Activation of phospholipase C via adenosine receptors provides synergistic signals for secretion in antigen-stimulated RBL-2H3 cells. Evidence for a novel adenosine receptor

5'-(N-Ethyl)carboxamidoadenosine (NECA), an analog of adenosine, transiently stimulated a rat tumor mast cell (RBL-2H3 cells) to cause a release of inositol phosphates and an increase in levels of Ca2+ in the cytosol. It failed, however, to stimulate a sustained uptake of 45Ca2+ or secretion. The effects of other agents that act on P1- or P2-purinergic receptors suggested that NECA and other adenosine agonists acted via a novel subtype of adenosine membrane receptor. Although the order of potency of agonists was characteristic of A2-adenosine receptors, there was no indication of the involvement of adenylate cyclase, and antagonists such as isobutylmethylxanthine, 8-phenyltheophylline, and 8-p-sulfophenyltheophylline inhibited the responses to either NECA or antigen. The fact that stimulation of inositol phospholipid hydrolysis by NECA in washed, permeabilized RBL-2H3 cells was blocked by pertussis toxin as well as by cholera toxin suggested instead that the NECA-sensitive receptor activated phospholipase C via a G-protein. In contrast to NECA, antigen stimulation resulted in a pertussis toxin-resistant, sustained hydrolysis of inositol phospholipids, increases in free intracellular Ca2+, accelerated influx of 45Ca2+, and secretion from RBL-2H3 cells. In combination with NECA, all responses to antigen were markedly enhanced, and the enhancement was selectively blocked by pertussis toxin. The ability of antigen, but not NECA, to provoke secretion may be dependent primarily on the sustained activation of a cholera toxin-sensitive Ca2+ influx pathway that serves to amplify stimulatory signals for secretion. These studies also suggested that phospholipase C could be activated through different G-proteins via different receptors within the same cell.     

Treatment with dexamethasone down-regulates IgE-receptor-mediated signals and up-regulates adenosine-receptor-mediated signals in a rat mast cell (RBL-2H3) line

As reported by other workers, the treatment of rat basophilic leukemia RBL-2H3 cells with dexamethasone resulted in a marked decrease in responsiveness to Ag-stimulation. All responses measured, which included hydrolysis of inositol phospholipids, increase in concentration of cytosolic Ca2+, release of arachidonic acid and the secretion of serotonin, were suppressed, but once the cells were permeabilized the inhibitory actions of dexamethasone were no longer apparent. This suggested that all the necessary components of the stimulatory cascade were intact in the dexamethasone-treated cells. The measurement of phospholipase C activity in cell extracts and studies with phorbol ester also indicated that the cells contained a normal complement of phospholipase C and protein kinase C activity. We had previously shown that both Ag and the adenosine analog, 5'-(N-ethylcarboxamide)-adenosine, can activate phospholipase C, but they do so through different G proteins. Interestingly, the activation of phospholipase C by 5'-(N-ethylcarboxamide)-adenosine and the ensuing stimulatory events were markedly enhanced in dexamethasone-treated cells. The treatment with dexamethasone thus did not result in direct suppression of effector systems, but instead resulted in the selective modulation of the coupling between receptors and the effector systems by mechanisms that require soluble cytosolic factors.     

Assessment of IgE-receptor function through measurement of hydrolysis of membrane inositol phospholipids. New insights on the phenomena of biphasic antigen concentration-response curves and desensitization

Previous studies have shown that hydrolysis of membrane inositol phospholipids in rat basophilic leukemia (RBL-2H3) cells depended on the rate and extent of the aggregation of receptors of IgE. This response was used as an experimental probe to study the role of IgE receptors in initiating stimulatory and inhibitory processes within the cell. The response was amplified markedly by increasing the concentration of external Ca2+ from 0 to 1 mM, but the concentration required to support half-maximal response varied from less than 0.1 mM for the most potent cross-linking reagent, DNP24BSA (24 molecules of DNP attached to 1 molecule of BSA) to 0.5 mM for the least potent reagent, aggregated OVA. The dependency of phosphoinositide hydrolysis on external Ca2+ was reduced to zero once hydrolysis of inositol phospholipids was underway but secretion of histamine remained totally dependent on the presence of 0.5 to 1 mM external Ca2+. The stimulatory response persisted as long as receptors remained aggregated but it was modulated by a biochemical process, possibly the activation of protein kinase C, that targeted specifically aggregated receptors, or an associated protein. For example, when cells had become desensitized to high concentrations of one Ag, a normal response could be evoked with a second Ag. Also cells that had become desensitized could be reactivated by permeabilizing the cells. Interestingly, bell-shaped Ag dose-response curves, which were characteristic for both the phosphoinositide and secretory responses, were transformed to sigmoid-shaped curves once cells were permeabilized and dialyzed.     

Quantitative relationships between aggregation of IgE receptors, generation of intracellular signals, and histamine secretion in rat basophilic leukemia (2H3) cells. Enhanced responses with heavy water

RBL-2H3 cells (a tumor analog of rat mast cells) have plasma-membrane receptors that bind immunoglobulin E, which when aggregated, initiate degranulation. As in other systems, secretion is preceeded by enhanced hydrolysis of inositol phospholipids and by a rise in intracellular Ca2+. Unlike the responses of many other cells, however, both of these earlier events require extracellular Ca2+. The relationship of these events to each other and to the subsequent secretory process is thus unclear. By exposing cells to covalent oligomers of IgE one can demonstrate substantial increases in secretion of histamine by increasing the concentration and size of the oligomers or by using heavy water (D2O) in the medium. We have used such maneuvers to examine the quantitative relationships between aggregation of the receptors and the breakdown of inositol phospholipids, the increase in cytosolic Ca2+ and secretion. Our principal findings were: all treatments that increased secretion, correspondingly increased the changes that precede degranulation. These early events correlated with the degree of aggregation of the receptors even when the stimulatory conditions resulted in maximal secretion. Although the results were insufficient to prove that the hydrolysis of inositol phospholipids is required for the rise in cytosolic Ca2+, the studies with D2O and other observations supported this view. Since a plasma-membrane ion channel for Ca2+ has been implicated in the IgE-mediated rise in cytosolic Ca2+ in RBL 2H3 cells, this in turn suggests a heretofore undescribed role for hydrolysis of inositol phospholipids.     

Studies of protein kinase C in the rat basophilic leukemia (RBL-2H3) cell reveal that antigen-induced signals are not mimicked by the actions of phorbol myristate acetate and Ca2+ ionophore

Exogenous activators of protein kinase C such as PMA in combination with a Ca2+ ionophore (A23187), cause secretion in rat basophilic (RBL-2H3) cells,but they do so through stimulatory signals that are not the same as those generated by Ag or oligomers of IgE. On the one hand, the synergy between PMA and A23187 and the suppression of Ag-mediated signals (hydrolysis of inositol phospholipids and rise in concentration of cytosolic Ca2+) by PMA were totally dependent on protein kinase C. The loss of synergistic and inhibitory actions of PMA, for example, correlated with the loss of protein kinase C (as determined by immunoblotting techniques) when cells were continuously exposed to PMA. Furthermore, the permeabilization of RBL-2H3 cells resulted in the loss of both protein kinase C and the inhibitory action of PMA, but both were retained if cells were exposed to PMA before permeabilization Ag-induced secretion, on the other hand, was not as dependent on the presence of protein kinase C. The potent inhibitor of this enzyme, staurosporine, which blocked completely the secretory response to the combination of PMA and A23187, did not inhibit Ag-induced secretion except at concentrations (greater than 10 nM) that inhibited Ag-stimulated hydrolysis of inositol phospholipids as well. Also RBL-2H3 cells still showed some secretory-response (approximately 25% of normal) to Ag when cells were depleted (greater than 98%) of protein kinase C by prolonged treatment with PMA. Previous studies have indicated that the secretory response to PMA and A23187 is much lower than that elicited by Ag when the concentrations of stimulants were matched to give the same increase in concentrations of cytosolic Ca2+.     

Identification of variants of the basophilic leukemia (RBL-2H3) cells that have defective phosphoinositide responses to antigen and stimulants of guanosine 5'-triphosphate-regulatory proteins

Antigen immunoglobulin E-mediated secretion of histamine from RBL-2H3 cells is associated with substantial hydrolysis of membrane inositol phospholipids and a rise in the concentration of cytosol Ca2+ (calcium signal). Such responses differed among cloned variant lines of the RBL-2H3 cell line from undetectable (1A3 bromodeoxyuridine-resistant (BUDRR), 2B1 BUDRR, and 1B3 BUDRR lines) to about 80% of those in the parent RBL-2H3 cells. In all but one line (1B3 thioguanine-resistant (TgR)), the intensities of the phosphoinositide response and of the calcium signal were correlated with the secretory response. The 1B3 TgR line had no detectable calcium signal (as measured by quin 2 fluorescence or uptake of 45Ca2+) but paradoxically showed modest rates of hydrolysis of inositol phospholipids and of secretion. The responses of the 1B3 TgR line were, however, dependent on the presence of external Ca2+ ions. The induction of secretion with antigen, therefore, was invariably associated with the hydrolysis of inositol phospholipids, but it was not necessarily associated with a change in concentration of cytosol Ca2+. All antigen unresponsive clones could secrete when synergistic signals were induced by exposure to the Ca2+ -ionophore, A23187 and the phorbol ester, 12-O-tetradecanoylphorbol 13-acetate. These lines, otherwise, had immunoglobulin E receptors and had no obvious defect in their capacity to synthesize the inositol phospholipids or in their phenotypic expression of phospholipase C as measured in cell extracts. One finding of possible relevance to the role of guanosine 5'-triphosphate-regulatory proteins in the activation of phospholipase C was the inability of one antigen-nonresponsive line to respond to NaF (in intact cells) or to guanosine 5'-(3-O-thio)triphosphate (in electrically permeabilized cells).     

Certain inhibitors of protein serine/threonine kinases also inhibit tyrosine phosphorylation of phospholipase C gamma 1 and other proteins and reveal distinct roles for tyrosine kinase(s) and protein kinase C in stimulated, rat basophilic RBL-2H3 cells.

Various inhibitors of phospholipases and serine/threonine kinases were used to determine whether activation of these enzymes was necessary for Ag-induced exocytosis in rat basophilic RBL-2H3 cells. Several inhibitors, however, inhibited events other than those intended in stimulated RBL-2H3 cells. Staurosporine and KT5926, inhibitors of protein kinase C and myosin L chain kinase, respectively, suppressed, in a dose-dependent manner, hydrolysis of inositol phospholipids, release of arachidonic acid, and exocytosis in cells stimulated with Ag or Ca(2+)-ionophore, A23187. Such generalized inhibition could also be induced in permeabilized cells with several peptide inhibitors of tyrosine kinases. All the above inhibitors suppressed Ag-induced tyrosine phosphorylation of several proteins, including phospholipase C gamma 1, and this suppression correlated with the inhibition of hydrolysis of inositol phospholipids and exocytosis. Three inhibitors of protein kinase C, Ro31-7549, calphostin C, and a peptide inhibitor, did not inhibit the tyrosine phosphorylation of proteins but selectively blocked exocytosis, presumably, by inhibiting protein kinase C. Thus, both tyrosine phosphorylation of proteins and the activation of protein kinase C were necessary events for hydrolysis of inositol phospholipids and exocytosis.     

The rise in concentration of free Ca2+ and of pH provides sequential, synergistic signals for secretion in antigen-stimulated rat basophilic leukemia (RBL-2H3) cells

Ag stimulation of rat basophilic leukemia (RBL-2H3) cells results in hydrolysis of inositol phospholipids, a transient increase in concentration of cytosol Ca2+ [( Ca2+]i), a gradual increase in cytosolic pH (pHi) and the activation of protein kinase C. To determine whether all these changes serve as signals for secretion, studies were conducted with cells permeabilized with streptolysin O in which pHi and [Ca2+]i could be varied independently of each other and enzyme activities could be manipulated. At resting pHi (approximately 7.0) and [Ca2+]i (0.1 microM), the permeabilized cells showed little secretory response to Ag. At resting pHi, elevated levels of Ca2+ (0.33 microM) were required for maximal secretory response to Ag. At a pHi of 7.4, however, 0.1 microM [Ca2+]i was sufficient to sustain near maximal responses to Ag. Therefore, a small increase of [Ca2+]i to 0.33 microM was required to initiate secretion, but once the pHi was elevated secretion could be sustained at near basal levels of [Ca2+]i. Since elevating the [Ca2+]i and pHi, by themselves promoted little secretion, another potentiating signal must have been generated by antigen stimulation. This signal was possibly transduced via hydrolysis of inositol phospholipids and protein kinase C. Even with an elevated [Ca2+]i (0.33 microM) the hydrolysis of the phospholipids and secretion stimulated by Ag were inhibited by guanosine 5'(2-O-thio)diphosphate and neomycin. Furthermore, both protein-kinase C and the secretory response to Ag were lost after permeabilized cells were washed but both were retained if cells were exposed to PMA before permeabilization.     

Depletion of guanine nucleotides with mycophenolic acid suppresses IgE receptor-mediated degranulation in rat basophilic leukemia cells

In RBL-2H3 rat basophilic leukemia cells, Ag that crosslink IgE-receptor complexes stimulate the turnover of inositol phospholipids, the mobilization of Ca2+ from intra- and extracellular sources, the release of serotonin and other substances from granules and the transformation of the cell surface from a microvillous to a lamellar architecture. This study explores the role of GTP-binding proteins (G proteins) in the control of these biochemical and functional responses. We report that incubating RBL-2H3 cells for 4 h with 10 microM mycophenolic acid (MPA), an inhibitor of de novo GTP synthesis, reduces GTP levels by over 60% and causes an average reduction of 50% in Ag-stimulated serotonin release. This inhibition of secretion is associated with a 50% decrease in the rate of 45Ca2+ influx in MPA-treated cells. In contrast, Ag-stimulated inositol trisphosphate production is only slightly reduced, indicating that the phosphatidylinositol-specific phospholipase C can be activated by Ag in GTP-depleted cells. The membrane responses to IgE receptor cross-linking are unaffected by incubating cells with MPA. Exogenous guanine or guanosine protects the GTP pools in MPA-treated cells and permits normal ion transport and secretory responses to Ag; adenine does not. These results implicate a guanine nucleotide-binding protein in the control of IgE receptor-dependent signal transduction in RBL-2H3 cells. This protein may particularly control the Ca2+ influx pathway that is essential for secretion.     

Pertussis toxin pretreatment reveals differential effects of adenosine analogs on IgE-dependent histamine and peptidoleukotriene release from RBL-2H3 cells

The effects of adenosine (A) and the nonmetabolizable adenosine analogs, N-ethylcarboxamidoadenosine (NECA), L-phenylisopropyladenosine (L-PIA), D-PIA and 2-chloroadenosine (2CHA) were examined on the IgE-dependent mediator release from RBL-2H3 cells, a model for mast-cell function. Adenosine and the adenosine analogs failed to influence mediator release from cells, previously sensitized with monoclonal anti-TNP mouse immunoglobulin E (anti-TNP IgE), when added alone. When added prior to conjugated trinitrophenol-ovalbumin (TNP-OVA), adenosine and the adenosine analogs (10(-8)-10(-4) M) significantly potentiated the release of both histamine (marker for degranulation) and peptidoleukotrienes (LT) (marker for de novo synthesized mediators). The effects were concentration-dependent with the potency order being L-PIA greater than NECA greater than A greater than D-PIA, 2CHA. The stimulatory effect on both histamine and LT release were reversed by prior treatment of the cells with pertussis toxin but not by the purinoceptor antagonists, theophylline and 8-phenyltheophylline, nor adenosine uptake blockers. At higher concentrations (above 10(-5) M), adenosine and adenosine analogs were also inhibitory on LT but not on histamine release. This inhibition was more evident on pertussis-toxin-treated cells in which there was no effect of adenosine or adenosine analogs on histamine release, but a concentration-dependent inhibition of IgE-dependent LT release. These findings demonstrate that adenosine analogs have two distinct mechanisms on mediator release from RBL-2H3 cells; a stimulatory effect on both histamine and LT release, mediated via a pertussis-toxin-sensitive G protein and an inhibitory effect on LT release via a pertussis-toxin-insensitive pathway. An abstract of this work has been published.     

Studies with transfected and permeabilized RBL-2H3 cells reveal unique inhibitory properties of protein kinase C gamma.

To characterize protein kinase C (PKC) gamma, an isozyme found exclusively in brain and spinal cord, its cDNA was introduced into basophilic RBL-2H3 cells that lack this isozyme. The expression of PKC gamma significantly attenuated antigen-induced responses including hydrolysis of inositol phospholipids, increase in cytosolic calcium, and secretion of granules but enhanced antigen-induced release of arachidonic acid. Instead of a sustained increase in cytosolic calcium, antigen now induced calcium oscillations; possibly as a consequence of suppression of the phospholipase C activity and incomplete emptying of internal calcium stores. In addition, PKC gamma appeared to inhibit activation of other PKC isozymes because phorbol 12-myristate 13-acetate failed to act synergistically with the Ca(2+)-ionophore on secretion. This was confirmed in other studies where PKC gamma was shown to suppress the transduction of stimulatory signals by other isozymes of PKC on provision of these isozymes to PKC-depleted permeabilized cells. The studies in total indicated that only PKC gamma was capable of inhibiting both early and distal signals for secretion including those signals transduced by endogenous isozymes of PKC.     

Receptor-mediated release of inositol 1,4,5-trisphosphate and inositol 1,4-bisphosphate in rat basophilic leukemia RBL-2H3 cells permeabilized with streptolysin O

Antigen-mediated exocytosis in intact rat basophilic leukemia (RBL-2H3) cells is associated with substantial hydrolysis of membrane inositol phospholipids and an elevation in concentration of cytosol Ca2+ ([ Ca2+i]). Paradoxically, these two responses are largely dependent on external Ca2+. We report here that cells labeled with myo-[3H]inositol and permeabilized with streptolysin O do release [3H]inositol 1,4,5-trisphosphate upon stimulation with antigen or guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) at low (less than 100 nM) concentrations of free Ca2+. The response, however, is amplified by increasing free Ca2+ to 1 microM. The subsequent conversion of the trisphosphate to inositol 1,3,4,5-tetrakisphosphate is enhanced also by the increase in free Ca2+. Although [3H]inositol 1,4,5-trisphosphate accumulates in greater amounts than is the case in intact cells, [3H]inositol 1,4-bisphosphate is still the major product in permeabilized cells even when the further metabolism of [3H]inositol 1,4,5-trisphosphate is suppressed (by 77%) by the addition of excess (1000 microM) unlabeled inositol 1,4,5-trisphosphate and the phosphatase inhibitor 2,3-bisphosphoglycerate. It would appear that either the activity of the membrane 5-phosphomonoesterase allows virtually instantaneous dephosphorylation of the inositol 1,4,5-trisphosphate under all conditions tested or both phosphatidylinositol 4-monophosphate and the 4,5-bisphosphate are substrates for the activated phospholipase C. The latter alternative is supported by the finding that permeabilized cells, which respond much more vigorously to high (supraoptimal) concentrations of antigen than do intact RBL-2H3 cells, produce substantial amounts of [3H]inositol 1,4-bisphosphate before any detectable increase in levels of [3H]inositol 1,4,5-trisphosphate.     

The Action of Adenosine Analogs on PC12 Cells

Abstract: PC12 cells, a nerve growth factor–responsive clone of rat pheochromocytoma, contain a membrane–bound adenylate cyclase, which can be activated by adenosine analogs. The characteristics of the cyclase response indicate the presence of stimulatory adenosine receptors. Adenosine analogs also produce a marked increase in the ornithine decarboxylase levels of the cells, and the characteristics of this response suggest that it is linked to the adenylate cyclase–stimulatory adenosine receptors. The ornithine decarboxylase response elicited by 5'- N -ethyIcarboxamideadenosine (NECA), a potent stimulatory adenosine analog, is synergistic with that produced by nerve growth factor. Differentiation of the cells with nerve growth factor, however, does not substantially alter either the response of cyclase to the adenosine analog or the magnitude of the adenosine–evoked ornithine decarboxylase response. Treatment of the cells with NECA produces an increase in the phosphorylation of a specific non–histone nuclear protein. While causing little or no morphological alteration by itself, NECA is synergistic with nerve growth factor in producing neurite outgrowth in PC12 cells. NECA does not cause an induction of acetylcholinesterase in the cells, nor does it appear to affect the induction of this enzyme by nerve growth factor.     

Cyclic GMP stimulates inositol phosphate production in cultured pituitary cells: possible implication to signal transduction

Addition of the stable and permeable analog 8-bromo cyclic GMP (8-BR-cGMP) to myo-[2-3H]inositol prelabeled cultured rat pituitary cells results in enhanced formation of [3H]-myo-inositol monophosphate (IP1). The stimulatory effect of the cyclic nucleotide analog is additive to the effect of Li+, which accumulates IP1 via inhibition of inositol 1-monophosphatase, and also to the effect of gonadotropin releasing hormone (GnRH) which stimulates the formation of IP1, as well as that of inositol 1,4-bisphosphate (IP2) and inositol 1,4,5-trisphosphate (IP3) via enhanced hydrolysis of polyphosphoinositides. Many Ca2(+)-mobilizing hormones acting via phosphoinosite turnover also stimulate cGMP formation. The cyclic nucleotide might then serve as a modulator by further hydrolysis of phosphoinositides needed for protein kinase C activation.     

Cardiotoxin from cobra venom increases the level of phosphatidylinositol 4-monophosphate and phosphatidylinositol kinase activity in two cell lines

In rat basophilic leukemia-2H3 (RBL-2H3) and Madin-Darby canine kidney (MDCK) cells, cardiotoxin from cobra venom induced a marked decrease in the level of [3H] phosphatidylinositol and a corresponding increase in the level of [3H]phosphatidylinositol 4-monophosphate over the course of 20 min as demonstrated in cells that had been labeled to equilibrium with [3H]inositol. The effect was dependent on the concentration (5-30 micrograms/ml) of the toxin. In plasma membrane-enriched fractions isolated from the two cell lines, the cardiotoxin enhanced the endogenous activity of phosphatidylinositol kinase especially at temperatures above 14 degrees C. In RBL-2H3 cells, cardiotoxin also induced release of substantial amounts of histamine and lactate dehydrogenase. The release of histamine, but not of lactate dehydrogenase, was totally dependent on external calcium and this release probably represented an exocytotic response of the cells to cardiotoxin. Although, initially, treatment with the toxin did not impair antigen-induced hydrolysis of inositol phospholipids or prevent the antigen-induced rise in the concentration of cytosol Ca2+, prolonged exposure to the toxin did result in a progressive loss of responsiveness of RBL-2H3 cells to antigen.     

Adenosine Inhibits Histamine-Induced Phosphoinositide Hydrolysis Mediated via Pertussis Toxin-Sensitive G Protein in Human Astrocytoma Cells

The effect of adenosine on phosphoinositide hydrolysis was examined in 1321N1 human astrocytoma cells. Adenosine, L-N6-phenylisopropyladenosine (L-PIA), and 5'-(N-ethylcarboxamido)adenosine (NECA) inhibited histamine-stimulated accumulation of inositol phosphates in a concentration-dependent manner. The potency order of adenosine analogues for inhibition of inositol phosphate accumulation was L-PIA greater than adenosine greater than NECA, a finding indicating that A1-class adenosine receptors are involved in the inhibition. The reduction in inositol phosphate accumulation by L-PIA was blocked by an adenosine receptor antagonist, 8-phenyltheophylline. Stimulation of A1-class adenosine receptors inhibited isoproterenol-stimulated cyclic AMP accumulation as well as histamine-induced inositol phosphate accumulation. Both inhibitory effects were blocked by pretreatment of the cells with pertussis toxin [islet-activating protein (IAP)]. L-PIA also inhibited guanosine 5'-(gamma-thio)triphosphate (GTP gamma S)-stimulated accumulation of inositol phosphates in membrane preparations, and 8-phenyl-theophylline antagonized the inhibition. L-PIA could not inhibit GTP gamma S-induced accumulation of inositol phosphates in IAP-treated membranes. Gi/Go, purified from rabbit brain, inhibited GTP gamma S-stimulated accumulation of inositol phosphates in a concentration-dependent manner in membrane preparations. These results suggest that stimulation of A1-class adenosine receptors interacts with the IAP-sensitive G protein(s), resulting in the inhibitions of phospholipase C as well as adenylate cyclase in human astrocytoma cells.     

Loss of secretory response of rat basophilic leukemia (2H3) cells at 40 degrees C is associated with reversible suppression of inositol phospholipid breakdown and calcium signals

Antigen-induced stimulatory signals as well as histamine secretion from the RBL-2H3 cells were found to be highly temperature dependent. There was no hydrolysis of inositol phospholipids, increase in cytosol calcium concentration (calcium signal), or secretion upon antigen stimulation at temperatures below 20 degrees C. At higher temperatures (i.e., 20 to 37 degrees C), all responses increased in extent with increase in temperature. Temperatures of 38 degrees C or higher, however, resulted in a marked decline in all responses, until no responses were observed at 40 to 42 degrees C. As indicated by the decay in calcium signal, the duration of response was also temperature dependent. The response was of long duration at 30 to 32 degrees C, but it became progressively more transient as the temperature was increased from 32 to 40 degrees C. The effects of low or high temperature were fully reversible. For example, in the presence of antigen, stimulatory signals immediately appeared once the temperature was decreased from 40 to 37 degrees C. Although the diminished responses could be explained, in part, by a reduction in rates of IgE receptor aggregation and phospholipase C activity, the reductions were insufficient to account for complete loss of activity at 40 degrees C. We conclude that generation of intracellular signals in 2H3 cells is blocked by quite small elevations in temperature above 37 degrees C, possibly as consequence of changes in membrane fluidity.     

Adenosine Inhibition of Histamine-Stimulated Inositol Phospholipid Hydrolysis in Mouse Cerebral Cortex

The effects of adenosine on inositol phospholipid hydrolysis in mouse cerebrocortical slices were examined. Despite having no effect alone, adenosine and some structural analogues inhibited histamine-stimulated accumulation of inositol phosphates in a concentration-dependent manner. The responses to carbachol, noradrenaline, 5-hydroxytryptamine, and elevated KCl levels were unaffected. The effect of adenosine was on the maximal response to histamine rather than on its EC50. Several adenosine antagonists competitively blocked the inhibition due to adenosine. The results are discussed in relation to the previously reported enhancement of histamine-stimulated hydrolysis of inositol phospholipids in guinea pig brain.     

Identification and characterization of leukotriene D4 receptors and signal transduction processes in rat basophilic leukemia cells

The leukotriene D4 (LTD4) receptor on rat basophilic leukemia (RBL-1) cell membranes was characterized using a radioligand binding assay. [3H]LTD4 binding to RBL-1 membrane receptors was stereoselective, specific, and saturable. The binding affinity and maximum binding density of [3H]LTD4 to RBL-1 membrane receptors were 0.9 +/- 0.2 nM and 800 +/- 125 fmol/mg protein, respectively. Binding of [3H]LTD4 to the receptors was enhanced by divalent cations (Ca2+, Mg2+, and Mn2+) and inhibited by guanine nucleotides and sodium ions, specifically, indicating that a guanine nucleotide-binding protein may regulate the agonist-receptor interaction. LTD4, LTE4 agonist and antagonist analogs competed with the radioligand in binding to the RBL-1 LTD4 receptors. The binding affinities of these analogs correlated with (a) those determined from the guinea pig lung LTD4 receptors and (b) the pharmacological activities in smooth muscle contraction. LTD4 and related agonists also induced time- and concentration-dependent phosphatidylinositol hydrolysis in RBL-1 cells. The LTD4 induction of inositol 1-phosphate was potent, stereoselective, specific, and was blocked by LTD4 receptor antagonists. The rank order potency of agonist-induced inositol 1-phosphate formation in RBL-1 cells was equivalent to the receptor binding affinity determined using either RBL-1 cell or guinea pig lung membranes. These studies have demonstrated the G protein coupled LTD4 receptors on RBL-1 cell membranes. Binding of agonists to the receptor may activate the G protein-regulated phospholipase C to induce hydrolysis of phosphatidylinositol. The hydrolytic products of phosphatidylinositol, possibly inositol trisphosphate and diacylglycerol, may be the intracellular messengers for LTD4 receptors in RBL-1 cells.     

Calcium influx in a rat mast cell (RBL-2H3) line. Use of multivalent metal ions to define its characteristics and role in exocytosis

An increase in concentration of cytosolic Ca2+ ([Ca2+]i) is associated with an accelerated influx of 45Ca2+ when cultured RBL-2H3 cells are stimulated with either antigen or analogs of adenosine although these agents act via different receptors and coupling proteins (Ali, H., Cunha-Melo, J.R., Saul, W.F., and Beaven, M.A. (1990) J. Biol. Chem. 265, 745-753). The same mechanism probably operates for basal Ca2+ influx in unstimulated cells and for the accelerated influx in stimulated cells. This influx had the following characteristics. 1) It was decreased when cells were depolarized with high external K+; 2) it was blocked by other cations (La3+ greater than Zn2+ greater than Cd2+ greater than Mn2 = Co2+ greater than Ba2+ greater than Ni2+ greater than Sr2+) either by competing with Ca2+ at external sites (e.g. La3+ or Zn2+) or by co-passage into the cell (e.g. Mn2+ or Sr2+); and 3) the inhibition of influx by K+ and the metal ions had exactly the same characteristics whether cells were stimulated or unstimulated even though influx rates were different. The dependence of various cellular responses on influx of Ca2+ was demonstrated as follows. The stimulated influx of Ca2+, rise in [Ca2+]i, and secretion, could be blocked in a concentration-dependent manner by increasing the concentration of La3+, but concentrations of La3+ (greater than 20 microM) that suppressed influx to below basal rates of influx markedly suppressed the hydrolysis of inositol phospholipids (levels of inositol 1,4,5-trisphosphate were unaffected). Some metal ions, e.g. Mn2+ and Sr2+, however, supported the stimulated hydrolysis of inositol phospholipid and some secretion in the absence of Ca2+. Thus a basal rate of influx of Ca2+ was required for the full activation of inositol phospholipid hydrolysis, but in addition an accelerated influx was necessary for exocytosis.