Evidence for control of mast cell granule protease in situ by low pH

The second order rate constant, k2, for the inhibition of mast cell protease I by phenylmethanesulfonyl fluoride (PMSF) is lower for intact mast cells and isolated granules with intact membranes than for granules stripped of their membranes and suspended in medium at pH 7.1. In order to test the hypothesis that the decreased activity of the protease in intact granules is attributable to low pH, two agents capable of lowering pH in intracellular compartments similar to mast cell granules were tested. Ammonium chloride increased k2 of the protease in isolated granules with intact membranes and mast cells and wash out of the salt partially reversed this effect. Treatment of cells with nigericin also substantially increased the rate of protease inactivation by PMSF. These results are consistent with the proposal that the observed k2 is determined in whole or part by a low pH of the granule in situ or isolated with intact membranes. If the low k2 in situ is solely dependent on low pH, then the rate of protease inhibition can be utilized as an endogenous probe of granule pH. On this basis we have estimated the pH of the intracellular granule as 5.2 and that of the isolated granule with its membrane intact as 6.0. The value for the pH of granules in situ is lower than that previously estimated, and we have considered possible bases for this discrepancy.     

Does intracellular histamine mediate mast cell histamine release?

Previously, we demonstrated that through binding a novel intracellular receptor of microM affinity (HIC), histamine mediates, and the HIC antagonist N,N-diethyl-2-[4-(phenylmethyl)phenoxy]ethanamine. HCl (DPPE) inhibits, platelet aggregation and serotonin granule secretion; the latter response is dependent upon the same processes that mediate histamine release from mast cell granules. We now show that, as for platelet serotonin release, DPPE blocks concanavalin A-stimulated mast cell histamine release with a potency (IC50 = 30 microM) greater than the H1-antagonist, pyrilamine (IC50 = 150 microM) or the H2-antagonist cimetidine (IC50 = 5 mM), correlating with rank order of potency to inhibit 3H-histamine binding in rat brain membranes and liver microsomes. We postulate that histamine release from mast cells is mediated at HIC by second messenger intracellular histamine. However, unlike platelets, mast cells do not appear to rely on newly synthesized histamine. Rather, as for calcium, histamine may be mobilized from bound stores to mediate histamine secretion.     

Measurement of the internal pH of mast cell granules using microvolumetric fluorescence and isotopic techniques

The intragranular pH of isolated mast cell granules was measured. Because of the minute amounts of isolated granules available, two techniques were developed by modifying aminoacridine fluorescence and [14C]methylamine accumulation techniques to permit measurements with microliter sample volumes. Granule purity was demonstrated by electron microscopy, ruthenium red exclusion, and biochemical (histamine, mast cell granule protease) analysis. The internal pH was determined to be 5.55 +/- 0.06, indicating that the pH environment within mast cell granules is not significantly different from that of previously studied granule types (i.e., chromaffin, platelet, pancreatic islet, and pituitary granules). Collapse of the pH gradient by NH+4 was demonstrated with both techniques. No evidence of Cl-/OH- or specific cation/H+ transport was found, and major chloride permeability could not be unequivocably demonstrated. Ca2+ and Cl- at concentrations normally present extracellularly destabilized granules in the presence of NH+4, but this phenomenon does not necessarily indicate a role for these ions in the exocytotic release of granule contents from intact cells. The pH measurement techniques developed for investigating the properties of granules in mast cells may be useful for studying other granules that can be obtained only in limited quantities.     

Proton NMR studies of nucleotide and amine storage in the dense granules of pig platelets

1H NMR measurements have been conducted at 360 MHz on isolated pig platelet dense granules. Resonances of the H8, H2 protons of the adenine ring, H1' protons of the ribose moiety, and the aromatic hydrogens of 5-hydroxytryptamine (5HT) have been identified in spectra of intact dense granules. Like the 31P resonances of the nucleotides contained in the dense granules (U?urbil et al., 1984), the line widths and the intensities of these resonances were sensitive to sample temperature and osmolarity of the suspension medium. Their chemical shifts indicate that 5HT in the granule interior is predominantly bound to the nucleotides through ring-stacking interactions. Association of 5HT with the nucleotides was also confirmed by the presence of intermolecular nuclear Overhauser effect (NOE) between 5HT and nucleotide protons. Large and negative intermolecular NOE's observed among the nucleotide H8, H2 and H1' protons, together with upfield shifts undergone by these protons within the dense granules, demonstrate that the nucleotides form a complex where they are in close proximity of each other. The formation of this complex apparently does not require the presence of amines since removal of 5HT and histamine did not change the chemical shifts of the nucleotide protons. From T1 and T2 data, rotational correlation time of 4 ns was calculated for the nucleotides in the dense granule interior at 35 degrees C. A resonance tentatively identified as H2 of histamine was found to shift upon manipulation of the intragranular pH; it was used as an indicator of pH changes within the granule interior during 5HT uptake and showed that 5HT accumulation increases the intragranular pH. These results demonstrate that 5HT is first taken up in response to the inside acidic pH gradient across the granule membrane and is subsequently sequestered in a matrix formed by the divalent cations and the nucleotides.     

Determination of the intracellular pH of intact erythrocytes by 1H NMR spectroscopy

A method is described for determining the intracellular pH of intact erythrocytes by ¹H NMR. The determination is based on the pH dependence of the chemical shifts of resonances for carbon-bonded protons of an indicator molecule (imidazole) in intact cells. The imidazole is introduced into the erythrocytes by incubation in an isotonic saline solution of the indicator. The pH dependence of the chemical shifts of the imidazole resonances is calibrated from ¹H NMR spectra of the imidazole-containing red cell lysates whose pH is varied by the addition of acid or base and measured directly with a pH electrode. To reduce in intensity or eliminate the much more intense envelope of resonances from the hemoglobin, the ¹H NMR measurements are made by either the spin-echo Fourier transform technique or by the transfer-of-saturation by cross-relaxation method.     

Mast cell clones: a model for the analysis of cellular maturation

Cloned mouse mast cells resemble, by ultrastructure, immature mast cells observed in vivo. These mast cell clones can be grown in the absence of any other cells, facilitating direct investigations of their biochemistry and function. We find that cloned mast cells express plasma membrane receptors (Fc epsilon R) that bind mouse IgE with an equilibrium constant (KA) similar to that of normal mouse peritoneal mast cells. In addition, cloned mast cells do not display detectable la antigens and cannot enhance lg secretion when added to lymphocyte cultures or mediate natural killer lysis. In the presence of 1 mM sodium butyrate, cloned mast cells stop dividing and acquire abundant electron-dense cytoplasmic granules similar to those of mature mast cells. Their histamine content increases concomitant with cytoplasmic granule maturation and may exceed that of untreated mast cells by 50- fold. Unlike peritoneal mast cells, cloned mast cells incorporate 35SO4 into chondroitin sulfates rather than heparin. These findings demonstrate that, unlike fully differentiated mouse peritoneal mast cells, cloned immature mouse mast cells contain no heparin and low levels of histamine. In addition, they establish that high-affinity Fc epsilon R are expressed early in mast cell maturation, well before completion of cytoplasmic granule synthesis and mediator storage.     

Colocalization of heparin and histamine in the intracellular granules of test cells from the invertebrate Styela plicata (Chordata-Tunicata)

In most ascidian species the oocytes are surrounded by two types of accessory cells called follicle cells and test cells. Test cells are located on the periphery of oocytes and remain in the perivitelline space during egg development until hatching. Heparin and histamine were previously described in the test cells of the ascidian Styela plicata. In the present study, electron microscopy techniques were used to characterize the ultrastructure of the S. plicata test cells and to localize heparin and histamine in these cells. Test cells contain several intracellular granules with unique ultrastructural features. They are formed by elongated filaments composed of serial globules with an electron-lucent circle, containing a central electron-dense spot. Immunocytochemistry showed that heparin and histamine colocalize at the border of granule filaments in the test cell. Compound 48/80, a potent secretagogue of heparin-containing mast cells, also induced degranulation of test cells. According to these results, we suggest that test cells represent ancient effector cells of the innate immunity in primitive chordates.     

Simultaneous detection of histamine release and lactate production in rat mast cells induced by compound 48/80 using 1H NMR.

1H NMR spectroscopy was used to evaluate histamine release and lactate production in intact mast cells isolated from rats. The resonance lines of the aromatic histamine protons in mast cells, detected by the selective spin-excitation technique, were broader and located in a lower magnetic field than those in free histamine solution. When exocytosis of mast-cell granules was induced by compound 48/80, free histamine appeared, with a corresponding decrease in the amount of histamine in the mast cells; the lactate signal was also detected in the spectrum. On the addition of compound 48/80, there was a further release of histamine from mast cells, accompanied by further production of lactate. This result indicates that the mechanisms which induce the exocytosis of granules, and/or the events following exocytosis, activate glycolysis.     

Ionic control of the size of the vesicle matrix of beige mouse mast cells

Isolated matrices of the giant secretory vesicles of mast cells of the beige mouse were reliably produced by the osmotic lysis of isolated vesicles. These matrices maintained their form, and their sizes were easily measured using Nomarski optics. The size of the matrix depended on the ionic composition of the bathing solution. The physiologically relevant ions, histamine and serotonin, contracted the matrix. Multivalent cations condensed the matrix relative to univalents. Ag+, acid pH (below 5), and basic pH (above 9) expanded the matrix. In the presence of 10 mM histamine, lowering the pH from 9 to 5 contracted the matrix more than can be attributed to the pH-dependent matrix contraction in zero histamine. The nontitratable organic cation, dimethonium, contracts the matrix with little effect of pH in the range of 5-9. These results suggest that histamine acts as a matrix contractor in the divalent form. The dose-response (contraction) relation for histamine was gradual from micromolar to 316 mM (millimolar) histamine. Experiments with mixtures of histamine and sodium show antagonistic effects on the matrix but are inconsistent with either a model where ions compete for identical sites or a parallel model where ions interact with separate independent sites. In vigorous histamine washoff experiments, the half time for vesicle expansion in 10(-4) M pH buffer was approximately 4 s; in isotonic NaCl solution, it was 0.5 s. When 1 M histamine was presented to closely apposed matrices, fusion resulted. The matrix material returned to its initial shape after being mechanically deformed with a glass probe. These results suggest that the matrix size is controlled by its ion exchange properties. The matrix expansion can quantitatively account for the vesicular size increase observed upon exocytosis (as a postfusional event) and the osmotic nonideality of intact vesicles. The mechanical expansion is probably significant in the widening of the exocytotic pore and the dispersal of the vesicular contents.     

Simultaneous detection of histamine release and lactate production in rat mast cells induced by compound 48/80 using H NMR

¹H NMR spectroscopy was used to evaluate histamine release and lactate production in intact mast cells isolated from rats. The resonance lines of the aromatic histamine protons in mast cells, detected by the selective spin-excitation technique, were broader and located in a lower magnetic field than those in free histamine solution. When exocytosis of mast-cell granules was induced by compound 48/80, free histamine appeared, with a corresponding decrease in the amount of histamine in the mast cells; the lactate signal was also detected in the spectrum. On the addition of compound 48/80, there was a further release of histamine from mast cells, accompanied by further production of lactate. This result indicates that the mechanisms which induce the exocytosis of granules, and/or the events folowing exocytosis, activate glycolysis.     

Cytofluorometric quantitation of 5-hydroxytryptamine and heparin in individual mast cell granules.

Cytofluorometric quantitation of 5-hydroxytryptamine (5-HT) and heparin in individual mast cell granules is described. The technique is based on micromanipulation of intact mast cells reacted with formaldehyde or stained with Berberine sulfate and the use of a cytofluorometer equipped with a sensitive peak detecting device. The quantities of 5-HT and heparin contained in mast cell granules which are of the order of 10(-16) and 10(-13) g, respectively were expressed as relative fluorescence guanta. The results of measurements on representative samples of mast cell granules indicate that all granules contain heparin as well as 5-HT, and that there are large variations in both 5-HT and heparin content within the granule populations of individual cells. A dose dependent increase in 5-HT content in both cells and individual mast cell granules occurred 24 hr after the injection of 10--50 mg L-5-hydroxytryptophan/kg intraperitoneally. There was no evidence for an increase in the heparin content of granules or cells, indicating that a new synthesis of granular macromolecules is not required for the 5-HT uptake. The results further suggest that 5-HT may be stored initially in a cytoplasmic extragranular pool and then taken up in the mast cell granules.     

Colocalization of heparin and histamine in the intracellular granules of test cells from the invertebrate Styela plicata (Chordata-Tunicata)

In most ascidian species the oocytes are surrounded by two types of accessory cells called follicle cells and test cells. Test cells are located on the periphery of oocytes and remain in the perivitelline space during egg development until hatching. Heparin and histamine were previously described in the test cells of the ascidian Styela plicata. In the present study, electron microscopy techniques were used to characterize the ultrastructure of the S. plicata test cells and to localize heparin and histamine in these cells. Test cells contain several intracellular granules with unique ultrastructural features. They are formed by elongated filaments composed of serial globules with an electron-lucent circle, containing a central electron-dense spot. Immunocytochemistry showed that heparin and histamine colocalize at the border of granule filaments in the test cell. Compound 48/80, a potent secretagogue of heparin-containing mast cells, also induced degranulation of test cells. According to these results, we suggest that test cells represent ancient effector cells of the innate immunity in primitive chordates.     

Kinetics of release of serotonin from isolated secretory granules. II. Ion exchange determines the diffusivity of serotonin.

We measured the efflux of 5-hydroxytryptamine (5-HT, serotonin) from an intact secretory granule extracted from the mast cell of the beige mouse. The efflux was measured with amperometry after rupture of the granule membrane was triggered by electroporation. We determined the diffusivity of 5-HT within the secretory granule to be 2.0 x 10(-8) cm2 s(-1) when the granule is in contact with a physiological saline and found that this diffusivity depends on the valence of the cation in the external electrolyte. There is a fivefold increase in the diffusion coefficient of 5-HT determined in CsCl (150 mM, pH 7.2) at 3.7 x 10(-8) cm2 s(-1) compared to that determined in histamine dihydrochloride (Hi, 100 mM at pH 4.5) at 0.7 x 10(-8) cm2 s(-1). We found that the rate of expansion of the granule matrix observed in physiological medium correlates with the efflux of 5-HT, and that the rate of swelling of the matrix and the efflux depend on the microviscosity within the granule matrix and not the bulk viscosity of the external solution. The low diffusivity of 5-HT (approximately 500-fold less than in the bulk), the observation that the valence of the counterion affects this diffusivity, and the relationship between the volume changes of the matrix and the efflux suggest that 5-HT is released from the granule by ion exchange. We discuss the implications of this result for exocytotic release in mast cells and propose that an ion exchange mechanism could control the rate of release in other secretory systems.     

Immunologic release of heparin from purified rat peritoneal mast cells.

High m.w. [35S]heparin, labeled in vivo or in vitro, was released from purified rat mast cells by challenge with rabbit anti-rat F(ab')2, guinea pig anti-rat IgE, or calcium ionophore. The released and the residual heparin were isolated by Dowex 1 chromatography and were of comparable size by Sepharose 4B gel filtration. The majority of the released heparin was found by differential centrifugation to be granule-associated. Net percentage of mast cell heparin release, quantitated by metachromasia after isolation on Dowex 1 chromatography, correlated in a linear fashion with net percentage of histamine release, with heparin exhibiting a threshold requirement for onset of release. The correlation of histamine and high m.w. heparin release provides chemical support for the conclusion of others from ultrastructural studies that mast cell activation by immunologic means or by the calcium ionophore results in secretion of the whole granule.     

Rat mast cell tryptase

Rat mast cell tryptase is located largely if not totally in the cell's secretory granules. When the active site reagent [3H]diisopropyl fluorophosphate was used to label tryptase and chymase simultaneously, the ratio of tryptase:chymase active sites was determined to be 0.05. In comparison to chymase and tryptase in other species and chymase in the rat, rat tryptase is poorly bound to the granule matrix as evidenced by (1) its release parallel to histamine on induction of secretion and (2) its appearance in the supernatant when isolated granules were stripped of their membranes with hypotonic medium. Tryptase on release from the granule is moderately stable at a pH of 5.0 but unstable at pH 7.5, the pH that the enzyme encounters on secretion from the cell. These several properties indicate that the role of rat mast cell tryptase extracellularly is likely to differ greatly from that of chymase.     

31P and 35Cl nuclear magnetic resonance measurements of anion transport in human erythrocytes

The exchange of anions across the erythrocyte membrane has been studied using 31P nuclear magnetic resonance (NMR) to monitor inorganic phosphate influx and 35Cl NMR to monitor chloride ion efflux. The 31P NMR resonances for intracellular Pi and extracellular Pi could be observed separately by adjusting the initial extracellular pH to 6.4, while the intracellular pH was 7.3. The 35Cl NMR resonance for intracellular Cl- was so broad as to be virtually undetectable (line width greater than 200 Hz), while that of extracellular Cl-is relatively narrow (line width of about 30 Hz). The transports of Pi and Cl-were both totally inhibited by 4,4'-diisothiocyanostilbene-2,2'-disulfonate, a potent inhibitor of the band 3 protein. Since the 31P resonance of Pi varies with pH, intra- and extracellular pH changes could also be determined during anion transport. The extracellular pH rose and intracellular pH fell during anion transport, consistent with the protonated monoanionic H2PO4-form of Pi being transported into the erythrocyte rather than the deprotonated dianionic HPO24-form. The rates of Cl-efflux and Pi influx were determined quantitatively and were found to be in close agreement with values determined by isotope measurements. The Cl-efflux was found to coincide with the influx of the monoanionic H2PO4-form of Pi.     

The molecular basis for the storage and release of histamine in rat mast cell granules

The proposal that mast cell histamine is stored in a histamine-heparin or histamine-zinc-heparin complex is criticised. In the first place, both heparin and zinc are present in amounts sufficient to bind only a minor part of the histamine stored. Secondly, it is evident from titration studies on the granules that the ester sulphate groups of heparin, which are essential for the binding of histamine, are already occupied since they are involved in the binding between heparin and basic protein in the complex that forms the matrix of the basophil granules. This protein-heparin complex is the histamine-binding material. It has the properties of a weak cation exchange resin, that binds organic and inorganic cations unselectively. Histamine release from mast cells is a simple cationic exchange between histamine and inorganic cations (mainly sodium) which takes place in granules which become exposed to the extracellular medium during the exocytotic process. The possibility that other release processes are also based on ionic exchange is briefly discussed.     

Carboxypeptidase A in mouse mast cells. Identification, characterization, and use as a differentiation marker

By using a conventional spectrophotometric assay with hippuryl-L-phenylalanine as the substrate, 10(6) BALB/c mouse serosal mast cells possessed 1.5 +/- 0.43 U (mean +/- SE, n = 5, range = 0.48 to 2.5) of carboxypeptidase A activity, while T cell factor-dependent, mouse bone marrow-derived mast cells (BMMC) had barely detectable levels of 0.01 +/- 0.001 U/10(6) cells (mean +/- SE, n = 3). In order to characterize the carboxypeptidase A present in the BMMC, a sensitive assay was developed that used angiotensin I as the substrate and reverse phase-high performance liquid chromatography to separate and quantify production of the cleavage product des-leu-angiotensin I. Using this assay, mouse BMMC carboxypeptidase A had a neutral to basic pH optimum and hydrolyzed angiotensin I with a Km of 0.78 mM. The antigen-induced net percent release of carboxypeptidase A from IgE-sensitized BMMC was proportional to that of the secretory granule component beta-hexosaminidase which indicates a secretory granule location for the exopeptidase. As defined by exclusion during Sepharose CL-2B chromatography, carboxypeptidase A was exocytosed as a greater than 1 X 10(7) m.w. complex bound to proteoglycans. Because BMMC cocultured with mouse skin-derived 3T3 fibroblasts are known to undergo an increase in histamine content and biosynthesis of 35S-labeled heparin proteoglycans, carboxypeptidase A activity was measured during BMMC/fibroblast coculture for 0 to 28 days. The carboxypeptidase A activity increased progressively during 28 days of co-culture from 0.004 +/- 0.002 U/10(6) starting BMMC (mean +/- SE, n = 3) to 0.36 +/- 0.10 U/10(6) co-cultured mast cells. These findings indicate that carboxypeptidase A, a neutral protease, is exocytosed from the secretory granules of mouse mast cells bound to proteoglycan and is increased during the in vitro differentiation of mouse BMMC from mucosal-like mast cells to serosal-like mast cells.     

ELECTRON MICROSCOPE OBSERVATIONS ON COMPOUND 48/80-INDUCED DEGRANULATION IN RAT MAST CELLS : Evidence for Sequential Exocytosis of Storage Granules

In vitro degranulation of rat mast cells was studied at different intervals ranging from 10 to 60 sec after adding the histamine liberator, compound 48/80 (0.4 µg/ml, 17°C). The ultrastructural changes were followed by electron microscopy, and parallel assays were made to determine the histamine released. In addition, the extracellular tracers lanthanum and hemoglobin (demonstrated by its peroxidative activity) were applied to mast cells to follow communication of the extracellular space with the cavities formed during degranulation. After a lag period of 10 sec, degranulation started in the most peripherally located granules. The perigranular membrane fused with the plasma membrane, resulting in a pore bridged by a thin diaphragm. This was followed by rupture of the diaphragm and extrusion of the granule matrix (exocytosis). The process advanced towards the cell interior by fusion and opening of the deeper situated granules to the formerly opened granule cavities. At the end of the process, the cell was filled by a system of complicated cavities containing a number of altered granules. Extracellular tracers have shown that these intracellular cavities were in unbroken communication with the extracellular space from the very beginning of their formation. Both lanthanum and hemoglobin were found to be adsorbed to the limiting membrane of the cavities and bound to altered mast cell granules. In contrast, no tracer substance was present in nondegranulating mast cells. Degranulation of mast cells by compound 48/80 is regarded as a sequential exocytosis, a process similar to that described for some exocrine gland cells. All the "intracellular" cavities, formed by degranulation, were shown to communicate with the extracellular space; consequently, granules lying in these cavities must be considered as biologically extracellular. The present findings support the view that histamine is released from the granule matrix by the extracellular ionic milieu.     

Actions of histamine on muscle and ganglia of the guinea pig gallbladder

Histamine is an inflammatory mediator present in mast cells, which are abundant in the wall of the gallbladder. We examined the electrical properties of gallbladder smooth muscle and nerve associated with histamine-induced changes in gallbladder tone. Recordings were made from gallbladder smooth muscle and neurons, and responses to histamine and receptor subtype-specific compounds were tested. Histamine application to intact smooth muscle produced a concentration-dependent membrane depolarization and increased excitability. In the presence of the H(2) antagonist ranitidine, the response to histamine was potentiated. Activation of H(2) receptors caused membrane hyperpolarization and elimination of spontaneous action potentials. The H(2) response was attenuated by the ATP-sensitive K(+) (K(ATP)) channel blocker glibenclamide in intact and isolated smooth muscle. Histamine had no effect on the resting membrane potential or excitability of gallbladder neurons. Furthermore, neither histamine nor the H(3) agonist R-alpha-methylhistamine altered the amplitude of the fast excitatory postsynaptic potential in gallbladder ganglia. The mast cell degranulator compound 48/80 caused a smooth muscle depolarization that was inhibited by the H(1) antagonist mepyramine, indicating that histamine released from mast cells can activate gallbladder smooth muscle. In conclusion, histamine released from mast cells can act on gallbladder smooth muscle, but not in ganglia. The depolarization and associated contraction of gallbladder smooth muscle represent the net effect of activation of both H(1) (excitatory) and H(2) (inhibitory) receptors, with the H(2) receptor-mediated response involving the activation of K(ATP) channels.