Diurnal Variation in Cyclic Nucleotide Levels in Normal and Phorbol Myristate Acetate Treated Mouse Epidermis

0022-202X/80/7404-0224$02.00/0 THE JOURNAL OF INVE STIGATIVE DERMATOLOGY,

Vol. 74, No. 4

74:224-225, 1980

Printed in U.S.A.

Copyright © 1980 by The Williams & Wilkins Co.

Diurnal Variation in Cyclic Nucleotide Levels in Normal and Phorbol Myristate Acetate Treated Mouse Epidermis SEYMOUR

J.

GARTE,

PH.D., AND SIDNEY BELMAN, PH.D.

Institute of Environmental M edicine, New York University Medical Center, New York, NY U.S.A.

6% trichloroacetic acid. Homogenates were filtered through 0.45 P.M Millipore ftlters and the ftltrates were extracted 3 times with 5 volumes of ether. Cyclic AMP and cyclic GMP were assayed by radioimmune assay as previously described [5,6] using antisera and [' 2°I]-labelled antigens purchased from Collaborative Research (Waltham Mass.). Samples for cGMP assay were purified by column chromatography [11]. Cyclic nucleotide phosphodiesterase (Sigma, St. Louis, Mo.) treatment of samples was used to demonstrate assay specificity [5]. Epidermal homogenates were assayed for DNA by a fluorometri c procedW'e [12].

The diurnal variations in cyclic AMP, cyclic GMP and cyclic GMP/cyclic AMP were determined in mouse epidermis. Slight (60%) diurnal fluctuations were observed in the levels of cyclic AMP and cyclic GMP/cyclic AMP while cyclic GMP showed a more substantial (250%) variation. All 3 parameters showed peaks between 10 AM and 2 PM. The tumor promoter phorbol myristate acetate had no effect on the variation in either cyclic AMP or cyclic GMP, but did appear to reduce the extent of diurnal variation in cyclic GMP I cyclic AMP ratios. The diurnal variation in epidermal mitosis in these mice showed a maximum at 2 PM and a minimum at 10 PM to 2 AM.

Mitotic Index Shaved mice were given i.p. injections of 0.1 mg colchicine in 0.2 mI water, 4 hr before sacrifice by cervical dislocation. The skins were excised, fixed in 10% formalin, sectioned at 5 /lM and stained with hematoxylin/eosin. Five animals were used for ~ach time point and at least 1000 cells were scored (using coded slides to avoid bias) in each epidermis.

The role of cyclic nucleotides in the regulation of epidermal proliferation in normal [1] ' and psoriatic [2] skin has been intensively investigated. Marks and Grimm [3] reported a substantial diurnal variation in mouse epidermal cyclic AMP (cAMP) levels which was inversely correlated with mitotic activity. This diurnal variation was suppressed by prior treatment of epidermis with the carcinogen benzo(a)pyrene [4]. We have studied the effects of the potent skin tumor promoter and hyperplastic agent phorbol myristate acetate (PMA) on cyclic nucleotide metabolism in mouse epidermis [5-7]. Among other effects PMA has been shown to inhibit f3-adrenergic stimulation of adenyl cyclase [7-9], to suppress the increase of cAMP due to ischemia [5], and to increase cGMP levels [5,6]. Epidermal mitosis is believed to be under f3-adrenergic hormone regulation based on in vitro and in vivo studies of Bullough [10]. It was therefore of interest to determine the diurnal levels of cAMP, cGMP and cGMP IcAMP ratios and the effects of PMA on these parameters.

RESULTS AND DISCUSSION

MATERIALS AND METHODS Fe~ale

HallCR (Sprague Dawley) mice aged 8 weeks were exposed to artifiCial llght (6 AM-6 PM) and darkness. Animals were shaved 3-4 days before sac rifice and those showing hair regrowth or wounding were not used. The backs of mice were treated with 0.2 ml acetone or 0.2 mI acetone containing 10 /lg PMA (Dr. P. Borchert, Chemical Carcinogenesis, Eden Prairie, Minn.). Animals were sacrificed 12 hr after treatment by c~rvical dislocation, immediately immersed in liquid rutrogen, and the epIdermis was harvested by scraping with a scalpel as has been previously described [5].

Cyclic Nucleotide Assay Each sample consisted of epidermis from 1 mouse. The frozen tissue samples were homogenized for 1 min in a Polytron P-10 at top speed in Manuscript received August 16, 1979; accepted for publication November 6, 1979. T.his work was supported by Grant No. CA18536, awarded by the NatIOnal Cancer Institute, DHEW, and is part of Center Programs supported by Grant No. ES00260 from the National Institute of Environmental Health Science, and Grant No. CA13343 from the National Cancer Institute. Reprint requests to: Dr. S. J. Garte, Dept. of Environmental Medicine, New York Univ. Medical Center, 550 First Ave., New York, NY 10016. Abbreviations: cAMP: cyclic AMP cGMP: cyclic GMP PMA: phorbol myristate acetate

A significant diurnal variation in cGMP levels in mouse epidermis was found with a 2.5 fold higher value at 10 AM than at 10 PM (Fig IE). The diurnal variation in cAMP (Fig lA) was less marked (60 % increase at 6 AM over 10 PM). Our data agree with previous work [3,4] with respect to the time course of cAMP diurnal variation. However we did not find the !luge differences reported by Marks and Grimm [3]. A possible source of this discrepancy was the fact that we did not excise the skin before removal of epidermis, thus avoiding artifacts due to ischemia [13]. To test the possibility that the ischemic effect exhibits a diurnal variation we sacrificed mice at 10 AM and 10 PM, and surgically removed the back skins, before freezing and scraping. The cAMP levels from excised (ischemic) skin were 2.4 and 2.9 times those of controls at 10 AM and 10 PM respectively. The discrepancy between our data and those of Marks and Grimm is therefore not related to the ischemic effect. The diurnal variations in cGMP and in the cGMP/cAMP ratio (shown in Fig 2) in mouse epidermis show the same time pattern of maximum and minimum as does cAMP. The variation in cGMP IcAMP between 10 AM and 10 PM is ca 60%. Maximum inhibition of f3-adrenergic responsiveness in mouse epidermis is found when PMA is applied 12 hr before sacrifice [8]. Treatment of mouse skin with PMA 12 hI' before sacrifice did not dramatically affect the diurnal variation in either cyclic nucleotide (Fig 1). However the tumor promoter does appear to reduce the extent of variation in the cGMP Ic AMP ratios (Fig 2), with significant increases at 2 PM and 10 PM. This result is similar to the effect ofbenzo(a)pyrene on the diurnal variation of cAMP observed by Verma and Murray [4]. Because of conflicting reports in the literature [14,15] we determined the diurnal variation in epidermal mitotic index for our Ha/IGR mice, as shown in Fig 3. The mitotic rate is highest at 2 PM and lowest in the evening. Vorrhees et al have postulated an inhibitory role for cAMP and a stimulatory role for cGMP at the G2 phase of the cell cycle [16]. Our data, which show the peak of mitosis occurring 4 hr after the peak of cGMP IcAMP ratio is consistent with this interpretation, although the precise relationship between these parameters and the cell cycle is still unclear.

224

April 1980

DIURNAL VARIATION IN NORMAL AND TREATED MOUSE EPIDERMIS

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FIG 1. Diurnal variation in mouse epiderm al cyclic nucleotides. Mice were treated with 0.2 ml aceto ne (e---e) or 10 IJ.g PMA (t,.- - - t,. ) in 0.2 ml acetone 12 hours before sacrifice. A, cyclic AMP; B, cyclic GMP. Data represent means ± SE from groups of 5 mice. Asterisk denotes statistically significant (P < 0.10) differences by Students t-test.

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FIG 2. Diurnal variatio n in cGMP/cAMP ratio in mo use epidermis. Legend same as Fig 1. Each point represents the mean ± SE of cG MP / cAMP ratios determined individually from 5 mice.

The d ata presented su ggest that levels of cGMP a r e more sensitive to diurnal variation t h an th ose of cAMP in mouse epidermis . Our data also s uggest th at t h e mechanism responsible for epidermal cyclic nucleotide diurnal variation is n ot {3a dre n ergic hormone d epe nde n t since PMA h ad n o effect on diurn a l cyclic AMP levels, and PMA is known to in terfere with ,B-adrenergic stimulation of ade nyl cyclase [5,8,9]. This conclus ion was also reached by M arks a nd Grimm [3]. On the other h a nd th e loss of diurnal variation in cGMP /cAMP ratios cause d by PMA may be s ignifican t with respect to the interference of carcinogens [4] and t umor promo tors [7-9] with normal cyclic nucleo tide metabolism. We than k Karen B. Fennikoh for excellent technical assistance.

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FIG 3. D iurna l variation in mito tic index. For det.ails see Materials and Methods. Data represe nt means ± SE from 5 mice/ group.

REFERENCES 1. Halprin KM : Cyclic nucleotides and epidermal cell proliferation. J Invest Dermatol 66:339-343, 1976 2. Voorhees JJ, Colburn NH, Stawiski M, Duell EA, Haddox · M, Goldberg ND: Imbala nced cyclic AMP a nd cyclic GMP levels in the rapidly dividing incompletely differentiated epide rmis of psoriasis, Control of Proliferation in Animal Cells. Edited by B Clarkso n and R Baserga. Cold Sprin g Har bor, New York, 1974, pp 635- 648 3. Marks F, Grimm W: Diurnal flu ctuation and ,B-adrenergic elevation of cyclic AMP in mouse epiderm is in vivo. Nature 240:178-179, 1972 4. Verma AK, Murray AW: The effect of benzo(a) pyrene on the basa l and isoproterenol-stimulated levels of cyclic adenosine, 3',5' monophosphate in mouse epidermis. Cancer Res 34:3408-3413, 1974 5. Belman S, Troll W, Garte SJ: Effect of phorbol myristate acetate on cyclic nucleotide. levels in mouse epidermis. Cancer Res 38: 2978- 2982, 1978 6. Garte SJ, Belman S: Effects of multiple phorbol myristate acetate treatments on cyclic nucleotide levels in mouse epidermis. Biochern Biophys Res Comm 84:489-494 , 1978 7. Garte SJ, Belman S: Phorbol myristate acetate uncoupl es ,B-adrenergic receptors from adenyl cyclase in mouse epidermis: Antagonism by butyric acid. Proc Am Assoc Cancer Res 20:52, 1979 8. Grimm W, Marks F: Effect of tumor promoters on the normal and Isoproterenol elevated level of adenosine 3'5'-cyclic monophosphate in mouse epidermis in vivo. Ca nce r Res 34:3128-3134, 1974 9. Mufso n RA , Simsiman RC, Boutwell RK: The effect of the phorbol ester tu mor promoters on the basal and catechola mine stimul ated levels of cyclic adenosine 3',5' -mo nophosphate in mouse skin and epidermis in vivo. Cancer Res 37:665-669, 1977 10. Bullough WS: Stress a nd epidermal mitotic activity. 1. The effects of the adrenal hormones. J Endoc rinol 8:265-274 , 1952 11. Mmad F, Manganiello V, Va ughan M: A simple sensitive protei nbinding assay for guanosine 3',5' -mo nophosphate. Proc Nat! Acad Sci 68:736-739, 1971 12. Switzer BR, Summer GK: A Modified Fluorometric M icromethod for DNA. Clin Chim Acta 32:203-206, 1971 13. Yoshikawa K, Adachi K , Halprin KM , Levine V: Cyclic AMP in skin: Effects of acute ischemia. Br J Dermatol 92:249-254, 1975 14. Tvermyr EMF: Circadia n rhythms in epiderma l mitotic activity. Virchows Arch Abt B Zellpath 2:318-325, 1969 15. Bullough WS: Mitotic activity in the adult male mo use Mus musculu.s. T he diurnal cycles and their relation to waking and sleeping. Proc Roy Soc B135:212-232, 1948 16. Voorhees JJ , Duell EA, Stawiski M, Harrell ER: Cyclic nucleotide metabolism in norma l and p roliferating epidermis. Adv Cyclic Nucleotide Res 4:117-160, 1974