Acta physiol. scand. 1975. 95. 133-141 From the Department of Pharmacology, Karolinska Institutet,Stockholm, Sweden
Intracellular Distribution of Amines Taken up by Rat Mast Cells In Vitro BY ANDERSBERGENDORFF Received 27 March 1975
Abstract BERGENDORFF, A. Intracellular distribution of amines taken up by rat mast cell in vitro. Acta physiol. scand. 1975. 95. 133-141, Mast cells isolated from rat peritoneal and pleural cavities were incubated in uifro with radioactivelylabelled histamine (Hi), 5-hydroxytryptamine (5-HT), dopamine (DA), noradrenaline (NA), tyramine (TA), phenylethylamine (PhEA), tryptamine (TrpA), ephedrine (Eph) or amphetamine (Arnph). All these amines were taken up by the mast cells. The dose-response curves for the compound 48/80-induced release of endogenous Hi and for the various amines taken up by the cells were compared. The release curves for Hi, 5-HT, DA, NA and TA were found to be similar to that for endogenous Hi, while those for PhEA, TrpA, Eph and Amph were different from that for endogenous Hi. The uptake of Hi, 5-HT, DA, PhEA, TrpA and Eph into granules in mast cells was studied. Membrane-bound granules were obtained by sonication of mast cells incubated with the respective arnine, followed by differential centrifugation. The amine content of these granules was then measured. Hi, 5-HT and DA were found to be mainly localized to the granules, while a smaller proportion of the PhEA, TrpA and Eph was found there, the rest being located extragranularly. The present results suggest that, when taken up by rat mast cells, even amines which are not endogenous to the cells are stored in the same way as the endogenous amines Hi and 5-HT.
Many authors (Furano and Green 1964, Cabut and Haegermark 1966, Jansson 1970, FriskHolmberg and Uvnas 1972 and Heisler and Uvnas 1972) have shown that rat mast cells can take up not only the amines which occur in them naturally-histamine (Hi) and 5hydroxytryptamine (5-HT)-but also noradrenaline (NA) and dopamine (DA). Granules isolated from water-lysed rat mast cells have been shown to bind various amines in a rather unselective manner (Bergendorff and Uvnas 1973). The main constituent of the granule matrix is a heparin-protein complex (Bergqvist, Samuelsson and Uvnas 1971) which has the properties of a weak cation exchange material. The binding sites on the complex are probably carboxyl groups (Uvnas, Aborg and Bergendorff 1970), to which the amines are linked ionically. Since granules isolated in the above way are devoid of an intact membrane, there is no barrier to ion movements between the granules and the surrounding medium. The object of the present study was to determine if the unselective cation storage mechanism found in granules (see above) was also operative inside the mast cell. Two separate 133
134
ANDERS BERGENDORFF
approaches were used to investigate to what extent various amines, both endogenous and exogenous to rat mast cells, taken up by the cells were stored in the granules: a) Compound 48/80 is known to release the endogenous amines Hi and 5-HT from mast cells in a dosedependent manner by a process involving exocytosis of amine-containing granules. Consequently, the release curves for the two amines are almost identical (Moran, Uvnas and Westerholm 1962). This being so, other amines taken up by cell ought to be released in parallel with the endogenous amines if they are localized to the granules. b) Differential centrifugation of ultrasonically disintegrated mast cells yield a mixture of electron dense granules having an intact membrane, and swollen, less electron dense granules which are devoid of a limiting membrane (Anderson ei al. 1974). The granules surrounded by an intact membrane can retain their amines even when suspended in a cation-containing medium (e.g. isotonic NaCl). This means that amines taken up by the cells and stored in the granules can be demonstrated directly. In the present in vitro study all the amines investigated were taken up by the cells and stored to a greater or lesser extent in the granules.
Methods Isolation of mast cells Mixed cell suspensions were obtained from the peritoneal and pleural cavities of male Sprague-Dawley rats weighing 350450 g and the mast cells were isolated according to the method described by Thon and Uvnas (19661, as modified by Uvnas, Aborg and Bergendorff (1970). The method was recently published in detail by Anderson e f a/. (1974). Isolation of granules Isolated mast cells were sonicated (MSE, 100 Watt) in 0.34 M sucrose solution at 4 p m for 5 s and then subjected to differential centrifugation at 350 x g for 10 min and 3 000 x g for 20 min, all procedures being carried out at 4°C. The 3 000 x g precipitate, containing a mixture of granules with an intact membrane and granules devoid of an intact membrane, was suspended in 0.15 M NaCl adjusted to p H 7 with Siirensen phosphate buffer and recentrifuged (3 000 x g , 20 min, 4°C). The intensity and duration of sonication used was found t o give the largest proportion (about 5 0 % ) of granules with a n intact membrane (Anderson et al. 1974), Anderson and Uvnas in press). Suspension of the mixed granule precipitate in 0.15 M NaCl at p H 7 releases all amines stored in the granules devoid of an intact membrane by cation exchange (Aborg, Novotn); and Uvnas 1967, Uvnas, Aborg and Bergendorff 1970, Bergendorff and Uvnas 1972, 1973). Thus the amines remaining in the granule mixture will be localized to granules with a n intact membrane. Charging of mast cells with amines Isolated mast cells were pooled and suspended in a buffered salt-albumin solution (154 mM NaCl; 2.7 mM KCI; 0.9 mM CaCl,; 10% Sorensen phosphate buffer, p H 7, and 1 mg human serum albumin/ml), hereafter called the incubation medium. Unlabelled amine and the corresponding 14C-labelled amine were added to the mast cell suspension. The concentration of mast cells was usually 0.5-0.7 x lo8 cells/ml, but was occasionally as low as 0.25 x lo8 cells/ml. The level of radioactivity was usually 1pCi/ml. In those experiments in which low amine concentrations were used the radioactivity was limited t o 0.5 or 0.05 pCi/ml. The amine concentrations used in the uptake studies varied between and 3mM, depending on the uptake of the amine (Fig. 1-4). In the release studies, solutions containing 0.1-0.15 m M of the amines were used, except for 5-HT and DA where concentrations of 0.005 m M and 0.02 m M respectively were used. At these concentrations the release of endogenous Hi induced by compound 48/80 was not affected (Fig. 8 and 9). The incubation of mast cells with amines was carried out in a shaking water-bath at 37°C for 90 min.
AMINES IN MAST CELLS
135
After incubation, the cells were centrifuged down at 350 x g for 10 min. The cells were washed in the incubation medium a t room temperature until the radioactivity in the wash was close to background values (3 washes, each of 10 ml). Amine assays
In mast ceI1s. Cells loaded with the 14C-labelled amine under investigation were suspended in M HCI and heated in a boiling water-bath for 5 min. Aliquots were taken for the assay of histamine and radioactivity (for calculations see below) and, in a few cases, for paper chromatography.
In granules. Granules from mast cells loaded with the l4C-labe1led amine under investigation were prepared as described above. The precipitated granules were suspended in lo-' M HCl and heated in a boiling water-bath for 5 min in order to release the amines stored in the granules with an intact membrane. Aliquots were taken for the assay of histamine, protein and radioactivity. For calculations see below. Quantitative determination of unlabelled histamine. Histamine was assayed by the method of Shore et ai. (1959) by direct condensation with o-phthaldialdehyde, as described by Bergendorff and UvnLs (1972). Quantitative determination of ''C-labelled amines. The radioactivity in aliquots of test samples and the corresponding 14C-labelled amine incubation solution was measured using a Tri-carb@liquid scintillation spectrometer, Model 3375 (Packard Instrument Co.), with Instagela as scintillation fluid. Calculations. At the beginning of each experiment, aliquots of the mast cell suspension were taken for the assay of endogenous histamine and for cell counting. From these results the histamine content per los cells was calculated. At the end of each experiment, aliquots were taken from each sample for the determination of radioactivity and endogenous histamine. Assuming that the Hi-content of intact mast cells does not change during the incubation and washing procedures (checked in 3 expts.), the number of intact mast cells present at the end of the experiment was calculated from the endogenous Hi-content at that time and the Hi-content per loo cells a t the start of the experiment. Data obtained from the above calculations enable a direct comparison to be made between the amount of amine taken up by cells and by membrane-bound granules. Release studies with compound 48/80 Mast cells were incubated with the 14C-labelled amine under investigation and washed free from external radioactivity as described above. The amine-loaded cells were exposed to different concentrations of compound 48/80 (0.1 to 30pg per ml), for 10 min at 37"C, and centrifuged down at 350 x g for 10 min. Aliquots of the supernatant and the sediment were taken for the determination of radioactivity and histamine. Radio paper chromatography
Ascending paper chromatography was carried out using Whatman No. 1 paper and the following solvent system-n-butanol/acetic acid/water (12 :3 :5). Reference chromatograms with "C-labelled and unlabelled amines were run in parallel. The chromatograms of the unlabelled amines were stained with ninhydrin. The radioactive chromatograms were cut into 1 cm strips which were cut into smaller pieces and placed in counting vials with 10 ml of Instagel@scintillation fluid and the radioactivity was measured as described above. Determination of ether water partition coefficient 10 pl of 14C-labelled amine solution (1 pCi) was added to 1 ml of 0.1 M sodium phosphate buffer, p H 7.4 and 1 ml of ether saturated with this buffer. The two phases were thoroughly mixed and then allowed to separate. Aliquots were taken from each phase and the 14C-activitywas recorded.
Materials Histamine-(ring-2-14C) dihydrochloride, sp. act. 59 mCi/mmole; tyramine-l1J4C hydrochloride, sp. act. 42 mCi/mmole; dopamine-14C hydrochloride, sp. act. 59 mCi/mmole; DL-n~radrenaline-(carbinol-~~C) DL-bitartrate sp. act. 41.2 mCi/mmole; The Radiochemical Centre, Amersham, England. 5-Hydroxytryptamine-2-14C binoxalate, sp. act. 27.5 mCi/mmole; tryptamine-2-14C bisuccinate, sp. act. 60 mCi/ mmole; p-phenylethylamine-l-14Chydrochloride, sp. act. 7 mCi/mmole; New England Nuclear, Boston,
136
ANDERS BERGENDORFF UPTAKE
0.01-1
Fig. 1. Uptake by mast cells of 5-HT (A-A), D A (+-+I TA (X-X), N A (W-m) and Hi (0-0).
,
1v3
16'
I
104 10-1 CONC. AMlNE
3mM
Mass., USA. D L -E ~ he drine - l- l~C hydrochloride, sp. act. 20 mCi/mmole; D-amphetamine-7J4C sulphate, sp. act. 15.5 mCi/mmole; Commissariat a I'Energie Atomique, Gif-Sur-Yvette, France. Ficoll; AB Pharmacia, Uppsala, Sweden, human serum albumin (free from preservatives), AB Kabi, Striingnas, Sweden. All other substances were obtained from the usual commercial sources.
Abbreviations Amph, amphetamine; DA, dopamine; Eph, ephedrine; Hi, histamine; 5-HT, 5-hydroxytryptamine; NA, noradrenaline; PhEA, p-phenylethylamine; TA, tyramine; TrpA, tryptamine.
Results Uptake of amines into mast cells
All the amines investigated were taken up by isolated mast cells (Fig. 1 and 2). There were, however, large differences between the uptake curves for the different amines. None of the amines produced any Hi release in the concentrations studied. Light microscopic observations did not reveal any morphological changes in the mast cells.
UPTAKE 10
-5
1
Intracellular Distribution of Amines Taken up by Rat Mast Cells In Vitro BY ANDERSBERGENDORFF Received 27 March 1975
Abstract BERGENDORFF, A. Intracellular distribution of amines taken up by rat mast cell in vitro. Acta physiol. scand. 1975. 95. 133-141, Mast cells isolated from rat peritoneal and pleural cavities were incubated in uifro with radioactivelylabelled histamine (Hi), 5-hydroxytryptamine (5-HT), dopamine (DA), noradrenaline (NA), tyramine (TA), phenylethylamine (PhEA), tryptamine (TrpA), ephedrine (Eph) or amphetamine (Arnph). All these amines were taken up by the mast cells. The dose-response curves for the compound 48/80-induced release of endogenous Hi and for the various amines taken up by the cells were compared. The release curves for Hi, 5-HT, DA, NA and TA were found to be similar to that for endogenous Hi, while those for PhEA, TrpA, Eph and Amph were different from that for endogenous Hi. The uptake of Hi, 5-HT, DA, PhEA, TrpA and Eph into granules in mast cells was studied. Membrane-bound granules were obtained by sonication of mast cells incubated with the respective arnine, followed by differential centrifugation. The amine content of these granules was then measured. Hi, 5-HT and DA were found to be mainly localized to the granules, while a smaller proportion of the PhEA, TrpA and Eph was found there, the rest being located extragranularly. The present results suggest that, when taken up by rat mast cells, even amines which are not endogenous to the cells are stored in the same way as the endogenous amines Hi and 5-HT.
Many authors (Furano and Green 1964, Cabut and Haegermark 1966, Jansson 1970, FriskHolmberg and Uvnas 1972 and Heisler and Uvnas 1972) have shown that rat mast cells can take up not only the amines which occur in them naturally-histamine (Hi) and 5hydroxytryptamine (5-HT)-but also noradrenaline (NA) and dopamine (DA). Granules isolated from water-lysed rat mast cells have been shown to bind various amines in a rather unselective manner (Bergendorff and Uvnas 1973). The main constituent of the granule matrix is a heparin-protein complex (Bergqvist, Samuelsson and Uvnas 1971) which has the properties of a weak cation exchange material. The binding sites on the complex are probably carboxyl groups (Uvnas, Aborg and Bergendorff 1970), to which the amines are linked ionically. Since granules isolated in the above way are devoid of an intact membrane, there is no barrier to ion movements between the granules and the surrounding medium. The object of the present study was to determine if the unselective cation storage mechanism found in granules (see above) was also operative inside the mast cell. Two separate 133
134
ANDERS BERGENDORFF
approaches were used to investigate to what extent various amines, both endogenous and exogenous to rat mast cells, taken up by the cells were stored in the granules: a) Compound 48/80 is known to release the endogenous amines Hi and 5-HT from mast cells in a dosedependent manner by a process involving exocytosis of amine-containing granules. Consequently, the release curves for the two amines are almost identical (Moran, Uvnas and Westerholm 1962). This being so, other amines taken up by cell ought to be released in parallel with the endogenous amines if they are localized to the granules. b) Differential centrifugation of ultrasonically disintegrated mast cells yield a mixture of electron dense granules having an intact membrane, and swollen, less electron dense granules which are devoid of a limiting membrane (Anderson ei al. 1974). The granules surrounded by an intact membrane can retain their amines even when suspended in a cation-containing medium (e.g. isotonic NaCl). This means that amines taken up by the cells and stored in the granules can be demonstrated directly. In the present in vitro study all the amines investigated were taken up by the cells and stored to a greater or lesser extent in the granules.
Methods Isolation of mast cells Mixed cell suspensions were obtained from the peritoneal and pleural cavities of male Sprague-Dawley rats weighing 350450 g and the mast cells were isolated according to the method described by Thon and Uvnas (19661, as modified by Uvnas, Aborg and Bergendorff (1970). The method was recently published in detail by Anderson e f a/. (1974). Isolation of granules Isolated mast cells were sonicated (MSE, 100 Watt) in 0.34 M sucrose solution at 4 p m for 5 s and then subjected to differential centrifugation at 350 x g for 10 min and 3 000 x g for 20 min, all procedures being carried out at 4°C. The 3 000 x g precipitate, containing a mixture of granules with an intact membrane and granules devoid of an intact membrane, was suspended in 0.15 M NaCl adjusted to p H 7 with Siirensen phosphate buffer and recentrifuged (3 000 x g , 20 min, 4°C). The intensity and duration of sonication used was found t o give the largest proportion (about 5 0 % ) of granules with a n intact membrane (Anderson et al. 1974), Anderson and Uvnas in press). Suspension of the mixed granule precipitate in 0.15 M NaCl at p H 7 releases all amines stored in the granules devoid of an intact membrane by cation exchange (Aborg, Novotn); and Uvnas 1967, Uvnas, Aborg and Bergendorff 1970, Bergendorff and Uvnas 1972, 1973). Thus the amines remaining in the granule mixture will be localized to granules with a n intact membrane. Charging of mast cells with amines Isolated mast cells were pooled and suspended in a buffered salt-albumin solution (154 mM NaCl; 2.7 mM KCI; 0.9 mM CaCl,; 10% Sorensen phosphate buffer, p H 7, and 1 mg human serum albumin/ml), hereafter called the incubation medium. Unlabelled amine and the corresponding 14C-labelled amine were added to the mast cell suspension. The concentration of mast cells was usually 0.5-0.7 x lo8 cells/ml, but was occasionally as low as 0.25 x lo8 cells/ml. The level of radioactivity was usually 1pCi/ml. In those experiments in which low amine concentrations were used the radioactivity was limited t o 0.5 or 0.05 pCi/ml. The amine concentrations used in the uptake studies varied between and 3mM, depending on the uptake of the amine (Fig. 1-4). In the release studies, solutions containing 0.1-0.15 m M of the amines were used, except for 5-HT and DA where concentrations of 0.005 m M and 0.02 m M respectively were used. At these concentrations the release of endogenous Hi induced by compound 48/80 was not affected (Fig. 8 and 9). The incubation of mast cells with amines was carried out in a shaking water-bath at 37°C for 90 min.
AMINES IN MAST CELLS
135
After incubation, the cells were centrifuged down at 350 x g for 10 min. The cells were washed in the incubation medium a t room temperature until the radioactivity in the wash was close to background values (3 washes, each of 10 ml). Amine assays
In mast ceI1s. Cells loaded with the 14C-labelled amine under investigation were suspended in M HCI and heated in a boiling water-bath for 5 min. Aliquots were taken for the assay of histamine and radioactivity (for calculations see below) and, in a few cases, for paper chromatography.
In granules. Granules from mast cells loaded with the l4C-labe1led amine under investigation were prepared as described above. The precipitated granules were suspended in lo-' M HCl and heated in a boiling water-bath for 5 min in order to release the amines stored in the granules with an intact membrane. Aliquots were taken for the assay of histamine, protein and radioactivity. For calculations see below. Quantitative determination of unlabelled histamine. Histamine was assayed by the method of Shore et ai. (1959) by direct condensation with o-phthaldialdehyde, as described by Bergendorff and UvnLs (1972). Quantitative determination of ''C-labelled amines. The radioactivity in aliquots of test samples and the corresponding 14C-labelled amine incubation solution was measured using a Tri-carb@liquid scintillation spectrometer, Model 3375 (Packard Instrument Co.), with Instagela as scintillation fluid. Calculations. At the beginning of each experiment, aliquots of the mast cell suspension were taken for the assay of endogenous histamine and for cell counting. From these results the histamine content per los cells was calculated. At the end of each experiment, aliquots were taken from each sample for the determination of radioactivity and endogenous histamine. Assuming that the Hi-content of intact mast cells does not change during the incubation and washing procedures (checked in 3 expts.), the number of intact mast cells present at the end of the experiment was calculated from the endogenous Hi-content at that time and the Hi-content per loo cells a t the start of the experiment. Data obtained from the above calculations enable a direct comparison to be made between the amount of amine taken up by cells and by membrane-bound granules. Release studies with compound 48/80 Mast cells were incubated with the 14C-labelled amine under investigation and washed free from external radioactivity as described above. The amine-loaded cells were exposed to different concentrations of compound 48/80 (0.1 to 30pg per ml), for 10 min at 37"C, and centrifuged down at 350 x g for 10 min. Aliquots of the supernatant and the sediment were taken for the determination of radioactivity and histamine. Radio paper chromatography
Ascending paper chromatography was carried out using Whatman No. 1 paper and the following solvent system-n-butanol/acetic acid/water (12 :3 :5). Reference chromatograms with "C-labelled and unlabelled amines were run in parallel. The chromatograms of the unlabelled amines were stained with ninhydrin. The radioactive chromatograms were cut into 1 cm strips which were cut into smaller pieces and placed in counting vials with 10 ml of Instagel@scintillation fluid and the radioactivity was measured as described above. Determination of ether water partition coefficient 10 pl of 14C-labelled amine solution (1 pCi) was added to 1 ml of 0.1 M sodium phosphate buffer, p H 7.4 and 1 ml of ether saturated with this buffer. The two phases were thoroughly mixed and then allowed to separate. Aliquots were taken from each phase and the 14C-activitywas recorded.
Materials Histamine-(ring-2-14C) dihydrochloride, sp. act. 59 mCi/mmole; tyramine-l1J4C hydrochloride, sp. act. 42 mCi/mmole; dopamine-14C hydrochloride, sp. act. 59 mCi/mmole; DL-n~radrenaline-(carbinol-~~C) DL-bitartrate sp. act. 41.2 mCi/mmole; The Radiochemical Centre, Amersham, England. 5-Hydroxytryptamine-2-14C binoxalate, sp. act. 27.5 mCi/mmole; tryptamine-2-14C bisuccinate, sp. act. 60 mCi/ mmole; p-phenylethylamine-l-14Chydrochloride, sp. act. 7 mCi/mmole; New England Nuclear, Boston,
136
ANDERS BERGENDORFF UPTAKE
0.01-1
Fig. 1. Uptake by mast cells of 5-HT (A-A), D A (+-+I TA (X-X), N A (W-m) and Hi (0-0).
,
1v3
16'
I
104 10-1 CONC. AMlNE
3mM
Mass., USA. D L -E ~ he drine - l- l~C hydrochloride, sp. act. 20 mCi/mmole; D-amphetamine-7J4C sulphate, sp. act. 15.5 mCi/mmole; Commissariat a I'Energie Atomique, Gif-Sur-Yvette, France. Ficoll; AB Pharmacia, Uppsala, Sweden, human serum albumin (free from preservatives), AB Kabi, Striingnas, Sweden. All other substances were obtained from the usual commercial sources.
Abbreviations Amph, amphetamine; DA, dopamine; Eph, ephedrine; Hi, histamine; 5-HT, 5-hydroxytryptamine; NA, noradrenaline; PhEA, p-phenylethylamine; TA, tyramine; TrpA, tryptamine.
Results Uptake of amines into mast cells
All the amines investigated were taken up by isolated mast cells (Fig. 1 and 2). There were, however, large differences between the uptake curves for the different amines. None of the amines produced any Hi release in the concentrations studied. Light microscopic observations did not reveal any morphological changes in the mast cells.
UPTAKE 10
-5
1
