Analogs of adenosine 3',5'-cyclic phosphate. II. Synthesis and enzymatic activity of derivatives of 1,N6-ethenoadenosine 3',5'-cyclic phosphate

The reactions of chloroacetaldehyde with adenosine 3′,5′-cyclic phosphate, and with several analogs modified at C₈ of the purine ring or C₅, of the sugar, lead to the corresponding 1,N⁶-etheno derivatives^d. Similar reactions using other 2-bromoaldehydes or phenacyl bromide give 1,N⁶-ethenonucleotides substituted at the α- or β-positions of the etheno bridge respectively. The ability of these compounds to activate the protein kinases from rabbit muscle and calf brain has been evaluated over a wide range of concentrations. While no derivative proved to be more active than adenosine 3′,5′-cyclic phosphate itself using the enzyme from rabbit muscle, a wide spectrum of activities was found using that from calf brain.     

Investigation of the adenosine 3', 5'-cyclic phosphate binding site of pig brain histone kinase with the aid of some analogues of adenosine 3', 5'-cyclic phosphate.

2'-O-Chloroacetyl cyclic AMP, 2'-O-acrylyl cyclic AMP and N-6, 2'-O-diacrylyl cyclic AMP were synthesized by the reaction of cyclic AMP with chloroacetic and acrylic anhydrides, respectively. Selective O-deacylation of N-6, 2'-O-diacrylyl cyclic AMP yielded N-6 -monoacrylyl cyclic AMP. In the reaction of gamma-mercaptobutyric acid with 8-bromo cyclic AMP, 8-(gamma-carboxypropylthio) cyclic AMP was obtained. The compounds synthesized and other cyclic AMP analogues (8-bromo cyclic AMP and adenosine 3', 5'-cyclic sulphate) were tested for ability to interact with the highly purified pig brain histone kinase. All compounds under study were found to be activators of the enzyme. The highest activating potency was manifested by 8-bromo cyclic AMP and 8-(gamma-carboxypropylthio) cyclic AMP; adenosine 3', 5'-cyclic sulphate was the least potent in this respect. All compounds were shown to inhibit binding of cyclic [-3-H]AMP to histone kinase. The inhibition was competitive with respect to cyclic AMP in all cases. All compounds, except for 2'-O-chloroacetyl cyclic AMP may indicate the formation of a covalent bond between this analogue and the enzyme. These findings suggest that an active site of the regulatory subunit of the histone kinase contains at least three specific areas responsible for cyclic AMP binding.     

Purification and characterization of an adenosine cyclic 3':5' monophosphate-dependent protein kinase from human erythrocyte membrane.

A cAMP dependent protein kinase was extracted from human erythrocyte membrane with hydrosoluble fraction and partially purified by ammonium sulfate-precipitation and DEAE-cellulose chromatography. The pH of optimal activity is 6.5; the enzyme has an absolute requirement of Mg²⁺ ions at the concentration of 10 mM and is strongly inhibited by Ca²⁺. It uses ATP as phosphate donor with a Km of 3.7 × 10^?6 M. Cyclic AMP stimulates the activity with an apparent Ka of 5 × 10^?8 M; cIMP and cGMP also acts as activators. Enzyme activity is thermolabile and not protected by Mg ATP complex. The enzyme purified from erythrocyte membrane is a type I protein-kinase as proven by DEAE cellulose chromatography and dissociation of the subunits in presence of NaCl 0.5 M and histone.     

An adenosine 3':5'-monophosphate-dependent protein kinase from the human erythrocyte membrane. Purification and characterization.

An adenosine 3':5'-monophosphate-dependent protein kinase (ATP:protein phosphotransferase, EC 2.7.1.37) has been isolated from the human erythrocyte memebrane and the phosphotransferase activity exhibited by this enzyme has been purified 800-fold. In concentrated solutions, the membrane-derived protein kinase undergoes aggregation with a concomitant loss in observed phosphotransferase activity. This loss of activity can be restored by means of inducing deaggregation. The phosphotransferase activity of the protein kinase is virtually obliterated in the presence of high (300 mM) concentrations of sodium chloride. This effect is also reversible. The pH optimum for the phosphotransferase reaction that is catalyzed by the membrane-derived protein kinase is approximately 8. Micromolar concentrations of cAMP are optimal with respect to promoting the phosphotransferase reaction. Initial velocity and product inhibition studies were conducted on the cAMP-independent protein kinase derived from the cAMP-dependent enzyme. These studies indicate that the phosphotransferase reaction proceeds by a sequential kinetic mechanism.     

1,N6-etheno-2-aza-adenosine 3',5'-monophosphate: a new fluorescent substrate for cycle nucleotide phosphodiesterase

1,N⁶-etheno-2-aza-adenosine 3′,5′-monophosphate (cyclic 2-aza-?-AMP) has been shown to be a sensitive and an efficient substrate for the assay of cyclic-nucleotide phosphodiesterase. The relative activity is 75% compared to cyclic AMP. Two K_m values of 503 and 15 μm were observed with the beef heart enzyme.     

Differential modulation by N4, 2'-O-dibutyryl cytidine 3':5'-cyclic monophosphate of neutrophil activation.

The cyclic pyrimidine nucleotide, cCMP, is an endogenous substance in mammalian cells but little is known on its functional role. We studied the effects of cCMP, its cell-permeant analogue, N4,2'-O-dibutyryl cytidine 3':5'-cyclic monophosphate (Bt2cCMP), and of butyrate on superoxide (O2-) formation and cytosolic Ca2+ [( Ca2+]i) in human neutrophils. Bt2cCMP inhibited O2- formation and the rise in [Ca2+]i induced by a chemotactic peptide at submaximally effective concentrations. O2- formation induced by platelet-activating factor was potentiated by Bt2cCMP, whereas the cyclic nucleotide had no effect on the rise in [Ca2+]i induced by this agonist. Bt2cCMP enhanced O2- formation induced by tau-hexachlorocyclohexane at a submaximally effective concentration. O2- formation stimulated by complement C5a, concanavalin A, NaF, A 23187, phorbol myristate acetate and arachidonic acid was not affected by Bt2cCMP. cCMP was less effective than Bt2cCMP to inhibit fMet-Leu-Phe-induced O2- formation, and butyrate was without effect on any of the functional parameters studied. Our data show that a cell-permeant analogue of cCMP differentially inhibits and potentiates activation of human neutrophils.     

Stimulation of calcium uptake in platelet membrane vesicles by adenosine 3',5'-cyclic monophosphate and protein kinase.

The events involved in platelet shape change, aggregation, the release reaction and contraction are thought to be mediated by the availability of Ca2+. Increased cytoplasmic calcium, released from intracellular stores, triggers platelet activity, and increased concentration of adenosine 3',5'-cyclic monophosphate (cyclic AMP) inhibits platelet alterations. We have studied the hypothesis that cyclic AMP may regulate the level of platelet cytoplasmic calcium by stimulating calcium removal by a membrane system. Such a hypothesis would be consistent with the reversibility of most manifestations of platelet activation. Human platelets were sonicated and unlysed platelets, mitochondria and granules were removed by centrifugation at 19 000 X g. Electron microscopy shows that the sediment, after centrifugation of the supernatant at 40 000 X g consists to a large extent of membrane vesicles. Such preparations actively concentrate calcium, as measured by the uptake of 45Ca, and also have the maximal calcium-stimulated ATPase activity. Optimal calcium uptake requires ATP and oxalate, and release of calcium from loaded vesicles was stimulated by the calcium ionophore A23187 and inhibited by LaCl3. These data indicate that calcium was being actively concentrated within membrane vesicles. After washing of such preparations in the absence of ATP, their capacity to take up Ca2+ is reduced to an initial value of 2.8 nmol/mg protein per min. In the presence of 2 - 10(6) M cyclic AMP to which was added a protein kinase preparation from human platelets, up to a 3-fold increase of this rate of uptake was observed. These results suggest that in platelets, as in muscle, cyclic AMP is a regulatory factor in the control of cytoplasmic calcium. Although the cyclic nucleotide may have still other functions, it appears likely that the well-known inhibition of many platelet activities by high intracellular cyclic AMP concentrations is directly linked to the stimulation of the removal of Ca2+ from the cytoplasm.     

Studies on the uptake and metabolism of adenosine 3':5'-cyclic monophosphate and N6,O2-dibutyryl 3':5'-cyclic adenosine monophosphate in sea urchin eggs

Structurally abnormal mitochondria were found in Candida utilis cells grown in the presence of either high copper concentrations or copper deficiency as compared with cells grown in standard media (copper 60 μg/l). In cells grown under conditions of copper deficiency the average size of the mitochondria is 0.6 μm compared with 0.2 μm of the normal cell, and the cristae have an abnormal appearance. In cells grown in the presence of high copper concentrations the mitochondrial size is up to 2–3 μm with poorly developed cristae and abnormal appearance of the matrix area.     

Neutrophil dysfunction in guanosine 3',5'-cyclic monophosphate-dependent protein kinase I-deficient mice

The regulation of neutrophil functions by Type I cGMP-dependent protein kinase (cGKI) was investigated in wild-type (WT) and cGKI-deficient (cGKI-/-) mice. We demonstrate that murine neutrophils expressed cGKIalpha. Similar to the regulation of Ca2+ by cGKI in other cells, there was a cGMP-dependent decrease in Ca2+ transients in response to C5a in WT, but not cGKI-/- bone marrow neutrophils. In vitro chemotaxis of bone marrow neutrophils to C5a or IL-8 was significantly greater in cGKI-/- than in WT. Enhanced chemotaxis was also observed with cGKI-/- peritoneal exudate neutrophils (PE-N). In vivo chemotaxis with an arachidonic acid-induced inflammatory ear model revealed an increase in both ear weight and myeloperoxidase (MPO) activity in ear punches of cGKI-/- vs WT mice. These changes were attributable to enhanced vascular permeability and increased neutrophil infiltration. The total extractable content of MPO, but not lysozyme, was significantly greater in cGKI-/- than in WT PE-N. Furthermore, the percentage of MPO released in response to fMLP from cGKI-/- (69%) was greater than that from WT PE-N (36%). PMA failed to induce MPO release from PE-N of either genotype. In contrast, fMLP and PMA released equivalent amounts of lysozyme from PE-N. However, the percentage released was less in cGKI-/- (approximately 60%) than in WT (approximately 90%) PE-N. Superoxide release (maximum velocity) revealed no genotype differences in responses to PMA or fMLP stimulation. In summary, these results show that cGKIalpha down-regulates Ca2+ transients and chemotaxis in murine neutrophils. The regulatory influences of cGKIalpha on the secretagogue responses are complex, depending on the granule subtype.     

Human erythrocyte proteins associated with adenosine 3',5'-cyclic monophosphate action.

Two adenosine 3',5'-cyclic monophosphate (cyclic-AMP)-binding protein factors (molecular weight 230,000) have been partially purified from human erythrocytes. One of these proteins seems to be different from the cyclic-AMP-binding component of the cyclic-AMP-dependent protein kinases. These protein factors are also capable of binding adenosine. We present data also on two forms of cyclic-AMP-dependent protein kinases (ATP: protein phosphotransferase, EC 2.7.1.37) partially purified from the cytosol of normal human erythrocytes. Kinase I has been classified as type I enzyme on the basis of its activation when preincubated with protamine, histone or NaCl. The substrate specificities of the two kinases and many of their kinetic parameters are rather similar. Their subunit structure is reminiscent of that of kinases obtained from other sources. The catalytic subunit of both enzymes reversibly cross-react with the regulatory subunit of kinase I from the rabbit red blood cell.     

Cholera toxin-sensitive 3',5'-cyclic adenosine monophosphate and calcium signals of the human dopamine-D1 receptor: selective potentiation by protein kinase A.

The signal transduction pathways of the dopamine-D1 receptor were investigated in two cell types stably transfected with the human D1 receptor cDNA, rat pituitary GH4C1 cells (GH4-hD1), and mouse Ltk-fibroblast cells (L-hD1). In both GH4-hD1 and L-hD1 cell lines, stimulation of the dopamine-D1 receptor induced a marked increase in cAMP accumulation. In addition, dopamine potentiated activation of L-type voltage-dependent calcium channels in a cAMP-dependent manner in GH4-hD1 cells. However, in L-hD1 cells, dopamine increased cytosolic free calcium concentrations ([Ca++]i) by mobilization of intracellular calcium rather than by calcium influx. This effect was correlated with a dopamine-induced enhancement of phospholipase C activity in L-hD1 cells. Pretreatment (24 h) with cholera toxin (CTX) was used to maximally activate the GTP-binding protein (G protein) Gs, causing a maximal elevation of cAMP levels and uncoupling the D1 receptor from Gs. The described actions of dopamine in both cell lines were abolished by pretreatment with CTX, indicating that CTX substrates (e.g. Gs) may mediate these actions. The blockade by CTX was not due to CTX-induced elevation of cAMP, since pretreatment with forskolin or 8-bromo-cAMP to activate cAMP-dependent protein kinase did not inhibit dopamine actions nor alter basal [Ca++]i. Pretreatment (1-3 h) of L-hD1 cells with forskolin (10 microM) or 8-bromo-cAMP (5 mM) altered neither the basal activity of phospholipase C nor basal [Ca++]i in L-hD1 cells but greatly enhanced the dopamine-induced increase of phosphatidyl inositol turnover and [Ca++]i. From these results we conclude that: 1) the dopamine-D1 receptor induces multiple and cell-specific signals, including elevation of cAMP levels in both GH and L cells, cAMP-dependent activation and potentiation of opening of L-type voltage-dependent calcium channel in GH cells, and a novel phosphatidyl inositol-linked mobilization of cellular calcium in L cells; 2) coupling of the D1 receptor to these responses involves CTX-sensitive proteins, possibly Gs; and 3) acute preactivation of cAMP-dependent protein kinase can markedly enhance, rather than attenuate, certain pathways of dopamine-D1 transmembrane signaling.