Roles of intracellular Ca2+ and cyclic AMP in mast cell histamine release induced by radiographic contrast media
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- Saito, M., Itoh, Y., Yano, T. et al. Naunyn-Schmiedeberg's Arch Pharmacol (2003) 367: 364. doi:10.1007/s00210-003-0706-7
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Mast cell histamine release is considered to be associated with the etiology of anaphylactoid reactions to iodinated radiographic contrast media (RCM). In the present study, the effects of various ionic and non-ionic RCM on histamine release from mast cells were compared, and the possible mechanisms of the histamine release were subsequently determined. Both ionic (ioxaglate and amidotrizoate) and non-ionic (iohexol, ioversol, iomeprol, iopamidol and iotrolan) RCM increased histamine release from the dissociated rat pulmonary cells, whereby ionic materials were more potent than non-ionic agents. There was no significant correlation between the extent of histamine release and the osmolarity of each RCM solution. In addition, hyperosmotic mannitol solution (1000 mOsm/kg) caused no marked histamine release. Thus, it is unlikely that the hyperosmolarity of RCM solutions contributes to the histamine release. RCM also stimulated, but to a lesser extent, the histamine release from rat peritoneal cells. The RCM-induced histamine release from both types of cells was inhibited by dibutyl cyclic AMP or combined treatment with forskolin and 3-isobutyl-1-methylxanthine. Corresponding to these results, RCM markedly reduced the cellular cyclic AMP content. On the other hand, the removal of intracellular but not the extracellular Ca2+ attenuated the RCM-induced mast cell histamine release. From these findings, it is suggested that the decrease in cellular cyclic AMP content and an increase in intracellular Ca2+ contribute at least in part to the RCM-induced mast cell histamine release.
KeywordsRadiographic contrast mediaPulmonary mast cellsPeritoneal mast cellsHistamineIntracellular calciumOsmolarity
Although radiographic contrast media (RCM) are increasingly used for radiography and magnetic resonance imaging, undesirable actions associated with intravascular injection of RCM such as local pain, thrombosis, phlebitis and edema (Lalli 1980; Barstad et al. 1996; Thomsen and Bush 1998) occur occasionally and no prevention or effective therapy for the adverse events has yet been established. Pulmonary edema is a serious life-threatening adverse event, although the incidence is rare. RCM-induced pulmonary edema is of the non-cardiogenic type and appears to be related to the increase in pulmonary vascular permeability that accompanies activation of an inflammatory cascade and release of a variety of chemical mediators (Bouachour et al. 1991). It has been considered that histamine release from mast cells or basophils contributes to the onset of the adverse reactions to RCM (Cogen et al. 1979; Pinet et al. 1988; Rodriguez et al. 2001). Laroche et al. (1998) have reported in patients with mild to severe adverse reactions to iodinated RCM that plasma concentrations of histamine and its metabolite N-methylhistamine increase in relation to the severity of the adverse reactions. Based on these facts, clinical trials have shown that premedication with histamine H1 receptor antagonists such as diphenhydramine is beneficial for the prevention of anaphylactoid reactions to RCM (Wittbrodt and Spinler 1994). We have shown in rats that the intravenous injection of ionic RCM causes severe pulmonary vascular hyperpermeability and edema (Sendo et al. 1999). Moreover, the RCM-induced pulmonary extravasation is reduced by the pretreatment with histamine H1 and H2 antagonists (Goromaru et al. in press). Therefore, mast cells are assumed to be the primary target of RCM for eliciting the acute inflammatory responses (Galli et al. 1993).
Many studies have demonstrated that RCM induce histamine release from isolated mast cells and basophils prepared from various tissues of a variety of species (Ennis 1982; Assem et al. 1983; Amon et al. 1989, 1990; Ennis et al. 1991; Baxter et al. 1993; Genovese et al. 1996; Peachell and Morcos 1998). However, little is known about the cellular mechanisms underlying RCM-induced histamine release. It has been reported that the histamine release is attributable to the hyperosmolarity of the RCM solutions, since hyperosmotic mannitol solution induces histamine release from basophils and mast cells (Baxter et al. 1993; Genovese et al. 1996; Peachell and Morcos 1998). In contrast, a lack of correlation between the osmolarity of RCM and histamine release from human lung mast cells has been reported by other investigators (Stellato et al. 1996). The ionic properties may also be associated with the RCM-induced histamine release, since non-ionic RCM are generally less potent than ionic compounds in eliciting histamine release (Ennis 1982; Salem et al. 1986a; Ennis et al. 1991; Rodriguez et al. 2001). On the other hand, Laroche et al. (1999) have reported an involvement of IgE-mediated mechanism in RCM-evoked histamine release, since specific IgE antibody against RCM is detected in plasma of patients.
The present study was designed to determine the possible mechanisms underlying the RCM-evoked mast cell histamine release. For this purpose, the effects of various RCM on histamine release from rat pulmonary cells were investigated in relation to the osmolarity of their solution. The roles of intracellular Ca2+ and cyclic AMP (cAMP) in the RCM-induced histamine release was subsequently determined.
Materials and methods
The present experiments were reviewed by the ethics committee for animal experiments at the Faculty of Medicine, Kyushu University, and the law (No.105) and notification (No.6) of the Japanese government.
Male Sprague-Dawley rats weighing 180–230 g were purchased from Kyudo Co. (Saga, Japan). Animals were maintained on a 12-h light/dark schedule (lights on at 7:00 am) at a temperature of 23±2°C with free access to food and water.
The iodinated RCM used in the present study were as follows: ioxaglate (Hexabrix 320; iodine, 320 mg iodine/ml; lot no. 94002; Mallinckrodt Medical, St. Louis, MO, USA), amidotrizoate (urographine 60%; iodine, 292 mg iodine/ml; lot no. 82154; Schering AG, Berlin, Germany), iohexol (Omnipaque 300; iodine 300 mg iodine/ml; lot no. KV78; Daiichi Pharmaceutical, Tokyo, Japan), iomeprol (Iomeron 400; iodine, 400 mg iodine/ml; lot no. 6YA11B; Eisai, Tokyo), iopamidol (Iopamiron 300; iodine, 300 mg iodine/ml; lot no. 61271 Schering AG, Berlin), ioversol (Optiray 350; iodine, 350 mg iodine/ml; lot no. 1010025 J; Mallinckrodt Medical, St. Louis, MO) and iotorolan (Isovist 300; iodine, 300 mg iodine/ml; lot no. 03015; Schering AG, Berlin,). D-Mannitol and 3-isobutyl-1-methylxanthine (IBMX) were obtained from Sigma Chemical (St. Louis, MO). Forskolin and 1,2-bis(ο-aminophenoxy)ethane-N,N,N',N'-tetra-acetic acid (BAPTA/AM) were obtained from Carbiochem-Novabiochem Co. (San Diego, CA, USA.) and Research Biochemicals International (Natick, MA, USA), respectively. Ethylene glycol-bis(β-aminoethyl ether)N,N,N',N'-tetraacetic acid (EGTA) was obtained from Nacalai tesque (Kyoto, Japan). All other chemicals were of reagent grade. RCM and mannitol were dissolved in Krebs-Ringer solution or Hank's balanced salt solution (HBSS: 137 mM NaCl, 5.36 mM KCl, 0.2 mM MgSO4, 0.34 mM Na2HPO4, 0.44 mM KH2PO4, 4.17 mM NaHCO3, 1.26 mM CaCl2 and 5.6 mM glucose).
Preparation of cell suspensions containing mast cells from rat lungs and peritoneal fluids
Rat pulmonary and peritoneal cell suspensions were prepared according to the method of Ali and Pearce (1985) and Sueyasu et al. (1997), respectively. Briefly, rats were anesthetized with ether and exsanguinated by cutting the carotid arteries. The chest was opened, and the lung was isolated and immersed in ice-cold Krebs-Ringer solution. Tissues were minced with scissors, then washed over gauze. The suspensions were incubated at 37°C for 90 min with 0.05% collagenase (type-I, 62.5 collagen digestion units, Sigma) under gentle agitation, then filtered through 70-μm Nylon mesh (Cell Strainer, Becton Dickinson Labware, NJ, USA). The filtrates were centrifuged (100×g for 8 min at 25°C) and washed 4 times with Krebs-Ringer solution containing 0.1% bovine serum albumin (BSA). The cell suspensions were finally filtered through 70-μm nylon mesh. Peritoneal cells were prepared as follows: 20 ml of HBSS was injected into the peritoneal cavity. After gentle massage of the abdomen for 90 s, the intraperitoneal fluid was collected with a plastic pipette. The cell suspensions were centrifuged at 100×g for 6 min at 25°C, and washed with 4 times with 0.1% BSA-containing HBSS. The number of pulmonary or peritoneal mast cells in the cell suspensions was counted after staining with o-toluidine blue, and diluted with 0.1% BSA-containing Krebs-Ringer solution or HBSS to a concentration of 1.5×105 cells/ml. The percentages of mast cells were 5.6±0.4% (mean ± SEM, n=3) for pulmonary cell suspensions and 10.8±1.1% (n=3) for peritoneal cell suspensions. In a set of experiments where the role of mast cell cyclic AMP in the regulation of ioxaglate-induced histamine release was examined, mast cells were purified from rat peritoneal fluids by the density gradient centrifugation with Percoll (Amersham Biosciences, Uppsala, Sweden), according to the method of Enerback and Svensson (1980). Briefly, 90% isoosmotic Percoll was prepared by mixing 9 volumes of Percoll with 1 volume of a 10-fold concentration of HBSS, followed by further dilution with HBSS to 70% Percoll. The peritoneal cell suspension (3 ml) obtained as described above were gently applied over 8 ml of 70% Percoll included in a plastic tube (CORNING 430789), and centrifuged at 100×g for 15 min. The cell pellets precipitated at the bottom of the tube were collected and re-suspended in HBSS. The cell suspensions were washed twice with HBSS by centrifugation at 45×g for 5 min. Finally, the cells were suspended in HBSS to a concentration of 2.0×104 cells/ml. The numbers of mast cells in the resultant cell suspensions were counted after staining with neutral red. The cell viability was more than 97% as assessed by trypan blue staining. The purity of mast cells was 91.9±1.3% (mean±SEM, n=3).
Measurement of osmolarity in RCM or mannitol solution
RCM and mannitol were dissolved in Krebs-Ringer solution, and the osmolarity was measured by using an osmometer (OM 801, Vogel Gmbh, Giessen, Germany). Briefly, the RCM or mannitol solution was transferred to 300-μl Eppendorf tube, and the osmolarity was measured by immersing the detection head of the apparatus into the solution. Calibration was made by using 300 mOsm/kg NaCl standard solution (Vogel Gmbh).
Measurement of histamine release
The histamine release was measured by mixing the cell suspensions (0.5 ml) with 0.5 ml Krebs-Ringer solution (pulmonary mast cells) or HBSS (peritoneal mast cells) containing various concentrations of RCM, and incubating for 10 min at 37°C. In a set of experiments where the effect of the removal of intracellular Ca2+ on the histamine release was measured, pulmonary cells were incubated for 30 min with 20 μM BAPTA/AM in normal Krebs-Ringer solution to be taken up and hydrolyzed to BAPTA by cellular esterases, then washed with the same buffer and incubated for 10 min in the presence of ioxaglate. To determine the role of extracellular Ca2+ in the histamine release, after preincubation of pulmonary cells for 5 min, the incubation medium was replaced by Krebs-Ringer solution devoid of Ca2+ but containing 1 mM EGTA and cells were incubated for 10 min in the presence of ioxaglate. The reaction was stopped by centrifugation of the mixture at 800×g for 5 min at 4°C. The supernatant was transferred to another tube and perchloric acid was added to a final concentration of 0.4 M, since RCM interfered the assay for histamine in the neutral solution but not in the solution added with 0.4 M perchloric acid. The pellets were homogenized with 0.4 M perchloric acid. The histamine concentrations in the supernatant and cell homogenate were determined by ion-pair high performance liquid chromatography (HPLC) coupled with post-column fluorescent derivatization, as described previously (Itoh et al. 1992). The histamine release was expressed as the percentage of histamine in the supernatant to the total (supernatant + cells). It represents the gross value, i.e., the basal release was not subtracted.
Measurement of cAMP in cell suspensions and purified mast cells
The concentration of cAMP in peritoneal and pulmonary cell suspensions and purified peritoneal mast cells was measured according to the method of Weiss et al. (1985). Briefly, the pulmonary and peritoneal cells or purified mast cells were incubated for 10 min at 37°C (5% CO2/95% air mixture) with Krebs-Ringer solution or HBSS in the presence of 0.5 mM IBMX and various concentrations of ioxaglate. The reaction was terminated by the addition of 1 ml ice-cold 0.4 N perchloric acid. Cells were homogenized, and centrifuged at 10,000×g for 10 min. The cAMP content was determined by an enzymatic immunoassay using a cAMP enzyme immunoassay kit (Amersham).
Data are shown as the mean ± SEM. Comparisons of data among multiple groups were performed by one-way analysis of variance followed by Dunnett's test (StatView; Abacus Concepts, CA, USA). Statistical significance was defined as P<0.05.
Comparative effects of various RCM on histamine release from rat pulmonary cells
Lack of involvement of hyperosmolarity in RCM-induced mast cell histamine release
Comparison of the effect of ioxaglate on histamine release between rat pulmonary and peritoneal cells
Effect of cAMP on the ioxaglate-induced mast cell histamine release
Effect of ioxaglate on cAMP accumulation in pulmonary and peritoneal cells
Effects of the removal of extracellular or intracellular Ca2+ on ioxaglate-induced histamine release from pulmonary cells
In the present study, a variety of ionic as well as non-ionic RCM produced significant increases in histamine release from rat pulmonary cells, although the extent was less marked in non-ionic RCM. It has been reported that the osmolarity contributes largely to the RCM-induced histamine release from basophils as well as mast cells (Baxter et al. 1993; Genovese et al. 1996; Peachell and Morcos 1998). The rank order of osmolarities of various RCM solutions (150 mg iodine/ml) used in the present study was different from that in producing histamine release. Particularly, ioxaglate produced the most marked histamine release, while showing the second lowest osmolarity among seven different RCM tested. Thus, there was no significant correlation between the osmolarity and histamine release. Moreover, mannitol solution even at an osmolar concentration of 1,000 mOsm/kg caused no marked histamine release from rat pulmonary cells. Taken together, it is unlikely that osmolarity is substantially involved in the RCM-mediated mast cell histamine release. Our present findings are not consistent with the previous data showing a major contribution of osmolarity to RCM-induced histamine release. This may be due partly to the difference in concentration of mannitol between the present study and others. Although hyperosmolar mannitol solution is reported to cause histamine release from basophils and mast cells, it requires high concentrations (1,000–2,000 mM) to produce significant histamine release (Baxter et al. 1993; Peachell and Morcos 1998). In the present study, the concentration of mannitol was chosen to correspond to the osmolarity of RCM solution used for the measurement of histamine release. On the other hand, Genovese et al. (1996) reported that mannitol induces histamine release from human basophils but to a lesser extent from mast cells. Peachell and Morcos (1998) also demonstrated that mannitol (1,000 mM) produce histamine release to a different degree from human lung mast cells, skin mast cells and basophils. Thus, the difference in the type of mast cells or species differences may cause such a variation in the involvement of osmolarity in RCM-induced histamine release.
Ionic RCM generally possess a higher risk of causing adverse reactions than non-ionic materials (Salem et al. 1986b; Rodriguez et al. 2001). It has also been reported that the ionic RCM produce a more marked histamine release than non-ionic agents (Salem et al. 1986a; Ennis et al. 1982, 1991; Rodriguez et al. 2001), as observed in the present study (ioxaglate and amidotrizoate). Therefore, the ionization of the molecules may enhance the secretory action on mast cells.
In the present study, pulmonary mast cells were more sensitive to ioxaglate than peritoneal mast cells in liberating histamine. Our data were generally consistent with those reported by Amon et al. (1990), who showed that rat pulmonary mast cells are more sensitive to RCM than peritoneal mast cells but that the maximal release is less marked in pulmonary (13% of total histamine) than in peritoneal mast cells (30%). At present, we do not know the precise reason for the difference in the maximal response between their data and ours. The extremely low value (<2%) for the basal histamine release from pulmonary mast cells reported by them may cause such an inconsistency.
It has been reported that the incidence of adverse reactions related to airway functions is high after intravenous injection of RCM (Shehadi et al. 1975; Ansell et al. 1980; Eloy et al. 1991). We have already reported in rats that ionic RCM causes pulmonary vascular hyperpermeability and edema within 10 min after intravenous injection (Sendo et al. 1999, 2000; Tominaga et al. 2001). Moreover, we recently found that histamine H1 and H2 antagonists inhibit the ioxaglate-induced the extravasation in the rat lung determined 10 min after ioxaglate injection (Goromaru et al. 2002). In the present study, a marked histamine release was observed during 10-min of incubation with RCM. Therefore, the greater response to RCM in releasing histamine from pulmonary mast cells may be related to the adverse events associated with the pulmonary function.
The cellular mechanisms underlying the histamine release induced by RCM remain to be elucidated. It has been demonstrated that agents that elevate intracellular cAMP, including β-adrenergic agonists, forskolin and 3-isobutyl-1-methylxanthine, or cAMP analog DBcAMP, inhibit the histamine release from mast cells induced by various secretagogues such as anti-IgE, compound 48/80, neurotensin and concanavalin A (Shores and Mongar 1980; Rossie and Miller 1982; Undem et al. 1985; Shichijo et al. 1999). Likewise, in the present study, DBcAMP was found to inhibit the ioxaglate-induced histamine release from rat peritoneal mast cells. Moreover, the enhancement of endogenous cAMP content after stimulation of adenylate cyclase and inhibition of the degradation of the nucleotide significantly reduced the ioxaglate-induced histamine release. Interestingly, ioxaglate at concentrations that produced a marked histamine release significantly decreased the accumulation of mast cell cAMP. Taken together, it is suggested that a reduction of cellular cAMP levels contributes at least in part to the RCM-evoked histamine release from mast cells.
On the other hand, Peachell and Morcos (1998) have shown that amidotrizoate-induced histamine release from human mast cells and basophils is not dependent on extracellular Ca2+ but temperature-dependent. Consistent with their results, in the present study, the ioxaglate-induced histamine release was not affected by the removal of extracellular Ca2+. However, it was noteworthy that the removal of intracellular Ca2+ by chelation with BAPTA/AM significantly reduced the ioxaglate-induced mast cell histamine release. These findings suggest that RCM stimulate the release of Ca2+ from intracellular Ca2+ stores, which leads to the mast cell degranulation.
In conclusion, a variety of RCM stimulated histamine release from rat pulmonary cells, in which the action of ionic RCM was more pronounced than that of non-ionic materials. There was no significant correlation between the osmolarity of RCM solution and the histamine release. In addition, hyperosmotic mannitol solution did not increase histamine release. The histamine-releasing action of ioxaglate was more marked in pulmonary than in peritoneal cells. The ioxaglate-induced histamine release was reduced by DBcAMP or the elevation of endogenous cAMP content. Consistent with these findings, ioxaglate lowered the accumulation of mast cell cAMP. On the other hand, the removal of intracellular but not extracellular Ca2+ attenuated the ioxaglate-induced histamine release from pulmonary cells. Therefore, it is suggested that the reduction in cellular cAMP content and Ca2+ release from intracellular stores contribute largely to mast cell histamine release induced by RCM.
This research was supported in part by Grant-in-Aid for Scientific Research (C:13672390) from the Ministry of Education, Science, Sports and Culture, Japan.