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PubMed1981

Mechanical response of rat myocardium to dibutyryl cyclic AMP in relation to effects of alpha-and beta-adrenoceptor stimulators.

Dibutyryl cyclic AMP, and alpha- and beta-adrenoceptor stimulators are all able to elicit inotropic effects. alpha- and beta-Adrenoceptor stimulation are known to change each myocardial contraction-relaxation cycle differently. In order to elucidate the myocardial function of cyclic AMP the effects of dibutyryl cyclic AMP on the contraction-relaxation cycle of isolated rat heart papillary muscle were examined and compared to the effects of alpha-and beta-adrenoceptor stimulation, respectively. Dibutyryl cyclic AMP (in the presence of propranolol) increased developed tension (Tmax) by 18%, rate of tension rise (T'max) by 46%, rate of tension fall (T'min) by 62% and onset-rate of relaxation (T"min) by 136%. These changes in the contraction-relaxation cycle were strikingly similar to those produced by isoprenaline (beta-adrenoceptor stimulation). The response to dibutyryl cyclic AMP, however, developed much more slowly than did the response to isoprenaline. The latter effect was associated with cyclic AMP elevation in a way indicating a trigger function for cyclic AMP. The alpha-adrenoceptor stimulation (by phenylephrine combined with propranolol), however, increased measures both for contraction and for relaxation by about the same degree, and the effects occurred without changes of cyclic AMP contents. Phenylephrine alone (combined alpha-and beta-adrenoceptor stimulation) elicited a substantial cyclic AMP elevation but gave mechanical effects only slightly different from the pure alpha-adrenergic response. Thus cyclic AMP effects did not seem to be fully expressed in this case. As a whole, the results indicate that the effects of both dibutyryl cyclic AMP and of isoprenaline are mediated by the cyclic AMP-system while alpha-adrenoceptor stimulation involves other mechanisms.

Adrenergic alpha-Agonists↗
PubMed1979

Mode of stimulation by injection of cyclic AMP and external acidification of the sodium efflux in barnacle muscle fibres.

1. A study has been made in single barnacle muscle fibres of the effect of micro-injected pure protein kinase inhibitor (PKI) on the response of the Na efflux to injection of cyclic AMP and external acidification. 2. (i) Injection into fibres of 1.6 x 10(-4) M-pure PKI is without effect on the resting Na efflux. (ii) Injection of 1.6 x 10(4) M-pure PKI before 0.03 M-cyclic AMP causes a marked reduction in the magnitude of the response of the Na efflux to the nucleotide. The same is true when 10(-4) M-cyclic AMP is injected after PKI. (iii) Injection of partially pure catalytic subunits causes a sustained stimulation of the ouabain-insensitive Na efflux, which is almost completely reversed by injecting PKI. (iv) Injection of 100 mM-EGTA before PKI fails to alter the lowered response of the ouabain-insensitive Na efflux to injection of 10(-4) M-cyclic AMP. (v) Ouabain (10(-4) M) when applied following the injection of 10(-4) M-cyclic AMP causes a drastic fall in the stimulated Na efflux. 3. (i) Injection of 1.6 x 10(-4) M-pure PKI before or after external acidification fails to abolish or reduce the stimulatory response to acidification. (ii) Injection of 1.6 x 10(-4) M-pure PKI before acidification practically abolishes the response of the ouabain-insensitive Na efflux to 0.03 M-cyclic AMP in the presence of acidification. (iii) Radioimmunoassay of total cyclic AMP and cyclic GMP content in single fibres before and after acidification shows no appreciable alteration in nucleotide content following acidificiation. (iv) Injection of 100 mM-EGTA before acidification enhances the stimulatory response to acidification. (v) External application of Dantrolene (10(-5) M) fails to alter the size of the stimulatory response to acidification. 4. (i) Prior external application of 5 x 10(-4) M-benzolamide results in a marked reduction in the magnitude of the response of the ouabain-insensitive Na efflux to the injection of 3 x 10(-4) M-cyclic AMP. (ii) Benzolamide totally abolishes the response of the ouabain-insensitive Na efflux to the injection of catalytic subunits. 5. The evidence brought forward is compatible with the view that (a) The mechanism by which cyclic AMP stimulates the Na efflux involves activation by cyclic AMP of the cyclic AMP-dependent protein kinase system, and hence release of the catalytic subunit, and (b) the mechanism by which external acidification leads to stimulation of the Na efflux involves activation of a benzolamide-sensitive system, possibly carbonic anhydrase, rather than the adenyl cyclase system. The actions of cyclic AMP and catalytic subunits on the Na efflux are closely linked to activation of the benzolamide sensitive system.

Animals↗
PubMed1992

The inhibitory action of cyclic AMP on responses to carbachol dependent on calcium stores in rat gastric smooth muscle.

1. The effects of cyclic AMP on contraction and Ca(2+)-activated K+ currents induced by carbachol (CCh), caffeine and inositol 1,4,5-trisphosphate (InsP3) were examined in intact and skinned smooth muscle fibres and in whole-cell voltage-clamped smooth muscle cells of the rat stomach. Intracellular Ca2+ level, [Ca2+]i, was monitored in intact muscle fibres loaded with Fura-2. 2. In intact muscle fibres, dibutyryl cyclic AMP, 8-bromo-cyclic AMP and forskolin inhibited a phasic contraction induced by CCh (100 microM) much more extensively than that induced by caffeine (30 mM) in Ca(2+)-free solution containing 2 mM-EGTA. A rise in [Ca2+]i evoked by CCh was also reduced by dibutyryl cyclic AMP. 3. In skinned muscle fibres, InsP3 (40 microM) produced a contraction of amplitude similar to that evoked by caffeine (30 mM) in Ca(2+)-free solution containing 0.05 mM-EGTA. Cyclic AMP suppressed the InsP3-induced contraction to a much greater extent than that induced by caffeine. 4. In cells voltage-clamped at 0 mV, CCh (100 microM) evoked a transient Ca(2+)-activated outward K+ current in 61% of cells tested. After wash-out of CCh, caffeine (10 mM) evoked a similar K+ current in all cells. In cells loaded with cyclic AMP (100 microM), the percentage of cells responding to CCh was reduced to 26% and the magnitude of current response tended to decrease. Cyclic AMP caused a small increase in the caffeine-induced K+ current. 5. An outward current was elicited immediately after the patch membrane was ruptured at a holding potential of 0 mV, using a patch pipette containing InsP3 (40 microM), in 76% of cells tested. In cells treated with dibutyryl cyclic AMP, the percentage of cells responding to InsP3 was reduced to 50% and the magnitude of current response tended to decrease. 6. In intact muscle fibres loaded with Fura-2, the relationship between [Ca2+]i and tension development shifted to the right in the presence of dibutyryl cyclic AMP. In skinned muscle fibres, cyclic AMP shifted the pCa-tension relation to the right, suggesting that cyclic AMP inhibits the contractile machinery directly. 7. These results suggest that the inhibitory effect of cyclic AMP on muscarinic receptors mediating both the contraction and the Ca(2+)-activated K+ current, is partly due to the inhibition of InsP3-induced Ca2+ release from intracellular stores in rat gastric smooth muscle cells.

Animals↗
PubMed1973

Regulation of adrenal ornithine decarboxylase by adrenocorticotropic hormone and cyclic AMP.

Adrenal ornithine decarboxylase activity was stimulated in a dose-related manner after administration of ACTH or dibutyryl ((6)N-2'-O-dibutyryl) cyclic AMP to hypophysectomized rats. Little effect was observed for 2 h, but striking increases in enzyme activity were observed 4 h after administration of these substances. Effects of ACTH and dibutyryl cyclic AMP were not secondary to stimulation of steroidogenesis, since hydrocortisone had no effect on adrenal ornithine decarboxylase although it did stimulate activity of the enzyme in the liver and kidney.ACTH, given subcutaneously to hypophysectomized rats, induced striking increases in adrenal cyclic AMP levels within 15-30 min with a fall towards the base line in 1 h. Increases in ornithine decarboxylase activity lag several hours after this endogenous cyclic AMP peak, in contrast to the stimulatin of steroidogenesis by the nucleotide that requires only 2-3 min. After graded doses of ACTH, increases in adrenal cyclic AMP levels at 30 min were paralleled by proportional increases in adrenal ornithine decarboxylase activity 4 h after hormone treatment. Whereas maximal levels of adrenal steroidogenesis have been observed at tissue cyclic AMP levels of 6 nmol/g. ACTH is capable of inducing increases in nucleotide levels up to 200 nmol/g or more. These high tissue levels of cyclic AMP, although unneccessary for maximal steroidogenesis, appear to stimulate adrenal ornithine decarboxylase activity. Several results in addition to the time lag in the stimulation of ornithine decarboxylase activity suggest a mechanism involving accumulation of the enzyme or some factor needed for its activity rather than direct activation of the enzyme by cyclic AMP. Thus, the addition of cyclic AMP directly to the ornithine decarboxylase assay mixture in vitro was without stimulatory effect. In addition, actinomycin D or cycloheximide in doses sufficient to block adrenal RNA and protein synthesis, respectively inhibited the stimulation of ornithine decarboxylase activity by ACTH in vivo. An adrenocortical cancer was found to maintain ornithine decarboxylase activity at very high levels, but did so at much lower cyclic AMP levels than those of ACTH-stimulated adrenals. It is concluded that ACTH stimulates adrenal ornithine decarboxylase activity and that this effect may be mediated by cyclic AMP. However, cyclic AMP be mediated by appear to be a determinant of the high level of enzyme activity found in adrenocortical cancer.

Adrenal Gland Neoplasms↗
PubMed1975

Stimulation of osmotic water flow in toad bladder by prostaglandin E1. Evidence for different compartments of cyclic AMP.

The effect of prostaglandin E1 (PGE1) on osmotic water flow across toad bladder and cyclic AMP content of the mucosal epithelial cells has been determined under basal conditions and in the presence of either theophylline or antidiuretic hormone (ADH); Under basal conditions and with PGE1 concentrations from 10(-8) to 10(-5) M no evidence of stimulation of water flow was observed, and with 10(-7) M PGE1 a significant inhibition was foundmcyclic AMP content under control conditions was 8 pmol/mg protein. It was 9 at 10(-8) M PGE1, 13 at 10(-7) M, 16 at 10(-6) M, and 23 at 10(-5) M. In the presence of theophylline, 10(-8) and 10(-7) M PGE1 inhibited the theophylline-induced water flow as expected. In contrast, 10(-6) and 10(-5) M PGE1 enhanced the rate of water flow. Theophylline increased cyclic AMP content from 8 to 18 pmol/mg protein. PGE1 in the presence of theophylline caused marked increases in cyclic AMP content; The content was 23 at 10(-7) M, 41 at 10(-6) M, and 130 at 10(-5) M; Thus PGE1 stimulates theophylline-induced water flow at cyclic AMP concentrations somewhere between 23 and 41 pmol/mg. Further evidence along these lines was obtained from experiments in which the effects of PGE1 on ADH-induced water flow were studied. Inhibitory effects of PGE1 were not observed at concentrations of PGE1 which raised the level of intracellular cyclic AMP to 30 pmol/mg protein or higher. These results were obtained despite the fact that all four concentrations of PGE1 tested were found capable of inhibiting ADH-induced water flow under appropriate conditions or, in other words, were inhibiting the adenylate cyclase controlling water flow, Thus the increase in cyclic AMP content in response to PGE1 is not derived from this enzyme. Thus the stimulation of water flow by PGE1 in the presence of theophylline is thought to be caused by cyclic AMP spilling over from one compartment to the water flow compartment. No evidence was obtained to directly suggest spillover into the sodium transport compartment. Furthermore evidence is discussed to suggest that most of the cyclic AMP generated in the tissue does not originate from the enzyme controlling sodium transport. As cyclic AMP-stimulated water flow and sodium transport are thought to occur in one cell type, the granular cells, distinct pools of cyclic AMP are thought to be present in one and the same cell type. Thus one pool controls water flow and one controls sodium transport. With high concentrations of PGE1 in the presence of theophylline or high concentrations of ADH, the adenylate cyclase responsible for water flow is inhibited; However, PGE1 can stimulate a tissue adenylate cyclase to sufficiently high levels that cyclic AMP spills over into the "water flow compartment" and thus stimulates water flow.

Animals↗
PubMed1975

Studies of the elevated extracellular concentration of cyclic AMP in uremic man.

This study was designed to elucidate the mechanism of elevation of plasma cyclic AMP in uremic man. Plasma cyclic AMP was measured in 15 normal subjects and in 18 patients with severe renal failure. In some members from both groups the kinetic parameters of the metabolism of extracellular cyclic AMP were measured. Plasma cyclic AMP was elevated from 23 nM in control subjects to 59 nM in uremic patients, regardless of the presence or absence of the kidneys or parathyroid glands. A single pass of uremic blood through a Kiil hemodialyzer decreased plasma cyclic AMP from 58 to 30 nM. The clearance of cyclic AMP by the dialyzer correlated directly with the blood flow passing through the machine. Hemodialysis for 6 h decreased plasma cyclic AMP levels in the systemic circulation by only 12%. Studies with tritiated cyclic AMP revealed a plasma clearance rate of 624 ml/min in normal subjects and of 344 ml/min in patients with uremia. Such a large decrease in plasma clearance rate cannot be explained by a failure of urinary excretion of cyclic AMP and suggests impairment of "metabolic clearance." In addition, the "plasms production rate" of cyclic AMP was 65% higher in patients with renal failure than in normal subjects. It is concluded that the elevation of plasma cyclic AMP in uremic man is due to a combination of: (a) lack of urinary excretion, (b) decreases metabolic clearance, and (c) increased production of plasma cyclic AMP.

Adult↗
PubMed1975

[Effect of cyclic AMP and Ca++ on steroidogenesis by rat adrenal mitochondrial fraction. Studies on the mechanism of ACTH action (I) (author's transl)].

Despite the accumulation of a number of studies, the mechanism of action of ACTH remains to be clarified. Although it is now clear that cyclic AMP acts as a intracellular mediator of ACTH action, the mechanism of its action on the stimulation of steroidogenesis is not known. The present studies were carried out to test the hypothesis that cyclic AMP might act directly on adrenal mitochondrial fraction to stimulate the metabolism of cholesterol to pregnenolone and progesterone, and to determine whether Ca++ might modulate the action of cyclic AMP. Adrenal mitochondria were obtained from male Sprague-Dawley rats pretreated with dexamethasone. Steroidogenesis by the mitochondrial fraction from cholesterol-4-14C (0.2-0.25 muCi, 3.6-4.5 mmumole/sample) were measured in a system containing 20 mM tris-HCl buffer (pH 7.4), 11.5 mM NaCl, 15.4 mM KCl, 70 mM sucrose, 10 mM sodium succinate and mitochondrial fraction (0.16-0.22 mg protein/sample). Incubations were performed at 37 degrees C, with shaking, in the presence or absence of cyclic AMP, cyclic GMP and cycloheximide. After incubation, the medium was extracted with chloroform, and the extracts were analyzed by thin-layer chromatography. And the radioactivity of the separated steroids was measured. The products from cholesterol-4-14C were mainly pregnenolone and progesterone, and the other products were almost negligible. Cyclic AMP effected the formation of pregnenolone and progesterone by mitochondria. Cyclic AMP exerted its effect even at low concentrations (5 X 10(-6) approximately 5 X 10(-5)M), which was presumably near the intracellular level. On the other hand, cyclic GMP (5 X 10(-5) M) failed to enhance steroidogenesis. The effect of Ca++ on the action of various concentrations (5 X 10(-6) approximately 3 X 10(-3) M) of cyclic AMP was also clearly demonstrated. Addition of Ca++ (1 mM) to the incubation medium intensified the stimulatory effect of cyclic AMP in each concentration. And in the presence of Ca++, the most effective level of cyclic AMP was shifted from 5 X 10(-4) approximately 3 X 10(-3)M to the lower concentration (5 X 10(-5)M). In addition, cyclic AMP action was modified by the changes in the concentration of Ca++ in the medium. At concentration of 10(-6) M of Ca++, steroid formation of mitochondria was maximally activated by cyclic AMP. These observations suggest that cyclic AMP enhances steroidogenesis by acting directly on adrenal mitochondria to stimulate pregnenolone and progesterone formation from cholesterol, and that Ca++ plays a significant role in its action.

Adrenal Glands↗
PubMed2006

Ecto- and cytosolic 5'-nucleotidases in normal and AMP deaminase-deficient human skeletal muscle.

In skeletal muscle, adenosine monophosphate (AMP) is mainly deaminated by AMP deaminase. However, the C34T mutation in the AMPD1 gene severely reduces AMP deaminase activity. Alternatively, intracellular AMP is dephosphorylated to adenosine via cytosolic AMP 5'-nucleotidase (cN-I). In individuals with a homozygous C34T mutation, cN-I might be a more important pathway for AMP removal. We determined activities of AMP deaminase, cN-I, total cytosolic 5'-nucleotidase (total cN), ecto-5'-nucleotidase (ectoN) and whole homogenate 5'-nucleotidase activity in skeletal muscle biopsies from patients with different AMPD1 genotypes [homozygotes for C34T mutation (TT); heterozygotes for C34T mutation (CT); and homozygotes for wild type (CC): diseased controls CC; and normal controls CC]. AMP deaminase activity showed genotype-dependent differences. Total cN activity in normal controls accounted for 57+/-22% of whole homogenate 5'-nucleotidase activity and was not significantly different from the other groups. A weak inverse correlation was found between AMP deaminase and cN-I activities (r2=0.18, p<0.01). There were no significant differences between different groups in the activities of cN-I, whole homogenate 5'-nucleotidase and ectoN, or in cN-I expression on Western blots. No correlation for age, fibre type distribution and AMPD1 genotype was found for whole homogenate nucleotidase, total cN and cN-I using multiple linear regression analysis. There was no gender-specific difference in the activities of whole homogenate nucleotidase, total cN and cN-I. The results indicate no changes in the relative expression or catalytic behaviour of cN-I in AMP deaminase-deficient human skeletal muscle, but suggest that increased turnover of AMP by cN-I in working skeletal muscle is due to higher substrate availability of AMP.

5'-Nucleotidase↗
PubMed1980

Cyclic AMP release from normal human thyroid slices in response to thyrotrophin.

Slice preparations of normal human thyroid tissue were incubated in vitro with TSH. The cyclic AMP contents of slices were determined at intervals up to 120 min, and cyclic AMP in the incubation medium was also estimated for each incubation period. Slice cyclic AMP levels were related both to incubation time and TSH dose. In response to 10 mU TSH/ml, slice cyclic AMP levels were maximal within 60 min, and were not significantly changed at 120 min. Cyclic AMP was detectable in the medium within 10 min of slice exposure to TSH, and increased throughout the initial 60 min of incubation. Cyclic AMP release during this period was dependent on both TSH dose and incubation time. Between 60-120 min, however, cyclic AMP release partially lost its TSH dose-dependency, and 0.5-5.0 mU TSH/ml were equipotent with respect to the final medium cyclic AMP level attained. Slices incubated without TSH released only small amounts of cyclic AMP, and maximal levels were attained within 20 min. In contrast to the adenylate cyclase response of thyroid membrane preparations, which was stimulated by NaF, suggesting that cyclic AMP release was not a result of the stimulation of damaged cells. These findings demonstrate the importance of cyclic AMP release from human thyroid slices, following in vitro exposure to TSH, and suggest that, after incubation periods such as are used for the functional biodetection of thyroid stimulators, the magnitude of cyclic AMP release may be of quantitative significance.

Adenylyl Cyclases↗
PubMed1982

Cyclic AMP-binding proteins in human blood platelets detected by photoaffinity labelling.

Cyclic AMP inhibits platelet aggregation induced by physiological agents. 8 Azido [32P]cyclic AMP (N3 cyclic AMP) has been utilized as a photoaffinity probe to define the cyclic AMP-binding proteins present in unperturbed human platelets and their subcellular fractions. Specificity of cyclic AMP binding was determined by contrasting binding in the presence and absence of excess unlabelled cyclic AMP, cyclic GMP and 5'-AMP. Binding was unaffected by 5'-AMP and obliterated by cyclic AMP. Four major species of binding proteins, 49 000, 42 000, 39 000, 37 000, were obtained in all platelet fractions (crude homeogenate, cytosol, membranes and granules). Two-dimensional gel electrophoresis of platelet cytosol resolved the major molecular weight species into 15 specific cyclic AMP binding proteins of four molecular weight classes differing by charge density. These studies suggest that platelets contain an array of specific cyclic AMP-binding proteins which may function in hemostatic regulation.

Affinity Labels↗
PubMed1992

Dynamic aspects of AMPs in the kidney in physiological conditions--their metabolism and turnover.

The past few decades have witnessed a number of studies on AMPs in the kidney. Almost all of these studies, however, have dealt with the quantitative and qualitative analysis of AMPs in the kidney using biochemical techniques, whereas the dynamic aspect of AMPs in the kidney has been overlooked and its metabolism and turnover have never been emphasized. On the other hand, the kidneys removed from humans and experimental animals revealed a wide variety in interstitial tissue in the medulla microscopically and in the papilla of the kidney macroscopically. The reasons for these variations in the medulla, its mechanism and the matrical component have not yet been clarified. In an effort to clarify these points, we herein describe our study on the dynamic aspect of AMPs in kidney histologically in relation to their biological turnover which we employed several staining methods. Our results revealed that there was a very active turnover of AMPs present in the kidney confirming that the kidney plays an important role physiologically in AMP metabolism. The degree of the turnover of AMPs in the kidney varies from case to case. In the active phase, AMPs are excreted in the glomerulus and reabsorbed in the proximal portion of the collecting tubules. Most of the absorbed AMPs are finally drained through the lymphatic vessels in situ and a few are excreted into the urine. Contrary cases have been demonstrated, however, only were a few AMPs were noted throughout the nephrons in the kidney. AMP turnover in the kidney is unexpectedly remarkable. The medulla, which has been considered less important, in fact, plays a key role in AMP metabolism.

Alcian Blue↗
PubMed1991

Transition-state analysis of a Vmax mutant of AMP nucleosidase by the application of heavy-atom kinetic isotope effects.

The transition state of the Vmax mutant of AMP nucleosidase from Azotobacter vinelandii [Leung, H. B., & Schramm, V. L. (1981) J. Biol. Chem. 256, 12823-12829] has been characterized by heavy-atom kinetic isotope effects in the presence and absence of MgATP, the allosteric activator. The enzyme catalyzes hydrolysis of the N-glycosidic bond of AMP at approximately 2% of the rate of the normal enzyme with only minor changes in the Km for substrate, the activation constant for MgATP, and the Ki for formycin 5'-phosphate, a tight-binding competitive inhibitor. Isotope effects were measured as a function of the allosteric activator concentration that increases the turnover number of the enzyme from 0.006 s-1 to 1.2 s-1. The kinetic isotope effects were measured with the substrates [1'-3H]AMP, [2'-2H]AMP, [2'-2H]AMP, [9-15N]AMP, and [1',9-14C, 15N]AMP. All substrates gave significant kinetic isotope effects in a pattern that establishes that the reaction expresses intrinsic kinetic isotope effects in the presence or absence of MgATP. The kinetic isotope effect with [9-15N]AMP decreased from 1.034 +/- 0.002 to 1.021 +/- 0.002 in response to MgATP. The [1'-3H]AMP isotope effect increased from 1.086 +/- 0.003 to 1.094 +/- 0.002, while the kinetic isotope effect for [1',9-14C, 15N]AMP decreased from 1.085 +/- 0.003 to 1.070 +/- 0.004 in response to allosteric activation with MgATP. Kinetic isotope effects with [1'-14C]AMP and [2'-2H]AMP were 1.041 +/- 0.006 and 1.089 +/- 0.002 and were not changed by addition of MgATP.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Monophosphate↗
PubMed1988

The induction, desensitization and de-induction of tyrosine aminotransferase by 8-bromo-cyclic AMP in rat hepatoma cells.

Addition of 1-3 mM-8-bromo-cyclic AMP to monolayer cultures of H-4 rat hepatoma cells resulted in a rapid but short-lived increase in tyrosine aminotransferase (EC 2.6.1.5) activity. The transient nature of this induction is due to desensitization to 8-bromo-cyclic AMP. Throughout this time course of induction and desensitization, removal of 8-bromo-cyclic AMP resulted in a rapid and significant decrease in tyrosine aminotransferase activity, a process referred to as 'de-induction' in this study. We showed that the changes in tyrosine aminotransferase activity in its induction, desensitization and de-induction by 8-bromo-cyclic AMP were directly attributable to changes in the synthesis rate of the protein, and the amount of translatable and hybridizable mRNA encoding for tyrosine aminotransferase (mRNATAT). We further showed that this desensitization was specific to cyclic AMP. First, only active analogues of cyclic AMP and agents which increased cellular concentrations of cyclic AMP elicited this desensitization. Second, the desensitized cells were refractory only to the effects of 8-bromo-cyclic AMP; dexamethasone and insulin induced the tyrosine aminotransferase activity in the 8-bromo-cyclic AMP-desensitized cells in a manner similar to that of the controls. Studies on the metabolism of 8-bromo-cyclic AMP suggest that neither its degradation nor the accumulation of its primary metabolite, 8-bromoadenosine, played a significant role in modulating the expression of tyrosine aminotransferase during the time course of action of 8-bromo-cyclic AMP. These results provide evidence for a specific pretranslational mode of action of cyclic AMP in the control of tyrosine aminotransferase expression in its desensitization and de-induction, in addition to the early phase of induction.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1987

Affinity labeling of the allosteric site of fructose 1,6-bisphosphatase with an AMP analog.

D-Fructose 1,6-bisphosphatase [EC 3.1.3.11, FBPase] is one of the key enzymes in glyconeogenesis and its activity is controlled by various effectors such as substrate, AMP and ATP. To analyze this complex regulation system, we tried an affinity labeling of FBPase with an AMP derivative, since AMP is a potent allosteric inhibitor of this enzyme. The results obtained are as follows. 1. To determine the functional groups which are essential for AMP as an inhibitor, inhibitory activities of some AMP derivatives were examined. These derivatives modified at the purine ring or phosphate group lost the activity while one modified at the ribose ring retained the ability to inhibit FBPase. This shows that an affinity labeling reagent should be an AMP derivative in which the ribose ring is modified. 2. 2',3'-Dialdehyde AMP (dial-AMP) was prepared by periodate oxidation of AMP and was reacted with FBPase. Under appropriate conditions, 1 mol of the reagent was incorporated per mol of enzyme subunit with a concomitant loss of enzyme activity. The reaction was prevented by the presence of AMP but not of ATP. The heat-stability, the kinetic parameters and the UV-absorption spectrum of the modified enzyme were all the same as those of native FBPase in the presence of AMP. Thus it was concluded that the allosteric AMP site in FBPase was modified specifically.

Adenosine Monophosphate↗
PubMed2000

Pharmacokinetics and safety of single intravenous infusions of the adenosine agonist, AMP 579, in patients with end-stage renal insufficiency.

The pharmacokinetics of an adenosine agonist (AMP 579) were characterized in patients with end-stage renal disease compared to sex- and age-matched healthy volunteers. All study participants were administered single AMP 579 doses of 50 micrograms/kg as a 6-hour, constant-rate intravenous infusion. Serial blood samples were obtained for measurement of plasma AMP 579 concentration, and predose samples were collected for determination of AMP 579 plasma protein binding. The safety of AMP 579 administration in renally impaired patients also was evaluated. AMP 579 was rapidly cleared from the systemic circulation in all subjects as plasma concentrations were below the limit of detection by 2 to 4 hours after terminating the infusion. Noncompartmental analysis yielded mean values for the plasma AMP 579 concentration at the end of the 6-hour infusion (C6 h) of 9.6 and 10.5 ng/mL and for systemic clearances (Cl) of 0.91 and 0.72 L/h/kg in renally impaired patients and healthy volunteers, respectively. Mean volumes of distribution (Vss) in the renally impaired and healthy volunteers were 0.92 and 0.84 L/kg, and terminal elimination half-life values (t1/2) were 1.61 and 1.33 hours, respectively. The extent to which AMP 579 is bound to plasma protein was not altered in renally impaired patients since the free fractions were 4.0% and 3.4% for renally impaired and healthy volunteers, respectively. It was concluded that the pharmacokinetic parameters of AMP 579 were similar in both groups. The 6-hour AMP 579 infusion was generally well tolerated by both renal patients and healthy volunteers. There were no serious adverse events, and there were only two mild adverse events in 1 renally impaired patient judged possibly related to the study drug that quickly resolved. There were no clinically significant changes in laboratory values or clinical evaluations during the study. There was a slight increase in heart rate during the infusion of similar magnitude for both the renal patients and healthy volunteers. These data suggest that AMP 579 may be administered to renally impaired patients with minimal cardiovascular effects and adverse events. These results in end-stage renal patients (worst-case scenario) indicate that dose adjustment in patients with renal insufficiency of any degree is not indicated in future studies of AMP 579.

Adult↗
PubMed2003

[Studies on physico-chemical properties and hypoglycemic activity of complex polysaccharide AMP-B from Atractylodes macrocephala Koidz].

AIM: To isolate a complex polysaccharide (AMP-B) from Atractylodes macrocephala Koidz and study its phtsico-chemical properties and hypoglycemic activity. METHODS: The root of Atractylodes macrocephala K. was extracted with water and precipitated with ethanol, dialyzed against water and freeze-dried to get the crude polysaccharides (AMP). A complex polysaccharide (AMP-B) was isolated and purified on DEAE-cellulose column. The model of diabetes rats was established with alloxan injection through the tail vein. Male rats were divided into 5 groups: the normal group, the control group, and three AMP-B-fed groups. Measuring the blood glucose, water and food consumption, thymus and pancreas index, and studying cut sections of pancreas tissues. RESULTS: AMP-B is a complex-polysaccharide, elemental analysis of AMP-B shown C 32.84%, H 5.68%, and N 1.79%. The neutral polysaccharide content of AMP-B was 50.3%, uronic acid was 40.4%, and protein was 11.5%. Monosaccharide composition of AMP-B was determined by GC, AMP-B composed of Glc, Gal, Man, Ara and Rha in a molar ratio of 3.0:2.5:1.3:3.5:1.0. AMP-B was found to reduce blood glucose level in alloxan-diabetic rats markedly at doses of 50, 100 and 200 mg.kg-1 by ig, but no effect in normal rat. AMP-B was found to decrease the consumption of water and food, recover pancreas damage of diabetic rats obviously, inhibited the atrophy of thymus and pancreas of the diabetic rats induced by alloxan. CONCLUSION: AMP-B showed significant hypoglycemic effect on the experimental hyperglycemias rats induced by alloxan.

Animals↗
PubMed1988

Interaction of fructose 2,6-bisphosphate and AMP with fructose-1,6-bisphosphatase as studied by nuclear magnetic resonance spectroscopy.

The interaction of AMP and fructose 2,6-bisphosphate with rabbit liver fructose-1,6-bisphosphatase has been investigated by proton nuclear magnetic resonance spectroscopy (1H NMR). The temperature dependence of the line widths of the proton resonances of AMP as a function of fructose-1,6-bisphosphatase concentration indicates that the nucleotide C2 proton is in fast exchange on the NMR time scale while the C8 proton is exchange limit. The exchange rate constant, koff, has been calculated for the adenine C8 proton and is 1900 s-1. Binding of fructose 6-phosphate and inorganic phosphate, or the regulatory inhibitor, fructose 2,6-bisphosphate, results in a decrease in the dissociation rate constant for AMP from fructose-1,6-bisphosphatase, as indicated by the sharpened AMP signals. A temperature dependence experiment indicates that the AMP protons are in slow exchange when AMP dissociates from the ternary complex. The rate constant for dissociation of AMP from the enzyme.AMP.fructose 2,6-bisphosphate complex is 70 s-1, 27-fold lower than that of AMP from the binary complex. These results are sufficient to explain the enhanced binding of AMP in the presence of fructose 2,6-bisphosphate and, therefore, the synergistic inhibition of fructose-1,6-bisphosphatase observed with these two regulatory ligands. Binding of fructose 2,6-bisphosphate to the enzyme results in broadening of the ligand proton signals. The effect of AMP on the binding of fructose 2,6-bisphosphate to the enzyme has also been investigated. An additional line width broadening of all the fructose 2,6-bisphosphate protons has been observed in the presence of AMP. The assignment of these signals to the sugar was accomplished by two-dimensional proton-proton correlated spectra (two-dimensional COSY) NMR. From these data, it is concluded that AMP can also affect fructose 2,6-bisphosphate binding to fructose-1,6-bisphosphatase.

Adenosine Monophosphate↗
PubMed1977

Synthesis of AMP analogs and their use for studies on the allosteric site of rabbit muscle glycogen phosphorylase b.

In order to obtain a better understanding of the allosteric site of rabbit muscle phosphorylase b, nine AMP analogs having a bulky hydrophobic benzene ring were synthesized and tested for activity as activators or inhibitors. N6-Benzyl-AMP derivatives activated phosphorylase b to the same extent as AMP but bound to the enzyme more tightly than AMP. N6-p-nitrobenzyl-AMP had the highest affinity (Ka = 7.7 X 10(-7) M) for the AMP site. In an attempt to irreversibly modify the allosteric site of phosphorylase b, N6-p-bromoacetaminobenzyl-AMP was synthesized. Phosphorylase b was maximally activated upon incorporation of 1.0 mol of N6-p-bromoacetaminobenzyl-AMP per enzyme subunit, and its activity was approximately 90% of that of native phosphorylase b measured in the presence of AMP. The modified enzyme showed characteristics (e.g., kinetic parameters, stability, solubility, inhibition by glucose-6-phosphate, and state of aggregation) quite similar to those observed for the native enzyme in the presence of AMP. These results indicate that the AMP site of phosphorylase was specifically labeled by N6-p-bromoacetaminobenzyl-AMP. The nature of the allosteric site of phosphorylase b is discussed based on the results obtained.

Adenosine Monophosphate↗