The influence of tetradecanoyl-phorbol-acetate (TPA) on histamine release from isolated rat mast cells

A detailed investigation of the influence of tetradecanoyl-phorbol-acetate (TPA) on isolated rat mast cells was undertaken in order to explore the possible involvement of protein kinase C in histamine release. TPA alone could induce histamine release in a medium without calcium, whereas 1 mM CaCl2 suppressed the release. TPA in combination with a low concentration of the ionophore A23187 induced a considerable histamine release. Preincubation with TPA followed by incubation with the ionophore induced a similar release at low concentrations of TPA (less than or equal to 2.5 nM) whereas the response was reduced at higher concentrations of TPA. The inhibition after preincubation with TPA was almost at a maximum within 2 min and was due to a decreased rate of release. TPA could also increase antigen-induced histamine release. After preincubation the potency of low concentrations of TPA increased, whereas higher concentrations (50 nM) became inhibitory. The effects of preincubation were almost fully expressed after 2 min and were not due to altered kinetics of the release. The interaction of oleoylacetylglycerol (OAG) with the ionophore A23187 and with antigen resembled that of TPA, but OAG was considerably less potent. Preincubation with TPA was inhibitory to the histamine release induced by compound 48/80, particularly in the absence of calcium. The release induced by TPA and the ionophore or antigen was calcium-dependent and energy-requiring, and the effects of TPA persisted after washing the cells before exposure to antigen or the ionophore. Preincubation with the protein kinase C inhibitor isoquinolinesulfonyl-methylpiperazine (H7) slightly enhanced the histamine release induced by the combination of TPA and the ionophore.(ABSTRACT TRUNCATED AT 250 WORDS)     

Synergistic effect of palmitoylcarnitine and the ionophore A23187 on isolated rat mast cells

Dl-palmitoylcarnitine in combination with low concentrations (0.2 microM) of the ionophore A23187 induced a pronounced non-cytotoxic histamine release, with maximal response at 10 microM palmitoylcarnitine and a lower response at 20 microM. The concentration-response curve was shifted to the right when the mast cells were preincubated with palmitoylcarnitine before exposure to the ionophore. Palmitoylcarnitine alone was without effect at concentrations below 20 microM but cytotoxic at higher concentrations. The response to the combination of palmitoylcarnitine and the ionophore was highly sensitive to changes in the ionophore concentration. The results indicate that conditions allowing physicochemical interactions between the two drugs led to greatest potency and effectiveness of palmitoylcarnitine. Preincubation with the phorbol ester TPA potentiated the response. The release induced by the combination of palmitoylcarnitine and the ionophore was completely inhibited by low concentrations of the flavonoid phloretin (IC50 of 0.5 - 2 microM) whereas the protein kinase inhibitor H-7 enhanced the response. The synergistic response to palmitoylcarnitine and the ionophore and its affection by phloretin and H-7 resembles previous findings with TPA and the ionophore. Although not conclusive the results indicate that palmitoylcarnitine can stimulate mast cells by activation of protein kinase C.     

Ether lipid (AMG) exhibits both synergistic and inhibitory interactions with the ionophore A23187 in mast cell histamine release.

Antineoplastic ether lipids with the structure 1-O-long-chain-alkyl-2-O-methylglycero-3-phosphocholine (AMG-PC) have direct tumour cytotoxic as well as immunomodulatory effects. Their tumouricidal action has been related to protein kinase C inhibition by the dialkylglycerol metabolite (AMG). The present investigation explores the influence of AMG (1-O-hexadecyl-2-O-methyl-sn-glycerol) on histamine release from isolated rat mast cells, which have a well-characterized response to protein kinase C activators. AMG could both enhance and antagonize responses to the ionophore A23187 and to A23187 in combination with the phorbol ester TPA. The synergistic effect was maximum at 2-5 microM AMG and could increase the response to A23187 more than 10-fold. Maximal inhibitory effect was found after preincubation with 20 microM AMG, irrespective of the ionophore concentration and the presence of TPA. The synergistic effect of AMG was dependent on energy and calcium, indicating non-cytotoxic mechanisms. The interaction between AMG and A23187 resembles previous findings with TPA and suggests an activation of protein kinase C.     

Inhibitory effect of phloretin on histamine release from isolated rat mast cells

The ability of the flavonoid phloretin to inhibit histamine release from rat mast cells varied considerably with the releasing agent investigated. The response to the combination of the ionophore A23187 and the phorbol ester TPA and to suboptimal concentrations of the ionophore (0.5 microM) was potently inhibited (IC50 about 5 microM), whereas phloretin was less potent against responses to the ionophore (1 microM) (IC50 of 17 microM), to antigen alone and in combination with TPA (IC50 of 30-50 microM), to TPA in the absence of calcium (IC50 of 50 microM) and to compound 48/80 in the absence and presence of calcium (IC50 of 60-90 microM). The inhibition by phloretin at concentrations above 10 microM was partly counteracted by glucose (5 mM) indicating effects on oxidative metabolism. The flavonoid quercetin was equally potent in inhibiting histamine release induced by antigen, the ionophore at different concentrations and in combination with TPA (IC50 of 20 microM). Although not conclusive, the results are consistent with an inhibition of protein kinase C by phloretin at concentrations below 10 microM. At higher concentrations unspecific actions become apparent and phloretin therefore seems to be of limited utility as a probe for signal-pathways in cell responses.     

The roles of calmodulin and protein kinase C in histamine secretion from mast cells.

The effect of a new calmodulin-antagonist, 5-iodo-1-C8, with a high selectivity for calmodulin in comparison to protein kinase C, has been investigated on histamine secretion from mast cells. It has been found to be much more sensitive for the inhibition of histamine secretion than the earlier calmodulin-antagonists, trifluoperazine and W7. The effect of four inhibitors of protein kinase C, viz. staurosporine, K252a, tamoxifen and sphingosine, has also been studied on histamine secretion from mast cells. All of them caused dose-dependent inhibition of histamine secretion induced by the three secretagogues used: antigen, compound 48/80 and the calcium ionophore A23187. K252a was tested against histamine release, induced by the stimulation of protein kinase C alone with the phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA) or the synthetic diacylglycerol, 1-oleoyl-2-acetyl-rac-glycerol (OAG). In both the cases K252a caused dose-dependent inhibition of histamine release. Staurosporine was also tested against TPA and was found to inhibit the release induced by it. Potentiation and inhibition (modulation) of secretagogue-induced histamine release by simultaneous protein kinase C stimulation with TPA or OAG have been demonstrated before. The potentiation and inhibition are shown to be antagonized by staurosporine. The observations point to the involvement of both calmodulin and protein kinase C in the histamine secretion process from mast cells.     

Effect of 12-O-tetradecanoylphorbol-13-acetate (TPA) on substance P-induced histamine release from rat peritoneal mast cells.

12-O-tetradecanoylphorbol-13-acetate (TPA, 1 to 30 ng/ml) produced a dose-related inhibition of substance P (SP)-induced histamine release from rat peritoneal mast cells. TPA itself induced some histamine release over this concentration range (maximum release about 20% of total). Maximum inhibition of SP-induced release by TPA required preincubation with TPA for at least 10 min. The inhibitory action of TPA was observed in the absence as well as in the presence of extracellular calcium (0.4 mM). Inhibition of diacylglycerol kinase by R 59022 or of diacylglycerol lipase by RHC 80267 reduced SP-induced histamine release. Oleolylacetylglycerol (OAG, 50 microM) inhibited histamine release induced by SP but was less potent than TPA. It is concluded that protein kinase C activation in rat peritoneal mast cells is associated with inhibition of SP-induced histamine release.     

Influence of staurosporine, a more selective derivative CGP 41,251 and an inactive analogue CGP 42,700 on histamine release from isolated rat mast cells.

The influence of staurosporine, a potent but nonselective inhibitor of protein kinase C, on rat mast cell histamine release, was compared with that of two derivatives, CGP 41,251 with a high degree of selectivity for protein kinase C and the related CGP 42,700 which is without activity. Staurosporine was a more potent inhibitor of mast cell responses than CGP 41,251, in accordance with their reported potencies. CGP 42,700 was investigated in the same concentration range as CGP 41,251 and served as a control for unspecific effects. Antigen induced histamine release was more effectively inhibited by staurosporine than by CGP 41,251, and responses to compound 48/80 were only modestly affected by both drugs. Responses to the ionophore A23187 were unaffected by staurosporine whereas CGP 41,251 was an effective inhibitor at suboptimal ionophore concentrations. In contrast, responses to combinations of the phorbol ester TPA and subthreshold concentrations of the ionophore could be potently inhibited by staurosporine but were under certain conditions moderately enhanced by lower concentrations of the drug, whereas CGP 41,251 was only inhibitory. Except for a slight inhibition of ionophore responses CGP 42,700 was without effect. The results demonstrate that the actions of staurosporine cannot be ascribed solely to inhibition of protein kinase C, whereas the influence of CGP 41,251 appears to be consistent with an inhibition of this kinase.     

Modulation of mast cell responses to adenosine by agents that alter protein kinase C activity

The acute incubation of mouse bone marrow-derived mast cells with low concentrations of agents known to activate protein kinase C [phorbol myristate acetate (PMA), 1,2-dioctanoyl-sn-glycerol (diC8), and 1-oleoyl-2-acetyl-glycerol (OAG)] caused an enhancement of beta-hexosaminidase release stimulated by the calcium ionophore A23187. Higher concentrations of protein kinase C activators tended to inhibit A23187- or antigen-induced preformed mediator release. All concentrations studied induced a striking mast cell hyporesponsiveness to the mediator release augmenting effect of adenosine. Agents that have been reported to block protein kinase C activity [1-(5-isoquinolinesulfonyl)-2-methyl-piperazine dihydrochloride (H-7) and sphingosine] demonstrated diverse responses in this system. Up to 100 microM H-7 failed to affect mast cell beta-hexosaminidase release in the presence or absence of PMA and secretagogue. Sphingosine (10 microM) was a potent inhibitor of antigen- or A23187-induced mediator release as well as adenosine responsiveness. Sphingosine also blocked the effects of PMA noted above in a dose-dependent fashion. The generation of leukotriene C4 (LTC4) by stimulated mast cells surprisingly was not affected by concentrations of diC8 that significantly inhibited granule-associated mediator release. Translocation of protein kinase C activity from the cytosol to the mast cell membrane was evident in cells briefly pretreated with A23187, adenosine alone, and diC8 in the presence of Tyrode's buffer, A23187, or adenosine. These findings lend further support to the contention that signal transduction from mast cell adenosine receptors to processes that regulate degranulation may involve protein kinase C.     

Expression of adhesion molecules by sphingosine 1-phosphate and histamine in endothelial cells

We investigated the effects of sphingosine 1-phosphate and histamine on the expression of vascular cell adhesion molecule-1 (VCAM-1), intercellular adhesion molecule-1 (ICAM-1) and E-selectin, and their signaling pathways in human umbilical vein endothelial cells. Sphingosine 1-phosphate increased the mRNA and protein level of VCAM-1, and the mRNAs of E-selectin and ICAM-1. The effects of sphingosine 1-phosphate were inhibited by the pertussis toxin and the respective inhibitors (10 μM 1-[6-[[(17β)-3-methoxyestra-1,3,5(10)-trien-17-yl]amino]hexyl]-1H-pyrrole-2,5-dione (U73122) for phosphoinositide-specific phospholipase C; 10 μM 4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)-1H-imidazole (SB203580) for p38 mitogen-activated protein kinase (MAPK); 1 μM 12-(2-cyanoethyl)-6,7,12,13-tetrahydro-13-methyl-5-oxo-5H-indolo(2,3-a)pyrrolo(3,4-c)-carbazole (Gö6976) for the α form of protein kinase C (PKC-α)), but not by a PKC-δ inhibitor (1 μM rottlerin). Histamine, which alone showed no effect, enhanced the sphingosine 1-phosphate-induced expressions via histamine H₁ receptor. The histamine response decreased by U73122 and rottlerin, but not by SB203580 and Gö6976. The effects of sphingosine 1-phosphate with and without histamine were abolished by the higher concentrations of PKC inhibitors and in the PKC-depleted cells. Sphingosine 1-phosphate and histamine alone stimulated phosphorylation of p38 MAPK in a phosphoinositide-specific phospholipase C-dependent but not in a PKCs-independent manner. These findings suggest that sphingosine 1-phosphate-induced expression of adhesion molecules was mediated by phosphoinositide-specific phospholipase C and preferentially by PKC-α and p38 MAPK, and the histamine response was mediated by phosphoinositide-specific phospholipase C and PKC-δ in human umbilical vein endothelial cells.     

The effects of the protein kinase C inhibitors staurosporine and H7 on the IgE dependent mediator release from RBL 2H3 cells.

RBL 2H3 cells, a model for mast cell function, sensitized with rat IgE, released histamine and peptidoleukotrienes (LT) in response to rabbit anti-rat IgE in a concentration-dependent manner. The calcium ionophore, A23187 also stimulated the release of both mediators but to a greater extent. The protein kinase C activator, 12-O-tetradecanoyl phorbol-13-acetate (TPA) failed to influence mediator release when added alone, but when added with either A23187 or anti-IgE, TPA significantly enhanced the release of both histamine and LT. The effects of anti-IgE, TPA and A23187 were completely inhibited by prior addition of the protein kinase C inhibitors staurosporine and 1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride (H7) but not by N-(2-guanidinoethyl)-5-isoquinoline-sulfonamide dihydrochloride (HA1004), a compound which has similar potency to H7 as an inhibitor of some protein kinases but is less potent as a protein kinase C inhibitor. Although other explanations are possible, these results support the hypothesis that the release of histamine and leukotrienes from RBL 2H3 cells resulting from the cross bridging of the IgE receptors, is dependent on activation of protein kinase C.     

The influence of ether lipid (AMG) on histamine release from isolated rat mast cells: synergistic interaction with TPA indicates protein kinase C activation.

We have previously shown that the ether lipid AMG (1-O-hexadecyl-2-O-methyl-sn-glycerol) has both synergistic and inhibitory effects on mast cell responses to the ionophore A23187. The present investigation showed only inhibitory effects of AMG in antigen- and compound 48/80-induced histamine release. Both enhancement and inhibition were noted in responses to the combination of A23187 and the phorbol ester TPA, at 2-5 microM and 10-20 microM and 10-20 microM AMG, respectively, as found earlier with A23187 alone. The synergistic response to AMG in combination with A23187 resembles that with TPA but required higher concentrations of A23187. The flavonoid phloretin was a potent inhibitor of the response to combinations of AMG and A23187. A pronounced synergistic interaction between AMG and TPA was found at very low concentrations of A23187. Our results do not provide much information about mechanisms involved in the inhibitory effect of AMG although some competition relating to protein kinase C activity might participate. The synergistic interactions indicate that AMG can activate protein kinase C but in a manner different from the phorbol ester.     

Influence of CGS 9343B, an inhibitor of calmodulin activity, on histamine release from isolated rat mast cells.

Investigations of calmodulin involvement in cell responses has been complicated by the lack of selective calmodulin antagonists. A novel inhibitor, CGS 9343B, reportedly without influence on protein kinase C, is used in the present study of mast cell responses. The histamine release induced by antigen and compound 48/80 in the presence of calcium was enhanced by 10-20 microM CGS 9343B and inhibited by higher concentrations. Only inhibitory effects on the response to compound 48/80 in the absence of calcium and to the ionophore A23187 were observed, the latter being inhibited by 20 microM CGS 9343B. The influence on responses to combinations of the phorbol ester TPA and the ionophore A23187 was more complex, giving rise to enhancement at lower and inhibition at higher concentrations of CGS 9343B in a manner which depended on the experimental conditions. Unlike previously used calmodulin antagonists, CGS 9343B is devoid of detergent effects and without serious metabolic interference. The inhibitor seems useful to reveal differences in the mechanisms involved in responses to various histamine liberators. Our results conform with an inhibition of calmodulin by CGS 9343B but are at present inconclusive.     

Multiple effects of staurosporine, a kinase inhibitor, on thymocyte functions. Comparison with the effect of tyrosine kinase inhibitors.

The effects of staurosporine, a protein kinase inhibitor, on the signal transduction and proliferation of thymocytes were studied. Signal transduction in response to Concanavalin A (Con A) as well as Concanavalin A (Con A)-induced augmentation of [3H]inositol incorporation into phospholipids were inhibited by staurosporine (> or = 10(-8) M). Staurosporine inhibited thymocyte proliferation in response to Con A in the presence or absence of the phorbol ester, phorbol myristate acetate (TPA) (10 nM). This inhibition was observed regardless of whether staurosporine was added together with Con A or 3 hr later. High concentrations of staurosporine (> 10(-8) M) inhibited thymocyte proliferation induced by the calcium ionophore A23187 and the phorbol ester TPA, whereas lower concentrations of the inhibitor (< or = 10(-8) M) enhanced thymidine incorporation in response to these activators. This dual effect of staurosporine was also observed in the presence of the staurosporine-related kinase inhibitor, K252a. In contrast, the tyrosine kinase inhibitor, tyrphostin AG490, inhibited the response to A23187 and TPA at all concentrations of the inhibitor and no augmentation was seen. Interleukin 2 (IL-2)-driven mitogenesis in IL-2-dependent cells was also inhibited by staurosporine. We suggest that the inhibition of thymocyte proliferation by staurosporine results from inhibition of both protein kinase C and tyrosine kinase: the augmentation of the response to A23187 and TPA results from inhibition of protein kinase C. Inhibition of signal transduction as well as inhibition of IL-2-driven mitogenesis result from inhibition of tyrosine kinase.     

The actions of retinal and retinoic acid on histamine release from rat peritoneal mast cells

Histamine release from rat peritoneal mast cells induced by anti-IgE was potentiated by the retinoids: retinoic acid 2-10 microM and retinal 1-5 microM. Retinal also produced a concentration-dependent increase in anti-IgE-stimulated 45Ca uptake by these mast cells. A similar potentiating action of both retinoids was observed on histamine release induced by 12-O-tetradecanoylphorbol-13-acetate (TPA). For both anti-IgE- and TPA-induced histamine release, the potentiating effect of the two retinoids was only observed in the presence of extracellular calcium. In contrast, histamine release induced by compound 48/80 was inhibited by retinal 1-5 microM and by retinoic acid 10-50 microM and the inhibition was the same in the presence as in the absence of extracellular calcium 1 mM. Histamine release induced by calcium and the calcium ionophore A 23187 was inhibited by retinoid acid 2-10 microM and by retinal 10 microM. Inhibitions of compounds 48/80-induced histamine release by cromoglycate and by retinal were additive. It is concluded that while the actions of retinoids on rat peritoneal mast cells are consistent with the inhibition of protein kinase C, another action of these compounds, unrelated to this enzyme, may explain the data.     

Influence of TMB-8 and calmidazolium on phorbol ester promoted histamine release from isolated rat mast cells.

The effects of TMB-8 and calmidazolium were investigated on mast cell responses believed to be mediated by protein kinase C, i.e. histamine release induced by TPA (tetradecanoyl-phorbol-acetate) in combination with sub-threshold concentrations of the ionophore A23187 and with antigen. Inhibition with both drugs was found in the same concentration range as observed earlier and could be counteracted by glucose, indicating an impaired oxidative energy production. Hence, the test drugs do not reveal protein kinase C selectivity.     

Role of Ca2+/phospholipid-dependent protein kinase in catecholamine secretion from bovine adrenal medullary chromaffin cells

The role of Ca2+/phospholipid-dependent protein kinase (protein kinase C) in catecholamine secretion from bovine adrenal medullary chromaffin cells was examined using four protein kinase C inhibitors: polymyxin B, sphingosine, staurosporine, and 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H-7). For this purpose, digitonin-permeabilized chromaffin cells were used. Secretion of catecholamines from these cells was stimulated by the addition of micromolar amounts of exogenous free Ca2+. 12-O-Tetradecanoylphorbol-13-acetate (TPA) and arachidonic acid, activators of protein kinase C, enhanced the catecholamine secretion evoked by Ca2+. But phorbol-12, 13-diacetate, a phorbol ester analog that does not activate protein kinase C, had no effect on Ca2(+)-evoked secretion. Polymyxin B at a low concentration (1 microM) abolished the enhancement of secretion by TPA or arachidonic acid without affecting the secretion evoked by Ca2+. However, polymyxin B at higher concentrations (10-100 microM) greatly reduced Ca2+-evoked catecholamine secretion. Sphingosine 10 microM-1 mM), Staurosporine (100 nM-1 microM, and H-7 (100-500 microM) inhibited TPA- or arachidonic acid-enhanced secretion but not Ca2(+)-evoked secretion. In cells in which protein kinase C was down-regulated by TPA, specific binding of [3H]phorbol-12,13-dibutyrate to the cells almost disappeared and the enhancement of secretion by TPA was no longer observed, whereas Ca2(+)-evoked secretion was maintained. These results strongly suggest that protein kinase C is not essential for the Ca2(+)-dependent catecholamine secretion from bovine adrenal chromaffin cells, but acts instead as a modulator.     

H-7, a protein kinase C inhibitor, inhibits phorbol ester-caused ornithine decarboxylase induction but fails to inhibit phorbol ester-caused suppression of epidermal growth factor binding in primary cultured mouse epidermal cells

12-O-tetradecanoylphorbol-13-acetate (TPA) induced ornithine decarboxylase (ODC) and suppressed 125I-epidermal growth factor (EGF) binding in primary cultured mouse epidermal cells. TPA (30 nM)-caused ODC induction was almost completely blocked by 30 microM H-7 [1-(5-isoquinolinylsulfonyl)-2-methylpiperazine], a well known protein kinase C inhibitor, but the same concentration of H-7 failed to restore the 125I-EGF binding suppressed by TPA (10 nM). On the other hand, sphingosine, another protein kinase C inhibitor, blocked not only TPA-caused ODC induction but also TPA-caused suppression of 125I-EGF binding. Concentration-response curves of sphingosine for these two TPA-caused cellular responses were almost identical. 1,2-Diacylglycerols such as 1,2-dioctanoylglycerol (30-300 microM) and 1-oleoyl-2-acetylglycerol (OAG) (30-300 microM) mimicked TPA actions. Similar to the case of TPA, suppression of 125I-EGF binding by OAG was barely inhibited by H-7, whereas sphingosine was more effective in inhibiting the OAG-caused suppression of 125I-EGF binding than was H-7. In TPA (50 nM)-pretreated epidermal cells, TPA (10 nM) failed to suppress 125I-EGF binding. H-7 (30 microM) did not affect TPA (30 nM)-caused translocation of protein kinase C. These results clearly demonstrate the differential inhibition by H-7 of the TPA-caused cellular responses and indicate that TPA-caused suppression of 125I-EGF binding to epidermal cells is mediated through protein kinase C function, which is barely inhibited by H-7.     

Effect of the anti-neoplastic agents edelfosine (ET-18-OCH3), ilmofosine (BM 41.440) and the hexadecylphosphocholine analogues D-20133 and D-21266 on histamine release from isolated rat mast cells

The ether phospholipid AMG-PC (1-O-hexadecyl-2-O-methyl-rac-glycero-3-phosphocholine, ET-16-OCH3) affects rat mast cell responses in a dual manner. A powerful synergistic interaction with the ionophore A23187 and the phorbol ester TPA indicated an involvement of mechanisms relating to activation of protein kinase C. In contrast, the related hexadecylphosphocholine (miltefosine) only causes inhibition. Here, the investigation is extended to include the antineoplastic ether phospholipids ET-18-OCH3 (edelfosine) and BM 41.440 (ilmofosine) as well as the heterocyclic hexadecylphosphocholine analogues D-20133 and D-21266. The four test drugs had an influence very similar to that of AMG-PC on mast cell responses to selected secretagogues, i.e., they both enhanced and inhibited antigen-induced histamine release whereas only inhibition was observed with compound 48/80. They significantly amplified the response to A23187 alone as well as in combination with TPA and, under certain conditions, inhibitory effects were observed with ET-18-OCH3, D-20133 and D-21266 but not with BM 41.440. The latter was more effective in enhancing A23187 mediated responses and had a wider concentration range of activity than the other three drugs. D-20133 and D-21266 influenced mast cells in a manner distinct from that of hexadecylphosphocholine and may share cellular targets with the ether phospholipids. The results raise speculation of an involvement of mast cells in the immunomodulatory action of these drugs.     

Histamine release induced by histone and phorbol ester from rat peritoneal mast cells

Histone 10 to 50 micrograms/ml released histamine from rat peritoneal mast cells in the absence of extracellular calcium. Extracellular calcium, 1 mM produced a slight shift of the histone dose-response curve to the right. In the absence of extracellular calcium, the histamine release-response to combined stimulation with histone and substance P was saturable. Neither histone nor substance P elicited any further response when one of these agonists alone was already eliciting a maximum response, although the cells were capable of a greater degree of histamine release in the presence of compound 48/80. In the presence of extracellular calcium, substance P at a concentration not by itself producing a response, acted synergistically with histone to induce histamine release. The substance P antagonist, SP-A, inhibited histamine release by histone. The phorbol ester, TPA, released histamine in a dose-dependent manner and this response was inhibited by SP-A. It is suggested that substance P and histone interact with a common site to release histamine and the role of protein kinase C in this release mechanism is discussed.     

Inhibition of basophil histamine release by methotrexate

Basophil leukocytes in whole blood from 4 healthy donors, 4 atopic patients, and 10 female patients operated for breast-cancer were preincubated from 1 to 20 hrs alone or in the presence of methotrexate (MTX) or MTX and folinic acid. After preincubation, the basophil leukocytes were challenged with anti-IgE, allergens or the calcium ionophore A23187 in the presence of 25 ng/ml TPA (12-o-tetradecanoyl-phorbol-13-acetate). A 9-hr preincubation with MTX produced significant inhibition of histamine release (greater than 20%) at 500-50 micrograms/ml. This effect increased up to 20 hrs of incubation, displaying maximal activity (100% inhibition) at 500 micrograms/ml, but even submicrogram concentrations (0.5 microgram/ml) produced significant inhibition. The addition of folinic acid did not alter the inhibition. It is concluded that MTX with or without the addition of folinic acid is a potent inhibitor of histamine release induced by anti-IgE, allergens, and A23187 combined with TPA. Like glucocorticoids the mechanism of action of MTX may be linked to arachidonate metabolism, but may interrupt earlier steps in prostaglandin synthesis.