Early transcriptomic response of mouse adrenal gland and Y-1 cells to dexamethasone.

Glucocorticoids have short- and long-term effects on adrenal gland function and development. RNA sequencing (RNA-seq) was performed to identify early transcriptomic responses to the synthetic glucocorticoid, dexamethasone (Dex), in vitro and in vivo. In total, 1711 genes were differentially expressed in the adrenal glands of the 1-h Dex-treated mice. Among them, only 113 were also considered differentially expressed genes (DEGs) in murine adrenocortical Y-1 cells treated with Dex for 1 h. Gene ontology analysis showed that the upregulated DEGs in the adrenal gland of the 1-h Dex-treated mice were highly associated with the development of neuronal cells, suggesting the adrenal medulla had a rapid response to Dex. Interestingly, only 4.3% of Dex-responsive genes in the Y-1 cell line under Dex treatment for 1 h were differentially expressed under Dex treatment for 24 h. The heatmaps revealed that most early responsive DEGs in Y-1 cells during 1 h of treatment exhibited a transient response. The expression of these genes under treatment for 24 h returned to basal levels similar to that during control treatment. In summary, this research compared the rapid transcriptomic effects of Dex stimulation in vivo and in vitro. Notably, adrenocortical Y-1 cells had a transient early response to Dex treatment. Furthermore, the DEGs had a minimal overlap in the 1-h Dex-treated group in vivo and in vitro.

The Herbicide Atrazine Potentiates Angiotensin II-Induced Aldosterone Synthesis and Release From Adrenal Cells.

Atrazine is one of the most commonly used pre-emergence and early post-emergence herbicides in the world. We have shown previously that atrazine does not directly stimulate the pituitary or adrenal to trigger hormone release but acts centrally to activate a stress-like activation of the hypothalamic-pituitary-adrenal axis. In doing so, atrazine treatment has been shown to cause adrenal morphology changes characteristic of repeated stress. In this study, adrenals from atrazine treated and stressed animals were directly compared after 4 days of atrazine treatment or restraint stress. Both atrazine and stressed animals displayed reduced adrenocortical zona glomerulosa thickness and aldosterone synthase (CYP11B2) expression, indicative of repeated adrenal stimulation by adrenocorticotropic hormone. To determine if reduced CYP11B2 expression resulted in attenuated aldosterone synthesis, stressed and atrazine treated animals were challenged with angiotensin II (Ang II). As predicted, stressed animals produced less aldosterone compared to control animals when stimulated. However, atrazine treated animals had higher circulating aldosterone concentrations compared to both stressed and control groups. Ang II-induced aldosterone release was also potentiated in atrazine pretreated human adrenocortical carcinoma cells (H295R). Atrazine pretreated did not alter the expression of the rate limiting steroidogenic StAR protein or angiotensin II receptor 1. Atrazine treated animals also presented with higher basal blood pressure than vehicle treated control animals suggesting sustained elevations in circulating aldosterone levels. Our results demonstrate that treatment with the widely used herbicide, atrazine, directly increases stimulated production of aldosterone in adrenocortical cells independent of expression changes to rate limiting steroidogenic enzymes.

Expression of Rasd1 in mouse endocrine pituitary cells and its response to dexamethasone.

Dexamethasone-induced Ras-related protein 1 (Rasd1) is a member of the Ras superfamily of monomeric G proteins that have a regulatory function in signal transduction. Rasd1, also known as Dexras1 or AGS1, is rapidly induced by dexamethasone (Dex). While prior data indicates that Rasd1 is highly expressed in the pituitary and that the gene may function in regulation of corticotroph activity, its exact cellular localization in this tissue has not been delineated. Nor has it been determined which endocrine pituitary cell type(s) are responsive to Dex-induced expression of Rasd1. We hypothesized that Rasd1 is primarily localized in corticotrophs and furthermore, that its expression in these cells would be upregulated in response to exogenous Dex administration. Rasd1 expression in each pituitary cell type both under basal conditions and 1-hour post Dex treatment were examined in adult male mice. While a proportion of all endocrine pituitary cell types expressed Rasd1, a majority of corticotrophs and thyrotrophs expressed Rasd1 under basal condition. In vehicle treated animals, approximately 50-60% of corticotrophs and thyrotrophs cells expressed Rasd1 while the gene was detected in only 15-30% of lactotrophs, somatotrophs, and gonadotrophs. In Dex treated animals, Rasd1 expression was significantly increased in corticotrophs, somatotrophs, lactotrophs, and gonadotrophs but not thyrotrophs. In Dex treated animals, Rasd1 was detected in 80-95% of gonadotrophs and corticotrophs. In contrast, Dex treatment increased Rasd1 expression to a lesser extent (55-60%) in somatotrophs and lactotrophs. Corticotrophs of the pars intermedia, which lack glucocorticoid receptors, failed to display increased Rasd1 expression in Dex treated animals. Rasd1 is highly expressed in corticotrophs under basal conditions and is further increased after Dex treatment, further supporting its role in glucocorticoid negative feedback. In addition, the presence and Dex-induced expression of Rasd1 in endocrine pituitary cell types, other than corticotrophs, may implicate Rasd1 in novel pituitary functions.

Pet Food-Associated Dietary Exogenous Thyrotoxicosis: Retrospective Study (2016-2018) and Clinical Considerations.

Dietary exogenous thyrotoxicosis is infrequently observed in pet food. A retrospective evaluation of pet food investigations (PFI) was conducted for 17 dogs, including review of medical records, dietary and environmental exposure interviews, food testing, and regulatory action. Five PFIs occurring between 2016 and 2018 involved 7 food products including 2 food types, jerky treats or canned food, made from beef or bison. The dogs' serum thyroid hormone concentrations were evaluated before and after diet change. The foods were tested for active thyroid hormones and hormone precursors using high performance liquid chromatography with inductively coupled plasma mass spectrometry detection. The foods were also examined microscopically. Serum thyroid hormone concentrations of thyroxine (T4) varied depending on the food type consumed. Dogs that consumed dried jerky containing greater T4 concentrations often had increased serum T4 concentrations, whereas dogs that consumed canned products containing greater and 3,4,5- and 3,5,3'-triiodothyronine (T3) concentrations often had decreased serum T4 concentrations. After the diets were changed, serum T4 and T3 concentrations normalized at 1 month. Seven foods containing beef or bison had iodine concentrations greater than 11 mg/kg, and iodine speciation identified variable concentrations of iodide, T4, T3, monoiodotyrosine (MIT), and di-iodotyrosine (DIT). Thyroid gland was found in microscopic sections from one finished food and one ingredient, gullet. FDA performed Health Hazard Evaluations to categorize the exposure risk, and 5 foods were recalled for which the product packaging had not been discarded. Dietary exogenous thyrotoxicosis should be considered in dogs exhibiting clinical signs compatible with hyperthyroidism, especially if consuming beef-based food. A thyroid panel that includes serum iodine, coupled with a thorough feeding history can aid in diagnosis. Thyrotoxicosis is typically reversible after removing the contaminated food from the diet.

Effect of EDTA on measurement of cortisol and thyroxine by chemiluminescent enzyme immunoassay in dogs.

The addition of ethylenediamine tetra-acetic acid (EDTA) to serum can affect the measurement of cortisol by chemiluminescent enzyme immunoassay (CEIA); addition of magnesium chloride (MgCl2) may reverse the effects. However, similar characteristics for thyroxine (T4) measurement are unknown. We measured cortisol and T4 in paired EDTA-anticoagulated plasma and serum samples from 50 dogs. Additionally, both hormones were measured in 15 samples of each type after the addition of MgCl2. Samples were collected under routine clinical conditions; therefore, specific EDTA concentrations in plasma samples were unknown. Cortisol and T4 values were significantly different comparing plasma and serum samples in the absence of MgCl2. For cortisol and T4, EDTA-plasma concentrations were 51.2% and 43.7% higher than serum, respectively (p < 0.001 for both). The addition of MgCl2 to plasma significantly decreased the measured cortisol concentrations (p < 0.001) but not T4 (p = 0.44). After addition of MgCl2, cortisol concentrations in EDTA-plasma were no longer significantly different from serum, whereas T4 concentrations in EDTA-plasma remained significantly different from serum. In the clinical setting in which tubes may be underfilled, use of EDTA-plasma significantly increases the measured concentration of cortisol and T4 obtained by CEIA. Addition of MgCl2 to EDTA-plasma can overcome the effects of EDTA when measuring cortisol, but not T4. Thus, T4 should not be measured in EDTA-plasma.

Low-dose ACTH stimulation testing in dogs suspected of hypoadrenocorticism.

BACKGROUND: Low-dose ACTH stimulation testing would lower cost and may increase sensitivity for identification of partial ACTH deficiency. HYPOTHESIS: (1) The low-dose ACTH stimulation test will provide comparable results to the standard-dose ACTH stimulation test in dogs suspected of hypoadrenocorticism and (2) partial ACTH deficiency exists in dogs and can result in chronic, intermittent gastrointestinal signs. ANIMALS: Thirty-one client-owned dogs suspected of having hypoadrenocorticism. METHODS: Prospective study. Dogs suspected of having hypoadrenocorticism received 1 µg/kg cosyntropin IV for the first ACTH stimulation test; the second test was performed 4 h later and dogs received 5 µg/kg cosyntropin IV. Blood samples were obtained pre-ACTH and 1 hour post-ACTH for each dose (4 measurements total). Samples for endogenous ACTH measurement were obtained at the time of initial blood collection. RESULTS: No significant difference was observed in the basal cortisol concentration before administration of a 1 µg/kg versus before a 5 µg/kg dose of cosyntropin (P = .544). For dogs suspected of having hypoadrenocorticism, the ACTH-stimulated cortisol concentrations in response to both doses of ACTH were equivalent (90% confidence interval [CI], 80.5-97.2%; P = .04). No cases with partial ACTH deficiency were identified conclusively. CONCLUSIONS AND CLINICAL IMPORTANCE: A 1 µg/kg dose of cosyntropin is equivalent to a 5 µg/kg dose of cosyntropin for screening dogs suspected of hypoadrenocorticism. The existence of partial ACTH deficiency was not identified in this small group of dogs.

Characterization of Activation of the Hypothalamic-Pituitary-Adrenal Axis by the Herbicide Atrazine in the Female Rat.

Atrazine (ATR) is a commonly used pre-emergence and early postemergence herbicide. Rats gavaged with ATR and its chlorometabolites desethylatrazine (DEA) and deisopropylatrazine (DIA) respond with a rapid and dose-dependent rise in plasma corticosterone, whereas the major chlorometabolite, diaminochlorotriazine (DACT), has little or no effect on corticosterone levels. In this study, we investigated the possible sites of ATR activation of the hypothalamic-pituitary-adrenal (HPA) axis. ATR treatment had no effect on adrenal weights but altered adrenal morphology. Hypophysectomized rats or rats under dexamethasone suppression did not respond to ATR treatment, suggesting that ATR does not directly stimulate the adrenal gland to induce corticosterone synthesis. Immortalized mouse corticotrophs (AtT-20) and primary rat pituitary cultures were treated with ATR, DEA, DIA, or DACT. None of the compounds induced an increase in ACTH secretion or potentiated ACTH release in conjunction with CRH on ACTH release. In female rats gavaged with ATR, pretreatment with the CRH receptor antagonist astressin completely blocked the ATR-induced rise in corticosterone concentrations, implicating CRH release in ATR-induced HPA activation. Intracerebroventricular infusion of ATR, DEA, and DIA but not DACT at concentrations equivalent to peak plasma concentrations after gavage dosing resulted in an elevation of plasma corticosterone concentrations. However, ATR did not induce c-Fos immunoreactivity in the paraventricular nucleus of the hypothalamus. These results indicate that ATR activates the HPA axis centrally and requires CRH receptor activation, but it does not stimulate cellular pathways associated with CRH neuronal excitation.

Excess EDTA interferes with cortisol measurement using a solid-phase, chemiluminescent enzyme immunoassay.

Hormone assays that use a solid-phase, automated, chemiluminescent enzyme immunoassay (CEIA) with an alkaline phosphatase-tagged hormone or antibody as a reporter are performed on serum or EDTA plasma in our laboratory. CEIA cortisol results appeared to increase in the presence of excess EDTA. We investigated the effect of the addition of different amounts of EDTA on cortisol concentrations in pooled canine serum samples. The recommended EDTA plasma concentration of 4.1 mmol/L (1.8 mg/mL) did not alter cortisol concentrations when added to serum pools; however, the addition of ≥5.1 mmol/L (2.25 mg/mL) of EDTA increased apparent concentrations of cortisol. Supplementation of serum samples with MgCl2 to 5 mmol/L reversed the effect of EDTA up to a concentration of ~8.1 mmol/L (3.6 mg/mL). Our findings show that CEIA cortisol results on EDTA plasma can be artificially increased if the EDTA concentration exceeds 5.1 mmol/L.

Effect of laparotomy on the pituitary-adrenal axis in dogs.

OBJECTIVE To assess effects of major abdominal surgery on serum cortisol and aldosterone and plasma canine ACTH (cACTH) concentrations. ANIMALS 39 healthy dogs undergoing laparotomy during veterinary student surgical laboratories. PROCEDURES Blood samples were obtained before and at completion of surgery. Serum cortisol and aldosterone and plasma cACTH concentrations were measured by use of validated radioimmunoassays. Changes in concentrations (postoperative concentration minus preoperative concentration) were calculated. Data were analyzed by use of the Wilcoxon signed rank test, Pearson correlation analysis, and Mann-Whitney rank sum test. RESULTS Cortisol, aldosterone, and cACTH concentrations increased significantly from before to after surgery. Although cortisol and aldosterone concentrations increased in almost all dogs, cACTH concentrations decreased in 6 of 32 (19%) dogs. All dogs had preoperative cortisol concentrations within the reference range, but 24 of 39 (62%) dogs had postoperative concentrations above the reference range. A correlation between the change in cACTH concentration and the change in cortisol concentration was not detected. CONCLUSIONS AND CLINICAL RELEVANCE Laparotomy caused a significant increase in serum cortisol and aldosterone concentrations. In most dogs, but not all dogs, plasma cACTH concentrations increased. Lack of correlation between the change in cACTH concentration and the change in cortisol concentration suggested that increased postoperative cortisol concentrations may have been attributable to ACTH-independent mechanisms, an early ACTH increase that caused a sustained cortisol release, or decreased cortisol clearance. Further studies are indicated to evaluate the effects of various anesthetic protocols and minimally invasive surgical techniques on the stress response.

Iodine speciation in dog foods and treats by high performance liquid chromatography with inductively coupled plasma mass spectrometry detection.

An analytical method for determination of the iodine species 3-monoiodotyrosine (MIT), iodide, 3,5-diiodotyrosine (DIT), 3,5-diiodothyronine (3, 5-T2), 3,5,3'-triiodothyronine (T3), and thyroxine (T4) in dog foods and treats is reported. Iodine speciation was carried out using a HPLC method capable of both anion-exchange and reversed-phase retention coupled with inductively coupled plasma mass spectrometry detection (LC-ICP-MS). The method was evaluated by the analysis of the iodine species concentrations in twelve dog foods and treats following enzymatic digestion. The concentrations of MIT, iodide, DIT, T3, and T4 in the samples ranged from 0.64-59.5µg/g, 0.86-4.05µg/g,

Exogenous thyrotoxicosis in dogs attributable to consumption of all-meat commercial dog food or treats containing excessive thyroid hormone: 14 cases (2008-2013).

OBJECTIVE: To describe findings in dogs with exogenous thyrotoxicosis attributable to consumption of commercially available dog foods or treats containing high concentrations of thyroid hormone. DESIGN: Retrospective and prospective case series. ANIMALS: 14 dogs. PROCEDURES: Medical records were retrospectively searched to identify dogs with exogenous thyrotoxicosis attributable to dietary intake. One case was found, and subsequent cases were identified prospectively. Serum thyroid hormone concentrations were evaluated before and after feeding meat-based products suspected to contain excessive thyroid hormone was discontinued. Scintigraphy was performed to evaluate thyroid tissue in 13 of 14 dogs before and 1 of 13 dogs after discontinuation of suspect foods or treats. Seven samples of 5 commercially available products fed to 6 affected dogs were analyzed for thyroxine concentration; results were subjectively compared with findings for 10 other commercial foods and 6 beef muscle or liver samples. RESULTS: Total serum thyroxine concentrations were high (median, 8.8 µg/dL; range, 4.65 to 17.4 µg/dL) in all dogs at initial evaluation; scintigraphy revealed subjectively decreased thyroid gland radionuclide in 13 of 13 dogs examined. At ≥ 4 weeks after feeding of suspect food or treats was discontinued, total thyroxine concentrations were within the reference range for all dogs and signs associated with thyrotoxicosis, if present, had resolved. Analysis of tested food or treat samples revealed a median thyroxine concentration for suspect products of 1.52 µg of thyroxine/g, whereas that of unrelated commercial foods was 0.38 µg of thyroxine/g. CONCLUSIONS AND CLINICAL RELEVANCE: Results indicated that thyrotoxicosis can occur secondary to consumption of meat-based products presumably contaminated by thyroid tissue, and can be reversed by identification and elimination of suspect products from the diet.

Amino acid differences in cat adrenocorticotropin account for the inability of a human-based immunoradiometric assay to detect the molecule in cat plasma.

A commercial immunoradiometric assay kit designed for the measurement of endogenous adrenocorticotropin (ACTH) concentrations in human plasma does not detect the molecule in plasma samples from cats. It was hypothesized that the inability of the assay to detect the molecule was related to variation(s) in the amino acid sequence of cat ACTH, compared with human ACTH. Cat ACTH complementary DNA was cloned from pituitary tissue and sequenced. The deduced structure showed amino acid differences from the human molecule with cat ACTH having a valine instead of alanine at amino acid 32 and a threonine instead of alanine at amino acid 34. Cat and human ACTH were synthesized along with 2 modified peptides containing alanine substitutions at cat ACTH 32 and 34. Only the human ACTH was detected using the commercial kit, indicating that an epitope recognized by one of the antibodies in the assay requires the presence of 2 alanines near the C-terminus of the molecule.

Inoculation of dogs with a recombinant ACTH vaccine.

OBJECTIVE: To determine whether inoculation of healthy dogs with a recombinant peptide containing 3 copies of ACTH would result in the production of antibodies against ACTH and whether this would affect pituitary-adrenocortical function. ANIMALS: 8 healthy dogs. PROCEDURES: A recombinant peptide consisting of 3 copies of ACTH fused to a T-helper cell epitope was produced in Escherichia coli. The protein was inoculated into 4 dogs at 4-week intervals (total of 3 inoculations/dog). Four control dogs received inoculations of PBS solution mixed with adjuvant. Blood samples were collected for determination of antibody titers against ACTH and for measurement of basal and ACTH-stimulated plasma cortisol concentrations. RESULTS: Inoculation with the ACTH vaccine resulted in production of anti-ACTH antibodies in all 4 dogs. Titers were initially high but declined by 15 weeks after the initial inoculation. Basal cortisol concentrations were unaffected by inoculation with the ACTH vaccine. Plasma cortisol concentrations in response to ACTH stimulation were reduced at 12 weeks, but not at 15 weeks, after the first inoculation. CONCLUSIONS AND CLINICAL RELEVANCE: Inoculation of dogs with a recombinant ACTH vaccine resulted in the production of antibodies against the hormone. Anti-ACTH titers were initially high but were not sustained. The only detectable endocrine effect in treated dogs was a reduction in cortisol concentration in response to ACTH stimulation in 2 of 4 dogs at 12 weeks after the first inoculation. The effect of vaccine administration on the pituitary-adrenal system was subtle and transient.

Validation of a low-dose adrenocorticotropic hormone stimulation test in healthy neonatal foals.

OBJECTIVE: To determine the lowest ACTH dose that would induce a significant increase in serum cortisol concentration and identify the time to peak cortisol concentration in healthy neonatal foals. DESIGN: Prospective randomized crossover study. ANIMALS: 11 healthy neonatal foals. PROCEDURES: Saline (0.9% NaCl) solution or 1 of 4 doses (0.02, 0.1, 0.25, and 0.5 µg/kg [0.009, 0.045, 0.114, and 0.227 µg/lb]) of cosyntropin (synthetic ACTH) was administered IV. Serum cortisol concentrations were measured before and 10, 20, 30, 60, 90, 120, 180, and 240 minutes after administration of cosyntropin or saline solution; CBCs were performed before and 30, 60, 120, and 240 minutes after administration. RESULTS: Serum cortisol concentration was significantly increased, compared with baseline, by 10 minutes after cosyntropin administration at doses of 0.1, 0.25, and 0.5 µg/kg. Serum cortisol concentration peaked 20 minutes after administration of cosyntropin at doses of 0.02, 0.1, and 0.25 µg/kg, with peak concentrations 1.7, 2.0, and 1.9 times the baseline concentration, respectively. Serum cortisol concentration peaked 30 minutes after cosyntropin administration at a dose of 0.5 µg/kg, with peak concentration 2.2 times the baseline concentration. No significant differences were detected among peak serum cortisol concentrations obtained with cosyntropin administration at doses of 0.25 and 0.5 µg/kg. Cosyntropin administration significantly affected the lymphocyte count and the neutrophil-to-lymphocyte ratio. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggested that in healthy neonatal foals, the lowest dose of cosyntropin to result in significant adrenal gland stimulation was 0.25 µg/kg, with peak cortisol concentration 20 minutes after cosyntropin administration.

Seasonal variation in results of diagnostic tests for pituitary pars intermedia dysfunction in older, clinically normal geldings.

OBJECTIVE: To determine whether seasonal variations exist in endogenous plasma ACTH, plasma α-melanocyte-stimulating hormone (α-MSH), serum cortisol, and serum insulin concentrations and in the results of a dexamethasone suppression test for older, clinically normal geldings in Alabama. DESIGN: Cohort study. ANIMALS: 15 healthy mixed-breed geldings (median age, 14 years). PROCEDURES: Sample collection was repeated monthly for 12 months. Dexamethasone (0.04 mg/kg [0.02 mg/lb], IM) was administered and cortisol concentrations were determined at 15 and 19 hours. Radioimmunoassays were used to measure ACTH, α-MSH, cortisol, and insulin concentrations at each testing time. Hormone concentrations were compared between months via repeated-measures ANOVA and correlated with age within each month. RESULTS: A significant time effect was found between months for α-MSH and insulin concentrations. Endogenous cortisol and ACTH concentrations remained within existing reference ranges. Significant correlations were detected between age and ACTH concentration for several fall and winter months and between age and insulin concentration for September. CONCLUSIONS AND CLINICAL RELEVANCE: Older horses have higher ACTH concentrations in several fall and winter months and higher insulin concentrations in September than do younger horses. Seasonally specific reference ranges are required for α-MSH and insulin concentrations, with significantly higher concentrations detected in the fall. Practitioners should be advised to submit samples only to local laboratories that can provide such reference ranges for their local geographic region.

Validation of a low-dose ACTH stimulation test in healthy adult horses.

OBJECTIVE: To determine the lowest ACTH dose that would induce a maximum increase in serum cortisol concentration in healthy adult horses and identify the time to peak cortisol concentration. DESIGN: Evaluation study. ANIMALS: 8 healthy adult horses. PROCEDURES: Saline (0.9% NaCl) solution or 1 of 4 doses (0.02, 0.1, 0.25, and 0.5 µg/kg [0.009, 0.045, 0.114, and 0.227 µg/lb]) of cosyntropin (synthetic ACTH) were administered IV (5 treatments/horse). Serum cortisol concentrations were measured before and 30, 60, 90, 120, 180, and 240 minutes after injection of cosyntropin or saline solution; CBCs were performed before and 30, 60, 120, and 240 minutes after injection. RESULTS: For all 4 doses, serum cortisol concentration was significantly increased, compared with the baseline value, by 30 minutes after administration of cosyntropin; no significant differences were detected among maximum serum cortisol concentrations obtained in response to administration of doses of 0.1, 0.25, and 0.5 µg/kg. Serum cortisol concentration peaked 30 minutes after administration of cosyntropin at a dose of 0.02 or 0.1 µg/kg, with peak concentrations 1.5 and 1.9 times, respectively, the baseline concentration. Serum cortisol concentration peaked 90 minutes after administration of cosyntropin at a dose of 0.25 or 0.5 µg/kg, with peak concentrations 2.0 and 2.3 times, respectively, the baseline concentration. Cosyntropin administration significantly affected WBC, neutrophil, and eosinophil counts and the neutrophil-to-lymphocyte ratio. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggested that in healthy horses, administration of cosyntropin at a dose of 0.1 µg/kg resulted in maximum adrenal stimulation, with peak cortisol concentration 30 minutes after cosyntropin administration.

Acute inhibition of carboxypeptidase E expression in AtT-20 cells does not affect regulated secretion of ACTH.

Carboxypeptidase E (CPE) is an exopeptidase that removes C-terminal basic amino acids from a variety of bioactive peptides. In addition to this role, data obtained in recent years has supported a potential function for CPE as a sorting receptor, helping direct peptides destined for regulated secretion from the trans-Golgi to granules in preparation for release. This possible sorting function was assessed using mouse AtT-20 cells, a well-established corticotroph cell line that synthesizes and releases POMC/ACTH in regulated fashion. Cells that were treated with siRNA to Cpe effectively suppressed CPE expression. ACTH was released in a regulated fashion from CPE-depleted cells in response to two secretagogues, 8-bromo-cyclic AMP and corticotrophin-releasing hormone. POMC/ACTH content of CPE-depleted cells was higher than that of control cells, but both released a similar percentage of ACTH content in response to secretagogue addition. Cells depleted of CPE generally secreted more high-molecular weight forms of POMC/ACTH under basal conditions than control cells; however, the CPE-depleted cells responded to a secretagogue by releasing newly synthesized ACTH 1-39 in a manner similar to controls. These results, whereby RNAi was used to acutely suppress CPE, do not support a role for this protein as necessary for or central to sorting of POMC/ACTH to the regulated secretory pathway in AtT-20 cells.

Effect of glucocorticoid administration on serum aldosterone concentration in clinically normal dogs.

OBJECTIVE: To evaluate the effects of oral administration of anti-inflammatory dosages of prednisone for 28 days on serum aldosterone, cortisol, and electrolyte concentrations in clinically normal dogs. ANIMALS: 10 dogs. PROCEDURES: On days 1 through 28, 5 dogs received prednisone (0.55 mg/kg, PO, q 12 h) and 5 dogs received similar treatments with a placebo (empty capsules). Serum cortisol and aldosterone concentrations before and after ACTH stimulation testing and serum electrolyte concentrations were measured before (day 0 [baseline]), during (days 7, 14, 21, and 28), and after (days 35 and 42) treatment. RESULTS: At baseline, variables did not differ between the 2 groups. Serum cortisol concentrations before and after ACTH stimulation testing did not change from baseline values in placebo-treated dogs. In prednisone-treated dogs, serum chloride and corrected chloride concentrations were significantly lower on days 7, 14, 21, and 28 and serum bicarbonate concentrations were significantly higher on days 14, 21, and 28, compared with baseline values. Serum cortisol concentrations before and after ACTH stimulation testing were significantly lower than baseline values during prednisone treatment. Serum aldosterone concentration after ACTH stimulation testing was significantly lower than baseline on day 35 (ie, 1 week after discontinuation of prednisone treatment) but returned to baseline by day 42 in prednisone-treated dogs. CONCLUSIONS AND CLINICAL RELEVANCE: Administration of anti-inflammatory dosages of prednisone caused significant changes in serum chloride, bicarbonate, and cortisol concentrations in clinically normal dogs. Although ACTH-stimulated serum aldosterone concentrations were unchanged from baseline during glucocorticoid administration, values decreased after treatment cessation but quickly returned to baseline values.

Prenylated Rab acceptor domain family member 1 is involved in stimulated ACTH secretion and inhibition.

Dexamethasone (Dex) inhibits stimulated adrenocorticotrophic hormone (ACTH) secretion in AtT-20 cells, a mouse corticotroph tumor cell line. Dexras1 protein expression is induced in corticotrophs by Dex. The function of Dexras1 is unknown; however, it may be involved in corticotrophic negative feedback. Here we report the identification of a Dexras1 interactor, prenylated Rab acceptor domain family member 1 (PRAF1), a protein that localizes to the Golgi complex, post-Golgi vesicles, and endosomes. We determined that amino acids 54-175 of PRAF1 are essential for interaction with Dexras1 and that specific point mutations located within this region enhance PRAF1-Dexras1 interactions. AtT-20 cells stably transfected with truncated or mutated PRAF1 constructs had altered responses to corticotrophin-releasing hormone and Dex, upregulated expression of the ACTH prohormone pro-opiomelanocortin (POMC), altered POMC processing, and altered Golgi complex morphology with decreased intra-Golgi and intracellular co-localization of PRAF1 and ACTH proteins. Our findings indicate that PRAF1 plays a novel role in ACTH stimulated secretion. We propose a model whereby Dexras1 interaction with PRAF1 may lock the sites necessary for PRAF1-Rab3A-VAMP2 interaction resulting in Dex-mediated inhibition of ACTH secretion.