Acetylcholine Releases ATP from Varicosities Isolated from Guinea Pig Myenteric Plexus

The effects of cholinergic agonists and antagonists on the release of ATP from isolated myenteric varicosities were studied using a firefly luciferin-luciferase technique. In a previous study, acetylcholine and nicotine released ATP from isolated myenteric varicosities, whereas the muscarinic agonist bethanechol did not. In the present study, release of ATP by acetylcholine was shown to be Ca2+ dependent. d-Tubocurarine competitively antagonized the release of ATP by either acetylcholine or nicotine. Maximal release of ATP by acetylcholine (10(-3) M) was approximately 24% that observed with the depolarizing drug veratridine (5 X 10(-5) M), suggesting either that not all of the varicosities capable of releasing ATP possess nicotinic receptors or that acetylcholine does not depolarize the varicosities to the degree that veratridine does. Tetrodotoxin slightly but significantly reduced ATP release induced by acetylcholine or nicotine, indicating some involvement of Na+ channels in the release process. Finally, 6-hydroxydopamine pretreatment produced a 48% reduction in the acetylcholine-evoked release of ATP, suggesting that much, but possibly not all, of the ATP release occurs from noradrenergic varicosities present in the preparation.     

A unique DR-related B cell differentiation antigen

The Ia or class II molecules in both mouse and man are the basis for the genetic control of the immune response. In addition to DR, other class II antigens have been described in man. We describe a new human Ia antigen K19, recognized by three monoclonal antibodies (HK-9, HK-19, and HK-20). This antigen has the general biochemical characteristics of an Ia antigen but is different from a DR antigen. Further, this antigen is found only on mature B lymphocytes and not on monocytes and activated T cells. Thus, this antigen may represent a new Ia-like molecule that is preferentially expressed on mature B cells.     

In vivo interactions of acrylonitrile with macromolecules in rats

The irreversible binding of [2,3-14C]acrylonitrile (VCN) to proteins, RNA and DNA of various tissues of male Sprague-Dawley rats after a single oral dose of 46.5 mg/kg (0.5 LD50) has been studied. Proteins were isolated by chloroform-isoamyl alcohol-phenol extraction. RNA and DNA were separated by hydroxyapatite chromatography. Binding of VCN to proteins was extensive and was time dependent. Radioactivity in nucleic acids was registered in the liver and the target organs, stomach and brain. DNA alkylation, which increased by time, was significantly higher in the target organs, brain and stomach (119 and 81 pmol/mg, respectively, at 24 h) than that in the liver. The covalent binding indices for the liver, stomach and brain at 24 h after dosing were, 5.9, 51.9 and 65.3, respectively. These results suggest that VCN is able to act as a multipotent carcinogen by alkylation of DNA in the extrahepatic target tissues, stomach and brain.     

Studies on chromatin-associated protein phosphokinase of submandibular gland from isoproterenol-treated rats

Water-soluble chromatin from rat submandibular gland nuclei was isolated, and had a DNA: RNA:protein ratio of 8:1:20. The spectral properties of this preparation were similar to those described for chromatins from other tissues. The rat submandibular gland chromatin possessed protein phosphokinase activity. It was able to incorporate ³²P from [γ-³²P]-ATP into chromatin proteins, and into dephospho-phosvitin. The chromatin-associated protein phosphokinase activity (measured with dephospho-phosvitin as substrate) required Mg²⁺, Na⁺ or K⁺ and dithiothreitol for optimal activity. A single injection of isoproterenol influenced the activity of this enzyme system, so that it was decreased at 2 h, showed a transient increase at 4 h, and a large increase at 10–16 h after the injection. This event appears to precede the increase in ribosomal RNA induced by Ipr [13]. By 48 h the chromatin-associated protein kinase returned to the normal control levels. These changes appeared to be commensurate with the corresponding alterations in the non-histone acidic protein complement of these chromatins. Actinomycin D or cycloheximide, when administered 30 min prior to isoproterenol, blocked the increase in chromatin-associated protein kinase at 4 as well as 10 h after the injection of isoproterenol. Injection of pilocarpine did not influence the chromatin-associated protein phosphokinase activity. Dichloroisoproterenol appeared to be antagonistic to the influence of isoproterenol in mediating changes in chromatin-associated protein kinase. The results suggest that the isoproterenol-induced increase in chromatin-bound protein phosphokinase which precedes the increase in RNA synthesis is related to the eventual onset of DNA synthesis in rat submandibular gland stimulated by isoproterenol. The chromatin-bound protein phosphokinase activity (or activities) may have a regulatory role on gene action, mediated through the control of phosphorylation of nuclear non-histone acidic proteins [26].     

Design and operation of a simple continuous-culture apparatus

A versatile and simple continuous-culture apparatus is described. With this apparatus, independent control of limiting growth factors and other nutrilites is possible and the conditions of each experiment are reproducible. In view of the synchronized speed of the feeder syringes, flow variation troubles are not encountered. The device allows the performance of growth experiments at different dilution rates simultaneously in a single run which makes the comparison of the results more reliable. The operation of the device has been tested successfully with a study of adenine deaminase induction in yeast.