucine concentration, and that it is not independently stimulated by hyperinsulinaemia. Type 1 diabetes is not associated with decreased leucine clearance. A general equation relating leucine concentration and clearance is proposed. These data support the view that peripheral leucine utilization is not decreased in type 1 diabetes mellitus.

Troponin T- and troponin I-like proteins in bovine vascular smooth muscle.

We have tested the hypothesis whether proteins with biochemical and immunochemical properties similar to those of troponin T (TnT) and troponin I (TnI) are expressed in bovine vascular smooth muscle (SM). Three monoclonal anti-TnT antibodies (TT-1, TT-2, and RV-C2) specific for the two isoforms of TnT present in the bovine cardiac muscle and two monoclonal antibodies (TI-1 and TI-5) reacting with cardiac TnI were used in this study. Anti-TnT antibodies were found to be unreactive with 1) skeletal and nonmuscle isoforms of glyceraldehyde-3-phosphate dehydrogenase, a glycolytic enzyme that shares some structural homologies with skeletal TnT, and 2) calponin, a TnT-like calmodulin/tropomyosin binding protein with some antigenic properties in common with TnT. When tested on SM extracts from aorta or coronary arteries by Western blotting, the anti-TnT antibodies were able to react exclusively with one or two polypeptides whose electrophoretic mobility corresponds to the cardiac TnT subunits. Similarly, anti-TnI antibodies specifically recognized a component in the aortic or coronary SM extracts with electrophoretic properties identical to the cardiac TnI. Immunofluorescence analysis performed on the vascular SM cells of bovine aorta, coronary arteries, and intramural branches of coronary vessels confirmed the existence of cardiac troponin immunoreactivity in these tissues. In addition, differences in the distribution of cardiac TnT- and TnI-like proteins were evidenced in nonvascular and vascular SM cells. This study shows for the first time that polypeptides with some structural properties in common with cardiac TnT and TnI can be found in the vascular SM system.

Cardiac troponin T in developing, regenerating and denervated rat skeletal muscle.

Fetal rat skeletal muscles express a troponin T (TnT) isoform similar to the TnT isoform expressed in the embryonic heart with respect to electrophoretic mobility and immunoreactivity with cardiac TnT-specific monoclonal antibodies. Immunoblotting analyses reveal that both the embryonic and the adult isoforms of cardiac TnT are transiently expressed during the neonatal stages. In addition, other TnT species, different from both cardiac TnTs and from the TnT isoforms expressed in adult muscles, are present in skeletal muscles during the first two postnatal weeks. By immunocytochemistry, cardiac TnT is detectable at the somitic stage and throughout embryonic and fetal development, and disappears during the first weeks after birth, persisting exclusively in the bag fibers of the muscle spindles. Cardiac TnT is re-expressed in regenerating muscle fibers following a cold injury and in mature muscle fibers after denervation. Developmental regulation of this TnT variant is not coordinated with that of the embryonic myosin heavy chain with respect to timing of disappearance and cellular distribution. No obligatory correlation between the two proteins is likewise found in regenerating and denervated muscles.

Actin and myosin expression during development of cardiac muscle from cultured embryonal carcinoma cells.

P19 embryonal carcinoma cells are multipotential stem cells that differentiate into striated muscle as well as some other cell types when aggregated and exposed to dimethyl sulfoxide (DMSO). Immunofluorescence experiments using monospecific antibodies indicated that the majority of muscle cells were mononucleate and contained four myosin isoforms normally found in cardiac muscle; atrial and ventricular myosin heavy chains, ventricular myosin light chain 1, and atrial myosin light chain 2. Northern blot analysis of RNA isolated from differentiating cultures indicated that cardiac actin and skeletal actin mRNAs were expressed at similar levels and with identical kinetics during the differentiation of P19-derived myocytes. These results demonstrate that most of the P19-derived myocytes are of the cardiac type and suggest that they closely resemble the cells of the early embryonic myocardium.

Troponin I switching in the developing heart.

Monoclonal antibodies identify two distinct isoforms of troponin I in rat cardiac muscle, one predominant in the embryonic and fetal heart and one predominant in the adult heart. The two isoforms can be resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, with apparent molecular weights of 27,000 and 31,500, respectively. The adult isoform is specifically recognized by a monoclonal antibody that is unreactive with the embryonic variant, while two other monoclonal antibodies recognize both isoforms. A monoclonal antibody to cardiac troponin T was used to isolate by affinity chromatography the troponin complex from adult and neonatal rat heart. Affinity purified troponin from neonatal heart was found to contain both the embryonic and adult isoforms of troponin I. Comparative immunoblotting analysis with different muscle tissues shows that embryonic troponin I is identical with respect to electrophoretic mobility and pattern of immunoreactivity to the major troponin I isoform found in adult slow skeletal muscle. Troponin I switching may be implicated in developmental changes involving Ca2+ and pH sensitivity of the contractile system and response to beta-adrenergic stimulation.

Three myosin heavy chain isoforms in type 2 skeletal muscle fibres.

Mammalian skeletal muscles consist of three main fibre types, type 1,2A and 2B fibres, with different myosin heavy chain (MHC) composition. We have now identified another fibre type, called type 2X fibre, characterized by a specific MHC isoform. Type 2X fibres, which are widely distributed in rat skeletal muscles, can be distinguished from 2A and 2B fibres by histochemical ATPase activity and by their unique staining pattern with seven anti-MHC monoclonal antibodies. The existence of the 2X-MHC isoform was confirmed by immunoblotting analysis using muscles containing 2X fibres as a major component, such as the normal and hyperthyroid diaphragm, and the soleus muscle after high frequency chronic stimulation. 2X-MHC contains one determinant common to 2B-MHC and another common to all type 2-MHCs, but lacks epitopes specific for 2A- and 2B-MHCs, as well as an epitope present on all other MHCs. By SDS-polyacrylamide gel electrophoresis 2X-MHC shows a lower mobility compared to 2B-MHC and appears to comigrate with 2A-MHC. Muscles containing predominantly 2X-MHC display a velocity of shortening intermediate between that of slow muscles and that of fast muscles composed predominantly of 2B fibres.

Myosin heavy-chain isoforms in human smooth muscle.

The myosin heavy-chain composition of human smooth muscle has been investigated by sodium dodecyl sulfate/polyacrylamide gel electrophoresis, enzyme immunoassay, and enzyme-immunoblotting procedures. A polyclonal and a monoclonal antibody specific for smooth muscle myosin heavy chains were used in this study. The two antibodies were unreactive with sarcomeric myosin heavy chains and with platelet myosin heavy chain on enzyme immunoassay and immunoblots, and stained smooth muscle cells but not non-muscle cells in cryosections and cultures processed for indirect immunofluorescence. Two myosin heavy-chain isoforms, designated MHC-1 and MHC-2 (205 kDa and 200 kDa, respectively) were reactive with both antibodies on immunoblots of pyrophosphate extracts from different smooth muscles (arteries, veins, intestinal wall, myometrium) electrophoresed in 4% polyacrylamide gels. In the pulmonary artery, a third myosin heavy-chain isoform (MHC-3, 190 kDa) electrophoretically and antigenically distinguishable from human platelet myosin heavy chain, was specifically recognized by the monoclonal antibody. Analysis of muscle samples, directly solubilized in a sodium dodecyl sulfate solution, and degradation experiments performed on pyrophosphate extracts ruled out the possibility that MHC-3 is a proteolytic artefact. Polypeptides of identical electrophoretic mobility were also present in the other smooth muscle preparations, but were unreactive with this antibody. The presence of three myosin heavy-chain isoforms in the pulmonary artery may be related to the unique physiological properties displayed by the smooth muscle of this artery.

Troponin T switching in the developing rat heart.

A monoclonal antibody specific for cardiac troponin T has been used to investigate troponin changes during development in the rat heart. Specificity of the antibody was determined by immunoblot analysis with purified bovine cardiac troponin. In the rat heart, immunoblot analysis shows that anticardiac troponin T reacts with a 42.5-kDa band in fetal ventricles and with a 41-kDa band in adult ventricles. The faster migrating troponin T is present in traces in the fetal heart and increases markedly during the first 2 weeks after birth, concomitantly with the progressive decrease of the slower migrating form that is no longer detectable in the adult. The pattern of reactivity of the monoclonal antibody is not modified by alkaline phosphatase pretreatment, suggesting that the antibody is not specific for a phosphorylated epitope. Conditions known to affect cardiac myosin composition, such as hypothyroidism and hypertrophy secondary to systemic hypertension, do not change the troponin T isoform profile of adult rat ventricles. The expression and accumulation of the adult isoforms of troponin T are not suppressed by propylthiouracil treatment of pregnant and nursing rats.

An embryonic-like myosin heavy chain is transiently expressed in nodal conduction tissue of the rat heart.

In the bovine nodal conduction tissue we have described the existence of a novel cardiac myosin isoform, immunologically related to the myosin types expressed during skeletal muscle development. Using different monoclonal antibodies specific for the embryonic and the neonatal skeletal myosin heavy chain types we investigated the myosin composition of the rat sino-atrial and atrio-ventricular nodes. We find that nodal conduction tissue fibers of the rat heart contain a distinct cardiac myosin isoform antigenically similar to the skeletal embryonic myosin heavy chain. The expression of this myosin isoform in nodal tissue appears to be developmentally regulated and partially controlled by thyroid hormone. Reactive cardiac fibers were detected in the nodal regions only during fetal development and a few days after birth, whereas very rare labelled fibers could be observed in the adult nodes. This myosin type does not represent a primordial cardiac myosin isoform since it was not detected in the embryonic heart before 13.5 days of gestation. When congenital hypothyroidism was induced in rats, the post-natal disappearance of reactive fibers in the nodal regions was delayed. On the other hand, hypothyroidism induced in the adult rats did not change the number of the reactive nodal fibers with respect to the euthyroid hearts.

Isomyosin changes after functional electrostimulation of denervated sheep muscle.

Isomyosin analyses by biochemical, immunochemical, and histochemical investigations have been carried out in five sheep following unilateral recurrent laryngeal nerve paralysis and direct functional electrostimulation of the denervated cricoarytenoid posterior muscle. Myosin light chains were identified by two-dimensional gel electrophoresis. Myosin heavy chains were analyzed by one-dimensional SDS-polyacrylamide gel electrophoresis. Slow myosin heavy chain was identified by orthogonal peptide mapping and immunochemistry. The stimulation effect at cellular level was determined using adenosine triphosphatase (ATPase) histochemistry. A dramatic increase of the type 1 fiber area (slow, fatigue-resistant fibers) could be seen after many weeks of an increasing regime of low-frequency direct electrical stimulation. Biochemically, the amount of slow myosin was always higher than in normal muscles. Some muscles were transformed almost completely to the slow type. At the time they were studied and with the methods employed, the expression of embryonic isomyosin was not observed. In conclusion, after numerous weeks of maintained functional activity, elicited by direct electrostimulation, the denervated muscle regionally showed areas of hypertrophy or at least lack of atrophy of slow myofibers without major signs of muscle damage.

Neonatal myosin heavy chains are not expressed in Ni-induced rat rhabdomyosarcoma.

Myosin heavy chain (MHC) composition of chemically-induced rhabdomyosarcoma (RMS) was analyzed by gel electrophoresis and Western blotting using a panel of monoclonal antimyosin antibodies specific for embryonic-, neonatal-, slow- and adult fast-type MHC isoforms. Myosin extracted from tumours and electrophoresed on 6%-sodium dodecyl sulfate (SDS)glycerol gels was found to migrate as three distinct MHC components. These polypeptides were present in different relative amounts in the five RMS studied. Western blotting experiments revealed that variable proportions of embryonic-, slow- and adult fast-, but not neonatal-type, MHC isoforms are consistently expressed in RMS. Indirect and double immunofluorescence procedures applied to cryosections of tumoral tissue showed that: (a) RMS cells were unreactive with antineonatal-type-MHC antibody, (b) the majority of neoplastic, desmin-positive, cells contained embryonic- as well as adult fast-type MHCs and (c) a minority of cells were labelled by anti-slow MHC antibody. The results of this study indicate that there is no obligatory sequence of MHC isoform expression in the molecular transition (emb----neo----adult) which occurs during rat skeletal myogenesis.

Embryonic and neonatal myosin heavy chain in denervated and paralyzed rat skeletal muscle.

Using immunofluorescence procedures with specific polyclonal and monoclonal antimyosin antibodies we have found that embryonic and neonatal myosin heavy chains (MHCs), which in rat skeletal muscle disappear during the first weeks after birth, are reexpressed in adult muscle after denervation. Reactivity for embryonic and neonatal MHCs was detected in some fibers as early as 3 days after denervation, became more evident by 7 days, and occurred exclusively in the type 2A fiber population. Paralysis of innervated muscles by tetrodotoxin block of the sciatic nerve also resulted in the reappearance of embryonic and neonatal MHCs in type 2A fibers. Significant variation in the degree of immunoreactivity was observed in different segments of the same muscle fiber, suggesting that coordination of muscle fiber nuclei in the control of myosin heavy chain gene expression is partially lost following denervation.

Myosin isoform expression in rat rhabdomyosarcoma induced by Moloney murine sarcoma virus.

Myosin isoform expression was analyzed in experimental rhabdomyosarcoma (RMS) using monoclonal antibodies (mAbs) and immunofluorescence techniques. Tumors induced by inoculating newborn rats with Moloney murine sarcoma virus (Mo-MSV) were examined 30-90 days after birth. Nine tumors and two lymph node metastases were studied by direct, indirect, and double immunofluorescence assays using a panel of five anti-myosin mAbs. The mAb BF-45 was specifically reactive with embryonic myosin heavy chain (MHC), mAb BF-34 was specific for a neonatal MHC epitope, mAb BF-B6 was directed against an epitope present in both embryonic and neonatal MHC, and mAbs BF-F3 and BF-32 detected epitopes present in adult MHC isoforms. Anti-desmin antibodies were also used for comparison. The results of this study show that: (1) the majority of neoplastic cells stained for desmin while only a minority of neoplastic cells were labeled by anti-myosin antibodies; (2) myosin positive tumor cells contained predominantly embryonic and neonatal MHC types but rare RMS cells reacted exclusively with anti-adult myosin antibodies; and (3) adult and embryonic MHC phenotypes were occasionally detected within the same tumor cell especially in RMS with the longest latencies. Together these results would suggest that the mechanism(s) regulating MHC gene expression in skeletal muscle cells can be altered by the transforming activity of Mo-MSV.