A different mode of action of potassium ions and veratridine on the formation of cyclic adenosine monophosphate in the cerebral cortex.

The levels of cyclic adenosine 3':5'-phosphate were determined in the rat cerebral cortex after topical application of KCl or veratridine solutions of various concentrations. Cyclic adenosine 3':5'-phosphate content was also measured in 100 μm frozen sections (1 mg wet wt.) of the cortex invaded by slow potential change of spreading depression or on the surface of which 24% (w/v) KCl solutions had been applied.Veratridine induced cyclic adenosine 3′:5′-phosphate accumulation only in the concentrations eliciting spreading depression, whereas a roughly linear correlation between potassium concentration and cyclic adenosine 3':5'-phosphate levels was found. In the appropriate range of concentrations (threshold for eliciting spreading depression potassium) appeared to act in a similar way as veratridine, i.e. by triggering spreading depression.The difference between the ways by which potassium and veratridine cause an accumulation of cyclic adenosine 3':5'-phosphate suggest a dual effect of potassium ions. First, they may affect ‘K⁺-sensitive elements’ in which formation of cyclic adenosine 3':5'-phosphate proceeds roughly linearly with increasing extracellular K⁺ concentration. Slight depolorization of these elements and/or their specific K⁺-receptors might activate the cyclic adenosine 3':5'-phosphate generating system. Second, K⁺ affects the cyclic adenosine 3′:5′-phosphate in higher concentration in a similar way as veratridine, i.e. by triggering spreading depression.     

Effects of an imprinting stimulus on adenylate cyclase and adenosine 3′:5′-phosphate in neonatal chick brain

Birds exposed to a visual imprinting stimulus show a complex pattern of changes in brain adenosine 3′:5′-phosphate concentration and adenylate cyclase specific activities as compared to several kinds of control. With increasing time of exposure there is an initial depression in adenosine 3′:5′-phosphate concentration followed by an elevation in the forebrain roof. There is also a tendency for all groups to show an initial generalized increase in adenosine 3′:5′-phosphate concentration which wanes with time. Adenylate cyclase specific activity is lowered after 30 min of exposure in the midbrain and elevated in the forebrain roof after 60 min of treatment.The results are discussed in relation to the hypothesis that adenosine 3′:5′-phosphate might have triggered the protein synthesis in the forebrain roof previously shown to be dependent upon visual imprinting stimulation. The results suggest that the effect on the adenosine 3′:5′-phosphate system is not consistent with this hypothesis but may be more related to translational control involving precursor availability.     

Purine salvage at the cholinergic nerve endings of the Torpedo electric organ: the central role of adenosine

The uptake and metabolism of adenosine, adenine, inosine and hypoxanthine were studied at the cholinergic nerve endings of the Torpedo electric organ. In isolated synaptosomes there is a linear uptake (measured up to 60 min) for adenosine and adenine at concentrations of 0.3 μM Uptake of adenosine exceeds that of adenine by a factor of 10. Adenosine is transported into synaptosomes via a saturable uptake system (K_m, 2 μM;V_max, ～- 30 pmols/min/mg protein). 2′-Deoxyadenosine is a competitive inhibitor of synaptosomal adenosine uptake. The nerve terminal possesses anabolic pathways for the formation of adenosine 5′-triphosphate from both adenosine and adenine. Adenosine becomes phosphorylated rapidly after entry into synaptosomes to form adenosine 5′-monophosphate; adenosine 5′-diphosphate and adenosine 5′-triphosphate were also major metabolites (70%). Adenine, inosine and hypoxanthine first accumulate in the synaptosomes. However, adenine leads to major formation of nucleotides (41% adenosine 5′-triphosphate after 60 min). Only traces of adenosine-3′:5′ cyclic monophosphate are formed from both adenosine and adenine. If adenosine 5′-triphosphate is added to a suspension of intact synaptosomes it becomes degraded to adenosine.We conclude that cholinergic nerve endings in the Torpedo electric organ possess an effective purine salvage system. Adenosine 5′-triphosphate released from either a pre- or a postsynaptic source would become degraded to adenosine in the extra-cellular medium and be re-used via an uptake system for renewed synthesis of adenosine 5′-triphosphate in nerve terminals.     

Phosphate excretion during 24 h of hypoxia in conscious rats

The objective of this study was to investigate renal phosphate excretion during 24 h of hypoxia in conscious rats fed by total parenteral nutrition. Wistar rats weighing 190 g were exposed to hypoxia (inspired oxygen fraction = 0.10) or normoxia (inspired oxygen fraction = 0.21) for 24 h in a normobaric chamber. Renal clearance and hormonal studies were performed. The results showed a greater fractional excretion of phosphate (5.37 pL 0.07%, P < 0.05) and hypophosphataemia (7.40 pL 0.12 mg dL^-1, P < 0.01) in hypoxic rats (n = 10) than in normoxic rats (n = 13; 3.50 pL 0.37% and 8.02 pL 0.16 mg dL^-1, respectively). In addition, during hypoxia there was a significant decrease in the excretion of urinary adenosine 3′,5′-cyclic monophosphate per glomerular filtrate (2.97 pL 1.27 nmol dL^-1, P < 0.005), a parameter of the renal action of parathyroid hormone, and a stable level of serum parathyroid hormone (10.2 pL 2.6 ng mL^-1) (cf. normoxia: 8.57 pL 0.70 nmol dL^-1 and 8.0 pL 1.7 ng mL^-1, respectively). However, creatinine clearance and the renal adenosine triphosphate level, both of which affect adenosine 3′,5′-cyclic monophosphate excretion, were not different between the two groups. These data suggest that exposure of conscious rats to 24 h of hypoxia causes renal hyporesponsiveness to physiological levels of parathyroid hormone, which is manifested as a decrease in adenosine 3′,5′-cyclic monophosphate excretion. Phosphaturia is not a direct net effect of hypoxia and secondary hypocapnia on renal phosphate transport, which is known to be regulated by parathyroid hormone through adenosine 3′,5′-cyclic monophosphate.     

Festphasensynthese von oligonucleotiden, 11. Verwendung eines neuartigen hydrophilen perlpolymerisats als träger

New hydrophilic swellable and lowly crosslinked bead copolymers consisting of dimethylaminocarbonylethylene or -propylene and alkoxycarbonylethylene or -propylene repeating units were prepared starting from activated esters of acrylic acid ( 8a–10a ) or methacrylic esters ( 8b–10b ) and N,N-dimethylacrylamide ( 12a ). This “hydrophylic Merrifield carriers” were applied for polymer-supported oligodeoxynucleotide syntheses. They were found to be swellable in a wide range of solvents. The first deoxynucleoside was attached to the carrier via its 5′-hydroxyl group by an ester bond. The cleavage of the bond between carrier and oligonucleotide was performed with aqueous sodium hydroxide under conditions which do not attack the N-protecting groups attached to the oligonucleotide chain. The acid labile 1-ethoxyethyl group was used as 3′-protecting group for the phosphate component (N-protected deoxynucleoside-5′-phosphate). Some suitable protected deoxynucleosides were synthesized for anchoring and deoxynucleoside-5′-phosphates for chain elongation. In a condensation reaction according to the diester method, a dinucleoside monophosphate ( 7 ) was prepared and, as a consequence of the flexibility of the polymeric backbone, the synthesis of longer oligonucleotides should be feasible. The alkali labile anchoring of the oligonucleotides to the carrier can allow to avoid wrong sequences by simple acylation of the non-reacted 3′-OH groups with acetic anhydride after each condensation step.     

Adenosine receptor subtypes in the airways responses to 5'-adenosine monophosphate inhalation of sensitized guinea-pigs

Background Endogenous adenosine levels are raised in the lungs during asthma attacks. 5′‐adenosine monophosphate (5′‐AMP) inhalation in asthmatics causes bronchoconstriction and in sensitized guinea‐pigs induces early (EAR) and late asthmatic responses (LAR), airway hyper‐reactivity (AHR) and inflammatory cell recruitment to the lungs. Objective The aim of this study was to investigate the roles of A₁, A_2A, A_2B and A₃ adenosine receptors in these responses to inhaled 5′‐AMP in sensitized guinea‐pigs. Comparisons were made with the effect of dexamethasone treatment on 5′‐AMP‐induced responses. Methods Functional airways responses to inhaled 5′‐AMP (3 and 300 mm ) of actively sensitized, conscious guinea‐pigs were determined by whole‐body plethysmography following administration of selective adenosine receptor antagonists or their vehicles. AHR to inhaled histamine (1 mm ) and inflammatory cell influx in bronchoalveolar lavage fluid were determined. Results 5′‐AMP at 3 mm caused an immediate bronchoconstriction (EAR), whereas 300 mm caused bronchodilatation. Both responses were followed at 6 h by a LAR, together with inflammatory cell influx and AHR to histamine. The A_2A receptor antagonist, ZM241385, further enhanced cell influx after 5′‐AMP inhalation (3 and 300 mm ), and blocked the immediate bronchodilator response to 300 mm 5′‐AMP, exposing an EAR. The A_2B receptor antagonist, MRS1706 (in the presence of ZM241385), inhibited the LAR, AHR and cell influx, following inhalation of 5′‐AMP (300 mm ). The A₃ receptor antagonist, MRS1220, inhibited 5′‐AMP‐induced inflammatory cell influx. The A₁ receptor antagonist, DPCPX (in the presence of ZM241385), inhibited the EAR following 5′‐AMP inhalation (300 mm ). Dexamethasone inhibited the LAR, AHR and cell influx following inhalation of 5′‐AMP (300 mm ). Conclusion All four adenosine receptor subtypes play various roles in the airways responses to inhaled 5′‐AMP in sensitized guinea‐pigs.     

Sequenzspezifische Cooligokondensation von Nucleinsäure-Bausteinen mit Affinitäts-Schutzgruppen, 1. Desoxyoligonucleotide mit 3′-Ribouridin-Terminus

The paper describes a new method for the preparation of oligonucleotide fragments, which can be used as building blocks for polynucleotide synthesis. It essentially uses nucleic acid constituents of the general formula X? A, pB and pC? Y in a chemical cooligocondensation, A, B and C being nucleosides, X and Y blocking groups which confer to the protected oligonucleotide chain a certain affinity (“affinity blocking groups”). Examples of cooligocondensation reactions are given, in which a uridine unit is the 3′-terminal affinity blocking group. Thus, thymidylic acid is reacted with derivatives of ribouridylic acid, the two types of products (pdT), and (pdT),-rU are separated by chromatography on boronate columns and the individual chains are obtained pure by DEAE-cellulose or paper chromatography. Analogously, the cooligocondensation of thymidylic acid with O²′, O³′-(diacetylribouridyl-5′,3′-yl)-N-(acetyl)deoxycytidine-5′-phosphate gave sequences (pdT)_n? dC? rU. Using 5′-O-[p-anisyldimethyl]thymidine, thymidylic acid and O²′,O³′-diacetylribouridylic acid, the cooligocondensation of a three component-system was studied. Differences to the conventional stepwise synthesis and advantages of the cooligocondensation method are discussed.     

Effects of 3‘, 5’-Cyclic Adenosine Monophosphate, 5-Hydroxy-tryptamine,Noradrenaline and Theophylline on the Simultaneous Release of Peroxidase and Amylase from the Guinea Pig Submanibular Gland

Noradrenaline (NA), 5-hydroxytryptamine (5-HT) and N⁶,2-0′-dibutyryl cyclic adenosine monophosphate (DBcAMP) induced a parallel discharge of peroxidase and amylase from the guinea pig submandibular gland in vitro. Theophylline alone, at a concentration of 5 mM, had only little effect on enzyme secretion, but it markedly potentiated the effects of noradrenaline, 5-HT and DBcAMP at submaximal concentrations. Adenosine-3′-monophosphoric acid (3′-AMP) and adenosine-5′-monophosphoric acid (5′-AMP) were without effect on enzyme secretion. Maximal effects of NA and DBcAMP on enzyme release were obtained at concentrations of 0.01 mM and 3 mM respectively. Dose-response curves for the stimulation of peroxidase and amylase release by 5-HT revealed that this amine was a potent secretagogue at the concentrations of 0.1 and 0.3 mM but was less effective at higher concentrations. It is concluded that peroxidase and amylase are simultaneously secreted from the guinea pig submandibular gland, and that the release of both enzymes is mediated via cyclic AMP.     

Polyvinyl alcohol containing N-derivatives of cytosine,uracil, or adenosine-5′-phosphate as pendant groups

Polyvinyl alcohols (P?_n ca. 500) having a nucleotide or one of its analogs, such as adenosine-5′-phosphate, N-[(2′-dihydrogenphosphato)ethyl]-uracil and -cytosine, as pendant group were synthesized by condensation using DCC as dehydrating agent in refluxing DMF. The resulting polyvinyl alcohols showed a hypochromic effect up to 4% when mixed with denaturated RNA or DNA in aqueous solution. Low molecular weight PVA (P?_n = 8) containing nucleotide analogs showed no hypochromic effect with DNA or RNA.     

Phosphate and cyclic AMP excretion decreases during less than 12 hours of hypoxia in conscious rats

Hypocapnia is known to have an antiphosphaturic effect that overcomes the phosphaturic effect of hypoxia. The objective of this study was to examine whether conscious rats exposed to acute hypoxia show a decrease in phosphate excretion due to the concomitant hypocapnia. Wistar rats weighing 200 g were exposed to hypoxia (inspired oxygen fraction=0.10) or normoxia (inspired oxygen fraction=0.21) for 6 h; and rats were alternately exposed to hypoxia or normoxia every 12 h for a total 36 h. Renal clearance and hormone studies were performed. Rats exposed to 6 h of hypoxia (n = 11) showed significant hypophosphaturia and decreases in absolute and fractional excretion of phosphate (0.38±0.10 μg min^-1, mean ±SE, P<0.0001 and 0.59±0.15%, P<0.0001) as compared with normoxic rats (n = 11, 3.91±0.68 μg min-¹ and 5.62±0.85%). In addition, nephrogenous adenosine 3′,5′-cyclic monophosphate level per glomerular filtrate was significantly decreased (-0.87±0.64 nmol dL GF^-1, P<0.05) and plasma parathyroid hormone level was unchanged (45.2±9.5 pg mL-¹) after 6 h of hypoxia as compared with normoxic rats (4.03±1.83 nmol dL GF-¹ and 54.3±10.4 pg mL-¹). A parallel increase in urinary noradrenaline and a decrease in dopamine excretion was observed in rats after 6 h of hypoxia. The decreased phosphate and adenosine 3′,5′-cyclic monophosphate excretion during acute hypoxia were restored to normoxic levels by reoxygenation with 21% oxygen in the study of 12-h intermittent hypoxia. In summary, (1) hypoxia produced by inhalation of 10% oxygen for 12 h or less causes reduced phosphate and adenosine 3′,5′-cyclic monophosphate (cAMP) excretion by spontaneously breathing rats; (2) these effects are reversed by reoxygenation and (3) hypoxia elicits a parallel increase in noradrenaline excretion and a decrease in dopamine excretion. These data suggest that renal adrenergic and dopaminergic systems play important roles in hypophosphaturia during acute hypoxia in conscious rats.     

Effect of reserpine on cell proliferation and energy stores in the developing rat brain

Administration of reserpine resulted in a dose-dependent progressive reduction in the rate of [³H]thymidine incorporation into brain deoxyribonucleic acid. The depression reached a nadir of approximately 45% and 70% of the control levels in the forebrain and the cerebellum respectively at a dose of about 1.5 mg/kg. The half maximal effect in the forebrain was obtained with 0.1–0.2 mg/kg reserpine. Reserpine at a dose of 2.5 mg/kg had no effect on the cerebral concentrations of adenosine 5′-mono, di- or triphosphates, lactate and glutamate, while the levels of creating phosphate and glucose were significantly elevated.It seems, therefore, that the depression of cell proliferation in the developing brain is not mediated through an effect of reserpine on the availability of adenosine 5′-triphosphate. Since other side-effects of the drug can also be excluded the results are consistent with an action of reserpine on replicating cells either directly or in an indirect way via changes in transmitter balance and thus affecting regulatory processes involving neurohumoral receptors.     

A HS6ST2 gene variant associated with X-linked intellectual disability and severe myopia in two male twins

X-linked intellectual disability (XLID) refers to a clinically and genetically heterogeneous neurodevelopmental disorder, in which males are more heavily affected than females. Among the syndromic forms of XLID, identified by additional clinical signs as part of the disease spectrum, the association between XLID and severe myopia has been poorly characterized. We used whole exome sequencing (WES) to study two Italian male twins presenting impaired intellectual function and adaptive behavior, in association with severe myopia and mild facial dysmorphisms. WES analysis detected the novel, maternally inherited, mutation c.916G > C (G306R) in the X-linked heparan sulfate 6-O-sulfotransferase 2 (HS6ST2) gene. HS6ST2 transfers sulfate from adenosine 3′-phosphate, 5′-phosphosulfate to the sixth position of the N-sulphoglucosamine residue in heparan sulfate (HS) proteoglycans. Low HS sulfation levels are associated with defective optic disc and stalk morphogenesis during mammalian visual system development. The c.916G>C variant affects the HS6ST2 substrate binding site, and its effect was considered “deleterious” by in-silico tools. An in-vitro enzymatic assay showed that the HS6ST2 mutant isoform had significantly reduced sulphotransferase activity. Taken together, the results suggest that mutant HS6ST2 is possibly involved in the development of myopia and cognitive impairment, characteristics of the probands reported here.     

Release of adenosine 5′-triphosphate from synaptosomes from different regions of rat brain

The release of adenosine 5′-triphosphate by elevated extracellular concentrations of KC1 and by veratridine was determined in synaptosomal fractions prepared from different regions of rat brain. Following correction for yields of synaptosomes from the various regions, the relative distribution of K⁺-induced release was corpus striatum > cerebral cortex > medulla > hypothalamus > cerebellum. In contrast, the relative distribution of veratridine-induced release of adenosine 5′-triphosphate was medulla > corpus striatum > hypothalamus > cerebral cortex > cerebellum.From these findings, it was concluded that (1) depolarization-induced release of adenosine 5′-triphosphate was not distributed uniformly throughout the brain but varied from region to region, (2) the K⁺-induced release of adenosine 5′-triphosphate which is Ca²⁺-dependent, had a different regional distribution than the veratridine-induced release, which is greatest in Ca²⁺-free medium, and (3) the distribution of K⁺-induced release of adenosiae 5′-triphosphate did not correlate well with the known distribution of noradrenaline concentrations in rat brain, but did correlate to some extent with the distributions of 5-hydroxytryptamine, dopamine and especially acetylcholine, so that co-release of adenosine 5′-triphosphate with these transmitters may possibly occur.     

Error‐prone replication bypass of the imidazole ring‐opened formamidopyrimidine deoxyguanosine adduct

Addition of hydroxyl radicals to the C8 position of 2′‐deoxyguanosine generates an 8‐hydroxyguanyl radical that can be converted into either 8‐oxo‐7,8‐dihydro‐2′‐deoxyguanosine or N‐(2‐deoxy‐d ‐pentofuranosyl)‐N‐(2,6‐diamino‐4‐hydroxy‐5‐formamidopyrimidine) (Fapy‐dG). The Fapy‐dG adduct can adopt different conformations and in particular, can exist in an unnatural α anomeric configuration in addition to canonical β configuration. Previous studies reported that in 5′‐TGN‐3′ sequences, Fapy‐dG predominantly induced G → T transversions in both mammalian cells and Escherichia coli, suggesting that mutations could be formed either via insertion of a dA opposite the 5′ dT due to primer/template misalignment or as result of direct miscoding. To address this question, single‐stranded vectors containing a site‐specific Fapy‐dG adduct were generated to vary the identity of the 5′ nucleotide. Following vector replication in primate cells (COS7), complex mutation spectra were observed that included ～3–5% G → T transversions and ～14–21% G → A transitions. There was no correlation apparent between the identity of the 5′ nucleotide and spectra of mutations. When conditions for vector preparation were modified to favor the β anomer, frequencies of both G → T and G → A substitutions were significantly reduced. Mutation frequencies in wild‐type E. coli and a mutant deficient in damage‐inducible DNA polymerases were significantly lower than detected in COS7 and spectra were dominated by deletions. Thus, mutagenic bypass of Fapy‐dG can proceed via mechanisms that are different from the previously proposed primer/template misalignment or direct misinsertions of dA or dT opposite to the β anomer of Fapy‐dG. Environ. Mol. Mutagen. 58:182–189, 2017. © 2017 Wiley Periodicals, Inc.     

Synthesis and Sulfation of Glycosaminoglycans in Fibroblasts From a Patient with Lowe's Syndrome

Glycosaminoglycans of cultured normal skin fibroblasts and fibroblasts of a patient with Lowe's syndrome were labelled for 72 h with either [¹⁴C]-glucosamine or with ³⁵SO₄. For each culture, the incorporation was measured in total glycosaminoglycans per culture and in the glycosaminoglycans isolated from the intracellular, pericellular and extracellular pools. The synthesis of the sulfated glycosaminoglycans in the three pools and the total ³⁵SO₄ incorporation in the glycosaminoglycans of the two types of cultures were strictly comparable. However, Lowe's intracellular glycosaminoglycans were less sulfated than the corresponding normal ones. Undersulfated glycosaminoglycans were present in the pericellular pool of Lowe's cells, while hypersulfated ones were present in their extracellular pool. Degradation of the different pools with chondroitinases indicated that hyposulfated chondroitin 4- and 6-sulfates are present on the cell surface of Lowe's cells, where an increased amount of normally sulfated heparan sulfate may be demonstrated. This abnormal distribution of pericellular glycosaminoglycans in Lowe's cells has been described already.⁴ However, the demonstration that the total incorporation of ³⁵SO₄ is normal in Lowe's cells does not support the possibility that this abnormal distribution is the consequence of excessive hydrolysis of the phosphosulfate bond of adenosine 3′-phosphate 5′-phosphosulfate.     

Pre‐conditioning activates adenosine utilization in a cost‐effective way during myocardial ischaemia

During pre‐conditioning the interstitial concentration of adenosine, in contrast to lactate, presents a die‐away curve‐pattern for every successive episode of ischaemia. This die‐away pattern might not necessarily be attributed to diminished adenosine production. The present study was undertaken to investigate whether pre‐conditioning alters the metabolic turnover of adenosine as observed by the lactate production during ischaemia. Interstitial levels of metabolites in pre‐conditioned (n=21) and non‐preconditioned (n=21) porcine hearts were monitored with microdialysis probes inserted in both ischaemic and non‐ischaemic tissue in an open chest heart model. Three subgroups perturbated with either plain microdialysis buffer (control), buffer containing adenosine (375 μM ), or buffer containing deoxyadenosine (375 μM ) were studied. All animals were subjected to 90 min of equilibrium microdialysis before 40 min of regional myocardial ischaemia and 120 min of reperfusion. Pre‐conditioning consisted of four repetitive episodes of 10 min of ischaemia and 20 min of reperfusion. Significantly higher levels of inosine and lactate were found in the ischaemic tissue of the pre‐conditioned subgroup receiving adenosine (P < 0.05) compared with the other two subgroups receiving deoxyadenosine and plain buffer, respectively. This difference was only valid for pre‐conditioned ischaemic myocardium, and hence equal amounts of inosine and lactate were produced in the non‐preconditioned ischaemic myocardium regardless of the presence of adenosine or deoxyadenosine. In the non‐ischaemic myocardium baseline levels of metabolites were measured in all subgroups. Pre‐conditioning favoured degradation of exogenous adenosine to inosine successively ending up in enhanced lactate production. This was probably because of the involvement of the hexose monophosphate pathway in the pre‐conditioned ischaemic myocardium. This route may therefore be supplementary in energy metabolism as a metabolic flow can be started by adenosine ending up in lactate without initial adenosine 5′‐triphosphate (ATP) investment. Utilization of adenosine in this way may also explain the successive die‐away pattern of adenosine seen in consecutive pre‐conditioning cycles.     

Effect of apamin on the electrical responses of smooth muscle to adenosine 5′-triphosphate and to non-adrenergic,non-cholinergic nerve stimulation

Apamin (a polypeptide from bee venom) specifically and reversibly blocks inhibitory nonadrenergic nerve-muscle transmission and the hyperpolarizing action of adenosine 5′-triphosphate in the smooth muscle of the guinea-pig caecum and stomach. Adenosine evokes a slow hyperpolarization in these muscles, which is not blocked by apamin. In the presence of apamin the muscle cells of the caecum, in response to intramural stimulation, generate excitatory instead of inhibitory junction potentials; these potentials are resistant to cholinergic, adrenergic and serotoninergic blocking agents. Under these conditions exogenous adenosine 5′-triphosphate, but not adenosine, induces depolarization instead of hyperpolarization. In contrast to the caecum, in the smooth muscle of the stomach neither non-cholinergic excitatory junction potentials nor depolarization by adenosine 5′-triphosphate occur in the presence of apamin.The excitatory junction potentials and the depolarization evoked by adenosine 5′-triphosphate that occur in caecum smooth muscle in the presence of apamin are not blocked by indomethacin—a specific inhibitor of the biosynthesis of prostaglandins. Thus, at least in the smooth muscle of the intestine, there appear to be not only inhibitory but also previously unrecognized excitatory, purinergic nerve-muscle transmission.Apamin does not block the effects of adenosine 5′-triphosphate (contraction or relaxation) in smooth muscles of blood vessels, urinary bladder and uterus. It is suggested that there are two kinds of adenosine 5′-triphosphate receptors in the smooth muscles we have studied, namely those blocked by apamin (A₁) and those resistant to apamin (A₂). In the smooth muscles of the gastrointestinal tract activation of A₁ receptors induces inhibition while activation of A₂ receptors induces excitation. It is also suggested that these smooth muscles contain ‘slow’ apamin-resistant purinergic receptors which are sensitive mainly to adenosine. Activation of these receptors evokes slow hyperpolarization, which mediates inhibition.     

Trypanosoma brucei: The effect of glycerol on the anaerobic metabolism of glucose

Studies measuring the glycolytic intermediate and adenine nucleotide concentrations in Trypanosoma brucei metabolising glucose either aerobically or under conditions where glycerol-3-phosphate oxidase is inactive have shown the following: 1. Inhibition with 0.5 mM salicylhydroxamic acid (SHAM) accurately simulates anaerobic conditions in T. brucei; 2. On inhibition of respiring cells with 0.5 mM SHAM, the concentrations of most glycolytic intermediates decreases; they decrease further as the concentration of glycerol, an end product, increases. Only the concentration of sn-glycerol-3-phosphate is increased. This increase depends upon the method of preparation but is independent of time and glycerol concentration; 3. Glycerol formation from sn-glycerol-3-phosphate is coupled to the phosphorylation of another compound. The results of these studies are consistent with this compound being ADP; 4. The degree of inhibition of the anaerobic metabolism of glucose exerted by glycerol varies with the sn-glycerol-3-phosphate concentration, implying that the effect of glycerol is at the site of sn-glycerol-3-phosphate: ADP transphosphorylation.     

Inhibitor studies of phage T4 wild-type and mutant DNA polymerases V. A summary of kinetic and inhibition data

The DNA polymerases of phage T4 wild-type, the mutator mutant L98 and the antimutator mutant CB121 were purified about 100-fold free of foreign enzyme activities interfering with the polymerase assay. The enzymes were characterized as to thermostability, exonuclease activity and kinetic data with DNA template primer, deoxythymidine 5′-triphosphate, and a mixture of all four deoxynucleoside 5′-triphosphates. The effects of eight inhibitors of DNA synthesis on the three enzymes were determined (Schroeder and Jantschak 1978 and 1980, Jantschak and Schroeder 1980) and are compared here. The most selective inhibitor, pyridoxal 5′-phosphate, interacts with the active site of the polymerases while the two least discriminating drugs, distamycin A and actinomycin D, do not directly interact with the polymerases at all. The study was intended to test whether specific enzyme inhibitors elicit a differential response in temperature-sensitive structural variants of this enzyme and whether, in principle, structural variants of virus enzymes or the respective ts- mutants in vivo are suitable as a screening system for selective antiviral agents.     

Adenosine and nitric oxide in exercise‐induced human skeletal muscle vasodilatation

The vasoactive substances adenosine and nitric oxide (NO) are credible candidates in the local regulation of skeletal muscle blood flow. Adenosine and NO have both been shown to increase in skeletal muscle cells and interstitial fluid during exercise and the enzymes responsible for their formation, AMP 5′‐nucleotidase and NO synthase (NOS), have been shown to be activated upon muscle contraction. In vitro as well as in vivo evidence suggest that the contraction‐induced increase in interstitial adenosine concentration largely stems from extracellular formation via the membrane‐bound ecto‐form of AMP 5′‐nucleotidase. It remains unclear whether the exercise‐induced NO formation in muscle originates from endothelial NOS in the microvascular endothelium, or from neuronal NOS (nNOS) in nerve cells and muscle fibres. Functional evidence for the role of adenosine in muscle blood flow control stems from studies using adenosine receptor agonists and antagonsits, adenosine deaminase or adenosine uptake inhibitors. The majority of these studies have been performed on laboratory animals and, although the results show some discrepancy, the majority of studies indicate that adenosine does participate in the regulation of muscle blood flow. In humans, evidence is lacking. The role of NO in the regulation of skeletal muscle blood flow has mainly been studied using NOS inhibitors. Despite a large number of studies in this area, the role of NO for the contraction‐induced increase in skeletal muscle blood flow is uncertain. The majority, but not all, human and animal studies show that, whereas blockade of NOS reduces muscle blood flow at rest and in recovery from exercise, there is no effect on the exercise‐induced increase in muscle perfusion. Conclusive evidence for the mechanisms underlying the precise regulation of the multiphased increase in skeletal muscle blood flow during exercise and the role and potency of various vasoactive substances, remain missing.