Intracellular ion exchange between cytoplasmic potassium and granule histamine,an integrated link in the histamine release machinery of mast cells*

Rat peritoneal mast cells isolated by gradient centrifugation in Percoll were placed between two membrane filters in a Sartorius filter apparatus and superfused with isotonic balanced salt solutions or with deionized isotonic sucrose. Histamine was released according to ion exchange kinetics. Our explanation of the observed phenomena is as follows. The superfusion induces a flow of cytoplasmic K⁺ ions across the histamine-containing granules, resulting in an ion exchange K⁺←Hi⁺ ions at the histamine binding sites. The concomitant equimolar outflow of histamine and potassium is considered to be due to a functional interplay between two histamine pools, a release and a donor pool. As the result of the K⁺←SHi⁺ ion exchange at the histamine binding sites of the release pool, these sites become transiently occupied by K⁺ ions only to be immediately reoccupied by Hi⁺ ions from the donor pool. The observed equimolar outflows are consistent with a 1/1 molar ratio in the exchange between histamine and potassium ions. The essential role of cytoplasmic potassium in the histamine release mechanism is a new and important observation with possible implications not only as to histamine release in general (including so-called ‘spontaneous’ histamine release) but also as to the release of biogenic amines and other positively charged substances stored in granules in ionic linkage to the matrix.     

Cation exchanger properties of isolated rat peritoneal mast cell granules

The synthetic carboxylic cation exchanger resin Amberlite IRC-50 was charged with histamine by suspension in histamine-containing solution with admixture of [14C]histamine. Mast cell granules were isolated from mast cells suspended in isotonic sucrose. The release of histamine induced from the two materials by superfusion with isotonic NaCl and KCL solutions showed identical kinetics, in accordance with the view that the release of histamine is due to a cation exchange: Na+ (K+) in equilibrium Hi+ at carboxyl groups in the granule heparin-protein complex.     

Do superfused mast cells release histamine by endogenous cation exchange with potassium ions?

Isolated rat peritoneal mast cells released histamine on superfusion with isotonic salt solutions or isotonic deionized sucrose. The histamine release followed the kinetics of cation exchange characteristic of the release from similarly superfused isolated mast cell granules and histamine charged carboxylic resin IRC-50. The histamine release was accompanied by an efflux of potassium and ascribed to an endogenous cation exchange K⁺Hi⁺ occurring on the passage of outflowing potassium ions over histamine storing granules.     

Juxtacellular histamine concentration governs histamine release from rat peritoneal mast cells

Isolated rat peritoneal mast cells release histamine when superfused with isoosmotic salt or sucrose solutions. The release was ascribed by us to an intracellular ion exchange between potassium and histamine at granule sites, resulting from a flux of cytoplasmic potassium across the granules secondary to the disturbance of the ‘state of equilibrium’ at the cell surface caused by the superfusion (Uvnäs et al. 1989). In the present article is shown that the histamine releasing effect is counteracted by the addition of histamine to the superfusion fluid. The inhibition is concentration-dependent and accompanied by concomitant changes in the potassium efflux. A 50% inhibition of the histamine release requires an external histamine concentration of 40 μm and extrapolation of the equilibrium curve hints at a total inhibition at concentrations around 170 μm. The observations are taken to indicate that reduction of the juxtacellular histamine concentration caused by the superfusion disturbs the histamine equilibrium at the mast cell surface resulting in the activation of the histamine secretory mechanism. In other words, the secretory activity of the mast cell is checked by the juxtacellular concentration of histamine. When the juxtacellular histamine is removed e.g. on isolation procedures, other experimental situations such as superfusion, or by consumption in vivo the mast cell delivers histamine to restore the juxtacellular equilibrium.     

Juxtacellular histamine concentration governs histamine release from rat peritoneal mast cells.

Isolated rat peritoneal mast cells release histamine when superfused with isoosmotic salt or sucrose solutions. The release was ascribed by us to an intracellular ion exchange between potassium and histamine at granule sites, resulting from a flux of cytoplasmic potassium across the granules secondary to the disturbance of the 'state of equilibrium' at the cell surface caused by the superfusion (Uvn?s et al. 1989). In the present article is shown that the histamine releasing effect is counteracted by the addition of histamine to the superfusion fluid. The inhibition is concentration-dependent and accompanied by concomitant changes in the potassium efflux. A 50% inhibition of the histamine release requires an external histamine concentration of 40 microM and extrapolation of the equilibrium curve hints at a total inhibition at concentrations around 170 microM. The observations are taken to indicate that reduction of the juxtacellular histamine concentration caused by the superfusion disturbs the histamine equilibrium at the mast cell surface resulting in the activation of the histamine secretory mechanism. In other words, the secretory activity of the mast cell is checked by the juxtacellular concentration of histamine. When the juxtacellular histamine is removed e.g. on isolation procedures, other experimental situations such as superfusion, or by consumption in vivo the mast cell delivers histamine to restore the juxtacellular equilibrium.     

Catecholamines (CA) and adenosine triphosphate (ATP) are separately stored in bovine adrenal medulla, both in ionic linkage to granule sites, and not as a non-diffusible CA-ATP-protein complex

On superfusion of chromaffin granules from bovine adrenals with isotonic sodium and potassium salts, catecholamines and ATP were released in parallel and both in accordance with ion exchange kinetics. An artificial model was prepared by mixing a cationic (IRC-50) and an anionic (IR-4B) ion exchanger with COO- and NH+3 groups, respectively, as binding sites. This mixed ion exchanger showed in its storage and release of CA+ and ATP- striking similarities to the chromaffin granules. Within the pH range given for the interior of the granules--5.5-6--the artificial model even stored and released CA+ and ATP- within the same molar ratio as observed for the granules. We hypothesize that the chromaffin granule matrix in its storage and release functions operates as an amphoteric ion exchanger with COO- and NH+3 groups as the binding sites.     

Storage Properties of Rat Mast Cell Granules In vitro

Basophil granules were isolated from rat peritoneal and thoracic mast cells. The granules were depleted of their endogenous Hi and 5-HT. The uptake of Hi, 5-HT, TrpA, PhEA, TA, DA, NA, ACh, Na and Ca (for abbreviations see page 215) by the depleted granules, the release of Hi from Hi-recharged granules by Ca, Na and K and the competition between NA and Hi, ACh and NA, ACh and Hi for the granule storage sites were studied. The results support the hypothesis that these granule stores have the properties of a weak cation exchange resin. Furthermore it was found that amphetamine and ephedrine competed with NA for the storage sites The results indicate an unspecific granule storage of cations—organic and inorganic—differing only in their affinities for the binding sites.     

Catecholamines (CA) and adenosine triphosphate (ATP) are separately stored in bovine adrenal medulla,both in ionic linkage to granule sites,and not as a non-diffusible CA-ATP-protein complex*

On superfusion of chromaffin granules from bovine adrenals with isotonic sodium and potassium salts, catecholamines and ATP were released in parallel and both in accordance with ion exchange kinetics. An artificial model was prepared by mixing a cationic (IRC-50) and an anionic (IR-4B) ion exchanger with COO and NH⁺₃ groups, respectively, as binding sites. This mixed ion exchanger showed in its storage and release of CA⁺ and ATP^- striking similarities to the chromaffin granules. Within the pH range given for the interior of the granules - 5.5–6 - the artificial model even stored and released CA⁺ and ATP^- within the same molar ratio as observed for the granules. We hypothesize that the chromaffin granule matrix in its storage and release functions operates as an amphoteric ion exchanger with COO^- and NH⁺₃, groups as the binding sites.     

Differential effect of hypertonicity on FMLP-, anti-IgE- and Ca2+ ionophore A23187-induced histamine release from human basophil leukocytes.

Effects of different extracellular Na+ and K+ concentrations (respectively, 135, 155, 220, 260 mM NaCl, and 2.7, 20, 50, 100 mM KCl) on IgE-dependent and IgE-independent histamine release from human basophils were examined. High extracellular Na+ and K+ concentrations were shown to reduce N-formyl-methionyl-leucyl-phenyl-alanine- (FMLP), but not anti-IgE- or Ca2+ ionophore A23187-induced histamine release. A high extracellular Ca2+ (7.2 mM CaCl2) concentration increased basophil response to anti-IgE and FMLP. The enhancement of FMLP- but not of anti-IgE-induced histamine release was antagonized by high extracellular Na+ and K+ concentrations. When leukocytes were suspended in isotonic choline chloride solutions (choline is a nonpermeant monovalent cation), an enhancement of anti-IgE- and FMLP-induced histamine release was observed. This suggests that monovalent cations, namely Na+ ions, at physiological concentrations, downregulate histamine release from human basophils. At high choline chloride concentrations, FMLP-, but not anti-IgE-induced histamine release was inhibited. Thus, the reduction of FMLP-evoked histamine secretion from human basophils seems to be due to hypertonicity and not to the type of monovalent cation, either permeant or nonpermeant, contained in extracellular milieu. The different effects of a hypertonic solution on anti-IgE and FMLP-induced histamine release are probably related to the different cell activation pathways triggered by the two stimuli.     

Possible role of nerve impulse induced sodium ion flux in a proposed multivesicular fractional release of adrenaline and noradrenaline from the chromaffin cell

Based on own observations concerning a two-compartment storage of CA in the adrenal medulla and a cation exchange dependent release of CA from perfused chromaffin granules in vitro, and encouraged by recent reports from other laboratories about the importance of sodium ions for the CA release from the adrenal gland, we propose a modification of the current quantal theory of CA secretion. Instead of secretion of quanta, each quantum corresponding to the content of one vesicle, we envisage a concomitant fractional release of CA from multiple vesicles adjacent to the chromaffin cell membrane. The CA secretion should be the result of a cation exchange across the contact area between the plasma membrane and the granule membrane during the period of depolarization caused by the nerve impulse. The size of the released quanta should be determined by the nerve impulse induced sodium ion flux and the number of such ions which reach the CA binding ionic sites in the cation exchanger pool (the release pool) of the granules.     

Cation exchange--a common mechanism in the storage and release of biogenic amines stored in granules (vesicles)? III. A possible role of sodium ions in non-exocytotic fractional release of neurotransmitters

The matrices of the amine storing granules in mast cells, chromaffin cells and noradrenergic nerves show properties reminiscent of cation exchanger materials. In vitro, the amines are released from their granule storage sites on exposure of the granules to cations, e.g. sodium ions. The proposal is made that also in vivo the release of transmitter amines is the result of cation exchange Amine+ in equilibrium Na+ ions and that the release of transmitter amines occurs as a nonexocytotic fractional release engaging multiple granules instead of exocytotic emptying of a few. Some physiological and pharmacological implications of a fractional transmitter release are discussed.     

Quantitative correlation between histamine and35S release from isolated rat mast cells due to the beta-adrenergic blocking drug Kö 1124

An attempt was made to explain the mechanism of histamine release from isolated rat mast cells induced by the beta-adrenergic blocking drug Kö 1124. This drug at the highest concentration used released 12 times more histamine than most other investigated beta-blockers. The release of histamine with Kö 1124 was dose and temperature dependent. The maximal histamine release was at pH 8 and in the absence of calcium ions. Increased calcium concentration decreased histamine release significantly. The effect of Kö 1124 on histamine release from mast cells was inhibited only by cocaine and 2,4-dinitrophenol; other metabolic inhibitors were ineffective. The histamine release due to Kö 1124 was not followed by an equal release of³⁵S. Isoprenaline in equimolar concentration decreased histamine release induced by Kö 1124 significantly. The release of³⁵S-labelled granules was decreased or blocked by isoprenaline.     

Mechanism of histamine release induced by the ionophore X537A from isolated rat mast cells.

X537A released histamine from isolated histamine-retaining mast cell granules incubated at 37 degrees C in Tris-sodium (150 mM) or Tris-potassium (150 mM), but not in Tris-glucose (300 mM). The release was depressed at 0 degrees C. In contrast, decylamine released all histamine bound to the granules irrespective of the presence of monovalent cations in the incubation medium of temperature. X537A did not release histamine from an artificial heparin-protamine complex when incubated in deionized water. The mechanism of histamine release by X537A can be explained by the ability of the ionophore to carry monovalent cations across cellular membranes, hereby making the ions available for exchange with histamine bound to the granular matrix. This mechanism can be distinguished from that of agents triggering an exchange between cations and bound histamine through a calcium- and energy-dependent exocytotic process on the one hand and through membrane lysis on the other. Based on the observation that the ionophore was able to carry histamine into the bulk of an organic phase, various possibilities exist to explain how histamine escapes from the cells following release from intracellular granular stores.     

Degranulation and Histamine Release,Two Consecutive Steps in the Response of Rat Mast Cells to Compound 48/80

Isolated rat peritoneal mast cells were exposed to compound 48/80 in A isotonic salt solution and in B isotonic sucrose solution. In both cases degranulation and histamine release occurred. In A all the released histamine appeared dissolved in the suspension medium, the discharged granules having lost their histamine. In B the discharged granules were show to retain histamine. These granules released their histamine when suspended in a NaC1-containing medium. Depleted granules could be refilled with histamine by suspension in a histamine-containing medium and again depleted of the amine by exposure to sodium chloride. The proposal is made that the histamine release induced by compound 40/80 is a two stage process, a primary energy-requiring transport of histamine-containing granules to the outside of the mast cell and a secondary nonenergy-requiring physico-chemical process, an extracellular cation exchange in the shed granules between histamine and cations, mainly sodium in the tissue fluid.     

Potentiation of anaphylactic histamine release from isolated rat pleural mast cells by rat serum phospholipids.

Isolation of sensitized rat mast cells by density gradient centrifugation in Ficoll decreases the histamine release obtained when they are subsequently exposed to antigen. The histamine release from such isolated cells is potentiated by the addition of 2% boiled rat serum. This potentiation is dose-dependent and has a temperature optimum of about 25 degrees C. The potentiating activity was localized to the serum phospholipid fraction. Of the pure phospholipids studies (LPC, PC, PE, PI, PS and SM) only phosphatidylserine and lysophosphatidylcholine were found to potentiate the histamine release. The mechanism behind this potentiation is discussed and it is suggested that the potentiation by phosphatidylserine and lysophosphatidylcholine is due to a requirement of these phospholipids for the ion exchange (Na+, K+ and Ca++) or the adenylcyclase activity essential for the histamine release process.     

Studies on histamine metabolism in allergen-induced asthma

The excretion of histamine (Hi) and its metabolite methythistamine (MeHi)was determined in separated fractions of urine up to 12h alter standardized allergen provocations in 18 adult patients wild defined extrinsic bronchial asthma. The main histamine metabolite, methylimidazoleacetic acid (MelmAA), was measured in six of the patients.
After positive provocations (decrease in FEV₁ > 20%) the excretion of Hi was significantly increased during 3h and that of MeHi during 4h after challenge. Negative provocations (decrease in FEV₁ <20%) were not followed by any changes in the excretion of Hi and MeHi. MelmAA excretion increased in five out of six patients after positive provocation. It was calculated that the increased excretion of Hi and its metabolites after a positive provocation corresponded to a release of about 1 mg histamine in the body or about 1 μg/g lung tissue if all histamine was liberated in the lung.
Pretreatment with two anti-allergic drugs, disodium cromoglycate and ICI 74.917, giving significant allergen protection, resulted in a smaller increase of the excretion of both Hi and MeHi, indicating an inhibition of histamine release in vivo.     

Inhibition of antigen-induced histamine release by ouabain.

The effect of ouabain, a specific sodium-potassium dependent adenosine triphosphatase (Na+-K+-ATPase) inhibitor, on antigen-induced histamine release was studied using guinea pig lung fragments sensitized in vitro with rabbit antibodies against bovine serum albumin. Histamine was assayed spectrofluorometrically. When sensitized tissue had been preincubated with ouabain (less than or equal to 1.0 x 10(-4) M) for 10 min prior to antigenic challenge, release of histamine was significantly inhibited (maximum 54%, p less than 0.001, N=9, paired t test). The most significant inhibition was obtained near the optimal concentration of antigen. The inhibition was dependent on the length of preincubation (less than or equal to 20 min), and was partially reversible upon washing the tissue removing the ouabain. Ouabain did not seem to prolong the duration of the histamine release process. Increase in potassium ion (less than or equal to 1.1 x 10(-2)M) inhibited the histamine release and had additive effects to ouabain action. Dibutyryl cyclic AMP (less than or equal to 5 x 10(-3) M), which could enhance the release, strongly antagonized the inhibition. Glucose removal from the medium did not abolish the ouabain effect. The results seem to indicate that immunologic release of histamine is under the influence of the membrane Na+-K+-ATPase activity.     

The effect of beta-adrenergic blocking drugs and inhibitors of phosphodiesterase on histamine release from isolated mast cells

Differences in the histamine liberation from isolated rat mast cells after beta-adrenergic blocking drugs were demonstrated. In equimolar concentrations histamine release was induced by Kö 1124, Kö 1500, Kö 1560, Kö 1561 and propranolol. Alprenolol, oxprenolol, propranolol, and trimepranol significantly decreased the histamine release induced by compound 48/80. The release of granules from cells was inhibited quantitatively more than the release of histamine. This enabled us to surmise the selective effect of beta-adrenergic blocking drugs on cell membranes of mast cells. The possible mechanisms of release reaction are discussed.     

Receptor-operated, but not voltage-operated, calcium channels are involved in basophil leucocyte activation and histamine release

Transmembrane calcium flux is a critical step in basophil and mast cell activation and subsequent histamine release. This calcium flux is likely to take place through specialized membrane ion channels. Two types of calcium channels have been described so far: the first type is voltage operated and the second type is receptor operated. Depolarization of cell membrane by K+-rich solutions is followed by voltage-operated channel opening in excitable cells, such as smooth muscle cells. We evaluated whether high K+ extracellular concentrations can trigger basophil activation and histamine release. We found that human basophil leucocytes, showing a normal response to activating signals, such as anti-IgE antiserum and formylmethionine peptide, release no histamine when exposed to K+-rich media, alone or in combination with the K+ carrier valinomycin. These results are consistent with there being receptor-operated, but not voltage-operated, calcium channels in the basophil leucocyte plasma membrane.     

Evidence for intracellular histamine liberation in isolated rat mast cells

The beta-adrenoceptor blocking drug exaprolol liberated histamine from isolated rat mast cells in a dose- and time-dependent way. Histamine was liberated within seconds and was not followed by a parallel granule liberation. The inhibition of histamine liberation was induced with low temperature, low pH, high concentration of Ca²⁺, TTD, suramin and EDTA. Subcellular distribution of³H-exaprolol demonstrated a quantitative relationship between histamine depletion against exaprolol uptake in isolated rat mast cell granules. A nonspecific mechanism of action in the effect of exaprolol on mast cells is discussed. It is proposed that the drug acts on mast cells due to the direct and indirect ion exchange mechanism resulted in disproportion between histamine and granule liberation.