Activation of respiration of liver mitochondria in various metabolic states by cyclic 3′,5′-AMP

Cyclic 3,5-AMP (10^–6M) activates respiration of the liver mitochondria in all metabolic states and neither changes nor increases the rate of phosphorylation during oxidation of saturating concentrations of isocitrate and succinate. For the effect to be manifested, preincubation of the mitochondria or liver homogenate with cyclic AMP is necessary. The fifth fraction of serum albumin and EDTA do not abolish the effect. Noradrenalin (NA) increases mitochondrial respiration only on incubation with the homogenate. Effects of NA and cyclic AMP do not undergo summation, and the effect of the former is probably mediated by cyclic AMP. The results do not confirm the decisive role of uncoupling of respiration and phosphorylation or accumulation of the oxidation substrate, but instead they suggest activation of mitochondrial enzymes.Department of Biochemistry and Central Research Laboratory, Krasnoyarsk Medical Institute. (Presented by Academician of the Academy of Medical Sciences of the USSR S. S. Debov.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 85, No. 3, pp. 291–294, March, 1978.     

Effect of the dephosphorylated core of 2′,5′-oligoadenylate (2′,5′-ApApA) on electrical activity of the snail neuron

Bulletin of Experimental Biology and Medicine -     

Inhibition by somatostatin of adenosine-3′,5′-monophosphate stimulated hepatic ribosomal protein synthesis

Summary Somatostatin which inhibits the secretion of various pituitary and intestinal hormones has been suspected to exert these effects by inhibiting adenosine-3,5-monophosphate accumulation in the respective endocrine gland. Our results, obtained by cell free protein synthesis and by sedimentation through sucrose gradients of ribosomes, prepared from rat liver after incubation with cyclic AMP and/or somatostatin also suggest this antagonism between somatostatin and cyclic AMP. In addition, they indicate that this antagonism is not restricted to endocrine tissues.Supported by a grant (M2-2777) of the Österreichischer Fonds zur Förderung der wissenschaftlichen Forschung     

The nonselective cation channel in the basolateral membrane of rat exocrine pancreas

Nonselective Ca²⁺-sensitive cation channels in the basolateral membrane of isolated cells of the rat exocrine pancreas were investigated with the patch clamp technique. With 1.3 mmol/l Ca²⁺ on the cytosolic side, the mean openstate probabilityP _o of one channel was about 0.5. In insideout oriented cell-excised membrane patches the substances diphenylamine-2-carboxylic acid (DPC), 5-nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB) and 3,5-dichlorodiphenylamine-2-carboxylic acid (DCDPC) were applied to the cytosolic side. These compounds inhibited the nonselective cation channels by increasing the mean channel closed time (slow block). 100 mol/l of NPPB or DPC decreasedP _o from 0.5 (control conditions) to 0.2 and 0.04, respectively, whereas 100 mol/l of DCDPC blocked the channel completely. All effects were reversible. 1 mmol/l quinine also reducedP _o, but in contrast to the abov mentioned substances, it induced fast flickering. Ba²⁺ (70 mmol/l) and tetraethylammonium (TEA⁺; 20 mmol/l) had no effects. We investigated also the stilbene disulfonates 4-acetamido-4-isothiocyanatostilbene-2,2-disulfonic acid (SITS), 4,4-diisothiocyanatostilbene-2,2-disulfonic acid (DIDS) and 4,4-dinitro-2,2-stilbenedisulfonate (DNDS). 10 mol/l SITS applied to the cytosolic side increasedP _o from 0.5 to 0.7 and with 100 mol/l SITS the channels remained nearly permanently in its open state (P _o1). A similar activation of the channels was also observed with DIDS and DNDS. These effects were poorly reversible. The stilbene disulfonates acted by increasing the channel mean open time. When the channel was inactivated by decreasing bath Ca²⁺ concentration to 0.1 mol/l, addition of 100 mol/l of SITS had no effect. Similarly, reducing bath Ca²⁺ concentration from 1.3 mmol/l in presence of 100 mol/l SITS (channels are maximally activated) to 0.1 mol/l, inactivated the channels completely. These results demonstrate, that SITS can only activate the channels in the presence of Ca²⁺. SITS had no effects, when applied to the extracellular side in outside out patches. In summary, the substances DPC, NPPB and DCDPC inhibit nonselective cation channels, where DCDPC has the most potent and NPPB the smallest effect; whereas SITS, DIDS and DNDS activate the channel when applied from the cytosolic side in the presence of Ca²⁺ ions.     

Renal responsiveness to parathyroid hormone in a case of nonfamilial hypophosphatemic osteomalacia

Summary Plasma immunoreactive parathyroid hormone level, urinary excretion of adenosine cyclic 3,5-monophosphate (cyclic AMP) and the sensitivity of the renal tubule to calcium infusion and to parathyroid extract were investigated in a patient with nonfamilial hypophosphatemic osteomalacia. Plasma immunoreactive parathyroid hormone concentration was normal and basal urinary excretion of cyclic AMP was increased. Renal cortical adenylate cyclase, as measured by urinary cyclic AMP excretion, was certainly as sensitive to exogenous parathyroid extract as in normal subjects. After a previous calcium infusion, a greater parathyroid-hormone-sensitive component of phosphorus transport in the kidney was present than in two control subjects. Our results indicate that in nonfamilial hypophosphatemic osteomalacia the renal tubule could be hyperresponsive to parathyroid hormone.This work was supported by a grant (n^o 20,463) from the Belgian Nationaal Fonds voor Geneeskundig Wetenschappelijk Onderzoek     

Effects of inhibitors of enzymatic and cellular pH-regulating systems on central sympathetic chemosensitivity

Previous studies in cats using isolated NaClCO₂ perfusion of the lower brainstem demonstrated an intrinsic chemosensitivity of sympathoexcitatory bulbospinal neurones within the rostroventrolateral medulla (RVLM). In the present experiments, the effects of inhibitors of enzymatic and cellular systems, known to be involved in pH regulation, were investigated. Isolated perfusion of the lower brainstem with CO₂-enriched solutions was performed and preganglionic sympathetic nerve activity (SNA) was recorded. Drugs were locally injected into the left RVLM with glass micropipettes. Perfusion of the RVLM with CO₂enriched solutions over a period of 15 s induced a marked increase in SNA. The magnitude of absolute changes in SNA during perfusion depended on the level of basal SNA before perfusion. Microinjections of 4,4-diisothiocyanatostilbene-2,2-disulphonic acid (DIDS) and acetazolamide (ACZ) induced a marked rise in basal SNA, whereas diethylpyrocarbonate (DEPC) and ethylisopropylamiloride (EIPA) had no significant effect on basal SNA. After application of DIDS and DEPC, the peak change in SNA due to perfusion of the RVLM with CO₂-enriched solutions was slightly diminished. Furthermore, neither ACZ nor EIPA produced any significant influence on the slope, peak change and time course of the increase in SNA compared with control perfusions. We conclude that the enzymatic and cellular carrier systems tested in this study are not or only slightly involved in central sympathetic chemosensitivity.     

Comparison of the 5′ Leader Sequences of North American Isolates of Reference and Field Strains of Porcine Reproductive and Respiratory Syndrome Virus (PRRSV)

The 5 leader is documented to be an important regulatory element in many (+) ssRNA virus genome. To understand the significance of the 5 leader RNA of PRRSV, we determined the complete leader sequences of fifteen different North American strains of PRRSV and predicted their secondary structures. Viruses analysed included three reference strains and nine field strains originating from different geographic locations. To further examine the leader region, one of the field strains was adapted to grow in tissue culture, and three clones were isolated. We also predicted the secondary structures of two European strains based on their published sequences. The predicted RNA secondary structures of the leader sequences suggested the existence of three conserved domains formed by the 5 region of the leader among the North American strains, two of which were conserved in the European strains. A variable structural domain was predicted from the 3 region of the leader sequences of the North American strains, where all tissue culture-adapted isolates were characterized by a stem-loop while field isolates were characterized by an internal bulge within the stem-loop.     

Negative inotropic effect of cyclic GMP in cardiac fiber fragments

Summary The force of spontaneously beating cardiac cellular fragments obtained from mice heart by homogenization was recorded in presence of cyclic guanosine –3.5-monophosphate (cGMP) and cyclic 8-bromguanosine –3.5-monophosphate in concentrations of 3×10^–6 M –33×10^–6 M. The nucleotide decreased the force and reduced the rate of spontaneity. Eventually the preparation became quiescent. It is thought that this nucleotide either reduces the capacity to sequester calcium or affects its release from the sarcotubular system.     

Cyclische Nucleotide als intracelluläre Überträgerstoffe bei Hormonwirkungen

Zusammenfassung Hormone dienen als extracelluläre Informationsüberträger zwischen ihrem Bildungsort, einer endokrinen Drüse, und den Zellen, deren Funktion sie regulieren. Durch die Reaktion des Hormons mit den an der Zellmembran gelegenen Receptoren wird die Aktivität der mit diesen eng verknüpften Adenyl-Cyclase beeinflußt. Die meisten Hormone erhöhen in ihrem Zielorgan die Aktivität dieses Enzyms und führen hierdurch zu einem raschen Anstieg der intracellulären Konzentration von Adenosin-3:5-monophosphat (Ado-3:5-P). Dieses cyclische Nucleotid wird durch eine spezifische Phosphodiesterase zu Adenosin-5-monophosphat abgebaut. Auch die Aktivität dieses Enzyms bestimmt die intracelluläre Ado-3:5-P-Konzentration, die im Vergleich zu der anderer Nucleotide sehr gering ist.Ado-3:5-P beeinflußt als zweiter, intracellulärer Überträgerstoff die Aktivität zahlreicher Schlüsselenzyme. Die Ado-3:5-P-Konzentration bestimmt hierdurch das Gleichgewicht verschiedener Stoffwechselwege zueinander und damit die Reaktion einer Zelle auf eine hormonale Stimulierung. An einer Reihe von Enzymen wird die durch Ado-3:5-P bedingte Aktivitäts-Änderung durch einen gleichartigen Mechanismus bewirkt. Das cyclische Nucleotid stimuliert Proteinkinasen, die eine Phosphatgruppe des ATP auf verschiedene Proteine übertragen und hierdurch deren Eigenschaften verändern können. So steigt bei Phosphorylierung durch eine Ado-3:5-P-stimulierbare Proteinkinase die Aktivität der Triglyceridlipase und der Glykogen-Phosphorylase-b-kinase an, dagegen nimmt die Aktivität der Glykogen-Synthetase ab; durch Phosphorylierung von Histonen kann deren Repressorcigenschaft vermindert und die Synthese von Enzymen gesteigert werden.In manchen tierischen Geweben wurde auch eine spezifisch durch Guanosin-3:5-monophosphat (Guo-3:5-P) stimulierbare Proteinkinase nachgewiesen. Dieses cyclische Nucleotid kommt wie Ado-3:5-P in allen Säugerorganen vor. Die Bildung von Guo-3:5-P aus GTP wird durch die Guanyl-Cyclase katalysiert, ein Ferment, das im Gegensatz zur Adenyl-Cyclase zum großen Teil nicht an die Zellmembranen gebunden ist. Die Konzentration von Guo-3:5-P in verschiedenen Geweben, im Blutplasma und im Urin wird durch Hormone beeinflußt. Es ist noch nicht bekannt, welche hormonalen Regulationen durch Guo-3:5-P vermittelt werden; dagegen ist bei vielen, rasch einsetzenden Hormonwirkungen die Beteiligung von Ado-3:5-P nachgewiesen worden.

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Abkürzungen Ado-3:5-P Adenosin-3:5-monophosphat - dAdo-3:5-P Desoxy-adenosin-3:5-monophosphat - Guo-3:5-P Guanosin-3 : 5-monophosphate - Nuc-3:5-P Nucleosid-3:5-monophosphat - NTP Nucleosidtriphosphat - NMP Nuclcosid-5-monophosphat - dATP Desoxyadenosintriphosphat - P_i anorganisches Phosphat - PP_i anorganisches Pyrophosphat - DNS Desoxyribonucleinsäure - RNS Ribonucleinsäure - r-RNS ribosomale RNS - m-RNS Boten-RNS - Glykogen-Synthetase UDP-Glucose--1,4-glucan--4-glucosyltransferase - ICSH interstitial-cell-stimulating hormone     

Effect of imidazole compounds on 3′,5′-AMP phosphodiestyerase activity

The effect of 24 imidazole compounds on activity on the phosphodiesterase (3, 5-AMP phosphohydrolase; EC 3.1.4.1) from rat brain and skeletal muscles ofRana temporaria was investigated. Imidazole compounds were shown to have both an activating and an inhibitory action on the enzyme. Imidazole itself and seven of its alkyl substitution products activated phosphodiesterase. Of the inhibitors, tetrachloro-2-trifluoromethyl-benzimidazole had the strongest action on the enzyme.Group for Biophysics of Synaptic Processes, I. M. Sechenov Institute of Evolutionary Physiology and Biochemistry, Academy of Sciences of the USSR, Leningrad. (Presented by Academician S. E. Severin.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 82, No. 9, pp. 1055–1059, September, 1976.     

Synchronizing action of cyclic AMP on the mitotic cycle of ehrlich's ascites carcinoma cells

The number of mitoses and of DNA-synthesizing cells in an Ehrlich's ascites carcinoma was studied during the 24 h after injection of dibutyryl cyclic 3,5-adenosine monophosphate. As a result of preprophase inhibition and, probably, of stimulation of entry of the cells into the synthetic period, a large number of cells commences mitosis simultaneously 8 h after the injection. The resulting synchronization of mitosis in the cell population of the tumor is evaluated.Department of General Biology, N. I. Pirogov Second Moscow Medical Institute. (Presented by Academician of the Academy of Medical Sciences of the USSR V. V. Kupriyanov.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 81, No. 1, pp. 66–68, January, 1976.