THE JOURNAL OF iNVESTIGATIVE DERMATOLOGY, 69:442-445, 1977 Vol. 69, No. 5 Copyright ttl 1977 by The Williams & Wilkins Co. Printed in U.S.A. EPIDERMAL ADENYLATE CYCLASE: STIMULATION OF THE HISTAMINE (H2) RECEPTOR BY TOLAZOLINE HAJIME hzu KA , 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, also 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-adre­ were floated for 5 or 10 min at. 37"C in Hank's medium nergic blocking agent has been used to treat containing various compounds to be tested. At least peripheral vascular diseases. Recently Yellin, two epidermal squares were used for each experiment. The cyclic AMP contents in the epidermal squares Sperow, and Buck fl ] reported that gastric acid were measured by Gilman's protein binding method 171 secretion and atrial stimulation of sinus rate in­ with minor modifications [6). The binding assay was duced by tolazoline were both antagonized by two done in duplicate. Protein was measured by the method specific histamine (H2) receptor antagonists, buri­ of Lowry et al 18). Chemicals and drugs were all mamide and metiamide. Since histamine (H 2) prepared fresh before each experiment and the pH of responses have been linked to the generation of the incubation media was adjusted to 7. Tolazoline cyclic AMP in some tissues [2,3), including epider­ hydrochloride, and phentolamine mesylate were ob­ mis [4], the data by Yellin, Sperow, and Buck tained from CIBA Pharmaceutical Co. (Summit, New prompted us to investigate the effect of tolazoline J ersey), epinephrine was the product of Parke Davis on epidermal adenylate cyclase. Our results re­ (Detroit, Michigan). Metiamide, cimetidine, and 4- methyl histamine were a kind gift from Dr. Brimble­ ported in this communication suggest that tolazo­ combe and Mr. Paul, Smith, Kline and French Lab. line can activate pig epidermal adenylate cyclase All other chemicals were purchased from Sigma Chem­ at the histamine (H2) receptor site and thereby ical Co. (St. Louis, Missouri). cause an accumulation of cyclic AMP. RESULTS MATERIALS AND METHODS Effect o(Tolazoline The ell.-perimental procedures were essentially the same as described previously [5). Skin slices were Tolazoline caused an increase in the intracellu­ taken from th e backs of domestic pigs by a keratome lar cyclic AMP level (Fig 1). The maximal increase (Storz Instruments, St. Louis. Mo. ) adjusted to a 0.3- was attained by 5 min and then gTaduaJJy de­ rnm setting without anesthetics. The skin thus ob­ creased over 30 min. The addition of theophylline tained was predominantly epidermis (80-90'*>· The greatly potentiated the effect of tolazoline, and slices were cut into 5 x 5 mm squares and preincubated the cyclic AMP level reached its maximal level in in Hank's medium for 15 min at 37"C to standardize about 10 min. Theophylline alone had little effect the initial cyclic AMP level in the epidermal squares on the cyclic AMP content. The effect of different [5,6]. After the preincubation, the epidermal squares concentrations of tolazoline is shown in Fig 2. The incubation medium contained 5 mM theophylline Manuscript received October 21, 1976; accepted for publication May 9, 1977. and the incubation was carried out for 10 min. This work was supported in part by grants from the The increase in the intracellular cyclic AMP level National Institute of Health (AM 17179) and the Der­ was dose-dependent and the maximal effect was matolo~ Foundation of Miami. Reprmt requests to: Dr. Adachi, Veterans Adminis­ obtained at about 3 mM. Lineweaver-Burk plots tration Hospital, 1201 Northwest 16th Street, Miami, (the insert of Fig 2) show the apparent Ka for Florida 33125. tolazoline at about 1 mM (1.34 x lQ- 3 M) . This Ka 442 Nov. 1977 TOLAZOLINE ACTIVATION OF ADENYLATE CYCLASE 443 4 50 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 nonthi­ 2 40 ourea analogue ofmetiamide), both specific antag­ 0 onists of the fH2 J receptor [9,10,11] and diphenhy­ c. dramine, a classical antihistamine which antago­ nizes 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 u shows the effects ofmetiamide, diphenhydramine, ,....u and propranolol (a J3-adrenergic receptor blocker) u on the stimulation of adenylate cyclase by tolazo­ line. 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. 0 10 20 30 m1nutes Interrelationship B etween the Effects of FIG 1. Time course of the effect of tolazoline on the Tolazoline, H istamine and Epinephrine intracellular cyclic AMP content. Details of the experi­ The effects of tolazoline, histamine, epineph­ mental condition are given in the "Materials and Meth­ ods" section. The concentration of tolazoline added was rine, and their combinations on cyclic AMP levels 2 mM and that of theophylline 5 mM (0 tolazoline only; are shown in the Table (A and Bl. In this experi­ !:::, tolazoline + theophylline; and X = control (no ment, all drugs were used at their saturation addition). concentrations (c.f. , Fig 2 and references 4 and 5). The cyclic AMP accumulation by the simultaneous addition of tolazoline and epinephrine was about I equal to the sum of the increases due to each drug <10 I 1irvJ 50 ~30 ~ 0.1 - co- c:: E ... (1.) 0..... 40 ~20·"' a. Q. 0.05 0"1 0.. E ::0: <: 30 ~ 10 "'(1.) u 0 ...,>. E a. 0 1xJ03 2xJQ3 3xJo3 4xlo3 1{sJ a.. 20 0 ~ 3 4 <( mM u u 10 Frc 2. The effect of tolazoline concentration. Tissues >- were incubated with various concentrations of tolazo­ u line for 10 min. Theophylline !5 mM) was added to all the incubation media. The insert shows Lineweaver­ Burk plots of the concentration curve. Frc 3. The effects of metiamide and cimetidine at value is much higher than that of histamine various concentrations. Cyclic AMP contents were where the apparent Ka was about 6 X w-'· M [4]. measured after the incubation with 3 mM tolazoline Phentolamine (mesylate), another a-adrenergic and various concentration of (H2) antagonists. Incuba­ tion was done for 10 min with 5 mM theophylline. te , blocking agent, had no effect on cyclic AMP accu­ metiamide. 0, cimetidine). Abscissa indicates the con­ mulation up to a concentration of 4 mM (data not centrations of (H2l antagonists, metiamide, and cime­ shown). tidine. 444 UZUKA ET AL Vol. 69, N o.5 0 The effect of tolazoline, epinephrine, histamine and 4- methy/-histamine on the cyclic AMP level in pig epidermis Cyclic AMP Additions (pmoles/mg protein) None 0 min 1.1 c: Qj 5 min 0.7 0 A. Tolazoline 25.1 a. 30 Epinephrine 17.8 00 Tolazoline & epinephrine 48.2 E A B. Tolazoline 29.3 A 4-Methyl-histamine 38.8 V'l -Q) Histamine 48.4 0 Tolazoline & 4-methyl-histamine 49.3 g_ 20 Tolazoline & histamine 53.0 45.4 a... 4-Methyl-histamine & histamine ::E Tolazoline & 4-methyl-histamine 52.0 < & hista mine ~ u Concentrations of drugs a dded were tolazoline = 5 >- u 10 mM , epinephrine = 50 p.M. histamine = 2 mM , a nd 4- methyl histamine = 1.6 mM. The incubation was car­ ried out for 5 min at 37•c. No phosphodiesterase inhib­ itors were added in this experiment. histamine) are not "additive". Furthermore, the O.uM lO .uM 100 JJM l mM stimulatory effect by tolazoline was not counter­ acted by propranolol, a ,a-adrenergic blocker. F1c 4 The effect of metiamide, diphenhydramine. and These results s uggest that tolazoline acts on the propra nolol at various concentrations. The experimen­ the tal condition was the same as in Fig 3 except that histamine receptor site which is distinct from different kinds of inhibitors were used. D = met1amide, ,a-adrenergic receptor site. Recently two different ); a n an­ a n antihistamine (H2 0 = diphenhydra mine, histamine receptors, (H 1l and C H ~). have been tihistamine (H,); 6. = propra nolol, a {3-adrenergic recognized. Since the histamine activation of skin blocker; X = control (no addition). adenylate cyclase is blocked by low concentration of metiamide but not by diphenhydramine [4], the added singly; i.e., the effects were additive.