0019~7227/92/1316-2659$03.00/0 Endocrinology Copyright CC 1992 hy The Endocrine
Vol. 131, No. 6 Printed m U.S.A.
Society
Corticotropin-Releasing Adrenocorticotropic Cerebrospinal Fluid Hyperadrenocorticism*
Hormone and Hormone Concentrations in of Dogs with Pituitary-Dependent
PETRA A. VAN WIJK, AD RIJNBERK, RONALD ELPETRA P. M. SPRANG, AND JAN A. MOL
J. M. CROUGHS,
GEORGE
Departments of Clinical Sciences of Companion Animals (P.A. W., A.R., E.P.M.S., (G. V.), Faculty of Veterinary Medicine, University of Utrecht, and the Department (R.J.M.C), University Hospital Utrecht, Utrecht, The Netherlands ABSTRACT
There is still some controversy concerning the question of whether Cushing’s disease in man is caused by a primary dysfunction of the pituitary or a hypothalamic disorder. In the latter option, excessive hypothalamic stimulation of pituitary corticotropes would cause or contribute to the genesis of POMC-secreting adenomas. In the present study cerebrospinal fluid (CSF) CRH levels and levels of ACTH and cortisol in CSF and plasma were measured in clinically healthy dogs, in dogs with pituitary-dependent hyperadrenocorticism (PDH), and in dogs with hyperadrenocorticism due to an adrenocortical tumor (ATH). In CSF from dogs with PDH, CRH concentrations (226.6 + 14.4 ng/ liter) were significantly (P < 0.05) lower than those in control dogs (309.5 + 20.3 rig/liter). In the dogs with ATH, CSF CRH concentrations
I
VOORHOUT,
J.A.M.) and Radiology of Endocrinology
(211.0 * 40.3 rig/liter) were in the range of those in PDH dogs. In dogs with ATH, CSF ACTH levels (13.0 + 3.0 rig/liter) were significantly (P < 0.05) lower than those in control dogs (63.4 + 3.5 rig/liter), whereas in dogs with PDH, the levels (116.8 + 47.5 rig/liter) were not different from those in the control group. In control dogs, the concentrations of CSF CRH and plasma ACTH were significantly correlated (r = 0.635; P < 0.01). This functional dependency appeared to he disturbed in dogs with PDH, as in these dogs CSF CRH concentrations did not correlate with plasma ACTH concentrations. It is concluded that continuous hyperstimulation of pituitary cortcotropes with hypothalamic CRH is probably not the cause of excessive ACTH secretion in dogs with pituitary-dependent hyperadrenocorticism. (Endocrinology 131: 2659-2662,1992)
fluences (4). Attempts to discriminate between a pituitary and a suprapituitary origin of the disease have included neuropharmacological interventions in the actions of the multiple transmitters involved in the regulation of ACTH secretion (5) as well as measurements of CRF in dog hypothalami. In the latter species, in which pituitary-dependent hyperadrenocorticism (PDH) is a common and well characterized entity (68), the results of these measurements have been somewhat conflicting. In an early study, CRF activity was low or undetectable in hypothalami of dogs with spontaneous PDH (9), whereas more recent immunoreactive CRH (i-CRH) determinations in paraventricular nuclei of dogs with PDH were not different from those obtained in healthy control dogs (10). In humans with PDH, the concentrations of i-CRH in cerebrospinal fluid (CSF) are lower than those in healthy controls (11) or patients with major depression (12). Here we report concentrations of i-CRH in CSF of 23 dogs with PDH and compare them with values in healthy control dogs and dogs with hyperadrenocorticism due to adrenocortical tumor.
N THE majority of patients with Cushing’s disease there is some type of pituitary adenoma. The absence of corticotroph hyperplasia in surrounding tissue and the favorable results of selective adenomectomy are used as arguments against primary hypothalamic dysfunction and seem to agree with the notion that the adenomas are the result of clonal expansion of genomically altered cells (1). However, investigations on the clonal composition of the adenomas, employing X-chromosome inactivation analysis, have revealed both monoclonal and polyclonal corticotroph adenomas (2). The latter may develop as cell lines of multiple foci of corticotroph hyperplasia or as an isolated, but heterogeneous, adenoma, both in response to increased stimulation from the hypothalamus (1). In addition, analyses of 24h secretion patterns of ACTH and cortisol have revealed hyperpulsatility in some cases in spite of the endogenous hypersecretion of cortisol (3, 4). The interindividual differences of these secretion patterns have been explained as different stages in the stepwise evolution of the disease, involving not only the pituitary, but also suprapituitary inReceived May 4, 1992. Address all correspondence and requests for reprints to: Dr. A. Rijnberk, Department of Clinical Sciences of Companion Animals, Faculty of Veterinary Medicine, University of Utrecht, P.O. Box 80.154, 3508 TD Utrecht, The Netherlands. * This work was supported by NW0 (Dutch Organization for Scientific Research) Grant 900-543-066.
Materials
and Methods
Animals Clinically healthy female dogs (n = 16; median age, 7 yr; range 6-11 yr) were used as a control group. The group of dogs with PDH had a
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median age of 10 yr (range, 5-13 yr) and comprised 13 female dogs (6 ovariohysterectomized) and 10 male dogs (1 castrated). The group of dogs with hyperadrenocorticism due to adrenocortical tumor (ATH) comprised 1 male and 4 females (1 ovariohysterectomized), and their median age was 8 yr (range, 7-14 yr). Apart from the characteristic signs and symptoms, such as polyuria, skin atrophy, and increased abdominal size (13), the diagnosis of hyperadrenocorticism was based upon elevated urinary corticoid/creatinine (C/C) ratios in two consecutive morning urine samples (14, 15). Immediately after collection of the second urine sample, the animals received three oral doses of 0.1 mg dexamethasone/kg BW at 8-h intervals. The next morning, a third urine sample was collected. When the C/C ratio in the third sample was less than 50% of the mean in the first two samples, the dog was categorized as being responsive to dexamethasone suppression, and PDH was diagnosed. In the nine dogs in which there was less than 50% suppression of the C/C ratio, i.e. the dexamethasoneresistent animals, the differentiation between PDH and ATH was based upon at least five measurements of basal plasma ACTH (16) and ultrasonography of the adrenal regions (17). In the five dogs with ultrasonographically demonstrated adrenocortical tumor, ACTH levels were low (mean + SEM, 23.0 + 3.5 rig/liter) compared to those in healthy dogs (99.0 t 13.6 rig/liter). In the four dogs with dexamethasoneresistent PDH, ACTH levels ranged from 49-193 rig/liter. At surgery, four dogs with ATH proved to have a unilateral adrenocortical tumor, and one dog had a bilateral tumor. The diameter of the tumors varied from lo-35 mm (mean f SEM, 24.7 + 2.7 mm).
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1992 No 6
For the validation of the CRH assay, canine hypothalamic tissue was homogenized with a Polytron (Brinkmann Instruments, Westbury, NY) in 5 vol 0.1 N HCl containing 0.001% (wt/vol) phenylmethylsulfonylfluoride, 0.05% (wt/vol) ascorbic acid, and 0.001% (vol/vol) fl-mercaptoethanol. After centrifugation for 30 min at 12,000 X g, the supernatant was Iyophilized. The extract was then dissolved in 30% (vol/vol) acetonitrile-0.1% (vol/vol) trifluoroacetic acid (TFA) and centrifuged for 5 min at 12,000 X g. The clear supernatant was separated by reverse phase HPLC (Fig. 1). One-milliliter fractions were lyophilized and then dissolved in 0.5 ml assay buffer before analysis. The hypothalamic CRH extract was eluted from the column at the position of hCRH, and it showed parallelism to the hCRH standard used in the RIA. No significant interference by other hypothalamic peptides was found in the HPLC fractions. ACTH was measured by RIA without extraction, according to the procedure of Arts et al. (22) and validated for the dog. Antiserum was obtained from IgG Corp. (Nashville, TN). The tracer was purchased from International CIS (St. Quentin-Yvelines, France), and the standard was obtained from the NIH (Bethesda, MD). The detection limit was 10 rig/liter. Interassay variation was 12%. Cortisol was measured by RIA (8, 15). The detection limit was 1 nmol/liter. Interassay variation was 8%. Statistical analysis. The results are presented as the mean ences among groups were tested with the Kruskal-Wallis by the Mann-Whitney test, as corrected by Bonferroni. significance was P < 0.05.
+ SEM. Differtest, followed The level of
Methods Samples. Between 0800-0900 h, CSF was collected from the cerebellomedullary cistern by suboccipital puncture (18, 19) after iv administration of 15 mg sodium thiopental/kg (Rhone Merieux, Lyon, France). CSF was collected in precooled polystyrene tubes. The samples were stored at -20 C until assayed. At the time of CSF sampling, blood samples were collected from the jugular vein in precooled EDTA-coated tubes. The samples were centrifuged at 4 C, and plasma was stored at -20 C until assayed. RIAs. To eliminate potential interfering binding proteins for CRH, CSF samples were extracted on solid phase extraction columns containing 500 mg Cis sorbent (J. T. Baker, Inc., Philipsburg, NJ). Columns were washed successively with 6 ml methanol, 6 ml 40% (vol/vol) acetonitrile0.17 M triethyl ammonium formate (TEAF; pH 3.2), and 6 ml 0.17 M TEAF. One milliliter of CSF was applied to the column and eluted by gravity alone. The columns were washed with 7 ml 0.17 M TEAF. i-CRH was eluted with 2.5 ml 50% (vol/vol) isopropanol-0.17 M TEAF. The extract was collected in polypropylene tubes and lyophilized using a Savant Speed-Vat concentrator (New Brunschwig Scientific, Hatfield, Herts, United Kingdom). Samples were stored at -20 C until assayed. The extraction efficiency was quantitated by the addition of 25,000 dpm [‘*‘I]Tyr-ovine (0) CRH to a CSF sample and amounted to 48 * 2% (mean + so; n = 4). Human (h) CRH antiserum (lot C13) was kindly donated by Dr. G. K. Stalla (Medizinische Klinik Innenstadt, University of Munich, Munich, Germany) (20). Tyr-oCRH (Sigma Chemical Co., St. Louis, MO) was radioiodinated with 12’1- by the chloramine-T method (21) to a specific activity of 200 &i/fig and used as a tracer. Tracer purification was achieved on Sep-Pak Cls columns by elution two times with l-ml aliquots of 0%, 25%, 50%, 75%, and 99% methanol in 0.1% (voI/voI) trifluoroacetic acid (TFA). CSF extracts were dissolved in 0.5 ml assay buffer, consisting of 67 rnM sodium phosphate (pH 7.4), 13 rnM EDTA, 0.25% (wt/vol) BSA, 0.1% (vol/vol) Triton X-100, and 2% (vol/vol) Trasylol. To 200 ~1 extract or hCRH standard solution (Peninsula Laboratories, Inc., Belmont, CA), 50 ~1 antiserum (diluted 1:16,000) were added and incubated overnight at 4 C. On the following day, 50 ~1 tracer were added (&SO00 dpm) and incubated for 30 h. The separation of bound and free hormone was achieved using second antibody-coated cellulose (Saccel, IDS, Boldon, Tyne and Wear, United Kingdom) according to the manufacturer’s protocol. The detection limit of the assay was 12 rig/liter. All samples were measured in a single assay, with a coefficient of variation of 8%. Results were corrected for the extraction efficiency.
Results CSF CRH concentrations in dogs with PDH (226.6 + 14.4 q/liter) were significantly (P < 0.05) lower than those in the control dogs (309.5 f 20.3 rig/liter; Fig. 2). In the dogs with ATH, the concentrations (211.0 + 40.3 rig/liter) were similar to those in the PDH dogs. CSF ACTH concentrations in dogs with ATH (13.0 + 3.0 rig/liter) were significantly (P < 0.05) lower than those in the control dogs (63.4 + 3.5 rig/liter). CSF ACTH levels in dogs with PDH (116.8 + 47.5 rig/liter) were not different from those in dogs of the control group. CSF cortisol values varied between 8-13% of the cortisol 100
6
60
=L 0) 1
6
60
z
z0
4
40
E d -l s
0
5
10
15
20
Fraction
(ml)
25
30
35
FIG. 1. HPLC analysis of canine hypothalamic extract. The extract was separated on a LiChrosper 100 RP-18 end-capped column (Merck, Darmstad, Germany) at a flow rate of 1 ml/min using a linear gradient of 30-60% acetonitrile in 0.01% (vol/vol) TFA (- - -). CRH immunoreactivity was assessed in lyophilized l-ml fractions. Absorbance was measured at 214 nm. In this procedure, hCRH was also eluted in fraction 25.
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CRH
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400
400 m
CONTROLm
PDH
m
ATH
300
300
c
%i c zs:
200
200
E c o .;
z 0
8 100
100
0
0 CRH c---
ACTH CORTISOL CSFh+-----PLASMA+
ACTH
CORTISOL
2. CRH concentrations in CSF, and ACTH and cortisol concentrations in CSF and plasma in control dogs, dogs with PDH, and dogs with ATH. Results are expressed as the mean + SEM. t, Statistically different from the control group (P < 0.05). FIG.
TABLE 1. Correlations for CRH, ACTH, and cortisol in CSF and plasma in the control group and the PDH group Plasma ACTH
Control group CSF CRH CSF ACTH CSF cortisol Plasma ACTH PDH group CSF CRH CSF ACTH CSF cortisol Plasma ACTH No correlations were found in
Plasma cortisol
0.64” 0.40 0.90*
0.37 0.09 0.37* 0.74*
0.05 0.14 0.63”
-0.37 0.03 0.72” 0.53
the ATH group.
aP < 0.01. *P < 0.001. concentrations in plasma. The differences in cortisol levels in CSF and plasma were not significant among the three groups. Correlations among the concentrations of CRH, ACTH, and cortisol in CSF and plasma were calculated for each group (Table 1). In the control group, a significant correlation of CSF CRH concentrations with the ACTH concentrations in plasma was found. Plasma concentrations of ACTH and cortisol were significantly correlated. Both the ACTH and cortisol concentrations in plasma were significantly correlated with the CSF cortisol levels. In the PDH group, the ACTH and cortisol concentrations in plasma were significantly correlated with CSF cortisol levels, but no correlation was found for the CSF CRH concentrations and plasma ACTH concentrations. There were no significant correlations for the ATH group. Discussion ACTH secretion is mediated by multiple factors of hypothalamic origin, including CRH and arginine vasopressin(23, 24). It is evident that hypothalamic CRH is the predominant central regulatory factor in the physiology of the hypothalamus-pituitary adrenocortical system in man. However, its
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role in the pathogenesisof pituitary-dependent hyperadrenocorticism is a matter of continuing debate (23). CRH neurons have a widespread distribution throughout the central nervous system (CNS), concordant with the functions of CRH. Consistent with a role for CRH as a hypothalamic releasing factor regulating pituitary-adrenocortical activity is the presence of CRH in high concentrations in the paraventricular nucleus (PVN) of the hypothalamus and in the median eminence of the rat (25). In rats, Beyer et al. (26) have shown that of mRNA for CRH of the same size synthesized in various CNS areas, only PVN mRNA responds to alterations of peripheral glucocorticoid status. Other hypothalamic areas, such as the supraoptic nucleus, do not respond with lower CRH mRNA levels after elevated glucocorticoid concentrations. They concluded that only CRF from the PVN is involved in control of the hypothalamicpituitary-adrenal axis. The present finding of low CSF CRH concentrations in canine PDH suggeststhat CRH releasedin the CSF is suppressible by sustained hypercortisolism. Similar findings in ATH, in which hypercortisolism is clearly derived from outsidethe CNS, further support the idea that centrally directed CRH secretion is suppressibleby sustainedhypercortisolism. The possibility that the lower level of CRH in PDH represents a disordered response to the stress of general anaesthesia and cisterna magna puncture characteristic of the diseaseis unlikely; thus, the reduced response of plasma ACTH and cortisol to the stress of insulin-induced hypoglycemia in patients with Cushing’s disease has been shown to be a phenomenon secondary to hypercortisolism per se(27). The results extend our previous findings of a reduced hypothalamic CRF content in dogs with PDH (9). In that study, CRF activity was measuredin blocks of hypothalamic tissueusing an indirect bioassay technique. More recently, Peterson et al. (10) found unaltered CRH concentrations of hypothalamic CRH in dogs with pituitary-dependent hyperadrenocorticism. These researchersused tissue from the PVN obtained by a micropunch technique, and CRH was measured by a direct RIA. Both the sampling methodology and the differencesin assayperformance may explain the different results. Peterson et al. (10) measured CRH specifically in the PVN, which doesnot seemto be the main location of hypothalamic CRH synthesis in the dog. In this species,Stolp et al. (28) found most of the cell bodies with immunoreactivity for CRH in the periventricular nucleus. This nucleus was certainly included in the blocks of hypothalamic tissueinvestigated by Meijer et al. (9). The present study confirms similar findings of reduced CSF CRH levels in human Cushing’s disease(11, 12) and suggeststhat the primary pathophysiology in PDH resides at the pituitary level. This is supported by the fact that in contrast to the control dogs, there was no correlation between CSF CRH levels and plasmaACTH levels in dogswith PDH. There seemsto be a discrepancy between dogs and man with regard to the ACTH concentrations in CSF. Kling et al. (12) have shown that in man, Cushing’sdiseaseis associated with suppressedCSF ACTH concentrations, indicating that the sourcesof CSF ACTH and plasma ACTH are different.
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The fact that in the control dogs there was no correlation between the levels of ACTH in plasma and those of ACTH in CSF suggests that the sources of ACTH in plasma and CSF are different in dogs also. The suppressed CSF ACTH concentrations in dogs with ATH can be explained by the negative feedback of the excess glucocorticoids on ACTH sources of suprapituitary origin. However, in contrast to hypercortisolism in man and dogs with ATH, no suppressed CSF ACTH levels were found in dogs with PDH, suggesting that in these dogs, CSF ACTH is at least in part of pituitary origin. In conclusion, both canine PDH and Cushing’s disease in man are probably not caused by a continuous hyperstimulation of pituitary corticotropes by hypothalamic CRH. Acknowledgments The authors gratefully acknowledge the enthusiastic contributions of Dr. L. J. Hellebrekers and Mr. G. Haalboom in anesthetizing the dogs, and Mrs. J. Wolfswinkel for technical assistance.
References 1. Grua JR, Nelson DH 1991 ACTH-producing pituitary tumors. Endocrinol Metab Clin North Am 20:319-362 EH, Allolio B, Katz DA, Berkman RA, Ali 2. Schulte HM, Oldfield IU 1991 Clonal composition of pituitary adenomas in patients with Cushing’s disease: determination by X-chromosome inactivation analysis. J Clin Endocrinol Metab 73:1302-1308 3. Van Cauter E, Refetoff S 1985 Identification of two subtypes of Cushing’s disease based on the analysis of episodic cortisol secretion. N Engl J Med 312:1343-1349 4. Schiirmeyer TH, Brabant G, Von zur Muhlen A 1990 Evidence that the hypothesis on a hypothalamic origin of Cushing’s disease is not an outdated concept: Ludecke DK, Chrousos G?, Tolis G (eds) ACTH, Cushing’s Syndrome, and Other Hypercortisolemic States. Raven Press, New York, p 105 5. Croughs RJM, Rijnberk A, Koppeschaar HPF 1990 Heterogeneity in Cushiner’s disease. Neth 1 Med 36:217-220 6. Peterson ME 1987 Pathophysiology of canine pituitary-dependent hyperadrenocorticism (canine Cushing’s disease). Front Horm Res 17:37-47 7. Orth DN, Peterson ME, Drucker WD 1988 Plasma immunoreactive proopiomelanocortin peptides and cortisol in normal dogs and dogs with Cushing’s syndrome: diurnal rhythm and responses to various stimuli. Endocrinology 122:1250-1262 8. Rijnberk A, Mol JA, Kwant MM, Croughs RJM 1988 Effects of bromocriptine on corticotrophin, melanotrophin and corticosteroid secretion in dogs with pituitary-dependent hyperadrenocorticism. J Endocrinol 118:271-277 9. Meijer JC, Mulder GH, Rijnberk A, Croughs RJM 1978 Hypothalamic corticotrophin releasing factor activity in dogs with pituitarydependent hyperadrenocorticism. J Endocrinol 79:209-213 ME, Palkovits M, Chiueh CC, Graves TK, Mezey E, 10. Peterson Vale W, Krieger DT 1989 Biogenic amine and corticotropin releasing factor concentrations in hypothalamic paraventricular nucleus and biogenic amine levels in the median eminence of normal dogs, chronic dexamethasone-treated dogs, and dogs with naturally-occurring pituitary-dependent hyperadrenocorticism (canine Cush-
in:
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ing’s disease). J Neuroendocrinol 1:169-171 11. Tomori N, Suda T, Tozawa F, Demura H, Shizume K, Mouri T 1983 Immunoreactive corticotropin-releasing factor concentrations in cerebrospinal fluid from patients with hypothalamic-pituitaryadrenal disorders. J Clin Endocrinol Metab 57:1305-1307 12. Kling MA, Roy A, Doran AR, Calabrese JR, Rubinow DR, Whitfield HJ, May Jr C, Post RM, Chrousos GP, Gold PW 1991 Cerebrospinal fluid immunoreactive corticotropin-releasing hormone and adrenocorticotropin secretion in Cushing’s disease and major depression: potential clinical implications. J Clin Endocrinol Metab 72:260-271 13. Rijnberk A, der Kinderen PJ, Thijssen JHH 1968 Spontaneous hyperadrenocorticism in the dog. J Endocrinol 41:397-406 14. Stolp R, Rijnberk A, Meijer JC, Croughs RJM 1983 Urinary corticoids in the diagnosis of canine hyperadrenocorticism. Res Vet Sci 34:141-144 15. Rijnberk A, Wees van A, Mol JA 1988 Assessment of two tests for the diagnosis of canine hyperadrenocorticism. Vet Ret 122:178-180 16. Rijnberk A, Mol JA, Rothuizen J, Bevers MM, Middleton DJ 1987 Circulating pro-opiomelanocortin-derived peptides in dogs with pituitary-dependent hyperadrenocorticism. Front Horm Res 17:48-60 17. Voorhout G, Rijnberk A, Sjollema BE, van den Ingh TSGAM 1990 Nephrotomography and ultrasonography for the localization of hyperfunctioning adrenocortical tumors in dogs, Am J Vet Res 51:1280-1285 18. Voorhout G 1990 Cisternography combined with linear tomography for visualization of the pituitary gland in healthy dogs. A comparison with computed tomography:Vet Radio1 31:88-7319. Voorhout G, Riinberk A 1990 Cisternograuhv combined with linear tomography for visualization of pituit& lesions in dogs with pituitary-dependent hyperadrenocorticism. Vet Radio1 31:74-78 20. Stalla GK, Stalla J, Schophol J, Werder von K, Miiller OA 1986 Corticotropin releasing factor (CRF) in humans: CRF stimulation in normals and CRF-radioimmunoassay. Horm Res 24:229-245 21. Hunter WM, Greenwood FC 1962 Preparation of iodine-131 labelled human growth hormone of high specific activity. Nature 194:495-496 22. Arts CJM, Koppeschaar HPE, Veeman W, Thijssen JHH 1985 A direct radioimmunoassay for the determination of adrenocorticotropit hormone (ACTH) and a clinical evaluation. Ann Clin Biochem 221247-256 23. Plotsky PM 1988 Central regulation of stimulus-induced ACTH secretion: characterization of hypophysial-portal plasma CRF and AVP concentrations profiles. In: Imura H (ed) Neuroendocrine Control of the Hypothalamo-Pituitary System. Karger, Base], p 31 24. Wittert GA, Crock, Donald RA, Gilford EJ, Boolell M, Alford FP, Espiner EA 1990 Arginine vasopressin in Cushing’s disease. Lancet 335:991-994 25. Bloom FG, Battenberg ELF, Rivier J, Vale W 1982 Corticotropin releasing factor (CRF) immunoreactive neurons and fibers in rat hypothalamus. Regul Peptides 4:43-48 26. Beyer HS, Matta SG, Sharp BM 1988 Regulation of the messenger ribonucleic acid for corticotropin-releasing factor in the paraventricular nucleus and other brain sites of the rat. Endocrinology 123:2117-2123 27. Croughs RJM, Timmermans H, Vingerhoeds ACM, Vermeulen A, Smals A, Kloppenburg PWC, Meyer JC 1977 Insulin stimulation tests in pituitary-dependent Cushing’s syndrome after complete adrenalectomy. Acta Endocrinol (Copenh) 86:578-582 28. Stolp R, Steinbusch HWM, Rijnberk A, Croughs RJM 1987 Organization of ovine corticotropin-releasing factor immunoreactive neurons in the canine hypothalamo-pituitary system. Neurosci Lett 74:337-342
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Vol. 131, No. 6 Printed m U.S.A.
Society
Corticotropin-Releasing Adrenocorticotropic Cerebrospinal Fluid Hyperadrenocorticism*
Hormone and Hormone Concentrations in of Dogs with Pituitary-Dependent
PETRA A. VAN WIJK, AD RIJNBERK, RONALD ELPETRA P. M. SPRANG, AND JAN A. MOL
J. M. CROUGHS,
GEORGE
Departments of Clinical Sciences of Companion Animals (P.A. W., A.R., E.P.M.S., (G. V.), Faculty of Veterinary Medicine, University of Utrecht, and the Department (R.J.M.C), University Hospital Utrecht, Utrecht, The Netherlands ABSTRACT
There is still some controversy concerning the question of whether Cushing’s disease in man is caused by a primary dysfunction of the pituitary or a hypothalamic disorder. In the latter option, excessive hypothalamic stimulation of pituitary corticotropes would cause or contribute to the genesis of POMC-secreting adenomas. In the present study cerebrospinal fluid (CSF) CRH levels and levels of ACTH and cortisol in CSF and plasma were measured in clinically healthy dogs, in dogs with pituitary-dependent hyperadrenocorticism (PDH), and in dogs with hyperadrenocorticism due to an adrenocortical tumor (ATH). In CSF from dogs with PDH, CRH concentrations (226.6 + 14.4 ng/ liter) were significantly (P < 0.05) lower than those in control dogs (309.5 + 20.3 rig/liter). In the dogs with ATH, CSF CRH concentrations
I
VOORHOUT,
J.A.M.) and Radiology of Endocrinology
(211.0 * 40.3 rig/liter) were in the range of those in PDH dogs. In dogs with ATH, CSF ACTH levels (13.0 + 3.0 rig/liter) were significantly (P < 0.05) lower than those in control dogs (63.4 + 3.5 rig/liter), whereas in dogs with PDH, the levels (116.8 + 47.5 rig/liter) were not different from those in the control group. In control dogs, the concentrations of CSF CRH and plasma ACTH were significantly correlated (r = 0.635; P < 0.01). This functional dependency appeared to he disturbed in dogs with PDH, as in these dogs CSF CRH concentrations did not correlate with plasma ACTH concentrations. It is concluded that continuous hyperstimulation of pituitary cortcotropes with hypothalamic CRH is probably not the cause of excessive ACTH secretion in dogs with pituitary-dependent hyperadrenocorticism. (Endocrinology 131: 2659-2662,1992)
fluences (4). Attempts to discriminate between a pituitary and a suprapituitary origin of the disease have included neuropharmacological interventions in the actions of the multiple transmitters involved in the regulation of ACTH secretion (5) as well as measurements of CRF in dog hypothalami. In the latter species, in which pituitary-dependent hyperadrenocorticism (PDH) is a common and well characterized entity (68), the results of these measurements have been somewhat conflicting. In an early study, CRF activity was low or undetectable in hypothalami of dogs with spontaneous PDH (9), whereas more recent immunoreactive CRH (i-CRH) determinations in paraventricular nuclei of dogs with PDH were not different from those obtained in healthy control dogs (10). In humans with PDH, the concentrations of i-CRH in cerebrospinal fluid (CSF) are lower than those in healthy controls (11) or patients with major depression (12). Here we report concentrations of i-CRH in CSF of 23 dogs with PDH and compare them with values in healthy control dogs and dogs with hyperadrenocorticism due to adrenocortical tumor.
N THE majority of patients with Cushing’s disease there is some type of pituitary adenoma. The absence of corticotroph hyperplasia in surrounding tissue and the favorable results of selective adenomectomy are used as arguments against primary hypothalamic dysfunction and seem to agree with the notion that the adenomas are the result of clonal expansion of genomically altered cells (1). However, investigations on the clonal composition of the adenomas, employing X-chromosome inactivation analysis, have revealed both monoclonal and polyclonal corticotroph adenomas (2). The latter may develop as cell lines of multiple foci of corticotroph hyperplasia or as an isolated, but heterogeneous, adenoma, both in response to increased stimulation from the hypothalamus (1). In addition, analyses of 24h secretion patterns of ACTH and cortisol have revealed hyperpulsatility in some cases in spite of the endogenous hypersecretion of cortisol (3, 4). The interindividual differences of these secretion patterns have been explained as different stages in the stepwise evolution of the disease, involving not only the pituitary, but also suprapituitary inReceived May 4, 1992. Address all correspondence and requests for reprints to: Dr. A. Rijnberk, Department of Clinical Sciences of Companion Animals, Faculty of Veterinary Medicine, University of Utrecht, P.O. Box 80.154, 3508 TD Utrecht, The Netherlands. * This work was supported by NW0 (Dutch Organization for Scientific Research) Grant 900-543-066.
Materials
and Methods
Animals Clinically healthy female dogs (n = 16; median age, 7 yr; range 6-11 yr) were used as a control group. The group of dogs with PDH had a
2659
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CRH
AND
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median age of 10 yr (range, 5-13 yr) and comprised 13 female dogs (6 ovariohysterectomized) and 10 male dogs (1 castrated). The group of dogs with hyperadrenocorticism due to adrenocortical tumor (ATH) comprised 1 male and 4 females (1 ovariohysterectomized), and their median age was 8 yr (range, 7-14 yr). Apart from the characteristic signs and symptoms, such as polyuria, skin atrophy, and increased abdominal size (13), the diagnosis of hyperadrenocorticism was based upon elevated urinary corticoid/creatinine (C/C) ratios in two consecutive morning urine samples (14, 15). Immediately after collection of the second urine sample, the animals received three oral doses of 0.1 mg dexamethasone/kg BW at 8-h intervals. The next morning, a third urine sample was collected. When the C/C ratio in the third sample was less than 50% of the mean in the first two samples, the dog was categorized as being responsive to dexamethasone suppression, and PDH was diagnosed. In the nine dogs in which there was less than 50% suppression of the C/C ratio, i.e. the dexamethasoneresistent animals, the differentiation between PDH and ATH was based upon at least five measurements of basal plasma ACTH (16) and ultrasonography of the adrenal regions (17). In the five dogs with ultrasonographically demonstrated adrenocortical tumor, ACTH levels were low (mean + SEM, 23.0 + 3.5 rig/liter) compared to those in healthy dogs (99.0 t 13.6 rig/liter). In the four dogs with dexamethasoneresistent PDH, ACTH levels ranged from 49-193 rig/liter. At surgery, four dogs with ATH proved to have a unilateral adrenocortical tumor, and one dog had a bilateral tumor. The diameter of the tumors varied from lo-35 mm (mean f SEM, 24.7 + 2.7 mm).
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For the validation of the CRH assay, canine hypothalamic tissue was homogenized with a Polytron (Brinkmann Instruments, Westbury, NY) in 5 vol 0.1 N HCl containing 0.001% (wt/vol) phenylmethylsulfonylfluoride, 0.05% (wt/vol) ascorbic acid, and 0.001% (vol/vol) fl-mercaptoethanol. After centrifugation for 30 min at 12,000 X g, the supernatant was Iyophilized. The extract was then dissolved in 30% (vol/vol) acetonitrile-0.1% (vol/vol) trifluoroacetic acid (TFA) and centrifuged for 5 min at 12,000 X g. The clear supernatant was separated by reverse phase HPLC (Fig. 1). One-milliliter fractions were lyophilized and then dissolved in 0.5 ml assay buffer before analysis. The hypothalamic CRH extract was eluted from the column at the position of hCRH, and it showed parallelism to the hCRH standard used in the RIA. No significant interference by other hypothalamic peptides was found in the HPLC fractions. ACTH was measured by RIA without extraction, according to the procedure of Arts et al. (22) and validated for the dog. Antiserum was obtained from IgG Corp. (Nashville, TN). The tracer was purchased from International CIS (St. Quentin-Yvelines, France), and the standard was obtained from the NIH (Bethesda, MD). The detection limit was 10 rig/liter. Interassay variation was 12%. Cortisol was measured by RIA (8, 15). The detection limit was 1 nmol/liter. Interassay variation was 8%. Statistical analysis. The results are presented as the mean ences among groups were tested with the Kruskal-Wallis by the Mann-Whitney test, as corrected by Bonferroni. significance was P < 0.05.
+ SEM. Differtest, followed The level of
Methods Samples. Between 0800-0900 h, CSF was collected from the cerebellomedullary cistern by suboccipital puncture (18, 19) after iv administration of 15 mg sodium thiopental/kg (Rhone Merieux, Lyon, France). CSF was collected in precooled polystyrene tubes. The samples were stored at -20 C until assayed. At the time of CSF sampling, blood samples were collected from the jugular vein in precooled EDTA-coated tubes. The samples were centrifuged at 4 C, and plasma was stored at -20 C until assayed. RIAs. To eliminate potential interfering binding proteins for CRH, CSF samples were extracted on solid phase extraction columns containing 500 mg Cis sorbent (J. T. Baker, Inc., Philipsburg, NJ). Columns were washed successively with 6 ml methanol, 6 ml 40% (vol/vol) acetonitrile0.17 M triethyl ammonium formate (TEAF; pH 3.2), and 6 ml 0.17 M TEAF. One milliliter of CSF was applied to the column and eluted by gravity alone. The columns were washed with 7 ml 0.17 M TEAF. i-CRH was eluted with 2.5 ml 50% (vol/vol) isopropanol-0.17 M TEAF. The extract was collected in polypropylene tubes and lyophilized using a Savant Speed-Vat concentrator (New Brunschwig Scientific, Hatfield, Herts, United Kingdom). Samples were stored at -20 C until assayed. The extraction efficiency was quantitated by the addition of 25,000 dpm [‘*‘I]Tyr-ovine (0) CRH to a CSF sample and amounted to 48 * 2% (mean + so; n = 4). Human (h) CRH antiserum (lot C13) was kindly donated by Dr. G. K. Stalla (Medizinische Klinik Innenstadt, University of Munich, Munich, Germany) (20). Tyr-oCRH (Sigma Chemical Co., St. Louis, MO) was radioiodinated with 12’1- by the chloramine-T method (21) to a specific activity of 200 &i/fig and used as a tracer. Tracer purification was achieved on Sep-Pak Cls columns by elution two times with l-ml aliquots of 0%, 25%, 50%, 75%, and 99% methanol in 0.1% (voI/voI) trifluoroacetic acid (TFA). CSF extracts were dissolved in 0.5 ml assay buffer, consisting of 67 rnM sodium phosphate (pH 7.4), 13 rnM EDTA, 0.25% (wt/vol) BSA, 0.1% (vol/vol) Triton X-100, and 2% (vol/vol) Trasylol. To 200 ~1 extract or hCRH standard solution (Peninsula Laboratories, Inc., Belmont, CA), 50 ~1 antiserum (diluted 1:16,000) were added and incubated overnight at 4 C. On the following day, 50 ~1 tracer were added (&SO00 dpm) and incubated for 30 h. The separation of bound and free hormone was achieved using second antibody-coated cellulose (Saccel, IDS, Boldon, Tyne and Wear, United Kingdom) according to the manufacturer’s protocol. The detection limit of the assay was 12 rig/liter. All samples were measured in a single assay, with a coefficient of variation of 8%. Results were corrected for the extraction efficiency.
Results CSF CRH concentrations in dogs with PDH (226.6 + 14.4 q/liter) were significantly (P < 0.05) lower than those in the control dogs (309.5 f 20.3 rig/liter; Fig. 2). In the dogs with ATH, the concentrations (211.0 + 40.3 rig/liter) were similar to those in the PDH dogs. CSF ACTH concentrations in dogs with ATH (13.0 + 3.0 rig/liter) were significantly (P < 0.05) lower than those in the control dogs (63.4 + 3.5 rig/liter). CSF ACTH levels in dogs with PDH (116.8 + 47.5 rig/liter) were not different from those in dogs of the control group. CSF cortisol values varied between 8-13% of the cortisol 100
6
60
=L 0) 1
6
60
z
z0
4
40
E d -l s
0
5
10
15
20
Fraction
(ml)
25
30
35
FIG. 1. HPLC analysis of canine hypothalamic extract. The extract was separated on a LiChrosper 100 RP-18 end-capped column (Merck, Darmstad, Germany) at a flow rate of 1 ml/min using a linear gradient of 30-60% acetonitrile in 0.01% (vol/vol) TFA (- - -). CRH immunoreactivity was assessed in lyophilized l-ml fractions. Absorbance was measured at 214 nm. In this procedure, hCRH was also eluted in fraction 25.
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CRH
AND
ACTH
400
400 m
CONTROLm
PDH
m
ATH
300
300
c
%i c zs:
200
200
E c o .;
z 0
8 100
100
0
0 CRH c---
ACTH CORTISOL CSFh+-----PLASMA+
ACTH
CORTISOL
2. CRH concentrations in CSF, and ACTH and cortisol concentrations in CSF and plasma in control dogs, dogs with PDH, and dogs with ATH. Results are expressed as the mean + SEM. t, Statistically different from the control group (P < 0.05). FIG.
TABLE 1. Correlations for CRH, ACTH, and cortisol in CSF and plasma in the control group and the PDH group Plasma ACTH
Control group CSF CRH CSF ACTH CSF cortisol Plasma ACTH PDH group CSF CRH CSF ACTH CSF cortisol Plasma ACTH No correlations were found in
Plasma cortisol
0.64” 0.40 0.90*
0.37 0.09 0.37* 0.74*
0.05 0.14 0.63”
-0.37 0.03 0.72” 0.53
the ATH group.
aP < 0.01. *P < 0.001. concentrations in plasma. The differences in cortisol levels in CSF and plasma were not significant among the three groups. Correlations among the concentrations of CRH, ACTH, and cortisol in CSF and plasma were calculated for each group (Table 1). In the control group, a significant correlation of CSF CRH concentrations with the ACTH concentrations in plasma was found. Plasma concentrations of ACTH and cortisol were significantly correlated. Both the ACTH and cortisol concentrations in plasma were significantly correlated with the CSF cortisol levels. In the PDH group, the ACTH and cortisol concentrations in plasma were significantly correlated with CSF cortisol levels, but no correlation was found for the CSF CRH concentrations and plasma ACTH concentrations. There were no significant correlations for the ATH group. Discussion ACTH secretion is mediated by multiple factors of hypothalamic origin, including CRH and arginine vasopressin(23, 24). It is evident that hypothalamic CRH is the predominant central regulatory factor in the physiology of the hypothalamus-pituitary adrenocortical system in man. However, its
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role in the pathogenesisof pituitary-dependent hyperadrenocorticism is a matter of continuing debate (23). CRH neurons have a widespread distribution throughout the central nervous system (CNS), concordant with the functions of CRH. Consistent with a role for CRH as a hypothalamic releasing factor regulating pituitary-adrenocortical activity is the presence of CRH in high concentrations in the paraventricular nucleus (PVN) of the hypothalamus and in the median eminence of the rat (25). In rats, Beyer et al. (26) have shown that of mRNA for CRH of the same size synthesized in various CNS areas, only PVN mRNA responds to alterations of peripheral glucocorticoid status. Other hypothalamic areas, such as the supraoptic nucleus, do not respond with lower CRH mRNA levels after elevated glucocorticoid concentrations. They concluded that only CRF from the PVN is involved in control of the hypothalamicpituitary-adrenal axis. The present finding of low CSF CRH concentrations in canine PDH suggeststhat CRH releasedin the CSF is suppressible by sustained hypercortisolism. Similar findings in ATH, in which hypercortisolism is clearly derived from outsidethe CNS, further support the idea that centrally directed CRH secretion is suppressibleby sustainedhypercortisolism. The possibility that the lower level of CRH in PDH represents a disordered response to the stress of general anaesthesia and cisterna magna puncture characteristic of the diseaseis unlikely; thus, the reduced response of plasma ACTH and cortisol to the stress of insulin-induced hypoglycemia in patients with Cushing’s disease has been shown to be a phenomenon secondary to hypercortisolism per se(27). The results extend our previous findings of a reduced hypothalamic CRF content in dogs with PDH (9). In that study, CRF activity was measuredin blocks of hypothalamic tissueusing an indirect bioassay technique. More recently, Peterson et al. (10) found unaltered CRH concentrations of hypothalamic CRH in dogs with pituitary-dependent hyperadrenocorticism. These researchersused tissue from the PVN obtained by a micropunch technique, and CRH was measured by a direct RIA. Both the sampling methodology and the differencesin assayperformance may explain the different results. Peterson et al. (10) measured CRH specifically in the PVN, which doesnot seemto be the main location of hypothalamic CRH synthesis in the dog. In this species,Stolp et al. (28) found most of the cell bodies with immunoreactivity for CRH in the periventricular nucleus. This nucleus was certainly included in the blocks of hypothalamic tissueinvestigated by Meijer et al. (9). The present study confirms similar findings of reduced CSF CRH levels in human Cushing’s disease(11, 12) and suggeststhat the primary pathophysiology in PDH resides at the pituitary level. This is supported by the fact that in contrast to the control dogs, there was no correlation between CSF CRH levels and plasmaACTH levels in dogswith PDH. There seemsto be a discrepancy between dogs and man with regard to the ACTH concentrations in CSF. Kling et al. (12) have shown that in man, Cushing’sdiseaseis associated with suppressedCSF ACTH concentrations, indicating that the sourcesof CSF ACTH and plasma ACTH are different.
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CRH
AND
ACTH
The fact that in the control dogs there was no correlation between the levels of ACTH in plasma and those of ACTH in CSF suggests that the sources of ACTH in plasma and CSF are different in dogs also. The suppressed CSF ACTH concentrations in dogs with ATH can be explained by the negative feedback of the excess glucocorticoids on ACTH sources of suprapituitary origin. However, in contrast to hypercortisolism in man and dogs with ATH, no suppressed CSF ACTH levels were found in dogs with PDH, suggesting that in these dogs, CSF ACTH is at least in part of pituitary origin. In conclusion, both canine PDH and Cushing’s disease in man are probably not caused by a continuous hyperstimulation of pituitary corticotropes by hypothalamic CRH. Acknowledgments The authors gratefully acknowledge the enthusiastic contributions of Dr. L. J. Hellebrekers and Mr. G. Haalboom in anesthetizing the dogs, and Mrs. J. Wolfswinkel for technical assistance.
References 1. Grua JR, Nelson DH 1991 ACTH-producing pituitary tumors. Endocrinol Metab Clin North Am 20:319-362 EH, Allolio B, Katz DA, Berkman RA, Ali 2. Schulte HM, Oldfield IU 1991 Clonal composition of pituitary adenomas in patients with Cushing’s disease: determination by X-chromosome inactivation analysis. J Clin Endocrinol Metab 73:1302-1308 3. Van Cauter E, Refetoff S 1985 Identification of two subtypes of Cushing’s disease based on the analysis of episodic cortisol secretion. N Engl J Med 312:1343-1349 4. Schiirmeyer TH, Brabant G, Von zur Muhlen A 1990 Evidence that the hypothesis on a hypothalamic origin of Cushing’s disease is not an outdated concept: Ludecke DK, Chrousos G?, Tolis G (eds) ACTH, Cushing’s Syndrome, and Other Hypercortisolemic States. Raven Press, New York, p 105 5. Croughs RJM, Rijnberk A, Koppeschaar HPF 1990 Heterogeneity in Cushiner’s disease. Neth 1 Med 36:217-220 6. Peterson ME 1987 Pathophysiology of canine pituitary-dependent hyperadrenocorticism (canine Cushing’s disease). Front Horm Res 17:37-47 7. Orth DN, Peterson ME, Drucker WD 1988 Plasma immunoreactive proopiomelanocortin peptides and cortisol in normal dogs and dogs with Cushing’s syndrome: diurnal rhythm and responses to various stimuli. Endocrinology 122:1250-1262 8. Rijnberk A, Mol JA, Kwant MM, Croughs RJM 1988 Effects of bromocriptine on corticotrophin, melanotrophin and corticosteroid secretion in dogs with pituitary-dependent hyperadrenocorticism. J Endocrinol 118:271-277 9. Meijer JC, Mulder GH, Rijnberk A, Croughs RJM 1978 Hypothalamic corticotrophin releasing factor activity in dogs with pituitarydependent hyperadrenocorticism. J Endocrinol 79:209-213 ME, Palkovits M, Chiueh CC, Graves TK, Mezey E, 10. Peterson Vale W, Krieger DT 1989 Biogenic amine and corticotropin releasing factor concentrations in hypothalamic paraventricular nucleus and biogenic amine levels in the median eminence of normal dogs, chronic dexamethasone-treated dogs, and dogs with naturally-occurring pituitary-dependent hyperadrenocorticism (canine Cush-
in:
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ing’s disease). J Neuroendocrinol 1:169-171 11. Tomori N, Suda T, Tozawa F, Demura H, Shizume K, Mouri T 1983 Immunoreactive corticotropin-releasing factor concentrations in cerebrospinal fluid from patients with hypothalamic-pituitaryadrenal disorders. J Clin Endocrinol Metab 57:1305-1307 12. Kling MA, Roy A, Doran AR, Calabrese JR, Rubinow DR, Whitfield HJ, May Jr C, Post RM, Chrousos GP, Gold PW 1991 Cerebrospinal fluid immunoreactive corticotropin-releasing hormone and adrenocorticotropin secretion in Cushing’s disease and major depression: potential clinical implications. J Clin Endocrinol Metab 72:260-271 13. Rijnberk A, der Kinderen PJ, Thijssen JHH 1968 Spontaneous hyperadrenocorticism in the dog. J Endocrinol 41:397-406 14. Stolp R, Rijnberk A, Meijer JC, Croughs RJM 1983 Urinary corticoids in the diagnosis of canine hyperadrenocorticism. Res Vet Sci 34:141-144 15. Rijnberk A, Wees van A, Mol JA 1988 Assessment of two tests for the diagnosis of canine hyperadrenocorticism. Vet Ret 122:178-180 16. Rijnberk A, Mol JA, Rothuizen J, Bevers MM, Middleton DJ 1987 Circulating pro-opiomelanocortin-derived peptides in dogs with pituitary-dependent hyperadrenocorticism. Front Horm Res 17:48-60 17. Voorhout G, Rijnberk A, Sjollema BE, van den Ingh TSGAM 1990 Nephrotomography and ultrasonography for the localization of hyperfunctioning adrenocortical tumors in dogs, Am J Vet Res 51:1280-1285 18. Voorhout G 1990 Cisternography combined with linear tomography for visualization of the pituitary gland in healthy dogs. A comparison with computed tomography:Vet Radio1 31:88-7319. Voorhout G, Riinberk A 1990 Cisternograuhv combined with linear tomography for visualization of pituit& lesions in dogs with pituitary-dependent hyperadrenocorticism. Vet Radio1 31:74-78 20. Stalla GK, Stalla J, Schophol J, Werder von K, Miiller OA 1986 Corticotropin releasing factor (CRF) in humans: CRF stimulation in normals and CRF-radioimmunoassay. Horm Res 24:229-245 21. Hunter WM, Greenwood FC 1962 Preparation of iodine-131 labelled human growth hormone of high specific activity. Nature 194:495-496 22. Arts CJM, Koppeschaar HPE, Veeman W, Thijssen JHH 1985 A direct radioimmunoassay for the determination of adrenocorticotropit hormone (ACTH) and a clinical evaluation. Ann Clin Biochem 221247-256 23. Plotsky PM 1988 Central regulation of stimulus-induced ACTH secretion: characterization of hypophysial-portal plasma CRF and AVP concentrations profiles. In: Imura H (ed) Neuroendocrine Control of the Hypothalamo-Pituitary System. Karger, Base], p 31 24. Wittert GA, Crock, Donald RA, Gilford EJ, Boolell M, Alford FP, Espiner EA 1990 Arginine vasopressin in Cushing’s disease. Lancet 335:991-994 25. Bloom FG, Battenberg ELF, Rivier J, Vale W 1982 Corticotropin releasing factor (CRF) immunoreactive neurons and fibers in rat hypothalamus. Regul Peptides 4:43-48 26. Beyer HS, Matta SG, Sharp BM 1988 Regulation of the messenger ribonucleic acid for corticotropin-releasing factor in the paraventricular nucleus and other brain sites of the rat. Endocrinology 123:2117-2123 27. Croughs RJM, Timmermans H, Vingerhoeds ACM, Vermeulen A, Smals A, Kloppenburg PWC, Meyer JC 1977 Insulin stimulation tests in pituitary-dependent Cushing’s syndrome after complete adrenalectomy. Acta Endocrinol (Copenh) 86:578-582 28. Stolp R, Steinbusch HWM, Rijnberk A, Croughs RJM 1987 Organization of ovine corticotropin-releasing factor immunoreactive neurons in the canine hypothalamo-pituitary system. Neurosci Lett 74:337-342
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