PTH and PTH-related peptide enhance steroid secretion from human adrenocortical cells

Giuseppina Mazzocchi1, Francesco Aragona2, Ludwik K. Malendowicz1, and Gastone G. Nussdorfer1

1 Section of Anatomy, Department of Human Anatomy and Physiology and 2 Department of Urology, University of Padua, I-35121 Padua, Italy


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Parathyroid hormone (PTH) and PTH-related peptide (PTH-RP) are two hypercalcemic hormones that share a common receptor subtype, the PTH/PTH-RP receptor. PTH and PTH-RP concentration dependently enhanced basal aldosterone and cortisol secretion from dispersed human adrenocortical cells, with a maximal effective concentration (~2-fold increase) of 10-8 M. The secretagogue effect of 10-8 M PTH or PTH-RP was abolished by the PTH/PTH-RP receptor antagonist [Leu11,D-Trp12]-PTH-RP-(7-34)-amide (10-6 M). PTH and PTH-RP (10-8 M) raised cAMP and inositol-triphosphate release by dispersed adrenocortical cells, and these effects were blocked by the adenylate cyclase inhibitor SQ-22536 (10-4 M) and the phospholipase C (PLC) inhibitor U-73122 (10-5 M), respectively. SQ-22536 (10-4 M) and U-73122 (10-5 M) partially inhibited aldosterone and cortisol response to 10-8 M PTH and PTH-RP; when added together, they abolished it. Similar results were obtained by using the protein kinase (PK)A and PKC inhibitors H-89 and calphostin C (10-5 M). It is concluded that PTH and PTH-RP exert a sizeable secretagogue action on the human adrenal cortex, probably acting through the PTH/PTH-RP receptor coupled with both adenylate cyclase/PKA- and PLC/PKC-dependent signaling cascades.

aldosterone; cortisol; protein kinases A and C


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

PARATHYROID HORMONE (PTH) and PTH-related peptide (PTH-RP) are two hypercalcemic hormones encoded by different genes, which possess similar intron-exon organization and are thought to have a common ancestral origin. Human PTH and PTH-RP display a remarkable homology in their amino acid composition (17) and share a common receptor subtype, called the PTH/PTH-RP receptor (22).

PTH and PTH-RP are included in the vasoactive intestinal peptide (VIP)-secretin-glucagon family of peptides, members of which (e.g., VIP and pituitary adenylate cyclase-activating peptide) are well known to modulate secretory activity of the adrenal cortex, acting in a paracrine manner (for a review, see Ref. 16). Accordingly, PTH-RP immunoreactivity has been detected in the cortex of the adult human adrenal gland (10) and in the adrenal cortex and medulla of human fetuses between the ages of 8 and 40 wk (2, 12). PTH-binding sites have been autoradiographically localized in the rat adrenal cortex (8), and the presence of PTH/PTH-RP receptor mRNA has been demonstrated in the adrenal gland of this species (24). A secretagogue action of PTH on dispersed rat (19, 20), mice (6), and bovine adrenocortical cells (8, 20) has been reported, but the possible effect of PTH-RP has not yet been studied. Moreover, no investigations were carried out using dispersed human adrenocortical cells.

Here we report findings strongly suggesting that both PTH and PTH-RP enhance steroid hormone secretion from human adrenocortical cells, through a common signaling mechanism involving the activation of both adenylate cyclase/protein kinase (PK)A and phospholipase C (PLC)/PKC cascades.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Reagents. Human PTH and PTH-RP, adrenocorticotropic hormone (ACTH), angiotensin II, and corticotropin-inhibiting peptide (CIP) were purchased from Peninsula Laboratories (St. Helens, UK). SQ-22536, U-73122, H-89, and calphostin C were obtained from Biomol Research Laboratories (DBA, Milan, Italy). Human [Leu11,D-Trp12]PTH-RP-(7-34)-amide was provided by Phoenix Pharmaceutical (Belmont, CA). Appropriate references for the effects of the above mentioned chemicals can be found in the catalogs of the purchasers. Medium 199 was obtained from Difco (Detroit, MI), and IBMX, human serum albumin (HSA), and other laboratory reagents were provided by Sigma Chemical (St. Louis, MO). RIA kits for aldosterone and cortisol were purchased from IRE-Sorin (Vercelli, Italy), and RIA kits for cAMP and D-myo-inositol-1,4,5-triphosphate (IP3) were obtained from Amersham Pharmacia (Aylesbury, UK).

Dispersed human adrenocortical cells. Dispersed cells were obtained from adrenal glands removed from 16 consenting patients undergoing unilateral nephrectomy with ipsilateral adrenalectomy for renal cancer. Starting from 2 wk before surgery, patients were kept on a normal diet; only patients not requiring medications to alter adrenal function were recruited. Adrenal tails, which do not contain medullary chromaffin tissue (14), were collected immediately after excision in the operating room, placed in Krebs-Ringer bicarbonate buffer with 0.2% glucose at 4°C, and immediately carried to our laboratory, where cell isolation was performed by sequential collagenase digestion and mechanical disaggregation. Our adrenocortical cell preparations were a mixture of zona glomerulosa (ZG) and zona fasciculata-reticularis (ZF/R) cells, and their contamination by stromal elements, as evaluated by phase microscopy, was virtually absent. The viability of isolated cells, as checked by the trypan blue exclusion test, was >90%. Adjacent sections of the adrenal glands underwent pathological evaluation and were found to be histologically normal. The study protocol was approved by the Padua Ethical Committee for Human Studies.

Steroid hormone secretion. Dispersed cells were put in medium 199 and Krebs-Ringer bicarbonate buffer with 2% glucose containing 5 mg/ml HSA and were incubated (105 cells/ml, in replicates of three each). The following incubation experiments were performed: 1) PTH or PTH-RP (from 10-12 to 10-6 M); 2) PTH or PTH-RP (10-8 M) and ACTH or ANG II (10-9 M) alone or in the presence of 10-6 M [Leu11,D-Trp12]PTH-RP-(7-34)-amide; 3) ACTH (10-9 M) and PTH or PTH-RP (10-8 M) alone or in the presence of 10-6 M CIP; 4) PTH or PTH-RP (10-8 M) alone or in the presence of 10-4 M SQ-22536 and/or 10-5 M U-73122; and 5) PTH or PTH-RP (10-8 M) alone or in the presence of 10-5 M H-89 and/or calphostin C. Incubations were carried out in a shaking bath at 37°C for 90 min in an atmosphere of 95% air-5% CO2, as previously described (14). The medium was collected and kept frozen at -80°C until hormonal assays. Aldosterone and cortisol were extracted from incubation media and purified by HPLC (14). Their concentrations were measured by RIA, with the ALDO-CTK2 (sensitivity, 5 pg/ml; intra- and interassay variations, 7.3 and 8.4%) and cortisol RIA (sensitivity, 30 pg/ml; intra- and interassay variations, 6.0 and 7.5%) kits.

cAMP and IP3 production. Dispersed cells were incubated, as described above, for 10 min with PTH or PTH-RP (10-8 M) alone or in the presence of 10-4 M SQ-22536 or 10-5 M U-73122. In the case of cAMP assay, the phosphodiesterase inhibitor IBMX (10-4 M) was added to prevent cAMP metabolism. cAMP was extracted by incubating the medium with 0.1 N HCl for 20 min at 4°C. The HCl extract was then neutralized, and cAMP concentration was determined by following the protocol of the Amersham Biotrak TRK 432 (sensitivity, 1 pmol/l; intra- and interassay variations, 5.3 and 6.6%). IP3 was extracted by the trichloroacetic acid method and purified by Amprep SAX-minicolumn chromatography, and its concentration was measured by RIA. The procedure followed the protocol of the Amersham Biotrak TRK 1000 (sensitivity, 2 pmol/l; intra- and interassay variations, 6.8 and 8.1%).

Statistics. Each incubation experiment was performed in quadruplicate by use of dispersed cells obtained from four different adrenal glands. Data obtained from each adrenal were averaged and expressed as the means ± SE of four separate experiments. The statistical comparison of results was performed using ANOVA, followed by the Duncan's Multiple Range Test. A value of P < 0.05 was considered significant.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

PTH and PTH-RP concentration dependently increased basal secretion of both aldosterone and cortisol from dispersed cells, minimal and maximal effective concentrations being 10-10 M and 10-8 M (Fig. 1). The potency (EC50) and efficacy (percent increase elicited by the maximal effective concentration) of PTH were slightly but significantly less than those of PTH-RP [(PTH vs. PTH-RP) for aldosterone: EC50, 1.1 ± 0.2 × 10-10 M vs. 2.3 ± 0.4 × 10-10 M (P < 0.02; n = 4); efficacy, 91 ± 8 vs. 137 ± 12% (P < 0.01; n = 4); for cortisol: EC50, 5.6 ± 0.6 × 10-10 M vs. 1.8 ± 0.3 × 10-10 M (P < 0.01; n = 4); efficacy, 97 ± 6 vs. 122 ± 9% (P < 0.05 ; n = 4)].


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Fig. 1.   Effects of parathyroid hormone (PTH, A) and PTH-related peptide (RP, B) on aldosterone and cortisol secretion from dispersed human adrenocortical cells. Data, expressed as pmol · 106 cells-1 · h-1, are means ± SE of 4 separate experiments. +P < 0.05 and *P < 0.01 vs. respective baseline (B) value.

CIP (10-6 M) abolished the secretory response of dispersed adrenocortical cells to 10-9 M ACTH (Fig. 2) but did not alter the response to 10-8 M PTH or PTH-RP (Fig. 3). Conversely, the secretagogue effect of PTH and PTH-RP was annulled by 10-6 M [Leu11,D-Trp12]PTH-RP (7-34)-amide (Fig. 3), which was unable to affect the response to 10-9 M ACTH or ANG II (data not shown).


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Fig. 2.   Effect of corticotropin-inhibitin peptide (CIP; 10-6 M) on 10-9 M ACTH-stimulated aldosterone and cortisol secretion from dispersed human adrenocortical cells. Data, expressed as pmol · 106 cells-1 · h-1, are means ± SE of 4 separate experiments. *P < 0.01 vs. respective baseline value (B); AP < 0.01 vs. respective control value.



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Fig. 3.   Effects of CIP (10-6 M) and the PTH/PTH-RP receptor antagonist (A) [Leu11,D-Trp12]PTH-RP-(7-34)-amide (10-6 M) on PTH- or PTH-RP (10-8 M)-stimulated aldosterone (top) and cortisol secretion (bottom) from dispersed human adrenocortical cells. Data, expressed as pmol · 106 cells-1 · h-1, are means ± SE of 4 separate experiments. *P < 0.01 vs. respective baseline value (B); AP < 0.01 vs. respective control value.

PTH and PTH-RP (10-8 M) evoked 3.0- to 3.5-fold rises in cAMP and IP3 release by dispersed adrenocortical cells (Fig. 4). cAMP response was blocked by 10-4 M SQ-22536, and IP3 response was annulled by 10-5 M U-73122 (Fig. 4). SQ-22536 (10-4 M) and U-73122 (10-5 M) partially inhibited the stimulating effect of 10-8 M PTH or PTH-RP on both aldosterone and cortisol secretion, and when added together they abolished it (Fig. 5). Similar results were obtained by using H-89 (10-5 M) and calphostin C (10-5 M; Fig. 6). These inhibitors did not affect basal hormonal production (Figs. 5 and 6).


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Fig. 4.   Effects of PTH and PTH-RP (10-8 M) on cAMP (top) and D-myo-inositol-1,4,5-triphosphate (IP3) production (bottom) from dispersed human adrenocortical cells, and their reversal by SQ-22536 (10-4 M) and U-73122 (10-5 M), respectively. Data, expressed as pmol · 106 cells-1 · min-1, are means ± SE of 4 separate experiments. *P < 0.01 vs. respective baseline value (B); AP < 0.01 vs. respective control value.



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Fig. 5.   Effects of SQ-22536 (10-4 M) and/or U-73122 (10-5 M) on 10-8 M PTH or PTH-RP-stimulated aldosterone (top) and cortisol secretion (bottom) from dispersed human adrenocortical cells. Data, expressed as pmol · 106 cells-1 · h-1, are means ± SE of 4 separate experiments. +P < 0.05 and *P < 0.01 vs. respective baseline value (B); aP < 0.05 and AP < 0.01 vs. respective control value.



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Fig. 6.   Effects of calphostin C (10-5 M) and/or H-89 (10-5 M) on 10-8 M PTH- or PTH-RP-stimulated aldosterone (top) and cortisol secretion (bottom) from dispersed human adrenocortical cells. Data, expressed as pmol · 106 cells-1 · h-1, are means ± SE of 4 separate experiments. +P < 0.05 and *P < 0.01 vs. respective baseline value (B); aP < 0.05 and AP < 0.01 vs. respective control value.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Our present findings show that PTH and PTH-RP exert a sizeable stimulatory action on basal steroid hormone secretion from human adrenocortical cells. The similarity of the effects of the two peptides strongly suggests that they act through a common receptor, the PTH/PTH-RP receptor (22). This contention is supported by the demonstration that the PTH/PTH-RP receptor antagonist [Leu11,D-Trp12]PTH-RP-(7-34)-amide abolished the secretory response of dispersed adrenocortical cells to both PTH and PTH-RP. Conceivably, both ZG and ZF/R cells are provided with this receptor, inasmuch as both aldosterone and cortisol production are enhanced. The biologically active region of PTH (the 1-34 sequence) displays a remarkable homology with that of ACTH-(1-39) (18). However, the possibility that PTH and PTH-RP may interfere with the receptors of ACTH, the most potent in vitro stimulator of both aldosterone and cortisol (for review, see Ref. 26), is unlikely because the ACTH-receptor antagonist CIP, at a concentration annulling the secretagogue action of the maximal effective concentration of ACTH, does not affect the secretory response to PTH and PTH-RP. The slightly minor effectiveness of PTH, compared with PTH-RP, in eliciting a secretory response by human adrenocortical cells may be explained by a lower affinity for PTH/PTH-RP receptors. This possibility, although requiring confirmation, appears to be in keeping with the fact that PTH binds not only PTH/PTH-RP receptors but also PTH2 and C-PTH receptors, whereas PTH-RP is selective for PTH/PTH-RP receptors (7, 22, 25).

Evidence indicates that PTH/PTH-RP receptors, by coupling to multiple G proteins, activate adenylate cyclase and PLC cascades (21). Our present findings clearly indicate that both PTH and PTH-RP stimulate basal steroid secretion from dispersed human adrenocortical cells through both adenylate cyclase/PKA- and PLC/PKC-dependent signaling mechanisms. In fact, 1) PTH and PTH-RP markedly raise cAMP and IP3 production by dispersed adrenocortical cells; 2) the secretagogue effect of PTH and PTH-RP is partially suppressed by either the adenylate cyclase inhibitor SQ-22536 or the PLC inhibitor U-73122 at concentrations able to abolish cAMP and IP3 responses of adrenocortical cells to both hormones, and it is annulled by the simultaneous exposure to the two inhibitors; 3) the PKA inhibitor H-89 and the PKC inhibitor calphostin C elicited suppressive effects similar to those of SQ-22536 and U-73122; and 4) as previously shown (11), none of these inhibitors induces significant changes in the basal steroid secretion over 90 min of static incubation, which lessens the likelihood that their effect may be due to a nonspecific toxic lesion of the steroidogenic machinery.

The possible role of the stimulating effect of PTH and PTH-RP in the physiological regulation of the human adrenal gland remains to be demonstrated. However, we wish to mention some pathological conditions in which our findings may become relevant. First, the aldosterone and cortisol secretagogue effect of PTH and PTH-RP could, at least in part, explain the mild hypertension frequently associated with primary hyperparathyroidism (for review, see Ref. 5). Second, PTH and PTH-RP were found to be highly expressed in an adrenal carcinoma producing pseudohyperparathyroidism and Cushing's syndrome (3). Finally, elevated serum concentrations of PTH and PTH-RP were observed in several patients bearing benign or malignant pheochromocytomas (1, 4, 13, 23), and PTH-RP mRNA was detected in several benign tumors (9, 13). The association of secreting pheochromocytomas with adrenocortical tumors and idiopathic hypercorticism has been described (for review, see Ref. 15), and our study could provide a clue to explain these observations.


    FOOTNOTES

Address for reprint requests and other correspondence: G. G. Nussdorfer, Dept. of Human Anatomy and Physiology, Section of Anatomy, Via Gabelli 65, I-35121 Padova, Italy (E-mail: ggnanat{at}ipdunidx.unipd.it).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 8 May 2000; accepted in final form 5 October 2000.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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Am J Physiol Endocrinol Metab 280(2):E209-E213
0193-1849/01 $5.00 Copyright © 2001 the American Physiological Society




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