Deletion of tyrosine hydroxylase gene reveals functional interdependence of adrenocortical and chromaffin cell system in vivo.

Catecholamines are produced in the medulla of the adrenal gland and may participate in the intraglandular regulation of its cortex. We analyzed the adrenal structure and function of albino tyrosine hydroxylase-null (TH-null) mice that are deficient in adrenal catecholamine production. Adrenal catecholamines were markedly reduced, and catecholamine histofluorescence was abrogated in 15-day-old TH-null mice. Chromaffin cell structure was strikingly altered at the ultrastructural level with a depletion of chromaffin vesicles and an increase in rough endoplasmic reticulum compared with wild-type mice. Remaining chromaffin vesicles lined up proximally to the cell membrane in preparation for exocytosis providing a "string-of-pearls" appearance. There was a 5-fold increase in the expression of proenkephalin mRNA (502.8 +/- 142% vs. 100 +/- 17.5%, P = 0.016) and a 2-fold increase in the expression of neuropeptide Y (213.4 +/- 41.2% vs. 100 +/- 59.9%, P = 0.014) in the TH-null animals as determined by quantitative TaqMan (Perkin-Elmer) PCR. Accordingly, immunofluorescence for met-enkephalin and neuropeptide tyrosine in these animals was strongly enhanced. The expression of phenylethanolamine N-methyl transferase and chromogranin B mRNA was similar in TH-null and wild-type mice. In TH-null mice, adrenocortical cells were characterized by an increase in liposomes and by tubular mitochondria with reduced internal membranes, suggesting a hypofunctional state of these steroid-producing cells. In accordance with these findings, plasma corticosterone levels were decreased. Plasma ACTH levels were not significantly different in TH-null mice. In conclusion, both the adrenomedullary and adrenocortical systems demonstrate structural and functional changes in catecholamine-deficient TH-null mice, underscoring the great importance of the functional interdependence of these systems in vivo.

Adrenal cortical activation in murine colitis.

BACKGROUND & AIMS: Proper adrenal glucocorticoid secretion is crucial in the course of inflammatory diseases. However, the function and structure of the adrenal glands have not been examined in inflammatory bowel diseases. METHODS: After induction of trinitrobenzene sulfonic acid (TNBS) colitis in SJL/J mice, plasma hormone and cytokine levels were measured, adrenal structure was analyzed by immunohistochemistry and electron microscopy, and adrenal cytokine/cytokine receptor expression were studied by RNase protection. RESULTS: Adrenals of colitic animals were enlarged and hypervascularized. These animals had a marked increase in plasma corticosterone levels during the course of colitis (270 +/- 34 vs. 16 +/- 11 ng/mL; P < 0.0001) but only a modest elevation of their concurrent adrenocorticotropin levels (57 +/- 13 vs. 29 +/- 9 pmol/L; NS). On electron microscopy, adrenocortical cells showed ultrastructural signs of marked stimulation, and intra-adrenal lymphocytes were frequently found in direct contact with these cells. Concurrent plasma levels of interleukin (IL)-6, the major cytokine activating the hypothalamic-pituitary-adrenal axis, were markedly increased (495 +/- 131 vs. 20 +/- 1.5 pg/mL; P < 0.0001), and this cytokine directly stimulated corticosterone secretion by adrenocortical cells in vitro. Intra-adrenal expression of IL-6 in animals with colitis was increased 80-fold, and the IL-6 receptor subunits IL-6R alpha and gp130 were present in the adrenal cells. Treatment of animals with neutralizing anti-IL-6 antibody reduced the TNBS-induced growth and activation of the adrenal cortices. CONCLUSIONS: Colitis is associated with a profound stimulation of adrenocortical cell function and glucocorticoid release. Direct immune-adrenal interactions seem to contribute to this activation of the adrenal glands during colitis.

Increased body fat mass and suppression of circulating leptin levels in response to hypersecretion of epinephrine in phenylethanolamine-N-methyltransferase (PNMT)-overexpressing mice.

Epinephrine is a major stress hormone that plays a central role in the control of metabolic function and energy homeostasis. To evaluate the role of epinephrine and the physiological and pathophysiological consequences of sustained elevation of epinephrine on metabolic and endocrine function, we studied several metabolic parameters and circulating leptin levels in a newly developed transgenic mouse model of phenylethanolamine-N-methyltransferase (PNMT) overexpression. A 100-fold overexpression of PNMT and subsequent elevation of epinephrine levels resulted in a marked suppression of circulating leptin levels in the transgenic animals (1.14 +/- 0.05 vs. 2.17 +/- 0.35 ng/ml; P < 0.01), which correlated negatively with plasma epinephrine (r = -0.82; P < 0.05), thus providing evidence for an inhibitory action of epinephrine on leptin production in vivo. In parallel, we found a marked increase in the body fat content of the transgenic animals (12.54 +/- 1.5 vs. 6.22 +/- 0.2%; P < 0.01) that was accompanied by enlarged adipocytes, indicating an increased lipid storage in PNMT transgenic mice. Interestingly, however, transgenic animals had normal body weight and did not exhibit major alterations in carbohydrate metabolism, as evidenced by analysis of random and fasted blood glucose levels, plasma insulin and C peptide levels, and insulin tolerance test. The metabolic alterations observed were not secondary to changes in food intake or increased activity of the hypothalamic-pituitary-adrenal axis, as there were no differences in these parameters. In summary, sustained primary overproduction of epinephrine resulted in suppression of plasma leptin levels and increased lipid storage in the PNMT transgenic mice. The concerted action of the sympathoadrenal system and reduced leptin may contribute to defending energy reservoirs while maintaining a normal body weight, which may be of vital importance under conditions of stress and energy deficiency.

Pharyngeal carriage of group C and group G streptococci and acute rheumatic fever in an Aboriginal population.

Acute rheumatic fever (ARF) and rheumatic heart disease (RHD) represent an autoimmune response to group A streptococcal pharyngitis. In the Aboriginal population of the Northern Territory of Australia, some of the highest rates of ARF in the world have been reported, although throat carriage rates of group A streptococcus in this population are extremely low and symptomatic group A streptococcal pharyngitis is uncommon; by contrast, carriage rates of group C and G streptococci are high. Therefore, we questioned the involvement of these groups in ARF and showed that they have the potential to elicit an autoimmune response that may trigger ARF.

Expression of adrenocortical steroidogenic acute regulatory (StAR) protein is influenced by chromaffin cells.

Cell-cell interactions are crucial role for the proper functioning of endocrine glands. We recently demonstrated that interactions of chromaffin and cortical cells are important for adrenocortical steroidogenesis. However, the molecular mechanisms have not been elucidated and it is unclear if this involves acute and/or chronic processes. By Northern analysis and the quantitative technique of TaqMan PCR we investigated whether chromaffin cells influence the regulation of StAR and the peripheral benzodiazepine receptor (PBR), both required for the rate-limiting step, the delivery of cholesterol to the inner mitochondrial membrane. StAR mRNA levels in bovine adrenocortical cells were increased by incubation with chromaffin cell-conditioned medium (CCM). Short-term treatment for 4 h resulted in a greater stimulation (229+/-29% of basal, mean+/-SEM) than did longer incubation times of 8 h and 5 days (159+/-13 and 177+/-24%). Neither short nor a long-term treatment affected PBR expression. Consistently, the major secretion of chromaffin cells, epinephrine dose-dependently stimulated StAR expression with no effect on PBR mRNA. In conclusion, adrenomedullary secretory products are not necessary for the maintenance of PBR expression but facilitate steroid biosynthesis by increasing StAR mRNA expression and therefore can account for an ACTH-independent regulation of the rate-limiting step in steroidogenesis.

Neurotransmitters and neuropeptides in the differential regulation of steroidogenesis in adrenocortical-chromaffin co-cultures.

Adrenocortical steroidogenesis is regulated in addition to a central regulation via the hypothalamus-pituitary-adrenal axis by intra-adrenal mechanisms involving the adrenal medulla. We could previously show that adrenocortical steroidogenesis is stimulated by co-culturing bovine adrenocortical cells with medullary chromaffin cells. This stimulation was due to soluble factors released from the chromaffin cells under basal, unstimulated conditions and involved the increased expression of P450 enzymes, StAR and de novo protein synthesis. In the present study we analyzed the differential regulation of the three cortical zones and characterized secretagogues involved in this stimulation. While cortisol and androstenedione release were increased 10 fold by incubation with chromaffin cell-conditioned medium, aldosterone secretion was not influenced. 80% of the stimulation proved to be due to adrenomedullary epinephrine, norepinephrine, ACTH, PACAP and PG-dependent mechanisms. Other adrenomedullary secretory products, serotonin, Met-enkephalin, Leu-enkephalin, galanin, CGRP, substance P, VIP or NPY did not stimulate steroidogenesis in this system. Our data show that adrenomedullary cells differentially regulate the three adrenocortical zones. This stimulation predominantly depended on epinephrine, norepinephrine, PACAP, and ACTH released from the chromaffin cells and prostaglandin-dependent mechanisms such as interleukin-1.

Leptin and its receptor in normal human gastric mucosa and in Helicobacter pylori-associated gastritis.

BACKGROUND: Leptin, a newly discovered weight-reducing hormone, is mainly produced in fat cells. Recently, this hormone has been reported to be produced in rat gastric mucosa cells. In the present study we analyzed the localization and expression of leptin and its receptors in normal human gastric mucosa and in patients with Helicobacter pylori-associated gastritis. METHODS: Plasma leptin levels and gastric mucosa leptin content were determined in 39 patients with dyspepsia. Cellular localization of leptin and of the signaling receptor (Ob-RL) were assessed by immunohistochemistry. Reverse transcriptase polymerase chain reaction (RT-PCR) for leptin receptor isoforms was performed on gastric epithelial cells isolated by laser-capture-microdissection. RESULTS: Leptin content of the corpus gastric mucosa in H. pylori-positive patients was significantly increased (4.6+/-1.2. n = 15) as compared with the H. pylori-negative group (27.5+/-0.5 pg/mg, n = 24, P = 0.006). The presence of leptin immunoreactivity was shown in the lower half of corpus epithelial glands. By RT-PCR no leptin mRNA was detectable in human gastric tissue. In contrast, expression of both Ob-R(L) and the leptin receptor isoforms could be detected in gastric epithelial cells. Leptin receptor protein was detected throughout the mucosa. CONCLUSIONS: Leptin itself is stored and secreted but not produced in human gastric mucosa. The functional receptor and all isoforms are present in human gastric mucosa. H. pylori-associated gastritis leads to significant increases in local leptin concentration in the gastric corpus.

Adrenomedullary function is severely impaired in 21-hydroxylase-deficient mice.

Deficiency of 21-hydroxylase (21-OH), one of the most common genetic defects in humans, causes low glucocorticoid and mineralocorticoid production by the adrenal cortex, but the effect of this disorder on the adrenomedullary system is unknown. Therefore, we analyzed the development, structure, and function of the adrenal medulla in 21-OH-deficient mice, an animal model resembling human congenital adrenal hyperplasia. Chromaffin cells of 21-OH-deficient mice exhibited ultrastructural features of neuronal transdifferentiation with reduced granules, increased rough endoplasmic reticulum and small neurite outgrowth. Migration of chromaffin cells in the adrenal to form a central medulla was impaired. Expression of phenylethanolamine-N-methyltransferase (PNMT) was reduced to 27 +/- 9% (P<0.05), as determined by quantitative TaqMan polymerase chain reaction, and there was a significant reduction of cells staining positive for PNMT in the adrenal medulla of the 21-OH-deficient mice. Adrenal contents of epinephrine were decreased to 30 +/- 2% (P<0. 01) whereas norepinephrine and dopamine levels were reduced to 57 +/- 4% (P<0.01) and 50 +/- 9% (P<0.05), respectively. 21-OH-deficient mice demonstrate severe adrenomedullary dysfunction, with alterations in chromaffin cell migration, development, structure, and catecholamine synthesis. This hitherto unrecognized mechanism may contribute to the frequent clinical, mental, and therapeutic problems encountered in humans with this genetic disease.

Expression of Ob receptor in normal human adrenals: differential regulation of adrenocortical and adrenomedullary function by leptin.

The major effects of leptin, an adipostatic hormone produced in fat tissue, are exerted through the hypothalamic-pituitary-adrenal axis and the systemic sympathetic/adrenomedullary system at the level of the central nervous system. Here, we examined the direct effects of leptin on the adrenal gland, a peripheral end organ of both the hypothalamic-pituitary-adrenal axis and the sympathetic/adrenomedullary system. As cortical and chromaffin tissues are intermingled in the human adrenal, we employed the novel technique of laser capture microdissection to analyze these systems separately. Functional full-length leptin receptor messenger ribonucleic acid and all human isoforms Ob219.1-3 were demonstrated by RT-PCR in both cortical and medullary tissue. Immunohistochemical staining of leptin receptor protein, however, demonstrated a strong signal only in the adrenal cortex, whereas there was weak positive staining in the medulla. Corticotropin (ACTH)-induced adrenal aldosterone, cortisol, and dehydroepiandrosterone secretion was inhibited by leptin in a concentration-dependent manner, whereas this hormone had no significant effect on catecholamine release by primary cultures of human adrenal chromaffin cells. Leptin itself was not expressed in human adrenal tissue, excluding a local paracrine or autocrine function of this peptide. In conclusion, this is the first report identifying functional leptin receptor in human adrenal tissue and showing a differential action of leptin on human adrenocortical and chromaffin hormone production. This peripheral action of leptin on the adrenal gland provides an additional important link between the human stress response and body weight regulation.

Human adrenocortical NCI-H295 cells express VIP receptors. Steroidogenic effect of vasoactive intestinal peptide (VIP).

VIP receptors are frequently overexpressed by various endocrine tumors. In this study the expression of VIP receptors in the human adrenocortical carcinoma cell line NCI-H295 and their involvement in the regulation of steroidogenesis was investigated. NCI-H295 cells express VIP1 and VIP2 receptors as demonstrated by RT-PCR, whereas they do not express VIP itself. The receptors are functionally coupled to steroidogenesis since VIP (10(-9) M to 10(-6) M) exerted a dose-dependent stimulatory effect on the release of aldosterone, cortisol, and DHEA. VIP increased ACTH-stimulated releases of aldosterone and cortisol. The proliferation rate of NCI-H295 cells was not affected by VIP. These data show that NCI-H295 cells express both forms of the VIP receptor and that VIP is involved in an ACTH-independent regulation of steroidogenesis in the adrenal tumor cells.

Basal steroidogenic activity of adrenocortical cells is increased 10-fold by coculture with chromaffin cells.

Historically, catecholamine-producing chromaffin cells and steroid-producing adrenocortical cells have been regarded as two independent endocrine systems that are united under a common capsule to form the adrenal gland. There is increasing evidence for bidirectional interactions, with regulatory influences of adrenocortical secretory products on adrenomedullary functions and vice versa. However, the direct involvement of chromaffin cells on the regulation and maintenance of cortical function has not yet been demonstrated. Therefore, we analyzed glucocorticoid secretion and P450 messenger RNA (mRNA) expression in bovine adrenocortical cells in cocultures with chromaffin cells compared with those in pure cortical cell cultures. Cortisol release from cortical cells in coculture with chromaffin cells was 10 times as high (mean +/- SEM, 1035 +/- 119%) as that from the same number of isolated cortical cells (100 +/- 11%). By a [3H]thymidine incorporation assay, it was demonstrated that this effect was not due to a higher proliferation rate. Northern analysis revealed an increasing expression of P450(17alpha) mRNA in the coculture from days 1-5, whereas in isolated cortical cells, P450(17alpha) mRNA decreased, leading to a 6-fold difference on day 5. Inhibitors of protein (cycloheximide) or RNA (actinomycin D) synthesis completely annulled the observed increase in cortisol release, indicating that de novo protein synthesis is required for this activation of adrenocortical steroidogenesis. Addition of the cyclooxygenase inhibitor indomethacin reduced the stimulatory effect, suggesting that this stimulation is in part mediated by PGs. Locally produced ACTH, catecholamines, and interleukin-1 accounted for 43% of the effect. Secretory products of chromaffin cells that act in concert are believed to be responsible for the stimulation of steroidogenesis in the coculture. The coculture system is an in vitro model that corresponds to the in vivo situation in the intact adrenal gland, where both endocrine cell systems are in close contact. Our data demonstrate the requirement of intraadrenal cellular communication for the full strength of the adrenocortical hormonal response.

Basal catecholamine and cortisol secretion in primary chromaffin cell cultures before and after purification and retroviral transfection.

We have investigated the effects of supraphysiological concentrations of catecholamines on glucocorticoid secretion in vitro. These effects were analyzed in adrenocortical cells shown to be present in chromaffin cell cultures as well as in cortical cells cocultured with transfected chromaffin cells that overproduce catecholamines. Cortisol release from residual cortical cells in chromaffin cell cultures was found to be 2.5 times higher than from isolated adrenocortical cells. Removal of the adrenocortical cells from the chromaffin cells resulted in an almost complete cessation of cortisol secretion. Catecholamine overproduction was achieved by transfecting chromaffin cells with the blank retroviral vector pSAM-EN. Coculture of adrenocortical cells with these transfected chromaffin cells further enhanced the stimulating effect of chromaffin cells on cortisol 2.3-fold compared to normal cocultures. In conclusion, cortical cells in chromaffin cell cultures secrete significant amounts of cortisol, which should be considered when evaluating the endocrine function of these cell cultures and which can be abolished by purification. The hormonal activity of adrenocortical cells is highly increased in an environment of catecholamine overproduction, which is of both basic and clinical importance.

Differential expression of prolactin receptor (PRLR) in normal and tumorous adrenal tissues: separation of cellular endocrine compartments by laser capture microdissection (LCM).

PRL stimulates adrenal steroidogenesis. In this study, we compared the PRLR expression in normal and tumorous adrenal tissues and investigated a potential proliferative effect of PRL in adrenal cells. mRNA expression of long and intermediate forms of PRLR was detected in both normal adrenal cortex as well as benign and malignant adrenal tumors and in the human adrenocortical carcinoma cell line NCI-H295. Molecular analysis of cells procured by LCM clearly demonstrated that PRLR mRNA is expressed in the adrenal cortex but not in the medulla. Immunostaining revealed PRLR protein in all three zones of the normal adrenal cortex. Furthermore, adrenal carcinomas and adenomas stained positive for the PRLR, while in phaeochromocytomas as in the normal adrenal medulla, no specific staining was observed. By WST-1 test, we could show that PRL (10(-7) M) decreased proliferation and viability of adrenal cells in primary cell culture suggesting that PRL is not a mitogenic factor of adrenocortical cells.

Evidence for a novel peripheral action of leptin as a metabolic signal to the adrenal gland: leptin inhibits cortisol release directly.

The crucial role of glucocorticoids in obesity and insulin resistance and the actions of the OB protein leptin on the hypothalamic-pituitary-adrenal (HPA) axis suggest that there is an important interaction of leptin with the glucocorticoid system. Therefore, we designed a study to test the effect of leptin directly on adrenocortical steroidogenesis. Primary cultures of bovine adrenocortical cells were incubated with increasing concentrations (10-1,000 ng/ml) of recombinant mouse leptin for 24 h, and the effects of leptin on basal and ACTH-stimulated cortisol secretion were determined. The accumulation of P450 17alpha mRNA following incubation with ACTH (10 nmol/l) and leptin (10-1,000 ng/ml) was analyzed by Northern blot. Adrenocortical cells were characterized by immunohistochemical staining for 17alpha-hydroxyprogesterone. Leptin (10-1,000 ng/ml) inhibited basal and ACTH-stimulated cortisol release. At a concentration that occurs in obese individuals in vivo (100 ng/ml), it reduced basal cortisol secretion to 52.7 +/- 37% (mean +/- SE). The rise in cortisol secretion following maximal ACTH stimulation (10 nmol/l) was blunted to 55.2 +/- 27%. At more physiological concentrations of ACTH (0.1 nmol/l), the inhibition of cortisol release by coincubation with low doses of leptin (10 ng/ml) was even more pronounced, leading to a reduction to 32.8% (1,248 +/- 134 vs. 410 +/- 157 nmol/l). Addition of OB protein (10-1,000 ng/ml) led to a dose-dependent reduction of ACTH-stimulated cytochrome P450 17alpha mRNA accumulation (from 80 to 45%), suggesting that leptin regulates adrenal steroidogenesis at the transcriptional level. These data clearly demonstrate that leptin inhibits cortisol production in adrenocortical cells and therefore appears to be a metabolic signal that directly acts on the adrenal gland.

Cellular communication in the neuro-adrenocortical axis: role of vasoactive intestinal polypeptide (VIP).

It is well established now that adrenocortical function, besides being regulated through systemic factors, is influenced by intra-adrenal mechanisms. In this context paracrine influences between the sympathoadrenal system and the adrenal cortex play an important role. As a prerequisite for these interactions, adrenal medullary cells and cortical cells are highly interwoven as revealed by immunohistochemistry. The potential role of VIP in the regulation of human adrenal steroidogenesis was now investigated in human adrenal cells in primary culture. The primary cultures contained both, cortical and chromaffin cells which were found to be in close cellular contact as revealed by immunocytochemistry. VIP enhanced cortisol secretion from adrenal cells in a dose-dependent manner with a maximal effect at 10(-7) M. VIP stimulated the release of dehydroepiandrosterone (DHEA), testosterone, androstenedione, and aldosterone significantly. The addition of propranolol, a beta-adrenergic antagonist, to the incubation medium attenuated VIP-induced corticosteroid secretion. It is concluded that VIP is a paracrine messenger in the human adrenal that could regulate adrenocortical function at least in part via catecholamines released from the medulla.

Functional aspects of the effect of prolactin (PRL) on adrenal steroidogenesis and distribution of the PRL receptor in the human adrenal gland.

Hyperprolactinemia is one of the most common disorders in endocrinology. A role for PRL on the human adrenal gland has been postulated in various clinical studies. We have demonstrated for the first time the expression of the PRL receptor in the human adrenal gland and in human adrenal primary cell cultures using PCR and immunohistochemical methods. Using immunostaining, we could detect the PRL receptor in all three zones of the adrenal cortex. Only weak staining was observed in the adrenal medulla. The influence of PRL on the secretion of cortisol, aldosterone, and androgens in human primary cell cultures was investigated. After stimulation with PRL (10(-7) mol/L), we measured increased concentrations of cortisol (155 +/- 9.8%; P < 0.005%), aldosterone (122 +/- 3.7%; P < 0.005), and dehydroepiandrosterone (121 +/- 8.6%; P < 0.05) in the cell supernatant. PRL did not affect the expression of messenger ribonucleic acid of cytochrome P45017 alpha in human adrenal cell cultures. In conclusion, we found the PRL receptor in the human adrenal gland. We postulate that PRL has a direct effect on adrenal steroidogenesis, thereby regulating adrenal function, which may be of particular relevance in clinical disorders with hyperprolactinemia.