Biochimica et Biophysica Acta, 448 (1976) 599-606

© Elsevier/North-Holland Biomedical Press BBA 77471

E F F E C T OF A N T I H I S T A M I N E S A N D C H L O R P R O M A Z I N E ON T H E C A L C I U M - I N D U C E D H Y P E R P O L A R I Z A T I O N OF T H E A M P H I U M A RED CELL M E M B R A N E

GEORGE G,~RDOSa, ULRIK V. LASSENb and LEON PAPEb aDepartment of Cell Metabolism, National Institute of Haematology and Blood Transfusion, Budapest (Hungary) and bZoophysiologieal Laboratory B, University of Copenhagen, Copenhagen (Denmark)

(Received March 8th, 1976)

SUMMARY 1. It has previously been demonstrated that an increase in extracellular Ca 2 + concentration induces a transient increase in K + permeability and associated hyperpolarization of the red cell membrane of the giant salamander, Amphiuma means. This phenomenon is analogous to the Ca2+-induced KC1 loss observed in ATPdepleted human red cells and red cell ghosts. 2. Histamine, which enhances the Ca2+-induced K + loss from depleted human red cells, is without effect on this Ca2+-induced hyperpolarization of Amphiuma red cells. 3. Promethazine (10 #M) and mepyramine (1 mM), which inhibit the Ca/+induced K + loss in depleted human red cells, also block the Ca2+-related hyperpolarization of Amphiuma erythrocytes. 4. Chlorpromazine (25 pM), despite being a weak antihistamine, is equally effective in blocking the Ca2+-induced hyperpolarization of Amphiuma red cells. 5. Ionophore A23187 causes a large and sustained Ca z +/K +-dependent hyperpolarization even in the presence of normal (1.8 mM) concentrations of Ca 2 +. This hyperpolarization is relatively insensitive to chlorpromazine and promethazine. 6. The inhibition o f the Ca u +-induced hyperpolarization of the Amphiuma red cell membrane by chlorpromazine and promethazine may be related to their properties as local anaesthetics.

INTRODUCTION Following the initial observation of Gfirdos [1, 2] that calcium induces a net K ÷ loss from ATP-depleted human red cells, an extensive literature dealing with this phenomenon has developed [3]. In an extension of these studies Gfirdos and Sz/tsz [4] reported that histamine potentiated the effect of calcium whereas antihistamines as well as the tranquilizer chlorpromazine [5] blocked the effect. Abbreviation: MOPS, morleholinopropane sulphonate.

600 In the presence of Ca 2+, 42K+ was rapidly taken up by ATP-depleted (Blum and Hoffman [6], G~irdos et al. [7]) and propranolol-treated (Manninen [8]) erythrocytes and temporarily reached a cellular concentration higher than the medium. Glynn and Warner [9] have shown, based on computations, that a temporary hyperpolarization could give rise to a similar accumulation of 42K+. It has been shown that giant red cells from the salamander Amphiuma means are transiently hyperpolarized following an increase in the extracellular Ca 2 + concentration (Lassen et al. [10]). This hyperpolarization is caused by an increase in K + permeability of the membrane. Thus the Ca 2 +-dependent K + permeability change in Amphiuma red cells is analogous to that seen in human red cells. Therefore, these cells provide a suitable experimental system for investigating the effect of drugs on the electrical properties of red cell membranes. This study describes the effects of histamine, antihistamines, and chlorpromazine on the Ca 2 +-induced hyperpolarization of the Amphiuma red cell membrane. MATERIALS AND METHODS Red cells were obtained from mature specimens of Amphiuma means by cardiac puncture. The blood samples were immediately washed in approx. 50 volumes of cold Ringer's solution containing 118 m M Na +, 2.5 m M K +, 124 m M CI-, 1.8 m M Ca 2+ and I0 m M morpholinopropane sulphonate buffer (MOPS) titrated to p H 7.2 ("normal Ringer's solution"). The cells were then incubated in the same medium until used for potential measurements. In order to bring the cells into the Ca 2+ induced hyperpolarized state, they were transferred rapidly to a Ringer's solution with the following composition: 95 m M Na +, 2.5 m M K +, 128 m M CI-, 15 mM Ca 2+ and 10 m M MOPS, p H 7.2 ("15 m M Ca 2÷ Ringer's solution"). For measurement of the effect of various drugs, cells were transferred from normal Ringer's solution to a solution with an increased Ca 2 + concentration and the appropriate drug. Membrane potentials were compared to those obtained in Ringer's solution containing the same Ca 2+ concentration without the drug. Measurements of membrane potentials of single cells were performed with conventional microelectrodes (filled with 3 M KCI) mounted in a piezoelectric transducer which advanced the electrode tip 1 #m within 50/~s for penetration of the membrane. This technique as well as temperature control of the measuring chamber has been described in detail previously [11 ]. Stock solutions of ionophore were prepared by dissolving the ionophore in a mixture of 1 : 9 acetone/ethanol (99 ~o)- The final concentration of acetone/ethanol in cell suspensions to which the ionophore was added was usually 0.5 )~, and never more than 1.0 ~ . RESULTS The distribution of measured potentials obtained from a representative group of ,4mphiuma red cells suspended in normal Ringer's solution is shown in Fig. 1A. The mean value of the potentials is --15 inV. When these cells are transferred to solutions with increased concentrations of Ca 2+ they undergo a transient hyperpolarization. The distribution of measured potentials recorded within the first three

601 %

A.

%

B.

!

60-

60

~0-

t,0

20-

20 r----'---v----

-20 CONTROL

-/,0

-60 rnV

-20

-t,0

-60mV

15 mM Ca

Fig. 1. The effect of an increase in extracellular concentration of Ca 2 + on the distribution of measured m e m b r a n e potentials in single Amphiuma red cells. A, Potential distribution in control Ringer's solution containing 1.8 m M Ca 2 ÷. B, Potential distribution after transferring cells from control Ringer's solution to Ringer's solution containing 15 m M Ca 2+. D a t a was obtained within the first three minnutes after transfer to 15 m M Ca 2+ Ringer's solution. Ordinate: fraction of cells having a potential within a 10 mV interval (indicated by the abscissa). 17 °C, p H 7.2.

min after cells are transferred to 15 m M Ca 2+ Ringer's solution is shown in Fig. lB. As can be clearly seen, the distribution is bimodal with the measured potentials being grouped around --20 and --40 to --70 mV. This distribution pattern has been discussed by Lassen et al. [10] in terms of an all-or-none effect of Ca 2 + on K + permeability of the Amphiumared cell membrane. In addition it has been shown that the threshold concentration of Ca 2+ required to induce hyperpolarization of these cells at p H 7.2 is 6 mM. As reported by Gfirdos and Szfisz [4] histamine enhances the Ca 2 +-induced K + loss from depleted human red cells. The equivalent effect of histamine in Amphiuma red cells would be an increase in the fraction of hyperpolarized cells under the influence of increased external Ca z+ concentration. Such an effect would be most clearly demonstrated in the presence of a threshold concentration of Ca 2 + (6 mM). Individual potential recordings obtained within the first three min after suspending cells in 6 m M Ca z + Ringer's solution with histamine were plotted as a function of histamine concentration in Fig. 2. No increase in the fraction of cells being hyperpolarized can be demonstrated with rising histamine concentrations in the range 0.05-2 mM. Although histamine is without apparent effect in the present system the effect of antihistamines was investigated. Cells were transferred from normal Ringer's solution to 15 m M Ca 2+ Ringer's solution plus 10 #M promethazine and potentials were measured within the first three min. Fig. 3 shows the relative distribution of the obtained potential values. Comparison of this figure with Fig. 1 indicates that there is almost complete blockage of the hyperpolarization associated with 15 m M Ca 2+. In the light of the above results membrane potentials were measured in 15 m M Ca 2 + Ringer's solution containing both 10 #M promethazine and 5 m M histamine. The results are shown in Fig. 4 and indicate that histamine is unable to prevent promethazine from blocking hyperpolarization. The typical antihistamines are substituted ethylamines of which promethazine is an example. Another drug of this type is mepyramine which is chemically very different from promethazine (see e.g. Goodman and Gilman [12]). 1.0 m M mepyramine employed under similar conditions to those

602 HISTAMINE CONCENTRATION

mM 0.I

0.5

I

5

10

L

i

i

i

J

.x -20 ¸

I

-40

I.

-60

mV Fig. 2. Measured m e m b r a n e potentials of single cells suspended in 6 m M Ca 2+ Ringer's solution as a function of extracellular histamine concentration. Potentials were measured within the first three minutes after transfer to the higher Ca 2+ concentration, Abscissa: histamine concentration in m M (log scale). Ordinate: membrane potential in mV. 17 °C, p H 7.2.

%

-20

-40

-60mV

15rnM Ca *10

p,M

PROMETHAZINE

Fig. 3. Distribution of measured membrane potentials of single cells suspended in 15 m M Ca 2÷ Ringer's solution with 10/*M promethazine. D a t a from the initial three minutes of exposure. Ordinate: fraction of cells with potentials in a 10 mV interval (indicated by the abscissa). 17 °C, p H 7.2.

603 m e n t i o n e d a b o v e resulted in t o t a l b l o c k a g e o f the Ca 2+ i n d u c e d h y p e r p o l a r i z a t i o n , whereas 0.1 m M m e p y r a m i n e was w i t h o u t significant effect. P r o m e t h a z i n e shares its p h e n o t h i a z i n e structure with a n u m b e r o f other p h a r m a c o l o g i c agents, one o f which is the a n t i p s y c h o t i c drug, c h l o r p r o m a z i n e . A l t h o u g h c h l o r p r o m a z i n e has at best a w e a k a n t i h i s t a m i n e action, it has a n u m b e r o f p h a r m a c o l o g i c effects in c o m m o n with p r o m e t h a z i n e a n d o t h e r local anaesthetic a m i n e s [12, 13]. T h e d i s t r i b u t i o n o f m e a s u r e d p o t e n t i a l s in the presence o f 10/~M c h l o r p r o m a z i n e is s h o w n in Fig. 5. It can readily be seen t h a t the a c t i o n o f chlorp r o m a z i n e in b l o c k i n g the C a Z ÷ - i n d u c e d h y p e r p o l a r i z a t i o n o f the Amphiuma r e d cell m e m b r a n e is similar t o t h a t o f p r o m e t h a z i n e . Fig. 6 is a plot o f individual m e a s u r e d p o t e n t i a l s as a f u n c t i o n o f c h l o r p r o m a z i n e c o n c e n t r a t i o n o f the m e d i u m (log scale). W h e n the c o n c e n t r a t i o n o f c h l o r p r o m a z i n e exceeds 1 0 / t M there is a t o t a l i n h i b i t i o n %

~0 20

-20

-40

-60

mY

15 mM Ca • 10 I~M PROMETHAZINE + 5 mM HISTAMINE

Fig. 4. Distribution of measured membrane potentials of single cells suspended in 15 mM Ca 2+ Ringer's solution containing both promethazine (10b, M) and histamine (5 raM). Data from the initial three minutes of exposure. Ordinate: fraction of cells with potentials in a 10 mV interval (indicated by the abscissa). 17 °C, pH 7.2.

% 80 60 40 20

-20

-40

- 6 0 mV

IS mM Co. ÷10pM CHLORPROMAZINE Fig. 5. Distribution of measured membrane potentials of single cells suspended in 15 mM Ca 2+ Ringer's solution with 10 #M chlorpromazine. Data from the initial three minutes of exposure. Ordinate: fraction of cells with potentials in a 10 mV interval (indicated by the abscissa). 17 °C, pH 7.2.

604

CHLORPROMAZINE CONCENTRATION klM ! =,

r -20

;

5

10

*

i

50 &-

100 J

;.

!

|

I

i

-t,O

:

i

-60

rnV Fig. 6. M e a s u r e d m e m b r a n e potentials o f single cells s u s p e n d e d in 15 m M Ca z + R i n g e r ' s solution as a f u n c t i o n o f extraeellular c h l o r p r o m a z i n e concentration. Abscissa: c h l o r p r o m a z i n e concentration in /~M (log scale). Ordinate: m e m b r a n e potential in inV. 17 °C, pI-I 7.2.

A 23187

%

25)JM CHLORPROMAZINE * A 23187

~o 20 -20

-t.o

-6Q -80 mV

-20

-t,0

-60

-80

rnV

50 tJM CHLORPROMAZINE 60

+A 23187

I -20

-/,0

-60

-80 mV

-20

o -40

-60

-80 mV

Fig. 7. D i s t r i b u t i o n o f m e a s u r e d m e m b r a n e potentials o f single cells s u s p e n d e d in R i n g e r ' s solution c o n t a i n i n g control c o n c e n t r a t i o n o f C a 2 + (1.8 m M ) and: A, 1.9 • 1 0 - 5 M divalent cation i o n o p h o r e A23187; B, 1.9 • 10 - s M A23187 p l u s 2 5 / t M c h l o r p r o m a z i n e ; C, 1.9 • 10 - 2 M A23187 plus 50,uM p r o m e t h a z i n e ; D, 1.9 • 1 0 - s M A23187 plus 50 # M c h l o r p r o m a z i n e . Ordinates: fraction o f cells with potentials in a 10 m V interval (indicated by the abscissae). 17 °C, p H 7.2.

605

of the hyperpolarization normally seen upon suspension of cells in Ringer's solution with 15 mM Ca 2+. The results described above, while clearly indicating that chlorpromazine blocks the Ca2+-induced hyperpolarization, do not make it possible to determine whether the drug acts to prevent entry of Ca 2+ or whether it blocks the internal action of Ca 2+ on the K + permeability mechanism. In an effort to distinguish between these modes of action, cells were exposed to the ionophore A23187 which has been shown to transport Ca 2 + across the plasma membrane [14]. The distribution of membrane potentials in Amphiuma red cells measured in normal Ringer's solution (1.8 mM Ca 2+) containing 1.9 • 10 -5 M A23187 is shown in Fig. 7A. It can be seen that practically all of the cells are hyperpolarized. This hyperpolarization which requires Ca 2+ in the suspending medium and is blocked by high external K + concentrations (unpublished data) closely resembles the hyperpolarization induced by an increase in extracellular Ca 2+ concentration (see Fig. 1B). The corresponding distribution of membrane potentials measured in cells suspended in Ringer's solution containing chlorpromazine or promethazine after having been first washed in A23187 Ringer's solution can be seen in Figs. 7B, C and D. These results indicate that chlorpromazine at a concentration of 50 #M resulted in a partial blockage of the ionophore induced hyperpolarization. This partial blockage may be due to inhibition by chlorpromazine of Ca 2+ transport via the ionophore. Promethazine at a concentration (50 #M) capable of totally blocking hyperpolarization elicited by 15 m M Ca 2+ left the hyperpolarization induced by A23187 unaffected. DISCUSSION The Ca2+-induced increase in K + permeability and associated hyperpolarization of the Amphiumared cell membrane is analogous to the CaZ+-dependent K + loss in depleted human red cells (Lassen et al. [10]). Gfirdos and Sz~isz [4] demonstrated that low concentrations of histamine (0.5 mM) stimulated, and higher concentrations of histamine inhibited the Ca2+-induced net K + loss from depleted human erythrocytes. Neither of these effects could be demonstrated in the Amphiuma red cell under the present experimental conditions. Sz~sz [15] has reported that the Ca 2+ influx rate is not increased by histamine in ATP-depleted human red cells. In the case of the Amphiuma cells used in the present study the intracellular concentrations of ATP are 3-5 times higher than that of non-depleted human red cells (unpublished observations). Therefore inadequate ATP levels cannot be the reason for the lack of a histamine effect in Amphiumared cells. In non-depleted human red cells a Ca2+-dependent K + loss can be provoked by the fl-blocking agent propranolol. Like histamine, this drug is also without effect on the membrane potential of the Amphiuma red cells (unpublished observations). There is however a marked inhibitory effect of antihistamines on the Ca 2+ induced hyperpolarization of the Amphiuma red cell membrane. The fact that promethazine is capable of inhibiting the hyperpolarization of these cell membranes, while histamine is without effect, raises the question as to whether this drug is acting as an "electrical membrane stabilizer" [13] (local anaesthetic) rather than solely as an antihistamine. Chlorpromazine which has a weak antihistamine effect is as effective as promethazine in inhibiting hyperpolarization. Both promethazine and chlorpromazine

606 are p h e n o t h i a z i n e derivatives a n d act as local anaesthetics [13]. C h l o r p r o m a z i n e is c a p a b l e o f b l o c k i n g the C a 2 +-induced h y p e r p o l a r i z a t i o n s a n d at high c o n c e n t r a t i o n s p a r t i a l l y abolishes the h y p e r p o l a r i z a t i o n caused by the i o n o p h o r e A23187. This o b s e r v a t i o n is consistent with the w o r k o f K w a n t a n d S e e m a n [16], as well as others [17], who have shown t h a t c h l o r p r o m a z i n e causes a d i s p l a c e m e n t o f m e m b r a n e b o u n d Ca 2 + f r o m the fixed negative sites. It is o f interest to note t h a t the c o n c e n t r a t i o n s o f c h l o r p r o m a z i n e t o prevent C a 2 + - i n d u c e d h y p e r p o l a r i z a t i o n in Amphiuma r e d cells are essentially the same as those required t o b l o c k nerve c o n d u c t i o n [13]. F o r the p u r p o s e s o f c o m p a r i s o n , if one assumes a u n i f o r m d i s t r i b u t i o n in the extracellular fluid o f an average person, i n t r a m u s c u l a r injection o f the lowest effective dose o f 100 m g c h l o r p r o m a z i n e [12] results in a p l a s m a c o n c e n t r a tion o f a b o u t 20 p M . ACKNOWLEDGEMENTS I o n o p h o r e A23187 was k i n d l y d o n a t e d by Ely Lilly a n d Co. C h l o r p r o m a z i n e was d o n a t e d b y N o v o Industries A/S. F u r t h e r m o r e , we a p p r e c i a t e the skillful technical assistance o f K i r s t e n A b e l a n d Pia A n t h o n s e n . F i n a l l y we wish to a c k n o w l e d g e the s t i m u l a t i n g discussions with Dr. I. Sz~isz c o n c e r n i n g various aspects o f the work. S u p p o r t e d b y G r a n t 511-1305 f r o m the D a n i s h Science R e s e a r c h Corencil. REFERENCES 1 G~irdos, G. (1958) Biochim. Biophys. Acta 30, 653-654 2 G~irdos, G. (1959)Acta Physiol. (Budapest) 15, 121-125 3 Riordan, J. R. and Passow, H. (1973) in Comparative Physiology (Bolls, L., Schmidt-Nielsen, K. and Maddrell, S. I-L P., eds.), pp. 543-581, North-Holland, Amsterdam 4 Gfirdos, G. and Szfisz, 1. (1968) Acta Biochim. et Biophys. Acad. Sci. Hung. 3, 13-27 5 Sz~isz,I. and G~irdos, G. (1974) FEBS Lett. 44, 213-216 6 Blum, R. M. and Hoffman, J. F. (1971) J. Membrane Biol. 6, 315-328 7 G~rdos, G., Sz~isz,I. and Sarkadi, B. (1975) in Biomembranes: Structure and Function (Gardos G. and Sz~sz, I., eds.), pp. 167-180, North-Holland, Amsterdam 8 Manninen, V. (1970) Acta Physiol. Scand. Suppl. 355, 1-76 9 Glynn, J. M. and Warner, A. E. (1972) Br. J. Pharmac. 44, 271-278 10 Lassen, U. V., Pape, L. and Vestergaard-Bogind, B. (1976) J. Membrane Biol. 26, 51-70 11 Lassen, U. V., Pape, L. and Vestergaard-Bogind, B. (1974) J. Membrane Biol. 18, 125-144 12 Goodman, L. S. and Gillman, A. (1970) in The Pharmacological Basis of Therapeutics, pp. 155-169, 635--644, Macmillan, London 13 Seeman, P. (1972) Pharrnac. Rev. 24, 583-655 14 Reed, P. W. and Lardy, H. A. (1972) in The Role of Membranes in Metabolic Regulation (Mildman, M. A. and Hanson, R. W., eds.), pp. 111-131, Academic Press, New York 15 Sz~isz, I. (1972) Acta Biochirn. et Biophys. Acad. Sci. Hung. 7, 335-339 16 Kwant, W. O. and Seeman, P. (1969) Biochirn. Biophys. Acta 193, 338-349 17 Bondani, A. and Karler, R. (1970) J. Cell Physiol. 75, 199-211

Effect of antihistamines and chlorpromazine on the calcium-induced hyperpolarization of the Amphiuma red cell membrane.

Biochimica et Biophysica Acta, 448 (1976) 599-606 © Elsevier/North-Holland Biomedical Press BBA 77471 E F F E C T OF A N T I H I S T A M I N E S A N...
396KB Sizes 0 Downloads 0 Views