Role of calcium in histamine release from rat mast cells activated by various secretagogues; intracellular calcium mobilization correlates with histamine release

Anti-IgE, con A or antigen caused an increase in the intracellular calcium concentration, [Ca2+]i, of mast cells. The increase occurred in two stages: a rapid initial rise caused by Ca-mobilization from intracellular Ca-stores and a second sustained rise caused by an influx of extracellular calcium (White, J.R., Pluznik, D.V., Ishizaka, K. & Ishizaka, T. (1985) Proc. Natl. Acad. Sci. U.S.A. 82, 8193-8197). The rapid initial rise was followed by a release of histamine, which seemed to coincide with the second rise. A23187 and compound 48/80 induced a rapid initial rise in [Ca2+]i, followed by a gradual decrease in [Ca2+]i, GMCHA-OPhBut, a specific pH 7 tryptase inhibitor (Muramatu, M., Ito, T., Takei, M. & Endo, K. (1988) Biol. Chem. Hoppe-Seyler 369, 617-625), strongly inhibited both the initial and second rises of [Ca2+]i, as well as histamine release by these secretagogues, and its effects on the initial rise were closely correlated with those on histamine release. Addition of GMCHA-OPhBut immediately after the initial rise strongly inhibited the second rise, thereby decreasing the final [Ca2+]i. These results strongly suggested a possible involvement of pH 7 tryptase, not only in Ca-mobilization leading to the initial rise in [Ca2+]i, but also in the second rise. Trapping of extracellular calcium by 3mM EGTA decreased both the initial rise in [Ca2+]i and histamine secretion induced by anti-IgE or con A; the magnitude of this effect depended on the time between induction and EGTA addition. Histamine release was closely correlated with the initial rise in [Ca2+]i. Similar results were obtained with A23187, but even 5 min after the addition of EGTA an initial rise of [Ca2+]i could still be induced, and histamine (30% of total histamine) was still released. However, A23187 did not induce a rise in [Ca2+]i in mast cells which had been exhaustively washed with Tyrode/Hepes solution containing 3mM EGTA, followed by suspension in the same solution. Even at 20 min after depletion of the extracellular calcium, compound 48/80 still caused an initial rise in [Ca2+]i to above half the maximal value, and histamine secretion was even less affected. The above results indicated that the initial rise in [Ca2+]i, due to Ca-mobilization, correlates with the histamine release promoted by the secretagogues described. On the other hand, isoproterenol strongly induced histamine secretion with no change of [Ca2+]i, while EGTA treatment prior to isoproterenol stimulation had no effect on histamine release, indicating a different secretion mechanism.     

Modulation of cyclic AMP in purified rat mast cells. III. Studies on the effects of concanavalin A and anti-IgE on cyclic AMP concentrations during histamine release.

Changes in rat mast cell cyclic adenosine 3',5' monophosphate (cAMP) concentrations during stimulation of histamine release by concanavalin A (con A) and anti-IgE were studied. Con A caused an increase in cAMP with a mean peak level at 20 sec of 232% of control (range 164% to 365%). Con A-stimulated cells demonstrated falls toward control levels after 20 sec, but generally remained above control for at least 5 min. By 10 min cAMP had returned to control values. The con A effect on cAMP occurred in the absence of phosphatidyl serine but was markedly inhibited by 5 mM alpha-methyl-D-mannose. Anti-IgE induced a less marked increase in cAMP (157% of control, range 110% to 540% of control) which reached a peak at 20 sec. Two monospecific goat anti-rat myeloma IgE antisera induced similar changes in cAMP whereas normal goat IgG had no effect. These peak values were followed by a rapid decrease in cAMP. Within 2 min the cAMP content of anti-IgE stimulated cells had fallen to levels well below control and remained below control levels from 45 sec to over 15 min. Histamine release in both systems began after the peak cAMP levels, during the period of rapid destruction of cAMP.     

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.     

Inhibitory effects of GMCHA-OPhBut on phospholipid methylation and histamine release in mast cells activated by concanavalin A, anti-IgE, and antigen

[3H]Methyl group incorporation and histamine secretion in rat mast cells induced by anti-IgE and con A were strongly inhibited by trans-4-guanidinomethylcyclohexanecarboxylic acid 4-tert-butylphenyl ester (GMCHA-OPhBut), a strong and specific inhibitor for pH 7 tryptase (Muramatsu et al. (1988) Biol. Chem. Hoppe-Seyler 369, 617-625) which is present in rat mast cells. The IC50s for these events were of the order of 10(-6) M. Addition of GMCHA-OPhBut after the maximal increase in [3H]methyl group incorporation in rat mast cells activated by con A and anti-IgE induced rapid reduction of the methylated phospholipid, and the later histamine release was strongly suppressed. Mast cells were prepared with Mg2+-free Tyrode-HEPES solution, and challenged with anti-IgE with or without Mg2+. With Mg2+, [3H]methyl group incorporation was enhanced, and histamine was secreted time-dependently. Without Mg2+, [3H]methyl group incorporation fell to one-third, whereas histamine secretion was not affected. These results were incompatible with the above results. From these results it was strongly suggested that a trypsin-like protease, probably pH 7 tryptase, is involved not only in the early events, such as activation of phosphatidylethanolamine methyltransferase I and/or II, but also in the late events such as histamine release, and phospholipid methylation is not associated with histamine secretion.     

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.     

Release of histamine and arachidonate from mouse mast cells induced by glycosylation-enhancing factor and bradykinin

Stimulation of normal rat splenic T cells with pertussigen (lymphocytosis-promoting factor from Bordetella pertussis) resulted in the release of a soluble factor that enhanced the assembly of N-linked oligosaccharides to IgE-binding factors during their biosynthesis. The glycosylation-enhancing factor (GEF) is a kallikrein-like enzyme and is purified by absorption to p-aminobenzamidine-Agarose followed by elution with benzamidine. Incubation of normal mouse mast cells with affinity-purified GEF or bradykinin, a product of cleavage of kininogen by kallikrein, resulted in the release of histamine and arachidonate from the cells. Passive sensitization of mast cells with mouse IgE antibody, followed by pretreatment of the cells with a suboptimal concentration of GEF, resulted in an enhancement of antigen-induced histamine release. It was found that GEF and bradykinin induced the same biochemical events in mast cells as those induced by bridging of IgE receptors. Both GEF and bradykinin induced phospholipid methylation and an increase in intracellular cyclic AMP (cAMP). Incorporation of 3H-methyl groups into phospholipids and intracellular cAMP levels both reached a maximum 30 sec after challenge with GEF or bradykinin, and then declined to base-line levels within 2 to 3 min. These biochemical events were followed by 45Ca influx and histamine release; 45Ca uptake reached a plateau value at 2 min, and histamine release reached a maximum at 5 to 8 min. The initial rise in cAMP induced by GEF (or bradykinin) was not inhibited by indomethacin, indicating that the activation of adenylate cyclase is not the result of prostaglandin synthesis. In both IgE-mediated and GEF-induced histamine release, inhibitors of methyltransferases, such as 3-deaza adenosine and L-homocysteine thiolactone, inhibited not only phospholipid methylation but also the cAMP rise and subsequent Ca2+ uptake and histamine release. The results indicate that GEF induces activation of methyltransferases and that phospholipid methylation is involved in the cAMP rise, Ca2+ uptake, and histamine release. The induction of the same biochemical events in the same sequence by bridging of IgE receptors and by GEF (bradykinin) supports the hypothesis that receptor bridging induces the activation of serine protease(s) and cleavage products of this enzyme in turn activate methyltransferases in mast cells.     

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.     

The human recombinant c-kit receptor ligand, rhSCF, induces mediator release from human cutaneous mast cells and enhances IgE-dependent mediator release from both skin mast cells and peripheral blood basophils.

The gene product of the steel locus of the mouse represents a growth factor for murine mast cells and a ligand for the c-kit proto-oncogene receptor, a member of the tyrosine kinase receptor class of oncogenes (for review, see O. N. Witte. 1990. Cell 63:5). We have studied the effect of the human recombinant c-kit receptor ligand stem cell factor (rhSCF) on the release of inflammatory mediators from human skin mast cells and peripheral blood basophils and compared its activity to that of rhIL-3, rhSCF (1 ng/ml to 1 microgram/ml) activated the release of histamine and PGD2 from mast cells isolated from human skin. Analysis by digital video microscopy indicated that purified human skin mast cells (84 +/- 5% pure) responded to rhSCF (0.1 to 1 microgram/ml) challenge with a rapid, sustained rise in intracellular Ca2+ levels that was accompanied by secretion of histamine. A brief preincubation (10 min) of mast cells with rhSCF (0.1 pg/ml to 1 ng/ml) significantly enhanced (100 +/- 35%) the release of histamine induced by anti-IgE (3 micrograms/ml), but was much less effective on IgE-mediated release of PGD2. In contrast, a short term incubation with rhSCF did not potentiate the secretion of histamine activated by substance P (5 microM). A 24-h incubation of mast cells with rhSCF did not affect the release of mediators induced by anti-IgE (3 micrograms/ml), probably due to receptor desensitization, rhSCF (1 ng/ml to 3 micrograms/ml) neither caused release of histamine or leukotriene C4 (LTC4) release from leukocytes of 14 donors, nor induced a rise in intracellular Ca2+ levels in purified (greater than 70%) basophils. Brief preincubation (10 min) of leukocytes with rhSCF (1 ng/ml to 3 micrograms/ml) caused an enhancement (69 +/- 11%) of anti-IgE-induced release of histamine that was significant at concentrations as low as 3 ng/ml (p less than 0.05), whereas it appeared less effective in potentiating IgE-mediated LTC4 release. In contrast, a prolonged incubation (24 h) with rhSCF (0.1 pg/ml to 100 ng/ml) did not enhance the release of histamine or LTC4 induced by anti-IgE (0.1 microgram/ml), whereas rhIL-3 (3 ng/ml) significantly potentiated the release of both mediators.(ABSTRACT TRUNCATED AT 400 WORDS)     

Characterization of primate bronchoalveolar mast cells. I. IgE-dependent release of histamine, leukotrienes, and prostaglandins

Large numbers of functional mast cells were obtained by bronchoalveolar lavage (BAL) of Macaca arctoides monkeys that had been infected with the nematode Ascaris suum. These lavage cells, of which 21% were mast cells, released histamine, LTC4, and PGD2 in a concentration-dependent fashion when challenged with ascaris antigen or antibody to human IgE. However, there was no release of histamine when these cells were challenged with compound 48/80. The amount of mediator released was highly dependent on the sensitivity of the cells to immunologic challenge, but was generally in the range of 2 to 5 micrograms histamine (30 to 70% of total), 20 to 80 ng LTC4, and 100 to 300 ng PGD2 per 10(6) mast cells when maximally challenged. Other eicosanoids measured were released only in much smaller quantities. Maximal values were 4 ng LTB4, 2 ng PGE2, and approximately 10 to 20 ng PGF2 alpha per 10(6) mast cells. The amount of LTC4 and PGD2 released correlated with the release of histamine, the calculated regression line indicating that 18 ng LTC4 and 50 ng PGD2 were released per microgram of histamine released. This correlation suggests that the majority of the LTC4 and PGD2 released was probably mast cell-derived. Further support for this conclusion was given by the observation that when lavage cells were fractioned on continuous Percoll gradients, the ability to release LTC4 and PGD2 on immunologic challenge coincided with the peak of mast cells.     

Guinea pig anaphylaxis: time course of changes in cyclic AMP and cyclic GMP.

The time course of changes in the levels of cyclic AMP and GMP and in histamine and prostaglandin release following the onset of anaphylaxis in guinea pig lung (sliced, minced and whole perfused) was studied. A parallel change in the cyclic nucleotide levels was found. There was an initial increase, followed by a decrease, and a subsequent greater and more prolonged increase. Significant histamine and prostaglandin release occurs at approximately the same time while prostaglandin metabolite is released later. Metiamide does not affect histamine or prostaglandin release but abolishes the initial cyclic nucleotide peak and delays the appearance of the secondary increase. The pattern of cyclic nucleotide changes in lung tissue thus appears to be different from that in isolated mast cells. The initial and late increases might be the response of two different cell populations to histamine.     

Cyclic nucleotide involvement in histamine release from mast cells--a reevaluation

Secretory events in cells in general are accompanied by increased levels of cyclic AMP. In mast cells, however, the pattern is reversed. Thus histamine release is associated with a fall in cAMP. It has been suggested that the lowered levels of cAMP lead to an increase in membrane permeability towards calcium and that an influx of such ions triggers the release mechanisms. It has further been reported that high levels of cAMP inhibit histamine release by decreasing the permeability. However, evidence has now accumulated indicating that this general concept is far too simplistic. Studies are reviewed which imply that there is little or no correlation between histamine release and intracellular levels of cyclic nucleotides. A new working hypothesis with respect to the role of these nucleotides in mast cell secretion is proposed.     

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.     

Biochemical analysis of glucocorticoid-induced inhibition of IgE-mediated histamine release from mouse mast cells

Pretreatment of mouse mast cells with 10(-7) to 10(-6) M dexamethasone (DM) during overnight sensitization with mouse IgE antibody resulted in inhibition of antigen-induced histamine release and degranulation. The inhibition of both degranulation and histamine release increased linearly with the duration of the treatment; maximal inhibition was obtained after approximately 16 hr with DM. The addition of DM to sensitized mast cells immediately before antigen challenge did not affect the antigen-induced histamine release. DM interacted directly with mast cells by binding to DM-specific cytoplasmic receptors. The treatment of mast cells with DM did not affect the binding of IgE to mast cells or intracellular cAMP levels. Bridging of cell-bound IgE anti-DNP antibody on mouse mast cells either by multivalent DNP-HSA or by anti-IgE induced phospholipid methylation at the plasma membrane and Ca++ influx into the cells. Pretreatment of mast cells with DM inhibited the antigen-induced phospholipid methylation and Ca++ uptake but failed to affect histamine release by Ca++ ionophore A23187. The results suggest that DM treatment inhibits histamine release by the inhibition of the early stage of biochemical processes leading to opening Ca++ channels but does not affect the process distal to Ca++ influx or the binding of IgE molecules to IgE receptors.     

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.     

Reduction in basal adenylate cyclase activity during the immunologic release of histamine from guinea pig lung.

Cyclic AMP has been implicated in the regulation of the immunologic release of histamine from lung and other tissues and cell types. The mechanism whereby intracellular levels of cAMP are altered during mediator release was investigated. Measurements of histamine, adenylate cyclase, and cAMP phosphodiesterase activities were made in actively and passively sensitized guinea pig lung after challenge with antigen. A transient decrease in basal adenylate cyclase activity occurred which returned to control levels after histamine release. There was no change in cAMP phosphodiesterase activity determined at substrate concentrations of 1 mM and 0.01 mM. The adenylate cyclase response did not occur under the following conditions: 1) incubation of nonsensitized lung with antigen, 2) incubation of sensitized lung with antigen in the absence of extracellular calcium, and 3) incubation of nonsensitized lung with compound 48/80. These observations indicate 1) the adenylate cyclase response and the immunologic release of histamine are intimately related, and 2) the reduction in intracellular levels of cAMP which have been reported to occur during immunologic histamine release are mediated via adenylate cyclase.     

Trypsin-induced increase in intracellular cyclic AMP of lymphocytes.

Trypsin increases intracellular levels of cylic AMP (cAMP) in lymphocytes. The trypsin-induced increase in cAMP is blocked by specific trypsin inhibitors and by high concentrations of different proteins. Several proteolytic enzymes from various sources, including other pancreatic proteases, do not cause an increase in cAMP under the same experimental conditions. Immobilized trypsin induced the same increase in cAMP as does free trypsin. The trypsin-induced rise in cAMP is not due to inhibition of cAMP phosphodiesterase, but consistent activation of adenylate cyclase by trypsin could not be demonstrated. The extent of the trypsin-induced increase in intracellular cAMP correlates with the type of the lymphocyte and with the state of maturity attained by the cells. Transformed lymphocytes and nonlymphoid cells do not react at all.     

Regulation of cyclic AMP levels in Arthrobacter crystallopoietes and a morphogenetic mutant.

The extracellular levels of cyclic AMP (cAMP), cAMP phosphodiesterase activity, and adenylate cyclase activity were measured at various intervals during growth and morphogenesis of Arthrobacter crystallopoietes. There was a significant rise in the extracellular cAMP level at the onset of stationary phase, and this rise coincided with a decrease in intracellular cAMP. The phosphodiesterase activity measured in vitro increased in the early exponential phase of growth as intracellular cAMP decreased, and, conversely, prior to the onset of stationary phase the phosphodiesterase activity decreased as the intracellular cAMP levels increased. Adenylate cyclase activity was greater in cell extracts prepared from cells grown in a medium where morphogenesis was observed. Pyruvate stimulated adenylate cyclase activity in vitro. A morphogenetic mutant, able to grow only as spheres in all media tested, was shown to have altered adenylated cyclase activity, whereas no significant difference compared to the parent strain was detectable in either the phosphodiesterase activity or the levels of extracellular cAMP. The roles of the two enzymes, adenylate cyclase and phosphodiesterase, and excretion of cAMP are discussed with regard to regulation of intracellular cAMP levels and morphogenesis.     

Differential calcium effects on prostaglandin D2 generation and histamine release from isolated rat peritoneal mast cells

We examined the role of Ca2+ mobilization in prostaglandin (PG) D2 generation and histamine release induced by A23187 from rat peritoneal mast cells. Both PGD2 generation and histamine release accompanied with 45Ca uptake were observed above 0.1 microM A23187. Although an increase of PGD2 generation was not exactly correlated with that of Ca2+ uptake, histamine release occurred in proportion to Ca2+ uptake. In contrast to PGD2 generation, below 0.1 microM A23187, about 20% of the total histamine was released without Ca2+ uptake and this response was inhibited by 10 microM 8-(N,N-diethylamino)-octyl-3,4,5-trimethoxybenzoate hydrochloride (TMB-8), which is an intracellular Ca2+ antagonist. However, TMB-8 had no effect on PGD2 generation. These results suggest that Ca2+ dependency of histamine release is clearly different from that of PGD2 generation, and that histamine release is induced by not only Ca2+ uptake but also intracellular Ca2+ mobilization.     

Intravascular anti-IgE challenge in perfused lungs: mediator release and vascular pressor response.

Intravascular application of goat anti-rabbit immunoglobulin E (IgE) was used to stimulate parenchymal mast cells in situ in perfused rabbit lungs. Sustained pulmonary arterial pressure rise was evoked in the absence of lung vascular permeability increase and lung edema formation. Early prostaglandin (PG) D2 and histamine release into the perfusate was documented, accompanied by more sustained liberation of cysteinyl leukotrienes (LT), LTB4, and PGI2. The quantities of these inflammatory mediators displayed the following order: histamine greater than cysteinyl-LT greater than PGI2 greater than LTB4 greater than PGD2. Pressor response and inflammatory mediator release revealed corresponding bell-shaped dose dependencies. Cyclooxygenase inhibition (acetylsalicylic acid) suppressed prostanoid generation, increased LT release, and did not substantially affect pressor response and histamine liberation. BW755 C, a cyclo- and lipoxygenase inhibitor, blocked the release of cysteinyl-LT and markedly reduced the liberation of the other inflammatory mediators as well as the pressor response. The H1-antagonist clemastine caused a moderate reduction of the anti-IgE-provoked pressure rise. We conclude that intravascular anti-IgE challenge in intact lungs provokes the release of an inflammatory mediator profile compatible with in situ lung parenchymal mast cell activation. Pulmonary hypertension represents the predominant vascular response, presumably mediated by cysteinyl-LT and, to a minor extent, histamine liberation.     

Dynamics of retinal waves are controlled by cyclic AMP

Waves of spontaneous activity sweep across the developing mammalian retina and influence the pattern of central connections made by ganglion cell axons. These waves are driven by synaptic input from amacrine cells. We show that cholinergic synaptic transmission during waves is not blocked by TTX, indicating that release from starburst amacrine cells is independent of sodium action potentials. The spatiotemporal properties of the waves are regulated by endogenous release of adenosine, which sets intracellular cAMP levels through activation of A2 receptors present on developing amacrine and ganglion cells. Increasing cAMP levels increase the size, speed, and frequency of the waves. Conversely, inhibiting adenylate cyclase or PKA prevents wave activity. Together, these results imply a novel mechanism in which levels of cAMP within an immature retinal circuit regulate the precise spatial and temporal patterns of spontaneous neural activity.