The response of plasma histamine to bronchoprovocation with methacholine, adenosine 5'-monophosphate, and allergen in atopic nonasthmatic subjects

To investigate the possible role of mast cell histamine release in mediating adenosine 5'-monophosphate (AMP)-induced bronchoconstriction, we have measured the histamine concentration in peripheral venous plasma following inhalation of methacholine, AMP, and allergen in concentrations sufficient to provoke mean maximum decreases in FEV1 of 42.8 +/- 2.2%, 46.5 +/- 3.9%, and 40.9 +/- 4.6%, respectively, in 10 atopic, nonasthmatic subjects. Mean baseline plasma concentrations of histamine were 0.25 +/- 0.02, 0.22 +/- 0.03, and 0.29 +/- 0.03 ng/ml on the methacholine, AMP, and allergen study days, respectively. Plasma histamine did not change following methacholine-induced bronchoconstriction, but increased in 9 out of 10 subjects to a mean maximum value of 0.78 +/- 0.15 ng/ml following inhalation of allergen (p less than 0.005). Following bronchial challenge with AMP, there was a significant elevation in plasma histamine in 9 out of 10 subjects to a mean maximum value of 0.39 +/- 0.03 ng/ml (p less than 0.005). We conclude that AMP-induced bronchoconstriction is associated with the enhanced release of histamine in the airways, probably from airway mast cells. However, the rise in plasma histamine, in being smaller than that occurring with a dose of allergen that provoked a similar degree of bronchoconstriction, suggests that additional mechanisms are operative in mediating the airways response to this nucleotide.     

Mechanisms of adenosine 5'-monophosphate catabolism in human erythrocytes

Uncertainties regarding the role of pyrimidine nucleotidase (PyrNase) in AMP catabolism were resolved by studies of erythrocytes from normal controls, controls with young mean cell ages, and patients with hereditary hemolytic anemia due to severe deficiency of PyrNase. Hemolysates from the latter exhibited undiminished capacity to dephosphorylate AMP over a broad range of pH, indicating that PyrNase was not directly involved. In each subject group, the rates of AMP dephosphorylation between pH 5.1 and 8.3 were indistinguishable from those of IMP, suggesting a potential role for AMP-deaminase, an erythrocyte enzyme that was stimulated by coformycin at pH 7.2. Quantitative analysis of catabolites in incubated hemolysates confirmed that AMP degradation preferentially occurred via deamination to IMP with subsequent dephosphorylation by another erythrocyte nucleotidase isozyme, deoxyribonucleotidase. Both AMP-deaminase and deoxyribonucleotidase have acidic pH optima with minimal activities at physiologic pH, suggesting that this pathway of AMP catabolism could accelerate depletion of the adenine nucleotide pool and thereby mediate the demise of senescent erythrocytes sequestered in the spleen.     

Measurement of plasma adenosine 3',5'-monophosphate.

Currently used methods for plasma cAMP measurements are either tedious (chromatographic preparation of sample) or potentially inaccurate (direct assay of plasma samples). A rapid, simple, and accurate competitive binding assay for plasma cAMP, which does not require chromatographic preparation of the sample, has been developed. This procedure prevents destruction of plasma cAMP by utilizing both theophylline and EDTA in the collection of the blood sample. Human plasma contains variable amounts of cAMP-binding activity which interfere with the measurement of cAMP by the standard competitive binding assay. Our assay procedure removes this binding activity by precipitation of plasma proteins with perchloric acid. The normal fasting value (+/- SD) of plasma cAMP using this technique is 17.6 +/- 4.3 pmol/ml, which is identical to values obtained by methods utilizing chromatographic purification of samples (18.3 +/- 3.0). The fasting plasma cAMP of patients with hyperparathyroidism is normal (16.2 +/- 3.4), but patients with maturity-onset diabetes mellitus have fasting values significantly below normal (12.3 +/- 2.4).     

Adenosine stimulates adenosine 3',5'-monophosphate and guanosine 3',5'-monophosphate accumulation in rat pinealocytes: evidence for a role for adenosine in pineal neurotransmission

Adenosine produces a concentration-dependent increase in pinealocyte cAMP (EC50, approximately 0.3 nM) and cGMP accumulation (EC50, approximately 0.7 nM). Maximal increases in both nucleotides are evident 10 min after treatment; 1 h later values return to pretreatment levels. Concentration-dependent effects on cAMP are also observed with N6-(L-2-phenylisopropyl)adenosine (EC50, approximately 0.75 nM), 5'-N-ethylcarboxy aminoadenosine (EC50, approximately 0.75 nM), and 2-chloroadenosine (EC50, approximately 2.0 nM); the EC50 values for stimulation of cGMP with these agents are higher by a factor of 2-10. In the case of 5'-N-ethylcarboxy amidoadenosine, the concentration-response curve is biphasic, with a significant effect evident within the range of 1-100 pM. The stimulatory nature of this response and the relative potency of the agonists tested are consistent with the involvement of an A2-like adenosine receptor. Comparison of adenosine and the selective beta-adrenergic agonist isoproterenol indicated that their maximal EC50 values were generally similar. Studies with antagonists revealed that both 8-(p-sulfophenyl)theophylline (1 microM) and the xanthine amine congener (8-[4-[[[(2-aminoethyl)carbonyl]methyl]oxy]phenyl]1,3- dipropylxanthine (1 microM) inhibited the effects of adenosine (1 nM to 1 microM), but xanthine amine congener was more potent; the latter was markedly effective at 0.1 nM, whereas 8-(p-sulfophenyl)theophylline was nearly ineffective at this concentration. It was also determined that pineal cells generate extracellular adenosine from extracellular ATP. ATP is thought to be released along with catecholamines during neurotransmission. Hence, these studies support the view that adenosine could participate in the transsynaptic regulation of pineal function.     

Catabolism of adenosine 5'-monophosphate in promastigotes of Leishmania tropica.

The specific activities of enzymes participating in AMP-catabolizing reactions in promastigotes of Leishmania tropica were determined. The following sequence of reactions is suggested: AMP leads to adenosine leads to adenine leads to hypoxanthine. The initial enzyme of this sequence, AMP nucleotidase, is apparently membrane-bound. The significance of AMP catabolism with respect to adenylate energy charge and a signal transfering mechanism is discussed. Adenine deaminase has been partially purified and characterized. Several purine analogues, inter alia allopurinol, proved to be inhibitors of the adenine deaminase catalyzed reaction.     

Time-dependent loss of adenosine 5'-monophosphate deaminase activity may explain elevated adenosine 5'-triphosphate levels in senescent erythrocytes

Senescent erythrocytes from rabbits were previously shown to have elevated levels of adenine nucleotides. The present study documents that aged red blood cells have a normal synthetic capacity for adenine nucleotides, as indicated by normal levels of adenosine kinase. However, senescent erythrocytes do have decreased levels of adenosine 5'-monophosphate deaminase, the critical enzyme involved in degrading adenine nucleotides. These circumstances of a normal synthetic capacity in the presence of decreased catabolic ability were observed previously in a human genetic deficiency of adenosine 5'-monophosphate deaminase; the red blood cells in these patients accumulate adenosine 5'- triphosphate as do senescent erythrocytes in rabbits.     

Effects of adenosine 3',5'-monophosphate, dibutyryl adenosine 3',5'-monophosphate, aminophylline, and imidazole on renal cellular calcium metabolism.

The effects of cAMP, dibutyryl cAMP (DBcAMP), aminophylline, and imidazole on total cell calcium, calcium transport, and distribution were studied in cultured kidney cells by kinetic analysis of 45Ca uptake and desaturation curves. Low concentrations of the cyclic nucleotides (10(-7) and 10(-5) M) increase the total cell calcium, all intracellular exchangeable pools, and calcium transport between all cellular compartments. Aminophylline (1 mM) has effects qualitatively similar to cAMP and DBcAMP, while imidazole has opposite effects. At concentrations of 15 and 40 mM, imidazole depresses the total cell calcium and the cellular exchangeable calcium. Compared to the effects of parathyroid hormone (PTH), the changes obtained with 10(-7) and 10(-5) M cAMP are relatively modest, but higher concentrations (10(-3) M) of both cAMP and DBcAMP produce stimulations as marked as with 15 ng/ml PTH. The most dramatic changes are seen in the mitochondrial calcium pool and in the mitochondrial calcium exchange, which increase between 20- and 40-fold. These experiments show that cAMP mimics the effect of PTH on kidney cells and support the theory that cAMP is the mediator of PTH action on renal cell calcium transport.     

Phenol-containing saline solution as a diluent for adenosine 5'-monophosphate in bronchial challenge testing

OBJECTIVES: To investigate the effect of dissolving adenosine 5'-monophosphate (AMP) with phenol-containing saline solution on the stability and the bronchoconstrictive properties of this indirect agonist. METHODS: Eleven subjects with asthma well controlled with short-acting inhaled beta2-agonists as required or with inhaled corticosteroids were studied. Bronchial challenge tests with AMP dissolved with either normal saline solution or saline solution containing 0.4% phenol were performed on separate days. Furthermore, to assess the potential influence of the phenol-containing saline solution on the stability of the bronchoconstrictor agent, AMP solutions in concentrations of 40 microg/mL and 400 microg/mL were prepared in saline solution and phenol-containing saline solution and, after 30 min, the AMP levels were determined by high-performance liquid chromatography (HPLC) assay. RESULTS: The geometric mean AMP provocative concentration causing a 20% fall in FEV1 (PC20) was 13.49 mg/mL (95% confidence interval [CI], 6.76 to 26.91) for the saline solution method, and AMP PC20 for the saline solution with phenol method was 8.91 mg/mL (95% CI, 3.39 to 23.44) [p = 0.18]. No significant differences were found between the concentrations of AMP made in saline solution compared to those made in phenol-containing saline solution measured by HPLC. CONCLUSION: These observations indicate that normal saline solution with or without phenol can be used as the diluent for AMP. However, since a potential risk with AMP of industrial sources is the bacterial contamination, adding a preservative such as phenol to a saline solution diluent might be recommended.     

Cyclic adenosine 3',5'-monophosphate modulation of mullerian duct regression

(Bu)2cAMP inhibits the action of testicular Mullerian inhibiting substance (MIS) in vitro, but it is unknown whether the intracellular nucleotide diminishes production of MIS by the testis or interferes with its action at the Mullerian duct. When added to the 14 1/2-day old rat embryo Mullerian duct in organ culture, (Bu)2cAMP (0.1 or 1.0 mM) inhibited regression caused by biologically active exogenous MIS fractions, as well as that produced by the fetal testis (1.0 mM). Dibutyryl cyclic guanosine 3',5'-monophosphate was ineffective against exogenous MIS fractions or the fetal testis. The phosphodiesterase inhibitor, methyl-isobutyl-xanthine, had the same inhibitory effect on the MIS-fraction (0.1 or 1.0 mM), as well as against MIS secreted from the fetal testis (1.0 mM). Theophylline at 1.0 mM inhibited the action of the MIS fraction. The presence of (Bu)2cAMP in the medium was required for at least the first 24 h of the 72-h incubation to significantly inhibit MIS action. These results indicate that intracellular cAMP inhibits the action of MIS at the Mullerian duct itself by a potentially reversible change in the cells. We speculate that cAMP may act by altering the state of differentiation in the cells, perhaps by mediating phosphorylation of intracellular (and extracellular) proteins.     

Regulation of adenosine 3',5'-monophosphate levels in the pancreatic B cell

The poor glucose-induced insulin release from single purified B cells has been attributed, in part, to the low cellular cAMP levels. The present study demonstrates that isolated B cells exhibit a markedly lower cAMP formation than B cells lodged in intact islets and suggest that this deficiency is caused by their separation from glucagon-containing A cells. cAMP levels in purified B cells are rapidly and potently elevated by glucagon from 10(-10) M on, reaching the values of islet B cells at 10(-9) M. In contrast, exogenous glucagon stimulates cAMP formation in isolated islets only from 3.10(-9) M on, suggesting that endogenously released glucagon is mainly responsible for the higher cAMP levels in islet B cells. Somatostatin counteracts glucagon-induced cAMP production in purified B cells and, therefore, has also the potential to mediate an intra-islet regulation of B cell functions. Neither insulin nor pancreatic polypeptide affect cAMP formation in pancreatic B cells. Glucose alone does not influence cAMP levels in purified B cells, but enhances glucagon-induced cAMP formation in these cells. The glucose-dependent increase in islet cAMP is therefore not considered as the nutrient-induced mediator for hormone release but as a minor amplification of the glucagon-dependent signal. Experiments on reaggregated islet cells permit the reconstruction of the events which regulate cAMP levels in isolated islets. Further support is hereby given to the hypothesis that a normal glucose-induced insulin release from intact islets requires the simultaneous synarchic participation of a nutrient-dependent and a hormone-dependent messenger system.     

Serotonin stimulates adenosine 3',5'-monophosphate accumulation in parathyroid adenoma

The effect of serotonin on cAMP accumulation in parathyroid adenoma tissue from patients with primary hyperparathyroidism was studied in vitro. Incubation with 10(-5) M serotonin elicited a marked increase (of 90--150%) in cAMP content in slices of parathyroid adenoma tissue. This stimulatory effect of serotonin was already apparent after 2 min of incubation; stimulation by serotonin was dose dependent, with the highest stimulation being achieved at 10(-4) M serotonin. The serotonin antagonists, methylsergide and cinanserin, in concentrations equimolar to serotonin completely blocked the stimulatory effect of serotonin on cAMP increase. The serotonin content in surgically removed parathyroid adenoma tissue, as determined by fluorometric assay, was 6.4 +/- 1.2 pmol/mg wet wt (approximately 0.8 x 10(-5) M). The present observations demonstrate that parathyroid adenoma tissue has a high content of serotonin, and serotonin stimulates cAMP accumulation in this tissue. Since cAMP acts as a mediator of parathyroid hormone (PTH) release, our results suggest that serotonin could be one of the factors regulating PTH secretion and/or contributing to PTH hypersecretion in various forms of primary hyperparathyroidism.