GH3 Cells Research Articles

Salts of short-chain fatty acids increase the activity of the large conductance, Ca<sup>2+</sup>-activated K<sup>+</sup> channels and reduce calcium oscillations in rat GH3 cells

The short-chain fatty acids such as acetic, propionic and butyric acids are microbiota metabolites that can exert a series of physiological effects both in the intestine and other organs, including the central nervous system. The present work aimed to examine the effects of sodium acetate, propionate, and butyrate on the activity of large conductance Ca2+ activated K+ channels and calcium oscillations in rat pituitary GH3 cells. It has been shown that fatty acids under study cause a dose-dependent increase in the amplitude of total outward potassium currents and these effects are prevented by tetraethylammonium, a Ca2+ activated K+ channel blocker, indicating the involvement of Ca2+ activated K+ channels in the effects of fatty acids. It is worthy of note that fatty acids increased open probability of single channels with no changes in the amplitude and the mean channel open time. In addition, fatty acids were associated with a significant reduction in the amplitude and frequency of Ca2+ oscillations in GH3 cells. An increase in potassium conductance and a decrease in the intracellular Ca2+ level can mediate the effects of short-chain fatty acids in various excitable structures, such as a relaxation of intestinal and vascular smooth muscle cells, hyperpolarization of neurons, and the regulation of hormone and neurotransmitter release.

Molnupiravir, a ribonucleoside antiviral prodrug against SARS-CoV-2, alters the voltage-gated sodium current and causes adverse events

Molnupiravir (MOL) is a ribonucleoside prodrug for oral treatment of COVID-19. Common adverse effects of MOL are headache, diarrhea, and nausea, which may be associated with altered sodium channel function. Here, we investigated the effect of MOL on voltage-gated Na+ current (INa) in pituitary GH3 cells. We show that MOL had distinct effects on transient and late INa, in combination with decreased time constant in the slow component of INa inactivation. The 50% inhibitory concentration (IC50) values of MOL for suppressing transient and late INa were 26.1 and 6.3 μM, respectively. The overall steady-state current-voltage relationship of INa remained unchanged upon MOL exposure. MOL-induced alteration of INa may lead to changes in physiological function through sodium channels. Apart from its effect on inhibiting RNA virus replication, MOL exerts inhibitory effects on plasmalemma INa, which might constitute an additional yet crucial underlying mechanism of its pharmacological activity or adverse events.

Salts of Short-Chain Fatty Acids Increase the Activity of Large Conductance Ca2+-Activated K+ Channels and Decrease Calcium Oscillations in Rat GH3 Cells

Salts of Short-Chain Fatty Acids Increase the Activity of Large Conductance Ca2+-Activated K+ Channels and Decrease Calcium Oscillations in Rat GH3 Cells

Bisphenol analogues induce thyroid dysfunction via the disruption of the thyroid hormone synthesis pathway

Bisphenol analogues are widely used in industrial and daily-use consumer products having imperfect thyroid hormones (THs) structures. Widespread exposure interferes with thyroid-related health outcomes in human. The mechanisms of disruption on TH synthesis and subsequent thyroid dysfunction by different bisphenol analogues remain unclear. Here, we evaluated bisphenol-induced thyroid endocrine disruption in C57BL/6 mice at doses of 0.002, 0.02, 2, and 20 mg/kg body weight/day (BW/d) for five consecutive weeks. Administration of 20 mg/kg BW/d bisphenol S (BPS) and 2 mg/kg BW/d tetrabromobisphenol S (TBBPS) significantly increased serum thyrotropin (TSH) levels to 1.21-fold and 1.20-fold of control group, respectively, indicating that bisphenols induced thyroid dysfunction in mice. Height of the thyroid follicle epithelium significantly increased to 1.27-, 1.24-, 1.26-, and 1.36-fold compared to control group with BPA, BPS, TBBPA, and TBBPS at 20 mg/kg BW/d, respectively, indicating impairment of the thyroid gland structure, and TBBPS showed potent effect. Exposure to bisphenol analogues of 0.02 mg/kg BW/d downregulated the protein expression levels of thyrotropin receptor, the sodium/iodide symporter, thyroperoxidase. The TH-dependent effects were further determined using the T-Screen assay at 10−11 M to 10−5 M concentrations. Bisphenol analogues significantly decreased TH-dependent GH3 cell proliferation, indicating the antagonistic activity of bisphenol analogues. The gene responsible for THs synthesis of thyrotropin releasing hormone receptor and TSH were upregulated, but downregulation of thyroid receptor β was observed. Our results suggest that bisphenol analogues distinctly induce thyroid dysfunction via TH synthesis, implying adverse effect of bisphenol analogues on TH homeostasis and subsequent physiological processes.

Estradiol and progesterone regulate NUCB2/nesfatin-1 expression and function in GH3 pituitary cells and THESC endometrial cells

ABSTRACT Estradiol (E2) and progesterone (P4) are essential sex steroid hormones that play critical roles in the pituitary gland and uterus. Recently, nesfatin-1, a polypeptide hormone that regulates appetite and energy homeostasis in the hypothalamus, was found to be expressed in the pituitary gland and uterus. In this study, we aimed to investigate the relationship between these two steroid hormones and the expression and function of nesfatin-1 in the pituitary gland and uterus using GH3 cells, a lacto-somatotroph cell line, and THESC cells, an endometrial stromal cell line. First, we verified the presence of nesfatin-1 and nesfatin-1 binding sites in GH3 and THESC cells. E2 increased the mRNA expression of NUCB2, the gene encoding the nesfatin-1 protein, in GH3 cells, while P4 had no significant effect. In THESC cells, NUCB2 mRNA expression was decreased by E2 but increased by P4. In addition, nesfatin-1 significantly increased growth hormone (GH) and prolactin (PRL) mRNA expression in GH3 cells, and E2 enhanced this effect. In THESC cells, nesfatin-1 significantly increased the mRNA expression of insulin-like growth factor binding protein 1 (IGFBP1) and PRL, which are decidualization marker genes, and P4 further enhanced this effect. These results suggest that nesfatin-1 may act as a local regulator of GH and PRL production in the pituitary gland and decidualization in the uterus, modulating its effects in response to E2 and P4.

Clinical and Proteomic-Based Molecular Characterizations of Invasive and Noninvasive Somatotroph PitNETs

Introduction: Somatotroph pituitary neuroendocrine tumours (PitNETs) are characterized by complex and variable biological behaviours with unpredictable patterns of growth and invasiveness. The molecular mechanisms and reliable predictors of biological markers of invasiveness remain unknown. Methods: Seventy-two acromegaly patients were consecutively enrolled. Data-independent acquisition-based proteomics and ingenuity pathway analysis were conducted between invasive and noninvasive somatotroph PitNETs. The expression of selected biomarkers was verified in PitNET tissue, and its correlation with various clinical indicators and outcomes of these tumours was assessed. The invasive phenotypes of GH3 cells were validated in vitro. Results: Patients with invasive somatotroph PitNETs were significantly younger at onset and diagnosis, with significantly higher secretion and faster growth and a lower long-term biochemical response rate than patients with noninvasive somatotroph PitNETs. Proteomic data were evaluated in a consecutively collected sample of 19 (10 invasive and 9 noninvasive somatotroph PitNETs) tumours and indicated a distinct proteomic pattern. The enriched and important pathways included IL-4, PDGF, PTEN, VEGF, PI3K/AKT, FAK, and other pathways that were significantly associated with tumour proliferation, migration, and invasion. High cathepsin Z (CTSZ) expression was found in invasive somatotroph PitNETs and significantly positively correlated with parameters of tumour invasion and growth. In Ctsz-overexpressing GH3 cells, cell proliferation, invasion, and migration were consequently increased. Conclusion: It is more difficult for patients with invasive somatotroph PitNETs to achieve remission than those with noninvasive somatotroph PitNETs, and proteomic data analysis has revealed the high expression of CTSZ as a contributing factor to invasive transformation and poor prognosis in somatotroph PitNETs for the first time.

The Methylation Analysis of the Glucose-Dependent Insulinotropic Polypeptide Receptor (GIPR) Locus in GH-Secreting Pituitary Adenomas.

The glucose-dependent insulinotropic polypeptide receptor (GIPR) is aberrantly expressed in about one-third of GH-secreting pituitary adenomas (GH-PAs) and has been associated with a paradoxical increase of GH after a glucose load. The reason for such an overexpression has not yet been clarified. In this work, we aimed to evaluate whether locus-specific changes in DNA methylation patterns could contribute to this phenomenon. By cloning bisulfite-sequencing PCR, we compared the methylation pattern of the GIPR locus in GIPR-positive (GIPR+) and GIPR-negative (GIPR-) GH-PAs. Then, to assess the correlation between Gipr expression and locus methylation, we induced global DNA methylation changes by treating the lactosomatotroph GH3 cells with 5-aza-2'-deoxycytidine. Differences in methylation levels were observed between GIPR+ and GIPR- GH-PAs, both within the promoter (31.9% vs. 68.2%, p < 0.05) and at two gene body regions (GB_1 20.7% vs. 9.1%; GB_2 51.2% vs. 65.8%, p < 0.05). GH3 cells treated with 5-aza-2'-deoxycytidine showed a ~75% reduction in Gipr steady-state level, possibly associated with the observed decrease in CpGs methylation. These results indicate that epigenetic regulation affects GIPR expression in GH-PAs, even though this possibly represents only a part of a much more complex regulatory mechanism.

Concerted suppressive effects of carisbamate, an anti-epileptic alkyl-carbamate drug, on voltage-gated Na+ and hyperpolarization-activated cation currents.

Carisbamate (CRS, RWJ-333369) is a new anti-seizure medication. It remains unclear whether and how CRS can perturb the magnitude and/or gating kinetics of membrane ionic currents, despite a few reports demonstrating its ability to suppress voltage-gated Na+ currents. In this study, we observed a set of whole-cell current recordings and found that CRS effectively suppressed the voltage-gated Na+ (INa) and hyperpolarization-activated cation currents (Ih) intrinsically in electrically excitable cells (GH3 cells). The effective IC50 values of CRS for the differential suppression of transient (INa(T)) and late INa (INa(L)) were 56.4 and 11.4 μM, respectively. However, CRS strongly decreased the strength (i.e., Δarea) of the nonlinear window component of INa (INa(W)), which was activated by a short ascending ramp voltage (Vramp); the subsequent addition of deltamethrin (DLT, 10 μM) counteracted the ability of CRS (100 μM, continuous exposure) to suppress INa(W). CRS strikingly decreased the decay time constant of INa(T) evoked during pulse train stimulation; however, the addition of telmisartan (10 μM) effectively attenuated the CRS (30 μM, continuous exposure)-mediated decrease in the decay time constant of the current. During continued exposure to deltamethrin (10 μM), known to be a pyrethroid insecticide, the addition of CRS resulted in differential suppression of the amplitudes of INa(T) and INa(L). The amplitude of Ih activated by a 2-s membrane hyperpolarization was diminished by CRS in a concentration-dependent manner, with an IC50 value of 38 μM. For Ih, CRS altered the steady-state I-V relationship and attenuated the strength of voltage-dependent hysteresis (Hys(V)) activated by an inverted isosceles-triangular Vramp. Moreover, the addition of oxaliplatin effectively reversed the CRS-mediated suppression of Hys(V). The predicted docking interaction between CRS and with a model of the hyperpolarization-activated cyclic nucleotide-gated (HCN) channel or between CRS and the hNaV1.7 channel reflects the ability of CRS to bind to amino acid residues in HCN or hNaV1.7 channel via hydrogen bonds and hydrophobic interactions. These findings reveal the propensity of CRS to modify INa(T) and INa(L) differentially and to effectively suppress the magnitude of Ih. INa and Ih are thus potential targets of the actions of CRS in terms of modulating cellular excitability.

Characterization of Stimulatory Action on Voltage-Gated Na+ Currents Caused by Omecamtiv Mecarbil, Known to Be a Myosin Activator.

Omecamtiv mecarbil (OM, CK-1827452) is recognized as an activator of myosin and has been demonstrated to be beneficial for the treatment of systolic heart failure. However, the mechanisms by which this compound interacts with ionic currents in electrically excitable cells remain largely unknown. The objective of this study was to investigate the effects of OM on ionic currents in GH3 pituitary cells and Neuro-2a neuroblastoma cells. In GH3 cells, whole-cell current recordings showed that the addition of OM had different potencies in stimulating the transient (INa(T)) and late components (INa(L)) of the voltage-gated Na+ current (INa) with different potencies in GH3 cells. The EC50 value required to observe the stimulatory effect of this compound on INa(T) or INa(L) in GH3 cells was found to be 15.8 and 2.3 µM, respectively. Exposure to OM did not affect the current versus voltage relationship of INa(T). However, the steady-state inactivation curve of the current was observed to shift towards a depolarized potential of approximately 11 mV, with no changes in the slope factor of the curve. The addition of OM resulted in an increase in the decaying time constant during the cumulative inhibition of INa(T) in response to pulse-train depolarizing stimuli. Furthermore, the presence of OM led to a shortening of the recovery time constant in the slow inactivation of INa(T). Adding OM also resulted in an augmentation of the strength of the window Na+ current, which was evoked by a short ascending ramp voltage. However, the OM exposure had little to no effect on the magnitude of L-type Ca2+ currents in GH3 cells. On the other hand, the delayed-rectifier K+ currents in GH3 cells were observed to be mildly suppressed in its presence. Neuro-2a cells also showed a susceptibility to the differential stimulation of INa(T) or INa(L) upon the addition of OM. Molecular analysis revealed potential interactions between the OM molecule and hNaV1.7 channels. Overall, the direct stimulation of INa(T) and INa(L) by OM is assumed to not be mediated by an interaction with myosin, and this has potential implications for its pharmacological or therapeutic actions occurring in vivo.

The novel polyfluoroalkyl benzenesulfonate OBS exposure induces cell cycle arrest and senescence of rat pituitary cell GH3 via the p53/p21/RB pathway

Sodium p-perfluorous nonenoxybenzene sulfonate (OBS), an economical alternative to perfluorooctane sulfonate (PFOS) in multiple industrial fields, is widely detected in the environment. The toxicity of OBS has received increasing attention. Pituitary cells are components of the endocrine system and act as vital regulators of homeostatic endocrine balance. However, the effects of OBS on pituitary cells remain unknown. The present study explores the effects of OBS (0.5, 5, and 50 μM) on GH3 rat pituitary cells after treatment for 24, 48, and 72 h. We found that OBS significantly inhibited cell proliferation in GH3 cells with remarkable senescent phenotypes, including enhanced SA-β-gal activity and expression of senescence-associated secretory phenotype (SASP)-related genes, cell cycle arrest, and upregulation of the senescence-related proteins γ-H2A.X and Bcl-2. OBS caused significant cell cycle arrest of GH3 cells at the G1-phase and concomitantly downregulated the expression of some key proteins for the G1/S transition, including cyclin D1 and cyclin E1. Consistently, the phosphorylation of retinoblastoma (RB), which plays a central role in regulating the cell cycle, was prominently reduced after OBS exposure. Furthermore, OBS notably activated the p53-p21 signalling pathway in GH3 cells, as evidenced by increased p53 and p21 expressions, enhanced p53 phosphorylation, and augmented p53 nuclear import. To our knowledge, this study is the first to reveal that OBS triggers senescence in pituitary cells via the p53-p21-RB signalling pathway. Our study demonstrates a novel toxic effect of OBS in vitro, and provides new perspectives for understanding the potential toxicity of OBS.

Thyroid endocrine disruption induced by [C8mim]Br: An integrated in vivo, in vitro, and in silico study

Conventional thyroid-disrupting chemicals (TDCs) such as polybrominated diphenyl ethers, polychlorinated biphenyls, and bisphenols perturb animal's thyroid endocrine system by mimicking the action of endogenous thyroid hormones (THs), since they share a similar backbone structure of coupled benzene rings with THs. 1-methyl-3-octylimidazolium bromide ([C8mim]Br), a commonly used ionic liquid (IL), has no structural similarity to THs. Whether it interferes with thyroid function and how its mode of action differs from conventional TDCs is largely unknown. Herein, zebrafish embryo-larvae experiments (in vivo), GH3 cell line studies (in vitro), and molecular simulation analyses (in silico) were carried out to explore the effect of [C8mim]Br on thyroid homeostasis and its underlying mechanism. Molecular docking results suggested that [C8mim]+ likely bound to retinoid X receptors (RXRs), which may compromise the formation of TH receptor/RXR heterodimers. This then perturbed the negative regulation of thyroid-stimulating hormone β (tshβ) transcription by T3 in GH3 cell line. The resulting enhancement of tshβ expression further caused hyperthyroidism and developmental toxicity in larval zebrafish. These findings provided a crucial aspect of the ecological risks of ILs, and presented a new insight into the thyroid-disrupting mechanisms for emerging pollutants that do not have structural similarity to THs.

NRF-2α and mitophagy underlie enhanced mitochondrial functions and biogenesis induced by T-2 toxin in GH3 cells.

T-2 toxin is a natural contaminant in grain cereals produced by species of Fusarium. Studies indicate that T-2 toxin can positively affect mitochondrial function, but the underlying mechanism is unclear. In this study, we examined the role of nuclear respiratory factor 2α (NRF-2α) in T-2 toxin-activated mitochondrial biogenesis and the direct target genes of NRF-2α. Furthermore, we investigated T-2 toxin-induced autophagy and mitophagy, and the role of mitophagy in changes in mitochondrial function and apoptosis. It was found that T-2 toxin significantly increased NRF-2α levels and nuclear localization of NRF-2α was induced. NRF-2α deletion significantly increased the production of reactive oxygen species (ROS), abrogated T-2 toxin-induced increases in ATP and mitochondrial complex I activity, and inhibited the mitochondrial DNA copy number. Meanwhile, With chromatin immunoprecipitation sequencing (ChIP-Seq), various novel NRF-2α target genes were identified, such as mitochondrial iron-sulphur subunits (Ndufs 3,7) and mitochondrial transcription factors (Tfam, Tfb1m, and Tfb2m). Some target genes were also involved in mitochondrial fusion and fission (Drp1), mitochondrial translation (Yars2) and splicing (Ddx55), and mitophagy. Further studies showed that T-2 toxin induced Atg5 dependent autophagy and Atg5/PINK1-dependent mitophagy. In addition, mitophagy defects increase ROS production, inhibit ATP levels and the expression of genes related to mitochondrial dynamics, and promote apoptosis in the presence of T-2 toxins. Altogether, these results suggest that NRF-2α plays a critical role in promoting mitochondrial function and biogenesis through regulation of mitochondrial genes, and, interestingly, mitophagy caused by T-2 toxin positively affected mitochondrial function and protected cell survival against T-2 toxin.

Receptor Binding-Induced Conformational Changes in Herpes Simplex Virus Glycoprotein D Permit Interaction with the gH/gL Complex to Activate Fusion.

Herpes simplex virus (HSV) requires four essential virion glycoproteins-gD, gH, gL, and gB-for virus entry and cell fusion. To initiate fusion, the receptor binding protein gD interacts with one of two major cell receptors, HVEM or nectin-1. Once gD binds to a receptor, fusion is carried out by the gH/gL heterodimer and gB. A comparison of free and receptor-bound gD crystal structures revealed that receptor binding domains are located within residues in the N-terminus and core of gD. Problematically, the C-terminus lies across and occludes these binding sites. Consequentially, the C-terminus must relocate to allow for both receptor binding and the subsequent gD interaction with the regulatory complex gH/gL. We previously constructed a disulfide bonded (K190C/A277C) protein that locked the C-terminus to the gD core. Importantly, this mutant protein bound receptor but failed to trigger fusion, effectively separating receptor binding and gH/gL interaction. Here, we show that "unlocking" gD by reducing the disulfide bond restored not only gH/gL interaction but fusion activity as well, confirming the importance of C-terminal movement in triggering the fusion cascade. We characterize these changes, showing that the C-terminus region exposed by unlocking is: (1) a gH/gL binding site; (2) contains epitopes for a group (competition community) of monoclonal antibodies (Mabs) that block gH/gL binding to gD and cell-cell fusion. Here, we generated 14 mutations within the gD C-terminus to identify residues important for the interaction with gH/gL and the key conformational changes involved in fusion. As one example, we found that gD L268N was antigenically correct in that it bound most Mabs but was impaired in fusion, exhibited compromised binding of MC14 (a Mab that blocks both gD-gH/gL interaction and fusion), and failed to bind truncated gH/gL, all events that are associated with the inhibition of C-terminus movement. We conclude that, within the C-terminus, residue 268 is essential for gH/gL binding and induction of conformational changes and serves as a flexible inflection point in the critical movement of the gD C-terminus.

The epigenetic EZH2/H3K27me3 axis modulates lactotroph tumor cell proliferation.

Interest in epigenetics has gained substantial momentum as a result of their identified role in the regulation of tumor progression as well as their ability to pharmacologically target genes. Pituitary neuroendocrine tumors (PitNETs) tend to be inactivated via epigenetic modification, and although emerging evidence has suggested a role for epigenetic factors in PitNET tumorigenesis, the degree to which these factors may be targeted by new therapeutic strategies still remains poorly understood. The objective of the present study was to examine the participation of the EZH2/H3K27me3 axis in the proliferation of lactotroph tumor cells. We demonstrated that the levels of EZH2 and H3K27me3 were increased in murine experimental prolactin (PRL) tumors with respect to a control pituitary, in contrast with the low p21 mRNA levels encountered, with an H3K27me3 enrichment being observed in its promoter region in a GH3 tumor cell. Furthermore, specific EZH2/H3K27me3 axis inhibition blocked the proliferation of primary tumor cell culture and GH3 cells, thereby making it an attractive therapeutic target for PRL PitNETs.

Transcriptome of GH-producing pituitary neuroendocrine tumours and models are significantly affected by somatostatin analogues

Pituitary neuroendocrine tumours (PitNETs) are neoplasms of the pituitary that overproduce hormones or cause unspecific symptoms due to mass effect. Growth hormone overproducing GH-producing PitNETs cause acromegaly leading to connective tissue, metabolic or oncologic disorders. The medical treatment of acromegaly is somatostatin analogues (SSA) in specific cases combined with dopamine agonists (DA), but almost half of patients display partial or full SSA resistance and potential causes of this are unknown. In this study we investigated transcriptomic landscape of GH-producing PitNETs on several levels and functional models—tumour tissue of patients with and without SSA preoperative treatment, tumour derived pituispheres and GH3 cell line incubated with SSA to study effect of medication on gene expression. MGI sequencing platform was used to sequence total RNA from PitNET tissue, pituispheres, mesenchymal stromal stem-like cells (MSC), and GH3 cell cultures, and data were analysed with Salmon—DeSeq2 pipeline. We observed that the GH-producing PitNETs have distinct changes in growth hormone related pathways related to its functional status alongside inner cell signalling, ion transport, cell adhesion and extracellular matrix characteristic patterns. In pituispheres model, treatment regimens (octreotide and cabergoline) affect specific cell proliferation (MKI67) and core functionality pathways (RYR2, COL8A2, HLA-G, ARFGAP1, TGFBR2). In GH3 cells we observed that medication did not have transcriptomic effects similar to preoperative treatment in PitNET tissue or pituisphere model. This study highlights the importance of correct model system selection for cell transcriptomic profiling and data interpretation that could be achieved in future by incorporating NGS methods and detailed cell omics profiling in PitNET model research.

Letrozole Decreased Testosterone-Induced Cell Proliferation and Prolactin Secretion also Increased Apoptosis in MMQ and GH3 Rat Prolactinoma Cell Lines.

Aromatase enzyme plays an essential role in estrogen-induced tumorigenesis. It is expressed in the normal pituitary and more significantly in prolactinoma tissues. The aim of this study was to investigate the effects of an aromatase inhibitor, letrozole, on MMQ and GH3 rat prolactinoma cell lines and evaluate the possible mechanism of action. MMQ and GH3 cells were characterized with demonstrating aromatase enzyme and estrogen receptor alpha expression by PCR and immunofluorescence staining. After dose optimization for testosterone (T) and letrozole (L), four groups were established: only the testosteron-treated group (T) to detect cell proliferation; only letrozole-treated group (L) to investigate apoptotic effects; testosterone and letrozole concomitant-treated group to demonstrate inhibition of testosterone induced cell proliferation with letrozole treatment s(T + L) and control group (C) with no treatment. The proliferation rate of cells was determined by WST-1. For the detection of apoptotic and necrotic cells, Annexin V and caspase-3 labeling was used. Prolactin and estrogen levels were measured with ELISA, and the mRNA expression of aromatase and Esr1 was also determined. Testosterone induced the proliferation of MMQ and GH3 cells and further increased prolactin and estradiol levels. Adding letrozole to testosterone resulted in decreased cellular proliferation and even induced apoptosis. Also, letrozole administration significantly decreased prolactin and estradiol levels. However, letrozole alone had no effects on proliferation and apoptosis. Gene expression of aromatase and Esr1 was also significantly decreased by letrozole treatment. This in vitro study demonstrated that treatment of testosterone proliferating cells with letrozole resulted in decreased prolactin levels and cell proliferation and induced apoptosis, and further loss of aromatase and Esr1 mRNA expression were observed. Although this is an in vivo study, the results showed unique and novel findings which may easily be adapted to clinical use for further verification.

Inhibition of Voltage-Gated Na+ Currents Exerted by KB-R7943 (2-[2-[4-(4-nitrobenzyloxy)phenyl]ethyl]isothiourea), an Inhibitor of Na+-Ca2+ Exchanging Process.

KB-R7943, an isothiourea derivative, has been recognized as an inhibitor in the reverse mode of the Na+-Ca2+ exchanging process. This compound was demonstrated to prevent intracellular Na+-dependent Ca2+ uptake in intact cells; however, it is much less effective at preventing extracellular Na+-dependent Ca2+ efflux. Therefore, whether or how this compound may produce any perturbations on other types of ionic currents, particularly on voltage-gated Na+ current (INa), needs to be further studied. In this study, the whole-cell current recordings demonstrated that upon abrupt depolarization in pituitary GH3 cells, the exposure to KB-R7943 concentration-dependently depressed the transient (INa(T)) or late component (INa(L)) of INa with an IC50 value of 11 or 0.9 μM, respectively. Likewise, the dissociation constant for the KB-R7943-mediated block of INa on the basis of a minimum reaction scheme was estimated to be 0.97 μM. The presence of benzamil or amiloride could suppress the INa(L) magnitude. The instantaneous window Na+ current (INa(W)) activated by abrupt ascending ramp voltage (Vramp) was suppressed by adding KB-R7943; however, subsequent addition of deltamethrin or tefluthrin (Tef) effectively reversed KB-R7943-inhibted INa(W). With prolonged duration of depolarizing pulses, the INa(L) amplitude became exponentially decreased; moreover, KB-R7943 diminished INa(L) magnitude. The resurgent Na+ current (INa(R)) evoked by a repolarizing Vramp was also suppressed by adding this compound; moreover, subsequent addition of ranolazine or Tef further diminished or reversed, respectively, its reduction in INa(R) magnitude. The persistent Na+ current (INa(P)) activated by sinusoidal voltage waveform became enhanced by Tef; however, subsequent application of KB-R7943 counteracted Tef-stimulated INa(P). The docking prediction reflected that there seem to be molecular interactions of this molecule with the hNaV1.2 or hNaV1.7 channels. Collectively, this study highlights evidence showing that KB-R7943 has the propensity to perturb the magnitude and gating kinetics of INa (e.g., INa(T), INa(L), INa(W), INa(R), and INa(P)) and that the NaV channels appear to be important targets for the in vivo actions of KB-R7943 or other relevant compounds.

A new insight into GH regulation and its disturbance from nutrition and autoimmune perspectives.

GH activates GH receptors, which activates IGF-1 in the liver through a cascade of processes. The GH/IGF-1 axis plays an important role in the regulation of metabolism. Insufficient GH secretion results in short stature in childhood, while adult GH deficiency (AGHD) is observed in adulthood. The early diagnosis of AGHD is important for early initiation of GH replacement therapy. This review described the regulatory mechanisms of GH signaling based on nutritional status and a novel disease concept pathogenesis that causes AGHD. GH-dependent IGF-1 production in the liver is regulated by a complex interplay between nutritional status, hormones, and growth factors. GH resistance is an adaptive response that enhances survival during starvation and malnutrition. Sirtuin 1 (SIRT1) negatively regulates GH-induced IGF-I production in the liver by directly inhibiting STAT5 activation, which causes GH resistance under starvation and malnutrition. The presence of autoantibodies is strongly associated with the disruption of immune tolerance in pituitary cells. Pituitary-specific transcription factors (PIT-1) are essential for the development, differentiation, and maintenance of GH, PRL, and TSH producing cells. However, the underlying mechanism that causes immune intolerance to PIT-1 remain unclear. The GH-IGF-1 system plays a pivotal role in growth, and the involvement of SIRT1 in this regulatory mechanism presents an intriguing perspective on the interplay between nutrient metabolism and lifespan. The discovery of the anti-PIT-1 pituitary antibody, a novel disease concept associated with AGHD, has provided valuable insights, which serves as a significant milestone towards unraveling the complete pathogenesis of the disease.

Thyroid hormone disrupting potentials of benzisothiazolinone in embryo-larval zebrafish and rat pituitary GH3 cell line

Benzisothiazolinone (BIT), one of the most widely used antimicrobial agents in consumer products, has frequently been detected in the water environment. The present study was conducted to determine the adverse effects of BIT on the thyroid neuroendocrine system of zebrafish embryos/larvae. Rat pituitary (GH3) cell line was employed to support the underlying mechanism of thyroid hormone disrupting effects. Significant coagulation and hatching delay were observed in embryos exposed to 30 μg/L of BIT, which in turn remarkably decreased hatchability and larval survival. In BIT-exposed larvae, tshβ, tshr, and trh genes were significantly upregulated along with a decrease in thyroxine and triiodothyronine content, indicating that BIT decreased thyroid hormones and increased thyrotropin-releasing hormone and thyroid stimulating hormone secretion through a feedback circuit. The downregulation of trα and deio2 genes in the zebrafish larvae suggests the inhibition of thyroid hormone receptors and deiodination. Similar to the results in zebrafish, upregulation of tshβ and downregulation of trα, trβ, deio1, and deio2 genes were observed in GH3 cells. Our observations suggest that BIT can decrease the level of thyroid hormones by influencing central regulation, receptor binding, and deiodination.

Evaluating the expression pattern of the opioid receptor in pituitary neuroendocrine tumors (PitNET) and the role of morphine and naloxone in the regulation of pituitary cell line growth and apoptosis

PurposeThe expression pattern of the opioid receptor (MOR) in pituitary neuroendocrine tumors (PitNET) and the possible effect of morphine and naloxone on GH3 cell growth and apoptosis were evaluated. MethodsThe 114 pituitary tissues including non-functioning, GH-producing and ACTH-producing PitNET and healthy cadaver pituitary tissues were included. The expression level of the MOR gene and protein was assessed using real-time PCR and Western blot. The association with patient demographic characteristics was assessed. Morphine and naloxone were applied to assess their possible pharmacological role in GH3 pituitary adenoma cell death. The cytotoxic effect, the apoptosis rate, the cell cycle distribution, the content of reactive oxygen species and the caspase 3 activity were measured. ResultsMOR gene levels increased significantly in pituitary neuroendocrine tumors (PitNET) compared to the healthy pituitary samples. The increased level of MOR gene expression was prominent in invasive functional and non-functional pituitary tumors. A consistent expression pattern was demonstrated for MOR protein levels in PitNET samples. A dose- and time-dependent reduction in the rate of GH3 pituitary cells was observed after morphine treatment with an IC50 of 483 µM after 24 h of incubation. Morphine induced early apoptosis, accumulation of cells in sub-G1 phase, increase in cellular ROS levels and caspase-3 activity. The observed effects of morphine were reversed after MOR blockade using 10 and 25 µM naloxone. ConclusionThe possible contributing role of the MOR in pituitary tumor cell growth and the putative pharmaceutical effect of morphine in pituitary neuroendocrine tumor cell death (PitNET) is illustrated