Role of adenosine A1 and A2 receptors in the regulation of aldosterone production in rat adrenal glands

Summary To investigate the roles of adenosine A₁ and A₂ receptors in the regulation of aldosterone production, we examined the effects of adenosine and adenosine agonists (N⁶-cyclohexyl adenosine; selective adenosine A₁ receptor agonist and 5-N-ethylcarboxamine adenosine; selective adenosine A₂ receptor agonist) on aldosterone and cyclic AMP production in rat adrenal capsular cells. Neither adenosine nor 5-N-ethylcarboxamine adenosine caused significant effects on basal aldosterone or cyclic AMP production. Also, adenosine (10^–3M) showed no consistent effects on aldosterone and cyclic AMP production induced by ACTH. On the other hand, N⁶-cyclohexyl adenosine exhibited a significant inhibition of basal aldosterone and cyclic AMP production at doses of 10^–4 M and 10^–3 M; furthermore, 10^–3 M N⁶-cyclohexyl adenosine inhibited aldosterone and cyclic AMP production stimulated by ACTH. These results suggest that adenosine A₁ receptors are coupled to and inhibit adenylate cyclase and may be involved in the inhibition of aldosterone production.     

Inhibition of prostaglandin-induced cyclic AMP accumulation in the rat anterior pituitary by alrestatin

Summary Alrestatin at 25–1×10^–4 M inhibited the accumulation of cyclic AMP induced by prostaglandin E₂, but not theophylline, in the rat anterior pituitary in vitro. Somatostatin, at lower concentrations, inhibited both; maximal inhibition of the prostaglandin effect was greater with alrestatin. As cyclic AMP is considered to be a mediator in induced-hormonal release, it appears from the present findings that alrestatin may be of potential use in altering hormonal release.The author acknowledges the technical assistance of Miss C. Pilapil.     

Zur Wirkung von Zn++ auf die Nukleinsäurebiosynthese von Aszites-Tumorzellen

Summary Ascites tumour cells have been employed to study the reactivity of Zn⁺⁺ on nucleic acid biosynthesis. 10^–4 M Zn⁺⁺ caused a selective inhibition of DNA synthesis of intact cells. The rate of RNA- and protein-biosynthesis, however, remained unchanged. The activity of DNA polymerase as well as DNA dependent RNA polymerase was strongly affected by Zn⁺⁺ in vitro.     

Die Wirkung von Spirolactonen und von Herzglykosiden auf den Natrium- und Kaliumtransport an Erythrocyten

Summary Experiments made on human red cells showed that spirolactones (SC 5233 and SC 9420) added to the sample as dry powder (10^–3 g/ml) exert an inhibitory action on the active transport of sodium and potassium across the cell membrane. Spirolactone at its saturation concentration in serum (1.5×10^–5 g/ml), however, was ineffective.The inhibitory action was not affected by aldosterone or hydrocortisone.Experiments involving exposure of red cells to spirolactone and a cardiac glycoside simultaneously led to the conclusion that these substances do not have the same mechanism of action. The inhibition caused by spirolactone added to the inhibition due to a supramaximal concentration of ouabain (5 × 10^–4).

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Diese Arbeit wurde unterstützt durch ein Stipendium der CIBA für naturwissenschaftliche, medizinische und technische Forschung.     

Effect of sodium octanoate on leucine incorporation into protein of rat liver slices and of Yoshida ascites hepatoma cells

Summary 7.38×10^–4 M octanoate does not significantly modify leucine incorporation into protein of rat liver slices, while in hepatoma cells a 19% inhibition has been noted.3.69×10^–3 M octanoate reduces leucine incorporation to about the same extent (71–76%) in both liver slices and hepatoma cells.     

Specific inhibition of a calcium dependent activation of brain cyclic AMP phosphodiesterase activity by vinblastine

Summary Vinblastine selectively inhibits the activation of brain cyclic AMP phosphodiesterase activity by Ca⁺⁺-protein activator (50% inhibition by 2×10^–5 M). This inhibitory effect was reversed by excessive amounts of the activator, whereas large quantities of Ca⁺⁺ caused only a slight suppression of the vinblastine effect. This result of vinblastine suggests a new site of its action and also suggests the possible role of protein activator, phosphodiesterase proteins or cyclic nucleotides in the previously known effects of vinblastine in vivo and in vitro.Our thanks to Drs A. R. Rhoads, C. B. Klee and R. A. Fergusson for analyzing PA preparations by gel electrophoresis and gel electrofocusing, and to Drs A. A. Suran and D. Soifer for their valuable critiques to this work. This work was partly supported by Biochemical Research Support Grant No. 5SO7RRO5361 to K.W. and Graduate Training Grant No. 5TO2GMO500002 to W.L.W. from NIH.     

Effect of enkephalins in the presence of the antibiotic bacitracin in the longitudinal muscle strip preparation from guinea-pig ileum

Summary The antibiotic bacitracin (5×10^–5–4×10^–4 M) increases the inhibition of the contractile response caused by both enkephalin release and direct application of Met-enkephalin 5×10^–7 M in the longitudinal muscle strip preparation from guinea-pig ileum. This effect is attributed to an inhibition of enkephalin degrading peptidases by bacitracin.     

Mechanism of inhibition of IgE-dependent histamine release from rat mast cells by xestobergsterol A from the Okinawan marine spongeXestospongia bergquistia

Histamine release from rat peritoneal mast cells induced by anti-IgE was essentially complete within 4–5 min. Xestobergsterol A and B, which are constituents of the Okinawan marine spongeXestospongia bergquistia Fromont, dose-dependently inhibited anti-IgE-induced histamine release from rat mast cells. The IC₅₀ values of xestobergsterol A and B for histamine release in mast cells activated by anti-IgE were 0.07 and 0.11 M, respectively. Anti-IgE stimulated PI-PLC activity in a mast cell membrane preparation. Xestobergsterol A dose-dependently inhibited the generation of IP3 and membrane-bound PI-PLC activity. Moreover, xestobergsterol A inhibited Ca²⁺-mobilization from intracellular Ca²⁺-stores as well as histamine release in mast cells activated by anti-IgE. On the other hand, xestobergsterol B did not inhibit the membrane-bound and cytosolic PI-PLC activity, IP3 generation or the initial rise in [Ca²⁺]_i in mast cells activated by anti-IgE. These results suggest that the mechanism of inhibition by xestobergsterol A of the initial rise in [Ca²⁺]_i, of the generation of IP3, and of histamine release induced by anti-IgE, was through the inhibition of PI-PLC activity.     

Vanadate: Non-selective inhibition of transepithelial transport of Na+, H+ and water

Summary In the isolated urinary bladder of the toad, 10^–5–10^–4M orthovanadate produces inhibition of the active transport of Na⁺ and H⁺ ions as well as of antidiuretic hormone-mediated osmotic flow of water. Since transport of H⁺ ions and osmotic water flow are not inhibited when (Na⁺+K⁺)-ATPase is inhibited by ouabain, biological actions of vanadate are not necessarily related to inhibition of (Na⁺+K⁺)-ATPase.This research was supported by grant AM-14915 from the National Institutes of Helath.     

Phosphoramidon enhances allatostatin-mediated inhibition of juvenile hormone biosynthesis in the corpora allta of the cockroach,Diploptera punctata

Use of the enkephalinase inhibitor phosphoramidon in the in vitro radiochemical assay for juvenile hormone biosynthesis enhanced allatostatin-mediated inhibition of hormone production by corpora allata of the cockroach,Diploptera punctata. Significant increases in inhibition in day 2 virgin female CA by AST 1 (at 10^–7 M) and AST 4 (10^–8–10^–7 M) were observed in the presence of phosphoramidon (10^–5M or greater). No significant increases in inhibition were seen in CA from day 6 mated females with AST 4 (10^–9–10^–7M) and phosphoramidon combined. Phosphoramidon alone had no effect on JH biosynthesis. Analysis of allatostatin content of the CA, as determined by ELISA, revealed that addition of phosphoramidon to the medium increased the endogenous allatostatin conten in CA of virgin and mated females. The similarity in primary structure between allatostatins and enkephalin-like peptides and their similar distribution makes it probable that phosphoramidon acts by preventing breakdown of allatostatins within the CA.     

The effect of cyclic AMP on Na+ and K+ transport systems in mouse macrophages

Summary Exogenous cyclic AMP (cAMP) inhibits the Na⁺, K⁺-cotransport system and stimulates the Na⁺, K⁺-pump and Na⁺, Ca²⁺ exchange in mouse macrophages. These effects are enhanced by inhibition of phosphodiesterase with methylisobutylxanthine (MIX). MIX alone showed little or no effect. A similar response was observed after stimulation of endogenous production of cAMP by isoproterenol.     

Antiproliferative effect of polyunsaturated fatty acids and interleukin-2 on normal and abnormal human lymphocytes

The polyunsaturated fatty acids (PUFAs), linoleic acid (LA), alpha linolenic acid (ALA), gamma linolenic acid (GLA), arachidonic acid (AA), docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA), showed inhibition of growth of both normal and abnormal (Molt-4) human lymphocytes, and inhibition was concentration-dependent. Interestingly, the production of the lymphokine Interleukin-2 (IL-2) was elevated in Molt-4 cells, but it was reduced in the normal human lymphocytes. Addition of GLA or IL-2 or a combination of both showed enhancement of SO ₂ ^– and of lipid peroxidation levels, which were significantly higher in Molt-4 cells than in the normal lymphocytes. Reduction of protein concentration was also observed in both types of cells during this treatment. The data showed that the antiproliferative effects of GLA and IL-2 may partly be exerted through the elevated production of superoxide free radicals and peroxidatin products. This is a novel finding and therefore, further exploitation of combinations of PUFAs and IL-2 may be a possible way of combating cancer cell growth.     

Macrophages derived from bone marrow modulate differentiation of myeloid dendritic cells

Dendritic cells (DC) are specialized antigen-presenting cells. Bone marrow monocytes have been widely used to generate murine myeloid DC. We found that mouse macrophages derived from bone marrow CD11b⁺ monocytes influenced the differentiation of these precursors into DC. Modulation of differentiation was demonstrated by the down-regulation of CD11c, CD40, and CD86 expression and by IL-12 production. DC differentiated in the presence of conditioned medium from bone marrow-derived macrophage culture (MCM) had impaired ability to stimulate proliferation of, and IFN- γ production by, allogeneic CD4⁺ T cells. This inhibition of DC differentiation was mainly mediated by secretory products from macrophages but not by cell-cell contact. MCM contained higher concentrations of macrophage-colony-stimulating factor (M-CSF), IL-10, and TGF- β1, whereas IL-6 remained unchanged compared with conditioned medium from fresh monocytes. M-CSF may be the major mediator in MCM inhibiting DC differentiation. This study demonstrates an important influence of bone marrow-derived macrophages on DC precursors during DC differentiation. Received 12 September 2006; received after revision 20 October 2006; accepted 13 November 2006     

Influence of cyclic nucleotides on protein synthesis in vascular smooth muscle

Summary The incorporation of leucice-¹⁴C into protein in bovine mesenteric arteries was augmented by cyclic GMP (10^–3 M) and decreased by cyclic AMP (10^–3 M). There was no effect of 5 AMP (10^–3 M). The phosphodiesterase inhibiting drugs theophylline (10^–3 M) and papaverine (5×10^–5 g/ml) both decreased the leucine-¹⁴C incorporation.We are indebted to Mrs.Lena Burlin for hear assistance. Finacial support has been provided by the Swedish State Medical Research Council (No. 04X-101X-4498).     

Hemmung der durch Proteuslipopolysaccharide gesteigerten Leukozytenemigration in vitro durch antiinflammatorische Corticosteroide

Summary The migration of leucocytes from the buffy coat of chicken blood is slightly but irregulatly inhibited by cortison. If migration of leucocytes is increased by proteus lipopolysaccharide, cortison in concentrations of 10^–4–10^–6, and other anti-inflammatory steroids, produce a pronounced and constant inhibition of leucocytic migration.     

Lymphocyte reactivity in Imuran-treated guinea-pigs and in vitro effect of colchicine

Summary Imuran treatment significantly diminished macrophage migration inhibition in immunized guinea-pigs and the percentage of E-rosette forming cells in all treated animals. 10^–6 M colchicine in vitro significantly increased the percentage of E-rosette forming cells in Imuran-treated animals. The results suggest possible alteration of the lymphocyte receptors after treatment with Imuran.This work was supported by the grants of the Republic Research Fund of Serbia.     

HCN2 channels in local inhibitory interneurons constrain LTP in the hippocampal direct perforant path

Neuronal hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are known to modulate spontaneous activity, resting membrane potential, input resistance, afterpotential, rebound activity, and dendritic integration. To evaluate the role of HCN2 for hippocampal synaptic plasticity, we recorded long-term potentiation (LTP) in the direct perforant path (PP) to CA1 pyramidal cells. LTP was enhanced in mice carrying a global deletion of the channel (HCN2^−/−) but not in a pyramidal neuron-restricted knockout. This precludes an influence of HCN2 located in postsynaptic pyramidal neurons. Additionally, the selective HCN blocker zatebradine reduced the activity of oriens-lacunosum moleculare interneurons in wild-type but not HCN2^−/− mice and decreased the frequency of spontaneous inhibitory currents in postsynaptic CA1 pyramidal cells. Finally, we found amplified LTP in the PP of mice carrying an interneuron-specific deletion of HCN2. We conclude that HCN2 channels in inhibitory interneurons modulate synaptic plasticity in the PP by facilitating the GABAergic output onto pyramidal neurons.     

Inhibition of glucose transport in human erythrocytes by 2,3-dioxoindole (Isatin)

10 mM isatin (2,3-dioxoindole) inhibited glucose influx into human erythrocytes by over 30%. The inhibition is of the competitive type, where the affinity constant (K_t) was increased from 5.71 (control) to 11.11 mM in the presence of isatin with no change in V_max (130 nmol/min/ml packed cells). The observed inhibition of sugar transport by isatin was not mediated through membrane–SH groups accessible to iodoacetate, iodoacetamide, DTNB, DNP or sodium arsenite. Isatin inhibited sugar transport in the presence of 2 mM harmaline, an alkaloid inhibitor of Na⁺, K⁺–ATP_ase activity. The inhibition was non additive which suggests that these two compounds interact with the same or a similar site on the erythrocyte membrane.     

Accessibility of a critical prion protein region involved in strain recognition and its implications for the early detection of prions

Human prion diseases are characterized by the accumulation in the brain of proteinase K (PK)-resistant prion protein designated PrP27 – 30 detectable by the 3F4 antibody against human PrP109 – 112. We recently identified a new PK-resistant PrP species, designated PrP^*20, in uninfected human and animal brains. It was preferentially detected with the 1E4 antibody against human PrP 97 – 108 but not with the anti-PrP 3F4 antibody, although the 3F4 epitope is adjacent to the 1E4 epitope in the PrP^*20 molecule. The present study reveals that removal of the N-terminal amino acids up to residue 91 significantly increases accessibility of the 1E4 antibody to PrP of brains and cultured cells. In contrast to cells expressing wild-type PrP, cells expressing pathogenic mutant PrP accumulate not only PrP^*20 but also a small amount of 3F4-detected PK-resistant PrP27 – 30. Remarkably, during the course of human prion disease, a transition from an increase in 1E4-detected PrP^*20 to the occurrence of the 3F4-detected PrP27 – 30 was observed. Our study suggests that an increase in the level of PrP^*20 characterizes the early stages of prion diseases. Received 17 October 2007; received after revision 5 December 2007; accepted 14 December 2007     

Melatonin antagonizes colchicine-induced mitotic arrest

Summary Melatonin, in concentrations up to 10^–3 M, showed no effect on mitosis in cultures of HeLa or KB cells. However, when melatonin at 10^–4 M was preincubated with HeLa cells prior to addition of 10^–7 M colchicine, a reduction in the mitotic index, in comparison to colchicine alone, was observed.This work was supported by PHS Grant No. CA 16425.