Regulation of phospholamban and troponin-I phosphorylation in the intact rat cardiomyocytes by adrenergic and cholinergic stimuli: roles of cyclic nucleotides,calcium, protein kinases and phosphatases and depolarization

Protein phosphorylation was investigated in [³²P]-labeled cardiomyocytes isolated from adult rat heart ventricles. The -adrenergic stimulation (by isoproterenol, ISO) increased the phosphorylation of inhibitory subunit of troponin (TN-I), C-protein and phospholamban (PLN). Such stimulation was largely mediated by increased adenylyl cyclase (AC) activity, increased myoplasmic cyclic AMP and increased cyclic AMP dependent protein kinase (A-kinase)-catalyzed phosphorylation of these proteins in view of the following observations: (a) dibutyryl-and bromo-derivatives of cyclic AMP mimicked the stimulatory effect of ISO on protein phosphorylation while (b) Rp-cyclic AMP was found to attenuate ISO-dependent stimulation. Unexpectedly, 8-bromo cyclic GMP was found to markedly increase TN-I and PLN phosphorylation. Both ₁- and ₂-adrenoceptors were present and ISO binding to either receptor was found to stimulate myocyte AC. However, the stimulation of the ₂-AR only marginally increased while the stimulation of ₁-AR markedly increased PLN phosphorylation. Other stimuli that increase tissue cyclic AMP levels also increased PLN and TN-I phosphorylation and these included isobutylmethylxanthine (non-specific phosphodiesterase inhibitor), milrinone (inhibits cardiotonic inhibitable phosphodiesterase, sometimes called type III or IV) and forskolin (which directly stimulates adenylyl cyclase). Cholinergic agonists acting on cardiomyocyte M₂-muscarinic receptors that are coupled to AC via pertussis toxin(PT)-sensitive G proteins inhibited AC and attenuated ISO-dependent increases in PLN and TN-I phosphorylation. Thein vivo PT treatment, which ADP-ribosylated G_i-like protein(s) in the myocytes, markedly attenuated muscarinic inhibitory effect on PLN and TN-I phosphorylation on one hand and, increased the -adrenergic stimulation, on the other. Controlled exposure of isolated myocytes to N-ethyl maleimide, also led to the findings similar to those seen following the PT treatment. Exposure of myocytes to phorbol, 12-myristate, 13-acetate (PMA) increased the protein phosphorylation, augmenting the stimulation by ISO, and such augmentation was antagonized by propranolol suggesting modulation of the -adrenoceptor coupled AC pathway by PMA. Okadaic acid (OA) exposure of myocytes also increased protein phosphorylation with the results supporting the roles for type 1 and 2A protein phosphatases in the dephosphorylation of PLN and TN-I. Interestingly OA treatment attenuated the muscarinic inhibitory effect which was restored by subsequent brief exposure of myocytes to PMA. While the stimulation of alpha adrenoceptors exerted little effect on the phosphorylation of PLN and TN-I, inactivation of alpha adrenoceptors by chloroethylclonidine (CEC), augmented -adrenergically stimulated phosphorylation. KCl-dependent depolarization of myocytes was observed to potentiate ISO-dependent increase in phosphorylation (incubation period 15 sec to 1 min) as well as to accelerate the time-dependent decline in this phosphorylation seen upon longer incubation. Verapamil decreased ISO-stimulated protein phosphorylation in the depolarized myocytes. Depolarization was found to have little effect on the muscarinic inhibitory action on phosphorylation. Prior treatment of myocytes with PMA, was found to augment ISO-stimulated protein phosphorylation in the depolarized myocytes. Such augmented increases were completely blocked by propranolol. Forskolin also stimulated PLN and TN-I phosphorylation. Prior exposure of myocytes to forskolin followed by incubation in the depolarized and polarized media showed that PLN was dephosphorylated more rapidly in the depolarized myocytes. The results support the view that both cyclic AMP and calcium signals cooperatively increase the rates of phosphorylation of TN-I and PLN in the depolarized cardiomyocytes during -adrenergic stimulation. The results raise the additional possibility that the calcium signal may regulate the dephosphorylation of PLN in the depolarized cell. While muscarinic attenuation of -adrenergic action on protein phosphorylation was mediated, in part, by decreased AC activity, and muscarinic inhibition of AC and protein phosphorylation was not detectably influenced by the depolarization, the evidence was seen that muscarinic stimulation of dephosphorylation mechanisms are intimately involved. The postulate that the simultaneous stimulation of ₁-adrenoceptors inhibits -adrenergic stimulation of PLN and TN-I phosphorylation is supported.     

Artificial seeding of the green seaweed Monostroma for cultivation

In Japan, the green seaweed Monostroma is an important source of humanfood. Monostroma nitidum Wittrock (Japanese name: hitoegusa) is cultivated in brackish waters and estuaries of central to southern Japan. The green seaweed Monostroma grows in the brackish water area in the upper part of the intertidal zone in the warm waters. Artificial seed culture began with the collection of many gametes in April. The resultant zygotes were allowed to adhere to plastic settlement boards (20 cm long and 10 cm wide). The zygoteboards were then cultured in tanks (1 ×2 ×0.5 m) with fertiliser in a controlled growth room (10–87 μmol photon m^-2s^-1). The cultivated zygotes on the board in the indoor tanks gradually increased in size from 10 to 40 μm in diameter during May to early August. Zygote growth became slowed at the end of August. The zygotesmatured in early September, and the plates were transferred into culture tanks in a dark room for dark treatment. Maturation of the zygote was promoted by providing dark conditions for two weeks. The production of a concentrated zoospore solution from the mature blades was achieved by adding fresh water at temperature 2–3 °C above that of the seeding vats. Zoospores were released in large numbers when exposed to strong irradiance of 100 μmol photon m^-2 s^-1 for 30 min. The zygotes produced flat unicellular fronds at the germling stage. The technology of artificial seed culture and zoospore release from the zygotes is based mainly on these experiments. This revised version was published online in August 2006 with corrections to the Cover Date.     

Test systems for biomonitoring based on membrane-bound enzyme complexes. IV. Assessment of genotoxic effects in an Ames test system with metabolic activation by the microsomal mono-oxygenases from fish livers

The study of mutagenic effect of 2-aminoantracene and benz(alpha)pyrene on Salmonella triphimurium TA 100 in the Ames test-system in the presence of postmitochondrial fractions S-9 from carp liver with 3-methylcholantrene induced by microsomal oxidation system has been carried out. The metabolic activity and cytochrome P450 contence in carp liver microsomes have been shown to concede considerably those in rats liver. But these characteristics are sufficient for the use of fraction S-9 from carp liver for the study of genotoxic effect of these xenobiotics in the Ames test-system. Several regimes of storage of S-9 preparations from carp liver have been compared. S-9 preparations frozen immediately after isolation preserve their metabolic activity with respect to 2-aminoantracene and benz(alpha)pyrene well.     

Computer analysis of two-dimensional gels

New methods and computer programs are described which enable one to analyze autoradiograms produced by two-dimensional gel electrophoresis. These programs are completely automatic with respect to finding spots resolved by such gels and quantitating the radioactivity in them. Semiautomatic programs have also been developed to match the spot patterns of different autoradiograms, and to follow the synthesis of any individual polypeptide through a series of gels.     

Electron-cytochemical localization of phospho(enol)pyruvate carboxylase (EC 4.1.1.31) in fungal cells

Summary New cytochemical method, based on biochemical experiments, was elaborated for the ultrastructural localization of phospho(enol)pyruvate carboxylase (EC 4.1.1.31). The procedure was used to study the saprophytic submerged mycelium of the ascomycetous fungusClaviceps purpurea Tul. producing clavine alkaloids. The pelleted mycelium was fixed in ice cold 3% glutaraldehyde in 50 mM cacodylate buffer pH 7.2 and washed repeatedly in the same cold buffer. The reaction mixture contained 100 mM Tris-HCl buffer pH 9.0, 10 mM phospho(enol)pyruvate, 30 mM sodium potassium tartrate, 3 mM Pb(NO₃)₂, 60 mM MgCl₂ and 30 mM NaHCO₃. Enzyme activity was localized in vacuoles, particularly inside lipid globules (spherosomes) and less frequently in membranous vesicles. Acetyl-CoA activated PEP-carboxylase both in cell free extracts and in the cytochemical staining. Aspartate inhibited the enzyme in the biochemical assay with coupled malate dehydrogenase system; the cytochemical reaction was not influenced, probably due to the interference of asparagine synthase (EC 6.3.1.1).     

Electron-microscopic localization of acetyl coenzyme A carboxylase in cells of Claviceps purpurea

Summary The ultrastructural localization of acetyl-CoA carboxylase activity was studied in two strains of the ascomycetous fungus Claviceps purpurea differing in the ergot alkaloid synthesis. Mycelia were harvested by centrifugation of saprophytic submerged cultures, fixed in cold 3% glutaraldehyde in 0.05 M cacodylate buffer pH 7.2 and washed repeatedly in the same buffer. The incubation medium of Yates et al. (1969) had to be modified in the molarity of ATP. The best results were obtained with a medium of the following composition: 50 mM cacodylate buffer pH 7.2, 4 mM ATP, 3.5 mM lead nitrate, 13.5 mM sodium citrate, 3.75 mM sodium bicarbonate, 1.25 mM manganese chloride, 0.4 mM acetyl-CoA and 2 mM biotin. The fixation is a prerequisite for a distinct localization. The enzyme activity was detected only in cells producing high amount of clavine alkaloids. It was confined to the membranes of endoplasmic reticulum and their derivatives: tonoplast of vacuoles, tiny vesicles and amorphous material inside vacuoles. The reaction product was very fine and localized in both leaflets of the membranes. The specificity of the reaction was confirmed by negative results in control preparations: boiled cells incubated in the complete medium, cells incubated in the medium supplemented with avidin or in the media from which either ATP, or acetyl CoA, or sodium bicarbonate, or biotin were omitted. It is suggested that the activity of acetyl-CoA carboxylase is linked to the synthesis of clavine alkaloid precursors which occurs in the endoplasmic reticulum and its derivatives.With technical assistance of J. Martínková     

Further studies on polyploid amphibians

The manner in which centromere regions of mitotic chromosomes are distributed with respect to the age of their DNA was studied. Cells of the Indian deer, Muntiacus muntjak, were grown in the presence of bromodeoxyuridine (BrdU) for two generations and stained with the fluorescent dye Hoechst 33258. Chromatids containing granddaughter DNA appear dim when compared with those containing grandparental DNA. The frequencies of the various anaphase patterns of bright and dim centromere regions were binomially distributed, indicating random distribution of chromatids with respect to the age of their DNA templates.     

Origin of sclerotia-like cells in submerged Claviceps purpurea producing clavine alkaloids

Ultrathin sectioning of submerged mycelium of Claviceps purpurea Tul. producing clavine alkaloids revealed yeast-like budding resulting in asexual sporesblastospores. These deciduous spores were born by extended hyphal cells and retained the same ultrastructure of cell organelles. Both the extended hyphae and the blastospores resembled the cells of ergot sclerotial tissue. A surface culture of C. purpurea Tul. producing no alkaloids was used as a reference.     

Heterogeneity of populations of the mollusc Bithynia inflata according to the degree of trematode infestation in opisthorchiasis foci

Results of studies of differences in the infection rate of natural populations of Bithynia inflata with trematodes (including Opisthorchis felineus) in the nidi of opisthorchiasis in the Sumsk and Chernigov Districts of the Ukrainian SSR and Tomsk District of the RSFSR are given. It has been shown that in the Ukrain over 1/3 of all populations of the specific intermediate host of opisthorchis does not take part in the circulation of the agent due to fundamentally different reasons: resistence to the infection and hyperinfection with parthenites of trematodes.