In vivo human myocardial metabolism during aerobic exercise by phosphorus-31 nuclear magnetic resonance spectroscopy

A few studies have been made in vivo on human myocardial energy metabolism. Hence, no discussion has taken place on metabolism during exercise or of training effects on metabolism. We examined human myocardial energy metabolism at rest and during exercise, and also training effects on the metabolism by phosphorus-31 nuclear magnetic resonance (³¹P NMR)-spectroscopy. Six sedentary male students (Cont) and six male long distance runners (Tr) were the subjects. Energy metabolism data were obtained from myocardium during rest and exercise by the region selection method using ³¹P NMR. Rotation of the legs while riding a bicycle, which was fitted with an ergometer we had made ourselves for NMR, imposed given exercise intensities. The heart rate was measured in a stationary phase during exercise. Although the heart rate at rest in the Tr group was significantly lower [Tr, 52.5 (SD 3.1) beat · min^–1; Cont, 67.1 (SD 2.9) beat · min^–1], no significant difference was observed in myocardial energy metabolism using the ³¹P NMR method [Tr, phosphocreatine/-adenosine 5-triphosphate (PCr/-ATP); 1.51 (SD 0.02); Cont, 1.51 (SD 0.01)]. When NMR measurements were investigated at two different intensities of exercise, heart rates in the Cont group were significantly higher by about 20 beat · min ^–1 than those in the Tr group at both exercise intensities, while no difference in energy metabolism was observed between the groups or between rest and exercise [Tr, 75.9 (SD 3.6), 88.3 (SD 3.7) beat · min^–; PCr/-ATP 1.51 (SD 0.03), 1.51 (SD 0.03); Cont, 95.9 (SD 2.4), 115.1 (SD 3.5) beat · min^–1 PCr/-ATP 1.51 (SD 0.01), 1.51 (SD 0.04)]. Thus, during submaximal exercise as employed in this study, it would seem that the high energy phosphate level normally observed during rest may still be maintained. From these results, the absence of change in the myocardial PCr: ATP ratio suggested that adenosine 5-diphosphate was not the primary regular of the increased metabolism needed to meet the higher cardiac workload during aerobic exercise in either group.     

Phosphorus-31 nuclear magnetic resonance study on the effects of endurance training in rat skeletal muscle

Summary To evaluate changes in muscle energetics following endurance training, we measured phosphorus-31 nuclear magnetic resonance (³¹P NMR) spectra on rat muscle in vivo before and after training in the same animals. The endurance training lasted for 3 months. The³¹P NMR spectra were obtained serially at rest, during exercise by electrical stimulation, and during recovery. Intramuscular phosphocreatine (PCr), inorganic phosphate (P_i, adenosine 5-triphosphate (ATP) and pH were determined from the NMR spectra. The ratio of PCr : (PCr + P_i) at rest showed no difference between the trained and control groups even after 3 months of training. During exercise, however, this ratio was significantly higher in the trained group than in the control group. The ratio also recovered more rapidly after exercise in the trained group. The intramuscular pH decreased slightly by approximately 0.1 pH unit during exercise but did not show a significant difference between the groups. These results indicated that endurance training of 3 months duration improved the ATP supply system in the muscle. They also demonstrated that³¹P NMR is a potent method for evaluating the effects of training in the same individuals.     

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     

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.     

Rapid refilling of Ca2+ stores in macrophages stimulated by ATP involves the sequential activation of phospholipase D and protein kinase C

Ca²⁺ movements between intracellular stores, the cytoplasm and external solution were analysed in murine peritoneal macrophages stimulated by various agonists. The Ca²⁺ content of intracellular stores was estimated from the amplitude of Ca²⁺-transients elicited by ionomycin applied in Ca²⁺-free solution. Both uridine 5-triphosphate (UTP) and platelet-activating factor (PAF) triggered the release of Ca²⁺ followed by a sustained influx, during which intracellular stores remained totally empty. In contrast, in the continuous presence of adenosine 5-triphosphate (ATP), Ca²⁺ was initially released and then rapidly sequestered again by the stores. ATP-induced store refilling was not related to cell depolarization or to an increase in the intracellular Na⁺ concentration (two specific consequences of ATP stimulation which are not induced by PAF and UTP). Store refilling was not caused by a signal that ATP would fail to induce (e.g. as a result of receptor desensitization), but was positively controlled by ATP, even in the simultaneous presence of a concentration of PAF which, on its own, would have caused a persistent store depletion. The hypothesis that the signal delivered by ATP involves the sequential activation of phospholipase D and protein kinase C is consistent with the present pharmacological evidence. However, although we found conditions in which Ca²⁺ stores did not refill in the presence of ATP, this maintained store depletion was not accompanied by a sustained Ca²⁺ response similar to that elicited by PAF or UTP, suggesting that store depletion is a condition which is necessary, but not sufficient, for inducing Ca²⁺ influx.     

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.     

Differential short-term desensitization to vasopressin,isoproterenol, glucagon,parathyroid hormone and calcitonin in the thick ascending limb of rat kidney

Short-term desensitization to hormone-induced cAMP accumulation was investigated in the medullary (MTAL) and the cortical (CTAL) thick ascending limbs of Henle's loop isolated by microdissection from the rat kidney. The following agonists were studied: vasopressin, glucagon and human calcitonin in the MTAL, and vasopressin, glucagon, human calcitonin, parathyroid hormone (PTH) and the -adrenergic agonist isoproterenol in the CTAL. Isolated tubules were preincubated in vitro for 60 min in the presence or absence of a maximal concentration of one of the five agonists (vasopressin 10 nM, glucagon 10 nM, calcitonin 100 nM, PTH 10 nM, isoproterenol 1 M). Desensitization induced by each agent to its own action was then quantified by measuring the amount of cAMP accumulating in the presence of the phosphodiesterase inhibitor isobutylmethylxanthine and the same agonist concentration as that used during preincubation. In the MTAL, as previously reported, preincubation with vasopressin led to a marked (80%–85%) desensitization to this hormone. A significant hormone self-induced desensitization of about 45% was also obtained with glucagon, but not with calcitonin. In the CTAL, the following order of potency to elicit desensitization was observed: vasopressin (80%) > isoproterenol (50%) > glucagon (30%) > PTH (20%, NS) > calcitonin (10%, NS). Thus, the magnitude of desensitization varied greatly from one hormone to another, but for a given hormone, was of roughly similar extent in both MTAL and CTAL. The strikingly different patterns of desensitization to vasopressin and calcitonin were confirmed by kinetics analysis of cAMP accumulation, which revealed that the action of the former hormone was much more rapidly blunted than that of the latter. It is concluded that vasopressin, isoproterenol and glucagon are all able to induce short-term desensitization in the thick ascending limb, although the extent of the process is specific for each agonist. The different degrees of desensitization to unrelated hormones may indicate variable capacities of the corresponding receptors for undergoing short-term desensitization.     

Fluorescence microplate assay for the detection of oxidative burst products in tobacco cell suspensions using 2′,7′-dichlorofluorescein

The technique of 2,7-dihydrodichlorofluorescin diacetate (H₂DCF-DA)-derived fluorescence was applied to measurements of the oxidative burst reaction in plant cell suspension cultures in an automatic fluorometric multiwell microplate assay. The developed procedure was found to be versatile and effective for the determination of the oxidative burst reaction in plant cell cultures. Using this assay, cumulative production of reactive oxygen intermediates may be monitored and recorded non-destructively on a real-time scale for a large number of samples at frequent and continual time intervals for time course experiments. Through the use of various inhibitors and inducers or elicitors of the oxidative burst in combination with H₂DCF-DA, this assay aids in the dissection of the signal transduction pathways and the determination of the origins of the oxidative burst in plant cells.     

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.     

A point mutation in the 5′-untranslated leader that affects translational activation of the mitochondrial COX 3 mRNA

The 613-base 5-untranslated leader (5-UTL) of the Saccharomyces cerevisiae mitochondrial COX 3 mRNA contains the target of an mRNA-specific translational activator complex composed of at least three nuclearly encoded proteins. We have genetically mapped a collection of cox 3 point mutations, using a set of defined COX 3 deletions, and found one to be located in the region coding the 5-UTL. The strain carrying this allele was specifically defective in translation of the COX 3 mRNA. Nucleotide-sequence analysis showed that the allele was in fact a double mutation comprised of a single-base insertion in the 5-UTL (T inserted between bases-428 and-427 with respect to the start of translation) and a G to A substitution at+3 that changed the ATG initiation codon to ATA. Both mutations were required to block translation completely. The effects of the ATG to ATA mutation alone (cox 3-1) had previously been analyzed in this laboratory: it reduces, but does not eliminate, translation, causing a slow respiratory growth phenotype. The T insertion in the 5-UTL had no detectable respiratory growth phenotype as a single mutation. However, the 5-UTL insertion mutation enhanced the respiratory defective phenotype of missense mutations in pet 54, one of the COX 3-specific translational-activator genes. This phenotypic enhancement suggests that the-400 region of the 5-UTL, where the mutation is located, is important for Pet54p-COX 3 mRNA interaction.     

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     

Adenosine-5′-triphosphate-induced sinus tachycardia mediated by prostaglandin synthesis via phospholipase C in the rabbit heart

Effects of adenosine 5-triphosphate (ATP) and adenosine on cardiac sinus pacemaker activity were examined in the rabbit heart. Electrocardiograms of hearts were recorded while using the Langendorff perfusion method. Both adenosine and ATP, added to the perfusate, slowed the sinus pacemaker activity in a concentration-dependent manner. But in about 40% of the cases, ATP higher than 300 M initially accelerated and then slowed the heart. The sinus slowing caused by adenosine and ATP was blocked by theophylline (a p₁ receptor antagonist) and disappeared in the hearts pre-treated with islet-activating protein (IAP). In contrast, the ATP-induced sinus acceleration was not affected by either theophylline or IAP. In about 75% of the IAP-treated hearts, ATP persistently accelerated the sinus pacemaker. In the remaining 25% of the hearts, ATP caused junctional tachycardia, which may have masked the ATP-induced sinus acceleration. Apamin specifically blocked the ATP-induced sinus acceleration, suggesting that P₂ receptors are involved. Among various adenine nucleotide analogues, the order of potency in inducing tachycardia in IAP-treated hearts is adenosine-5-[-thio]triphosphate > adenylyl imidodiphosphate > adenosine 5-[, -methylene]triphosphate = ATP > adenosine diphosphate = adenosine 5-[, -methylene]triphosphate. ATP-induced acceleration was partially blocked by indomethacin and aspirin (cyclooxygenase inhibitors), but not by nordihydroguaiaretic acid (a lipoxygenase inhibitor). These results suggest that cyclooxygenase and not lipoxygenase metabolites of arachidonic acid, e.g. prostaglandins, may be involved in the generation of tachycardia. Consistent with this notion, exogenously applied cyclooxygenase metabolites, prostaglandin E₂ and 6-keto-prostaglandin F₁, which are known to be produced by extracellular ATP in the rabbit heart [Schwartzman et al. (1981) Eur J Pharmacol 74: 167–173], accelerated the sinus rate. We also observed that the ATP-induced tachycardia was almost completely blocked by neomycin (a phospholipase C inhibitor). We suggest, therefore, that cardiac P₂ receptors may be coupled to prostaglandin synthesis via an IAP-insenstive stimulation of phospholipase C.     

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.     

Intracellular ADP activates ATP-sensitive K+ channels in vascular smooth muscle cells of the guinea pig portal vein

Vasodilatation following tissue ischemia is assumed to partially result from activation of ATP-dependent K⁺ channels (K_ATP). To assess the effect of cytosolic adenosine nucleotides, the balance of which depends on tissue pO₂, on K_ATP, we have measured steady state outward currents (SSC) by the whole-cell clamp technique in smooth muscle cells of the guinea pig portal vein at different concentrations of ATP and ADP in the pipette solution. Glibenclamide, a selective inhibitor of K_ATP, was used as a pharmacological tool. — With no nucleotides in the pipette solution (Ca²⁺-free), the SSC determined at +20 mV was unaffected by glibenclamide, while with 0.1 mM ATP or with 0.1 mM ADP, the SSC exhibited a glibenclamide-sensitive component indicating activation of K_ATP. At 5 mM ATP and no ADP, hardly any effect of glibenclamide on the SSC was detected, suggesting inhibition of K_ATP by this high concentration of ATP. With 0.1 mM ADP at 5 mM ATP however, activation of K_ATP was achieved. — At 10^–7 M Ca²⁺ in the pipette solution, an increased SSC was measured, but the responses to the nucleotides and/or glibenclamide were not modified. — These findings suggest that in vivo, ADP may be involved in the regulation of vascular K_ATP, linking tissue pO₂ with vascular tone and tissue perfusion.