Spontaneously active and ATPMg-dependent protein phosphatase activity in vascular smooth muscle

A spontaneously active (Mr greater than 350,000) and an ATPMg-dependent phosphatase (Mr congruent to 140,000) were identified in bovine aortic smooth muscle. The spontaneously active phosphatase was effective in dephosphorylating both phosphorylase a (240nmol32P/min/mg) and phosphorylated myosin light chains (1000nmol32P/min/mg). In contrast, the ATPMg-dependent phosphatase was only effective in dephosphorylating phosphorylase a (400nmol32P/min/mg). Phosphorylase phosphatase activity of the ATPMg-dependent enzyme was suppressed by the well-characterized modulator protein (inhibitor-2), whereas the activity of the spontaneously active enzyme was unaffected. The aortic spontaneously active phosphatase did not convert to an ATPMg-dependent form when it was stored at 4 degrees or incubated at 30 degrees C in either the presence or absence of modulator protein. These findings suggest that spontaneous and ATPMg-dependent phosphatase activities described in these studies are probably ascribable to different enzymes. Since both phosphorylase and myosin light chains are phosphorylated when smooth muscle contracts these phosphatases may participate in coordinating arterial contractility and metabolism.     

The ATPMg-dependent phosphatase is present in mammalian vascular smooth muscle

An ATPMg-dependent phosphorylase phosphatase was identified in vascular smooth muscle from bovine aorta. The smooth muscle enzyme, like the corresponding enzyme from striated muscle, exists as an inactive phosphatase (F_C-enzyme) which can be activated by a protein, F_A, in the presence of ATP and Mg²⁺. Moreover, smooth muscle F_C is activatable by skeletal muscle F_A and skeletal muscle F_C can be activated by smooth muscle F_A. The mode of activation of aortic F_C by aortic F_A is similar to that reported for the skeletal muscle proteins. In accord with earlier findings obtained with the skeletal muscle system, the activity of the aortic phosphatase is inhibited by a specific heat-stable modulator protein (previously called phosphatase inhibitor-2). Thus, the fundamental properties of arterial ATPMg-dependent phosphatase appear to be identical to those of its skeletal muscle counterpart which purportedly represents the major phosphorylase phosphatase in that tissue. Since glycogen phosphorylase is activated when vascular smooth muscle contracts, ATPMg-dependent protein phosphatase may participate in coordinating arterial metabolism and contractility.     

Isoform variants of troponin in skeletal and cardiac muscle cells cultured with and without nerves

Immunofluorescence microscopy shows that cultured skeletal and cardiac muscle cells of chicken embryos exhibit the same stainabilities with antibodies against skeletal and cardiac troponin components as do those in embryos. Muscle cells of each type cultured with motor or sympathetic nerves or in medium containing the nerve extract exhibit the same reactivities as do those in adult animals. Cardiac muscle cells incubated in the nerve-conditioned medium also change the form of troponin components to the adult type. It appears that the differentiation of individual muscle fibers to specific types is induced by nerves, and especially by the neurohumoral effect.     

Altered brain metabolism of testosterone is correlated with reproductive decline in aging quail

In aging quail, an increasing proportion of males show no sexual behavior. A decrease in the mean size of the tests, cloacal gland, and sternotracheal muscles is also observed. In both sexually active and inactive males, plasma testosterone decreases with age but more so in inactive birds. The behavioral and morphological changes observed during aging are correlated with shifts in the intracellular testosterone metabolism resulting in a change in the ratio of active versus inactive metabolites. In the hypothalamus there is a steady decrease with age of 5 beta-reductase activity in all birds and an increase in 5 alpha-reductase activity only in the birds which remain sexually active. In the cloacal gland, the 5 beta-reductase activity markedly increases with age but more so in the birds which become sexually inactive. These data support the notion that the effects of testosterone are controlled by enzymatic shifts which could modulate the sensitivity to the hormone at the cellular level.     

Channel open time of acetylcholine receptors on Xenopus muscle cells in dissociated cell culture

The kinetics of acetylcholine (ACh) receptor channels on cultured myotomal muscle cells from Xenopus embryos were studied by analyzing focally recorded membrane currents. The mean open time for receptor channels on embryonic muscle cells grown in dissociated cell cultures showed a time-dependent decrease similar to that seen in vivo. The changes in power density spectra are consistent with the hypothesis that the decrease results from the appearance of a class of ACh receptor with a short mean channel open time (0.7 msec) and a decrease in the proportion of receptors with a long mean channel open time (3 msec). The addition of dissociated neural tube cells to muscle cell cultures resulted in an unexpected increase in mean channel open time for ACh receptors in both synaptic and nonsynaptic regions. These studies demonstrate that ACh receptor function may be altered in cultured muscle cells.     

Cross-linking of fibronectin to sulfated proteoglycans at the cell surface.

Fibronectin is a major surface protein of normal animal cells but is absent from many transformed cells. Addition of fibronectin to transformed cells causes increased cell substrate adhesion and changes in the morphology and cytoskeleton of the cells. We have coupled fibronectin to photoactivable chemical cross-linkers and have added it to cells to identify those molecules to which it binds. In this way, fibronectin can be cross-linked to sulfated proteoglycans at the cell surface. The cross-linking is specific for fibronectin. The fibronectin-proteoglycan complex is sensitive to chondroitinase ABC and AC and to trypsin. Addition of fibronectin also affects binding of hyaluronic acid to the cells. These results suggest that fibronectin interacts with proteoglycans at the cell surface. The existence of such interactions may have implications for the role of fibronectin and proteoglycans in cell adhesion.     

Stimulation of adenylate cyclase by adenosine and other agonists in mesenteric artery smooth muscle cells in culture

An adenosine-sensitive adenylate cyclase has been characterized in cultured mesenteric artery smooth muscle cells. N-Ethylcarboxamide-adenosine (NECA), N-Methylcarboxamide-adenosine (MECA), L-N6-phenylisopropyladenosine (PIA) and 2-chloroadenosine (2-cl-Ado) all stimulated adenylate cyclase in a concentration dependent manner. NECA was the most potent analog (EC50, 1 microM), whereas PIA (EC50, 15 microM), 2-Cl-Ado (EC50, 15 microM) and MECA (EC50, 24 microM), were less potent and had efficacies relative to NECA of 0.61, 0.61 and 0.65, respectively. Adenosine showed a biphasic effect: stimulation at lower concentrations and inhibition at higher concentrations, whereas 2' deoxyadenosine only inhibited adenylate cyclase activity. The stimulatory effect of NECA on adenylate cyclase was dependent on metal ion concentration and was blocked by 3-isobutyl-l-methylxanthine (IBMX) and 8-phenyltheophylline (8-PT). Adenylate cyclase from these cultured cells was also stimulated by other agonists such as epinephrine, norepinephrine, prostaglandins, dopamine, NaF and forskolin. The stimulation of adenylate cyclase by isoproterenol, epinephrine and norepinephrine was blocked by propranolol but not by phentolamine. On the other hand, phentolamine, propranolol and flupentixol all inhibited dopamine-stimulated adenylate cyclase activity. In addition, the stimulation by an optimal concentration of PIA was additive or almost additive with maximal stimulation caused by catecholamines and prostaglandins. These data indicate the presence of adenosine (Stimulatory "Ra"), catecholamine and prostaglandin receptors in mesenteric artery smooth muscle cells and suggest that these agents may exert their physiological actions through their interaction with their respective receptors coupled to adenylate cyclase.     

Arachidonate metabolism in bovine gallbladder muscle

Incubation of [1-¹⁴C]arachidonic acid (AA) with homogenates of bovine gallbladder muscle generated a large amount of radioactive material having the chromatographic mobility of 6-keto-PGF_1α (stable product of PGI₂) and smaller amounts of products that comigrated with PGF_2α and PGE₂. Formation of these products was inhibited by the cyclooxygenase inhibitor indomethacin. The major radioactive product identified by thin-layer chromatographic mobility and by gas chromatography - mass spectrometric analysis was found to be 6-keto-PGF_1α. The quantitative metabolic pattern of [1-¹⁴C]PGH₂ was virtually identical to that of [1-¹⁴C]AA. Incubation of arachidonic acid with slices of bovine gallbladder muscle released labile anti-aggregatory material in the medium, which was inhibited by aspirin or 15-hydroperoxy-AA.These results indicate that bovine gallbladder muscle has a considerable enzymatic capacity to produce PGI₂ from arachidonic acid.     

Synthesis of type IV collagen and laminin in cultures of skeletal muscle cells and their assembly on the surface of myotubes

The synthesis of two components of the basal lamina, laminin and type IV collagen, and their extracellular deposition on the surface of myotubes was studied in cultures of embryonic mouse and quail skeletal muscle cells and in the rat myoblast cell line L6. Production of type IV collagen and laminin by myoblasts and muscle fibroblasts was demonstrated by incorporation of radioactive amino acids into proteins and by immunoprecipitation with specific antibodies and electrophoretic analysis of labeled proteins. Immunofluorescence staining experiments revealed strong intracellular reactions with antibodies to laminin and type IV collagen in mononucleated myogenic and fibrogenic cells. Cells of fibroblast-like morphology showed a more intense staining than bipolar, spindle-shaped cells which perhaps represented postmitotic myoblasts. Myotubes did not show detectable intracellular staining. The formation of a basal lamina on myotubes was indicated by the deposition of laminin and type IV collagen on the surface of myotubes as viewed by immunofluorescence examination of unfixed cells. Staining for extracellular laminin was stronger in mass cultures than in myogenic clones, suggesting that secretion and deposition of components of the basal lamina on the myotube surface are complex processes which may involve cooperation between myogenic and fibrogenic cells.     

Temporal changes in embryonic nerve cell recognition: correlate with cholinergic development in aggregate cultures

Chick embryo retina and optic tectum cells can be dissociated into single cells and then reaggregated in suspension cultures to give highly organized and differentiated aggregates. These aggregates show a degree of cholinergic differentiation that is characteristic of each cell type; the low activity of choline acetyltransferase in the optic tectum aggregates probably reflects the condition of natural deafferentation inherent in the culture situation. It is possible, in this respect, to study the retina-tectum interaction in vitro by preparing coaggregates including both types of cells. When coaggregates are prepared from tectum and retina cells of the same developmental age, the activity of choline acetyltransferase measured in the coaggregates is consistently higher than would be expected from the simple addition of the activities of the component cells, pointing to some kind of metabolic synergism between retinal and tectal cells. As for acetylcholinesterase, this synergism occurs only under special circumstances, and it is generally less marked. No synergism was observed when retina and tectum cells of different developmental age were coaggregated, suggesting the existence of a temporal control of neuronal interaction specificity. On the other hand, the synergism is only observed between neuronal systems that are known to establish synaptic connections during normal in vivo development: No interaction could be detected when either retinal or tectal cells were combined with telencephalon, cerebellum, or liver cells. Experimental evidence is presented suggesting that the retina-tectum interaction depends on intimate cell-cell contact, and it is not mediated by freely diffusible molecules. Neurotransmission-related metabolic studies in coaggregates seem to offer a promising tool to study recognition-interaction phenomena in groups of neurons establishing synaptic links during development.     

Ligand-induced alterations in the reactivity of sulfhydryl groups and the structure of bovine liver glutamate dehydrogenase

The fluorogenic probe 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-C1) was employed as an environmentally sensitive reporter label for free sulfhydryl groups of bovine liver glutamate dehydrogenase. A maximum of six -SH groups per subunit was titrated in Tris and borate buffers (pH 7.8) in 2 h but there was no reaction in the presence of phosphate buffer. The rate and extent of -SH reactivity was changed significantly by one of the substrates and some allosteric effectors. Adenosine nucleotides, NADH, and α-ketoglutarate promoted conformational alterations in glutamate dehydrogenase such that -SH groups were rendered virtually unreactive in [enzyme-ADP], [enzyme-NADH], and [enzyme-α-ketoglutarate]binary complexes. GTP, a negative allosteric modulator, showed no effect on -SH exposure. Measurements of protein circular dichroism spectra and catalytic activity in conjunction with -SH reactivity demonstrated a direct relationship between structural stability, biological activity, and ligand-induced conformational changes. The ligands that strongly protected the enzyme from reaction with NBD-C1 concomitantly maintained its structural and functional integrity.     

The synthesis and deployment of filamin in chicken skeletal muscle

During myogenesis in vitro the actin-binding protein filamin is present in myoblasts and early fused cells and is associated with α-actinin-containing filament bundles, as judged by double immunofluorescence using antibodies specific for these two proteins. Approximately one day after cell fusion, yet before the development of a-actinin-containing Z line striations, filamin disappears from the cells. Later in myogenesis, several days after the appearance of α-actinin-containing Z line striations, filamin reappears and accumulates in the cells. Double immunofluorescence with antibodies to filamin and vimentin (or desmin) reveals that the newly appearing filamin localizes now to the myofibril Z line and is visible there shortly before vimentin or desmin becomes associated with the Z line. Immunofluorescent localization of filamin in isolated chicken skeletal myofibrils and Z disc sheets indicates that filamin has the same distribution as desmin and vimentin; it surrounds each myofibril Z disc and forms honeycomb-like networks within each Z plane of the muscle fiber. Filamin may thus be involved in the transition of desmin and vimentin to the Z disc. Analysis of whole-cell extracts by SDS-polyacrylamide gel electrophoresis and by immunoautoradiography shows that filamin is present in myoblasts and in myotubes early after cell fusion. Concomitant with the absence of filamin fluorescence during the subsequent few days of myogenesis, the quantity of filamin is markedly reduced. During this time, metabolic pulse-labeling with ³⁵S-methionine reveals that the synthetic rate of filamin is also markedly reduced. As filamin fluorescence appears at the Z line, the quantity of filamin and its synthetic rate both increase. The removal of filamin from the cells suggests that filamin either may not be required, or may actually interfere with a necessary process, during the early stages of sarcomere morphogenesis. These results also indicate that the periphery of the Z disc is assembled in at least two distinct steps during myogenesis.     

Structural investigations of an arabinan from grape juice

An arabinan has been isolated from grape juice and purified by chromatography on polyamide and repeated ethanol precipitations. The structural identity of the arabinan was established by enzymatic degradation of the polysaccharide with a purified -L-arabinofuranosidase and methylation analysis. The results obtained suggest that the arabinan consists of an (1 → 5)-linked backbone of L-arabinofuranosyl residues to which sidechains of L-arabinose are attached in the 3-position.     

Peroxide induced activation of glycogen phosphorylase A activity in vascular smooth muscle

1-Phenoxy-2-propanone, 1-chloro-3-phenoxy-2-propanone, and 1-fluoro-3-phenoxy-2-propanone are competitive acetylcholinesterase inhibitors with K_I values of 30, 0.85, and 2.2 μM, respectively, compared to 2 mM for 4-phenyl-2-butanone. The substituent effect on inhibition suggests that these compounds bind by formation of a tetrahedral adduct and are transition state analogs.Other evidence supports this conclusion: N-benzyl-2-chloroacetamide and 1-phenoxy-2-propanol are poor inhibitors (K_I = 11 and >10 mM); 1-phenoxy-2-propanone and 1-chloro-3-phenoxy-2-propanone have K_I values 330 and 140 times smaller than K_m for corresponding substrates; and 1-chloro-3-phenoxy-2-propanone protects the enzyme against irreversible inhibition by CH₃SO₂F.     

Further characterization of the cells involved in leukocyte adherence inhibition with murine tumor extracts

The incidence of spontaneous primary lymphoma following adult thymectomy, irradiation, and bone marrow reconstitution was studied in SJL/J mice. A significantly higher incidence of generalized lymphoma was demonstrated in γ-irradiated and bone marrow-reconstituted (XBM) mice as compared to thymectomized, γ-irradiated, and bone marrow-reconstituted (ATx-XBM) mice. Including both localized and generalized lymphomas, ATx-XBM mice showed about half the incidence of XBM mice at 12 months of age. In comparison of XBM vs untreated controls, a much lower incidence of spleen involvement was noted in XBM mice than in untreated controls.     

Regulation of fibronectin biosynthesis by glucocorticoids in human fibrosarcoma cells and normal fibroblasts

When treated with the synthetic glucocorticoid dexamethasone, HT1080 human fibrosarcoma cells show changes in morphology, adhesion, and the extracellular matrix. Dexamethasone treatment results in a tenfold increase in the rate of fibronectin biosynthesis in HT1080 cells and a twofold increase in untransformed, normal human fibroblasts. Maximal induction levels are attained within one cell generation, while decay of the response requires several cell cycles. Pulse-chase studies showed that most of the newly synthesized fibronectin is secreted into the medium. The glucocorticoid antagonist, RU-486, blocks the dexamethasone-induced changes but does not alter the basal rate of fibronectin production. Therefore, fibronectin biosynthesis appears to be controlled by two distinct mechanisms--one, regulating basal rates of fibronectin production, which is transformation-sensitive and glucocorticoid-independent; and another, which is mediated by the glucocorticoid receptor, resulting in elevated rates of fibronectin biosynthesis upon dexamethasone treatment both in normal fibroblasts and in HT1080 cells.     

The association of suppressor cells with mononuclear cell aggregates in spleens of tumor-bearing mice

Spleen cell suspensions from mice with progressive B-16 melanoma consistently contained significant numbers of aggregates of mononuclear cells (MN-Agg), when compared to spleen cell suspensions from normal mice or mice in the early stages of tumor growth. Histological, histochemical and immunological characterization of the cells involved in MN-Agg from tumor-bearing mice indicated that aggregates were composed of macrophages and T and B lymphocytes. The formation of MN-Agg was dependent upon the macrophage content of the spleens of tumor-bearing mice since the appearance of MN-Agg correlated temporally with an increase in the number of splenic macrophages demonstrable in tumor-bearing animals. An antigen nonspecific suppressor cell was identified in the spleens of mice 15 days following the appearance of palpable B-16 tumor, and the appearance of the suppressor cell population closely correlated with the appearance of MN-Agg. Additionally, fractionation of MN-Agg-containing cell suspensions demonstrated that fractions highly enriched in MN-Agg were concomitantly enriched for suppressor cells. The suppressor cell associated with MN-Agg was a T lymphocyte since suppressor activity of MN-Agg could be abolished by treatment of MN-Agg with a rabbit anti-mouse brain serum and complement. It is proposed that the generation of suppressor cells in mice with B-16 melanoma may require specific interaction between macrophages and lymphocytes which is manifested in the spleens of tumor-bearing mice by the formation of MN-Agg.     

Purified desmin from adult mammalian skeletal muscle: a peptide mapping comparison with desmins from adult mammalian and avian smooth muscle.

Comparative one-dimensional peptide maps were prepared by the electrophoresis of digests derived from treatment of desmins with Ca²⁺-activated muscle protease, trypsin, $\text{Staphylococcus}\text{aureus}$ V8 protease, and cyanogen bromide. Desmins from adult mammalian skeletal and smooth muscles were very similar. Avian smooth muscle desmin, although homologous with respect to many peptides, was different from the mammalian smooth and skeletal desmins. The amino acid compositions of the three desmins were quite similar.