Evidence for an adenosine A2/Ra receptor on human basophils

5'-N-ethylcarboxamideadenosine (NECA) greater than 2-chloroadenosine greater than adenosine greater than (-)-N6-(R-phenyl-isopropyl)-adenosine greater than (+)-N6-(S-phenylisopropyl)-adenosine, in that order of potency, inhibited in vitro antigen-induced histamine release from human basophils in a dose-dependent fashion. Inhibition occurred only during the first stage of antigen-induced histamine release and the nucleosides failed to inhibit the release caused by the Ca2+ ionophore, A23187. 6-nitrobenzylthioinosine and dipyridamole, which inhibit adenosine uptake, and erythro-9-(2-hydroxy-3-nonyl)adenine, which blocks adenosine metabolism, did not impair the inhibition caused by NECA and adenosine. 8-phenyltheophylline and theophylline, two competitive antagonists of adenosine receptors, blocked the inhibition caused by NECA and adenosine. These data suggest that NECA and other adenosine analogs activate a specific cell surface adenosine receptor which possesses properties similar to those of an adenosine A2/Ra receptor.     

Inhibition of basophil histamine release by anti-inflammatory steroids. II. Studies on the mechanism of action

The glucocorticosteroids inhibit the IgE-dependent release of histamine by human basophils with an order of potency that very closely parallels that found in vivo (i.e., triamcinolone acetonide greater than dexamethasone greater than beta-methasone greater than prednisolone greater than hydrocortisone much greater than progesterone approximately tetrahydrocortisone approximately 0). The effect is seen after a 24-hr preincubation with nanomolar to micromolar concentrations of glucocorticoid. In contrast, release of histamine stimulated by the formyl methionine containing peptide f-met-leu-phe, the calcium ionophore A23187, and the tumor-promoting phorbol diester 12-O-tetradecanoylphorbol-13-acetate was not inhibited by 24-hr incubation with the potent glucocorticoid dexamethasone. Dexamethasone inhibited anti-IgE-induced histamine release without altering its rate, suggesting that the glucocorticoids do not inhibit histamine release by elevating the intracellular level of cAMP. Dexamethasone did not consistently alter either the total or occupied basophil IgE Fc receptor number, and therefore the glucocorticoid effect does not appear to be due to the modulation of cell surface Fc epsilon receptor content. These data indicate that steroid hormones inhibit basophil IgE-dependent activation through a specific glucocorticoid receptor. The mechanism by which they do so appears not to involve an elevation of cAMP or a shedding of cell surface Fc epsilon receptors. Further, because the glucocorticoids did not inhibit release initiated by the PLA2-dependent stimuli f-met-leu-phe, A23187 and TPA, the inactivation of IgE-dependent histamine release by glucocorticoids may not be the result of PLA2 inhibition.     

Preliminary identification and role of phosphodiesterase isozymes in human basophils.

We attempted to identify and establish the role of cyclic nucleotide phosphodiesterase (PDE) isozymes in human basophils by using standard biochemical techniques as well as describing the effects of isozyme-selective and nonselective inhibitors of PDE. The nonselective PDE inhibitors, theophylline and 3-isobutyl-1-methylxanthine, inhibited anti-IgE-induced release of histamine and leukotriene C4 (LTC4) from basophils. This inhibition was accompanied by elevations in cAMP levels. Rolipram, an inhibitor of the low Km cAMP-specific PDE (PDE IV), inhibited the release of both histamine and LTC4 from activated basophils and increased cAMP levels in these cells. In contrast, mediator release from basophils was not inhibited by either siguazodan or SK&F 95654, inhibitors of the cGMP-inhibited PDE (PDE III) or zaprinast, an inhibitor of the cGMP-specific PDE (PDE V). SK&F 95654 failed to elevate basophil cAMP in these experiments whereas zaprinast induced significant increases in cAMP content. The inhibitory effect of rolipram on mediator release was potentiated by siguazodan or SK&F 95654, but not by zaprinast. SK&F 95654 also enhanced the ability of rolipram to increase cAMP content. Forskolin, a direct activator of adenylate cyclase, inhibited IgE-dependent release of mediators from basophils and increased cAMP levels in these cells. These effects were enhanced by rolipram, but not by SK&F 95654 or zaprinast. The cell permeant analog of cAMP, dibutyryl cAMP, inhibited mediator release from these cells, a property not shared by either dibutyryl-cGMP or sodium nitroprusside, an activator of soluble guanylate cyclase. The presence of both PDE III and PDE IV was confirmed by partially purifying and characterizing PDE activity in broken cell preparations. Overall, these data lend support to the hypothesis that cAMP inhibits mediator release from basophils and suggest that the major PDE isozyme responsible for regulating cyclic AMP content in these cells is PDE IV, with a minor contribution from PDE III. However, the finding that zaprinast caused increases in cAMP without inhibiting mediator release indicates that cAMP accumulation is not invariably linked to an inhibition of basophil activation.     

Regulation of human basophil and lung mast cell function by cyclic adenosine monophosphate

Immunologic activation of purified human lung mast cells (HLMC) and basophils with anti-IgE induced histamine release but failed to elicit any changes in cAMP levels. In contrast, histamine release and monophasic rises in cAMP were observed in both rat peritoneal mast cells (RPMC) challenged with concanavalin A (73% enhancement over basal cAMP 20 sec after activation) and a cultured mouse bone marrow-derived mast cell (PT18 cell line) passively sensitized with dinitrophenol-specific IgE and stimulated with antigen (39% increase above basal at 15 sec). The adenylate cyclase activators isoprenaline, prostaglandin E2 (PGE2), and forskolin and the phosphodiesterase inhibitor isobutylmethylxanthine (IBMX) all induced elevations in cAMP levels in both basophils and HLMC. In basophils, PGE2 and isoprenaline produced approximately twofold increases in cAMP that were maximal at 1 min and decayed thereafter. Forskolin and IBMX produced threefold increases in cAMP that peaked 10 min after activation and persisted for up to 20 min. In HLMC, isoprenaline provoked a rapid monophasic fourfold increase in cAMP that was maximal at 1 min after addition. Levels of cAMP subsequently declined but remained significantly elevated over resting levels for up to 30 min. PGE2, forskolin, and IBMX all produced approximately threefold rises in HLMC cAMP that peaked around 5 min and persisted for 30 min. In both the basophil and HLMC, agonist-induced elevations in cAMP correlated well with the inhibition of mediator release. In basophils, the order IBMX greater than forskolin greater than PGE2 greater than isoprenaline held for both the inhibition of histamine and leukotriene C4 release and the augmentation of cAMP levels. In HLMC, individual agonists elevated cAMP levels to similar degrees and inhibited the release of histamine, leukotriene C4, and PGD2 to comparable extents, although the release of the arachidonate metabolites was generally more sensitive to the inhibitory actions of these agonists. These results suggest that elevations in cAMP, in both the basophil and HLMC, are associated with the inhibition of mediator release but not the initiation of the secretory process.     

Adenosine inhibits macrophage colony-stimulating factor-dependent proliferation of macrophages through the induction of p27kip-1 expression.

Adenosine is produced during inflammation and modulates different functional activities in macrophages. In murine bone marrow-derived macrophages, adenosine inhibits M-CSF-dependent proliferation with an IC50 of 45 microM. Only specific agonists that can activate A2B adenosine receptors such as 5'-N-ethylcarboxamidoadenosine, but not those active on A1 (N6-(R)-phenylisopropyladenosine), A2A ([p-(2-carbonylethyl)phenylethylamino]-5'-N-ethylcarboxamido adenosine), or A3 (N6-(3-iodobenzyl)adenosine-5'-N-methyluronamide) receptors, induce the generation of cAMP and modulate macrophage proliferation. This suggests that adenosine regulates macrophage proliferation by interacting with the A2B receptor and subsequently inducing the production of cAMP. In fact, both 8-Br-cAMP (IC50 85 microM) and forskolin (IC50 7 microM) inhibit macrophage proliferation. Moreover, the inhibition of adenylyl cyclase and protein kinase A blocks the inhibitory effect of adenosine and its analogues on macrophage proliferation. Adenosine causes an arrest of macrophages at the G1 phase of the cell cycle without altering the activation of the extracellular-regulated protein kinase pathway. The treatment of macrophages with adenosine induces the expression of p27kip-1, a G1 cyclin-dependent kinase inhibitor, in a protein kinase A-dependent way. Moreover, the involvement of p27kip-1 in the adenosine inhibition of macrophage proliferation was confirmed using macrophages from mice with a disrupted p27kip-1 gene. These results demonstrate that adenosine inhibits macrophage proliferation through a mechanism that involves binding to A2B adenosine receptor, the generation of cAMP, and the induction of p27kip-1 expression.     

Occupancy of adenosine receptors raises cyclic AMP alone and in synergy with occupancy of chemoattractant receptors and inhibits membrane depolarization.

We have recently demonstrated that adenosine, acting via adenosine A2 receptors, inhibits generation of superoxide anions (O2-) by stimulated neutrophils. To determine the mechanism(s) by which adenosine inhibits O2- generation stimulated by the chemoattractant N-formylmethionylleucylphenylalanine (FMLP), we examined cyclic AMP (cAMP) concentrations, stimulated membrane depolarization and Ca2+ movements. Neither adenosine nor 5'-N-ethylcarboxamidoadenosine (NECA), the most potent agonist at adenosine A2 receptors, increases neutrophil cAMP content. However in the presence of the non-methylxanthine phosphodiesterase inhibitor, Ro-20-1724, both adenosine and NECA elicit a reversible increase in intracellular cAMP concentration. The chemoattractant FMLP also elicits an increment in the neutrophil cAMP content. NECA, in the presence of Ro-20-1724, synergistically enhances the increment in cAMP following stimulation by FMLP. However Ro-20-1724 does not potentiate the inhibition of O2- generation by NECA. Unlike other agents which increase neutrophil cAMP concentrations, NECA, even in the presence of a phosphodiesterase inhibitor, only trivially inhibits degranulation. We also found that adenosine markedly inhibits stimulated membrane depolarization but does not affect the stimulated increment in free ionized intracellular calcium. Moreover, inhibition by adenosine of O2- generation does not vary with the concentration of extracellular calcium. These results fulfil the last criterion for the demonstration of an A2 receptor on human neutrophils, and indicate that adenosine occupies an A2 receptor on neutrophils to raise intracellular cAMP in synergy with occupancy of the FMLP receptor. The results reported here also indicate that cAMP is not the second messenger for inhibition of O2- generation by adenosine and its analogues.     

Effects of propentofylline on adenosine receptor activity in Chinese hamster ovary cell lines transfected with human A1, A2A, or A2B receptors and a luciferase reporter gene.

Propentofylline is neuroprotective in vivo, but its mechanism of action is not completely understood. Previously, propentofylline was shown to block adenosine transport processes, to inhibit three adenosine receptor subtypes, and to inhibit cAMP phosphodiesterase. We tested the effect of propentofylline on adenosine receptor function in Chinese hamster ovary (CHO) cells transfected with human adenosine A1, A2A, or A2B receptors and a luciferase reporter gene under control of a promoter sequence containing several copies of the cAMP response element. We investigated the concentration-dependent inhibitory effects of propentofylline on cAMP phosphodiesterase, adenosine transport processes, and adenosine A1, A2A, and A2B receptors. At concentrations > or = 1 mM, propentofylline increased luciferase activity probably as a result of inhibition of cAMP phosphodiesterase. Inhibition of [3H]adenosine uptake by propentofylline was concentration dependent, with IC50 values of 37-39 microM for the three cell types. Agonist-activated adenosine A1 receptors were antagonized by 100 microM propentofylline, but inhibition of agonist-stimulated A2A or A2B receptors was not observed. In contrast, A1 and A2A receptor mediated effects of adenosine were enhanced by propentofylline at concentrations of 1 and 100 microM, respectively. These data indicate that the net effects of propentofylline in vivo will be dependent on the concentrations of propentofylline and adenosine available and on the subtypes of adenosine receptors, phosphodiesterases, and nucleoside transporters present.     

PKC and cAMP positively modulate alkaline-induced exocytosis in the human mast cell line HMC-1

We study in HMC-1 the activation process, measured as histamine release. We know that ammonium chloride (NH(4)Cl) and ionomycin release histamine, and the modulatory role of drugs targeting protein kinase C (PKC), adenosine 3',5'-cyclic monophosphate (cAMP), tyrosine kinase (TyrK) and phosphatidylinositol 3-kinase (PI3K) on this effect. We used G?6976 (100 nM) and low concentration of GF 109203X (GF) (50 nM) to inhibit Ca(2+)-dependent PKC isozymes. For Ca(2+)-independent isozymes, we used 500 nM GF and 10 microM rottlerin (specifically inhibits PKCdelta). Phorbol 12-myristate 13-acetate (PMA) (100 ng/ml) was used to stimulate PKC, and genistein (10 microM) and lavendustin A (1 microM) as unspecific TyrK inhibitors. STI571 10 microM was used to specifically inhibit the activity of Kit, the receptor for stem cell factor, and 10 nM wortmannin as a PI3K inhibitor. Activation of PKC with PMA enhances histamine release in response to NH(4)Cl and ionomycin. PMA increases NH(4)Cl-induced alkalinization and ionomycin-induced Ca(2+) entry. Inhibition of PKCdelta strongly inhibits Ca(2+) entry elicited by ionomycin, but failed to modify histamine release. The effect of cAMP-active drugs was explored with the adenylate cyclase activator forskolin (30 microM), the inhibitor SQ22,536 (1 microM), the cAMP analog dibutyryl cAMP (200 microM), and the PKA blocker H89 (1 microM). Forskolin and dibutyryl cAMP do increase NH(4)Cl-induced alkalinization, and potentiate histamine release elicited by this compound. Our data indicates that alkaline-induced exocytosis is modulated by PKC and cAMP, suggesting that pH could be a modulatory signal itself.     

Functional compartments in rat mast cells for cAMP and calcium on histamine release

The crosstalk between 3', 5'-cyclic adenosine monophosphate (cAMP), intracellular calcium, and histamine release in rat mast cells using the stimulatory effect of three different drugs, thapsigargin, sodium fluoride (NaF), and compound 48/80 were studied. Each of these drugs induces histamine release by different mechanisms. The transducting pathways modulating cAMP and intracellular calcium levels were modified by using, cholera toxin (CTX) which ADP-rybosylates Gs-protein, pertussis toxin (PTX) which ADP-rybosylates Gi-protein, and okadaic acid (OA) which inhibits phosphatases 1 and 2a. Our results show that CTX increased cAMP levels and inhibited histamine release elicited by thapsigargin and compound 48/80. The inhibitory effect of CTX on histamine release was potentiated by OA in the presence of compound 48/80 but was decreased in the presence of thapsigargin. Calcium uptake was stimulated by NaF and compound 48/80. The previous treatment with OA increased calcium uptake when combined with compound 48/80 but not with NaF. Treatment with NaF highly stimulated calcium uptake and cAMP levels only when combined with OA and CTX. These results suggest that the modulatory effect of intracellular calcium and cAMP on histamine release depend more on the crosstalk of the activated signal transducting pathway than on the final level of calcium or cAMP, further supporting the theory that rat mast cells are divided into functionally distinct compartments.     

Inhibition by adenosine 3':5'-monophosphate of eicosanoid and platelet-activating factor biosynthesis in the mouse PT-18 mast cell.

A mouse spleen-derived mast cell line (PT-18) was employed to examine the mechanisms of adenosine 3':5'-monophosphate (cAMP)-mediated inhibition of antigen-induced lipid mediator biosynthesis. Specifically, we tested the hypothesis that increasing cAMP in mast cells inhibits lipid mediator biosynthesis by a mechanism independent of effects on histamine release (degranulation) or changes in cytosolic calcium concentration. Forskolin inhibited antigen-induced prostaglandin D2 (PGD2), leukotriene C4 (LTC4), and leukotriene B4 (LTB4) production by 30-50%. In contrast, forskolin had no inhibitory effect on antigen-induced increases in cytosolic calcium concentration, as monitored by the calcium indicator fura-2, or histamine release from the cells. The combination of the phosphodiesterase inhibitor isobutylmethylxanthine with forskolin inhibited the antigen-induced production of PGD2 and LTC4 by 90-100% and histamine release by about 60%. These responses were accompanied by a virtual abolition of the antigen-induced increase in cytosolic calcium. To test further the hypothesis that increasing cAMP can lead to inhibition of lipid mediator biosynthesis in the absence of effects on cytosolic calcium, we employed the calcium ionophores A23187 and ionomycin. Forskolin alone or in combination with isobutylmethylxanthine had no effect on ionophore-induced increases in cytosolic calcium but effectively inhibited leukotriene biosynthesis. In addition, increasing cyclic AMP led to an inhibition of ionophore-induced production of platelet-activating factor and liberation of arachidonic acid. These data suggest that a relatively modest increase in cAMP-dependent protein kinase activity in mast cells leads to inhibition of the lipase-catalyzed cleavage of arachidonic acid from membrane phospholipids in the absence of measurable effects on either histamine release or changes in cytosolic calcium concentration. This effect results in a selective inhibition of the biosynthesis of lipid mediators including LTC4, LTB4, PGD2, and platelet-activating factor.     

The role of agonists that activate adenylate cyclase in the control of cAMP metabolism and enzyme release by human polymorphonuclear leukocytes

An inverse relationship between cAMP content and effector function is ascribed generally to immune and inflammatory cells. Previous reports imply, however, that human polymorphonuclear leukocytes (PMN) are less responsive than other inflammatory cells to adenylate cyclase (AC) agonists. We therefore examined the effects of isoproterenol, prostaglandin E1 (PGE1), adenosine, and histamine on the adenosine 3',5'-monophosphate (cAMP) content of PMN and on particle-stimulated lysosomal enzyme release. For comparison, the effect of AC agonists on the cAMP content of human peripheral lymphocytes was evaluated in parallel. Although potent stimuli for cAMP accumulation in lymphocytes, the AC agonists produced only marginal increases in the cAMP content of PMN; this difference in responsiveness was independent of agonist concentration or length of incubation. Inhibition of lysosomal enzyme release by the AC agonists was likewise marginal (< 20%). The addition of theophylline with isoproterenol produced additive inhibition without significant cAMP increases. Hydrocortisone, which caused a small increase in the cAMP content, markedly potentiated the effects of AC agonists on the cAMP level in PMN; the synergistic increases in cAMP were accompanied by additive effects on lysosomal enzyme release. It is concluded that human lymphocytes and PMN exhibit differential sensitivity to AC agonists and that this difference may provide a basis for the selective modulation of individual PMN- or lymphocyte-mediated events.     

Modulation of cyclic AMP in purified rat mast cells. II. Studies on the relationship between intracellular cyclic AMP concentrations and histamine release.

Changes in intracellular and extracellular rat mast cell adenosine 3':5' monophosphate (cAMP) concentrations during stimulation of histamine release by 48/80 were studied. There was a rapid and progressive fall in intracellular cAMP beginning within 10 sec after the addition of 48/80. The lowest cAMP values were obtained at 10 min, with return to control levels by 30 min. The fall in cAMP was dose-related with progressive decreases in 10-min cAMP measurements as the 48/80 concentration was increased from 0.25 to 1.00 mug/ml. There was a graded increase in histamine release over the same concentration range. Attempts to demonstrate significant amounts of cAMP in the medium during 48/80 stimulation were unsuccessful, indicating that the changes in cAMP intracellularly are not due to altered cellular permeability. There was a general correlation between the ability of pharmacologic agents to sustain high intracellular levels of cAMP in the presence of 48/80, and inhibition of histamine release. Theophylline (20 mM) which increased cAMP levels 2- 3-fold prevented a detectable decrease in cAMP after 1 mug/ml 48/80 (measured at 10 min) and almost completely inhibited histamine release. Prostaglandin E1 (27 muM) also raised cAMP levels, decreased the 48/80-induced fall in cAMP (by 42%). Epinephrine increased mast cell cAMP levels, but did not prevent the subsequent 48/80-induced decrease in cAMP and did not inhibit histamine release. Carbamylcholine (1 nM), adenine (1 muM), and diazoxide (10 muM) lowered mast cell cAMP and potentiated 48/80 induced release. In view of previous studies from this laboratory indicating that 48/80 stimulates mast cell phosphodiesterase, it seems likely that the 48/80-induced fall in cAMP is due, at least in part, to increased cAMP destruction. Since agents which prevent the fall in cAMP inhibit histamine release, it is apparent that cAMP is an important part of the control mechanism of histamine secretion. On the other hand, it cannot be concluded that a decrease in cAMP alone is sufficient to produce a response since carbamylcholine, diazoxide, and adenine which lower cAMP do not alter histamine release unless 48/80 is also present.     

Characterization of a specific adenosine receptor on human lymphocytes.

We have examined the mechanism of action of adenosine, a naturally occurring nucleoside that has profound effects on lymphocyte function. Adenosine (0.01 micrometer to 10 micrometer) increased lymphocytes cAMP levels in a dose-dependent fashion with a maximal (10 micrometer) increase of about 4-fold, whereas adenine, guanosine, and inosine had no effect on lymphocyte cAMP levels at concentrations of 100 micrometer. Adenosine appears to act on the cell surface since 1) 2-chloroadenosine, a poorly metabolized adenosine analogue, was as active as adenosine and 2) dipyridamole, which markedly inhibited [3H]-adenosine uptake by human lymphocytes, did not affect adenosine-induced accumulation of cAMP. The specificity of the adenosine effect was established by showing that the methylxanthine derivatives, theophylline and 3-isobutyl-1-methylxanthine (IBMX), specifically block the accumulation of cAMP in lymphocytes induced by adenosine. Theophylline is a competitive inhibitor of the effect of adenosine, with an estimated dissociation constant of theophylline-receptor complex of about 6.3 X 10(-7) M. The results suggest that adenosine increases the intracellular cAMP content of lymphocytes as a result of its interaction with a specific membrane receptor which results in the activation of adenylate cyclase.     

Adenosine Receptors Activate Adenylate Cyclase and Enhance Secretion from Bovine Adrenal Chromaffin Cells in the Presence of Forskolin

Cells of the adrenal medulla release not only catecholamines but also high concentrations of neuropeptides and nucleotides. Chromaffin cells, like many neuronal cells, have a diversity of receptors: adrenergic receptors, peptide receptors, histamine receptors, and dopamine receptors. We recently reported that these cells have nucleotide receptors that can mediate inhibition of the secretory response. The present studies show that adenosine, in the presence of enabling concentrations of forskolin, can potently enhance response to nicotinic stimulation. Neither adenosine nor forskolin alone produces a significant effect. A marked rise in intracellular cyclic AMP (cAMP) concentration is associated with the enhancement of secretion caused by forskolin plus adenosine. A phosphodiesterase inhibitor, Ro 20-1724, used together with forskolin produces significant increases in both cellular cAMP content and catecholamine secretion. However, the adenosine agonist 5'-N-ethylcarboxyadenosine elevates cellular cAMP content in the presence of forskolin without having any positive effect on secretion. This finding suggests that the rise in cAMP level may not be the sole cause of the increase in secretion by adenosine.     

Neutrophils and mast cells. Comparison of neutrophil-derived histamine-releasing activity with other histamine-releasing factors

Human neutrophil-derived histamine-releasing activity (HRA-N) was partially purified and found to contain a heat-stable 1400 to 2300-Da fraction which caused human basophils and rat basophil leukemia cells (RBL) to degranulate. The capacity of HRA-N to activate basophils was not related to the gender or atopic status of the basophil donor, but was related to anti-IgE responsiveness. Several lines of evidence suggest that HRA-N and anti-IgE induce histamine release through distinctly different mechanisms: 1) the time course of HRA-N- and anti-IgE-induced RBL histamine release are different; 2) HRA-N causes histamine release from RBL with and without surface-bound IgE; 3) lactic acid stripping of IgE from human basophils reduces anti-IgE-induced histamine release, but has no consistent effect on HRA-N-induced histamine release; and 4) passive sensitization of lactic acid-stripped basophils with IgE restores anti-IgE-induced histamine release but not HRA-N-induced histamine release. Several histamine-releasing factors (HRF) were compared with HRA-N. Human nasal HRF (HRF-NW, crude and partially purified fractions of 15 to 30, 3.5 to 9, and less than 3.5 kDa), like HRA-N, caused equal histamine release from both native and IgE-sensitized RBL. However, only the 15- to 30-kDa fraction caused histamine release from human basophils in the doses tested. Mononuclear cell HRF (HRF-M, crude and a partially purified 25 kDa Mr fraction) and platelet HRF (HRF-P, crude preparation) failed to cause histamine release from either native or IgE-sensitized RBL but caused 30 +/- 5.5% and 20 +/- 10% net histamine release from human basophils, respectively. HRA-N and HRF-NW were both stable to boiling. These data, taken together, suggest that the capacity of HRA-N to induce RBL and human basophil histamine release and of HRF-NW to stimulate RBL histamine release is independent of IgE. The data further suggest that HRA-N and HRF-NW can be distinguished by size, and that they both differ from mononuclear cell HRF and platelet HRF. Thus, it appears that inflammatory cells generate a family of distinct HRF.     

Modulation of cyclic AMP in purified rat mast cells. I. Responses to pharmacologic, metabolic, and physical stimuli.

The cyclic adenosine 3', 5'-monophosphate (cAMP) content of isolated unstimulated mast cells and the changes induced by a variety of pharmacologic, metabolic, and physical stimuli were studied. A modified bovine serum albumin density gradient purification method consistently provided mast cell preparations which were 95% or more pure, without apparent damage, and a 73% recovery of the mast cells applied to the gradients. The measured cAMP in unstimulated mast cells was high, a mean of 16 picomoles per million cells. Moderate agitation or contact with glass increased cAMP content about 2-fold. When calcium was omitted from the medium mast cell cAMP was markedly elevated; incremental increases in added calcium ion concentration from 1 muM to 1 mM caused a linear decrease in cAMP content. Theophylline (3 to 20 mM) caused a dose-related increase in mast cell cAMP content, approximately 2-fold at 20 mM theophylline. Epinephrine (0.01 to 1 mM) caused a modest, dose-related increase in cAMP. In the presence of theophylline, epinephrine increased cAMP levels equal to or greater than the sum of the effects of the agents used individually. The increase in cAMP induced by epinephrine was completely inhibited by 100 muM propranolol and partially inhibited by 10 muM propranolol, thus suggesting that a beta adrenergic receptor is involved. Prostaglandin E1 (PGE1) and histamine (in the presence of theophylline) also raised cAMP. Carbamylcholine (1 nM) lowered cAMP 38%; Atropine (0.1 mM) inhibited the decrease in cAMP induced by 1 nM carbamylcholine by 83% indicating that a muscarinic receptor is involved. In these homogeneous single cell suspensions, therefore, cholinergic and beta adrenergic agents have opposing effects on cAMP levels. Diazoxide (10 muM) and adenine (1 muM) caused 37 and 32% decreases in cAMP, respectively. The availability of highly purified mast cells and the identification of agents which either decrease or increase cAMP content allows a direct examination of the role of cAMP in histamine release.     

Essential role of PI3-kinase and phospholipase A2 in Dictyostelium discoideum chemotaxis

Chemotaxis toward different cyclic adenosine monophosphate (cAMP) concentrations was tested in Dictyostelium discoideum cell lines with deletion of specific genes together with drugs to inhibit one or all combinations of the second-messenger systems PI3-kinase, phospholipase C (PLC), phospholipase A2 (PLA2), and cytosolic Ca(2+). The results show that inhibition of either PI3-kinase or PLA2 inhibits chemotaxis in shallow cAMP gradients, whereas both enzymes must be inhibited to prevent chemotaxis in steep cAMP gradients, suggesting that PI3-kinase and PLA2 are two redundant mediators of chemotaxis. Mutant cells lacking PLC activity have normal chemotaxis; however, additional inhibition of PLA2 completely blocks chemotaxis, whereas inhibition of PI3-kinase has no effect, suggesting that all chemotaxis in plc-null cells is mediated by PLA2. Cells with deletion of the IP(3) receptor have the opposite phenotype: chemotaxis is completely dependent on PI3-kinase and insensitive to PLA2 inhibitors. This suggest that PI3-kinase-mediated chemotaxis is regulated by PLC, probably through controlling PIP(2) levels and phosphatase and tensin homologue (PTEN) activity, whereas chemotaxis mediated by PLA2 appears to be controlled by intracellular Ca(2+).     

Adenosine-mediated inhibition of casein production by mouse mammary glands in culture

The present study was carried out to examine whether activation of adenosine receptors by adenosine analogues will affect casein production by mouse mammary epithelial cells. The morphogenesis and functions of epithelial tissue in the mammary gland are influenced by their surrounding adipocytes. Adipocytes are known to release adenosine into the extracellular fluid which can modulate cyclic-AMP levels in surrounding cells through binding to their adenosine receptors. To examine a possible paracrine effect of adenosine, the modulation of casein production in mammary explant culture and mammary epithelial cell (MEC) culture by adenosine receptor agonists has been investigated. We have observed that activation of the A₁-adenosine receptor subtype in mammary tissue by an adenosine analogue (—)N⁶-(R-phenyl-isopropyl)-adenosine (PIA) raised cAMP levels. PIA and another adenosine receptor agonist, isobutylmethylxanthine (IBMX), inhibited casein accumulation both in explants and in MEC cultures in the presence of lactogenic hormones, which suggests that PIA or adenosine can act directly on the epithelial cells. This inhibition does not appear to be caused by elevation of cAMP levels or phosphodiesterase activity. The inhibition of intracellular casein accumulation by PIA and IBMX in explant cultures can be reversed via treatment of pertussis toxin which is known to ADP-ribosylate GTP-binding Gαi-proteins, indicating that a Gi-protein-dependent pathway may be involved in this inhibition. The results also suggest that local accumulation of adenosine in the extracellular fluids of mammary glands is likely to inhibit the lactogenic response of mammary epithelial cells. © 1996 Wiley-Liss, Inc.     

Effects of guanine nucleotides on adenosine and glutamate modulation of cAMP levels in optic tectum slices from chicks.

Glutamate and adenosine both modulate adenylyl cyclase activity through interaction of their specific receptors with stimulatory or inhibitory G-proteins. Guanine nucleotides (GN), which modulate G-protein activity intracellularly, are also involved in the inhibition of glutamate responses, acting from the outside of the cells. We had previously reported that glutamate inhibits adenosine-induced cyclic AMP (cAMP) accumulation in slices obtained from the optic tectum of chicks. In the present study we investigated the interaction of GN with these two neurotransmitters and found that GN inhibit the inhibitory effect of glutamate on adenosine-induced cAMP accumulation and potentiate adenosine-induced cAMP accumulation. These effects were observed with 5'-guanylylimidodiphosphate (GppNHp) or GMP, but not with guanosine (the nucleoside). Besides, these interactions of GN occur via a metabotropic glutamate receptor (mGluR) sensitive to (1 S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid (1 S,3R-ACPD) but not to L-2-amino-4-phosphonobutyrate (L-AP4). These effects were partially modulated by a mGluR antagonist, (RS)-alpha-methyl-4-carboxyphenylglycine ((RS)M-CPG), and by an adenosine receptor antagonist, 8-phenyltheophylline. GN only potentiated the adenosine response when adenosine was acting through its receptor positively linked to adenylyl cyclase. Therefore, the data show that guanine nucleotides not only inhibit glutamate-induced responses, but also stimulate adenosine-induced responses, a fact that may contribute to the understanding of the physiological functions of guanine nucleotides.     

Induction of histamine secretion by polycations

Poly(arginine), poly(lysine) and poly(ornithine) induce histamine secretion from human basophil leukocytes in the concentration range 1--100 nmol/l. Histamine secretion induced by poly(arginine) requires extracellular calcium at 0.1--1 mmol/l. Strontium (1--10 mmol/l) will substitute for calcium. Lanthanum (30--90 nmol/l) inhibits histamine release induced by poly(arginine). Histamine secretion induced by poly(arginine) is inhibited by 1--30 mumol/l N-ethyl-maleimide, 0.3--3 mmol/l 2-deoxy-D-glucose, 0.3--3 mmol/l dibutyryl cyclic AMP, 0.3--3 mmol/l, adenosine 3'5'-cyclicphosphorothioate. The action of poly(arginine) is inhibited by pretreatment of basophils at 47 degrees C or with neuraminidase. 10 microgram/ml heparin inhibits the response to poly(arginine). Histamine releasing potency of the polymer amino acids is dependent on chain length of the peptide. Succinylated poly(lysine) is inactive. Monomer amino acids do not release histamine and do not inhibit the action of the polymers. Histones and protamine do not release histamine, nor do the peptides eledoisin and tuftsin. Putrescine, cadaverine, spermine and spermidine do not release histamine. Poly(glutamic acid), poly(aspartic acid) and poly(tyrosine) are also inactive. The IgE-mediated release of histamine appears to be independent of that mediated by poly(arginine).