Naunyn-Schmiedeberg's Arch Pharmacol (1993) 348:515-519 Naunyn-Schmiedeberg's Archivesof Pharmacology © Springer-Verlag 1993 Azelastine and allergen transduction signal in MC9 mast cells K. Fanous and R.P. Garay INSERM U2, Facult6 de M6dicine, 8 rue du G6n6ral Sarrail, F-94010 Cr6teil, France Received January 18, 1993/Accepted July 13, 1993 Summary. M C 9 mast cells, sensitized with monoclonal IgE antibody specific for 2,4-dinitrophenyl (DNP) group, were exposed to DNP-BSA and the pH and cytosolic calcium signals were recorded by using the fluorescent probes BCECF and Fura-2 respectively. DNP-BSA induced cell alkalinization was fully inhibited by azelastine with IC50 (1.6+0.5~tmol/1, mean_+SEM, n = 5) similar to that required to inhibit histamine release (1.4 ~tmol/1). Conversely, high azelastine concentrations ( > 100 ~tmol/ 1) were required to inhibit DNP-BSA-dependent cell calcium mobilization (IC50=200 ~tmol/1, n = 3). Amiloride, but not the H 1 histamine antagonist pyrilamine, was able to inhibit the DNP-BSA induced p H signal. In acidified mast cells, azelastine potently inhibited N a + : H + exchange activity (IC50 = 7.7 + 3.6 × 10- 6 M, mean + SEM, n = 3). Conversely, in mouse spleen lymphocytes azelastine was unable to inhibit the amiloride-sensitive p H signal induced by concanavalin A. In conclusion, the inhibition of histamine release by azelastine is not due to an interference with the cytosolic calcium signal. Conversely, azelastine potently antagonized the allergen-dependent Na + : H + exchange activation, suggesting an action on the protein kinase C signaling pathway. Key words: Azelastine - Mast cells - Cell membranes Antigen-induced mast cell activation is an essential event in allergic rhinitis and calcium mobilization seems to be involved in the biochemical mechanism by which bridging of IgE receptors triggers histamine secretion (for review see Metzger et al. 1986). Chand et al. (1983) found that azelastine antagonized the histamine release induced by the calcium ionophore A23187 in rat peritoneal mast cells (antagonism reversed by high concentrations of external calcium) and suggested that this drug acted via the inhibition of calcium mobilization. Direct evidence for such calcium antagonistic action was found by Nakamura et al. (1988) in guinea pig peritoneal macrophages, where azelastine inhibited the intracellular calcium mobilization induced by N-formylmethionyl-leucyl-phenylalanine (FMLP) and PAFacether. The above considerations pushed us to re-examine the calcium antagonistic action of azelastine in target cells for allergic rhinitis. Therefore, we sensitized MC 9 mast cells with a monoclonal IgE antibody specific for DNP. Then, we exposed the sensitized cells to D N P and followed calcium mobilization by using the fluorescent probe Fura-2. The low potency of azelastine on this model led us to investigate the protein kinase C signaling pathway (Nishizuka 1984). - Calcium - Histamine - N a + : H + exchanger - IgE Methods Introduction Azelastine is a new and very efficient antiallergic agent (Tasaka and Akagi 1979; Zechel et al. 1981) particularly in allergic rhinitis (for review see Szelenyi 1989). A m o n g other actions, azelastine has been reported to inhibit allergic induced histamine release from mast cells (Fields et al. 1981, Fisher and Schmutzler 1981; Katayama et al. 1981; Chand et al. 1983, 1985). Correspondence to." R. R Garay at the above address Mast cells. The MC9 line of mouse mast cells was obtained from American TypeCulture Collection (Rockville, Md., USA). This mast cell line was derived from fetal liver ceils of a (B6xA/J)F~ mouse. The cells have receptors for IgE and produce histamine and leukotrienes after sensitization and exposure to specific antigens (for details see Nabel et al. 1981). Mouse spleen lymphocytes. Spleens from C57 B6 black mice were removed and homogenizedfor 3 rain in a potter containing 12 ml of red cell lysis-buffer. The red cell lysis-buffer contained (mmol/1): NH4C1150 and Tris 17 (final pH adjusted to 7.2 with HC1). The suspension was filtered through nylon and diluted with a Hanks medium containing 5070of fetal calf serum. Then the cells were washed three times with the Hanks medium at room temperature. 516 One mouse spleen provided about 120x 10 6 cells. Of these about 60% were B lymphocytes and 40°7o T lymphocytes. Addition of trypan blue revealed that 95°70 of the cells excluded the dye. Cell culture. MC9 mouse mast cells were grown in conditioned medium derived from concanavalin A-stimulated splenocytes, according to the method of Razin et al. (1981) slightly modified. Briefly, spleen cells from Balb/c mice aged 2 months were suspended at a concentration of 5 × 106 cells/ml in Dulbecco's modified Eagle's medium (DME 1) supplemented with 2-mercaptoethanol (0.05 mmol/1), L-glutamine (2 mmol/1), heat-inactivated fetal bovine serum (4%) and concanavalin A (4 gg/ml). DME 2 medium contained L-arginine HC1 (32 mg/1), Lasparagine (36 mg/1), L-glutamine (2 mmol/1), folic acid (2 mg/1), nonessential amino acids (0.1 mmol/1), 2-mercaptoethanol (0.05 mmol/1) and heat inactivated fetal bovine serum (10%). The cell suspensions were incubated for 48h at 37°C in a 5% CO2-air mixture. Then, the supernatants were collected, centrifuged and filtered through a 0.22 gm Millipore filter. The filtered solutions were used as conditioned medium. MC9 mast cells were grown in flasks with a surface area o f 75 cm 2, containing a 1 : 1 mixture of conditioned medium and DME 2 (with 100 IU/ml penicillin and 100 gg/ml streptomycin). The flasks were incubated at 37 °C in an atmosphere containing 5 °7o CO 2. Cell concentration was maintained between 1.5 and 3 x 106 cells/ml. In actively growing cultures, the incubation medium was renewed every 2 - 3 days. Sensitization and antigen challenge. MC9 mast cells were sensitized during 1 h at 37 °C with mouse monoclonal IgE antibody specific for the 2,4-dinitrophenyl (DNP) group according to the method of White et al. (1985). Sensitized cells were washed three times and exposed to 50 ng/ml of DNP derivatives of bovine serum albumin (DNP-BSA). Measurement o f cell pH. Suspensions of MC9 mast cells or mouse spleen lymphocytes were centrifuged for 5min at 1500rpm and resuspended with Na-K Ringer medium containing 1 g/1 of bovine albumin. The Na-K Ringer's medium contained (mmol/1): NaCI 145, KC1 5, MgCI 2 1, CaC12 1, MOPS-Tris buffer (pH 7.4 at 37°C) 10 and glucose 5. An aliquot of the suspension was kept for using as control, unloaded cells. MC 9 mast cells or spleen lymphocytes were incubated with 1 gmol/1 of the fluorescent probe BCECF (in Ringer-albumin medium) for 15 min at 37 °C in the dark. Then the cell suspensions were centrifuged for 5 min at 1500 rpm, resuspended in Ringer-albumin medium and incubated for 15 min at 37 °C. The cell suspensions were centrifuged and resuspended in Ringer-albumin media. BCECF fluorescence was continuously recorded (period = 2 s) in a Shimadzu RF 5000 Spectrofluorimeter (Roucaire, V61izy-Villacoublay, France; excitation wavelength = 511 nm and emission = 525 nm). pH calibration was performed in the presence of 10 gmol/1 nigericin, in medium containing (mmol/1): KC1 135, NaC1 15, MgC12 1, CaC12 1, MOPS-Tris buffer 10 and glucose 5 (variable amounts of MOPS and Tris were added in order to obtain calibration media with the desired final pH). All measurements were performed in duplicates. Measurement o f cytosolic free calcium content. MC9 mast cells were washed and resuspended in Na-K Ringer-albumin medium containing 1 gmol/1 of Fura-2 AM. The cell suspensions were incubated for 15 min at 37°C in the dark. At the end of the incubation period, the cell suspensions were centrifuged for 5 min at 1500 rpm, resuspended in Ringer-albumin medium and incubated for 15 min at 37 °C. The cell suspensions were centrifuged and resuspended in Ringer-albumin medium. Fura-2 fluorescence was continuously recorded (period = 2 s) in a Shimadzu RF 5000 Spectrofluorimeter (excitation wavelengths = 355 and 380 nm and emission = 505 nm). All measurements were performed in duplicates. Measurement o f histamine release. In preliminar experiments with DNP-BSA-treated MC9 mast cells, we found that histamine concentrations were at the limit of detection by fluorimetry ( 1 0 - 5 0 nmol/1, measured by a fluorimetric method similar to that of Chand et al. (1983, 1985) in suspensions of 2 - 4 x 106 cells/tube). Therefore, we measured histamine release by using a 125I-histamine radioimmunoassay (Pharmacia methyl-histamine double antibody radioimmunoassay; Pharmacia Diagnostica AB, Uppsala, Sweden). Briefly, duplicates of sensitized MC9 mast cells were exposed to DNP-BSA for 30 min at 37 °C. Then, the cells were centrifuged and histamine contents were measured in the supernatants. All measurements were performed in duplicates. Figure 1 shows the displacement of bound 125I-histamine by cold histamine in this radioimmunoassay. It can be seen that the assay can detect histamine concentrations of 1 0 - 5 0 nmol/l (see also the section "action of compounds"). Measurement o f Na + : H + exchange activity in acidified M C 9 mast cells. The initial rate of amiloride-sensitive Li + uptake was taken as a marker of N a + : H + exchange activity (for details see Rosati et al. 1990). All measurements were performed in duplicate. MC9 mast cells were incubated for 30 min at 37 °C in a solution containing (mmol/1): NH4C125, NaC1 120, KC1 5, MgC12 1, CaC12 1, MOPS-Tris buffer (pH 7.4 at 37 °C) 10 and glucose 5. Then, the cells were washed with cold medium containing (mmol/1): N-methyl glucamine 145, KCI 5 and MOPS-Tris buffer (pH 7.4 at 4 °C) 10, and resuspended in Li + medium with and without 2 retool/1 of amiloride. The Li + medium contained (mmol/1): LiCl145, KC15, MgCI21, CaC121, MOPS-Tris buffer (pH7.4 at 37°C) 10, ouabain2, bumetanide 0.1 and glucose 5. The cells were incubated for 15 min at 37 °C (in control experiments we verified that internal Li + content was linearly increasing with time for at least 15 min). To stop the reaction, the cells were chilled at 4 ° C for 1 min and washed with cold medium containing (mmol/1): N-methyl glucamine 145, KC15 and MOPS-Tris buffer (pH 7.4 at 4 °C) 10. Then, 2 ml of Acationox 0.02% was added to the cell pellets. The tubes were frozen and thawed two times and sonicated. 0.5 ml of TCA 50% were added to the samples, which were then centrifuged for 10 min at 4000 rpm. The supernatants were removed and transferred into tubes for Li + analysis in an IL 457 atomic absorption spectrophotometer (Instrumentation Laboratory Inc., Wilmington, Mass., USA) and for K + analysis in an Eppendorf flame photometer (Eppendorf Ger~tebau Netheler, Hamburg, Germany). Li + and K + standards were prepared by dilution of commercial standards (Merck, Darmstadt, Germany) in water. Internal Li + contents were divided by the internal K + contents (Li + contents never exceeded 40070 of K + contents). Amiloride-sensitive Li + uptake was calculated from the difference in Li + contents between tubes with and without amiloride divided by the incubation time. Action o f compounds. Azelastine was provided by ASTA Medica (Frankfurt, Germany). Pyrilamine was obtained from SIGMA (distributed through Coger, Paris, France). The compounds were added from freshly prepared, concentrated stock solutions in DMSO. Azelastine was evaluated in a manner so as to generate concentration-response curves. The cells were preincubated for 5 to 15 rain with each azelastine concentration prior to addition of DNP or Concanavalin A. In control experiments we verified that, in the range of concentrations used, azelastine and pyrilamine were not able to interfere by themselves with the Fura-2 or the BCECF signals. Conversely, Fig. I shows that azelastine was able to displace bound ~25I-histamine in the radioimmunoassay. Therefore, histamine contents released by azelastine were subtracted from the measured total histamine contents. Results A l l e r g e n i n d u c e d cell c a l c i u m m o b i l i z a t i o n M C 9 m a s t cells w e r e s e n s i t i z e d w i t h I g E ( a n t i - D N P ) as described in Methods. Basal cytosolic free calcium cont e n t w a s 72_+ 8 n m o l / 1 ( m e a n + S E M , n = 9). E x p o s u r e t o DNP-BSA induced a rapid increase in cytosolic free calciu m levels ( 6 0 - 1 0 0 ° 7 0 o f b a s a l l e v e l s ) t h a t l a s t e d f o r a b o u t 4 min. 517 lOO Table 1. Lack of action of pyrilamine on allergen (DNP-BSA)-stimulated Na+: H + exchange activity in MC9 mast cells Bound e 80 125i.Histamin [% of control] ~ ~ [ ] ~ . r 60 4O 20 histamine ~ O I I -9 -8 ~ I I I -7 -6 -5 I I Displacement of bound 125I-histamine by cold histamine and by azelastine (Pharmacia methyl-histamine double antibody radioimmunoassay). Values are given as mean_+ SEM (n = 3). The assay can detect histamine contents in the range of those released by allergenstimulated MC 9 mast cells ( 1 0 - 5 0 nmol/1). High azelastine concentrations displaced bound lzsI-histamine, thus interfering with the assay Figure 2 shows concentration response curves for the action of azelastine on the DNP-BSA-dependent calcium signal. It can be seen that high concentrations of the c o m p o u n d ( > 100 gmol/1) were required to inhibit the calcium signal (ICs0--200 gmol/1, n = 4). Allergen induced cell alkalinization Basal cytosolic pH in sensitized MC 9 mast cells was between 6.90 and 7.05. Exposure to DNP-BSA induced a rapid increase in cytosolic pH, with initial rate of 0.116+_0.011 pH units/min (mean+_SEM, n = 8). Figure 2 shows the action o f azelastine on the initial rate of DNP-BSA-dependent cell alkalinization. It can be seen that the allergen induced pH signal was highly sensitive to azelastine (ICs0 = 1.6_+0.5 gmol/1, mean_+ SEM, n = 5). 120 [% of control] a l k 80 60 I n ~ DNP DNP + Amiloride 2 mmol/1 D N P + P y r i l a m i n e 100 gmol 0.129 _+0.009 0.005 _+0.003 ** 0.121_+0.005 n.s. experiments I I Fig. 1. ~ Alkalinization Values in this table are given as mean + SEM of 5 ** P < 0.0001 n.s., not significant (Student's t-test) -4 -3 Compound concentration log [mol/l] DNP-dependent phenomena 100 rate (pH units/rain) Condition histamine Table 1 shows that DNP-BSA-dependent cell alkalinization was fully inhibited by amiloride (2 retool/l), but not by the Ht antagonist, pyrilamine. Allergen induced histamine release Figure 2 shows the inhibition by azelastine of the histamine release from sensitized mast cells exposed to DNPBSA. It can be seen that the dose-response curve (ICs0 = 1.4_+0.5 ~mol/1, m e a n + S E M , n = 3) was very similar to that obtained for inhibition of the pH signal. Na + : H + exchange activity in acidified mast cells Na + : H + exchange activity in M C 9 mast cells was stimulated by cytosolic acidification (see Methods). Figure 3 shows concentration response curves for the action of azelastine on N a + : H + exchange activity. It can be seen that the mast cell Na + : H + exchanger was very sensitive to azelastine (ICs0 = 7.7 + 3.6 gmol/1, mean_+ SEM, n - - 3). Mouse spleen lymphocytes Concanavalin A (10 gg/ml) induced an increase in the cytosolic pH of mouse spleen lymphocytes, with initial alkalinization rate of 0.025 - 0.045 pH units/min. Table 2 shows that this pH change was sensitive to amiloride (2 mmol/1), confirming that it was due to Na + : H + exchange activation (see also Grinstein et al. 1987). Moreover, Table 2 shows that azelastine was unable to inhibit concanavalin A-dependent lymphocyte alkalinization. pH s i g l n a - - ~ l ~ ~ 10o 4o1 Na+:H+ exchange activity 80 2O [% of control] 0 , -8 i -7 , i -6 , .L ,I"~ , 6O i -5 -4 -3 Azelastine concentration log [mol/t] Fig. 2. Azelastine and allergen transduction signals in mast cells. High azelastine concentrations ( > 100 gmol/1) were required to inhibit DNPBSA-dependent cell calcium mobilization in sensitized MC9 mast cells (ICs0 ~ 200 ~tmol/1, n = 4). Conversely, cytosolic alkalinization was very sensitive to azelastine (ICs0 = 1.6_+0.5 I~mol/1, n = 5). The inhibition of the pH signal was superposable to the inhibition of histamine release. Basal cytosolic free calcium content was between 50 and 95 nmol/l and basal cytosolic pH was between 6.90 and 7.05. DNP-BSA induced a rapid 60-100070 increase in cytosolic free calcium levels (that lasted for about 4 min) and a cell alkalinization rate of 0.116_+0.011 pH units/min. Histamine release was 3 0 - 6 0 nmol/1/h 20 0 -7 -6 -5 Azelastineconcentration Fig. 3. -4 log [tool/l] Inhibition by azelastine of Na + : H + exchange activity in acidified MC9 mast ceils. Values represent m e a n + S E M (n = 3). It can be seen that azelastine was able to potently inhibit the mast cell Na + : H + exchange (IC50 = 7.7_+ 3.6 gmol/1) 518 Table 2. Lack of action of azelastine on concanavalin A induced lymphocyte alkalinization Condition Alkalinization rate (pH units/min) Concanavalin A 10 gg/ml Con A+Amiloride 2 mmol/1 Con A+Azelastine 100gmol/1 0.031 + 0.002 0.002_+0.00i** 0.036+0.003 n.s. Values in this table are given as mean + SEM of 3 experiments ** P < 0.0001 n.s., not significant (Student's t-test) Discussion Azelastine was a poor inhibitor of the cell calcium mobilization elicited by DNP-BSA in MC9 mast cells. Indeed, inhibition of the allergen-induced cytosolic calcium signal required azelastine concentrations two order of magnitude higher (ICs0 = 200 gmol/1) than those required to inhibit histamine release in the same cell preparation (Fig. 2), or in rat peritoneal mast cells (ICs0 = 4 - 5 gmol/1, Chand et al. 1983, 1985). Azelastine was also less potent to inhibit calcium mobilization in MC9 mast cells than to inhibit the calcium signal induced by PAF-acether in guinea pig peritoneal macrophages (ICs0 = 16 ~mol/1; Nakamura et al. 1988). Penner (1988) and others have shown that multiple signaling pathways can control the stimulus-secretion coupling in mast cells. In particular, calcium is not necessary for GTP[g-S]-induced secretion and increased cytosolic free calcium per se is not a sufficient stimulus for secretion under physiological conditions. Protein kinase C is known to stimulate Na + : H + exchange activity and increase the calcium sensitivity of a number of cellular processes (Nishizuka 1984; Grinstein et al. 1987; Grinstein and Dixon 1989). In sensitized mast cells, DNP stimulates protein kinase C activity (White et al. 1985). Moreover, phorbol esters, which activate protein kinase C, were unable to induce histamine release per se, but acted synergistically with additionally applied ionophores (Katakami et al. 1984; Heiman and Crews 1985). Therefore, protein kinase C has been suggested to increase the calcium sensitivity of the secretory process in mast cells. The rather negative results on cytosolic calcium with azelastine suggested that this compound was acting distally with respect to the calcium signal. Therefore, we explored the action of azelastine on the protein kinase C signaling pathway. Activation of protein kinase C in a number of cell systems is associated to the activation of the amiloridesensitive N a + : H + exchanger, which in turns alkalinizes the cytosol (Grinstein et al. 1987; Grinstein and Dixon 1989). This was confirmed in MC9 mast cells, where DNP-BSA induced cell alkalinization, a phenomena blocked by amiloride. Azelastine potently inhibited the allergen-induced pH signal, with ICs0 (1.6 gmol/1) similar to that required to inhibit histamine release (Fig. 2), thus suggesting an interference on the protein kinase C signaling pathway. Middleton et al. (1989) have shown that azelastine was unable to modify rat brain protein kinase C activity. Therefore, we investigated if azelastine was acting more distally, by inhibiting the pathway at the level of the Na + : H + exchanger. Therefore, we stimulated the activity of the exchanger by a protein kinase C-independent procedure, i.e., by increasing the internal H + concentration. Azelastine inhibited Na + : H + exchange activity under those conditions, further suggesting that it was acting distally with respect to protein kinase C. The inhibitory action of azelastine on the mast cell N a + : H + exchanger was intringuing because this drug has a very different pharmacological profile than amiloride. Therefore we tested azelastine in a well characterized immunological model of N a + : H + exchange activation, i.e., the cytosolic alkalinization induced by Concanavalin A in lymphocytes (Grinstein et al. 1987). Azelastine was unable to block this phenomena, clearly showing that, unlike amiloride, it was not a direct inhibitor of the Na + : H + exchanger. The above results suggested that azelastine was distally acting on the protein kinase C signaling pathway, via a receptor protein specific for mast cells and coupled to the N a + : H + exchanger. On the other hand, it is well known that azelastine is an H1 histamine receptor antagonist (reviewed in Szelenyi 1989) and that some Ht antagonists are able to inhibit histamine release (for review see Foreman and Rihoux 1992). However, the DNP-BSA induced mast cell alkalinization was insensitive to the Hi antagonist pyrilamine, thus suggesting that the above azelastine receptor protein was not an H1 histamine receptor protein. A causal and sequential link between the antagonism of Na + : H + exchange activation and the inhibition of histamine release by azelastine is unlikely because, among others, phorbol esters, which activate protein kinase C, were found to be unable to induce histamine release per se in mast cells (Katakami et al. 1984; Heiman and Crews 1985). Thus, further experiments are required in order to clarify the role of the protein kinase C / N a + : H + exchange signaling pathway in the inhibition of histamine release by azelastine in mast cells. In conclusion, the inhibition of histamine release by azelastine is not due to an interference with the cytosolic calcium signal. Conversely, azelastine is a potent inhibitor of the protein kinase C signaling pathway in allergen stimulated mast cells. This compound seems to act distally on this pathway, on a specific mast cell receptor, which is different from the Hi-histamine receptor and is coupled to the Na + : H + exchanger. A potential role for the protein kinase C signaling pathway in the mechanism of action of azelastine deserves further investigation. Acknowledgments. We are greatly indebted to M. Baehre, M. Gudenzi and I. Szelenyi (ASTA Medica, Frankfurt, Germany) for interest and discussion, to C. Carnaud (INSERM U 25, H6pital Necker, Paris, France) for the preparation of mouse spleen lymphocytes, to B. 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