Guinea pig eosinophil major basic protein (GMBP) as a potent histamine releaser. (I) Histamine releasing activity of GMBP and its chemical structure

When isolated guinea pig major basic protein (MBP) was applied to SDS-PAGE, it exhibited a single band with an apparent molecular weight of 11000. When rat peritoneal mast cells were exposed to MBP at concentrations higher than 0.3 μM, significant histamine release was elicited both in the presence and absence of extracellular Ca²⁺. The histamine releasing activity of MBP was more pronounced in a Ca-free medium than in the presence of Ca²⁺. When MBP was applied on reverse phase HPLC, two distinct peaks were observed. These two proteins exhibited identical molecular weights and pI values. Their amino acid compositions were very similar. It was therefore assumed that guinea pig MBP is composed of two subtypes, namely GMBP₁ and GMBP₂, and that both have similar histamine releasing activities. MBP elicited not only⁴⁵Ca uptake from the extracellular medium but also induced Ca²⁺ release from intracellular Ca stores.

     

Guinea pig eosinophil major basic protein as a potent histamine releaser. (II) Structure-activity relationships of GMBP1 and GMBP2

Based on the amino acid sequences of GMBP₁ and GMBP₂, several oligopeptides were synthesized and their histamine releasing activity from rat peritoneal mast cells was compared. Basic peptides having more than two amino acids in succession were effective in releasing histamine both in the presence and in the absence of extracellular Ca²⁺, although neutral and acidic peptides were not active. Histamine release caused by the active peptides was more pronounced in the absence of extracellular Ca²⁺ than in the presence of Ca²⁺. From the structure-activity relationships, it is suggested that the histamine releasing activity of these oligopeptides may be dependent not only on the basicity but also on the arrangement of basic amino acids residues, and that hydrophobic amino acids are probably also involved in histamine release.

     

Guinea pig eosinophil major basic protein as a potent histamine releaser. (II) Structure-activity relationships of GMBP1 and GMBP2

Based on the amino acid sequences of GMBP₁ and GMBP₂, several oligopeptides were synthesized and their histamine releasing activity from rat peritoneal mast cells was compared. Basic peptides having more than two amino acids in succession were effective in releasing histamine both in the presence and in the absence of extracellular Ca²⁺, although neutral and acidic peptides were not active. Histamine release caused by the active peptides was more pronounced in the absence of extracellular Ca²⁺ than in the presence of Ca²⁺. From the structure-activity relationships, it is suggested that the histamine releasing activity of these oligopeptides may be dependent not only on the basicity but also on the arrangement of basic amino acids residues, and that hydrophobic amino acids are probably also involved in histamine release.     

Ionic regulation of human basophil releasability. I. Inhibitory effect of copper

The effects of copper (CuSO₄ and CuCl₂) onin vitro histamine release from human basophils stimulated by anti-IgE and Ca²⁺ ionophore A23187 were evaluated. Both CuSO₄ and CuCl₂ caused a dose-related inhibition of histamine release, which was more pronounced on anti-IgE-than on Ca²⁺ ionophore-induced histamine release. The concentration which produced 50% inhibition of anti-IgE-induced histamine release was 1.3 M for CuSO₄ and 1.5 M for CuCl₂; the maximal inhibition of Ca²⁺ ionophore-induced histamine release was 33% for CuCl₂ (4 M) and 51% for CuSO₄ (16 M). The inhibitory effect on anti-IgE-induced histamine release persisted also when extracellular Cu²⁺ was removed by cell washing before IgE-induced histamine release persisted also when extracellular Cu²⁺ was removed by cell washing before stimulation, whereas no inhibition of Ca²⁺ ionophore-induced histamine release was found when extracellular Cu²⁺ was removed. The activity of Cu²⁺ was independent of any effects of deuterium oxide and colchicine, two agents known to interact with microtubules. Increased extracellular Ca²⁺ concentrations reduced the inhibitory effect of CuCl₂ on Ca²⁺ ionophore-induced histamine release, and Schild plot analysis demonstrated that Cu²⁺ ions are competitive antagonists of Ca²⁺ ions.These results indicate that Cu²⁺ ions in the micromolar range down-regulate anti-IgE- and Ca²⁺ ionophore-induced histamine release. Since Cu²⁺ concentration in human plasma is in the micromolar range (30 M with 10–30% of free Cu²⁺), it is conceivable that Cu²⁺ ions contribute to thein vivo regulation of histamine release from human basophils.     

Caffeine and histamine-induced oscillations of K(Ca) current in single smooth muscle cells of rabbit cerebral artery

In the present experiment, we characterized the intracellular Ca²⁺ oscillations induced by caffeine (1 mM) or histamine (1–3 M) in voltage-clamped single smooth muscle cells of rabbit cerebral (basilar) artery. Superfusion of caffeine or histamine induced periodic oscillations of large whole-cell K⁺ current with fairly uniform amplitudes and intervals. The oscillatory K⁺ current was abolished by inclusion of ethylenebis(oxonitrilo)tetraacetate (EGTA, 5 mM) in the pipette solution. Caffeine- and histamine-induced periodic activation of the large-conductance Ca²⁺-activated K⁺ [K_(Ca)] channel was recorded in the cell-attached patch mode. These results suggest that the oscillations of K⁺ current are carried by the K_(Ca) channel and reflect the oscillations of intracellular Ca²⁺ concentration ([Ca²⁺]_i). Ryanodine (1–10 M) abolished both caffeine- and histamine-induced oscillations. Caffeine- induced oscillations were abolished by the sarcoplasmic reticulum Ca²⁺-adenosine 5-triphosphatase (Ca²⁺-ATPase) inhibitor, cyclopiazonic acid (10 M), and a high concentration of caffeine (10 mM). Inclusion of heparin (3 mg/ml) in the pipette solution blocked histamine-induced oscillations, but did not block caffeine-induced oscillations. By the removal of extracellular Ca²⁺, but not by the addition of verapamil and Cd²⁺, the caffeine-induced oscillations were abolished. Increasing Ca²⁺ influx rate increased the frequencies of caffeine-induced oscillations. Spontaneous oscillations were also observed in cells that were not superfused with agonists, and had similar characteristics to the caffeine-induced oscillations. From the above results, it is concluded, that in smooth muscle cells of the rabbit cerebral (basilar) artery, ryanodine-sensitive Ca²⁺-induced Ca²⁺ release pools play key roles in the generation of caffeine- and histamine-induced intracellular Ca²⁺ oscillations.     

A postsynaptic inhibitory histamine H2-receptor in the mouse isolated vas deferens

The effect of histamine on adrenergic neurotransmission in the mouse isolated vas deferens preparation was investigated. Concentrations of histamine ranging from 0.2 to 650 M depressed, in a dose-related manner, not only the contractile response elicited by field stimulation but also the response caused by the addition of exogenous noradrenaline and acetylcholine. However, the release of [³H]-NA evoked by field stimulation or by high K⁺ remained unchanged in the presence of these concentrations of histamine. The inhibitory effect of histamine on the contractile responses caused by various stimuli was reduced or completely antagonized by cimetidine, a histamine H₂-receptor antagonist but not by mepyramine, a conventional antihistamine. The inhibitory effect of histamine was found to be inversely proportional to both the Ca²⁺ concentration in the bathing medium and to the frequency of field stimulation. Further, the inhibitory effect of histamine was markedly reduced when Mg²⁺ was omitted from the bathing medium. It is concluded that the mouse vas deferens preparation contains a post-junctional inhibitory H₂-receptor. The stimulation of H₂-receptors by histamine inhibits the contractile response of the vas deferens, possibly by decreasing the availability of Ca²⁺ required for contraction by depressing the influx of Ca²⁺.     

Antiallergic Effect of Epinastine (WAL 801 CL) on Immediate Hypersensitivity Reactions: (I) Elucidation of the Mechanism for Histamine Release Inhibition

Abstract

Epinastine caused an inhibition of histamine release from rat peritoneal mast cells induced by both antigen-antibody reaction and compound 48/80. Epinastine was similarly effective in inhibiting compound 48/80-induced histamine release not only from isolated rat peritoneal mast cells but also from rat mesenterial pieces. Also, histamine release from lung pieces obtained from actively sensitized guinea pigs after exposure to antigen challenge was markedly inhibited by epinastine. The drug was effective in inhibiting not only Ca²⁺ uptake into lung mast cells in actively sensitized guinea pigs but also Ca²⁺ release from the intracellular Ca store of rat peritoneal mast cells exposed to both compound 48/80 and substance P. No significant changes were observed in phosphodiesterase activity in rat peritoneal mast cells treated with epinastine, while adenylate cyclase activity was augmented by epinastine. Epinastine has no inhibitory effect on histamine release induced by Ca²⁺ or IP3 from permeabilized mast cells. However, the drug significantly and dose-dependently suppressed calmodulin activity suggesting that histamine release inhibition due to epinastine may be partly attributable to Ca²⁺-calmodulin dependent process(es). The drug caused no visible changes in thermodynamic behavior of lipids, either in order parameter or in differential scanning calorimetry, indicating that the drug has no influence on membrane fluidity.     

Ionic regulation of human basophil releasability. II. Non-releasing basophils are converted into releasing basophils in a low-Na+ medium

The effects of different extracellular Na⁺ and CV²⁺ concentrations on histamine release from human basophils were investigated. Isosmotic replacement of extracellular Na ⁺ either with choline ⁺, a non-permeant Na ⁺ analogue, or glucose significantly increased spontaneous and anti-IgE-induced histamine release. Basophils from 12 of 49 normal subjects, which were found not to release histamine upon challenge with an optimal dose of anti-IgE in a 135 mM NaCl buffer, were converted into releasing basophils when stimulation with anti-IgE was performed in a low-Na⁺ medium. The increase in Na ⁺ concentration in the extracellular medium was accompanied by a reduction in the magnitude of basophil response to anti-IgE, which was significantly more pronounced in non-releasers than in releasers (per cent inhibition by 70 mM NaCl 75.5 + 3.2 vs 43.5 + 9.0, P < 0.01). At higher Na⁺ concentrations a progressive and almost complete abrogation of histamine release was observed in non-releasers, but not in releasers (maximal per cent inhibition at 140 mM NaCl 97.3+1.3 vs 50.4 + 8.6). The Na⁺/H⁺ exchanger monensin had a dose-dependent inhibitory effect on anti-IgE-induced histamine release, and the concentration inhibiting 50% of histamine release was l.5 × 10^?7M. When basophils were challenged in the presence of different Na⁺ and C²⁺ concentrations, it was shown that the two cations have antagonistic effects, which is to say that they down-regulate and upregulate histamine release, respectively. Moreover, the requirement of extracellular Ca²⁺ was lowered in a low-Na⁺ medium. These results suggest that Na⁺ and Ca⁺ ions contribute with opposite effects to the modulation of basophil response to anti-IgE and that non-releasing basophils are converted into releasing basophils in a low-Na ⁺ medium.     

Contribution of Ca2+ inflow to quantal,phasic transmitter release from nerve terminals of frog muscle

Evoked quantal release from sections of frog endplates contained in an extracellular electrode has been investigated with Ca²⁺ inflow prevented by superfusing the extracellular space with a Ringer's solution containing Cd _e ²⁺ or with an intracellular, EGTA-buffered solution containing less than 0.1 M Ca _e ²⁺ . Pulse application and recording were by a perfused macro-patch-clamp electrode. The muscle outside the electrode (bath) was superfused with Ringer's solutions containing Cd _b ²⁺ to block Ca²⁺ inflow and normal (1.8mM) or elevated (10 mM) Ca _b ²⁺ . The depolarization level of the terminal during current pulses that generated maximal Ca²⁺ inflow was used as unit relative depolarization. Starting from a threshold above 0.5 relative depolarization, the average release increased by a factor of about 1000 with increasing depolarization, reaching a plateau above 1.2 relative depolarization. The high level of plateau release extended to at least a relative depolarization of 4, i.e. to about +200 mV. When Ca²⁺ inflow was prevented in the section of the terminal within the electrode, release was depressed strongly for relative depolarizations around 1, i.e. at potentials at which Ca²⁺ inflow is high. However, for large depolarizations (>1.5 relative units), the depression of release by block of Ca²⁺ inflow was weak or absent. The time course of release, measured in distributions of the delays of quanta after the depolarizing pulse, was unaffected by block of Ca²⁺ inflow. If the extra-electrode superfusion of Ca _b ²⁺ of the muscle was elevated to 10 mM and Cd _b ²⁺ was 0.1 mM or 0.5 mM, perfusion of the electrode with solutions below 0.1M Ca _e ²⁺ raised the average release paradoxically. With 0.5 mM Cd _b ²⁺ this paradoxical increase of release was, on average, 4-fold at 6 °C, and 19-fold at 16 °C. Quantal endplate currents recorded in less than 0.1 M Ca _e ²⁺ had slightly increased amplitudes, and decay time constants were prolonged by about 50%. The results are interpreted to support the Ca²⁺/voltage theory of release, which proposes that evoked, phasic release is controlled by both intracellular Ca²⁺ concentration and another membrane-depolarization-related factor. If the resting intracellular Ca²⁺ concentration is sufficiently high, large depolarizations can elicit release independent of the presence or absence of Ca²⁺ inflow.     

The effect of Ro 21-7634 on the antigen-induced production of bronchoconstrictive arachidonic acid metabolites in the guinea pig lung

Ro 21-7634 has previously been shown to inhibit histamine and SRS-A release from actively-sensitized guinea pig lung fragments upon antigen challenge. In the studies described herein, it was observed that Ro 21-7634 does not decrease SRS-A release but instead acts to inhibit the synthesis of this mediator. This was confirmed by studying SRS-A synthesisin vitro in rat peritoneal cells after challenge with ionophore A23187. In the peritoneal cell system, Ro 21-7634 exhibited an IC₅₀ of 500 M, in comparison with 5,8,11,14-eicosatetraynoic acid, phenidone and BW755C (IC₅₀'s of 2, 100, and 100 M, respectively). When studied at 10^–4 and 10^–3 M in perfused guinea pig lung, Ro 21-7634 inhibited antigen-induced thromboxane A₂ production by 68 and 96%, respectively. In this system, antigen is believed to induce thromboxane A₂ production through the release of histamine and SRS-A from lung tissue. These mediators then interact at receptor sites in the lung parenchyma to induce thromboxane A₂ synthesis. Ro 21-7634 could thus be inhibiting thromboxane A₂ production by preventing the release of histamine and synthesis of SRS-A in the perfused lung system. Such a mechanism is suggested by the fact that although Ro 21-7634 was effective in inhibiting antigen-induced thromboxane production, it was ineffective in inhibiting thromboxane A₂ production induced in the guinea pig lung system by the direct perfusion of histamine or SRS-A through the lung.     

Bradykinin and inositol 1,4,5-trisphosphate-stimulated calcium release from intracellular stores in cultured bovine endothelial cells

The relative importance of intracellular and extracellular Ca²⁺ in the release of endothelium-derived relaxing factor (EDRF) and the mechanisms involved in the release of intracellular Ca²⁺ were investigated in cultured bovine endothelial cells. The release of EDRF by bradykinin, determined by bioassay, was dose-dependent showing an EC₅₀ of 4×10^–10 M. The bradykinin-induced EDRF release from endothelial cells was maintained in the presence of extracellular Ca²⁺. However, in the absence of external Ca²⁺, bradykinin-induced EDRF release was both attenuated and transient. In cells loaded to isotopic equilibrium with⁴⁵Ca, bradykinin increased the⁴⁵Ca efflux into both calcium-containing and calcium-free solutions, with an EC₅₀ for the increase in⁴⁵Ca efflux induced by bradykinin of 1.3×10^–9 M. The involvement of an intracellular Ca²⁺ store and the participation of a second messenger in its release were investigated in saponin-permeabilized endothelial cells. In saponin-permeabilized cells, ATP-sensitive calcium uptake was Ca²⁺,Mg²⁺-ATPase-dependent. The ATP-sensitive uptake of calcium at different free Ca²⁺ concentrations showed at least two compartments involved in the uptake of Ca²⁺. The⁴⁵Ca uptake into the compartment with the lowest affinity and highest capacity could be inhibited by sodium azide, suggesting that this uptake was into mitochondria. The majority of the⁴⁵Ca uptake into the azide-insensitive store could be released by inositol-1,4,5-trisphosphate (IP₃). The IP₃-induced release was not affected by apyrase or exogenous GTP. The EC₅₀ for the release of Ca²⁺ by IP₃ was 1.0 M and was unaffected by an inhibitor of IP₃ breakdown (2,3-diphosphoglyceric acid). The results suggest that the release of EDRF is dependent on extracellular Ca²⁺ influx and the release of intracellular Ca²⁺. The release of calcium from one of the high affinity intracellular Ca²⁺ stores is mediated by the intracellular second messenger, IP₃.     

Studies of the mechanism of histamine release from lung tissue in vitro by cotton dust extracts

Using an in vitro incubation technique with human and pig lung, the histamine releasing activity of cotton dust extracts was examined. For both species, histamine release increased with increasing concentration of cotton dust in the medium. The maximum release obtained was about 26 and 15% for human and pig lung respectively at a concentration of 150 mg dust/ml and activity was optimal at 37°C. For human lung, there was a pH optimum for release at 7.4 but with pig lung this pH optimum was observed only at low concentrations of dust. Histamine release increased with increasing pH when pig lung was incubated with a high dust concentration (83 mg/ml). A Ca²⁺/Mg²⁺-free medium inhibited the release of histamine by cotton dust from pig lung. In the absence of glucose, potassium cyanide inhibited the release of histamine by dust from both human and pig lung. However, in the latter species, cyanide potentiated the effect of high dust concentrations. In pig lung, the histamine releasing activity of cotton dust was inhibited by 2,4-dinitrophenol (in a glucose-free medium), ninhydrin and N-ethylmaleimide. It is concluded that the release of histamine from lung tissue in vitro by cotton dust is due largely to an energy-dependent mechanism. However, at a dust concentration of 83 mg/ml, and particularly at an alkaline pH an energy-independent mechanism may also operate in pig lung. The results are discussed in relation to the occupational pulmonary disease, byssinosis.     

Recognition of the ‘calcium paradox’ and the effects of verapamil and gallopamil in human adenoidal mast cells

Human mast cells from adenoids show when resuspended in medium containing 10^–3 M CaCl₂ after their temporary exposure to Ca²⁺-free saline for about 20 min an irreversible reduction of responsiveness to a variety of stimuli: The histamine release induced by concanavalin A or ionophore A 23187 is only 30–50% of the one obtained in cells which were kept in 10^–3 M Ca²⁺ throughout the experiment. This phenomenon called calcium paradox can be almost entirely avoided if the cells are temporarily exposed to 10^–4 M Ca²⁺ instead of Ca²⁺-free saline. Number yields, average histamine contents of mast cells and the rate of the spontaneous histamine release are not affected by the transitory lack of Ca²⁺, nor is the histamine release enhancing effect of adenosine.At 10^–3 M Ca²⁺ concentration the calcium antagonists verapamil or gallopamil cause a significant inhibition of the Con A-induced histamine release only at concentrations much higher (10^–4 M) than those effective in smooth muscle preparations. The actions of both calcium antagonists were not affected by the presence of added extracellular adenosine.To whom requests for reprints should be addressed.     

Secretagogue effects on intracellular calcium in pancreatic duct cells

Regulation of intracellular free calcium ([Ca²⁺]_i) in single epithelial duct cells of isolated rat and guinea pig pancreatic interlobular ducts by secretin, carbachol and cholecystokinin was studied by microspectrofluorometry using the Ca²⁺-sensitive, fluorescent probe Fura-2. Rat and guinea pig duct cells exhibited mean resting [Ca²⁺]_i of 84 nM and 61 nM, respectively, which increased by 50%–100% in response to carbachol stimulation, thus demonstrating the presence of physiologically responsive cholinergic receptors in pancreatic ducts of both species. The carbachol-induced increase in [Ca²⁺]_i involved both mobilization of Ca²⁺ from intracellular stores and stimulation of influx of extracellular Ca²⁺. In contrast, neither cholecystokinin nor secretin showed reproducible or sizeable increses in [Ca²⁺]_i. Both rat and guinea pig duct cells showed considerable resting Ca²⁺ permeability. Lowering or raising the extracellular [Ca²⁺]_i led, respectively, to a decrease or increase in the resting [Ca²⁺]_i. Application of Mn²⁺ resulted in a quenching of the fluorescence signal indicating its entry into the cell. The resting Ca²⁺ and Mn²⁺ permeability could be blocked by La³⁺ suggesting that it is mediated by a Ca²⁺ channel.     

Mechanisms of vancomycin-induced histamine release from rat peritoneal mast cells

The mechanisms of vancomycin (VCM)-induced histamine release were studied with rat peritoneal mast cells. VCM (>1×10^–3 M) released histamine from the isolated mast cells in a dose-dependent and noncytotoxic manner. In the absence of extracellular Ca²⁺, the histamine release was reduced markedly. When the intracellular Ca²⁺ was depleted, it was further decreased. The Fura-2-loaded single mast cells showed a biphasic increase in the intracellular Ca²⁺ concentration ([Ca²⁺]_i) by VCM: the first transient and the second sustained components. In the absence of extracellular Ca²⁺, the transient component was unchanged, while the sustained component was eliminated completely. The IP₃ content in the mast cells increased within 10 s after the application of VCM. These results suggest that VCM releases histamine from rat peritoneal mast cells via an IP₃ production and increase in [Ca²⁺]_i.     

Physiological concentrations of zinc inhibit the release of histamine from human basophils and lung mast cells

We have previously shown that physiological concentrations of zinc (7×10^–6 M) inhibit the release of histamine from human basophil leukocytes (Maroneet al., J. Pharmacol. Exp. Ther. 217: 292, 1981). In these experiments we compared the effect of zinc chloride on the release of chemical mediators from human basophils and mast cells isolated from human lung. Preincubation (5 min, 37°C) of human basophils and lung mast cells with zinc chloride (10^–6–3×10^–5 M) caused dose-related inhibition of histamine and peptide leukotriene C₄ (LTC₄) release induced by anti-IgE. Increase Ca²⁺ concentrations (0.3 to 6 mM) in the extracellular medium completely reversed the inhibitory effect of zinc on anti-IgE-mediated histamine secretion. Zinc chloride was a competitive antagonist of the action of Ca²⁺ in histamine secretion induced by anti-IgE with a dissociation constant (Kd) of about 10^–5 M in both the basophil and mast cell systems. Thus physiological concentrations of zinc inhibit the release of histamine from human basophils and lung mast cells, presumably by blocking Ca²⁺ uptake induced by anti-IgE activation.     

The time course of intracellular calcium movements in single human umbilical vein smooth muscle cells

Intracellular Ca²⁺ ([Ca²⁺]_i) was measured in single isolated human umbilical vein smooth muscle cells. Stimulation with histamine, in the absence of external Ca²⁺, mobilised Ca²⁺ from intracellular stores. When repeated brief applications of agonist were used, the time to onset, amplitude and rate of rise of the Ca²⁺ transients were found to change. Two components could often be discerned in the rising phase of the transients, an initial slow pacemaker and a second faster and larger component. Following the first histamine-activated transient the basal level of [Ca²⁺]_i was invariably lower than that prior to stimulation. This lower value was maintained whilst the cell remained in Ca²⁺-free solution, but could be returned to a higher level if the cell was exposed to external Ca²⁺. When the mobilisation of the intracellular store was reduced to undetectable levels, re-exposure to Ca²⁺-containing medium reactivated responses. In the absence of external Ca²⁺, continuous application of histamine activated a series of transient increases in intracellular Ca²⁺, which decreased progressively in amplitude and rate of rise. The interval between transients also increased. These findings are discussed in terms of the activation of inositol trisphosphate-sensitive intracellular Ca²⁺ stores and their sensitivity to cytoplasmic Ca²⁺ and intrasarcoplasmic reticulum Ca²⁺.     

On the mechanism of histamine induced enhancement of the cardiac Ca2+ current

In guinea pig ventricular myocytes, the effect of histamine on the slow Ca²⁺ current (I_Ca) was studied and the following results were obtained: (1) Superfusion of cells with histamine resulted in a dose-dependent enhancement of the amplitude of I_Ca. The threshold concentration of histamine was 10^–8 M, half maximal increase occurred at 3×10^–7 M and maximal enhancement (about 3–4-fold) at 5×10^–6 M. (2) The histamine effect was greatly reduced by the H₂ antagonist cimetidine (10^–5 M) but only slightly by the H₁ antagonist diphenhydramine (10^–5M). (3) Effects of isoprenaline (ISP) and histamine at maximal effective concentrations on I_Ca were not additive, suggesting that both agents use the same intracellular pathway. Intracellular infusion of a blocker of the cAMP-dependent protein kinase, R_p-cAMPS (10^–4 M), prevented the histamine effect. (4) The involvement of GTP-dependent transducer proteins was studied by cell dialysis with several GTP derivatives. Intracellular application of the stable GDP-analogue, GDP--S, reduced the histamine effect on I_Ca, whereas the stable GTP analogue, GTP--S, mimicked the histamine effect.     

Role of the cytoskeleton in Ca2+ release from the intracellular Ca store of rat peritoneal mast cells

In order to study the role of the cytoskeleton in histamine release from mast cells, the effects of cytochalasin D, cholchicine and vinblastine on Ca²⁺ release from the intracellular Ca store induced by compound 48/80 were investigated by means of a video-intensified microscopy system. When the quin 2-loaded mast cells were stimulated by 0.35 g/ml of compound 48/80, a rapid increase in intracellular Ca²⁺ was observed. At concentrations higher than 10^–6 M, both colchicine and vinblastine pretreatments significantly inhibited the increase in intracellular Ca²⁺ concentrations caused by compound 48/80, although cytochalasin D had no effect. When permeabilized mast cells were exposed to potassium-antimonate solution, microtubules became attached to the endoplasmic reticulum, where many dots of Ca-antimonate were observed; in some areas, the microtubules interconnected the endoplasmic reticulum and granules in the mast cells. From the results of the present study, it was assumed that microtubules play some important role in the processes leading to Ca²⁺ release from the intracellular Ca store.     

The effect of N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7) on muscarinic receptor-induced Ca2+ mobilization in a human salivary epithelial cell line

We have investigated the effect of W-7, a calmodulin (CaM) antagonist, on Ca²⁺ mobilization in a human salivary epithelial cell line, HSG-PA, after muscarinic receptor stimulation. In a medium containing 1.5 mmol/l Ca²⁺, W-7 reduced both the maximum peak increase in cytosolic Ca²⁺ ([Ca²⁺]_i) which follows stimulation by carbachol (Cch, 100 mol/l) and the sustained nature of the response. Using an experimental approach which allows separate visualization of the intracellular Ca²⁺ release and extracellular Ca²⁺ entry phases, W-7 was shown preferentially to inhibit Ca²⁺ release. At 100 mol/l W-7, Cch-induced Ca²⁺ release was completely inhibited, but Cch-induced Ca²⁺ entry was partially (40%) maintained. This W-7 residual Ca²⁺ entry response was abolished when cells were depolarized with high K⁺ or gramicidin D. W-7 also substantially inhibited Cch-induced inositol trisphosphate (IP₃) production (75%). W-5, a less potent CaM antagonist than W-7, had markedly smaller effects on Cch-induced Ca²⁺ mobilization and IP₃ formation. W-7 (100 mol/ l) completely blocked (comparable to 10 mol/l atropine) the binding of the muscarinic antagonist [³H] quinuclidinyl benzilate (QNB) to muscarinic receptors on cell membranes, whereas Cch (at 100 mol/l) had minimal effects on ligand binding. W-7 and W-5 were equipotent in their ability to inhibit [³H] QNB binding. These results suggest that W-7 reduces Ca²⁺ mobilization in HSG-PA cells by a mechanism which likely involves the antagonism of a CaM regulatory step(s) but may also involve at least a partial blockade of the muscarinic receptor. In addition, in the presence of W-7, Ca²⁺ entry can occur via a receptor-operated Ca²⁺ pathway which is modulated by membrane potential.