Receptor mechanisms in fish chromatophores--VI. Adenosine receptors mediate pigment dispersion in guppy and catfish melanophores

Using the guppy, Lebistes reticulatus, and the siluroid catfish, Parasilurus asotus , the effects of purine and pyrimidine derivatives on the movement of melanophores were studied. All the substances tested did not aggregate pigment within melanophores. Adenosine and adenine nucleotides were very effective in dispersing melanosomes within the cell, although adenine itself lacked such action. Derivatives of other purines than adenine and of pyrimidines did not disperse melanosomes. The pigment dispersion induced by adenine derivatives was specifically antagonized by methylxanthines. It was concluded that adenosine receptors are present on the melanophore membrane, which take part in the darkening reaction of fishes.     

Extracellular ATP induces a nonspecific permeability of thymocyte plasma membranes

We have previously demonstrated that extracellular ATP can give medullary thymocytes the calcium message required for the induction of their blastogenesis, without mobilization of intracellular calcium. We describe here the effects of extracellular nucleotides on membrane permeability to monovalent and divalent cations in mouse thymocytes. Among all nucleotides tested, under physiological conditions, only ATP and, to a lesser extent, 2-methylthio-ATP, adenosine 5'-O-(3-thio-triphosphate), and ADP were able to depolarize thymocyte plasma membranes and to induce Na+ and Ca2+ influxes into thymocytes; other nonhydrolysable ATP analogs were only effective in the absence of Mg2+. The ATP-induced effects were inhibited in a dose-dependent manner by Mg2+ and greatly potentiated in its absence, which suggests that the tetrabasic ATP4 is probably the active species and that a phosphotransferase activity is not involved in its effects. There ATP-mediated changes in ion fluxes result from an increase in nonspecific permeability of thymocyte membranes, probably by pore formation. These ion flux changes might be responsible for the mitogenic induction of phorbol 12-myristate 13-acetate treated medullary thymocytes. The potency order for the adenine derivatives to affect these fluxes (ATP greater than ADP much greater than AMP greater than adenosine) suggests the presence of ATP specific receptors (P2 purinergic receptors) on thymocyte plasma membranes.     

Interaction of adenine derivatives with chloroplast membranes]

Structural changes of chloroplast fragments result in increased adsorption of adenine derivatives. The nativity of membranes is essential for AMP adsorption. Chloroplast fragments, containing CF1, adsorb adenine and adenosine by 30% and ADP by 50% more than the fragments, devoid of the enzyme. It is assumed that lipids may act as sorbents during nucleotides interaction with the chloroplast membrane.     

Adenosine in cerebral homeostatic role: appraisal through actions of homocysteine, colchicine, and dipyridamole

Mammalian neocortical tissues were incubated in [14C]adenine-containing fluids and their newly-synthesized adenine derivatives examined after periods of superfusion. Increased [K+] released adenine derivatives from the tissues, a release diminished by homocysteine. Homocysteine acted also to diminish the tissue content of adenosine plus its metabolites hypoxanthine and inosine, while increasing that of S-adenosylhomocysteine. Hypoxia also increased the tissue content and the output of adenosine plus its metabolites, and again homocysteine augmented the S-adenosylhomocysteine. Glutamic acid also increased tissue content and output of adenosine and derivatives, an action diminished by homocysteine and associated with augmented S-adenosylhomocysteine. Colchicine or dipyridamole did not prevent augmentation of S-adenosylhomocysteine by the reagents described; the sequence from adenosine phosphates to S-adenosylhomocysteine is concluded to be intracellular and not to involve extracellular formation of precursor adenosine. Adenosine displayed properties consistent with its being involved in two distinct categories of homeostasis, and also with its exerting an inhibitory tone in normal cerebral systems.     

Interaction between adenosine generated endogenously in neocortical tissues,and homocysteine and its thiolactone

[¹⁴C]Adenine derivatives in normal guinea pig or rat neocortical tissues maintained by superfusion included ATP, ADP and AMP collectively forming some 98% of the acid-extracted ¹⁴C; adenosine, inosine and hypoxanthine each at less than 0.5% and S-adenosylhomocysteine at about 0.1%. l-Homocysteine and/or its thiolactone increased only a little the S-adenosylhomocysteine. The superfusion fluid carried from the tissue per minute about 0.1% of its acid-extractable [¹⁴C]adenine derivatives. Electrical stimulation of the superfused tissue increased 10-fold its output of [¹⁴C]adenine derivatives and diminished the 5′-nucleotides in the tissue to 94% of the acid-extractable [¹⁴C]adenine derivatives, the remainder being adenosine, inosine and hypoxanthine with little change in S-adenosylhomocysteine. Homocysteine in the superfusion fluids now caused large increases in tissue S-adenosylhomocysteine, which became the preponderant non-nucleotide ¹⁴C-derivative when homocysteine was 0.1 mM or greater. The total [¹⁴C]adenine conversion to non-nucleotide derivatives then increased and the 5′-nucleotides fell to 88% of the total. It is concluded that concentration relationships observed in the action of homocysteine make it feasible that convulsive conditions and mental changes associated with administered homocysteine and with homocystinuria are due to cerebral adenosine concentrations being diminished through formation of S-adenosylhomocysteine. Adenosine is preponderantly depressant in cerebral actions; effects of the S-adenosylhomocysteine produced may also be relevant.     

Fluorometric Determination of Adenosine Nucleotide Derivatives as Measures of the Microfouling, Detrital, and Sedimentary Microbial Biomass and Physiological Status

Adenosine, adenine, cyclic adenosine monophosphate (AMP), AMP, nicotinamide adenine dinucleotide, adenosine diphosphate, and adenosine triphosphate (ATP) were recovered quantitatively from aqueous portions of lipid extracts of microfouling, detrital, and sedimentary microbial communities. These could be detected quantitatively in the picomolar range by forming their 1-N⁶-etheno derivatives and analyzing by high-pressure liquid chromatography with fluorescence detection. Lipid extraction and subsequent analysis allowed the simultaneous measurement of the microbial community structure, total microbial biomass with the quantitative recovery of the adenine-containing cellular components, which were protected from enzymatic destruction. This extraction and fluorescent derivatization method showed equivalency with the luciferin-luciferase method for bacterial ATP measurements. Quick-freezing samples in the field with dry ice-acetone preserved the ATP and energy charge (a ratio of adenosine nucleotides) for analysis at remote laboratories. The metabolic lability of ATP in estuarine detrital and microfouling communities, as well as bacterial monocultures of constant biomass, showed ATP to be a precarious measure of biomass under some conditions. Combinations of adenosine and adenine nucleotides gave better correlations with microbial biomass measured as extractable lipid phosphate in the detrital and microfouling microbial communities than did ATP alone. Stresses such as anoxia or filtration are reflected in the rapid accumulation of intracellular adenosine and the excretion of adenosine and AMP into the surrounding milieu. Increases in AMP and adenosine may prove to be more sensitive indicators of metabolic status than the energy charge.     

Adenine nucleotide metabolism in relation to purine enzymes in liver, erythrocytes and cultured fibroblasts.

To evaluate the regulation of adenine nucleotide metabolism in relation to purine enzyme activities in rat liver, human erythrocytes and cultured human skin fibroblasts, rapid and sensitive assays for the purine enzymes, adenosine deaminase (EC 2.5.4.4), adenosine kinase (EC 2.7.1.20), hyposanthine phosphoribosyltransferase (EC 2.4.28), adenine phosphoribosyltransferase (EC 2.4.2.7) and 5'-nucleotidase (EC 3.1.3.5) were standardized for these tissues. Adenosine deaminase was assayed by measuring the formation of product, inosine (plus traces of hypoxanthine), isolated chromatographically with 95% recovery of inosine. The other enzymes were assayed by isolating the labelled product or substrate nucleotides as lanthanum salts. Fibroblast enzymes were assayed using thin-layer chromatographic procedures because the high levels of 5'-nucleotidase present in this tissue interferred with the formation of LaCl3 salts. The lanthanum and the thin-layer chromatographic methods agreed within 10%. Liver cell sap had the highest activities of all purine enzymes except for 5'-nucleotidase and adenosine deaminase which were highest in fibroblasts. Erythrocytes had lowest activities of all except for hypoxanthine phosphoribosyltransferase which was intermediate between the liver and fibroblasts. Erhthrocytes were devoid of 5'-nucleotidase activity. Hepatic adenosine kinase activity was thought to control the rate of loss of adenine nucleotides in the tissue. Erythrocytes had excellent purine salvage capacity, but due to the relatively low activity of adenosine deaminase, deamination might be rate limiting in the formation of guanine nucleotides. Fibroblasts, with high levels of 5'-nucleotidase, have the potential to catabolize adenine nucleotides beyond the control od adenosine kinase. The purine salvage capacity in the three tissues was erythrocyte greater than liver greater than fibroblasts. Based on purine enzyme activities, erythrocytes offer a unique system to study adenine salvage; fibroblasts to study adenine degradation; and liver to study both salvage and degradation.     

Adenine derivatives as neurohumoral agents in the brain. The quantities liberated on excitation of superfused cerebral tissues

Adenine nucleotides of guinea-pig neocortical tissues were labelled by incubation with [(14)C]adenine and excess of adenine was then removed by superfusion with precursor-free medium. Adenine derivatives released from the tissue during continued superfusion, including a period of electrical stimulation of the tissue, were collected by adsorption and examined after elution and concentration. The stimulation greatly increased the (14)C output, and material collected during and just after stimulation had a u.v. spectrum which indicated adenosine to be a major component. The additional presence of inosine and hypoxanthine was shown by chromatography and adenosine was identified also by using adenosine deaminase. Total adenine derivatives released from the tissue during a 10min period of stimulation were obtained as hypoxanthine, after deamination and hydrolysis of adenosine and inosine, and amounted to 159nmol/g of tissue. This corresponded to the release of approx. 7pmol/g of tissue per applied stimulus. The hypoxanthine sample derived from superfusate hypoxanthine, inosine and adenosine was of similar specific radioactivity to the sample of inosine separated chromatographically, and each was of higher specific radioactivity than the adenine nucleotides obtained by cold-acid extraction of the tissue.     

Studies on purine transport and on purine content in vacuoles isolated from Saccharomyces cerevisiae.

The transport of purine derivatives into vacuoles isolated from Saccharomyces cerevisiae was studied. Vacuoles which conserved their ability to take up purine compounds were prepared by a modification of the method of polybase-induced lysis of spheroplasts. Guanosine greater than inosine = hypoxanthine greater than adenosine were taken up with decreasing initial velocities, respectively; adenine was not transported. Guanosine and adenosine transporting systems were saturable, with apparent Km values 0.63 mM and 0.15 mM respectively, while uptake rates of inosine and of hypoxanthine were linear functions of their concentrations. Adenosine transport in vacuoles appeared strongly dependent on the growth phase of the cell culture. The system transporting adenosine was further characterized by its pH dependency optimum of 7.1 and its sensitivity to inhibition by S-adenosyl-L-methionine. In the absence of adenosine in the external medium, [14C]adenosine did not flow out from preloaded vacuoles. However, in the presence of external adenosine, a very rapid efflux of radioactivity was observed, indicating an exchange mechanism for the observed adenosine transport in the vacuoles. In isolated vacuoles the only purine derivative accumulated was found to be S-adenosyl-L-homocysteine.     

Effects of adenosine and adenine nucleotides on synaptic transmission in the cerebral cortex.

Adenosine and the adenine nucleotides have a potent depressant action on cerebral cortical neurons, including identified corticospinal cells. Other purine and pyrimidine nucleotides were either weakly depressant (inosine and guanosine derivatives) or largely inactive (xanthine, cytidine, thymidine, uridine derivatives). The 5'-triphosphates and to a lesser extent the 5'-diphosphates of all the purine and pyrimidines tested had excitant actions on cortical neurons. Adenosine transport blockers and deaminase inhibitors depressed the firing of cortical neurons and potentiated the depressant actions of adenosine and the adenine nucleotides. Methylxanthines (theophylline, caffeine, and isobutylmethylxanthine) antagonized the depressant effects of adenosine and the adenine nucleotides and enhanced the spontaneous firing rate of cerebral cortical neurons. Intracellular recordings showed that adenosine 5'-monophosphate hyperpolarizes cerebral cortical neurons and suppresses spontaneous and evoked excitatory postsynaptic potentials in the absence of any pronounced alterations in membrane resistance or of the threshold for action potential generation. It is suggested that adenosine depresses spontaneous and evoked activity by inhibiting the release of transmitter from presynaptic nerve terminals. Furthermore, the depressant effects of potentiators and excitant effects of antagonists of adenosine on neuronal firing are consistent with the hypothesis that cortical neurons are subject to control by endogenously released purines.     

ADENINE NUCLEOTIDE-INDUCED CONTRACTION OF THE INNER MITOCHONDRIAL MEMBRANE : II. Effect of Bongkrekic Acid

In bovine heart mitochondria bongkrekic acid at concentrations as low as about 4 nmol/mg protein (a) completely inhibits phosphorylation of exogenous adenosine diphosphate (ADP) and dephosphorylation of exogenous adenosine triphosphate (ATP), (b) completely reverses atractyloside inhibition of inner membrane contraction induced by exogenous adenine nucleotides, and (c) decreases the amount of adenine nucleotide required to elicit maximal exogenous adenine nucleotide-induced inner membrane contraction to a level which appears to correspond closely with the concentration of contractile, exogenous adenine nucleotide binding sites Bongkrekic acid at concentrations greater than 4 nmol/mg protein induces inner membrane contraction which seems to depend on the presence of endogenous ADP and/or ATP. The findings appear to be consistent with the interpretations (a) that the inner mitochondrial membrane contains two types of contractile, adenine nucleotide binding sites, (b) that the two sites differ markedly with regard to adenine nucleotide affinity, (c) that the high affinity site is identical with the adenine nucleotide exchange carrier, (d) that the low affinity site is accessible exclusively to endogenous adenine nucleotides and is largely unoccupied in the absence of bongkrekic acid, and (e) that bongkrekic acid increases the affinity of both sites in proportion to the amount of the antibiotic bound to the inner membrane.     

The adsorption of nucleotides and polynucleotides on montmorillonite clay

The binding of adenine derivatives to Na(+)-montmorillonite increases in the order 5'AMP, 3'-AMP, 5'ADP < adenosine < purine, adenine. With the exception of cytosine, cytosine derivatives bind less strongly than the corresponding adenine derivatives in the order 5'-CMP < cytidine < cytosine. There is little difference in the binding of uracil derivatives and these compounds bind less strongly than the corresponding adenine analogs. It is concluded that the adenine ring in adenine derivatives is protonated by the acidic montmorillonite surface and binding is a consequence of the electrostatic interaction between the protonated base and the negative charges on the surface of the montmorillonite. Different binding trends were observed with Cu(2+)-montmorillonite with AMP binding more strongly than adenosine and UMP binding more strongly than uridine. It is concluded that ligation to the Cu2+ is a major force in the binding of nucleotides to Cu(2+)-montmorillonite and are not readily washed from the clay. Factors contributing to the binding are discussed. Watson-Crick hydrogen bonding of 5'-AMP to poly(U) and 5'GMP to poly(C) was observed when the homopolymers are bound to the surface of the clay. No association of 5'-UMP to poly(U) bound to clay was detected. The possible role of montmorillonite clays in the prebiotic formation of RNA is discussed.     

The metabolism of adenine derivatives in different parts of the brain of the rat, and their release from hypothalamic preparations on excitation.

Five enzymes concerned with the metabolism of adenine derivatives were assayed in seven regions of the rat brain. A region which included the hypothalamus had the highest AMP deaminase and adenosine deaminase activities, while its 5'-nucleotidase activities were relatively low. The enzymes named and also the uptake of [14C]adenine by incubated tissue samples were more active with hypothalamic than with neocortical tissues. On superfusion with glucose-bicarbonate saline after assimilating [14C]adenine, the hypothalamic tissues released about 0.2 per cent of their 14C content per minute. This release was increased fourfold with electrical excitation but the presence of 0.25 muM tetrodotoxin prevented most of this increase. The compounds released during superfusion and electrical stimulation were preponderantly hypoxanthine, inosine, and adenosine, with only small amounts of adenine nucleotides. The output of all these compounds increased during the period of stimulation and also the proportion of adenine nucleotides increased when stimulation was carried out in the presence of tetrodotoxin. The output of the nucleotides and adenosine increased more promptly when stimulated than did that of the other compounds named. The results are discussed in terms of the metabolic roles of the enzymes concerned. and in relation to whether the enzymes are acting on intracellular or extracellular substrates.     

Acid-soluble purine and pyrimidine derivatives of growing, encysting and encystment-inhibited amoebae

The intracellular acid-soluble purine and pyrimidine derivatives of myxamoebae-swarm cells of Physarum flavicomum were investigated during growth, microcyst formation, and during adenine-inhibition of encystment, using high performance liquid chromatography (HPLC). We also studied the incorporation of exogenous radioactive adenine into the acid soluble purine derivatives and S-adenosyl-sulphur compounds separated by HPLC. The most abundant ribonucleoside monophosphate was AMP in the growing and 15 h encysting cells (NC), while it was UMP in the 15 h adenine-inhibited cells (AIC). ADP was the nucleoside diphosphate present in the greatest quantity in the growing and NC cells but it was CDP in the AIC. The nucleoside triphosphate in highest concentration was ATP, UTP, and GTP in growing, NC, and AIC, respectively. Guanosine was the most abundant nucleoside in all cells. The nucleobase occurring in greatest concentration was cytosine, cytosine and guanine, and adenine in the growing, NC, and AIC, respectively. The AMP content in the 15 h AIC was 2.1-fold higher than that of adenosine. The 15 h NC had the lowest adenylate energy charge, a value of 0.54 +/- 0.02, while the values for growing cells and the AIC were 0.62 +/- 0.02 and 0.76 +/- 0.01, respectively. [14C]-Adenine labelling studies (15 h) revealed the occurrence of purine nucleotide interconversion, as the label was detected not only in adenosine, AMP, ADP, ATP, but also in guanine, guanosine, GMP, GDP, GTP, as well as, in inosine monophosphate and xanthosine monophosphate. The percentage incorporation of the radiolabelled adenine into AMP was higher than into adenosine. An increased intracellular level of guanine nucleotides is associated with the inhibition of encystment. The extracellular adenine, rather than internal adenine sources, appears to be the primary precursor of nucleotide for S-adenosylmethionine synthesis during adenine-inhibition of encystment.     

5'-adenine mononucleotides in preparations from guinea pig cerebral cortex. Accumulation, translocation and release.

Isolated nerve terminals (synaptosome beds) were prepared from the neocortex of guinea pig and their ability to accumulate and release adenine nucleotides was studied. Synaptosome beds prelabelled with [14C]adenosine released newly synthesized [14C] adenine derivatives on superfusion. Electrical stimulation and K+ depolarization gave augmented output of both [14C] adenine derivatives and lactate from the preparations. Action of metabolic inhibitors on this output was examined. During incubation and superfusion, the synaptosomes displayed glycolysis and synthesis of ATP. Supply of adenine derivatives to the nerve terminals also occurred by translocation from other parts of the tissue.     

Production of a precursor to the pyrimidine moiety of thiamine.

The supernatant fluid from cultures of Escherichia coli W-11, a pur E mutant, prevented the inhibition of growth of E. coli B in a medium containing adenine or adenosine. Adenine inhibition was prevented more readily than adenosine inhibition. More than 90% of the biological activity of the supernatant fluid was recovered in the anionic fraction after treatment with Dowex-50 (NH4+). The cationic fraction, containing large amounts of 5-aminoimidazole ribonucleoside (AIRS), did not prevent adenine inhibition. The W-11 supernatant fluid was shown by bioautography to contain only one compound that prevented adenine inhibition. Proliferating and non-proliferating cultures produced only one compound that prevented adenine inhibition. The compound was shown to be an intermediate (int-1) in the biosynthesis of the pyrimidine moiety of thiamine, Int-1 was stable during sterilization at 121 C for 15 min, during concentration by either flask evaporation or lyophilization, and after storage for several days at 4 C or at -- 20 C. Int-1 was distinguishable from other known derivatives or intermediates of the pyrimidine moiety. A scheme is presented that illustrates the proposed relationship between int-1 and the synthesis of thiamine.     

The metabolism of adenine derivatives in different parts of the brain of the rat,and their release from hypothalamic preparations on excitation

Five enzymes concerned with the metabolism of adenine derivatives were assayed in seven regions of the rat brain. A region which included the hypothalamus had the highest AMP deaminase and adenosine deaminase activities, while its 5'-nucleotidase activities were relatively low. The enzymes named and also the uptake of [¹⁴C]adenine by incubated tissue samples were more active with hypothalamic than with neocortical tissues. On superfusion with glucose-bicarbonate saline after assimilating [¹⁴C]adenine, the hypothalamic tissues released about 0.2% of their ¹⁴C content per minute. This release was increased fourfold with electrical excitation but the presence of 0.25 μUM tetrodotoxin prevented most of this increase. The compounds released during superfusion and electrical stimulation were preponderantly hypoxanthine, inosine, and adenosine, with only small amounts of adenine nucleotides. The output of all these compounds increased during the period of stimulation and also the proportion of adenine nucleotides increased when stimulation was carried out in the presence of tetrodotoxin. The output of the nucleotides and adenosine increased more promptly when stimulated than did that of the other compounds named. The results are discussed in terms of the metabolic roles of the enzymes concerned, and in relation to whether the enzymes are acting on intracellular or extracellular substrates.     

Uptake, transport and metabolism of cytokinin in moss protonema

Uptake, transport and metabolism of cytokinin in the protonemaof Funaria hygrometrica were studied using labelled kinetin(6-furfurylamino [8-¹⁴C]-purine). All cells of the protonema,chloronema and caulonema, were able to take up kinetin, whichwas carried in the symplastic transport system from cell tocell. Radioactivity was especially accumulated in growing cellsof the protonema. Kinetin was metabolized immediately afteruptake. While only very little kinetin (less than 1%) remainedas free kinetin and one part was immobilized in chromatographicseparation [e.g. attached to proteins and incorporated intonucleic acids (17)], most of the remaining kinetin was metabolizedto adenine derivatives. Exogenously supplied adenosine changedthe metabolism of kinetin. In the caulonema, adenosine reducedthe turnover of kinetin to other adenine derivatives and enhancedthe content of labelling in the start fraction. Thus adenosinecan stimulate cytokinin-dependent bud formation in moss protonema. (Received November 24, 1977; )     

Adenine nucleotides are required for activation of rat atrial natriuretic peptide receptor/guanylyl cyclase expressed in a baculovirus system.

Atrial natriuretic peptide (ANP) binds to a transmembrane receptor having intrinsic guanylyl cyclase activity; this receptor has been designated GC-A. Binding of ANP to GC-A stimulates its catalytic activity, resulting in increased production of the second messenger, cyclic GMP. Here we show that GC-A can be expressed in insect cells using a recombinant baculovirus and that the expressed protein retained its abilities to bind ANP and to function as an ANP-activated guanylyl cyclase. In addition, GC-A produced in insect cells was absolutely dependent on the presence of adenine nucleotides for activation by ANP. Millimolar concentrations of ATP were required for optimal activation. The relative potencies of various nucleotides for activation was adenosine 5'-O-(thiotriphosphate) greater than ATP greater than ADP, adenosine 5'-(beta, gamma-imino)triphosphate greater than ADP beta S. AMP had no effect. These studies suggest that binding of an adenine nucleotide, most likely to the protein kinase-like domain of GC-A, is absolutely required for ANP activation. Regulation of guanylyl cyclase activation by adenine nucleotides represents a novel mechanism for the modulation of signal transduction, possibly analogous in some respects to the role of guanine nucleotides and G proteins in the regulation of adenylyl cyclase activity.