Cytochemical demonstration of cyclic 3′,5′ AMP phosphodiesterase in different tissue of migratory locust (Locusta migratoria migratorioides R.F.)

Summary The localization of cyclic 3,5-AMP phosphodiesterase was studied by ultrastructural cytochemical methods in the various tissues of Locusta migratoria. Large amounts of electron dense, granular reaction products were detectable on the surface of the corpora allata and the corpus cardiacum, bound to the basal lamina. In the protocerebral neuropile rather large amounts of reaction products were observed in the processes of the glial cells. A significant number of lead phosphate deposit was found to occur on the membrane of certain large axons, the microtubules of the axons, furthermore on the membrane of several terminals. Reaction product was also observable in certain terminals, bound to the synaptic vesicles and the mitochondria. At the same time, electron dense deposits were not detectable at all on the surface of cerebral neurons. In the case of myocardium. reaction product was only found on the basal lamina and the extracellular surface of the sarcolemma.On the basis of our results it can be stated that the cyclic 3,5-AMP phosphodiesterase is detectable by cytochemical methods in different tissues of Locusta migratoria and presumably it fulfils the task of the extracellular cAMP level regulation.     

Cyclic 3',5'-nucleotide phosphodiesterase; cytochemical localization in rat renomedullary interstitial cells.

The localization of cyclic 3', 5' -nucleotide phosphodiesterase activity in rat renal papillae was examined by utilizing cytochemical methods. Renal medullary interstitial cells had predictable phosphodiesterase activity predominantly on the cytoplasmic border of dilated cisternal membranes. Cells of the collecting tubule and loop of Henle contained diffuse reaction product. Capillaries had reaction product localized in pinocytic vesicles. Addition of theophylline resulted in no deposition of reaction product in interstitial cells and in cells of the collecting tubule and loop of Henle, suggesting an inhibition of phosphodiesterase activity. Since the membranes of dilated cisternae of renal medullary interstitial cells have been shown to be related to prostaglandin synthesis and probably to the anti-hypertensive function of these cells, the finding of phosphodiesterase activity on these membranes suggests a possible role of cyclic AMP in these two functions.     

Repressible extracellular phosphodiesterases showing cyclic 2'',3''- and cyclic 3'',5''-nucleotide phosphodiesterase activities in Neurospora crassa.

Two molecular species of repressible extracellular phosphodiesterases showing cyclic 2',3'- and cyclic 3',5'-nucleotide phosphodiesterase activities were detected in mycelial culture media of wild-type Neurospora crassa and purified. The two molecular species were found to be monomeric and polymeric forms of an enzyme constituted of identical subunits having molecular weights of 50,000. This enzyme had the same electrophoretic mobility as repressible acid phosphatase. The enzyme designated repressible cyclic phosphodiesterase showed pH optima of 3.2 to 4.0 with a cyclic 3',5'-AMP substrate and 5.0 to 5.6 with a cyclic 2',3'-AMP substrate. Repressible cyclic phosphodiesterase was activated by MnCl2 and CoCl2 with cyclic 2',3'-AMP as substrate and was slightly activated by MnCl2 with cyclic 3',5'-AMP. The enzyme hydrolyzed cyclic 3',5'- and cyclic 2',3'-nucleotides, in addition to bis-rho-nitrophenyl phosphate, but not certain 5' -and 3'-nucleotides. 3'-GMP and 3'-CMP were hydrolyzed less efficiently. Mutant strains A1 (nuc-1) and B1 (nuc-2), which cannot utilize RNA or DNA as a sole source of phosphorus, were unable to produce repressible cyclic phosphodiesterase. The wild type (74A) and a heterocaryon between strains A1 and B1 produced the enzyme and showed growth on orthophosphate-free media containing cyclic 2',3'-AMP or cyclic 3',5'-AMP, whereas both mutants showed little or no growth on these media.     

A phosphate-binding histidine of binuclear metallophosphodiesterase enzymes is a determinant of 2',3'-cyclic nucleotide phosphodiesterase activity

Binuclear metallophosphoesterases are an enzyme superfamily defined by a shared fold and a conserved active site. Although many family members have been characterized biochemically or structurally, the physiological substrates are rarely known, and the features that determine monoesterase versus diesterase activity are obscure. In the case of the dual phosphomonoesterase/diesterase enzyme CthPnkp, a phosphate-binding histidine was implicated as a determinant of 2',3'-cyclic nucleotide phosphodiesterase activity. Here we tested this model by comparing the catalytic repertoires of Mycobacterium tuberculosis Rv0805, which has this histidine in its active site (His(98)), and Escherichia coli YfcE, which has a cysteine at the equivalent position (Cys(74)). We find that Rv0805 has a previously unappreciated 2',3'-cyclic nucleotide phosphodiesterase function. Indeed, Rv0805 was 150-fold more active in hydrolyzing 2',3'-cAMP than 3',5'-cAMP. Changing His(98) to alanine or asparagine suppressed the 2',3'-cAMP phosphodiesterase activity of Rv0805 without adversely affecting hydrolysis of bis-p-nitrophenyl phosphate. Further evidence for a defining role of the histidine derives from our ability to convert the inactive YfcE protein to a vigorous and specific 2',3'-cNMP phosphodiesterase by introducing histidine in lieu of Cys(74). YfcE-C74H cleaved the P-O2' bond of 2',3'-cAMP to yield 3'-AMP as the sole product. Rv0805, on the other hand, hydrolyzed either P-O2' or P-O3' to yield a mixture of 3'-AMP and 2'-AMP products, with a bias toward 3'-AMP. These reaction outcomes contrast with that of CthPnkp, which cleaves the P-O3' bond of 2',3'-cAMP to generate 2'-AMP exclusively. It appears that enzymic features other than the phosphate-binding histidine can influence the orientation of the cyclic nucleotide and thereby dictate the choice of the leaving group.     

Effects of adenosine 5'-monophosphate and adenosine 3',5'-cyclic monophosphate on l cells.

The adenine nucleotides, 5'-AMP and 3',5'-cyclic AMP block L cells in the S-phase of the cell cycle. The intracellular level of cyclic AMP is reduced after incubation of cells with 5'-AMP, and rates of uridine transport are increased after incubation with either 5'-AMP or cyclic AMP. On the contrary, cyclic AMP levels are increased and uridine transport decreased in cells treated with an inhibitor of the cyclic AMP phosphodiesterase. This inhibitor partially reverses the growth-inhibitory effect of cyclic AMP, indicating that a breakdown product is the effective inhibitor of growth. The inhibition of cell growth induced by the adenine nucleotides is prevented by uridine, suggesting that the block in S is due to a lack of availability of pyrimidines.     

Cyclic 3',5'-AMP phosphodiesterase in Physarum polycephalum. I. Chemotaxis toward inhibitors and cyclic nucleotides.

The effect of several inhibitors of the enzyme cyclic 3',5'-AMP phosphodiesterase as chemoattractants in Physarum polycephalum was examined. Of the compounds tested, 4-(3-butoxy-4-methoxybenzyl)-2-imidazolidinone (Roche 20-1724/001) and 1-ethyl-4-(isopropylidinehydrazino)-1H-pyrazolo-(3,4-b)-pyridine-5-carboxylic acid ethyl ester, hydrochloride (Squibb 20009) were the most potent attractants. 3-Isobutyl-1-methyl xanthine, theophylline, and morin (a flavanoid) were moderate attractants and sometimes gave negative chemotaxis at high concentrations. Cyclic 3',5'-AMP was an effective, but not potent attractant. A repellent effect following the positive chemotactic action was sometimes observed with cyclic 3',5'-AMP at concentrations as high as 1 . 10(-2) M. Dibutyryl cyclic AMP appeared to be a somewhat more potent attractant than cyclic 3',5'-AMP. The 8-thiomethyl and 8-bromoderivatives of cyclic AMP, which are poorly hydrolyzed by the phosphodiesterase, were not attractants in Physarum. Possible participation of cyclic 3',5'-AMP in the directional movement in P. polycephalum is discussed.     

A new histo- and cytochemical method for demonstration of cyclic 3',5'-nucleotide phosphodiesterase activity in retinal rod photoreceptor cells of the rat

Cyclic 3',5'-mononucleotide phosphodiesterase (cyclic nucleotide PDEase) activity was studied histo- and cytochemically in the retinal rod photoreceptor cells of the rat by means of a newly developed technique utilizing the intrinsic 5' nucleotidase activity instead of an exogenous 5' nucleotidase source (snake venom). Cyclic GMP and was used as a substrate, the intense activity of phosphodiesterase (PDEase) was distributed over the entire rod outer segments; reaction product was observed on the plasmalemma and on the disk membranes of the outer segments. A slight reaction was also observed on the plasmalemma of the inner segments. However, no precipitate was found in the perinuclear and synaptic regions of the rod photoreceptors. In contrast, when cyclic AMP was utilized as a substrate, a moderate reaction was seen in the synaptic region of the plexiform layer. The intensity of the reaction in the outer segments was much reduced in comparison to the results with cyclic GMP. The enzyme activity was almost completely inhibited by 2 mM 3-isobutyl-1-methylxanthine (IBMX) or 2 mM theophylline, which were potent inhibitors of PDEase. To confirm the propriety of our new cytochemical method, the localization of 5' nucleotidase was also studied utilizing 5' AMP or 5' GMP as substrates. In contrast to the activity of cyclic nucleotide PDEase, the activity of 5' nucleotidase was distributed on all membranes of the photoreceptors from the synaptic outer plexiform layer to the tip of outer segments.(ABSTRACT TRUNCATED AT 250 WORDS)     

Expression of the 2',3'-cAMP-adenosine pathway in astrocytes and microglia

Many organs express the extracellular 3',5'-cAMP-adenosine pathway (conversion of extracellular 3',5'-cAMP to 5'-AMP and 5'-AMP to adenosine). Some organs release 2',3'-cAMP (isomer of 3',5'-cAMP) and convert extracellular 2',3'-cAMP to 2'- and 3'-AMP and convert these AMPs to adenosine (extracellular 2',3'-cAMP-adenosine pathway). As astrocytes and microglia are important participants in the response to brain injury and adenosine is an endogenous neuroprotectant, we investigated whether these extracellular cAMP-adenosine pathways exist in these cell types. 2',3'-, 3',5'-cAMP, 5'-, 3'-, and 2'-AMP were incubated with mouse primary astrocytes or primary microglia for 1 h and purine metabolites were measured in the medium by mass spectrometry. There was little evidence of a 3',5'-cAMP-adenosine pathway in either astrocytes or microglia. In contrast, both cell types converted 2',3'-cAMP to 2'- and 3'-AMP (with 2'-AMP being the predominant product). Although both cell types converted 2'- and 3'-AMP to adenosine, microglia were five- and sevenfold, respectively, more efficient than astrocytes in this regard. Inhibitor studies indicated that the conversion of 2',3'-cAMP to 2'-AMP was mediated by a different ecto-enzyme than that involved in the metabolism of 2',3'-cAMP to 3'-AMP and that although CD73 mediates the conversion of 5'-AMP to adenosine, an alternative ecto-enzyme metabolizes 2'- or 3'-AMP to adenosine.     

Effect of adenosine 3′,5′-(cyclic)-monophosphate on the synthesis of progestational steroids by rabbit ovarian tissue in vitro

1. Adenosine 3',5'-(cyclic)-monophosphate (3',5'-AMP) stimulates the synthesis of progestational steroids by rabbit ovarian tissue in vitro. 2. Other adenosine phosphates fail to increase steroidogenesis. 3. The ratio of 20alpha-hydroxypregn-4-en-3-one to progesterone, the maximal response of the tissue, and the responses of separated corpora lutea and interstitial tissue produced by luteinizing hormone are closely paralleled by 3',5'-AMP. 4. In tissues maximally stimulated by luteinizing hormone, 3',5'-AMP fails to produce an additional response. 5. The addition of theophylline, an inhibitor of phosphodiesterase, potentiates the effects of 3',5'-AMP and also luteinizing hormone. 6. The results obtained suggest that 3',5'-AMP is a mediator of the action of luteinizing hormone on progestational steroid synthesis by rabbit ovarian tissue.     

Properties of cyclic nucleotide phosphodiesterase of the rabbit myometrium in various functional states

Chromatography on DEAE-cellulose of a soluble sulfate-precipitated fraction of cyclic nucleotide phosphodiesterase from rabbit myometrium revealed two 3':5'-GMP and 3':5'-AMP-hydrolase activities. 3':5'-GMP phosphodiesterase (fraction I) was eluted with 0.15-0.23 M NaCl, while 3':5'-AMP phosphodiesterase (fraction II) with 0.2-0.35 M NaCl. 3':5'-GMP phosphodiesterase hydrolyzed 3':5'-GMP with Km = 14 microM and V = 5.25 nmol . min . mg of protein, while 3':5'-AMP phosphodiesterase hydrolyzed both cyclic nucleotides with Km for 3':5'-GMP equal to 12 microM and V = 1.33 nmol . min . mg of protein; the Km value for 3':5'-AMP was 3.6 and 30.5 microM, respectively; the corresponding values of V were 0.28 and 0.97 nmol . min . mg of protein. In late pregnancy, the level of the 3':5'-AMP hydrolase activity of rabbit myometrium was significantly elevated in parallel with an increase in V, predominantly for the enzyme with a low affinity for 3':5'-AMP. The 3':5'-GMP hydrolase activity and V were largely decreased for both phosphodiesterase fractions; the Km value for fraction I was also diminished. During labour, the rate of 3':5'-AMP hydrolysis by myometrium phosphodiesterase was decreased down to the level typical of functional rest. The rate of 3':5'-GMP hydrolysis during the same period by fraction I remained at a low level, i. e., as in pregnancy, while that of fraction II was increased up to the level typical of functional rest.(ABSTRACT TRUNCATED AT 250 WORDS)     

CYTOCHEMICAL LOCALIZATION OF 5''-NUCLEOTIDASE IN SUBCELLULAR FRACTIONS ISOLATED FROM RAT LIVER : I. The Origin of 5''-Nucleotidase Activity in Microsomes

A procedure has been developed for the cytochemical localization of 5'-nucleotidase in isolated, unfixed, rat liver microsomes. Membranes were incubated with adenosine 5'-phosphate (5'-AMP) and Pb(NO₃)₂ and then isolated on sucrose density gradients: all the phosphate released was recovered with the membranes by this procedure. Adenosine 2'-phosphate (2'-AMP) and adenosine 3', 5'-cyclic phosphate (3',5'-AMP) were shown to be competitive inhibitors, but not substrates, for purified 5'-nucleotidase and were employed to determine the specificity of the cytochemical reaction. It was found that the incubation conditions for the cytochemical assay did not affect the specificity of 5'-nucleotidase. Microsomes incubated as controls with Pb²⁺, or Pb²⁺ and 2'-AMP or 3',5'-AMP were of the same density, although slightly denser than microsomes incubated without Pb²⁺, and were unassociated with lead precipitate when examined by electron microscopy; microsomes incubated with Pb²⁺ and 5'-AMP were much denser and were stained heterogeneously with lead phosphate when examined by electron microscopy. Precipitates formed artificially from Pb²⁺ and inorganic phosphate did not resemble the reaction product. Microsomes were, therefore, separated on sucrose gradients and the subfractions were examined cytochemically. Lead precipitates were associated with the majority of rough-surfaced vesicles, and the reaction product was distributed heterogeneously in all fractions. Vesicles which stained like the membranes of the bile canaliculi in isolated plasma membranes were observed in the lightest subfraction. The reaction product was localized on the outside surface of the microsomal membranes, and was solubilized by low concentrations of ethylenediaminetetraacetic acid. It is concluded that 5'-nucleotidase is present in the endoplasmic reticulum and that the microsome fraction contains, in addition, vesicles derived from the plasma membrane.     

Cytochemical localization of adenylate cyclase in the various tissues of Locusta migratoria (migratorioides R.F.)

Summary The ultrastructural cytochemical procedure to demonstrate adenyl cyclase in mammalian organs was used in insects. After several modifications, an utilizable method was applied for the detection of the enzyme in the various tissues. Adenylate cyclase which can be stimulated with octopamine was localized on the membrane of the glial cells and the axolemma of certain large axons in the insect brain. Adenylate cyclase which could be activated by NaF and isoproterenol was also demonstrated in the lipid droplets of glial cells of the brain. With the simultaneous application of NaF and isoproterenol, rather strong adenylate cyclase activity could be detected on the surface of the corpora allata cells both in the cells situated on the glandular surface and the central part of the gland. In contrast in the corpus cardiacum enzyme activity was only observable on the basal lamina of the glandular surface. An appreciable amount of reaction product, indicating the presence of the enzyme, could be found on the surface of the lipid droplets in the fat body situated near the glandular tissues. In the heart muscle, reaction product referring to enzyme activation could not be demonstrated with the help of the methods applied.     

Ca2+-calmodulin-activated cyclic nucleotide phosphodiesterase from the rabbit myometrium

Two forms of soluble phosphodiesterase of cyclic nucleotides separating by DEAE-cellulose ion-exchange chromatography and not only differing in physicochemical and catalytic parameters but also differently regulated by calmodulin are found in the doe myometrium. Calmodulin with 10(-7)-10(-5) M concentrations of Ca2+ promotes the two-fold activation of the 3':5'-AMP (but not of 3':5'-GMP) hydrolysis by the first form of phosphodiesterase. Trifluoperazine (10 microM) lowers the activating action of calmodulin. The second form of soluble phosphodiesterase is not sensitive to the action of both calmodulin and Ca2+. 3':5'-GMP (10 microM) inhibits the 3':5'-AMP hydrolysis by the first form of phosphodiesterase; calmodulin exerts no effect on this process. The data obtained testify to the possible participation of Ca2+ and calmodulin in Ca2+-calmodulin-dependent phosphodiesterase regulation of the content of cyclic nucleotides (3':5'-AMP, in particular) in the doe myometrium.     

Platelet cyclic 3':5'-nucleotide phosphodiesterase released by thrombin and calcium ionophore.

Contact of rat platelets with thrombin or the divalent cation ionophore A-23187, in the presence of extracellular calcium, resulted in the secretion of adenosine 3':5'-monophosphate (cyclic AMP) and guanosine 3':5'-monophosphate (cyclic GMP) phosphodiesterases. Significant association of calcium with platelets occurred during platelet surface contact with thrombin. Thrombin concentration to induce association of calcium virtually agreed with that to release the enzyme. The finding that A-23187 (5 to 20 muM) also provoked a rapid and marked association of extracellular calcium with platelets suggests that calcium mobilization into the intracellular environment may account, at least in part, for this association between platelet and calcium. Two different phosphodiesterases, a relatively specific cyclic AMP and a relatively specific cyclic GMP phosphodiesterase were secreted from platelets into the plasma in soluble form. The amounts of the phosphodiesterases secreted were dose- or time-dependent on thrombin (0.1 to 2 units) or A-23187 (5 to 20 muM) within 30 min. The enzyme release by thrombin was completely inhibited by heparin but the release by A-23187 was not. The two phosphodiesterases secreted seemed to correspond to the two enzymes isolated from platelet homogenates in many respects. Rat platelets contained, at least, three cyclic 3':5'-nucleotide phosphodiesterases, namely, two relatively specific cyclic AMP phoshodiesterases and a relatively specific cyclic GMP phosphodiesterase which were clearly separated from each other by Sepharose 6B or DEAE-cellulose column chromatography or sucrose gradient centrifugation. The two platelet cyclic AMP phosphodiesterase (Mr = 180,000 and 280,000) had similar apparent Km values of 0.69 and 0.75 muM with different sedimentation coefficient values of 4.9 S and 7.1 S, respectively. They did not hydrolyze cyclic GMP significantly. A cyclic GMP phosphodiesterase (Mr - 260,000) exhibited abnormal kinetics for cyclic GMP with an apparent Km value of 1.5 muM and normal kinetics for cyclic AMP with a Km of 300 muM. The properties of a platelet cyclic AMP phosphodiesterase (Mr = 180,000) and a platelet cyclic GMP phosphodiesterase were found to agree with those of the two phosphodiesterases released from platelets by thrombin or A-23187. Depletion of extracellular calcium by an addition of citrate, EDTA, or ethylene glycol bis(beta-aminoethyl ether)-N,N'-tetraacetic acid (EGTA) to the blood or platelet suspension resulted in a loss of the activity of the smaller form of platelet cyclic AMP phosphodiesterase (Mr = 180,000) and addition of calcium restored the activity of this cyclic AMP phosphodiesterase. Thus, calcium seemed to be involved in the mechanism of an occurrence of this smaller form of cyclic AMP phosphodiesterase as well as the secretion of this enzyme. Contact of human platelets with thrombin also resulted in the secretion of cyclic nucleotide phosphodiesterase which was dependent on the concentration of calcium. No species difference was observed in this respect.     

Effect of benzimidazole derivatives and levamisole (phenylimidothiazole) on cyclic 3':5'-AMP phosphodiesterase from pig brain

The effects of phenylimidothiazole and some benzimidazole derivatives on phosphodiesterase of cyclic 3':5'-AMP (cAMP) from pig brain were studied. Depending on their concentrations the compounds tested activated or inhibited the enzyme activity. A correlation between the effects of benzimidazoles and phenylimidothiazole which is known to exert an immunomodulating action was observed.     

Induction of phosphodiesterase by cyclic adenosine 3':5'-monophosphate in differentiating Dictyostelium discoideum amoebae.

Cyclic adenosine 3':5'-monophosphate added to the starvation media of Dictyostelium discoideum amoebae induces both intracellular and extracellular phosphodiesterase activities of these cells. The induced enzyme activity appears several hours earlier than that in starved cells which have not been induced with cyclic nucleotide. In both cases, the appearance of enzyme is inhibited by cycloheximide, and actinomycin D, and daunomycin. The KmS for the extracellular enzyme(s) of nucleotide-induced and uninduced control cells are identical. The induction of enzyme activity seems specific for cyclic adenosine 3':5'-monophosphate since cyclic guanosine 3':5'-monophosphate, as well as other nucleotides, have no effect. No differences in the activity or excretion of either N-acetylglucosaminidase or the inhibitory of the extracellular phosphodiesterase are observed between cyclic adenosine 3':5'-monophosphate-induced and control cells. A direct activation of phosphodiesterase by cyclic adenosine 3':5'-monophosphate can be excluded, since the addition of this nucleotide to cell lysates has no effect on the enzyme activity.     

rac-Glycerol 1:2-cyclic phosphate 2-phosphodiesterase, a new soluble phosphodiesterase of mammalian tissues.

1. A soluble phosphodiesterase is present in mammalian tissues which rapidly hydrolyses enantiomorphs of rac-glycerol 1:2-cyclic phosphate, producing rac-glycerol 1-phosphate. 2. The enzyme has been purified up to 1700-fold by a combination of acetone precipitation and chromatography on DEAE-Sephadex A-50, Sephadex G-150 and hydroxyapatite. 3. The Km with glycerol cyclic phosphate as substrate is 7.2 mM, and the pH optimum broad (6.9--7.5). The molecular weight (by gel filtration) of the enzyme is approx. 35500. 4. The phosphodiesterase has no requirement for Ca2+ or Mg2+, but is stimulated by reducing agents (cysteine, dithiothreitol) and Fe2+. 5. The purified phosphodiesterase preparation also hydrolysed 3':5'-cyclic AMP, producing 5'-AMP exclusively, and 2':3'-cyclic AMP, forming 3'-AMP and 2'-AMP in the ratio 7:3. Bis-(p-nitrophenyl) phosphate was slowly hydrolysed, but other phosphodiesters tested were not attacked. 6. The phosphodiesterase is inhibited by theophylline and o-phenanthroline. It is inhibited by Pi and by a variety of phosphomonoesters, of which certain aromatic primary phosphates are particularly effective.     

The 2',3'-cAMP-adenosine pathway

Our recent studies employing HPLC-tandem mass spectrometry to analyze venous perfusate from isolated, perfused kidneys demonstrate that intact kidneys produce and release into the extracellular compartment 2',3'-cAMP, a positional isomer of the second messenger 3',5'-cAMP. To our knowledge, this represents the first detection of 2',3'-cAMP in any cell/tissue/organ/organism. Nuclear magnetic resonance experiments with isolated RNases and experiments in isolated, perfused kidneys suggest that 2',3'-cAMP likely arises from RNase-mediated transphosphorylation of mRNA. Both in vitro and in vivo kidney experiments demonstrate that extracellular 2',3'-cAMP is efficiently metabolized to 2'-AMP and 3'-AMP, both of which can be further metabolized to adenosine. This sequence of reactions is called the 2',3'-cAMP-adenosine pathway (2',3'-cAMP → 2'-AMP/3'-AMP → adenosine). Experiments in rat and mouse kidneys show that metabolic poisons increase extracellular levels of 2',3'-cAMP, 2'-AMP, 3'-AMP, and adenosine; however, little is known regarding the pharmacology of 2',3'-cAMP, 2'-AMP, and 3'-AMP. What is known is that 2',3'-cAMP facilitates activation of mitochondrial permeability transition pores, a process that can lead to apoptosis and necrosis, and inhibits proliferation of vascular smooth muscle cells and glomerular mesangial cells. In summary, there is mounting evidence that at least some types of cellular injury, by triggering mRNA degradation, engage the 2',3'-cAMP-adenosine pathway, and therefore this pathway should be added to the list of biochemical pathways that produce adenosine. Although speculative, it is possible that the 2',3'-cAMP-adenosine pathway may protect against some forms of acute organ injury, for example acute kidney injury, by both removing an intracellular toxin (2',3'-cAMP) and increasing an extracellular renoprotectant (adenosine).     

Regulation of urocaninase activity in the liver: role of 3',5'-AMP]

A dependence of rat liver urocaninase activity on the agents affecting the adenylate cyclase system was studied in vitro and in vivo. Urocaninase is considerably activated after the injection of glucagone, NaF, theophylline and 3',5'-AMP. Under conditions optimal for the protein kinase activity of phosphorylase the urocaninase of liver extracts was activated 7-fold on the average. The nezyme retains its activity after gel-filtration through Sephadex G-25 and is capable of inactivation in the presence of Mg2+ and of reactivation after addition of ATP and 3',5'-AMP. These data suggest a possibility of regulation of mammalian liver urocaninase activity by 3',5'-AMP-dependent phosphorylation of the enzyme. Derivatives of hypoxanthine (theophylline and caffeine) in concentration 10(-4) M activate urocaninase in liver extracts 2--3 and 1.5-fold respectively. The activation is probably not due to the 3',5'-AMP phosphodiesterase inhibition, since another phosphodiesterase inhibitor--papaverine--has no activating effect on urocaninase.     

Effect of training and physical load on 3':5'-AMP content and activity fo the enzymes of its metabolism in rat muscle

It is established tha in skeletal muscles of dormant animals under the influence of training the 3':5'-AMP content and the activity of adenylate cyclase increase; that of phosphodiesterase remains unchanged. A long physical load causes no changes in the 3':5"-AMP content in the skeletal muscles of the trained rats as compared to its content in the intact animals but evokes a decrease as compared to its level in the trained rats muscles. The activity of adenylate cyclase in the skeletal muscles of the trained rats under long physical load lowers, that of 3':5'-AMP phosphodiesterase also decreases but to a less extent. The found changes in the 3':5'-AMP metabolism in the trained animals skeletal muscles evidence for a possible participation of the 3':5'-AMP system in development of the organism adaptation to higher physical loads.