Differential antimitogenic effectiveness of atrial natriuretic peptides in primary versus subcultured rat aortic smooth muscle cells: Relationship to expression of ANF-C receptors

Previous studies have shown that atrial natriuretic peptides inhibit mitogenesis in subcultured aortic smooth muscle cells by a mechanism that appears to be mediated via the C-type or “clearance” receptor. In the current study, we have compared the antimitogenic effect of these peptides in serum-stimulated primary aortic smooth muscle cell cultures and in subcultured cells. A series of atrial peptides, including rANF_99–126, rANF_103–126, and rANF_103–125, were only poorly antimitogenic in serum-stimulated primary cultures, whereas des[Cys¹⁰⁵, Cys¹²¹] rANF_104–126 which binds selectively to the ANF-C receptors had no antimitogenic activity. In contrast, in subcultured cells (between subcultures 3 and 25), rANF_99–126, rANF_103–126, rANF_103–126, Cys¹¹⁶rANF_102–116, and des[Cys¹⁰⁵, Cys¹²¹]rANF_104–126 inhibited serum-induced [³H]thymidine incorporation (IC₅₀ in the range of 10–50 nM), with maximal inhibition of 40–70%. The lack of antimitogenic activity in primary cultures did not appear to be related to the lack of cGMP elevation elicited by atrial peptides or to an inherent insensitivity to the action of antimitogens, because primary cultures were responsive to the cGMP-elevating effect of atrial peptides and the cells were more rather than less sensitive to the antimitogenic effect of the nitric-oxide-vasodilator, SNAP, as compared to subcultured cells. Analysis of the affinity and binding capacity of freshly isolated aortic membranes, and primary or secondary cultures for [¹²⁵I]rANF_99–126, revealed that the number of ANF receptors increased by tenfold, following subculture. Moreover, subcultured cells contained receptors with increased binding affinity for peptide analogues selective for the ANF-C-type type receptor. Covalent cross-linking studies with (¹²⁵I)rANF_99–126 confirmed that membranes prepared from fresh aortae predominantly expressed the ANF-A/guanylate cyclase receptor, whereas in subcultured cells the predominantly cross-linked protein was the ANF-C-type receptor, with receptors in primary cultures occupying an intermediate position. These results suggest that the binding and antimitogenic activity of atrial peptides in aortic smooth muscle cells depends on the phenotypic state of these cells. Moreover, the increased antimitogenic potency of atrial peptides in secondary cultures may reflect increased expression of the ANF-C-type receptors. © 1993 Wiley-Liss, Inc.     

Vesicular Quantal Size Measured by Amperometry at Chromaffin, Mast, Pheochromocytoma, and Pancreatic β-Cells

Abstract: Amperometric detection of exocytosis at single chromaffin cells has shown that the distribution of spike areas, or quantal size, is dependent on the volume and catecholamine concentration of individual secretory vesicles. The present work offers an alternate, simplified model to analyze the current spikes due to single exocytotic events. When the cube root of these spike areas is plotted as a histogram, a Gaussian distribution is obtained for chromaffin cells and also mast, pheochromocytoma, and pancreatic β-cells. It was found that the relative SD of these distributions is similar to that for the vesicular radii, which also have a Gaussian distribution in all four cell types. In addition, this model was used to evaluate conditions where the quantal size of individual events was altered. When chromaffin cells were maintained in culture for <6 days, spikes of approximately double the quantal size were obtained on repeated exposure to 60 m M K⁺. The results suggest a heterogeneous distribution of catecholamine-containing vesicles at later days in culture is responsible for this alteration.     

Clearance receptor-binding atrial natriuretic peptides inhibit mitogenesis and proliferation of rat aortic smooth muscle cells.

The current studies were designed to explore the effects of C-receptor-binding atrial natriuretic peptide analogues on serum-induced mitogenesis in cultured rat aortic smooth muscle cells. To this end, rANF99-126 and a series of truncated (rANF103-126, rANF103-125), ring-deleted (des[Gln116, Ser117, Gly118, Leu119, Gly120]rANF102-121-NH2 (c-ANF) and linear des(Cys105, Cys121)rANF104-126 peptide analogues were used. The latter two peptides have been reported to be selective for the ANF-C receptor. In cells subcultured between passage 3 to 19, rANF99-126, rANF103-126, and rANF103-125 concentration-dependently (0.1-1000 nM) inhibited serum-induced (3H) thymidine incorporation with maximal inhibition observed at 1 microM for each peptide (approximately 40, 31 and 56%) respectively. Furthermore, des[Cys105, Cys121]rANF104-126 inhibited serum-induced (3H)thymidine incorporation concentration-dependently without altering basal or elevated cellular cAMP or cGMP levels. Moreover, the reduction in thymidine incorporation was associated with inhibition of serum-induced clonal cell proliferation. In contrast, c-ANF failed to inhibit serum-induced mitogenesis, yet at a concentration of 100 nM it antagonized the antimitogenic effects of des[Cys105, Cys121]rANF104-126 or rANF99-126 without having any effect on basal or elevated cellular cyclic nucleotide levels. We conclude that the antimitogenic effect of atrial peptides is mediated through interaction with the ANF-C receptor and may be independent of changes in cellular cyclic nucleotide levels.     

Elimination and subsequent metabolism of circulating hyaluronic acid in the fetus

Hyaluronic acid differs from other glycosaminoglycans in its lack of covalently linked peptide, absence of sulphate groups, and the exceptional size of its single-chain polymers. These differences can be related to its distinct physical and functional properties, and may be pertinent to its greater abundance in early tissue development. In mature animals, the turnover of hyaluronic acid in tissues is reflected at least partly in the blood stream. The metabolism of circulating hyaluronic acid was therefore studied in fetal sheep after intravenous injection of [3H]acetylhyaluronic acid. Between 95% and 99% was removed within 6 min. Plasma radioactivity decayed by first-order kinetics, with a half-life between 0.8 and 1.25 min. The rate of elimination did not vary materially with hyaluronic acid fractions of widely disparate average Mr or with fetal age between 70 and 120 days. 3H2O was detected in plasma within 8-10 min. Labelled material found in urine from 10 min onward included polymers greater than or equal to 70,000 Mr, which indicates that urine may be a source of hyaluronic acid in amniotic fluid. Elimination from the plasma took place mainly in the liver, where labelled material was largely recovered in small metabolic residues as early as 28 min after injection. These were shown by high pressure liquid chromatography (h.p.l.c.) to include water, acetate, N-acetylglucosamine and a fraction tentatively identified as N-acetylglucosamine 1-phosphate. Tritium radioactivity was detected in hepatic lipids but not those of the spleen. Estimated plasma turnover was in the order of 10 micrograms/min per kg body weight. This is about 3-10 times that in adult animals and is consistent with an increased inflow of hyaluronic acid generated during the maturation of developing tissues.     

Spontaneous rosette-forming lymphocytes in sheep lymph. A marker for antigen-binding cells

Type O Rh positive human red blood cells (HRBC), native or treated with one of three enzymes (papain, trypsin, or neuraminidase), were labeled with ⁵¹Cr and then sensitized with anti-Rh immune globulin. These cells served as targets in antibody-dependent cellular cytotoxicity (ADCC) for unfractionated human mononuclear cells (MC), MC depleted of monocytes by adhesion to plastic, and MC enriched for monocytes. Enzyme-treated HRBC were lysed with greater efficiency in ADCC than native HRBC. This was explained by the finding that the enzyme modified HRBC were lysed both by lymphocytes and monocytes, whereas native HRBC were lysed only by monocytes. The lysis of native HRBC was strongly inhibited by small amounts of human serum or free IgG. In contrast, the lysis of enzyme-treated HRBC was considerably more resistant to inhibition by human serum or free IgG. The enhanced lysis of enzyme-treated HRBC could not be the result of increased binding of antibody to the target cells, since augmented lysis was observed both for HRBC sensitized before neuraminidase treatment as well as for HRBC sensitized after neuraminidase treatment. These results suggest that the surface charge on target cells plays a critical role in determining which classes of leukocytic effector cells are active in ADCC systems.     

1H, 13C, 15N backbone NMR assignments of the Staphylococcus aureus small multidrug-resistance pump (Smr) in a functionally active conformation

The plasmid-encoded small multidrug resistance pump from S. aureus transports a variety of quaternary ammonium and other hydrophobic compounds, enhancing the bacterial host’s resistance to common hospital disinfectants. The protein folds as a homo-dimer of four transmembrane helices each, and appears to be fully functional only in lipid bilayers. Here we report the backbone resonance assignments and implied secondary structure for ²H¹³C¹⁵N Smr reconstituted into lipid bicelles. Significant changes were observed between the chemical shifts of the protein in lipid bicelles compared to those in detergent micelles.     

Preventing ascidian fouling in aquaculture: screening selected allelochemicals for anti-metamorphic properties in ascidian larvae

Fouling by ascidians causes major stock losses and disrupts production in marine aquaculture, especially bivalve aquaculture. Currently, no cost effective solution exists despite the testing of many prospective control techniques. This study examined a range of allelochemicals suspected to inhibit metamorphosis in marine larvae. Five allelochemicals were screened in a larval metamorphosis bioassay using Ciona savignyi Herdman to determine their potential as a remedy for ascidian fouling in bivalve aquaculture. Three of the compounds tested inhibited ascidian larval metamorphosis and increased mortality at low concentrations. These were radicicol (99% inhibition of metamorphosis [IC??], 0.8 μg ml?1; 99% lethal concentration [LC??], 2.5 μg ml?1; 99% lethal time [LT??], 7.0 days), polygodial (IC??, 0.003 μg ml?1; LC??, 0.9 μg ml?1; LT??, 6.4 days), and ubiquinone-10 (IC??, 3.2 μg cm?2; LC??, 14.5 μg cm?2; LT??, 5.6 days; expressed as μg cm?2 due to insolubility in water and ethanol). While spermidine significantly affected metamorphosis and mortality of C. savignyi, the effect was insufficient to achieve inhibition in 99% of larvae over the 7-day timeframe of the assay. Muscimol did not affect metamorphosis or mortality at the concentrations tested. The present study demonstrates that radicicol, polygodial and ubiquinone-10 have potential for future development in antifoulant formulations targeted towards the inhibition of metamorphosis in ascidian larvae, while spermidine and muscimol appear unsuitable.