THE JOURNAL OF iNVESTIGATIVE DERMATOLOGY , 69:442-445, 1977 Copyright ttl 1977 by The Williams & Wilkins Co.

Vol. 69, No. 5 Printed in U.S.A.

EPIDERMAL ADENYLATE CYCLASE: STIMULATION OF THE HISTAMINE (H2)

RECEPTOR BY TOLAZOLINE

HAJIME hzuKA, M.D. , KENJJ ADACHI , M.D .. PH.D., KENNETH M. HALPRIN, M.D., AND VICTOR LEVINE, B.Sc.

Veterans Administration Hospital and the Department of Dermatology. University of Miami School of Medicine, Miami. Florida, U.S. A .

Tolazoline (2-benzyl-2-imidazolineJ activated adenylate cyclase in pig epidermal slices resulting in the accumulation of cyclic AMP. This effect was highly potentiated by thl:! addition of the cyclic AMP-phosphodiesterase inhibitor, theophylline. Specific histamine (H2) receptor inhibitors (metiamide and cimetidine) completely blocked the tolazoline activation of adenylate cyclase. At low concentrations (10-100 J..I.M). a histamine (H ,) receptor inhibitor (diphenhydramine) and a ,a-adrenergic blocker (propranolol) did not inhibit this effect. The stimulation of cyclic AMP formation by the combination of tolazoline and histamine was about the same as the stimulation by histamine alone (nonadditive), whereas the stimulatory effects by tolazoline and epinephrine were additive. These data suggest that tolazoline, an ex-adrenergic blocker, a lso activates adenylate cyclase at the histamine (H2) receptor site which is distinct from the ,8-adrenergic receptor site. Another ex-adrenergic blocker, phentolamine, did not have this effect.

Tolazoline, a peripheral vasodilator and ex-adre­nergic blocking agent has been used to treat peripheral vascular diseases. Recently Yellin, Sperow, and Buck fl ] reported that gastric acid secretion and atrial stimulation of sinus rate in­duced by tolazoline were both antagonized by two specific histamine (H2) receptor antagonists, buri­mamide and metiamide. Since histamine (H 2)

responses have been linked to the generation of cyclic AMP in some tissues [2,3), including epider­mis [4], the data by Yellin, Sperow, and Buck prompted us to investigate the effect of tolazoline on epidermal adenylate cyclase. Our results re­ported in this communication suggest that tolazo­line can activate pig epidermal adenylate cyclase at the histamine (H2) receptor site and thereby cause an accumulation of cyclic AMP.

MATERIALS AND METHODS

The ell.-perimental procedures were essentially the same as described previously [5). Skin slices were taken from th e backs of domestic pigs by a keratome (Storz Instruments, St. Louis. Mo.) adjusted to a 0.3-rnm setting without anesthetics. The skin thus ob­tained was predominantly epidermis (80-90'*>· The slices were cut into 5 x 5 mm squares and preincubated in Hank's medium for 15 min at 37"C to standardize the initial cyclic AMP level in the epidermal squares [5,6]. After the preincubation, the epidermal squares

Manuscript received October 21, 1976; accepted for publication May 9, 1977.

This work was supported in part by grants from the National Institute of Health (AM 17179) and the Der­matolo~ Foundation of Miami.

Reprmt requests to: Dr. Adachi, Veterans Adminis­tration Hospital, 1201 Northwest 16th Street, Miami, Florida 33125.

442

were floa ted for 5 or 10 min at. 37"C in Hank's medium containing various compounds to be tested. At least two epidermal squares were used for each experiment. The cyclic AMP contents in t he epidermal squares were measured by Gilman's protein binding method 171 with minor modifications [6). The binding assay was done in duplicate. Protein was measured by the method of Lowry et al 18). Chemicals and drugs were all prepared fresh before each experiment and the pH of the incubation media was adjusted to 7. Tolazoline hydrochloride, and phentolamine mesylate were ob­tained from CIBA Pharmaceutical Co. (Summit, New J ersey), epinephrine was the product of Parke Davis (Detroit, Michigan). Metiamide, cimetidine, and 4-methyl histamine were a kind gift from Dr. Brimble­combe and Mr. Paul , Smith, Kline and French Lab. All other chemicals were purchased from Sigma Chem­ical Co. (St. Louis, Missouri).

RESULTS

Effect o(Tolazoline

Tolazoline caused an increase in the intracellu­lar cyclic AMP level (Fig 1). The maximal increase was attained by 5 min and then gTaduaJJy de­creased over 30 min. The addition of theophylline greatly potentiated the effect of tolazoline, and the cyclic AMP level reached its maximal level in a bout 10 min. Theophylline alone had little effect on the cyclic AMP content. The effect of different concentrations of tolazoline is shown in Fig 2. The incubation medium contained 5 mM theophylline and the incubation was carried out for 10 min. The increase in the intracellular cyclic AMP level was dose-dependent and the maximal effect was obtained at about 3 mM. Lineweaver-Burk plots (the insert of Fig 2) show the apparent Ka for tolazoline at about 1 mM (1.34 x lQ- 3 M) . This Ka

Nov. 1977

50

2 40 0

c.

u

u ,.... u

0

4

~------- ..

10 20 30 m1nutes

FIG 1. Time course of the effect of tolazoline on the intracellular cyclic AMP content. Details of the experi­mental condition are given in the "Materials and Meth­ods" section. The concentration of tolazoline added was 2 mM and that of theophylline 5 mM (0 tolazoline only; !:::, tolazoline + theophylline; and X = control (no addition).

I <10 I

~30 ~ co-E

... "' ~20· Q.

0.. ::0: <: ~ 10

u >. ...,

0

mM

1irvJ 0.1 -

0.05

0 1xJ03 2xJQ3 3xJo3 4xlo3 1{sJ

3 4

Frc 2. The effect of tolazoline concentration. Tissues were incubated with various concentrations of tolazo­line for 10 min. Theophylline !5 mM) was added to all the incubation media. The insert shows Lineweaver­Burk plots of the concentration curve.

value is much higher than that of histamine where the apparent Ka was about 6 X w-'· M [4]. Phentolamine (mesylate), another a-adrenergic blocking agent, had no effect on cyclic AMP accu­mulation up to a concentration of 4 mM (data not shown).

TOLAZOLINE ACTIVATION OF ADENYLATE CYCLASE 443

Effect of Histamine (H 1) and (H 2 ) Inhibitors

To assess the characteristics of the receptor for tolazoline, two specific histamine antagonists were used: metiarnide and cimetidine (a nonthi­ourea analogue ofmetiamide), both specific antag­onists of the fH2J receptor [9,10,11] and diphenhy­dramine, a classical antihistamine which antago­nizes the activation of the (H ,) receptor. Figure 3 shows the effect of increasing concentrations of metiarnide and cimetidine on the accumulation of cyclic AMP induced by 3 mM of tolazoline. Both metiamide and cimetidine clearly blocked the stimulatory effect of tolazoline in a dose-dependent manner. The 10 50 (concentration of the drug giving 50~ of its maximal inhibition) of metiamide and cimetidine were about 1 - 2 !J-M. Figure 4 shows the effects ofmetiamide, diphenhydramine, and propranolol (a J3-adrenergic receptor blocker) on the stimulation of adenylate cyclase by tolazo­line. At low inhibitor concentrations (below 100 !J-M) only metiamide was able to block the effect of tolazoline. At high inhibitor concentrations (above 100 !J-M) diphenhydramine and propranolol also inhibited the effect.

Interrelationship B etween the Effects of Tolazoline, H istamine and Epinephrine

The effects of tolazoline, histamine, epineph­rine, and their combinations on cyclic AMP levels are shown in the Table (A and Bl. In this experi­ment, all drugs were used at their saturation concentrations (c.f. , Fig 2 and references 4 and 5). The cyclic AMP accumulation by the simultaneous addition of tolazoline and epinephrine was about equal to the sum of the increases due to each drug

50

c:: (1.)

0 ..... a. 40

0"1 E

"' 30 (1.)

0 E a.

a.. 20 ~ <(

u

u 10 >-u

Frc 3. The effects of metiamide and cimetidine at various concentrations. Cyclic AMP contents were measured after the incubation with 3 mM tolazoline and various concentration of (H2) antagonists. Incuba­tion was done for 10 min with 5 mM theophylline. te , metiamide. 0, cimetidine). Abscissa indicates the con­centrations of (H2l antagonists, metiamide, and cime­tidine.

444 UZUKA ET AL

0

c: Qj

0

a. 30 00

E A -V'l A

Q)

0

g_ 20 a... ::E < ~ u >-u 10

O.uM lO .uM 100 JJM l mM

F1c 4 The effect of metiamide, d iphenhydramine. and propra nolol at various concentrations. The experimen­tal condition was the same as in Fig 3 except that different kinds of inhibitors were used . D = met1amide, a n antihistamine (H2); 0 = diphenhydra mine, a n an­tihistamine (H,); 6. = propra nolol , a {3-adrenergic blocker; X = control (no addition).

added singly; i.e., the effects were additive. On the other hand, the cyclic AMP accumulation by any combination of tolazoline and <H2 ) agonists, histamine or 4-methyl-histamine, does not exceed the stimulation due to histamine alone, i.e., the effects were nonadditive. The significance of the additive or nonadditive effects of drugs is dis­cussed below.

DISCUSSION

The present studies indicate that tolazoline can activate epidermal adenylate cyclase resulting in the a ccumulation of cyclic AMP. Since this effect was highly potent iated by the addition of theo­phylline, the accumulation of cyclic AMP by tola­zoline was not due to phosphodiesterase inhibition but to the stimulation of adenylate cyclase. We have previously shown [ 4] that the cyclic AMP increase in skin by the simultaneous addition of histamine and epinephrine was about equal to the sum of the increase due to each drug added singly, i.e. , histamine and epinephrine showed the "additive" effect. The data suggest the occur­rence of separate receptor sites for each drug, i.e., this histamine and ,a-adrenergic receptors. Pres­ent studies show that the stimulatory effects of tolazoline and epinephrine are "additive" but those of tolazoline and histamine (or 4-methyl-

Vol. 69, N o.5

The effect of tolazoline, epinephrine, histamine and 4-methy/-histamine on the cyclic AMP level in pig

epidermis

None 0 min 5 min

Additions

A. Tolazoline Epinephrine Tolazoline & epinephrine

B. Tolazoline 4-Methyl-histamine Histamine Tolazoline & 4-methyl-histamine Tolazoline & histamine 4-Methyl-histamine & histamine Tolazoline & 4-methyl-histamine

& hista mine

Cyclic AMP (pmoles/mg

protein)

1.1 0.7

25.1 17.8 48.2 29.3 38.8 48.4 49.3 53.0 45.4 52.0

Concentrations of drugs added were tolazoline = 5

mM, epinephrine = 50 p.M. histamine = 2 mM, a nd 4-methyl histamine = 1.6 mM. The incubation was car­ried out for 5 min at 37•c. No phosphodiesterase inhib­itors were added in this experiment.

histamine) are not "additive". Furthermore, the stimulatory effect by tolazoline was not counter­acted by propranolol, a ,a-adrenergic blocker. These results suggest that tolazoline acts on the histamine receptor site which is distinct from the ,a-adrenergic receptor site. Recently two different histamine receptors, (H 1l and CH~). have been recognized. Since the histamine activation of skin adenylate cyclase is blocked by low concentration of metiamide but not by diphenhydramine [4], the histamine adenylate cyclase system in skin is defined as histamine CH 2 J receptor site. The pres­ent studies also showed that the tolazoline activa­tion was completely blocked by metiamide <and also by cimetidine), but not by diphenhydramine at the low concentration. The results are consist­ent with a view that the tolazoline action on skin adenylate cyclase is mediated by the histamine CH2) receptor site.

The effect of tolazoline on the histamine cH2)

receptor appears to be not strong enough to cause full activation. The maximal activation by tolazo­line was much less than that produced by hista­mine. In our epidermal system the addition of histamine caused further accumulation of cyclic AMP even after the maximal stimulation by tola­zoline. Also, the activation constant for histamine (60 MM) was much lower tha n that of tolazoline ( 1

mM). These facts suggest that the "histamine­like" action of tolazoline is a weak one and proba­bly due to a weak affinity of tolazoline to the <H2)

receptor site. The weak affinity of tolazoline may also explain the apparently nonspecific inhibition of tolazoline stimulation by diphenhydramine and propranolol at high concentrations (1 mM).

Among the four receptor sites for activation of skin adenylate cyclase, ,a-adrenergic [5], prosta­glandin E [12], h istamine (H2) 14], and adenosine

Nou. 1977

[13), tolazoline appears to act through the hista­mine (H2 ) receptor. This study again stresses the multiple sites of action of drugs affecting the epidermal cyclic AMP system.

REFERENCES 1. Yellin TO, Sperow JW, Buck SH: Antagonism of

tolazoline by hlstamine H2-receptor blockers. Na­ture 235:561-563, 1975

2. Dousa TP, Code CF: Effect of histamine and its methyl derivatives on cyclic AMP metabolism in gastric mucosa and its blockade by an H2 receptor antagonist. J Clin Invest 53:334-337, 1974

3. McNeil JH, Verma SC: Blockade by burimamide of the effects of histamine and histamine a nalogs on cardiac contractility, phosphorylase activa­tion and cyclic adenosine monophosphate. J Pharmac Exp Ther 188:180-188, 1974

4. Iizuka H, Adachi K, Halprin KM, Levine V: His­tamine (H~> receptor adenylate cyclase system in pig skin (epidermis). Biochim Biophys Acta 473:150-157, 1976

5. Yoshikawa K, Adachi K, Halprin KM, Levine V: The effects of catecholamine and related com­pounds on the adenylate cyclase system in the

TOLAZOLINE ACTIVATION OF ADENYLATE CYCLASE 445 epidermis. Br J Derrnatol 93:29-36, 1975

6. Yoshikawa K, Adachi K, Halprin KM, Levine V: Cyclic AMP in skin: Effects of acute ischemia. Br J Derma to] 92:249-254, 1975

7. Gilman AG: A protein binding assay for adenosine 3 ', 5' cyclic monophosphate. Proc Nat] Acad Sci USA 67:305-312, 1970

8. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ: Protein measurement with folin phenol reagent. J Bioi Chern 193:265-275, 1951

9. Ash ASF, Schild HO: Receptors mediating some actions of histamine. Br J Pharmac Cbemother 27:427-439, 1966

10. Black JW, Duncan AM, Durant CJ, Ganellin CR, Parsons EM: Definition and antagonism of rus­tamine H2-receptors. Nature 236:385-390, 1972

11. Brimblecombe RW, Duncan WAM, Durant GJ , Emmett JC, Ganellin CR, Parsons ME: Cimeti­dine-A non-thiourea H2-receptor antagonist. J lnl Med Res 3:86-92, 1975

12. Adachi K, Yoshikawa K. Halprin KM, Levine V: Prostaglandins and cyclic AMP in epidermis. Br J Dermatol 92:381-388, 1975

13. lizuka H , Adacru K, Halprin KM, Levine V: Aden­osine and adenine nucleotides stimulation of skin (epidermal) adenylate cyclase. Biochim Biophys Acta 444:685-693, 1976