Expression of vasopressin receptors in ACTH-independent macronodular bilateral adrenal hyperplasia causing Cushing's syndrome: molecular, immunohistochemical and pharmacological correlates.

Cortisol secretion in ACTH-independent macronodular adrenal hyperplasia (AIMAH) causing Cushing's syndrome can be controlled by illegitimate receptors. The aim of the present study was to characterize the molecular, immunohistochemical, and pharmacological profiles of vasopressin receptors in cells derived from three patients with AIMAH (H1-H3), in order to evaluate the role of ectopic vasopressin receptors in the physiopathology of hypercortisolism. Expression of mRNAs encoding the vasopressin receptor types (V(1a), V(1b), and V(2)) were analyzed by RT-PCR in adrenal tissues. The presence of V(1a) and V(2) receptors was studied by immunohistochemistry on adrenal sections. The pharmacological profiles of vasopressin receptors involved in the control of cortisol secretion were investigated using the V(1a) receptor antagonist SR49059 and the V(2) receptor agonist [deamino-Cys(1), Val(4), D-Arg(8)]-vasopressin on cultured cells. The V(1a) receptor protein was present and functional in H1 and H3 tissues, whereas the V(1b) receptor was not expressed in any of the tissues. RT-PCR experiments revealed that V(2) receptor mRNAs were detected in the three tissues. In contrast, immunohistochemical and cell incubation studies showed that the V(2) receptor was involved in the stimulatory effect of AVP on cortisol secretion in H1 and H2, but not in H3 cells. Taken together, these data show that expression of functional ectopic V(2) receptors and repression of eutopic V(1a) receptor can coexist in some hyperplastic corticosteroidogenic tissues. They also reveal that immunohistochemical and incubation studies are essential for the characterization of ectopic receptors actually involved in the control of cortisol secretion by AIMAHs.

Effects of serotonin and vasopressin on cortisol production from an adrenocortical tumor causing subclinical Cushing's syndrome.

In dexamethasone-suppressed healthy volunteers, the serotonin4 (5-HT4) receptor agonist cisapride and lysine vasopressin [LVP, an analog of arginine vasopressin (AVP)] have no influence on plasma cortisol levels (PCL). In contrast, cisapride and AVP have been shown to stimulate cortisol secretion in patients with adrenal tumor or bilateral adrenal hyperplasia and Cushing's syndrome. In this report, we describe a case of adrenocortical adenoma causing subclinical Cushing's syndrome. Cisapride and terlipressin, a precursor of LVP, both induced an increase in PCL reaching +88% and +100%, respectively, without any significant variation of plasma ACTH levels. In vitro experiments were conducted to investigate the effects of 5-HT and AVP on cortisol production from cultured tumor cells and normal adrenocortical cells. 5-HT and AVP both induced a dose-dependent increase in cortisol production from cultured tumor cells. Comparison of the data obtained with tumor and normal cells, respectively, showed that 5-HT was more efficient to stimulate steroidogenesis in adenomatous than normal cells. Concurrently, the efficacy and potency of AVP were both higher in tumor than normal cells. Collectively, these results show that the abnormal in vivo responses of the adrenocortical adenoma to cisapride and LVP could be ascribed to an increased sensitivity of the tumor tissue to 5-HT and AVP. The data also suggest that the adrenocortical tumor overexpressed eutopic 5-HT4 and V1 receptors.

Variable expression of the V1 vasopressin receptor modulates the phenotypic response of steroid-secreting adrenocortical tumors.

We studied the putative role of the vasopressin receptors in the phenotypic response of steroid-secreting adrenocortical tumors. A retrospective analysis of a series of 26 adrenocortical tumors responsible for Cushing's syndrome (19 adenomas and 7 carcinomas) showed that vasopressin (10 IU, i.m., lysine vasopressin) induced an ACTH-independent cortisol response (arbitrarily defined as a cortisol rise above baseline of 30 ng/mL or more) in 7 cases (27%). In comparison, 68 of 90 patients with Cushing's disease (76%) had a positive cortisol response. We then prospectively examined the expression of vasopressin receptor genes in adrenocortical tumors of recently operated patients (20 adenomas and 19 adrenocortical carcinomas). We used highly sensitive and specific quantitative RT-PCR techniques for each of the newly characterized human vasopressin receptors: V1, V2, and V3. The V1 messenger ribonucleic acid (mRNA) was detected in normal adrenal cortex and in all tumors. Its level varied widely between 2.0 x 10(2) and 4.4 x 10(5) copies/0.1 microgram total RNA, and adenomas had significantly higher levels than carcinomas, although there was a large overlap. Among the 6 recently operated patients who had been subjected to the vasopressin test in vivo, the tumor V1 mRNA levels were higher in the 4 responders (9.5 x 10(3) to 5.0 x 10(4)) than in the 2 nonresponders (2.0 x 10(2) and 1.8 x 10(3)). One adenoma that had a brisk cortisol response in vivo, also had in vitro cortisol responses that were inhibited by a specific V1 antagonist. In situ hybridization showed the presence of V1 mRNA in the normal human adrenal cortex where the signal predominated in the compact cells of the zona reticularis. A positive signal was also present in the tumors with high RT-PCR V1 mRNA levels; its distribution pattern was heterogeneous and showed preferential association with compact cells. RT-PCR studies for the other vasopressin receptors showed a much lower signal for V2 and no evidence for V3 mRNA. We could not establish whether the V2 mRNA signal observed in normal and tumoral specimens was present within adrenocortical cells or merely within tissue vessels. We conclude that the vasopressin V1 receptor gene is expressed in normal and tumoral adrenocortical cells. High, and not ectopic, expression occurs in a minority of tumors that become directly responsive to vasopressin stimulation tests.

Vasopressin-responsive adrenocortical tumor in a mild Cushing's syndrome: in vivo and in vitro studies.

We report a case of a Cushing's syndrome caused by an autonomously secreting unilateral adrenocortical tumor, characterized by a clinically and biologically mild hypercortisolemic state and an unusual response pattern to vasopressin. Laboratory tests showed normal early morning plasma cortisol and 24-h urinary cortisol excretion, but lack of nycthemeral variations and suppressed plasma ACTH. Urinary cortisol excretion was not suppressed by either the low dose or the high dose dexamethasone test. Injection of lysine vasopressin, (10 IU, im) induced a marked increase in plasma cortisol, without an elevation of plasma ACTH. Computed tomography scan revealed an adrenocortical mass of the left gland with a contralateral atrophic gland. Removal of the tumor led to complete remission of Cushing's symptoms. In vitro studies were then performed to investigate the effect of arginine vasopressin (AVP) on calcium mobilization in cultured tumor cells using a microfluorimetric technique. Application of AVP in the vicinity of the cells induced a rapid and marked increase in the intracellular calcium concentration. Preincubation of the cells with the V1 vasopressin receptor antagonist [d(CH2)5,Tyr(OMe)2]AVP totally suppressed the AVP-induced stimulation of intracellular calcium concentration. Reverse transcription followed by polymerase chain reaction of tumor ribonucleic acid with specific oligonucleotides amplified high levels of V1 receptor signal compared with normal adrenocortical ribonucleic acid. Specific oligonucleotides for the V2 or V3 receptors amplified only a faint signal. This is the first report describing a mild case of Cushing's syndrome caused by an AVP-sensitive cortisol-producing adenoma. The direct effect of AVP on cultured tumor cells was mediated through the V1 type of vasopressin receptor, similar to that previously characterized in normal human fasciculata cells, suggesting that the tumor expressed an eutopic V1 AVP receptor and exhibited overresponsiveness to AVP.

Vasopressin stimulates cortisol secretion from human adrenocortical tissue through activation of V1 receptors.

It has previously been shown that arginine vasopressin (AVP) exerts a direct stimulatory action on rat adrenocortical cells. In the present study, we have investigated the possible effect of AVP on cortisol secretion by normal human adrenocortical tissue. The occurrence of endogenous AVP in the human adrenal gland has been studied by means of the indirect immunofluorescence technique. The presence of AVP-containing cells was observed in both cortex and medulla. The action of AVP on corticosteroidogenesis has been investigated in vitro using a perifusion system technique coupled to a specific RIA for cortisol. Graded doses of AVP (from 10(-11)-10(-9) M) increased cortisol secretion in a dose-dependent manner (ED50, 4.5 x 10(-11) M). AVP also induced a significant stimulation of cortisol release from acutely dispersed adrenocortical cells. Prolonged administration of AVP (3 h) induced a rapid and transient increase in cortisol output, followed by a gradual decline in cortisol secretion. Repeated pulses of AVP, given at 90-min intervals, resulted in reproducible stimulations of cortisol output. Selective agonists and antagonists have been used to determine the type of receptor involved in the response of adrenocortical cells to AVP. Oxytocin at doses up to 10(-7) M had virtually no effect on cortisol secretion. The stimulatory effect of AVP was blocked by the V1 antagonist [beta-mercapto-beta,beta-cyclopentamethylene propionyl 1, OMe-Tyr2, Arg8]AVP. In contrast, the V2 antagonist [d(CH2)5D-Phe2,Ile4,Ala9-NH2]AVP did not affect the response of the adrenal gland to AVP. The selective V2 agonist [deamino-Cys1,D-Arg8]AVP did not mimic the stimulatory effect of AVP on cortisol secretion. Taken together, these results suggest that AVP, locally released by intracortical cells, may act as a paracrine factor to stimulate adrenal steroidogenesis in man. The effect of AVP on cortisol secretion appears to be mediated through activation of typical V1 receptors.