Acta Physiol Scand 1991, 142, 173-180
ADONIS
000167729 100107K
Juxtacellular histamine concentration governs histamine release from rat peritoneal mast cells B. U V N A S , C. -H. ABORG, L. L Y S S A R I D E S and L. G. DANIELSSON" Department of Pharmacology, Karolinska Institutet, and * Department of Analytical Chemistry, T h e Royal Institute of Technology, Stockholm, Sweden
Uwiis, B., ABORG,C. -H., LYSSARIDES, L. & DANIELSSON, L. G. 1991. Juxtacellular histamine concentration governs histamine release from rat peritoneal mast cells. Acta Physiol Scand 142, 173-180. Received 12 December 1990, accepted 22 January 1991. ISSN 0001-6772. Department of Pharmacology, Karolinska institutet, and Department of Analytical Chemistry, The Royal Institute of Technology, Stockholm, Sweden. 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 (Uvnas 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 ,LLM and extrapolation of the equilibrium curve hints at a total inhibition at concentrations around 170 p ~ . 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. Key words : histamine release, inhibition of histamine release, intracellular ion exchange, juxtacellular histamine equilibrium, kinetics of histamine release, mast cells, potassium release from mast cells, release equations.
Mast cell granules have the properties of a weak cation exchange resin. In vitro they release their histamine when exposed to monovalent cations and they do so according to the same kinetics as characteristic of cation exchangers with carboxyl groups as the ionic binding sites. Mast cell degranulation is generally assumed to be a prerequisite for the release of histamine, the release occurring as an ion exchange in expelled granules on their exposure to the cat ions Correspondence : Professor Borje Uvnas, Department of Pharmacology, Karolinska institutet, Box 60400, S-104 01 Stockholm, Sweden.
(mainly Na+ ions) in the extracellular fluid. However, we recently reported on a role for cytoplasmic potassium in the machinery of histamine release from mast cells (Uvnas et al. 1989). Superfusion of isolated rat peritoneal mast cells with salt or sucrose solutions, isoosmotic with physiological saline, induced an efflux of histamine accompanied with a simultaneous, approximately equimolar efflux of potassium. T h e kinetics of the histamine release suggested to us that the release was the result of an intracellular ion exchange H A ' e K ' at granule storage sites, brought about by an intracellular potassium ion flux across the
173 S
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174
B. Uzinis et al.
granules d u e to contractile forces, induced by the superfusion of the mast cells. In our previous paper (Uvnas et al. 1989) we ventured the hypothesis that the histamine efflux from superfused mast cells was due t o a disturbance of the existing 'state of equilibrium ' a t the mast cell surface. I n the present paper will be described a concentration-dependent inhibition of the histamine efflux o n the addition of histamine in p~ concentrations t o the superfusion fluid.
$1 A T E R I .A I, S AN 11 M E T 1 3 0 D S The techniques used for isolation and superfusion of mast cells and mast cell granules, as well as for the characterization of the kinetics of the histamine and
F = rclrucd histamine (tonc.i F
FW-kXml
or fhe rtraighl In F
i
In
line:
F( - k h l
--
hl
H i fioe-keVZml
60
--
i
Hmsx
or 0 1 3lniphI line:
In H
i
In Bo-ka * L m l
Fig. 1. Equations used for identification of release kinetics. Left : release from ionically bound store. Right : release from non-ionicall>- bound ('free') store.
potassium releases have been described in previous reports (Uvnas et al. 1985, 1986, 1989) and are therefore only briefly dealt with here. Peritoneal mast cells from five rats were isolated by gradient centrifugation in Percoll, transferred to a Sartorius filter apparatus and spread as homogenously as possible on a filter not allowing granules and cells to pass through (Schleicher and Schuell NL 16, 0.2 p m thick). T h e number of cells averaged j.106 ml-'. This filter did not contain or take up potassium, a prerequisite for quantitative studies on the relationship between the histamine and potassium effluxes. When not otherwise stated the mast cells were in the present experimental series superfused with 0.32 M deionized sucrose at a rate of0.2 m\ min-l, at a pH around 7 and at room temperature. Histamine was routinely determined with the modified method of Shore et al. (1959) as simplified by Uvnas ct a / . (1989) and potassium with atomic emission spectrometry. For determination of histamine in peritoneal and pleural fluids the complete extraction procedures of Shore et al. were used in order to remove contamination with histidine. Releuse kinetics. The kinetics of histamine and potassium effluxes were determined and characterized as described previousll- (Uvnas et a / . 1989). Under the given experimental conditions release or as data satisfying the equation B = B,e-"R\ expressed in its linear form In B = In B,-K,j C nil (B,,= B,,,as, the term previously used by us) were taken as evidence for release on ion exchange basis (R stands for the remaining content of releasable histamine in the granules during the course of the experiment, B,,,c,hfor the estimated maximal binding
17Fo
150
280
+
a I k I 5
E A
30
U
10 I
I
I
I
0
1
2
3
C ml
I
4
I
0
2
dc ml
-
Fig. 2. Superfusion of mast cells with 0.32 hi deionized sucrose, pH 7, 0.19 ml min-'. (a) release curve, (b) release values plotted according to ion exchange equation F = e , e - " ~ '"". \
175
Extracellular histamine controls histamine release
A E\CFo 1700
r I
r
r I
1000
+z a
E
+
a
I
I
'0
600
To
E
E _c,
I
_r.
44
IL
LL
100 0
I
2
C mi
: 0
\5
1
2
C mi
-
Fig. 3. Superfusion of nylon filter, soaked in 12 mM histamine solution, with 0.32 M deionized sucrose, p H 7,0.19 ml min-l. (a) release curve, (b) release values plotted according to 1st order equation F = F,e-"'"'' capacity of the granules for histamine andoCml for the cumulated effluent volume (Uvnas & Aborg 1984, Uvnas et al. 1985). Since only part of the total histamine store was available for immediate release, the amount of releasable histamine (by us denoted R,,,) had to be estimated in each experiment. For this purpose graphical plotting was performed by the use of the two equations below both found to give agreeing values : (1 a) the general equation;
1
-
R
=
1
1
Cml
Rmax
K-+-
(1 b) the empirical equation ; R = Rmax- K d F , where R stands for the cumulated release of histamine (CAR), RmSx for the estimated maximal release of histamine, F for the concentration of histamine in the effluents. T h e values for B required for the equation above were then obtained by subtracting the measured release values from the estimated Rmaxvalue
( B = Rmax-R). To avoid these indirect ways Of characterizing the release kinetics one of us (C. -H Aborg) introduced an equation based directly on the release values for or in histamine. The equation reads F = Foe-"F' its logarithmic form In F = In 4- K F z / Zml. T h e validity of this equation not only for histamine release but also for the release of catecholamines will be dealt with in a later communication. In this paper we will only state the fact that the values for Rmaxobtained by
integral calculation from the equation above agree with the corresponding R,,, values obtained from the equations (1 a) and (1 b). The integral calculation for R,,, is given in the appendix at the end of this paper. Comments on the characterization of release kinetics. The efflux of a substance from a 'free', non-ionically bound depot occurs according to a first order process, i.e. it satisfies the equationF = ~ l e - K z mor ' in its logarithmic form In F = In F, - KZml, where F stands for the concentration of histamine measured in the eluates. The equation expressing a release due to ion exchange thus differs from the equation of the first order expressing a release from a 'free' depot only in that way that the release process in the first case (ion exchange release) is directly correlated with the square root values of the eluation volume but in the second case ('free' depot release) is correlated directly to the eluate volume as such (Fig. 1). T h e difference between the two equations might seem trivial but when based on quantitatively reliable release figures they have with their specificity and reliability in our hands proven to be indispensable tools in characterizing release kinetics. The applicability of the two equations to characterize the kinetics of release processes is illustrated in Figures 2 and 3 which demonstrate the release of histamine from mast cells superfused with 0.32 M deionized sucrose, i.e. release by ion exchange (Fig. 2a) and the release of histamine by similar superfusion of a filter soaked with histamine, i.e. release from a 'free' depot (Fig. 3a). The release curves as such do not give any kinetic information to the unexperienced eye but plotting according to the kinetic equations clearly 8-2
B. Uvnas et al.
176
i5a
150,
-
r
1-
A
+.-
I
-
100
E
+.-
:
2 5 pM Hi'
Y
a
I
-0 E
v
5 0 pM Hi+
u.
50
98 pM Hi'
0
1
2
3
4
0
1
C ml
2
3
4
Cml
-
Fig. 4. Inhibition of histamine release from superfused mast cells by addition of histamine to the superfusion fluid (isoosmotic, 0.32 M, deionized sucrose, pH 7, 0.2 ml min-', room temperature). No HA, x 25 ,LlM HA, A 50 p HA, 98 p~ HA. (a) cumulative curves, (b) concentration curves. identify release from ionically (Fig. 2b) and nonionicall~(Fig. 3b) bound histamine depot (in mast cells and in filter paper respectively). Abbreviations. HL4 histamine; Hi histamine (in some of the Figs.) ;NA noradrenaline (norepinephrine, I-arterenol); and PhEA phenylethylamine. Matertals. Percoll (Pharmacia Sweden) ; Histamine dihydrochloride (Sigma) ; L-Arterenol free base (Sigma);/I-Phenylethylamine free base (Sigma); ( k ) Isoproterenol hydrochloride (Sigma); and Nylon filter Schleicher and Schuell NL 16, 0.2 pm thick.
RESULTS
E@ux of histamine and potassium In accordance with previous reports superfusion of mast cells with iso-osmotic deionized sucrose was observed to elicit a transient release of histamine, in the present experimental series corresponding to 10-1 5 yoof the total histamine store. The typical release pattern, expressed as cumulation and concentration curves, emerges from Figure 4a & b. The release of histamine was accompanied by an equimolar efflux of potassium (Fig. 5 ) and ran according to ion exchange kinetics as expressed by the equation (Fig. 6). B = B,eTKB'
Inhibitory action of external histamine (2) On e@ux of histamine. The addition of histamine to the superfusion medium caused a concentration dependent decline of the efflux of histamine (Fig. 4a, & b). Noticeable was the fact that the remaining histamine efflux still ran according to ion exchange kinetics (Fig. 6), suggesting that ion exchange HA+ K+ was still instrumental in the release. T h e degree of inhibition could therefore be estimated from the R,,, values calculated for the histamine release obtained with the respective inhibitory histamine concentrations. (b) On e@ux of potassium. As reported previously and illustrated in Figure 5, the histamine efflux from superfused mast cells was accompanied by a concomitant and equimolar outflow of potassium. In other words, the molar ratio HA'/K+ was 1. On addition of histamine in inhibitory concentrations to the superfusion fluid the ratio declined below 1, approaching zero values at the end of the secretory response, i.e. the potassium efflux exceeded the histamine efflux. Under the assumption that the histamine release response remaining at inhibitory concentrations of histamine still occurred as an 1 to 1 ion exchange (R,,+ = R,+) the decline of the
F=
Extracellular histamine controls histamine release
177
30.
100
20
/
K-1.05
+.-
I
a
10.
Y so
0
R
I 0
10
20
100
~ (nmol) +
30
Fig. 7. Inhibition of histamine release from superfused mast cells (isoosmotic 0.32 M deionized sucrose, pH 7,0.2 ml min-', room temperature) with 25 ,UM Fig. 5. Molar ratio between histamine and potassium histamine in superfusion fluid. Measured total potrelease from loe superfused mast cells. Effect of assium efflux R,+ during histamine release histamine added to the superfusion fluid (isoosmotic, ;).-( constructed equimolar efflux of pot0.32 M deionized sucrose, pH 7,0.2 ml min-', room .-). assium and histamine R,t = RHA+ (temperature). No HA, x 25 ,UM HA, A 50 ,UM HA. R,tR, , + = 'Excess' potassium outflow satisfies Note the decline of molar ratio HA+/K+ from 1 in control without HA towards zero in the presence of 1st order equation. external histamine. K = molar ratio between histamine and potassium in effluents. Kc(nrnol)
N
N
h
+.I
2
0 pM Hi+
.-
molar ratio HA+/K+ would mean an 'excess' efflux of potassium not involved in the histamine release. If so, this efflux of potassium not taking part in the ion exchange should not be expected to obey ion exchange kinetics. I n fact, plotting the potassium release values obtained by subtracting the values for the potassium taking part in the ion exchange process (which were assumed to be equimolar to the corresponding histamine release values) from the values for total potassium efflux (R,+ -RHA+in Fig. 7) revealed that the 'excess' potassium outflow did not obey ion exchange kinetics but ran according to first order kinetics. Thus, it was possible to calculate R,, values. for both ionically and non-ionically released potassium from their respective equations (Fig. l).
-
0
2
Fig. 6. Inhibition of histamine release from superfused mast cells by addition of histamine to the superfusion fluid (isoosmotic, 0.32 M deionized sucrose, p~ 7, 0.2 m~ temperature), Note that even under inhibitory influence the persistent histamine release occurs according to ion exchange kinetics.
Quantitative considerations With our isolation and sampling procedures small variations between individual experiments in number of mast cells and amounts of releasable histamine could not be avoided. Since, however, the histamine release Was quantitatively correlated to the potassium content of the mast cell (Uvnas et al. 1989) we used the potassium
178
R. 1:i’nds et al.
figures as reference values for quantitative estimations of the concentration-dependence of the inhibitory action of external histamine. Plotting the release of histamine (expressed as R,,,,,HA+) in per cent of total release of potassium (expressed as R,,,,,K-) as described above, versus the square root values of the concentrations of external histamine yielded a straight line correlation (Eq. 1 b). Taking the histamine release in the absence of external histamine as 100°,,, an external histamine concentration of about 40 p~ was required for about 50°,, reduction of the histamine efflux. lstrapolation of the linear equilibrium c u r x towards the abscissa suggested a total inhibition of the histamine efflux at external histamine concentrations around 170 /ILL T h e inhibitory action was specific for histamine at least so far that other amines tested (X.4, PhEA, and isoproterenol) were noninhibitory within the effective histamine concentration range.
1) 1 s c L-s s I 0 N In a previous paper w-e reported on some novel observations concerning the mechanism behind the release of histamine from rat peritoneal mast cells. When superfused with isoosmotic salt or sucrose solutions the mast cells delivered histamine to the superfusion media. T h e efflux of histamine was not due to a ‘simple leakage’ from the superfused cells, on the contrary the efflux kinetics suggested a release process on ion exchange basis. We found that the histamine outflow was accompanied with an equimolar outflow of potassium and in fact, the availability of cytoplasmic potassium *-as a prerequisite for the histamine release response. After emptying of the releasable potassium store the mast cells did not deliver any further histamine in spite of the f k t that most of the histamine was still left in the cells. According to our interpretation the superfusion of the mast cells disturbed a prevailing state of equilibrium at the mast cell surface, leading to the release of histamine. T h i s secretory response was considered to be the result of an inrrucellulur ion exchange at granule sites between histamine and cytoplasmic potassium, H A + e K + ions. I n other words, histamine was released not by extracellular but by intracellular cation exchange at granule sites, i.e. a histamine release without degranulation of
the mast cell. Intracellular ion exchange between histamine and cytoplasmic potassium was considered by us to be an integrated link in the histamine release machinery of the mast cells.
Jiisiacelliilar histarnine concentration modulates histamine release I n the present paper we have described the inhibitor!. action of histamine added to the superfusion medium on the histamine efflux from superfused mast cells. T h e mere fact that histamine added to the superfusion fluid counteracted the histamine release induced by the superfusion suggested to us that the removal of extracellular histamine was the destabilizing factor initiating the secretory activity of the mast cells. T h e inhibitory action of extracellular histamine was specific so far that no inhibitory effects were seen with comparable concentrations of three other amines tried (NA, PhEA, isoproterenol). T h e inhibitory action of histamine was concentration-dependent, inhibition being exerted in pM concentrations. Extrapolation in Figure 8 suggested a total inhibition of histamine release at extracellular concentrations of histamine around 1 7 0 p M . I n other words, with a juxtacellular concentration of that order of magnitude the secretory activity of a mast cell should come to a standstill. Unfortunately, we d o not know the histamine concentration close to a mast cell in z i z ~ ~Preliminary . studies on the histamine concentration gradients between suspensions of mast cells in minute volumes of various size yielded extrapolation values consistent with juxtracellular histamine concentrations sufficient to check the secretory activity of the mast cell (to be published). Furthermore, the peritoneal and pleural fluids have histamine concentrations around 30-10 p (unpublished observations), in other words concentrations able to modulate histamine secretion. T o sum up, our observations are consistent with the view that the secretory activity of‘ the resting mast cell is kept under control by the juxtacellular concentration of histamine. When this juxtacellular histamine is removed, e.g. on isolation procedures or other experimental situations, e.g. superfusion, or by consumption in z.iz.o, the disturbed equilibrium at the cell surface will activate the mast cell to release histamine. T h e so called ‘spontaneous ’ histamine releasc
Extracellular histamine controls histamine release
1w
\
\ \ \
\ \
P
+
179
\ \
I
I
seen in connection with various experimental conditions may to a large extent have such an explanation. In case the secretory activity of the mast cell is determined by the juxtacellular histamine equilibrium between intra- and extracellular histamine there is a need for a signalling system for initiating the cell to react to disturbances in this equilibrium. So far specific histamine receptors in the mast cell have not been described. But even if the mast cell response is merely a compensatory phenomenon with an efflux of histamine in order to re-establish juxtracellular histamine equilibrium, there is still need for some ‘sensory’ device to induce a coordinated secretory machinery. Of special interest in the present paper is the mechanism behind the intracellular potassium ion fluxes K+ ininducing the ion exchange HA+ strumental in the histamine release process. As illustrated in Figures 5 and 8 the histamine was released according to ion exchange kinetics, histamine and potassium leaving the cell in equimolar amounts. The two ions may use the same exit, according to our hypothetical scheme (Fig. 9) the openings of the channels containing
Fig. 9. Schematic picture of inhibition by external histamine of histamine release from superfused mast cells. (a) On superfusion without histamine in the superfusion fluid all potassium ions pass across the histamine-storing granules and exchange with histamine. Both ions leave the cell with a molar ratio of 1 to 1. (b) On superfusion with histamine in the superfusion fluid only part of the outpassing potassium ions exchanges with histamine. Histamine release is reduced and ‘excess’ potassium bypasses the histamine release mechanism via opened potassium passages.
the histamine-carrying granules (Padawer 1970). In the presence of extracellular histamine in inhibitory concentrations the histamine secretion was reduced but still ran according to ion exchange kinetics. Assuming a persistent 1 : 1 molar ratio in the H A f = K + ion exchange a diminished ratio would mean an appearance of ‘excess’ potassium not taking part in the histamine release process. Accordingly, we observed that when histamine release was reduced there was a concomitant increase of a potassium efflux of 1st order, i.e. a flux of potassium not K+ exchange. Whether involved in HA+* this ‘excess’ potassium escaped via newly opened channels, as suggested in Fig. 9 b, or passed as
180
B. Uvnas et al.
‘an overflow ’ via the granule-containing channels together with the released histamine, remains to be established.
APPENDIX Determination of R,,,
F = Foe-K REFERENCES
\
from :
1ml
C m l = I/ F, and K are determined from semilogarithmic plotting of F against .\/ V
lox lox
PADAWAR, J. 1970. The reaction of rat mast cells to 2 polylysine. 3, Cell Biol 41, 332-372. F d r = Fo e - K \ ud v = F,SHORE, P.A., BURKHALTER, A. & COHNJR, V.H. 1959, Ec’ A method for the fluorimetric assay of histamine in z‘ = re in F,, tissues. 3 Pbarmacol Exp Therap 127, 182-186. CTVNAS, B. & ABORG, C.H. 1984. Cation exchange - a Fdz! common mechanism in the storage and release of biogenic amines in granules (vesicles?) 11. Comparative studies on sodium induced release of biogenic amines from the synthetic weak cation exchangers Amberlite IRC-50 and Duolite CS-100 = the area vup, F, F, and from biogenic (granule enriched) materials. Acta Phy$ol Scand 120, 87-97. For R,,, the triangle v2.F2/2 is added. UYNAS, B., ABORG,C.-H., LYSSARIDES, L. & THYBERG, J. 1985. Cation exchanger properties of isolated rat peritoneal mast cell granules. Acta Physiol Scand 125, 25-3). UVNAS,B., ABORG, G H . , LYSSARIDES, L. & DANIELSr SON, L.-G. 1989. Intracellular ion exchange between i C cytoplasmic potassium and granule histamine, an PO0 integrated link in the histamine release machinery of mast c5lls. Acta Physiol Scand 136, 309-320. To UVNAS,B., ABORG,C.-H., LYSSARIDES, L., THYBERG, E J. & DANIELSSON, L.-G. 1986. Rat mast cells superfused with isotonic solutions release histamine 100 probably via intracellular exchange K ’ e Hi+ ions. Acta Physiol Scand 128, 657-658.
1:
-E
0
ADONIS
000167729 100107K
Juxtacellular histamine concentration governs histamine release from rat peritoneal mast cells B. U V N A S , C. -H. ABORG, L. L Y S S A R I D E S and L. G. DANIELSSON" Department of Pharmacology, Karolinska Institutet, and * Department of Analytical Chemistry, T h e Royal Institute of Technology, Stockholm, Sweden
Uwiis, B., ABORG,C. -H., LYSSARIDES, L. & DANIELSSON, L. G. 1991. Juxtacellular histamine concentration governs histamine release from rat peritoneal mast cells. Acta Physiol Scand 142, 173-180. Received 12 December 1990, accepted 22 January 1991. ISSN 0001-6772. Department of Pharmacology, Karolinska institutet, and Department of Analytical Chemistry, The Royal Institute of Technology, Stockholm, Sweden. 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 (Uvnas 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 ,LLM and extrapolation of the equilibrium curve hints at a total inhibition at concentrations around 170 p ~ . 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. Key words : histamine release, inhibition of histamine release, intracellular ion exchange, juxtacellular histamine equilibrium, kinetics of histamine release, mast cells, potassium release from mast cells, release equations.
Mast cell granules have the properties of a weak cation exchange resin. In vitro they release their histamine when exposed to monovalent cations and they do so according to the same kinetics as characteristic of cation exchangers with carboxyl groups as the ionic binding sites. Mast cell degranulation is generally assumed to be a prerequisite for the release of histamine, the release occurring as an ion exchange in expelled granules on their exposure to the cat ions Correspondence : Professor Borje Uvnas, Department of Pharmacology, Karolinska institutet, Box 60400, S-104 01 Stockholm, Sweden.
(mainly Na+ ions) in the extracellular fluid. However, we recently reported on a role for cytoplasmic potassium in the machinery of histamine release from mast cells (Uvnas et al. 1989). Superfusion of isolated rat peritoneal mast cells with salt or sucrose solutions, isoosmotic with physiological saline, induced an efflux of histamine accompanied with a simultaneous, approximately equimolar efflux of potassium. T h e kinetics of the histamine release suggested to us that the release was the result of an intracellular ion exchange H A ' e K ' at granule storage sites, brought about by an intracellular potassium ion flux across the
173 S
ACT 142
174
B. Uzinis et al.
granules d u e to contractile forces, induced by the superfusion of the mast cells. In our previous paper (Uvnas et al. 1989) we ventured the hypothesis that the histamine efflux from superfused mast cells was due t o a disturbance of the existing 'state of equilibrium ' a t the mast cell surface. I n the present paper will be described a concentration-dependent inhibition of the histamine efflux o n the addition of histamine in p~ concentrations t o the superfusion fluid.
$1 A T E R I .A I, S AN 11 M E T 1 3 0 D S The techniques used for isolation and superfusion of mast cells and mast cell granules, as well as for the characterization of the kinetics of the histamine and
F = rclrucd histamine (tonc.i F
FW-kXml
or fhe rtraighl In F
i
In
line:
F( - k h l
--
hl
H i fioe-keVZml
60
--
i
Hmsx
or 0 1 3lniphI line:
In H
i
In Bo-ka * L m l
Fig. 1. Equations used for identification of release kinetics. Left : release from ionically bound store. Right : release from non-ionicall>- bound ('free') store.
potassium releases have been described in previous reports (Uvnas et al. 1985, 1986, 1989) and are therefore only briefly dealt with here. Peritoneal mast cells from five rats were isolated by gradient centrifugation in Percoll, transferred to a Sartorius filter apparatus and spread as homogenously as possible on a filter not allowing granules and cells to pass through (Schleicher and Schuell NL 16, 0.2 p m thick). T h e number of cells averaged j.106 ml-'. This filter did not contain or take up potassium, a prerequisite for quantitative studies on the relationship between the histamine and potassium effluxes. When not otherwise stated the mast cells were in the present experimental series superfused with 0.32 M deionized sucrose at a rate of0.2 m\ min-l, at a pH around 7 and at room temperature. Histamine was routinely determined with the modified method of Shore et al. (1959) as simplified by Uvnas ct a / . (1989) and potassium with atomic emission spectrometry. For determination of histamine in peritoneal and pleural fluids the complete extraction procedures of Shore et al. were used in order to remove contamination with histidine. Releuse kinetics. The kinetics of histamine and potassium effluxes were determined and characterized as described previousll- (Uvnas et a / . 1989). Under the given experimental conditions release or as data satisfying the equation B = B,e-"R\ expressed in its linear form In B = In B,-K,j C nil (B,,= B,,,as, the term previously used by us) were taken as evidence for release on ion exchange basis (R stands for the remaining content of releasable histamine in the granules during the course of the experiment, B,,,c,hfor the estimated maximal binding
17Fo
150
280
+
a I k I 5
E A
30
U
10 I
I
I
I
0
1
2
3
C ml
I
4
I
0
2
dc ml
-
Fig. 2. Superfusion of mast cells with 0.32 hi deionized sucrose, pH 7, 0.19 ml min-'. (a) release curve, (b) release values plotted according to ion exchange equation F = e , e - " ~ '"". \
175
Extracellular histamine controls histamine release
A E\CFo 1700
r I
r
r I
1000
+z a
E
+
a
I
I
'0
600
To
E
E _c,
I
_r.
44
IL
LL
100 0
I
2
C mi
: 0
\5
1
2
C mi
-
Fig. 3. Superfusion of nylon filter, soaked in 12 mM histamine solution, with 0.32 M deionized sucrose, p H 7,0.19 ml min-l. (a) release curve, (b) release values plotted according to 1st order equation F = F,e-"'"'' capacity of the granules for histamine andoCml for the cumulated effluent volume (Uvnas & Aborg 1984, Uvnas et al. 1985). Since only part of the total histamine store was available for immediate release, the amount of releasable histamine (by us denoted R,,,) had to be estimated in each experiment. For this purpose graphical plotting was performed by the use of the two equations below both found to give agreeing values : (1 a) the general equation;
1
-
R
=
1
1
Cml
Rmax
K-+-
(1 b) the empirical equation ; R = Rmax- K d F , where R stands for the cumulated release of histamine (CAR), RmSx for the estimated maximal release of histamine, F for the concentration of histamine in the effluents. T h e values for B required for the equation above were then obtained by subtracting the measured release values from the estimated Rmaxvalue
( B = Rmax-R). To avoid these indirect ways Of characterizing the release kinetics one of us (C. -H Aborg) introduced an equation based directly on the release values for or in histamine. The equation reads F = Foe-"F' its logarithmic form In F = In 4- K F z / Zml. T h e validity of this equation not only for histamine release but also for the release of catecholamines will be dealt with in a later communication. In this paper we will only state the fact that the values for Rmaxobtained by
integral calculation from the equation above agree with the corresponding R,,, values obtained from the equations (1 a) and (1 b). The integral calculation for R,,, is given in the appendix at the end of this paper. Comments on the characterization of release kinetics. The efflux of a substance from a 'free', non-ionically bound depot occurs according to a first order process, i.e. it satisfies the equationF = ~ l e - K z mor ' in its logarithmic form In F = In F, - KZml, where F stands for the concentration of histamine measured in the eluates. The equation expressing a release due to ion exchange thus differs from the equation of the first order expressing a release from a 'free' depot only in that way that the release process in the first case (ion exchange release) is directly correlated with the square root values of the eluation volume but in the second case ('free' depot release) is correlated directly to the eluate volume as such (Fig. 1). T h e difference between the two equations might seem trivial but when based on quantitatively reliable release figures they have with their specificity and reliability in our hands proven to be indispensable tools in characterizing release kinetics. The applicability of the two equations to characterize the kinetics of release processes is illustrated in Figures 2 and 3 which demonstrate the release of histamine from mast cells superfused with 0.32 M deionized sucrose, i.e. release by ion exchange (Fig. 2a) and the release of histamine by similar superfusion of a filter soaked with histamine, i.e. release from a 'free' depot (Fig. 3a). The release curves as such do not give any kinetic information to the unexperienced eye but plotting according to the kinetic equations clearly 8-2
B. Uvnas et al.
176
i5a
150,
-
r
1-
A
+.-
I
-
100
E
+.-
:
2 5 pM Hi'
Y
a
I
-0 E
v
5 0 pM Hi+
u.
50
98 pM Hi'
0
1
2
3
4
0
1
C ml
2
3
4
Cml
-
Fig. 4. Inhibition of histamine release from superfused mast cells by addition of histamine to the superfusion fluid (isoosmotic, 0.32 M, deionized sucrose, pH 7, 0.2 ml min-', room temperature). No HA, x 25 ,LlM HA, A 50 p HA, 98 p~ HA. (a) cumulative curves, (b) concentration curves. identify release from ionically (Fig. 2b) and nonionicall~(Fig. 3b) bound histamine depot (in mast cells and in filter paper respectively). Abbreviations. HL4 histamine; Hi histamine (in some of the Figs.) ;NA noradrenaline (norepinephrine, I-arterenol); and PhEA phenylethylamine. Matertals. Percoll (Pharmacia Sweden) ; Histamine dihydrochloride (Sigma) ; L-Arterenol free base (Sigma);/I-Phenylethylamine free base (Sigma); ( k ) Isoproterenol hydrochloride (Sigma); and Nylon filter Schleicher and Schuell NL 16, 0.2 pm thick.
RESULTS
E@ux of histamine and potassium In accordance with previous reports superfusion of mast cells with iso-osmotic deionized sucrose was observed to elicit a transient release of histamine, in the present experimental series corresponding to 10-1 5 yoof the total histamine store. The typical release pattern, expressed as cumulation and concentration curves, emerges from Figure 4a & b. The release of histamine was accompanied by an equimolar efflux of potassium (Fig. 5 ) and ran according to ion exchange kinetics as expressed by the equation (Fig. 6). B = B,eTKB'
Inhibitory action of external histamine (2) On e@ux of histamine. The addition of histamine to the superfusion medium caused a concentration dependent decline of the efflux of histamine (Fig. 4a, & b). Noticeable was the fact that the remaining histamine efflux still ran according to ion exchange kinetics (Fig. 6), suggesting that ion exchange HA+ K+ was still instrumental in the release. T h e degree of inhibition could therefore be estimated from the R,,, values calculated for the histamine release obtained with the respective inhibitory histamine concentrations. (b) On e@ux of potassium. As reported previously and illustrated in Figure 5, the histamine efflux from superfused mast cells was accompanied by a concomitant and equimolar outflow of potassium. In other words, the molar ratio HA'/K+ was 1. On addition of histamine in inhibitory concentrations to the superfusion fluid the ratio declined below 1, approaching zero values at the end of the secretory response, i.e. the potassium efflux exceeded the histamine efflux. Under the assumption that the histamine release response remaining at inhibitory concentrations of histamine still occurred as an 1 to 1 ion exchange (R,,+ = R,+) the decline of the
F=
Extracellular histamine controls histamine release
177
30.
100
20
/
K-1.05
+.-
I
a
10.
Y so
0
R
I 0
10
20
100
~ (nmol) +
30
Fig. 7. Inhibition of histamine release from superfused mast cells (isoosmotic 0.32 M deionized sucrose, pH 7,0.2 ml min-', room temperature) with 25 ,UM Fig. 5. Molar ratio between histamine and potassium histamine in superfusion fluid. Measured total potrelease from loe superfused mast cells. Effect of assium efflux R,+ during histamine release histamine added to the superfusion fluid (isoosmotic, ;).-( constructed equimolar efflux of pot0.32 M deionized sucrose, pH 7,0.2 ml min-', room .-). assium and histamine R,t = RHA+ (temperature). No HA, x 25 ,UM HA, A 50 ,UM HA. R,tR, , + = 'Excess' potassium outflow satisfies Note the decline of molar ratio HA+/K+ from 1 in control without HA towards zero in the presence of 1st order equation. external histamine. K = molar ratio between histamine and potassium in effluents. Kc(nrnol)
N
N
h
+.I
2
0 pM Hi+
.-
molar ratio HA+/K+ would mean an 'excess' efflux of potassium not involved in the histamine release. If so, this efflux of potassium not taking part in the ion exchange should not be expected to obey ion exchange kinetics. I n fact, plotting the potassium release values obtained by subtracting the values for the potassium taking part in the ion exchange process (which were assumed to be equimolar to the corresponding histamine release values) from the values for total potassium efflux (R,+ -RHA+in Fig. 7) revealed that the 'excess' potassium outflow did not obey ion exchange kinetics but ran according to first order kinetics. Thus, it was possible to calculate R,, values. for both ionically and non-ionically released potassium from their respective equations (Fig. l).
-
0
2
Fig. 6. Inhibition of histamine release from superfused mast cells by addition of histamine to the superfusion fluid (isoosmotic, 0.32 M deionized sucrose, p~ 7, 0.2 m~ temperature), Note that even under inhibitory influence the persistent histamine release occurs according to ion exchange kinetics.
Quantitative considerations With our isolation and sampling procedures small variations between individual experiments in number of mast cells and amounts of releasable histamine could not be avoided. Since, however, the histamine release Was quantitatively correlated to the potassium content of the mast cell (Uvnas et al. 1989) we used the potassium
178
R. 1:i’nds et al.
figures as reference values for quantitative estimations of the concentration-dependence of the inhibitory action of external histamine. Plotting the release of histamine (expressed as R,,,,,HA+) in per cent of total release of potassium (expressed as R,,,,,K-) as described above, versus the square root values of the concentrations of external histamine yielded a straight line correlation (Eq. 1 b). Taking the histamine release in the absence of external histamine as 100°,,, an external histamine concentration of about 40 p~ was required for about 50°,, reduction of the histamine efflux. lstrapolation of the linear equilibrium c u r x towards the abscissa suggested a total inhibition of the histamine efflux at external histamine concentrations around 170 /ILL T h e inhibitory action was specific for histamine at least so far that other amines tested (X.4, PhEA, and isoproterenol) were noninhibitory within the effective histamine concentration range.
1) 1 s c L-s s I 0 N In a previous paper w-e reported on some novel observations concerning the mechanism behind the release of histamine from rat peritoneal mast cells. When superfused with isoosmotic salt or sucrose solutions the mast cells delivered histamine to the superfusion media. T h e efflux of histamine was not due to a ‘simple leakage’ from the superfused cells, on the contrary the efflux kinetics suggested a release process on ion exchange basis. We found that the histamine outflow was accompanied with an equimolar outflow of potassium and in fact, the availability of cytoplasmic potassium *-as a prerequisite for the histamine release response. After emptying of the releasable potassium store the mast cells did not deliver any further histamine in spite of the f k t that most of the histamine was still left in the cells. According to our interpretation the superfusion of the mast cells disturbed a prevailing state of equilibrium at the mast cell surface, leading to the release of histamine. T h i s secretory response was considered to be the result of an inrrucellulur ion exchange at granule sites between histamine and cytoplasmic potassium, H A + e K + ions. I n other words, histamine was released not by extracellular but by intracellular cation exchange at granule sites, i.e. a histamine release without degranulation of
the mast cell. Intracellular ion exchange between histamine and cytoplasmic potassium was considered by us to be an integrated link in the histamine release machinery of the mast cells.
Jiisiacelliilar histarnine concentration modulates histamine release I n the present paper we have described the inhibitor!. action of histamine added to the superfusion medium on the histamine efflux from superfused mast cells. T h e mere fact that histamine added to the superfusion fluid counteracted the histamine release induced by the superfusion suggested to us that the removal of extracellular histamine was the destabilizing factor initiating the secretory activity of the mast cells. T h e inhibitory action of extracellular histamine was specific so far that no inhibitory effects were seen with comparable concentrations of three other amines tried (NA, PhEA, isoproterenol). T h e inhibitory action of histamine was concentration-dependent, inhibition being exerted in pM concentrations. Extrapolation in Figure 8 suggested a total inhibition of histamine release at extracellular concentrations of histamine around 1 7 0 p M . I n other words, with a juxtacellular concentration of that order of magnitude the secretory activity of a mast cell should come to a standstill. Unfortunately, we d o not know the histamine concentration close to a mast cell in z i z ~ ~Preliminary . studies on the histamine concentration gradients between suspensions of mast cells in minute volumes of various size yielded extrapolation values consistent with juxtracellular histamine concentrations sufficient to check the secretory activity of the mast cell (to be published). Furthermore, the peritoneal and pleural fluids have histamine concentrations around 30-10 p (unpublished observations), in other words concentrations able to modulate histamine secretion. T o sum up, our observations are consistent with the view that the secretory activity of‘ the resting mast cell is kept under control by the juxtacellular concentration of histamine. When this juxtacellular histamine is removed, e.g. on isolation procedures or other experimental situations, e.g. superfusion, or by consumption in z.iz.o, the disturbed equilibrium at the cell surface will activate the mast cell to release histamine. T h e so called ‘spontaneous ’ histamine releasc
Extracellular histamine controls histamine release
1w
\
\ \ \
\ \
P
+
179
\ \
I
I
seen in connection with various experimental conditions may to a large extent have such an explanation. In case the secretory activity of the mast cell is determined by the juxtacellular histamine equilibrium between intra- and extracellular histamine there is a need for a signalling system for initiating the cell to react to disturbances in this equilibrium. So far specific histamine receptors in the mast cell have not been described. But even if the mast cell response is merely a compensatory phenomenon with an efflux of histamine in order to re-establish juxtracellular histamine equilibrium, there is still need for some ‘sensory’ device to induce a coordinated secretory machinery. Of special interest in the present paper is the mechanism behind the intracellular potassium ion fluxes K+ ininducing the ion exchange HA+ strumental in the histamine release process. As illustrated in Figures 5 and 8 the histamine was released according to ion exchange kinetics, histamine and potassium leaving the cell in equimolar amounts. The two ions may use the same exit, according to our hypothetical scheme (Fig. 9) the openings of the channels containing
Fig. 9. Schematic picture of inhibition by external histamine of histamine release from superfused mast cells. (a) On superfusion without histamine in the superfusion fluid all potassium ions pass across the histamine-storing granules and exchange with histamine. Both ions leave the cell with a molar ratio of 1 to 1. (b) On superfusion with histamine in the superfusion fluid only part of the outpassing potassium ions exchanges with histamine. Histamine release is reduced and ‘excess’ potassium bypasses the histamine release mechanism via opened potassium passages.
the histamine-carrying granules (Padawer 1970). In the presence of extracellular histamine in inhibitory concentrations the histamine secretion was reduced but still ran according to ion exchange kinetics. Assuming a persistent 1 : 1 molar ratio in the H A f = K + ion exchange a diminished ratio would mean an appearance of ‘excess’ potassium not taking part in the histamine release process. Accordingly, we observed that when histamine release was reduced there was a concomitant increase of a potassium efflux of 1st order, i.e. a flux of potassium not K+ exchange. Whether involved in HA+* this ‘excess’ potassium escaped via newly opened channels, as suggested in Fig. 9 b, or passed as
180
B. Uvnas et al.
‘an overflow ’ via the granule-containing channels together with the released histamine, remains to be established.
APPENDIX Determination of R,,,
F = Foe-K REFERENCES
\
from :
1ml
C m l = I/ F, and K are determined from semilogarithmic plotting of F against .\/ V
lox lox
PADAWAR, J. 1970. The reaction of rat mast cells to 2 polylysine. 3, Cell Biol 41, 332-372. F d r = Fo e - K \ ud v = F,SHORE, P.A., BURKHALTER, A. & COHNJR, V.H. 1959, Ec’ A method for the fluorimetric assay of histamine in z‘ = re in F,, tissues. 3 Pbarmacol Exp Therap 127, 182-186. CTVNAS, B. & ABORG, C.H. 1984. Cation exchange - a Fdz! common mechanism in the storage and release of biogenic amines in granules (vesicles?) 11. Comparative studies on sodium induced release of biogenic amines from the synthetic weak cation exchangers Amberlite IRC-50 and Duolite CS-100 = the area vup, F, F, and from biogenic (granule enriched) materials. Acta Phy$ol Scand 120, 87-97. For R,,, the triangle v2.F2/2 is added. UYNAS, B., ABORG,C.-H., LYSSARIDES, L. & THYBERG, J. 1985. Cation exchanger properties of isolated rat peritoneal mast cell granules. Acta Physiol Scand 125, 25-3). UVNAS,B., ABORG, G H . , LYSSARIDES, L. & DANIELSr SON, L.-G. 1989. Intracellular ion exchange between i C cytoplasmic potassium and granule histamine, an PO0 integrated link in the histamine release machinery of mast c5lls. Acta Physiol Scand 136, 309-320. To UVNAS,B., ABORG,C.-H., LYSSARIDES, L., THYBERG, E J. & DANIELSSON, L.-G. 1986. Rat mast cells superfused with isotonic solutions release histamine 100 probably via intracellular exchange K ’ e Hi+ ions. Acta Physiol Scand 128, 657-658.
1:
-E
0
