Epidermal dexamethasone receptors in dogs with confirmed hyperadrenocorticalism, hypothyroidism or undiagnosed hormonal alopecia

Researchin VeterinaryScience1991, 51, 335-338

Epidermal dexamethasone receptors in dogs with confirmed hyperadrenocorticalism, hypothyroidism or undiagnosed hormonal alopecia A. H. M. VAN DEN BROEK, Department of Veterinary Clinical Studies, W. L. STAFFORD,

Department of Preclinical Veterinary Sciences, University of Edinburgh, Royal (Dick) School of Veterinary Studies, Summerhall, Edinburgh EH9 1QH

Low capacity, high affinity [3H] dexamethasone binding receptors were identified in cytosolic preparations of the skin (mean number 42.0 ± 25.2 fmol mg-1 protein, apparent dissociation constant (1 nM ± 0.23) of clinically normal dogs. No [3H] dexamethasone binding was observed in the skin of nine out of 10 dogs with confirmed spontaneous hyperadrenocorticism or in the skin of three out of six dogs with undiagnosed hormonal aiopecia. A reduction was detected in the number of [3H] dexamethasone binding receptors in the skin of one dog with confirmed hypothyroidism. This study provides evidence for the susceptibility of canine glucocorticoid receptors to down regulation by imbalances of endogenous hormones, particularly increased glucocorticoid concentrations.

STUDIES of glucocorticoid administration in mice following adrenalectomy (Svec et al 1989) and in rats following experimental induction of hypothyroidism and, or, thyroid hormone administration (Morishige 1982, Lesney et al 1987, Lu et al 1988) provide evidence for the regulation of these receptors by glucocorticoid and thyroid hormone concentrations. In man administration of exogenous glucocorticoids (Shipman et al 1983, Griese et al 1988), but not alterations in endogenous glucocorticoids which accompany hyperadrenocorticism (HAG) (Kontula et al 1980) appear to influence glucocorticoid receptors on mononuclear cells. In dogs HAC is frequently accompanied by cutaneous manifestations such as non-pruritic, bilaterally symmetrical alopecia, pigmentary distUrbances, hyperextensibility and hypotonicity (Muller et al 1989). Some of these signs, particularly non-pruritic, bilaterally symmetrical alopecia and pigmentary disturbances, also occur in dogs with other hormonal imbalances (Scott 1990).

The present study was undertaken to investigate [3H] dexamethasone binding in the skin of dogs with confirmed spontaneous HAC and compare this with [3HI dexamethasone binding in the skin of dogs with bilaterally symmetrical alopecia due to hypothyroidism or undiagnosed hormonal imbalances. Materials and methods

Chemicals 1,2,4,6,7-3H-dexamethasone was purchased from Amersham International. Non-radioactive dexamethasone, tris (hydroxymethyl) aminomethane and DL-dithiothreital were o b t a i n e d from Sigma Chemical Company and all other chemicals from BDH Chemicals.

Animals Clinically normaldogs. Skin and liver samples were obtained from seven adult dogs presented for euthanasia. Although their hormonal status was not determined the dogs were clinically normal and had received no medication for at least three months before euthanasia. Dogs with hyperadrenocorticism. These comprised a group of 10 adult dogs which had exhibited clinical signs typical of HAC (polyphagia, polydipsia, polyuria, symmetric alopecia, pendulous abdomen) for at least three months before presentation. The diagnosis was confirmed by the adrenocorticotrophic hormone (ACTH) stimulation test or dexamethasone suppression test (Feldman 1983) and response to treatment with mitotane.

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Dogs with bilaterally symmetrical alopecia. The

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A. H. M. van den Broek, W. L. Stafford

diagnosis of hypothyroidism was confirmed in one adult dog by the thyroid stimulating hormone stimulation test (Thoday 1990) and response to treatment. In six adult dogs with undiagnosed, non-seasonal, hormonal alopecia histopathology of skin biopsies was compatible with an endocrinopathy. However, the ACTH stimulation test, dexamethasone screening test and thyroid stimulating hormone stimulation test gave results within normal limits. In addition serum oestradiol, progesterone and testosterone concentrations were within normal limits. A growth hormone response test was not carried out. The breeds represented in this group were Airedale terrier, boxer, doberman pinscher (two), crossbred terrier and Yorkshire terrier. While a seasonal growth hormone deficiency has been reported in the boxer and doberman pinscher (Muller et al 1989), an apparently non-seasonal growth hormone deficiency has been documented only in the Airedale (Shanley and Miller 1987).

Collection of skin samples Immediately after euthanasia with intravenous pentobarbitone sodium (clinically normal dogs) or following sedation with a combination of xylazine and ketamine (all other dogs) the sublumbar skin was clipped and, if necessary, shaved. An ellipse of skin (4 cm by 2 cm) was excised, the subcutaneous fat scraped away and a portion was placed in formol saline and submitted for histopathological examination, The remainder was snap-frozen in liquid nitrogen. All subsequent procedures were carried out in a cold room at 4°C.

Buffer The buffer consisted of 0-01 M tris (hydroxymethyl) aminomethane, 0.002 M MgClz6H20, 0.005 M dithiothreitol, 0.01 M sodium molybdate, 0.01 M trisodium citrate, 2H20, 10 per cent glycerol, pH 7 . 2 5 at 21°C. The dithiothreitol, sodium molybdate and trisodium citrate were added three hours before use.

Preparation of cytosol Skin samples were diced and then pulverised by hammering in a stainless steel mortar chilled with liquid nitrogen. Approximately 500 mg of pulverised skin was transferred to a glass test tube, 1-8 ml of buffer added and tissue homogenates were prepared using two 20-second bursts of a polytron homogeniser at speed setting 10. Throughout this phase the glass test tube was surrounded by ice and the two bursts

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Dexamethasone(nM) FIG 1: Epidermal dexamethasone binding in normal dog 7. • Total binding,[] Non-specificbinding,• Specific binding

were separated by an interval of 60 seconds. Subsequently a further 4 ml of buffer was added to the skin homogenates and these were centrifuged at 15,000 g, f o r l 0 minutes at 2°C. The supernatant was removed and a sample of resultant cytosol was frozen for protein determination.

Receptor analysis Following the method of Smith et al (1983), 200/~1 portions of cytosol were mixed in triplicate with 100/zl of a series of solutions of 3H-dexamethasone to give final dexamethasone concentrations in the range 1-7 to 6"7 nm to ensure saturation conditions (Fig 1). Parallel tubes were set up containing an additional 200 times excess of unlabelled dexamethasone to assess non-specific binding. After a 20 hour incubation, 300/zl of 0- 6 dextran coated charcoal was added and the contents of the tubes mixed by vortex and remixed after seven minutes. After a total incubation of 15 minutes the charcoal was pelleted by centrifugation at 2500 g for I0 minutes. The supernatant was decanted into vials, scintillation fluid added and the radioactivity counted in an LKB 1218 Rockbita liquid scintillation counter (counting efficiency 25 to 30 per cent). Duplicate analyses gave a relative standard deviation of 5"9 per cent (number of receptors) and 5" 6 per cent (apparent Kd).

Histopathological examination of skin samples Histopathology of skin samples confirmed removal of the dermal tissue and of the lower (intradermal) segment of the hair follicles and adnexa.

Glucocorticoid receptors in the skin of dogs 0"04-

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Results

Results for individual dogs are given in Table 1.

Clinically normal dogs

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The mean number of specific dexamethasone binding receptors detected was 42.0 ± 25.2 fmol m g protein (range: 18 to 73 fmol mg -~ protein) and the mean apparent dissociation constant (Kd app) 1 ± 0-23 nM (range: 0.7 to 1.4 nM).

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Specific [3HI dexamethasone binding was detected (number of binding sites 12.6 fmol mg-1 protein, Kd app 0"7 nM) in only one of the 10 cases.

FIG 2: Scatchard plot of data f r o m normal dog 7

Dogs with bilaterally symmetrical alopecia A nalys& of cytosolic protein Cytosolic protein concentration was determined using bicinchoninic acid reagent (Pierce Chemical Company) (Smith et al 1985).

Analysis of data The Scatchard plot was used to analyse the data and from the linear relationships to determine the apparent dissociation constant (Kd app) and number of specific binding sites (Fig 2).

In the case of hypothyroidism the number of receptors and Kd app detected were, respectively, 21-6 fmol mg -~ protein and 3.0 nM. No specific [3H] dexamethasone binding was detected in the boxer, one of the doberman pinschers or the Yorkshire terrier with undiagnosed bilaterally symmetrical alopecia. In the remaining three cases the number of binding sites and Kd app were, 21-7 fmol m g - l protein and 1-9 nM (Airedale terrier); 23 fmol m g - I protein and 1 nM (crossbred terrier); 35 fmol m g - l protein and 4 nM (doberman pinscher).

Discussion TABLE 1: Apparent Kd and number of specific dexamethasone binding receptors detected in the epidermis of normal dogs, dogs

with hyperadrenocorticism and dogs with bilaterally symmetrical alopecia

Kd app (nM) Normal dogs 1 2 3 4 5 6 7 Dogs w i t h HAC Cases 1-9 Case 10 Dogs with bilaterally symmetrical alopecia Case 1 -- hypothyroid Case 2 -- undiagnosed Case 3 -- undiagnosed Case 4 -- undiagnosed Cases 5-7

I .1 0.9 1 .1 1 -4 1.2 0.9 0.7

Number of specific binding sites (fmol mg 1 protein) 73 18 18 22 70 62 31

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None detected 12.6

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21 .6 21 . g 23.0 35.0 None detected

This study demonstrates the presence of high affinity, low capacity dexamethasone binding receptors in the skin of dogs. The number (42-0 fmol mg -1 ± 25-2) and Kd app (1-0 n m ± 0"23) of these receptors in clinically normal dogs appears to be similar to that (50 fmol mg -~ protein, Kd app 1.1 nM) documented in the epidermis of man (Epstein and Bonifas 1982) and the epidermis of neonatal mice (40 fmol mg -~ protein, Kd app 3.4 nM) (Epstein and Munderloh 1981). Failure, in this present study, to detect [3H] dexamethasone binding in the skin of nine out of 10 dogs with Confirmed spontaneous HAC and the reduction in number of receptors (12"6 fmol mg -1 protein) in the skin of the remaining case provides evidence for a profound down regulation of cutaneous glucocorticoid receptors in dogs by endogenous glucocorticoids. Though more pronounced this observation is consistent with the reported down regulation of glucocorticoid receptors in the liver of mice (Svec et al 1989) and in human mononuclear ceils (Shipman et al 1983, Griese et al 1988) following administration of exogenous glucocorticoid. However, studies of

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A. H. M. van den Broek, IV. L. Stafford

mononuclear cells (Kontula et al 1980, Pardes et al 1989) and cultured skin fibroblasts (Nawata et al 1984) from cases of spontaneous human HAC failed to detect any difference from normal in glucocorticoid receptor numbers or apparent Kd. Although reported differences in glucocorticoid receptor response in man to endogenous and exogenous glucocorticoids may be attributed to the duration of glucocorticoid excess, this does not explain the conflicting observations made in spontaneous human HAC and the present study of spontaneous canine HAC. These differences may reflect a species or tissue variation in susceptibility of glucocorticoid receptors to endogenous glucocorticoids and require further investigation. Reductions in the number, but no change in apparent Kd, of glucocorticoid receptors have been documented in lungs (Morishige 1982), liver (Lesney et al 1987) and pancreas (Lu et al 1988) of rats with experimentally induced hypothyroidism. However, no significant reduction in the number of [3H] dexamethasone binding receptors was detected in the skin of the hypothyroid dog though the apparent Kd (3 nM) was higher than the range (0.7 to 1.4 nM) documented in the clinically normal dogs. This suggests that though thyroid hormone may modulate glucocorticoid receptor activity in dogs its influence is not as great as in rats. Further studies of hypothyroid dogs are necessary to confirm this observation. Although possible, it is unlikely that the absence of [3HI dexamethasone binding in three of the dogs with undiagnosed bilaterally symmetrical alopecia indicates the presence of HAC undetected by the ACTH stimulation test or dexamethasone screening test. Though the possible role of growth hormone deficiency in dexamethasone binding needs to be investigated, the observations in these th?ee dogs suggest that in the apparent absence of systemic hormone imbalance receptor function may be abnormal. Such abnormalities of receptor function may be involved in the genesis of some currently undiagnosed symmetrical alopecias of dogs.

References EPSTEIN, E. R. & BONIFAS, J. M. (1982) Glucocorticoid receptors o f normal human epidermis. Journal ofln vestigative Dermatology 78, 144-146 EPSTEIN, E. R. & MUNDERLOH, N. H. (1981) Glucocorticoid receptors of mouse epidermis and dermis. Endocrinology 108, 703-711

FELDMAN, E. C. (1983) Comparison of ACTH response and dexamethasone suppression as screening tests in canine hyperadrenocorticism. Journal of the American Veterinary Medical Association 182, 506-510 GRIESE, M., KUSENBACH, G., LUSEBRING, K., KOSTER, W., ROTH, B. & REINHARDT, D. (1988) Glucocorticoid receptors in mononuclear blood cells and their correlation to endogenous and exogenous corticoids in healthy and asthmatic children. European Journal of Paediatrics 147, 490-495 KONTULA, K., PELKONEN, R., ANDERSSON, L. & SIVULA, A. (1980) Glucocorticoid receptors in adrenocorticoid disorders. Journal of Clinical Endocrinology and Metabolism 51,654-657 LESNEY, A. M., BENMILOUD, M., BEFORT, N. & BEFORT, J. J. (1987) In vitro evidence that hypothyroidism modifies glucocorticoid receptors. Molecular and Cellular Endocrinology 52, 1-10 LU, R. B., LIEBENTHAL, E. & LEE, P. C. (1988) Regulation of rat pancreatic glucocorticoid receptors by thyroxine during development. Endocrinology 123, 2235-2241 MORISHIGE, W. K. (1982) Thyroid hormone influences glucocorticoid receptor levels in the neonatal rat lung. Endocrinology 111, 1017-1019 MULLER, G. H., KIRK, R. W. & SCOTT, D. W. (1989) Small Animal Dermatology. 4th edn. Philadelphia, W. B. Saunders. pp 575-657 NAWATA, H., HIGUCHI, K., HIGASHIZMA, M., KATO, K. Y. & IBAYASHI, H. (1984) Glucocorticoid receptors in cultured skin fibroblasts of normal and adrenocorticoid disorders. Endocrinologica Japonica 31,109-116 PARDES, E. M., DE YAMPEY, J. W., SOTO, R. J., MOSES, D. F. & DE NICOLA, A. F. (1989) A correlative study between glucocorticoid receptor levels in human mononuclear leucocytes and biochemical data in Cushing's disease. Acta Endocrinologica 120, 55-61 SCOTT, D. W. (1990) Seasonal flank alopecia in ovariohysterectomised dogs. Cornell Veterinarian 80, 187-195 SHANLEY, K. J. & MILLER, W. H. (1987) Adult-onset growth hormone deficiency in ~ibling Airedale terriers. Compendium of Continuing Education for the Practicing Veterinarian 9, 1076-1082 SHIPMAN, G. F., BLOOMFIELD, C. D., GAJL-PECZALSKA, K. J., MUNCK, A. U. & SMITH, K. A. (1983) Glucocorticoids and lymphocytes. III. Effects of glucocorticoid administration on lymphocyte glucocorticoid receptors. Blood 61, 1986-1990 SMITH, K., SHUSTER, S. & RAWLINS, M. (1983) Characterisation of the glucocorticoid receptor in rat skin. Journal of Endocrinology 96, 229-239 SMITH, P. K., KROHN, R. I., HERMANSON, G. T., MALLIA, A. K., GARTNER, F. H., PROVENZANO, M. D., FUKIMOTO, E. K. , GROEKE, N. M., OLSON, B. J. & KLENK, D. C. (1985) Measurement of protein using bicinchoninic acid. Analytical Biochemistry 150, 76-85 SVEC, F., GORDON, S. & TATE, D. (1989) Glucocorticoid receptor regulation: the effects of adrenalectomy, exogenous glucocorticoid and stress on hepatic receptor numbers in male and female mice. Biochemical Medicine and Metabolic Biology 41, 224-233 THODAY, K. L. (1990) The Thyroid Gland. In Hormones of Small Animal Endocrinology. Ed M. Hutchison. Cheltenham, British Small Animal Veterinary Association. pp 25-58

Received April 30, 1991 Accepted July 17, 1991