Histamine (H2) receptor-adenylate cyclase system in pig skin (epidermis)

150

Biochimica el Biophysica Acta, 437 (1976) 150--157

@ Elsevier Scientific Publishing Company, Amsterdam -- Prin'ted in The Netherlands

BBA 27938 HISTAMINE (H2) RECEPTOR-ADENYLATE CYCLASE SYSTEM IN PIG SKIN (EPIDERMIS)

HAJIME IIZUKA, KENJI ADACHI, KENNETH M. HALPRIN and VICTOR LEVINE Dermatology Service, Miami Veterans Administration Hospital and Department of Dermatology, University o f Miami School o f Medicine, Miami, Fla. 33125 (U.S.A.)

(Received January 6th, 1976)

Summary Histamine activated adenylate cyclase in pig skin {epidermal) slices, resulting in the accumulation of cyclic AMP. This effect was highly potentiated by the addition of cyclic AMP-phosphodiesterase inhibitors (theophylline, papaverine). A specific H~ receptor inhibitor (metiamide) inhibited the effect of histamine completely, while other antihistamines (diphenhydramine, acetophenazine, perphenazine, fluphenazine, promethazine) inhibited the effect of histamine to various lesser degrees. It has been shown that both epinephrine and prostaglandin E stimulate epidermal adenylate cyclase. Our data using specific blocking agents indicate that histamine, epinephrine and prostaglandin E2 act independently on the epidermal adenylate cyclase system.

Introduction

It has been well established that various peptide hormones and biogenic amines act on specific receptor sites on the cell membrane and activate the adenylate cyclase system. This causes an intracellular accumulation of cyclic AMP, which as the second messenger in the cell, regulates the cell metabolism to produce the specific response required by the original messenger at the cell surface. In skin {epidermis), we have shown that epinephrine and prostaglandins act independently on their respective specific receptor sites of adenylate cyclase [1,2]. In this comYnunication we report that histamine is a third specific activator of skin (epidermal) adenylate cyclase. Materials and Methods Skin slices were taken by a keratome adjusted to a 0.3 mm setting without the use of anesthesia. The skin thus obtained was predominantly epidermis

151 {80--90%) with a little contamination by dermis (10--20%). The slices were kept in Hank's medium at 4°C until use. The slices were then cut into 5 X 5 mm squares and preincubated in the same medium for 15 min at 37°C. This preincubation procedure is necessary to standardize the initial cyclic AMP level in the epidermal pieces [2,3]. After the preincubation two pieces of epidermal slices were randomly selected and floated with their keratin layers up in Hank's media containing different concentrations of the drugs and chemicals to be tested. After incubation at 37°C in a water bath (for 5 min unless otherwise stated), they were quickly frozen between two plates of dry ice. The cyclic AMP contents in these skin slices were measured by Gilman's protein-binding m e t h o d [4] with minor modification [3,5]. The binding assay was done in duplicate. The cyclic AMP accumulated after the histamine activation was completely digested by the commercial cyclic AMP-phosphodiesterase. Protein concentration was measured by the m e t h o d of Lowry et al. [6]. Chemicals and drugs were all prepared fresh before each experiment and the pH of the media was adjusted to 7. Epinephrine was the product of Parke Davis (Detroit, Mich.), perphenazine and acetophenazine dimaleate of Schering (Bloomfield, N.J.) fluphenazine dihydrochloride of White Lab. (Kenilworth, N.J.), and promethazine hydrochloride of Wyeth (Philadelphia, Pa.). Metiamide was a kind gift from Dr. Brimblecombe, Smith, Kline and French Lab., U.K. All other chemicals were purchased from Sigma Chemical Co. (St. Louis, Mo.). Results

Effect of histamines The changes in the intracellular content of cyclic AMP in response to histamine are shown in Fig. 1. The intracellular cyclic AMP concentration increases rapidly, reaches its maximal level by 5 min and gradually decreases over 60 min. This effect was greatly potentiated by the addition of theophylline (5 mM) or another p o t e n t cyclic AMP-phosphodiesterase inhibitor, papaverine at 250 pM {Table I). Papaverine alone or theophylline alone has little effect on the cyclic AMP content. This potentiation effect by theophylline and papaverine reaches its saturating level at 5 mM and 250 pM respectively. The combination of 5 mM theophylline and 250 pM papaverine with histamine shows no further increase in the cyclic AMP level beyond that given by theophylline alone. The data suggest that theophylline and papaverine act at the same site (i.e. the phosphodiesterase enzyme). The effect of different concentrations of histamine is showns in Fig. 2. The increase in cyclic AMP was dose dependent and was linear up to a histamine concentration of about 50 pM, then the rate of stimulation decreased. The maximal effect was obtained at 200 pM. Lineweaver-Burk plots (the insert in Fig. 2) show the apparent K a for histamine was about 6 X 10 -s M.

Effects of antihistamines The effects of antihistamines are summarized in Table II. All antihistamines showed various degrees of inhibition of the accumulation of cyclic AMP upon the action of histamine. Among them an ethanolamine derivative (diphenhydra-

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153 TABLE I THE EFFECTS OF THEOPHYLLINE

AND PAPAVERINE

T h e c y c l i c A M P i n c r e a s e d u r i n g t h e 5 m i n i n c u b a t i o n p e r i o d is s h o w n . T h e r e s u l t s are t h e a v e r a g e o f two experiments. Additions

Cyclic AMP (pmol/mg protein)

None 0 min 5 rain Theophylline 5 mM Papaverine 250 pM H i s t a m i n e 1 0 0 ~zM Histamine 100 pM + papaverine 250 #M 500 pM + theophylline 5 raM 10 m M + p a P a v e r i n e 2 5 0 ~tM + theophylline 5 mM

2.2 1.0 4.3 3.6 36.1 61.1 65.0 79.3 85.5 77.5

mine) had the weakest and the recently developed H2 receptor inhibitor (metiamide) had the strongest inhibitory effect. Phenothiazines * (perphenazine, fluphenazine, acetophenazine, promethazine) had various degrees of inhibitory effect, and within this category promethazine was the strongest inhibitor. Since the above comparison was made only at 1 mM each of the drugs, the inhibitory effects of 3 representative antihistamines were studied at various concentrations (Fig. 3). Diphenhydramine had a relatively small inhibitory effect; i.e. the concentration increase from 10 pM to 1 mM did not cause much inhibition. On the contrary, a strong inhibitory effect was observed with metiamide, which completely blocked the effect of histamine at the concentration of 100 pM. The effect of acetophenazine had a midway effect of both other drugs.

T A B L E II THE EFFECTS OF ANTIHISTAMINES

O N T H E C Y C L I C A M P A C C U M U L A T I O N IN P I G E P I D E R M I S

Additions

Cyclic AMP (pmol/mg protein)

None 0 rain 5 rain Histamine (100 pM) + p e r p h e n a z i n e (1 r a M ) + f l u p h e n a z i n e (1 r a M ) + a c e t o p h a n a z i n e (1 m M ) + p r o m e t h a z i n e (1 m M ) + d i p h e n h y l d r a m i n e (1 r a M ) + m e t i a r a i d e (1 r a M )

1.0 2.5 41.0 18.3 22.5 8.5 4.2 25.6 1.8

* P a l m e r a n d M a n i a n [ 7 ] r e p o r t e d t h a t p h e n o t h i a z i n e d e r i v a t i v e s m i g h t h a v e a d i r e c t i n h i b i t o r y act i o n o n t h e a d e n y l a t e c y c l a s e s y s t e m in r a t b r a i n .

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F i g . 3. T h e e f f e c t s o f a n t i h i s t a m i n e s a t v a r i o u s c o n c e n t r a t i o n s . T h e e x p e r i m e n t a l c o n d i t i o n w a s t h e s a m e as i n T a b l e I I e x c e p t t h a t d i f f e r e n t c o n c e n t r a t i o n s o f a n t i h i s t a m i n e s w e r e a d d e d . X = d i p h e n h y d r a m i n e ; = a c e t o p h e n a z i n e ; a n d :) = m e t i a m i d c .

Interrelationship glandin E2

between the effects of histamine, epinephrine and prosta-

The effects of histamine, epinephrine, and their combination on the cyclic AMP levels are shown in Table III. In this experiment, histamine and epinephrine were used at their saturation concentrations (cf. Fig. 2 and ref. 2). The simultaneous addition of both drugs caused a greater increase of cyclic AMP than the single addition of either drug alone. This increase in cyclic AMP by the

T A B L E III THE EFFECTS OF EPINEPHRINE, PROPRANOLOL, ltISTAMINE, G L A N D I N E 2 ON T H E C Y C L I C A M P L E V E L IN P I G E P I D E R M I S

METIAMIDE

AND

PROSTA-

D e t a i l s o f e x p e r i m e n t a l c o n d i t i o n s are as i n t h e t e x t . C o n c e n t r a t i o n s o f t h e d r u g s a d d e d t o t h e m e d i a w e r e e p i n e p h r i n e = 5 0 p M , p r o p r a n o l o l = 5 0 p M , h i s t a m i n e = 1 raM, m e t i a m i d e = 5 0 0 /~M, p r o s t a g l a n d i n E 2 = 5 0 # M . R e s u l t s are t h e a v e r a g e o f t w o o r t h r e e e x p e r i m e n t s . N o c y c l i c A M P p h o s p h o d i e s t e r a s e i n h i b i t o r s were a d d e d in these series of e x p e r i m e n t s . Additions

Cyclic AMP (pmol/mg protein)

None 0 rain 5 rain E p i n e p h r i n e (A) H i s t a m i n e (B) S u m (A + B) Epinephrine + histamine Epinephrine + mettamide Histamine + propranolol Histamine + metiamidc Prostaglandin E 2 Prostaglandin E 2 + metiamide Metiamide

1.2 0.8 18.1 33.3 51.4 54.6 16.5 33.0 0.8 8.2 7.5 1.0

155 addition of the two drugs was about equal to the sum of the increases due to epinephrine and histamine added singly. Table III also shows that the effect of epinephrine is not inhibited by the addition of metiamide, a drug which completely blocked the effect of histamine. Conversely, the activation by histamine was not inhibited by the addition of propranolol (a potent/~-adrenergic blocker) which inhibited the effect of epinephrine completely. Metiamide had little effect on the cyclic AMP increase caused by prostaglandin E2. Metiamide alone had little effect on the accumulation of cyclic AMP.

The effects of related compounds The following related compounds were tested at 1 mM each concentration except for 48/80 at 100 pg/ml (data not shown). C o m p o u n d 48/80 (a potent histamine releaser) had little effect on the cyclic AMP concentration. Aminoguanidine (a p o t e n t histaminase inhibitor) had also little effect. Histidine (a precursor of histamine) slightly increased the concentration of cyclic AMP, but imidazole acetate (the degradation product) had no effect. Other monoam~nes such as dopamine or serotonin showed a slight effect on the concentration of cylcic AMP. Since imidazole is known as a cyclic AMP phosphodiesterase stimulator, we incubated the epidermal slices with histamine (100 pM) and imidazole (1 mM) together. The simultaneous addition of histamine and imidazole caused only slightly less cyclic AMP accumulation than the addition of histamine alone did. Imidazole does not appear to be a phosphodiesterase stimulator in this pig skin system. Discussion

Histamine-adenylate cyclase system in pig epidermis It is apparent that histamine can cause a rapid accumulation of cyclic AMP in epidermis. This is the consequence Of the activation of adenylate cyclase by histamine. The possibility that the accumulation of cyclic AMP was due to inhibition of cyclic AMP-phosphodiesterase by histamine is very unlikely. If this was a mechanism of action of histamine, in the presence of other phosphodiesterase inhibitors such as theophylline and papaverine histamine should have less effect; instead it gave a further increase in the cyclic AMP level. In addition, histamine showed no effect on the phosphodiesterase activity when it was assayed with skin homogenate as enzyme source (ref. 8 and also our unpublished observation). Is the histamine-adenylate cyclase system specific and independent of the epinephrine- or prostaglandin E-adenylate cyclase system? Our data summarized in Table indicate that the histamine receptor is specific and independent. Namely (1) the cyclic AMP increase due to the simultaneous addition of histamine and epinephrine is equal to the sum of the increases due to the single addition of histamine and epinephrine (at their respective saturation concentrations). (2) The effect of histamine was not inhibited by the ~-adrenergic blocker (propranolol). (3) The cyclic AMP increase due to epinephrine addition was not inhibited by an antihistamine (metiamide), which completely inhibited the activation by histamine. (4) The cyclic AMP accumulation caused by prostagtandin E2 was not inhibited by metiamide.

156 Recently, two different histamine receptors, H~ and H2, have been recognized [9--11]. The H~ receptor is antagonized by classical antihistamines such as diphenhydramine, pyrilamine, and their analogs. It is responsible for the contraction of bronchial and ileal smooth muscle. The H~ receptor is not affected by the antagonist of diphenhydramine series but is specifically antagonized by metiamide and burimamide, both of which have been recently synthesized as thiourea derivatives of histamine. The H2 receptor mediates gastric acid secretion and arterial rate stimulation. Our data strongly suggest that the histamine receptor of pig epidermis is so-called "H2"

Possible physiological significance of the histamine H2-adenylate cyclase sytem of epidermis Our studies have shown that histidine, a precursor of histamine, did not cause an increase in cyclic AMP. Furthermore 48/80, a histamine releaser, did not cause an increase in cyclic AMP. These studies indicate that the epidermis can neither synthesize nor release enough histamine to cause an increase in the epidermal cyclic AMP level. One would suppose then that under normal physiological conditions histamine does not activate epidermal adenyl cyclase. The bulk of the histamine in skin resides in mast cell granules in the dermis [12,13]. The histamine content of the dermis is substantial (10--20 pg/gm wet weight) [14]. If released this might allow a concentration of about 100 pM histamine to reach the epidermis, an a m o u n t which is sufficient to stimulate cyclic AMP formation. Therefore, in an abnormal physiological state characterized by the release of histamine from dermal mast cells, one might expect a rise in epidermal cyclic AMP. What effect might such an increase in epidermal cyclic AMP have? The evidence on this point is contradictory. Voorhees et al. [15] reported that in mouse ear skin slices, histamine caused an acceleration of cells from G2 into mitosis. This is peculiar since epinephrine and dibutyryl cyclic AMP (which also presumably act like histamine to increase the intracellular cyclic AMP level) caused an inhibition of G2. Flaxman et al. [16] using human skin in an in vitro model reported that dibutyryl cyclic AMP, epinephrine, and histamine inhibited cells in the G2 phase of the cell cycle. Eaglstein and Weinstein [17] reported that the intradermal injection of histamine into humans led to a slight increase in cells which incorporated tritiated thymidine 48 h later. This question then cannot be answered at present. Perhaps one should also cite the seeming lack of any epidermal change in the wheals of an urticarial reaction, probably the most potent and dramatic of histamine release phenomena in the skin. Acknowledgements This work was supported in part by Grant AM 17179 of National Institutes of Health and Dermatology Foundation of Miami.

157 References 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17

Adachi0 K., Yoshikawa, K., Halprin, K.M. and Levine, V. (1975) Br. J. Dermatol. 9 2 , 3 8 1 - - 3 8 8 Yoshikawa, K., Adachi, K., Halprin, K.M. and Levine, V. (1975) Br. J. Dermatol. 93, 29--36 Yoshikawa, K., Adachi, K., Halprin, K.M. and Levine, V. (1975) Br. J. Dermatol. 92, 249--254 Gilman, A.G. (1970) Proc. Natl. Acad. Sci. U.S. 6 7 , 3 0 5 - - 3 1 2 Yoshikawa, K., Adachi, K., Levine, V. and Halprin, K.M. (1975) Br. J. Dermatol. 92, 241--248 Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J. (1951) J. Biol. Chem. 193, 265--275 Palmer, G.C. and Manian, A.A. (1974) Neuropharmacology 1 3 , 6 5 1 - - 6 6 4 Marks, F. and Raab, I. (1974) Biochim. Biophys. Acta 334, 368--377 Ash, A.S.F. and Schild, H.O. (1966) Br. J. Pharmacol. Chemother. 27,427---439 Black, J.W., Duncan, W.A.M., Durant, C.J., GaneUin, C.R. and Parsons, E.M. (1972) Nature 236, 385--390 Black, J.W., Durant, C.J., E m m e t t , J.C. and GaneUin, C.R. (1974) Nature 248, 65--67 Riley, J. and West, G.B. (1952) J. Physiol. Lond. 119, 15--23 Riley, J. and West, G.B. (1953) J. Physiol. Lond. 120, 528--537 R o t h m a n , S. (1954) in Physiology and Biochemistry of the Skin, p. 96, The University of Chicago Press, Chicago Voorhees, J.J., Duell, D.A., Bass, L.J., PoweU, J.A. and Harrell, E.R. ( 1 9 7 2 ) J . Invest. Dermatol. 59, 11 4--120 F l a x m a n , B.A. and Harper, R.A. (1975) J. Invest. DermatoL 65, 52--59 Eaglstein, W.H. and Weinstein, G.D. (1975) J. Invest. Dermatol. 64, 386--389