Epidermal adenyl cyclase of human and mouse

Epidermal adenyl cyclase of human and mouse A study of the atopic Tee-Ping

state

lee, Ph.D., William

W. Busse, M.D., and Charles

E. Reed, M.D.

Madison, Wis.

The level of cyclic AMP (CAMP) in normal human epidermis and mouse skin is elevated by catecholamin.cs added in vi&o. After pertussis vaccination, the deny1 cylase of mouse skin responds less to adrenergio agents. The defect induced by the pertussis treatment is the noncompetitbe type of inhibition. Skin from patients with the atopic eczema has considerably higher initiul concentration of CAMP than normal skin. No significant difference was noted betwren the responsiveness of adenyl cyclase of skin obtained from normal or wzematous individuals. It nppears that the defective inhibition by catecholamzines of epidermd mitosis in eczema is not the result of defective response of adenyl cyolase.

Beta adrenergic agonists inhibit epidermal mitosis both in vitro and in vivo.lp 2 We have previously reported that this response is deficient in patients with atopic eczema.3 Catecholamines failed to inhibit epidermal mitosis both in the involved and the noninvolved epidermis. Since these studies were conducted in vitro on excised skin, we concluded the defect was in the skin itself, and not in the autonomic innervation. This observation provided additional indirect, evidence of impaired P-adrenergic receptor function in atopy. Cyclic AMP (CAMP) has been considered as a “second messenger” for various hormones. In skin, the hormonal regulation both of the metabolism of glycogen and of mitosis appears to be mediated by cAMP.~ The studies we now report were designed to investigate a possible defect in the adenyl cyclase system of the epidermis in atopic eczema. Skin from Bacillus pertfusis-treated mice was also used since the pertussisvaccinated animals have served as laboratory models for atopic disease.5’ G METHODS Hairless mice weighing 25 Gm. were sacrificed by cerebral dislocation and the skin dissected free of fat and loose connective tissues. The sheet of skin was mounted on a firm surface; replicate circles (5 mm. diameter) of skin were punched with an Osborne arch punch. Circles of skin were then suspended in ICrebs-Ringer bicarbonate buffer. Sheets of human skin, measuring 20 x 60 x 0.3 mm., were obtained with a keratotome as

From the Department of Medicine, University of Wisconsin Medical School. Supported by United States Public Health Service Grant No. AI10404-03. Received for publication Aug. 6, 1973. Reprint requests to: Tee-Ping Lee, Ph.D., Allergy Laboratory, 433 SMI, 1300 University Ave., Madison, Wis. 53706. Vol. M, No. 5, pp. SLY-687

284

Lee, Busse, and

J. ALLERGY CLIN.

Reed

TABLE 1. Effect of catecholamines

on CAMP content

Addition

None (control) 1-Norepinephrine

I

I-Isoprot,erenol

TABLE II. Effect of catecholamines

on CAMP content

Addition

I-Epinephrine I-Norepinephrine Each value represents in duplicate.

CAMP (ph1olas/20

I

0.1 1.0 10.0 100.0 1.0 1.0 average

from

t + rf I! + k t -c 2

skin plug)

0.15 0.18 0.51 0.31 0.10 0.11 0.38 0.32 0.27

one mouse containing

in human CAMP

4 to fi pieces

epidermis ipmoler/mm.3

epidermis)

0.22 0.41 1.49 1.71 1 .BR 0.93 0.50

PM ,nM ,uM /LM PM pM

of 2 samples

mm.’

1.57 3.36 4.99 5.96 4.28 5.30 6.42 4.55 6.10

Each value represents mean 5 S.E. of 3 samples of skin. Each sample was analyzed in triplicate.

None (control) 1 Isoproterenol

of mouse skin

lJ.1 pM 1.0 ,JM 10.0 /LM 0.1 $I 1.0 /JM 10.0 uM 10.1 ;IM I.0 uM

I-Epinephrine

IMMUNOL. MAY 1974

from

one person.

Each

sample

was analyzed

described previously.:% The sheets of skin were sliced with McIlwain Tissue Chopper (Brinkmann) to give slices sized 1.0 x 20 x 0.3 mm. The skin samples were distributed into conical centrifuge tubes containing 0.9 ml. of Krebs-Ringer bicarbonate buffer. The test agents were added in a volume of 0.1 ml. buffer and the tubes incubated at 37” (‘. for 4 minutes. Then the medium was removed and the tubes containing the pieces of skin were immersed in a boiling water bath for 10 minutes to stop the reaction. Water, 0.5 ml., was added to each tube, ant1 the samples were autoclaved for 15 minutes at 123” Ci. After cooling, 2 ml. of ethanol was added to each tube, and the skin was homogenized. Then the protein was removed l)y centrifugation. The supernatant containing CAMP was removed and dried under nitrogen. After dissolving in water, aliquots of samples were assayed for CAMP l)y saturation assay method. The binding protein for CAMP was prepared from bovine adrenal cortex.: The concentration of CAMP is expressed in terms of area, rather than weight or protein content, because of variation in thickness of sampleq ant1 amount of collagen or keratin present.

Case

selection

Epidermal samples were obtainetl from X subjects, agetl 1X to Xi years old, without a history of hay fever, asthma, eczema, or other skin diseases. The 5 patients with chronic atopic eczema did not have hap fever or asthma; they ranged in age from 17 to 24 years old. The tissue was obtained from the vol.ar surface of the forearm. At the time the samples were taken the skin of the atopic subjeots appeared normal, though there was lichenification at adjacent areas of the wrist or antecubital fossa. The biopsies had similar histologic appearance.:{

RESULTS

Isoproterenol produced significant increase of level of CAMP at lo-’ M, the lowest concentration tested. Maximum stimulation was obtained with lO+ of

VOLUME 53 NUMBER 5

Epidermal

TABLE III. Effect of various

/3-adrenergic

antagonists

on the level

I None Ccontrol~ Propra;lolol ’ Hutoxamine Prado101 Phent,olamine Each value duplicate.

represents

mm.’

I

0

1.50 10 100 10 100 10 100 10 100 average

TABLE IV. Effect of pertussis

,JM PM uM ;LM pM gM UM ;rM

treatment

from

Addition

In the absence of theophyllinc None (control) Tsoprotcrenol 0.1 ,uM Tsoproterenol 1.0 PM Tsoproterenol 10.0 pM

0.99 1.34 3.51 3.40

+ + + +

0.13* 0.27 0.52 0.21

In the presence of 4 mM theophylline None (control) 1.07 + 0.14 Tsoproterenol 0.1 ,.LM 2.33 2 0.19 Tsoproterenol Isoproterenol

1.0 ,JM 10.0 PM

4.20 1.80 1.20 2.10 1.20 3.30 3.90 4.00 4.00

of epidermal

Normal

3.94 + 0.65 4.25 + 0.44

skin

skin plug)

1.35 1.50 1.20 1.50 1.50 1.50 one mouse. Each sample was assayed

on the responsiveness

CAMP tpmoles/skin Plug)

285

1 -Isaproterenol (1.0 pM)

1.80 1.80

of 2 samples

cyclase

of CAMP in mouse

CAMP lpmoler/20

I

Addition

adenyl

adenyl

in

cyclase

Pertussis Per cent stimulation

CAMP (pmoler/skin plug)

t f f 2

Per cent stimulation

35 255 243

1.04 1.24 2.16 1.84

118

1.46 A 0.24 1.58 + 0.07

r

268 297

2.67 + 0.52 3.63 ? 0.63

8; 149

*Each value represents mean -C 8.E. of 4 mice. Each sample contained and was analyzed in triplicate.

0.04 0.30 0.44 0.25

19 108 77

3 to 4 pieces of skin

isoproterenol (Tables I and II). In addition to isoproterenol, epinephrine and norepinephrine also caused increase in level of CAMP. The potency of these adrenergic agents in elevating CAMP levels parallels their potencies in inhibitThe ing DNA synthesis,3 i.e., isoproterenol > epinephrine > norepinephrine. mouse epidermal adenyl cyclase system is inhibited by propranolol and butoxamine and is not inhibited by practolol nor phentolamine (Table III), suggesting that the adenyl cyclase is associated with a p-2 type adrenergic receptor. In pertussis-treated mice the basal level of CAMP in epidermis is elevated and there was reduced responsiveness to catecholamines. The reduced response induced by pertussis vaccination is not reversed by the presence of 4 mM theophylline, an inhibitor of cyclic nucleotide phosphodiesterase (Table IV), suggesting that the defect is at the site of adenyl cyclase rather than an increase of the activity of phosphodiesterase. Similarly, the level of CAMP in atopic epidermis is considerably higher than that of normal skin. However, the responsiveness of adenyl cyclase system in atopic epidermis is normal (Table V) .

286

Lee, Busse, and

TABLE V. Comparsion epidermis

J. ALLERGY CLIN.

Reed of CAMP content

and adenyl

cyclase

I I

Addition Sone

Jsoproterenol 1.0 ,&f Per cent stimulation by lsoprotercnol

1.0 f&l

response

CAMP Normal

of normal

(pmole~/~

(8)*

0.19 f 0.09 il ‘*46 7t 0.26 496

IMMUNOL. MAY 1974

and atopic

epidermis) Atopic

(5)*

0.36 2 0.14 I .4ri f 0.42 400

t

Each value represents mean ? RF:. *Tuml)er of patients studied. t?;o difference from normal patients. Discussion

Several lines of evidence indicate that CAMP may be involved in the glycogen metabolism and cell division of epidermis. 2, 4. s In the present studies, WC have found that the effectiveness of adrenergic agents in elevating levels of CAMP parallels their activities in inhibiting epidermal DNA synthesis. The stimulation of adenyl cyclase and inhibition of DNA synthesis by catecholamines are both of ,8-Z type adrenergic receptor, thus providing further evidence that the intracellular levels of CAMP may regulate the cpidermal DSA synthesis. It has been proposed that abnormal responses to P-adrenergic agonists found in the atopic state are the result of defects in adenyl cyclase system.” The level of cAMP in leukocytes from asthmatic patients does not increase when stimulated with catecholamines.“, lo In the present studies, we have found that the basal level of CAMP is elevated in the epidermis of atopic eczema. Similarly, there is the greater concentration of the cyclic nucleotide in the epidermis of the B. pertzcssis-vaccinated mouse, though the difference is not as striking as in the human skin. This increase of CAMP in atopic epidermis might be the reflection of a higher activity of adenyl cyclase in epidermal membrane,l’ or of decreased phosphodirsterase. llolla and associatesZ2 reported normal phosphodiestcrasc. so the former seems more likely. In the B. pertussis mouse epidermis, there was further CAMP elevation following p-adrenergic stimulation, though reduced compared to the control mouse epidermis. The normal maximal level of cAIMP following adrenergic stimulation could be achieved by increasing concentration of adrenergic agents, suggesting that beta blockade by pertussis vaccination is noncompetitive. In the case of atopic epidermis, however, the beta adrenergic response OSthe adenyl cyclase system was comparable to that of normal epidermis. Several possible explanations are available for the discrepancies between the failure to inhibit epidermal DNA synthesis by catecholamines and the apparent, normal response of the adenyl cyclase system to catecholamines in the skin of patients with atopic eczema. The initial elevated levels of CAMP may be high enough to inhibit the DNA synthesis maximally so that further increase of CAMP by catecholamines has no additional inhibitory effect-or the skin preparation contains several cell types. Basal cells are the only ones undergoing DNA synthesis, but it is possible that adenyl cyclase of basal cells responds differently than other cells, and that an over-all normal response of the skin could obscure a reduced response of this single cell type.

VOLUME 53 NUMBER 5

Epidermal

adenyl

cyclase

287

REFERENCES 1 Bullough, W. S.: A study of the hormonal reactions of epidermal mitotic activity in vitro. III. Adrenaline, Exp. Cell. Res. 9: 108, 1955. 2 Bullough, W. S.: Control of mitotie activity in adult mammalian tissues, Biol. Rev. 37: 307, 1962. 3 Carr, R. H., Busse, W. W., and Reed, C. E.: Failure of catecholamines to inhibit epidermalmitosis in vitro, 5. ALLERGYCLIN.~MM~JNOL.~~: 255,1973. stimulation of en4 Powell, J. A., Duel& E. A., and Voorhees, J. J.: Beta adrenergic dogenous epidermal cyclic AMP formation, Arch. Dermatol. 104: 359, 1971. 5 Reed, .C. E.: Pertussm sensitization as an animal model for the abnormal bronchial sensitivity of asthma, Yale J. Biol. Med. 40: 507, 1968. 6 Szentivanyi? A.: Effect of bacterial products and adrenergic blocking agents on alImmunological diseases, Boston, 1971, Little, lergic reactions, in Samter, M., editor: Brown & Company. 7 Brown, B. L., Albano, J. D. M., Ekins, R. P., Sgherzi, A. M., and Tampion, W.: A simple and sensitive saturation assay method for the measurement of CAMP, Biochem. J. 121: 561, 1971. cyclic AMP inhibition of 8 Voorhees, J. J., Duell, E. A., and Kelsey, W. A.: Dibutyryl epidermal cell division, Arch. Dermatol. 105: 354, 1972. 9 Logsdon, P. J., Middleton, E., Jr., and Coffey, R. G.: Stimulation of leukocyte adenyl cyclase by hydrocortisone and isoproterenol in asthmatic and nonasthmatic sub.jects, J. ALLERGYCLIN.IMMUNOL.~O: 45,1972. 10 Parker, C. W., and Smith, J. W.: Alterations in cyclic AMP metabolism in human bronchial asthma. I. Leukocyte responsiveness to /3-adrenergic agents, J. Clin. Invest. 52: 48. 1973. 11 Mier, P. D., and Urselmann, E.: The adenyl cyclase of skin. II. Adenyl cyclase levels in atopic dermatitis, Br. J. Dermatol. 83: 364, 1970. 12 Holla, S. W., Hollman, E. P., Mier, P. D., Staak, W. J., Urselmann, E., and Warndorff, J. A. : Adenosine-3’:5’-cyclic monophosphate phosphodiesterase in skin. II. Levels in atopic dermatitis, Br. J. Dermatol. 86: 147, 1972.

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