Influence of pyrethroids and piperonyl butoxide on histamine release from isolated rat mast cells.

OBJECTIVE AND DESIGN: Pyrethroids are insecticides with low acute toxicity in mammals but their world-wide use for domestic and occupational purposes has caused concern about the risks of long-term exposure. The mammalian toxicity of pyrethroids is related to disturbances of membrane function in neuronal tissues whereas their influence on nonneuronal tissues is poorly understood. Recently, selected pyrethroids were shown to affect the function of rat synaptosomal and leukocyte membranes similarly. The present investigation explores to what extent their influence on the function of intact cells, i. e. isolated rat mast cells, correlates with these membrane interactions. MATERIALS AND TREATMENT: Permethrin and the more water soluble esbiol (S-bioallethrin), both type I pyrethroids, and cyfluthrin, type II, used alone and together with the enhancing substance piperonyl butoxide (PBO) at concentration ratios of 1:5 and 1:10, were tested for influence on histamine release induced by compound 48/80 without and with calcium. RESULTS: Permethrin (5-10 microM) caused a 10-25 % inhibition of the histamine release in the absence of calcium but did not affect the response with calcium present and had no interaction with PBO. Esbiol (10 microM) was an effective inhibitor on its own, with 70 and 45% inhibition in the absence and presence of calcium, respectively, and caused virtually complete inhibition in a synergistic interaction with PBO. The effect of esbiol could partly be ascribed to inhibition of oxidative energy production. Cyfluthrin was inactive at concentrations up to 10 microM. PBO alone (50 microM) caused some inhibition, in particular in the absence of calcium (ca. 25 %). CONCLUSIONS: The results relating to mast cell histamine release reveal both similarities and differences with the influence of the pyrethroids on cell membrane activities. They indicate that not solely membrane interactions but also additional or alternative targets are involved in the effects of pyrethroids on mammalian tissues. Moreover, the pronounced effects of a brief cell exposure suggest that long-term exposure can be hazardous.

Similar effects of ether phospholipids, PAF and lyso-PAF on the Ca(2+)-ATPase activity of rat brain synaptosomes and leukocyte membranes.

The present study is an extension of our previous work with the antineoplastic ether phospholipid ET-18-OCH3 (edelfosine), which was shown to affect the activity of the Ca(2+)-ATPase of rat brain synaptosomes and peritoneal leukocyte membranes. The effect of ET-18-OCH3 was compared with that of the 16-carbon chain analogue ET-16-OCH3 as well as with the structurally related 16- and 18-carbon PAFs (platelet-activating factors) and lyso-PAFs. In addition, the two alkylphosphocholines D-20166 and D-21266 (perifosine) were included in the investigation. The influence of all of the compounds followed the same pattern, i.e., the Ca(2+)-ATPase activity of the synaptosomes was increased over a relatively narrow concentration range (peak at 20-30 microM) and that of the leukocyte membranes was inhibited in a concentration-dependent manner by 10-50 microM concentrations of the drugs. Ether phospholipids with an 18-carbon chain at C-1 were more potent than those with a 16-carbon chain. All of the compounds increased the activity of the synaptosomal ATPase to the same extend (ca. 50%). With the exception of lyso-PAF, all inhibited the enzyme activity of leukocyte membranes by 60-70%, whereas lyso-PAF was less effective (ca. 50% inhibition). The concentration range of activity for PAF and lyso-PAF indicates that their effect on the enzyme activity was caused by receptor-independent mechanisms. The ether phospholipids and alkylphosphocholines are suggested to act by accumulating in the membranes and thereby altering the character of the lipid environment of the enzyme rather than by a direct interaction with the Ca(2+)-ATPase.

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.

Influence of probes for calcium-calmodulin and protein kinase C signalling on the plasma membrane Ca2+-ATPase activity of rat synaptosomes and leukocyte membranes.

The influence of selected inhibitors of calcium signalling on the plasma membrane Ca2+-ATPase activity of rat synaptosomes and peritoneal leukocyte membranes was studied. The calmodulin inhibitor calmidazolium was an efficient inhibitor (50%) of the synaptosomal Ca2+-ATPase activity in a manner competitive with phosphatidylserine. The inhibition by CGS 9343B (30%) was not counteracted by phosphatidylserine. The intracellular calcium antagonist TMB-8 and the protein kinase inhibitor staurosporine and the derivatives CGP 41251 and CGP 42700 hardly affected the synaptosomal Ca2+-ATPase activity. The flavonoid quercetin was a more effective inhibitor of the ATPase activity of synaptosomal than of leukocyte membranes. Phloretin, at relatively high concentrations, caused only a modest inhibition of synaptosomes. The protein kinase C inhibitor sphingosine was a weak inhibitor of the synaptosomal but an effective inhibitor of the leukocyte membrane Ca2+-ATPase activity. The antineoplastic ether phospholipids BM 41.440 (ilmofosine) and ET-18-OCH3 (edelfosine) effectively inhibited the leukocyte membranes whereas the ATPase activity of synaptosomes was significantly increased by 20 microM and slightly inhibited by higher concentrations of these agents. The analogue hexadecylphosphocholine (miltefosine) did not affect the ATPase activity of the synaptosomes and only inhibited that of the leukocyte membranes at concentrations above 20 microM. These results show that several test substances of current interest affect the activity of the plasma membrane Ca2+-ATPase. The effects depend on the origin of the membranes. The investigation does not permit a distinction between direct effects on the enzyme and an interference with its membrane environment although the latter is indicated for the ether phospholipids.

Influence of DPPE on histamine release from isolated rat mast cells.

A second messenger function for histamine has been proposed based on the effects of the anti-estrogen drug DPPE (N,N-diethyl-2-(4-(phenylmethyl)phenoxy) ethanamine.HCl). The ability of DPPE to inhibit concanavalin A-induced histamine release led to the present investigation of its influence on the mast cell response to a wider selection of secretagogues. DPPE was an efficient inhibitor of antigen-induced release, while responses to compound 48/80 were virtually unaffected. Responses to the ionophore A23187 could be enhanced as well as inhibited, whereas the influence of DPPE on the combination of the ionophore and the phorbol ester TPA was variable and small. These results seem to exclude an involvement of a DPPE-sensitive histamine-mediated signal system of common importance in mast cell 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.

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.

Effects of the ether phospholipid AMG-PC on mast cells are similar to that of the ether lipid AMG but different from that of the analogue hexadecylphosphocholine.

The ether lipid AMG (1-O-hexadecyl-2-O-methylglycerol) has previously been shown to have both enhancing and inhibitory effects on histamine release from isolated rat mast cells. In addition, a synergistic interaction with the phorbol ester TPA indicated protein kinase C activation. In the present investigation the effects of the related ether phospholipid AMG-PC (1-O-hexadecyl-2-O-methylglycero-3-phosphocholine) and the analogue hexadecylphosphocholine (miltefosine (HPC) are compared with those of AMG. HPC had only inhibitory effects, in contrast to AMG-PC which acted similarly on histamine release induced by compound 48/80, but had dual influence on responses to antigen and the ionophore A23187 and mainly exhibited a synergistic interaction with combinations of the ionophore and TPA. The influence of AMG-PC differed from that of AMG in some aspects whereas the character of its synergistic interaction with A23187 resembled that of AMG, in particular after pre-incubation with TPA. The results show that HPC does not share all biological activities of the antineoplastic ether lipids. They indicate that the effects of AMG-PC under appropriate conditions may be due to activation of protein kinase C but do not permit a distinction between direct effects and indirect through metabolic conversion to AMG.

Sphingosine inhibition and promotion of histamine release from isolated rat mast cells.

Sphingosine inhibited the histamine release induced by antigen, compound 48/80 with and without calcium, and the combination of TPA and the ionophore A23187. The inhibition occurred in the concentration range 1-3 microM, where no sign of cytotoxicity was noted. Preincubation for 5-10 min was needed for inhibition, and the effect persisted after washing of the cells. No inhibition was found with optimal concentrations of the ionophore or with TPA present during the preincubation. Sphingosine in combination with suboptimal concentrations of the ionophore could induce a considerable histamine release. This response was dependent on energy and was potently inhibited by the flavonoid phloretin. After preincubation with TPA, sphingosine exerted a pronounced potentiation of the response to very low concentrations of the ionophore. The findings regarding inhibitory effects of sphingosine do not seem to be compatible with a selective action on protein kinase C. The ability to synergize with the ionophore and to potentiate the effect of preincubation with TPA resembles previous findings with palmitoylcarnitine and suggests that sphingosine can stimulate mast cells by 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.

Unusual effects of the ionophore A23187 at suboptimal concentrations in mast cell histamine release.

Certain peculiarities are observed in connection with histamine release induced by suboptimal concentrations of the ionophore A23187. The potency of the ionophore can increase by a factor of 3 when added to the incubation medium from solutions in either DMSO or buffer without added calcium as compared with addition from standard buffer. The effect was not due to the solvent per se but may relate to physicochemical conditions facilitating the ionophore's access to cellular domains.

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.

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.

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.

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 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)

On the isolation of mast cells from human adenoids and tonsils.

Human adenoids and tonsils were disintegrated mechanically and the cells dispersed by passage through a stainless-steel screen in EDTA-containing buffer. Collagenase digestion did not increase the yield of adenoidal cells. The mast cell content of the cell suspensions was in the range of 1-10 mast cells/10(4) cells with an estimated mean of 1-2 mast cells/10(4) cells, a value considerably below previous reports on adenoidal cell suspensions. The mast cell content was determined by staining with toluidine blue at low pH (to prevent interference by phagocytes). The mast cell count as assessed by alcian blue staining and by fluorescence microscopy after FITC-anti-human IgE binding was similar. Various attempts to enrich the cell suspension (i.e. by differential centrifugation, by gradient centrifugation on Ficoll or Ficoll-Hypaque and by velocity sedimentation at unit gravity) all gave negative results.

Effect of calmidazolium (R24571) on histamine release from isolated rat mast cells.

The effects of selected calmodulin-antagonists, i.e. calmidazolium (R24571), trifluoperazine, cis- and transflupenthixol, chlorpromazine, and imipramine, on rat mast cells and on mast cell histamine release were investigated. The drugs induced histamine release, apparently by cytotoxic effects, with a rank order of potency in accordance with their lipid solubility and with maximal release at calmidazolium (5 mumol/l), trifluoperazine (40 mumol/l), cis- and trans-flupenthixol (50 mumol/l), chlorpromazine (100 mumol/l), and imipramine (500 mumol/l). Inhibition of the histamine release induced by antigen, compound 48/80, and the ionophore A23187 was only observed with some of the drugs tested, with maximal inhibition at calmidazolium (2 mumol/l), chlorpromazine (25-50 mumol/l), and imipramine (100-250 mumol/l). The concentration-response curve for histamine release induced by calmidazolium was significantly shifted to the right by antigen (i.e. horse serum) in the medium and the addition of antigen was capable of immediately stopping the release induced by calmidazolium, indicating binding of calmidazolium by antigen. Similar effects on the actions of calmidazolium were observed with phosphatidylserine. The inhibition by calmidazolium of the histamine release induced by antigen, compound 48/80, and the ionophore A23187 was significantly counteracted by glucose in the medium. The findings do not confirm an involvement of calmodulin in the histamine release process in rat mast cells. The ability of calmidazolium to bind to proteins and phospholipids in the medium indicates multiple cellular targets for calmidazolium, and the observations with glucose suggest an impaired mitochondrial function to be of major significance.(ABSTRACT TRUNCATED AT 250 WORDS)

Binding of compound 48/80 by isolated rat mast cells in connection with histamine release.

The binding of compound 48/80 in connection with histamine release from isolated rat mast cells was investigated by a two-step incubation procedure. After incubation of mast cells with known concentrations of compound 48/80, the supernatants were collected and subsequently exposed to fresh mast cells. The response in the second incubation step provided a measure of the amount of compound 48/80 remaining in the supernatants. At noncytotoxic concentrations of the releasing agent, binding capacities for compound 48/80 of up to 4 micrograms/10(5) mast cells (4% w/v) were observed. The binding of compound 48/80 was of high affinity, giving mast cell concentrations of the compound exceeding that in the incubation medium by four orders of magnitude. The binding occurred rapidly with a substantial proportion bound within the first minute. These findings indicate that quantitation of exocytotic events by means of basic dyes may be compromised by competition for granular binding sites by basic releasing agents.