Feldmann, R. J. & Maibach, H. 1.(1967). Regional variations in pereutaneous penetration of

'4C cortisol in man. J. invest. Derm. 48, 181-183.

Gazith, J. & Sehaefer, H. (1977). 8-Methoxypsoralen: its isolation and gas-chromatographie

determination in aqueous solution and serum . Biochem. Med., 18, 102.

Kammerau, B., Klebe, U., Zesch, A. & Schaefer, H. (1976). Penetration, permeation and

resorption of8-methoxypsoralen. Arch. Derm. Res.. 255,31-42.

Kammerau. B., Zeseh, A. & Sehaefer, H. (1975). Absolute eoncentrations of dithranol and

triacetyl-dithranol in the skin layers after local treatment: in vivo investigations with four

different types ofpharmaceutieal vehicles. J. invest. Derm.. 64, 145-149.

Malkinson, F. D. (1968). Radioactive agents and radioisotopes in dermatology: investigative

applieations. In Pilisburg and Livingood Proceedings International Congress 0/

Dermatology; Int. Congr. Ser. 55, pp. 657--661. Amsterdam, New York, London,

Milan , Tokyo : Excerpta med. Found.

Pinkus, H. (1951). Examination ofthe epidermis by the strip method ofremoving homy layers.

J. invest. Derm.. 17, 383-386.

PRINCIPLES OF PERC UT ANEOUS ABSORPTION 403

Pinkus, H. (\952). Examination of the epidermis by the strip method 11. Biometrie data on

regeneration ofthe human epidermis. J. invest. Derm ., 19,431-447.

Schaefer, H., Zesch, A. & Stüttgen, G. (1977). Penetration, Permeation and Absorption of

Triamcinolone Acetonide in Normal and Psoriatic Skin. Arch. Derm . Res., 258,241-249.

Schaefer H., Zesch, A. & Stüttgen, G. (1980). Skin Permeability. Springer Verlag: Berlin,

Heidelberg, New York, in press.

Schalla, W., Schaefer, H., Kammerau, B. & Zesch, A. (1976) Pharmacokinetics of

8-methoxypsoralen after oral and local application. J. invest. Derm ., 66, 258-259.

Wolf, J. (1940). Das Oberflächenrelief der menschlichen Haut. Z. mikr-anat. Forschung, 47,

351.

Zesch, A., Nordhaus, R. & Schaefer, H. (1972). Zur Kontrolle des Hornschicht-abrisses durch

Widerstands messungen. Arch. Derm. Forsch., 242,398-402 .

Zesch, A. & Schaefer, H. (1973). Penetrationskinetik von radiomarkiertem Hydrocortison aus

verschiedenartigen Salbengrundlagen in die menschliche Haut in vitro. Arch. Derm .

Forsch, 246, 335-354.

Zesch, A. & Schaefer, H. (1975). Penetrationskinetik von radiomarkiertem Hydrocortison aus

verschiedenartigen Salbengrundlagen in die menschliche Haut 11, in vivo. Arch. Derm .

Forsch., 225,245-256.

BIOCHEMICAL PHARMACOLOGY OF

THEEPIDERMIS

E.A.DUELL

University ofMichigan Medical School,

Department ofDermatology and Biological Chemistry,

R6558 Kresge I Box 56,

Ann Arbor, MI 48109, USA

Introduction

The influence ofadenosine 3', 5'-monophosphate (cyclic AMP) on the proliferation

and differentiation of several cell types was indicated in the literature of the early

1970s (Pastan, Johnson & Anderson, 1975). An investigation of the cyclic AMP

system in the epidermis appeared to be a reasonable approach for the development of

new treatrnents for various proliferative skin diseases such as psoriasis. From the data

available in the literature, an increase in the cyclic AMP concentrations in the tissue

might decrease proliferation and induce differentiation.

The epidermis is the avascular portion ofthe skin and is composed offour distinct

layers, the stratum corneum, stratum granulosum, stratum spinosum and basallayer.

The outermost stratum corneum is composed of dead cells and serves as the main

barrier region for preventing material from entering the body through the skin . The

stratum granulosum is several cell layers thick and contains keratohyalin granules,

the differentiation product unique to the skin. The stratum spinosum contains cells

rich in tonofilaments and microtubules and this layer forms the major portion ofthe

epidermis. Only the basallayer retains the capacity to replicate and divide when the

epidermis is in its normal state . In the hyperproliferative state, suprabasal cells also

undergo cell division. The epidermis is separated from the dermis by the basal

lamina.

Agents that alter the cyclic AMP levels in the epidermis via increased adenylate

cyclase activity.

ß-adrenergic receptor agonists were the first class ofcompounds to be investigated as

possible agents for increasing the cyclic AMP concentration in the epidermis. The

agonists that are effective in increasing the cyclic AMP levels are isoprenaline

(Duell, Voorhees, Kelsey & Hayes, 1971; Marks & Rebien , 1972), noradrenaline

(Duell, 1980a), and adrenaline (Adachi, Yoshikawa, Halprin & Levine , 1975; Marks

& Rebien, 1972). Because of the possible therapeutic implications, it seemed

important to establish whether the ß-adrenergic receptor was primarily ßI or ß2in

nature.

The delineation ofthe type ofreceptor was based upon the order ofpotency ofthe

BIOCHEMICAL PHARMACOLOGY OF THE EPIDERMIS 405

agonist that is, isoprenaline (lP) > noradrenaline (NOR) > adrenaline (AD) , the use

ofrelatively specific antagonists that is, practolol as a 13 i-adrenoceptor blocking drug

and butoxamine as a !32-adrenoceptor blocking drug, and the use of a

ß2-adrenoceptor agonist salbutamol (Duell , 1980a). The {3-adrenergic antagonists,

butoxamine, practolol and propranolol had no efTect on the basal level of cyclic AMP

which was 6.9 ± l.l pmol cyclic AMP/mg epidermal protein.

A dose response relat ion exists between IP and NOR concentrations and the level

of cyclic AMP in the epidermal tissue after 5 min of incubation at 3TC. IP was

efTective at two log units lower in concentration than NOR, and the amount of cyclic

AMP formed was slightly lower even with the most effective concentration ofNOR

(Duell, 1980a). Ifthe antagonists (lO-5M) are added simultaneously with IP, the levels

ofcyclic AMP formed are returned to base level with propranolol, reduced somewhat

with butoxamine and slightly augmented with practolo!.

The addition of salbutamol in aseries of concentrations also produced a

corresponding increase in the cyclic AMP levels. The addition of 5 x 1O-5M

salbutamol to the tissue produced the maximum increase (3.8-fold) in the cyclic

AMP levels with this agonist (Duell , 1980a). The addition ofbutoxamine reduced the

cyclic AMP accumulation 60% while practolol had little or no effect on the

salbutamol induced increases in cyclic AMP. The above data is consistent with the

ß-adrenergic receptor in the epidermis being primarily 132 in nature.

Other agents that are known to increase the level of cyclic AMP in the epidermis

are : adenosine (Duell, 1980b; Iizuka , Adachi, Halprin & Levine, 1976a), histamine

(Iizuka, Adachi, Halprin & Levine, 1976b) and prostaglandins (Adachi et al.. 1975;

Aso, Orenberg & Farber, 1975). The efTect of increasing adenosine concentrations

and the cyclic AMP levels in the brain was first described by Sattin & Rall (1970).

Other characteristics ofthis response were the inhibition by methyl xanthines such as

theophylline and the increases of cyclic AMP by the addition of adenine nucleotides.

More recently Burnstock (1978) has categorized the receptors of the purnergic

nervous system as PI or P2. The agonist order of potency for the PI receptor is

adenosine .» AMP > ADP > ATP, which results in an increase in cyclic AMP

levels that can be blocked by methyl xanthines. The P2 receptor has a reverse order of

potency, is unafTected by methyl xanthines, does not involve increases in the cyclic

AMP levels and may be associated with the release ofprostaglandin E2.

The data obtained with the epidermis is consistent with a PI receptor. Increasing

concentrations ofadenosine from 5 x 10-4 to O.OIM produces a corresponding increase

in the cyclic AMP concentration to a maximum increase of 3-4-fold (Duell , 1980b).

At the lower concentrations of adenosine, the addition of 2 x 1O-3M theophylline

reduced the amount of cyclic AMP accumulated in the tissue after 5 min of incubation . The addition of cyclic nucleotide phosphodiesterase inhibitors such as

papaverine or Ro 20-1724 simultaneously with the addition of adenosine augumented the incre ase in cyclic AMP 2-3-fo ld in comparison to adenosine alone (25.1

pmol cycl ic AMP/mg protein). The addition of 5mMAMP increased the cyclic levels

to about 60% of the value achieved with 5mM adenosine. Adenine had no effect on

the cyclic AMP levels in the tissue . Thus the data is consistent with the epidermis

containing a receptor similar to the PI type.

The cell surface nature ofthe receptor is indicated by incubation of epidermal cells

in culture in the presence of both adenosine and dipyridimole with the subsequent

determination ofthe cyclic AMP levels in the cells . The addition ofO.OlmM dipyridimole blocks the uptake ofadenosine into the cells with minimal changes in the cyclic

AMP levels. The addition of adenosine plus dipyridimole to the cells increases the

cyclic AMP approximately 2-fold which is similar to that produced by the addition

of adenosine alone. Thus the adenosine efTect probably occurs at the cell surface

rather than altering the concentration of the adenine containing compounds by

uptake of adenosine inside the cell.

406 E.A.DUELL

The histamine receptor appears to be H2 since the increases in cyclic AMP due to

the addition of histamine can be blocked by the addition of the anti-histamine,

metiamide, (lizuka et al., 1976b). The prostaglandins (PG) that are most efTective in

elevating the cyclic AMP levels in the epidermis are EI and E2 (Adachi et al.. 1975;

Aso et al.. 1975). PGFza is relatively inefTective in altering the cyclic AMP levels in

the tissue.

Agents that alter the cyclic nucleotide levels in the epidermis by inhibiting cyclic

nucleotide phosphodiesterase (PDE) activity.

Two compounds that have proven to be good inhibitors of PDE in the epidermis are

Ro 20-1724 and papaverine (Rusin, Duell & Voorhees, 1978). A linear increase in

the cyclic AMP concentration with time occurs in the presence of 5 x IQ-4M

papaverine or 1Q-5 M Ro 20-1724. Substrate concentrations of2 x IQ--{;M cyclic AMP or

5 x IQ--{;M cyclic GMP were utilized to assay the soluble cyclic PDE activities in the

epidermis. Both activities were inhibited approximately 90% by the addition of 5 x

IQ-4M papaverine whereas only the cyclic AMP hydrolysis was inhibited by 5 x IQ-4M

Ro 20-1724 (Rusin et al., 1978). Approximately 80-90% of the cyclic PDE of the

epidermis is located in the 17,000 g supernatant fraction of the epidermal

homogenate.

Cyclic AMP system in psoriasis

The involved and uninvolved epidermal slices from psoriasis patients have been

incubated with the agonists that elevate the cyclic AMP levels via activation of

adenylate cyclase or the slices have been incubated with the cyclic PDE inhibitors in

order to elevate the cyclic AMP levels. Both the involved and uninvolved tissues

respond to the compounds but there are some difTerences. A decreased responsiveness ofthe involved tissue to PGEI or PGE2 has been reported (Adachi el al., 1975;

Aso et al., 1975) and a decreased responsiveness with adrenaline (lizuka, Adachi,

Halprin & Levine, 1978).

In aseries ofsix patients and six normal volunteers for each agonist, the response

ofthe tissue to IP, NOR, AD, PGE2 and histamine were determined. IP was the most

efTective agonist for the normal epidermis and resulted in a 5-6-fold increase in the

cyclic AMP levels . PGE2 was the least efTective and produced a 2-3-fold elevation in

the cyclic AMP levels . The baseline values ofO.16 pmol cyclic AMP/\lg DNA was

very constant. The baseline values for both the involved and uninvolved tissue

sampies were more variable and ranged from 0.26 ± 0.09 pmol cyclic AMP/\lg DNA

to 0.56 ± 0.21 pmol cyclic AMP/\lg DNA.

For the uninvolved tissue sampies, IP was most efTective (3-fold increase) and

histamine was the least efTective (40% increase). For the involved tissue sampies,

histamine was the most efTective (2-fold increasc) and PGE2 was the least efTective

(60% increase). The overall response ofthe psoriasis tissues was more variable from

patient to patient when compared to the response elicited from the normal volunteer

tissue sampies. The significance of the variations requires additional investigation

before a definitive statement can be made.

The addition of papaverine or Ro 20-1724 to epidermal slices obtained from

involved and uninvolved tissue elevated the cyclic AMP levels (Rusin et al.. 1978).

Papaverine increased the cyclic AMP levels almost 4-fold after 15 min ofincubation.

Ro 20-1724 increased the cyclic AMP levels in uninvolved area approximately

lQ-fold and in the involved areas approximately 7-fold. Either the Ro 20-1724

penetrates the tissue more efTectively or it may be exerting some other efTect in

addition to inhibiting cyclic nucleotide PDE activity.

BIOCHEMICAL PHARMACOLOGY OF THE EPIDERMIS 407

Both papaverine and Ro 20-1724 have been applied topically to the lesional areas

ofpsoriasis. Both compounds are efTective in improving the lesion, with Ro 20-1724

the more efficacious (Stawiski, Powell, Lang, Schork, Duell & Voorhees, 1975;

Stawiski, Rusin , Burns, Weinstein & Voorhees, 1979). Whether the improvement is

due to alterations in the cyclic nucleotide system alone or in conjunction with other

systems is unknown at this time .

Arachidonic acid metabolism in the epidermis

Arachidonic acid is probably released from the pool of phosphatidylcholine present

in the cell membranes of the epidermis by the action of phospholipase A2.

Arachidonic acid is the substrate for the enzymes lipoxygenase and cyclo-oxygenase,

The prostacylin and thromboxane pathways for the metabolism of arachidonic acid

are not detectable in the epidermal tissue. However the formation of PGE2, PG Fla

and 12-L-hydroxy-5,8 ,10,l4 eicosatetraenoic acid (l2-HETE) is increased in psoriatic epidermis (Hamrnarströrn, Harnberg, Samuelsson, Duell, Stawiski & Voorhees,

1975). The most dramatic increase was in HETE (86-fold) present in the involved

psoriasis areas. Free arachidonic acid was also elevated 26-fold in the involved areas.

In contrast there was only a 40% increase in PGE2 and a 86% increase in PGFla in

the lesional area.

The application of a topical glucocorticoid to a lesional area markedly decreased

the arachidonic acid and HETE accumulation in the treated lesional area compared

to the untreated lesion (Harnmarström, Harnberg, Duell, Stawiski, Anderson &

Voorhees, 1977). One ofthe many efTects ofglucocorticoid treatment is the reduction

in phospholipase A2 activity. Whether the improvement in lesions following

glucocorticoid treatment is due to its efTects on the formation of arachidonic acid or

HETE is unknown at the present time .

Effects of elevated cyclic AM P levels on primary cultures of epidermal cells

The primary epidermal cell cultures can be used efTectively as model systems for

studying the efTects of altered cyclic nucleotide metabolism on proliferation and

difTerentiation of epidermal cells. The early observations obtained from adult guinea

pig cells grown on plastic indicated that an elevation in the cyclic AM P levels in the

cultures resulted in decreased proliferation (Delescluse, Colburn, Duell & Voorhees,

1974). Similar results were obtained with explants from human tissue grown on glass

coverslips (Harper, Flaxman & Chopra, 1974a; Harper, Flaxman & Chopra, 1974b) .

In these studies 10-2 to IO-4M dibutyryl cyclic AMP were used . Other agonists such as

PGE2 or IP were also used to increase the cyclic AMP levels. Inhibitions ofproliferation of30-90% were obtained.

Two recent reports indicate that in culture systems utilizing primary epidermal

cultures derived from neonatal mice (Marcelo, 1979) or the cultures of astrain of

epidermal cells derived from newborn foreskin and grown on 3T3 feeder layers

(Green, 1978) are stimulated to proliferate by the addition oflO-3 to 10-4M cyclic AMP

derivatives. Thus the response ofthe cells to high levels of cyclic AMP varies with the

age and species ofstarting material.

To further complicate the efTects of cyclic AMP on epidermal proliferation was

both the inhibition and the stimulation of proliferation of adult epidermal cells

grown on collagen gels. At high 8-bromo AMP levels 10-3 to IO-6M, the proliferation

of the epidermal cells was inhibited whereas the cells were stimulated at concentrations of 10-8 to 1O-9M (Marcelo & Duell, 1979). The mechanisms by wh ich

cyclic AMP influences proliferation is unknown at this time and awaits further

investigation.

408 E.A. DUELL

Conclusions

The epidermis contains a ß-adrenergic receptor that appears to be primarily of the

ßz type as determined by measuring the increase of the cyclic AMP levels after

incubating the tissue with IP, NOR and sa lbutam ol. Butoxamine but not practolol

decreases the accumulation ofcyclic AMP in the tissue ifadded simultaneously with

the agonists.

The agents which increase the cyclic AMP level s by stimulating adenylate cyclase

are: adenosine, histamine and prostagJandins. The increases in cyclic AMP in the

epidermis due to the presence of adenosine were blocked by theophylline but

augmented by other cyclic nucleotide PDE inhibitors.

Approximately 80-90% of the cycl ic nucleotide PDE activity was present in the

soluble fraction. Ro 20-1724 inhibits onl y the cycli c AMP PDE acti vit y while

papaverine inhibits both cyclic AMP and cycli c GMP PDE activities.

Arachidonic acid is metabolized in the epidermis to form PGEz, PGFza , PGDz,

and 15-HETE. The formation of thromboxanes or prostacyclin does not seem to

occur in the epidermis.

References

Adachi , K., Yoshikawa, K., Halprin , K. & Levine, V. (1975). Prostaglandins and cyc1ic AMP in

epidermis. Brit. J. Derm., 92, 381-388.

Aso, K., Orenberg , E. & Farber, E. (1975). Reduced epidermal cyc1ic AMP accumulation

following prostagiandin stimulation: Its possible role in the pathophysiology ofpsoriasis. J.

invest. Derm., 65, 375-378.

Bumstock, G. (1978). A basis for distinguishing two types of purinergic receptors. In Cell

Membrane Receptors for Drugs and Hormones, ed Straub , R. W. & Bolis, L., pp. 107-118.

New York: Raven Press.

Delesc1use, c., Colbum, N., Duell, E. & Voorhees, J. (1974). Cyclic AMP--elevating agents

inhibit proliferation ofkeratinizing guinea pig epidermal cells. Differemiation, 2,343-35.0.

Duell, E. (1980a). Identification of a betaz-adrenergic receptor in mammalian epidermis.

Biochem. Pharmac., 29,97-101.

Duell, E. (1980b). Adenosine-induced alterations in the adenosine 3':5'-monophosphate levels

in mammalian epidermis . Mol. Pharmac.. 18,84-87.

Duell, E., Voorhees,J., Kelsey, W. & Hayes, M. (1971). Isoproterenol-sensitive adenyl cyc1ase in

a particulate fraction of epiderm is. Arch. Derm., 104,601-610.

Green , H. (1978). Cyc1ic AM? in relation to proliferation of the epidermal cell: A new view.

Cell, 15, 80I-8II.

Hamma rström, S., Hamberg, M., Duell, E., Stawiski, M., Anderson , T. & Voorhees, J. (1977).

Glucocortico id in inflammatory proliferative skin disease reduces arachidonic and

hydroxyeicosatetraenoic acids. Science. 197,994-996.

Hammarström, S., Hamberg, M., Samuelsson, B., Duell, E., Stawiski, M. & Voorhees,J. (1975).

Increased concentrations of nonesterified arach idonic acid, 12L-hydroxy-5,8,1O,14-

eicosatetraenoic acid, prostagiandin Ez, and prostagIandin Fz« in epidermis of

psoriasis. Proc. Nat. Acad. Sei. USA, 72, 5130-5134.

Harper, R., Flaxrnan, B. & Chopra, D. (1974a). EtTect of pharmacological agents on human

keratinoc yte mitosis in vitro I. Inhibit ion by adenine nuc1eotides. Proc. Soc. exp, Biol.

Med. , 146, 1032-1036.

Harper , R., Flaxman, B. & Chopra, D. (1974b). Mitotic response of normal and psoriatic

keratinoc ytes in vitro to compounds known to atTect intra-cellular cyc1 ic AMP. J. invest.

Derm..62, 384-3 87. .

lizuka , H., Adachi, K., Halprin , K. & Levine V. (19 76a). Adenosine and adenine nuc1eotides

stimulation ofskin (epidermal) aden ylate cyc1ase. Biochim. Biophys. Acta., 444, 685-693.

lizuka, H., Adachi , K., Halprin , K. & Levine, V. (1976b). Histamine (Hz) receptor-adenylate

cyc1ase system in pig skin (epidermis ). Biochim. Biophys. Acta, 437, 150-157.

lizuka, H., Adachi, K., Halprin , K. & Levine V. (1978). Cyc1ic AM? accumulation in psoriatic

skin: DitTerential responses to histarnine, AM?, and epinephrine by the uninvolved and

involved epidermis . J. invest. Derm., 70, 250-253.

BIOCHEMICAL PHARMACOLOGY OF THE EPIDERMIS 409

Marcelo, C. (1979). Differential effects of cAMP and cGMP on in vitro epidermal cell growth.

Exp. Cell Res.. 120,201-210.

Marcelo, C. & Duell, E. (1979). Cyclic AMP stimulates and inhibits adult human epidermal cell

growth. J. invest. Derm.. 72,279.

Marks, F. & Rebien W. (1972). The second messenger system of mouse epidermis I. Properties

and ß-adrenergic activation of adenylate cyclase in vitro. Biochim. Biophys. Acta. 284,

556-567.

Pastan, 1.,Johnson, G. & Anderson, W. (1975). Role of cyclic nucleotides in growth control. In

Annual Review ofBiochem istry. ed . Snell, E., Boyer, P., Meister, A. & Richardson, C; pp .

491-522. Palo Alto: Annual Reviews Inc.

Rusin, L., Duell, E. & Voorhees, J. (1978). Papaverine and Ro 20-1724 inh ibit cyclic nucleotide

phosphodiesterase activity and increase cyclic AMP levels in psoriatic epidermis in vitro. J.

invest. Derm., 71, 154-156 .

Sattin, A. & Rall, T . W. (1970). The effect of adenosine and adenine nucleotides on the cyclic

adenosine 3',5 '-phosphate content of guinea pig cerebral cortex slices . Mol. Pharmac.. 6,

\3-23.