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Original Article |
Third Department of Internal Medicine (T.M., A.M., T.S., J.H., H.D., H.M., K.Y.), Gifu University School of Medicine, Gifu 500-8705; Department of Internal Medicine (H.M.), Daiyukai Hospital, Ichinomiya, Aichi 491-8551; Department of Internal Medicine (N.Y.), Matsunami General Hospital, Kasamatsu, Gifu 501-6062; First Department of Internal Medicine (T.F.), Okayama University, Okayama 700-8558; and Department of General Medicine (M.M.), Gifu Prefectural Hospital, Gifu 500-8717, Japan
Address all correspondence and requests for reprints to: Tomoatsu Mune, M.D., Ph.D., Third Department of Internal Medicine, Gifu University School of Medicine, 40 Tsukasa-machi, Gifu 500-8705, Japan. E-mail: mune{at}cc.gifu-u.ac.jp.
Abstract
Arginine vasopressin (AVP) stimulates cortisol secretion through its vascular type V1a receptor in the adrenal glands, in addition to stimulating ACTH secretion through pituitary V3 receptor. Because hyper-response of plasma cortisol to vasopressin is documented in some patients with Cushing’s syndrome due to adrenal adenoma (CS) or ACTH-independent macronodular adrenocortical hyperplasia (AIMAH), we analyzed the expression of V1a, V2, V3 receptor and AVP mRNA in human adrenal tissues by quantitative competitive RT-PCR or real-time PCRs. V1a receptor mRNA levels (ratio against glyceraldehyde 3-phosphate dehydrogenase) were 0.378 ± 0.143 (mean ± SE) in preclinical CS (n = 5) and 0.630 ± 0.072 in AIMAH (n = 4), which were significantly higher than those (0.046 ± 0.012; n = 9) in control adrenals, whereas those in overt CS (0.143 ± 0.048; n = 10) or aldosterone-producing adenomas (0.069 ± 0.018; n = 12) were similar to control adrenals. Although ectopic expression of V2 or V3 receptor was detected in half of AIMAH cases, the absolute levels were low. Furthermore, V1a receptor mRNA levels in the adjacent adrenal glands (0.190 ± 0.039, n = 9) of aldosterone-producing adenomas were higher than those in control adrenals and in the corresponding tumor portions (0.079 ± 0.024). In contrast, there were no significant differences in AVP mRNA levels among these groups.
These results suggest that eutopic V1a receptor overexpression is involved in the etiology of AIMAH and a subset of adrenal adenomas causing overt or preclinical Cushing’s syndrome. Our results imply a possible association of V1a receptor expression with adrenal hyperplasia.
WHEREAS ACTH IS physiologically a main modulator of steroidogenesis in the adrenal cortex, arginine vasopressin (AVP) also directly stimulates cortisol secretion (1, 2). AVP actions are mediated by three different membrane-bound receptors. The renal V2 receptor coupled to A kinase mediates well-known antidiuretic action (3), and the pituitary-specific V3 (V1b) receptor coupled to C kinase stimulates ACTH secretion and potentiates corticotropin releasing factor-evoked ACTH release (4, 5). The vascular type V1a (V1) receptor coupled to phospholipase C is present in adrenocortical cells and mediates the stimulatory effects of AVP on aldosterone and cortisol secretion (2, 6). In addition, AVP itself is produced in the adrenal medulla and cortex, suggesting its autocrine or paracrine role (6, 7).
As discussed in a recent review (8), hyperresponsiveness of plasma cortisol to vasopressin has been reported in some patients with Cushing’s syndrome due to adrenal adenoma (CS) or ACTH-independent bilateral macronodular adrenocortical hyperplasia (AIMAH). We have previously reported a case of AIMAH in which insulin-induced hypoglycemia increased plasma cortisol, followed by elevation of plasma AVP under suppression of plasma ACTH (9). The patient exhibited exaggerated cortisol responses to small doses of AVP, and a V1a receptor antagonist, OPC-21268, partially suppressed urinary cortisol excretion in vivo and suppressed AVP-induced cortisol release from isolated AIMAH cells in vitro.
Because the above findings strongly suggested hypersensitivity of cortisol secretion to AVP in these tissues, we have now evaluated the mRNA expression levels of the V1a receptor together with those of V2 receptor, V3 receptor, and AVP in adrenal tissues including AIMAH samples. We have also analyzed the relationships of the expression levels to clinical and hormonal phenotypes.
Materials and Methods
Patients and adrenal tissues
The subjects were 37 patients with adrenal tumors, and their clinical and hormonal data with operative findings are summarized in Table 1
. Patients with aldosterone-producing adenomas (n = 12) had hypertension, hypokalaemic alkalosis, elevated plasma aldosterone concentrations and suppressed plasma renin activity. Patients with adrenal adenomas causing overt Cushing’s syndrome (n = 10) had elevated plasma cortisol and suppressed plasma ACTH levels with typical signs of Cushing’s syndrome. Patients with preclinical Cushing’s syndrome (n = 5) had no signs or symptoms of Cushing’s syndrome and normal basal plasma cortisol levels but had plasma cortisol levels that were not suppressible by 1 mg (<83 nM; 3 µg/dl) and 8 mg (<28 nM; 1 µg/dl) dexamethasone and plasma ACTH levels in the low normal range or suppressed.
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, together with the responses in plasma ACTH, cortisol and endogenous AVP to insulin-induced hypoglycemia in case 3. Case 4, a 60-yr-old Japanese who had been diabetic and hypertensive for 10 yr, was observed to have bilateral adrenal enlargement with multiple nodules by computed tomography in a medical check-up and was referred to our department. He was 158 cm in height, 68 kg in weight, and 192/100 mm Hg in blood pressure with no typical Cushingoid features. Serum potassium was normal (4.0 mEq/liter). The plasma levels of ACTH/cortisol were 1.4 pM/395 nM at 0800 h and less than 0.8/157 at 2000 h, and 1.0/119 and less than 0.8/105 after 1 mg and 8 mg dexamethasone, respectively. An iv administration of 100 µg CRH increased plasma cortisol from 328 to 635 nM and plasma ACTH from less than 0.8 to 3.0 pM, but this ACTH increase was subnormal. An iv injection of low dose (0.5 µg) ACTH-(1–24) caused an exaggerated cortisol response (179–1046 nM). Although AVP administration was not employed in this patient due to suspicious coronary heart disease, upright posture increased plasma cortisol as well as plasma AVP levels (cortisol, 317–436 nM; AVP, 1.7–2.7 pM) without changes in plasma ACTH levels. Furthermore, iv injection of 10 mg metoclopramide or oral administration of 10 mg cisapride, both of which are known as serotonin 5-HT4 receptor agonist, induced rises in plasma cortisol levels (323–419 nM and 276–497 nM, respectively) again without changes in plasma ACTH levels. However, meal, GnRH, or TSH-releasing hormone did not induce cortisol secretion. The serum dehydroepiandrosterone sulfate level (1.1 µM) was low compared with age-adjusted normal males. The 131I-iodocholesterol scintigraphy revealed increased bilateral adrenal uptake, and magnetic resonance imaging of the pituitary gland did not show any abnormal findings. Collectively, case 4 was diagnosed as having preclinical Cushing’s syndrome due to AIMAH. After total adrenalectomy, the hypertension and diabetes mellitus were ameliorated. All of these four patients with AIMAH had no family history of endocrine tumors, and the other endocrine neoplastic syndromes such as Carney complex or Multiple Endocrine Neoplasia were excluded by corresponding hormonal and radiological examinations.
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Biochemical and hormonal measurements
Blood for serum electrolytes and hormone determinations were drawn from fasting patients after resting for at least 30 min between 0800 h and 0900 h, and 24-h urine samples were collected in plain plastic containers without preservatives. Plasma ACTH and renin activity were measured using a two-site immunoradiometric assay kit (Mitsubishi Petrochemical Co. Ltd., Tokyo, Japan) and a Renin RIA Bead (Dainabot RI Laboratory, Tokyo, Japan), respectively. The plasma aldosterone concentration and urinary pH1 aldosterone excretion were measured using an Aldosterone-RIA Kit (Shionogi Pharmaceutical Co., Osaka, Japan), and plasma cortisol and urinary free cortisol excretion were measured using a direct RIA kit (INCSTAR Corp., Stillwater, MN).
Measurement of abundance of V1a receptor and AVP mRNA in adrenal tissues by competitive RT-PCR
We employed a previously described (12) quantitative competitive RT-PCR method to measure the mRNA levels of V1a receptor and AVP, because only a small amount of RNA could be obtained from some adrenal tissues. The mRNA values were normalized for glyceraldehyde 3-phosphate dehydrogenase (GAPDH), a housekeeping gene, to minimize variations in deoxyribonuclease digestion and RT between samples. Competitors for each cDNA were prepared following the PCR MIMIC KIT protocol (CLONTECH Laboratories, Inc., Palo Alto, CA). Gene-specific primers for each cDNA were as follows; V1a receptor (13): 5' GTG CAG AGC AAG CGG GTG TG 3' [sense, nucleotides (nt) 773–792] and 5' CGA GTC CTT CCA CAT ACC CGT 3' (antisense, nt 1192–1212), AVP (14): 5' CTG CGC TGC CAG GAG GAG AAC 3' (sense, nt 240–260) and 5' TCC AGC TGC GTG GCG TTG CTC 3' (antisense, nt 410–430), GAPDH (15): 5' TCA TCA TCT CTG CCC CCT CTG CTG 3' (sense, nt 482–497) and 5' GAC GCC TGC TTC ACC ACC TTC TTG 3' (antisense, nt 812–832). Quantitative competitive PCR was performed by addition of 2.5 pmol of a sense and an antisense primer to 0.5 µl of reverse-transcribed samples with 0.5 µl of a various range of 2.5 times serially diluted competitor in 5 µl of a previously described (16) PCR buffer and 0.25 U of Ex Taq DNA polymerase (TaKaRa Co., Osaka, Japan) for V1a receptor and GAPDH. For AVP, MasterAmp Taq DNA polymerase (Epicentre Technologies, Madison, WI) was used with premix buffer H supplied by the manufacturer. Samples were subjected to initial denaturation at 96 C for 2 min, followed by 35–50 cycles of 96 C denaturation for 20 sec and 65 C (V1a receptor and GAPDH) or 62C (AVP) annealing/extension for 30 sec. The PCR products were subjected to electrophoresis in 2.5% agarose gels, stained with ethidium bromide, and loaded into a Macintosh computer with a DC120 digital camera (Eastman Kodak Co., Rochester, NY). A representative result of the competitive RT-PCR derived from one sample is shown in Fig. 2. The band intensities were analyzed using NIH Image 1.60 (Rasband, W., NIH), and the amount of the competitor whose intensity was equal to the intrinsic template was calculated as the corresponding mRNA level by linear regression.
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The mRNA levels of V2 receptor, V3 receptor and GAPDH were measured by quantitative real-time PCRs with the LightCycler System using the FastStart DNA Master SYBER Green I kit (both from Roche Diagnostics) in 10 times diluted aliquots (2 µl) of above reverse transcribed samples. The PCR products were detected via intercalation of the fluorescent dye Sybr green. The primers used were as follows; V2 receptor (17): 5' GGC CAA GAC TGT GAG GAT GA 3' (sense, nt 1394–1413) and 5' ACA CGC TGC TGC TGA AAG AT 3' (antisense, nt 1680–1699), V3 receptor (18): 5' TCG GGT CAG CAG CAT CAA CA 3' (sense, nt 2293–2312) and 5' ACC CCC ACA GCA GGC AAG G 3' (antisense, nt 7970–7988), GAPDH: same primers as in the competitive PCR. Corresponding fragments amplified in the PCR buffer containing 3 mM Mg2+ and 4% dimethylsulfoxide for V2 receptor and GAPDH or 2 mM Mg2+ and 5% dimethylsulfoxide for V3 receptor with Ex Taq DNA polymerase (TaKaRa Co., Osaka, Japan) were gel-purified and quantitated using a DC120 digital camera and Kodak Digital Science 1D Image Analysis Software (Eastman Kodak Co.), yielding each standard to calculate the exact copy number of each mRNA in the samples. Standards were prepared by 10-fold serial dilutions and used over the range of 10 to 1,000,000 copies/µl. The protocol for V2 receptor and GAPDH included a 5-min denaturation at 95 C, followed by 45 cycles consisting of denaturation at 95 C for 10 sec, annealing at 68 C for 8 sec, and an extension phase at 72 C for 13 sec. The annealing and extension at 72 C for 25 sec was employed for V3 receptor. Fluorescence was measured at the end of the extension phase. The quality of the RT-PCR products was controlled by melting point curve analysis (representatives were shown in Fig. 3). The mRNA values calculated as copy numbers in each sample were corrected for the control sample (human kidney RT samples for V2 receptor, an RT sample derived from a pituitary adenoma causing Cushing’s disease for V3 receptor, and control adrenal RT samples for GAPDH) as an internal standard in each run of PCR, followed by normalization for the GAPDH mRNA level.
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Results were expressed as mean ± SE. The Bonferroni test was used to evaluate the differences between groups and P < 0.0014 was considered significant. We employed the Wilcoxon signed rank test to compare the mRNA levels between aldosterone-producing adenomas and the corresponding adjacent adrenal glands and the Pearson’s correlation analysis where appropriate, and P < 0.05 was considered significant.
RESULTS
V1a receptor mRNA expression levels
As shown in Fig. 4, the levels of V1a receptor mRNA (the ratio of V1a receptor/GAPDH) were 0.046 ± 0.012 (mean ± SE) in control adrenals, 0.069 ± 0.018 in aldosterone-producing adenomas, 0.045 and 0.005 in two tumors that secreted both aldosterone and cortisol, 0.143 ± 0.048 in cortisol-secreting adenomas resulting in overt Cushing’s syndrome, 0.063 and 0.070 in two pheochromocytomas and 0.117 and 0.004 in two adrenal carcinomas. The differences between these groups were not significant. However, the V1a receptor mRNA levels in adenomas with clinically autonomous cortisol secretion resulting in preclinical Cushing’s syndrome were 0.378 ± 0.143, which were higher than those in control adrenals and aldosterone-producing adenomas (P = 0.0002). Furthermore, the V1a receptor mRNA levels in AIMAH were 0.630 ± 0.072, which were clearly higher than those in the other groups (P < 0.0001) except the preclinical Cushing’s adenoma.
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AVP mRNA expression levels
AVP mRNA levels were 0.68 ± 0.14 (x 10-3, mean ± SE) in control adrenals, 1.04 ± 0.67 in aldosterone-producing adenomas, 0.80 and 0.03 in two tumors that secreted both aldosterone and cortisol, 1.11 ± 0.62 in cortisol-secreting adenomas resulting in overt Cushing’s syndrome, 0.78 ± 0.29 in adenomas with clinically autonomous cortisol secretion resulting in preclinical Cushing’s syndrome, 1.19 ± 0.59 in AIMAH and 0.03 and 0.04 in two pheochromocytomas. Adrenal carcinomas did not express detectable levels of AVP mRNA. AVP mRNA levels seemed to be low in two of two pheochromocytomas or carcinomas, but there were no significant differences in AVP mRNA levels among these groups.
V2 and V3 receptor mRNA expression levels
Using real-time PCRs, specific amplifications of V2 or V3 receptor mRNA were not detected in 9 control adrenals but observed in a few of adrenal tumorous tissues. V2 receptor mRNA was detected in 1 of 12 aldosterone-producing adenomas (no. 4, 0.054; the ratio of V2 receptor/GAPDH x 10-3), in 3 of 10 overt Cushing’s adenoma (no. 1, 0.007; no. 5, 0.023; and no. 9, 0.256), in two pheochromocytomas (0.162 and 0.454), and in two of four AIMAH (case 4, 0.017; and case 3, 2.173). Considering that V2 receptor mRNA level in kidney was 0.599 ± 0.110 (mean ± SE, n = 4), case 3 of AIMAH clearly had a high expression, though its absolute mRNA level was approximately 250 times fewer than that of V1a receptor.
V3 receptor mRNA was detected in 1 of 10 overt Cushing’s adenoma (no. 1, 0.212; the ratio of V3 receptor/GAPDH x 10-3) and in two of four AIMAH (case 1, 0.555; and case 2, 0.540). However, these levels were much fewer than the V3 receptor mRNA level in a pituitary adenoma causing Cushing’s disease (148.7).
Association of V1a receptor expression levels with clinical and biochemical phenotypes
In patients with overt Cushing’s syndrome, the tumor V1a receptor mRNA levels tended to correlate negatively with tumor volume (r = 0.57, P = 0.083, Fig. 5, left) but not with plasma cortisol or urinary free cortisol (Fig. 5, right) levels. No such correlations were detected in patients with preclinical Cushing’s syndrome or AIMAH (data not shown). In patients with aldosterone-producing adenomas, the V1a receptor mRNA levels did not correlate with tumor volume, plasma aldosterone concentration or 24 h urinary pH1 aldosterone excretion. There were no significant correlations between V1a receptor or AVP mRNA levels and clinical measures such as blood pressures or serum potassium levels (data not shown).
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In the present study, we have shown that the V1a receptor is highly expressed, nearly 10 times the control levels, in all 4 tissue samples derived from AIMAH. Ectopic expression of V2 or V3 receptor was detected in half of AIMAH cases, but the absolute levels were low. Together with the endocrinological data described above and reported previously (9, 10), the V1a receptor overexpression could mainly explain cortisol hypersensitivity to AVP in AIMAH. The V1a receptor mRNA levels were also high (three times more than the maximal value in control adrenals) in 2 of 5 adenomas derived from preclinical Cushing’s syndrome and one of 10 adenomas from overt Cushing’s syndrome. Unfortunately, AVP administration or postural tests were not employed in these patients, but V1a receptor overexpression might be involved in the etiology of tumorigenesis in a subset of adrenal adenomas causing preclinical or overt Cushing’s syndrome.
AIMAH is a rare cause of Cushing’s syndrome characterized by bilateral macronodular adrenal hyperplasia with autonomous cortisol secretion under suppressed ACTH. Previously described as giant or huge, the adrenal glands reach up to 200 g in combined weight (19, 20, 21, 22). The endocrinological abnormality described as Cushing’s syndrome is usually found with such larger adrenal lesions, suggesting that the cortisol production by each cell is low and that a massive increase in cell number is necessary to produce the apparent cortisol excess (11, 21). The size deviation of larger left adrenal glands compared with right ones, the smallest adrenal size in our case 4 presenting the preclinical Cushing’s stage, and a recently described transition from preclinical Cushing’s to overt Cushing’s syndrome (23) support this conception and also suggest a slower speed in adrenal proliferation. Morphologically, AIMAH tissues have numerous nodules composed of cord-forming clear cortical cells and nest-forming small compact cells (21, 22). Ultrastructurally, the greater majority of these cells showed poor development of smooth endoplasmic reticulum and mitochondria (21). An immunohistochemical and in situ hybridization study showed that the small compact cells expressed the 17
-hydroxylase essential for cortisol biosynthesis but not 3ß-hydroxysteroid dehydrogenase, whereas the clear cortical cells had the opposite pattern (22). This differential distribution of steroidogenic enzymes suggests that the pregnenolone supply from the clear cortical cells is necessary for the small compact cells to finally produce cortisol, and this ineffective corticosteroidogenesis might explain the relatively decreased cortisol production in AIMAH (11, 22). Although an antibody for the V1a receptor protein is not yet available, an in situ hybridization study of V1a receptor by Arnaldi et al. (24) demonstrated the presence of V1a receptor mRNA in compact cells of an adenoma and a carcinoma with a nonhomogeneous pattern, in addition to the normal adrenal cortex (mainly in the compact cells of the zona reticularis). As a next step, the precise localization of the observed V1a receptor overexpression in AIMAH will be necessary to clarify our present findings.
Lacroix et al. (25) have already showed the expression of V1a receptor expression in adrenal tissue from a female patient with AIMAH with extensive physiologic studies in vivo and in vitro. A recent clinical study documented cortisol response to AVP in 4 of 4 preclinical Cushing’s patients with AIMAH, with some having cortisol response to GnRH or serotonin 5-HT4 receptor agonists (26). Together with our present study, the V1a receptor appears to be confirmed as one of the eutopically overexpressed hormone receptors involved in the etiology of AIMAH. Because its bilateral adrenal lesions, one familial case (27) and abnormal vascular response to AVP (25) described in vasopressin-responsive AIMAH suggest genetic abnormalities such as constitutive activating mutation in its etiology, we sequenced the V1a receptor gene with a genomic DNA sample from case 3, but no mutation was identified in the whole coding region (data not shown).
Recently, Lacroix et al. (8) reviewed adrenal Cushing’s syndrome in detail from the viewpoints of abnormal and ectopic membrane hormone receptors. Considering previous reports (8, 26) and our present result, it would be logical to hypothesize that other multiple classes of hormone receptor are multifactorially involved. As regards eutopic hormone receptors, we preliminary examined the mRNA levels of the ACTH receptor, the effector system of a main secretagogue and a proliferative factor in adrenal cortex. They did not significantly differ between control adrenals and AIMAH (0.36 ± 0.06, n = 9, and 1.41 ± 0.69, n = 4; ratio against GAPDH), but 2 of the 4 AIMAH samples clearly had high expression (2.17 and 2.97). The ACTH receptor is known to be up-regulated by the ligand ACTH, so that the observed ACTH receptor overexpression in AIMAH with suppressed plasma ACTH might indicate that the regulatory mechanism of ACTH receptor expression is destroyed in AIMAH. This would, in part, explain the hyperresponsiveness of plasma cortisol to even low dose ACTH and suggest the trophic effect of ACTH on the adrenal proliferation. However, its pathophysiological significance is uncertain under suppressed plasma ACTH levels. Furthermore, we have to note that case 4 had medium responsiveness to hyperresponsiveness of plasma cortisol to two 5-HT4 receptor agonists as in a previous report (26), suggesting a possible overexpression of 5-HT4 receptor in this AIMAH tissue. Quantitation of 5-HT4 receptor mRNA in the present adrenal tissues is currently being investigated.
Ectopic hormone receptors are also implicated in the etiology of AIMAH (8). Food-dependent cortisol production in AIMAH (28, 29) is characterized by its female predominance and caused by ectopic expression of gastric inhibitory polypeptide receptor (30). However, because there were no meal-related cortisol responses in our four male patients with AIMAH (9, 10, 11 and present case 4), we have not measured the expression levels of gastric inhibitory polypeptide receptor in the present study. We undertook some preliminary experiments to try and measure the expression levels of ß2-adrenergic receptor (31, 32) and LH/human chorionic gonadotropin receptor (26, 33) in a part of our adrenal tissues by semiquantitative RT-PCR (after adjustment for quantitated GAPDH levels). However, none of the AIMAH or Cushing’s adenomas appeared to have a higher expression of the ß2-adrenergic receptor than the control adrenals (data not shown), and LH/human chorionic gonadotropin receptor mRNA has not been detected so far. The expression status of these eutopic or ectopic receptors and/or possible contributions of some transcription-regulatory factors should be further elucidated. In addition, heterogeneity in the pathogenesis of AIMAH must be considered.
Not only AIMAH, but also some adrenal Cushing’s adenomas, show cortisol hypersensitivity to AVP or lysine vasopressin (8, 32, 34). Perraudin et al. (35) showed high V1a receptor expression in a unilateral cortisol-secreting adenoma presenting mild Cushing’s syndrome. By a quantitative RT-PCR method, Arnaldi et al. (24) demonstrated a wide range of V1a receptor expression in 20 adrenal adenomas and 19 carcinomas, and the V1a receptor mRNA level was high in four positive cortisol responders compared with two nonresponders. In our study, the proportion that 1 of 10 overt Cushing’s adenomas had very high V1a receptor expression is comparable to a recent endocrinological report (32). Furthermore, the mean V1a receptor expression level in preclinical Cushing’s adenoma was high compared with the control adrenals. This might imply up-regulation by glucocorticoids because dexamethasone increases V1a receptor expression (36, 37). However, in our overt Cushing’s adenomas, the V1a receptor expression level tended to correlate negatively with tumor volume, which was shown to correlate positively with plasma or urinary cortisol in another series (38). Although this appears to be contradictory, our results suggest that normal Cushing’s adenomas might lose their V1a receptor expression with growth, whereas vasopressin-responsive tissues of Cushing’s subsets do not show such down-regulation. Further studies in a greater number of cases, including various ethnic groups, will be necessary to clarify the precise prevalence and racial difference of abnormal, either eutopic or ectopic, membrane hormone receptors in adrenal Cushing’s syndrome.
Interestingly, the expression levels of V1a receptor were high in the attached adrenals compared with tumorous tissues in aldosterone-producing adenomas. In aldosteronoma, the zona glomerulosa of the attached adrenal is expected to show atrophy due to the suppressed renin-angiotensin system, but the attached adrenals are known to exhibit paradoxical hyperplasia of zona glomerulosa in the absence of well-developed smooth endoplasmic reticulum and mitochondria (39). AVP stimulates the mitotic activity (40) and maintains the growth (41) of rat adrenocortical cells. Although the etiology of paradoxical hyperplasia is unknown, one can speculate that the higher expression of V1a receptor is related to hyperplasia with ineffective steroidogenesis. Again, elucidation of the localization of the V1a receptor expression will be necessary. This hypothesis of hyperplasia with ineffective steroidogenesis due to V1a receptor expression might also be involved in AIMAH and preclinical or overt Cushing’s syndrome.
AVP-immunoreactive cells are scattered in the adrenal cortex (6) as well as in the medulla (7), suggesting that AVP is released in the vicinity of steroidogenic cells and can modulate corticosteroid secretion. Considering that the ED50 value of AVP for stimulating cortisol secretion is reported to be around 100 pM (2), which is greater than the physiological plasma AVP concentration (2
4 pM), only supraphysiological or extremely pathological conditions could bring about high plasma AVP that can affect cortisol secretion. In this context, we have measured AVP mRNA levels in adrenal tissues to investigate its possible involvement in cortisol secretion and adrenal tumorigenesis as a paracrine system, in addition to evaluating the above-mentioned AVP effectors system. Our present study did confirm the presence of AVP mRNA in control adrenals and aldosterone-, cortisol- or catecholamine-secreting adrenal tissues except carcinomas, but no significant differences were observed among the groups. Of course, we cannot exclude the possibility that a stimulated AVP release by secretagogues such as CRF modulates the secretion of adrenal hormones (2, 42), but it is unlikely that intraadrenal AVP production per se is involved in the etiology of these adrenal disorders.
In conclusion, the eutopic V1a receptor overexpression might be involved in the etiology of AIMAH and a vasopressin-responsive subset of adrenal adenomas causing overt or preclinical Cushing’s syndrome. Our results also imply a possible association of V1a receptor expression with adrenal hyperplasia.
Acknowledgments
We gratefully acknowledge the following doctors, Drs. Yoshihito Takahashi and Takashi Deguchi (Department of Urology, Gifu University School of Medicine), Shoji Dodo (Seirei Hospital), Kazuo Kajita (Nagahama Red Cross Hospital), Shinobu Goto (Nagoya Memorial Hospital), Kazuhisa Takami (Kizawa Hospital), and Yoshiyuki Natsume (Hashima City Hospital), for providing the adrenal tissue samples and patients’ information. We also acknowledge Drs. Masao Takemura and Mitsuru Seishima (Department of Laboratory Medicine, Gifu University School of Medicine) for their help to set up the real-time PCRs.
Footnotes
This work was supported by grants for Disorders of the Adrenal Gland (1995–1998, 1999–2002) from the Ministry of Health, Labor and Welfare, Japan. There was a preliminary presentation of this work at the 79th Annual Meeting of The Endocrine Society, Minneapolis, Minnesota, June 11–14, 1997.
Abbreviations: AIMAH, ACTH-independent macronodular adrenocortical hyperplasia; AVP, arginine vasopressin; CS, Cushing’s syndrome due to adrenal adenoma; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; nt, nucleotides.
Received January 22, 2002.
Accepted September 6, 2002.
References
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); cortisol-secreting adenoma resulting in overt Cushing’s syndrome, PC (
); adenoma with clinically autonomous cortisol secretion resulting in preclinical Cushing’s syndrome, MAH (
); ACTH-independent macronodular hyperplasia. Note that the vertical axis in the left panel is linear in the lower but logarithmic in the higher range.