Differential effects of paraoxon on the M3 muscarinic receptor and its effector system in rat submaxillary gland cells

The effects of the organophosphorus anticholinesterase paraoxon on the binding of radioactive ligands to the M₃ subtype of the muscarinic receptor and receptor-coupled synthesis of second messengers in intact rat submaxillary gland (SMG) cells were investigated. The binding of [³H]quinuclidinyl benzilate ([³H]QNB) was most sensitive to atropine and the M₃-specific antagonist 4-DAMP followed by pirenzepine and least sensitive to the cardioselective M₂ antagonist AFDX116. This, and the binding characteristics of [³H]4-DAMP, confirmed that the muscarinic receptors in this preparation are of the M₃ subtype. Activation of these muscarinic receptors by carbamylcholine (CBC) produced both stimulation of phosphoinositide (PI) hydrolysis and inhibition of cAMP synthesis, suggesting that this receptor subtype couples to both effector systems. Paraoxon (100 μM) reduced B_max of [³H]4-DAMP binding from 27 ± 4 to 13 ± 3 fmol/mg protein with nonsignificant change in affinity, suggesting noncompetitive inhibition of binding by paraoxon. Like the agonist CBC, paraoxon inhibited the forskolininduced cAMP formation in SMG cells with an EC₅₀ of 200 nM, but paraoxon was > 500 fold more potent than CBC. However, while the inhibition by CBC was counteracted by 2 μM atropine, that by paraoxon was unaffected by up to 100 μM atropine. It suggested that this effect of paraoxon was not via binding to the muscarinic receptor. Paraoxon did not affect β-adrenoreceptor function in the preparation, since it did not affect the 10 μM isoproterenol-induced cAMP synthesis, which was inhibited totally by 10 μM propranolol and partially by CBC. Paraoxon had a small but significant effect on CBC-stimulated PI metabolism in the SMG cells. It is suggested that paraoxon binds to two different sites in these SMG cells. One is an allosteric site on the M₃ muscarinic receptor which affects ligand binding and may modulate receptor function. The other site may be on the G_i proteinadenylyl cyclase system, and produces CBC-like action, that is, inhibition of the forskolin-stimulated [³H]cAMP synthesis, and is unaffected by atropine inhibition of the muscarinic receptor. This adds to the complexity of paraoxon actions on muscarinic receptors and their effector systems.     

γ-Glutamylcysteinylserine — A New Homologue of Glutathione in Plants of the Family Poaceae*

In addition to glutathione (γ-GluCysGly), many species of the family Poaceae have another tripeptide which has the amino acid sequence γ-GluCysSer. This thiol was isolated from etiolated leaves of wheat (Triticum aestivum L. cv. Star). Its structure was elucidated by quantitative amino acid analysis after total hydrolysis and by partial hydrolysis with carboxypeptidase A and γ-glutamyltranspeptidase. The content of γ-GluCysSer in the leaves of T. aestivum is increased by incubation with sulfate and is severely diminished by incubation with buthionine sulfoximine, a specific inhibitor of γ-glutamylcysteine synthetase. Oxidized γ-GluCysSer is reduced by yeast glutathione reductase with a rate somewhat lower than for glutathione, but the new tripeptide is not a substrate of glutathione-S-transferase from equine liver. Besides homoglutathione (γ-GluCysßAla), a tripeptide found in plants of the order Fabales, the tripeptide γ-GluCysSer is the second homologue of glutathione detected in plants.     

Interferon-γ activates the cleavage of double-stranded RNA by bovine seminal ribonuclease

Bovine seminal ribonuclease (BS-RNase), a dimeric homologue of RNase A, cleaves both single- and double-stranded RNA and inhibits the growth of tumor cells. Its catalytic activity against double-stranded RNA, either homopolymeric ([³H]polyA/polyU) or mixed sequence, is enhanced by bovine or human recombinant interferon-γ (IFN-γ). Activation is seen with as little as 4–10 interferon units per assay. Enhancing the degradation of double-stranded RNA, an intermediate in the growth cycle of many viruses, could contribute to IFN-γ's ability to control cell growth and induce an antiviral state.     

Functional involvement of calcium in the homologous up-regulation of the 1,25-dihydroxyvitamin D3 receptor in osteoblast-like cells

In several cell types 1,25-dihydroxyvitamin D₃ (1,25(OH)₂D₃) causes up-regulation of its receptor. The present study demonstrates that in the osteoblast-like cell line UMR 106 this up-regulation is inhibited by two different calcium channel blockers (nitrendipine, verapamil). Also with chelating extracellular calcium (EGTA) and by inhibition of calcium release from intracellular stores (TMB-8) comparable results were obtained. These findings indicate that calcium is functionally involved in this cellular response to the steroid hormone 1,25(OH)₂D₃. Moreover, data obtained with EGTA show that the 1,25(OH)₂D₃ receptor level is closely regulated by the extracellular calcium concentration.     

The metabolism of 6-(2,3,4-trihydroxy-3-methylbutylamino) purine by soybean callus

6-(2,3,4-trihydroxy-3-methylbutylamino) purine (trihydroxyzeatin) applied to soybean callus is metabolised slowly. After 48 h only one peak of biological activity which co-eluted with the applied cytokinin was detected. When the callus was incubated on a medium which contained 10^–5 M trihydroxyzeatin, spiked with 8 {¹⁴C} trihydroxyzeatin, for 28 days, three peaks of biological activity and three peaks of radioactivity were detected. One of the biologically active and radioactive peaks co-eluted with zeatin. Another of the radioactive peaks co-eluted with N-(purin-6-yl) glycine. From the data obtained it apears that trihydroxyzeatin can be both oxidized and reduced by soybean callus. The potential to be converted to zeatin may explain why trihydroxyzeatin and its parent compound, which is usually rapidly metabolised by living material, are equally active in the soybean callus bioassay. From the radioactive data obtained it appears that trihydroxyzeatin is susceptible to oxidation to form N-(purin-6-yl) glycine.     

Induced parthenocarpy—a way of changing the levels of endogenous hormones in tomato fruits (Lycopersicon esculentum Mill.) 1. Extractable hormones

Ethylene, indol-3-acetic acid (IAA), gibberellin-like substances (GAs) and abscisic acid (ABA) were analysed in extracts from normal, seed-containing and parthenocarpic tomato fruits throughout fruit development. Parthenocarpic fruit growth was induced with an auxin (4-CPA), morphactin (CME) or gibberellic acid (GA₃) and compared with that of pollinated control fruits. Fruit growth was only affected by the treatment with GA₃, decreasing size and fresh weight by 60%. The peak sequence of hormones during fruit development was ethylene-GAs-IAA-ABA. Seeded fruits contained the highest levels of IAA and ABA but the lowest levels of GAs. Also, in seeded fruits, a high proportion of IAA and ABA was found in the seeds whereas this was not the case for GAs.Hormone levels of tomato fruits may be successfully, easily and reproducibly altered by inducing parthenocarpic fruit growth and thus eliminating development of seeds which are a major source of hormone synthesis. In spite of markedly changed hormone levels, there was no obvious relationship between fruit growth and extractable hormones per se. However, the results indicate that a high ratio of GAs: auxins is unfavourable for growth of tomato fruits.     

Enkephalin analogs and dermorphin in the conscious dog: Structure-activity relationships

Those structural features of enkephalins (ENK) responsible for in vitro organ bath and receptor binding activity have been investigated in detail in the conscious, chronically instrumented dog. Amide analogs of Leu⁵-ENK display reduced activity, which is restored by D-Ala² substitutions. N-terminal L-Tyr is required for full opiate activity. Although proven δ-receptor agonists do appear generally more active, distinctions made in vitro between μ and δ binding are not apparent in the complex hemodynamic responses which occur in the intact unanesthetized dog. The amphibian skin peptide dermorphin, which contains D-Ala², elevates heart rate, systemic arterial pressure, and induces vomiting with near maximal activity at a dose of 1.0 μg/kg; this activity is inhibited by naloxone. This activity, coupled with dermorphin's apparent presence in mammalian tissue, suggests that it may represent another peptide factor in cardiovascular regulation. In the conscious dog, ENK elevate heart rate and systemic arterial pressure; this activity does not appear to be fully explained by in vitro receptor models.     

Analysis of cellular interactions in density-dependent inhibition of 3T3 Cell proliferation

Subpopulations of different proliferative status are determined during cell-density dependent proliferation of 3T3 cells. From these data the probability of conversion of proliferative to quiescent cells is derived and found to correlate well with published data on binding of growth-inhibiting factors secreted from growth-inhibited cells.Based on material presented at the Symposium Intercellular Communication Stuttgart, September 16–17, 1982     

Stimulation of the insulin secretory mechanism following barium accumulation in pancreatic β-cells

Electrothermal atomic absorption spectroscopy was employed for measuring barium in β-cell-rich pancreatic islets microdissected from ob/ob-mice. Both the uptake and efflux of barium displayed two distinct phases. There was a 4-fold accumulation of barium into intracellular stores when its extracellular concentration was 0.26 mM. Unlike divalent cations with more extensive intracellular accumulation, the washout of Ba²⁺ was not inhibited by d-glucose. Ba²⁺ served as a substitute for Ca²⁺ both in maintaining the glucose metabolism after removal of extracellular Ca²⁺ and making it possible for glucose to stimulate insulin release. Furthermore, Ba²⁺ elicited insulin release in the absence of glucose and other secretagogues. The latter effect was reversible and was markedly potentiated under conditions known to increase the β-cell content of cyclic AMP. It is likely that the observed actions of Ba²⁺ are mediated by Ca²⁺, since Ca²⁺-dependent regulatory proteins, such as calmodulin, apparently cannot bind Ba²⁺ specifically.     

The quantum yield for CO2 uptake in C3 and C4 grasses

The quantum yield for CO₂ uptake was measured in C₃ and C₄ monocot species from several different grassland habitats. When the quantum yield was measured in the presence of 21% O₂ and 340 cm³ m^-3 CO₂, values were very similar in C₃ monocots, C₃ dicots, and C₄ monocots (0.045–0.056 mole CO₂ · mole^-1 quanta absorbed). In the presence of 2% O₂ and 800 cm³ m^-3 CO₂, enhancements of the quantum yield values occurred for the C₃ plants (both monocots and dicots), but not for C₄ monocots. A dependence of the quantum yield on leaf temperature was observed in the C₃ grass, Agropyron smithii, but not in the C₄ grass, Bouteloua gracilis, in 21% O₂ and 340 cm³ m^-3 CO₂. At leaf temperatures between 22–25°C the quantum yield values were approximately equal in the two species.