Dual in vitro effects of cortisol on cell turnover in the medaka esophagus via the glucocorticoid receptor

AimsCortisol is a glucocorticoid in mammals, but has both gluco- and mineralocorticoid activities in teleost fish. Our previous in vivo studies on osmoregulatory esophagi of euryhaline fish showed that epithelial apoptosis for the simple epithelium in seawater and cell proliferation for the stratified epithelium in fresh water are both induced by cortisol. The aim of the present study was to examine the mechanism of these dual cortisol effects on esophageal cell turnover.Main methodsWe developed a tissue culture method for the esophagus from euryhaline medaka (Oryzias latipes) and assessed cell proliferation and apoptosis in vitro in response to cortisol and 11-deoxycorticosterone (DOC), a recently identified agonist of the teleostean mineralocorticoid receptor.Key findingsEpithelial apoptosis, a well-established glucocorticoid function, was stimulated by treatment of the esophagus culture with 10 nM cortisol for 8 days, but no effects were seen at higher doses (100 and 1000 nM). In contrast, cell proliferation was induced by 1000 nM cortisol treatment for 8 days and this response was dose-dependent. Both effects were blocked by RU-486, a glucocorticoid receptor antagonist. DOC showed no significant effects at 10–1000 nM.SignificanceIn the esophageal epithelium in euryhaline fish, cortisol induces either apoptosis or cell proliferation via the glucocorticoid receptor, depending on the cortisol concentration. The glucocorticoid signaling may play a more important role than mineralocorticoid signaling in differentiation of the osmoregulatory esophagus in euryhaline fishes.     

Neuropeptide Y, an orexigenic hormone, regulates phagocytic activity of lizard splenic phagocytes

Present in vitro study in the wall lizard Hemidactylus flaviviridis, for the first time in ectothermic vertebrates, demonstrated the immunoregulatory role of neuropeptide Y (NPY) and its receptor-coupled downstream signaling cascade. NPY inhibited the percentage phagocytosis and phagocytic index of splenic phagocytes. The inhibitory effect of NPY on phagocytosis was completely antagonized by Y₂ and Y₅ receptor antagonists. This suggests that NPY mediated its effect on phagocytosis through Y₂ and Y₅ receptors. Further, NPY receptor-coupled downstream signaling cascade for NPY effect on phagocytosis was explored using the inhibitors of adenylate cyclase (SQ 22536) and protein kinase A (H-89). The SQ 22536/H-89 in a concentration-related manner decreased the inhibitory effect of NPY on phagocytosis. Further, an increase in intracellular cAMP level was observed in response to NPY. Taken together, it can be concluded that NPY via Y₂ and Y₅ receptor-coupled AC-cAMP-PKA pathway downregulated the phagocytic activity of lizard splenic phagocytes.     

Non-invasive administration of 17β-estradiol rapidly increases aggressive behavior in non-breeding,but not breeding,male song sparrows

17β-Estradiol (E₂) acts in the brain via genomic and non-genomic mechanisms to influence physiology and behavior. There is seasonal plasticity in the mechanisms by which E₂ activates aggression, and non-genomic mechanisms appear to predominate during the non-breeding season. Male song sparrows (Melospiza melodia) display E₂-dependent territorial aggression throughout the year. Field studies show that song sparrow aggression during a territorial intrusion is similar in the non-breeding and breeding seasons, but aggression after an intrusion ends differs seasonally. Non-breeding males stop behaving aggressively within minutes whereas breeding males remain aggressive for hours. We hypothesize that this seasonal plasticity in the persistence of aggression relates to seasonal plasticity in E₂ signaling. We used a non-invasive route of E₂ administration to compare the non-genomic (within 20 min) effects of E₂ on aggressive behavior in captive non-breeding and breeding season males. E₂ rapidly increased barrier contacts (attacks) during an intrusion by 173% in non-breeding season males only. Given that these effects were observed within 20 min of E₂ administration, they likely occurred via a non-genomic mechanism of action. The present data, taken together with past work, suggest that environmental cues associated with the non-breeding season influence the molecular mechanisms through which E₂ influences behavior. In song sparrows, transient expression of aggressive behavior during the non-breeding season is highly adaptive: it minimizes energy expenditure and maximizes the amount of time available for foraging. In all, these data suggest the intriguing possibility that aggression in the non-breeding season may be activated by a non-genomic E₂ mechanism due to the fitness benefits associated with rapid and transient expression of aggression.     

Rapid stimulatory effect of thyroxine on plasma membrane transport systems: Calcium uptake and neutral amino acid accumulation in immature rat testis

Although the biological effects of thyroid hormones are mediated by nuclear receptors (genomic mechanisms), interactions with receptors associated with the plasma membrane (non-genomic mechanisms) of target cells are not clear. In this study we investigated the rapid stimulatory effect of thyroxine (T₄) on ⁴⁵Ca²⁺ uptake as well as ionic currents and intracellular messengers involved in the stimulatory action of T₄ in amino acid accumulation in immature rat testes. Results indicated that 10^?9 M or 10^?6 M T₄ was able to increase immediately ⁴⁵Ca²⁺ uptake after 60 s of hormone exposure. These results indicate for the first time that voltage-dependent Ca²⁺ channels and ATP-dependent K⁺ channels can be seen as a set-point in the stimulatory effect of T₄ on amino acid accumulation. Apamin-sensitive small-conductance Ca²⁺-activated K⁺ channels (SK_Ca) and chloride channels were shown to be partially involved in this mechanism. The amino acid accumulation triggered by the PKC pathway suggests a functional link between different ion channel activities and the stimulatory effect of T₄ on amino acid accumulation. In conclusion, we show in this study a rapid and stimulatory effect of T₄ on calcium uptake and on amino acid accumulation, both events initiated at the plasma membrane, which strongly characterizes a non-genomic effect of T₄ in immature rat testes.     

Effect of resistance exercise on muscle steroid receptor protein content in strength-trained men and women

The purpose of this study was to examine the acute effect of resistance exercise (RE) on muscle androgen receptor (AR) and glucocorticoid receptor (GR) protein content. Fifteen resistance-trained men (n = 8; 21 ± 1 years, 175.3 ± 6.7 cm, 90.8 ± 11.6 kg) and women (n = 7; 24 ± 5 years, 164.6 ± 6.7 cm, 76.4 ± 15.6 kg) completed 6 sets of 10 repetitions of heavy squats. Blood samples were obtained before RE, after 3 and 6 sets of squats, and 5, 15, 30 and 70 min after RE. Muscle biopsies from the vastus lateralis were obtained before RE, and 10 min and 70 min after RE. Blood samples were analyzed for total and free testosterone concentrations and muscle samples were analyzed for AR and GR protein content. Circulating total testosterone increased significantly (p ≤ 0.05) in men and free testosterone increased in men and women with exercise. AR was significantly reduced at 70 min post-exercise in men and at 10 min post-exercise in women compared to pre-exercise. There were no changes in GR following RE, but GR was significantly higher in women compared to men. These findings support a current paradigm for stabilization followed by a reduction and then a rebound in the acute AR response to RE but demonstrate that gender differences exist in the timeline of the AR response.     

Spleen immune status is affected after acute handling stress but not regulated by cortisol in Eurasian perch,Perca fluviatilis

The effects of acute stress on immune status and its regulation by cortisol/corticosteroid receptors have received little attention in percids. To address that question, we investigated the physiological and immune responses of Eurasian perch, Perca fluviatilis to acute stress. We exposed immature perch to an 1-min exondation and measured at 1 h, 6 h, 24 h and 72 h post-stress: (1) stress-related parameters including plasma cortisol and glucose levels, (2) immune parameters in the plasma and in the spleen (complement, respiratory burst and lysozyme activity, total immunoglobulins; gene expression of lysozyme, complement unit 3, apolipoprotein A1 and 14 kDa, hepcidin and chemotaxin) (3) the corticosteroid receptors gene expression in the spleen after having cloned them. In addition, the in vitro effects of cortisol on the spleen immune parameters were also investigated.Plasma cortisol and glucose levels increased markedly 1 h post-stress and returned at basal levels after 24 h. P. fluviatilis mineralocorticoid receptor, but not glucocorticoid receptors, was significantly up-regulated both in vivo after the stress and in vitro by cortisol at a physiological concentration (100 ng/ml). The plasma immune parameters were not significantly affected by the stress. In contrast, spleno-somatic index, spleen lysozyme activity, lysozyme and hepcidin gene expression were depleted and total immunoglobulins increased along the whole time-course (1–72 h). But, these immune parameters were not regulated in vitro by cortisol at physiological or supra-physiological doses.Our results indicate that handling stress may affect spleen antibacterial defences without clear effects on circulating immune compounds and that the elevation of plasma cortisol after handling stress may not be related to the regulation of this splenic response.     

Glucocorticoid receptors on and in a unicellular organism,Cryptobia salmositica

This is the first report to our knowledge that demonstrates a functional steroid hormone receptor in a protozoon. The study used Cryptobia salmositica, a pathogenic haemoflagellate found in salmonid fishes. It has been previously shown that cortisol and dexamethasone (a synthetic glucocorticoid) enhanced the multiplication of C. salmositica under in vitro conditions indicating the presence of glucocorticoid receptors on/in the parasite. Also, the glucocorticoid receptor antagonist, mifepristone (RU486), inhibited the stimulatory effect of the two glucocorticoids on parasite multiplication. In the present study, we used an antibody (produced in a rabbit against glucocorticoid receptor protein) agglutination test and confocal microscopy with immunohistofluorescence staining to demonstrate cortisol-glucocorticoid receptor-like protein receptors on the plasma membrane and in the cytoplasm of the parasite. In two in vitro studies, the addition of 50 ng ml⁻¹ of RU486 was more effective in inhibiting parasite replication in cultures with 7,000 parasites ml⁻¹ than in cultures with 14,000 parasites ml⁻¹. Also, 100 ng ml⁻¹ of RU486/ml was more effective than 50 ng ml⁻¹ in inhibiting parasite multiplication in the 14,000 parasites ml^-1 cultures. These in vitro studies indicate that the number of binding sites on/in the parasite is finite. The findings may be important in future studies especially on steroid receptor signalling pathways and dissection of ligand–receptor interactions, and for evaluating the adaptations that develop in pathogens as part of the host–parasite interaction.     

Opioid and non-opioid receptor-mediated immunoregulatory role of Leucine-enkephalin in teleost Channa punctatus

Leucine-enkephalin (Leu-enk) is an endogenous opioid peptide and highly conserved throughout the vertebrates. Despite its conserved nature, the immunoregulatory property of Leu-enk is explored only in mammals. The present study describes the immunomodulatory role of Leu-enk in a lower vertebrate, spotted murrel Channa punctatus. Leu-enk increased the percentage phagocytosis and phagocytic index, though its stimulatory effect on phagocytosis markedly decreased at concentrations higher than 10^?9 M. Moreover, it had bell-shaped stimulatory effect also on the superoxide production by phagocytes. On the other hand, Leu-enk showed bimodal effects on nitrite release. The lower concentrations of Leu-enk produced inhibitory effect, while higher concentrations had stimulatory effect on nitrite release. Interestingly, the Leu-enk-induced increase in nitrite release was unaltered by non-selective opioid receptor antagonist though the same completely antagonized the inhibitory effect of Leu-enk on nitrite release and the stimulatory effect on phagocytosis and superoxide production. This suggests that the stimulatory effect of Leu-enk on nitrite production is mediated by the non-opioid receptor. Further, δ-opioid receptor was precisely seen involved in mediating the stimulatory effect of Leu-enk on phagocytosis and superoxide production, or inhibitory effect on nitrite release. It can be concluded that Leu-enk regulates the innate immune response of splenic phagocytes acting via both opioid and non-opioid receptor in the fish C. punctatus.     

Targeting the intersubunit cavity of Plasmodium falciparum glutathione reductase by a novel natural inhibitor: Computational and experimental evidence

Glutathione reductase (GR), a homodimeric FAD-dependent disulfide reductase, is essential for redox homeostasis of the malaria parasite Plasmodium falciparum and has been proposed as an antimalarial drug target. In this study we performed a virtual screening against PfGR, using the structures of about 170,000 natural compounds. Analysis of the two top-scoring molecules, TTB and EPB, indicated that these ligands are likely to interact with the homodimer intersubunit cavity of PfGR with high binding energy scores of −9.67 and −9.60 kcal/mol, respectively. Both compounds had a lower affinity for human GR due to differences in structure and electrostatic properties. In order to assess the putative interactions in motion, molecular dynamics simulations were carried out for 30 ns, resulting in TTB being more dynamically and structurally favored than EPB. A closely related compound MDPI 21618 was tested on recombinant PfGR and hGR, resulting in IC₅₀ values of 11.3 ± 2.5 μM and 10.2 ± 1.7 μM, respectively. Kinetic characterization of MDPI 21618 on PfGR revealed a mixed-type inhibition with respect to glutathione disulfide (K_i = 9.7 ± 2.3 μM) and an uncompetitive inhibition with respect to NADPH. Furthermore, MDPI 21618 was found to inhibit the growth of the chloroquine-sensitive P. falciparum strain 3D7 with an IC₅₀ of 3.2 ± 1.9 μM and the chloroquine-resistant Dd2 strain with an IC₅₀ of 3.2 + 1.6 μM. In drug combination assays with chloroquine, artemisinin, or mefloquine MDPI 21618 showed an antagonistic action, which might suggest partially overlapping routes of action. This study further substantiates research on PfGR as a potential antimalarial drug target.     

Action of ANP on the nongenomic dose-dependent biphasic effect of aldosterone on NHE1 in proximal S3 segment

The rapid (2 min) nongenomic effects of aldosterone (ALDO) and/or spironolactone (MR antagonist), RU 486 (GR antagonist), atrial natriuretic peptide (ANP) and dimethyl-BAPTA (BAPTA) on the intracellular pH recovery rate (pHirr) via NHE1 (basolateral Na⁺/H⁺ exchanger isoform), after the acid load induced by NH₄Cl, and on the cytosolic free calcium concentration ([Ca²⁺]_i) were investigated in the proximal S3 segment isolated from rats, by the probes BCECF-AM and FLUO-4-AM, respectively. The basal pHi was 7.15 ± 0.008 and the basal pHirr was 0.195 ± 0.012 pH units/min (number of tubules/number of tubular areas = 16/96). Our results confirmed the rapid biphasic effect of ALDO on NHE1: ALDO (10^?12 M) increases the pHirr to approximately 59% of control value, and ALDO (10^?6 M) decreases it to approximately 49%. Spironolactone did not change these effects, but RU 486 inhibited the stimulatory effect and maintained the inhibitory effect. ANP (10^?6 M) or BAPTA (5 × 10^?5 M) alone had no significant effect on NHE1 but prevented both effects of ALDO on this exchanger. The basal [Ca²⁺]_i was 104 ± 3 nM (15), and ALDO (10^?12 or 10^?6 M) increased the basal [Ca²⁺]_i to approximately 50% or 124%, respectively. RU 486, ANP and BAPTA decreased the [Ca²⁺]_i and inhibited the stimulatory effect of both doses of ALDO. The results suggest the involvement of GR on the nongenomic effects of ALDO and indicate a pHirr-regulating role for [Ca²⁺]_i that is mediated by NHE1, stimulated/impaired by ALDO, and affected by ANP or BAPTA with ALDO. The observed nongenomic hormonal interaction in the S3 segment may represent a rapid and physiologically relevant regulatory mechanism in the intact animal under conditions of volume alterations.     

Hair cortisol determination in sows in two consecutive reproductive cycles

Hair analysis has been proposed as a minimally invasive technique capable of furnishing information regarding the stress response during medium- and long-term periods. Bristle samples were collected from the rump region of sows at three key physiological phases (before delivery – BD; weaning time – WT; pregnancy diagnosis – PD) during consecutive reproductive cycles in order to test swine hair as a reliable matrix of cortisol evaluation. Cortisol was extracted from the bristles and assayed using radioimmunoassay. The highest mean hair cortisol concentrations were demonstrated (p < 0.001) at the PD time points (20.1 ± .95 and 16.29 ± 2.15 pg/mg). Moreover, cortisol was significantly higher (p < 0.001) at BD2 (10.48 ± 0.96 pg/mg) as compared to BD1 (5.17 ± 0.51 pg/mg) and WT1 (6.01 ± 0.47 pg/mg). The various physiological phases had a significant effect on cortisol concentration (p < 0.00001) with a higher cortisol concentration found during late pregnancy and lactation than in early-mid pregnancy. This could be due not only to the physiological hormonal status, but also to the different housing conditions (single crates vs. group housing). The season of the year was also observed to have an effect (p < 0.005), with the lowest cortisol concentration recorded during the hot season.     

Possibility of limiting the un-justified irradiation in 131I therapy of Graves' disease: A thyroid mass-reduction based method for the optimum activity calculation

ObjectiveIn Graves' disease therapy, the amount of ¹³¹I is usually decided following two different modalities: the administration of a fixed activity or of an activity individually calculated based on a fixed value of target absorbed dose. Although the effectiveness of each of these approaches is good (about 80% of patients cured), the ALARA principle must be applied avoiding the un-justified radioactivity to the patient himself, the people living/working near him and the environment. In this paper a new approach to the ¹³¹I therapy in Graves' disease, based on the optimum value of the final thyroid mass, is presented.Design97 Graves' disease patients (29 males) were randomly assigned into three groups (GR1, GR2, GR3). In two of them (GR1, GR3) the radioiodine administering activity was calculated based on two fixed thyroid absorbed dose values (100 Gy for GR1; 400 Gy for GR3), in GR2 it was calculated based on the desired final optimum thyroid mass value m_f = 0.24 m₀/U₀ResultsThe rate of cured patients are 48% (GR1), 97% (GR2) (z-test, p < 0.001) and 97% (GR3). The average activity administered to GR2 (393 ± 157 MBq) is lower than that administrered to GR3 patients (524 ± 201 MBq) (p = 0.007, two-tails unpaired t-test); the thyroid absorbed dose in GR2 (262 ± 78 Gy) is lower than in GR3 patients (407 ± 23 Gy) (p < 0.001, two-tails unpaired t-test).ConclusionOur results demonstrate that the thyroid-mass based approach optimizes the treatment avoiding an un-justified excess or a not-effective too low activity without time and resources consuming.     

Structure activity relationship modelling of milk protein-derived peptides with dipeptidyl peptidase IV (DPP-IV) inhibitory activity

Quantitative structure activity type models were developed in an attempt to predict the key features of peptide sequences having dipeptidyl peptidase IV (DPP-IV) inhibitory activity. The models were then employed to help predict the potential of peptides, which are currently reported in the literature to be present in the intestinal tract of humans following milk/dairy product ingestion, to act as inhibitors of DPP-IV. Two models (z- and v-scale) for short (2–5 amino acid residues) bovine milk peptides, behaving as competitive inhibitors of DPP-IV, were developed. The z- and the v-scale models (p < 0.05, R² of 0.829 and 0.815, respectively) were then applied to 56 milk protein-derived peptides previously reported in the literature to be found in the intestinal tract of humans which possessed a structural feature of DPP-IV inhibitory peptides (P at the N₂ position). Ten of these peptides were synthetized and tested for their in vitro DPP-IV inhibitory properties. There was no agreement between the predicted and experimentally determined DPP-IV half maximal inhibitory concentrations (IC₅₀) for the competitive peptide inhibitors. However, the ranking for DPP-IV inhibitory potency of the competitive peptide inhibitors was conserved. Furthermore, potent in vitro DPP-IV inhibitory activity was observed with two peptides, LPVPQ (IC₅₀ = 43.8 ± 8.8 μM) and IPM (IC₅₀ = 69.5 ± 8.7 μM). Peptides present within the gastrointestinal tract of human may have promise for the development of natural DPP-IV inhibitors for the management of serum glucose.     

Molecular docking studies and synthesis of novel bisbenzimidazole derivatives as inhibitors of α-glucosidase

A series of bisbenzimidazole derivatives starting from o-phenylenediamine and 4-nitro-o-phenylenediamine were prepared with oxalic acid. Most of the reactions were conducted using both the microwave and conventional methods to compare yields and reaction times. The operational simplicity, environmental friendly conditions and high yield in a significantly short reaction time were the major benefits. All substances’ inhibitory activities against α-glucosidase were evaluated. The results may suggest a significant role for the nature of bisbenzimidazole compounds in their inhibitory action against α-glucosidase. They showed different range of α-glucosidase inhibitory potential with IC₅₀ value ranging between 0.44 ± 0.04 and 6.69 ± 0.01 μM when compared to the standard acarbose (IC₅₀, 13.34 ± 1.26 μM). This has described a new class of α-glucosidase inhibitors. Molecular docking studies were done for all compounds to identify important binding modes responsible for inhibition activity of α-glucosidase.     

Effect of flavonoids on androgen and glucocorticoid receptors based on in vitro reporter gene assay

The effect of 32 flavonoids on androgen (AR) and glucocorticoid receptors (GR) was investigated using an MDA-kb2 human breast cancer cell line to predict potential AR and GR activities. Among them, 5-hydroxyflavone (7) had the highest AR antagonistic activity with an IC₅₀ value of 0.3 μM, whereas 6-methoxyflavone (11) had the highest induced luciferase activity with an EC₁₅₀ value of 0.7 μM. Genistein (2) and daizein (1) showed a sufficient increase of luciferase activities as their concentrations increased with EC₁₅₀ values of 4.4 and 10.1 μM, respectively. These findings provide evidence of a fundamental property of their structure–activity relationship with AR and/or GR.     

Gender Determines Serum Free Cortisol: Higher Levels in Men

Objective: Because only the free fraction of serum cortisol can readily access glucocorticoid receptors, we investigated whether or not a gender-related difference in serum free cortisol (FC) exists in the basal and adrenocorticotropic hormone (ACTH)-stimulated state.Methods: Serum total cortisol (TC) and FC were measured in 323 subjects (175 men; 148 women). Additionally, the low-dose 1-μg ACTH test was performed in 56 subjects (30 women, 26 men). Subjects were healthy volunteers, recruited in a preventive medicine screening program and an outpatient clinic.Results: Overall, basal serum TC and FC level were ~18 and ~33%, respectively, higher in men than in women (TC, 14.5 ± 0.33 μg/dL vs. 12.3 ± 0.33 μg/dL; P<.0001; FC, 0.68 ± 0.02 μg/dL vs. 0.51 ± 0.02 μg/dL; P<.0001). The higher FC in men relative to women was apparent across a wide age range (17 to 86 years) and persisted after adjustment for age and body mass index. The FC fraction (%FC, out of TC) was concordantly higher in men (5.4 ± 0.09% vs. 4.8 ± 0.3%; P = .046). FC was not related to the estimated menopausal status (women age below and above 47, 50, or 53 years). ACTH-stimulated FC levels were significantly higher in men compared to women, as reflected by the area under the response curve (49.4 ± 3.4 μg × min vs. 39.6 ± 2.2 μg × min; P = .0014).Conclusion: Gender is an unrecognized determinant of serum FC in humans. The possibility of lifelong exposure to the higher bioactive fraction of cortisol under basal conditions or daily stress involving ACTH stimulation should be further investigated in the context of gender-related phenotypic features such as “android” (visceral) fat deposition and longevity.Abbreviations:ACTH = adrenocorticotropic hormoneBMI = body mass indexCBG = cortisol-binding globulinFC = free cortisolHPA = hypothalamic-pituitary-adrenalTC = total cortisol     

Synthesis and investigation of dihydroxychalcones as calpain and cathepsin inhibitors

In order to identify potential calpain and cathepsin inhibitors we prepared 12 dihydroxychalcone analogues and tested their ability to inhibit μ-calpain, m-calpain, cathepsins B and L. In the calpain inhibition test, compound 10 exhibited the most active inhibitory activity against m-calpain with an IC₅₀ value of 25.25 ± 0.901 μM. With respect to inhibition of cathepsins B and L, compound 13 exhibited the most potent inhibitory activity on cathepsin L and moderate inhibitory activity on cathepsin B with IC₅₀ values of 2.80 ± 0.100 and 11.47 ± 0.087 μM, respectively. Our results suggest the possibility of developing dual calpain and cathepsin inhibitors by properly modulating structures and/or combining the essential aspects of the functional group effective for specific calpain and cathepsin inhibition.