Alpha-subunit mRNA Research Articles

Tolfenamic acid inhibits ROS-generating oxidase Nox1-regulated p53 activity in intrastriatal injection of malonic acid rats

It has been reported that wild-type p53-induced gene 1 (Wig1), which is downstream of p53, regulates the expression of mutant huntingtin protein (mHtt) in Huntington’s disease (HD) patients and transgenic mouse brains. Intrastriatal injection of malonic acid in rats is often used as a model to study the pathological changes of Huntington’s disease, and this model has the advantages of a fast preparation and low cost. Therefore, in this study, we used intrastriatal injections of 6 μM malonic acid in rats to evaluate the effect of tolfenamic acid on motor and cognitive deficits and the effect of 6 mg/kg and 32 mg/kg tolfenamic acid on p53 and its downstream targets, such as Wig1. The results showed that 32 mg/kg tolfenamic acid attenuated motor and spatial memory dysfunction, prevented Nox1-mediated reactive oxygen species (ROS) production, and downregulated the activity of p53 by increasing the phosphorylation level at the Ser378 site and decreasing the acetylation level at the Lys382 site. Tolfenamic acid reduced mouse double minute 2 (Mdm2), phosphatase and tensin homologue (Pten), P53-upregulated modulator of apoptosis (Puma) and Bcl2-associated X (Bax) at the mRNA level to inhibit apoptosis and downregulated sestrin 2 (Sesn2) and hypoxia inducible factor 1, alpha subunit (Hif-1α) mRNA levels to exert antioxidative stress effects. In addition, 32 mg/kg tolfenamic acid played a role in neuroprotection by decreasing the terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling (TUNEL)-positive cell numbers. However, there was no difference in the Wig mRNA level among all groups, and tolfenamic acid could not decrease the protein level of Wig1. In conclusion, tolfenamic acid inhibited the ROS-generating oxidase Nox1-regulated p53 activity and attenuated motor and spatial memory deficits in malonic acid-injected rats.

Single-cell multiplex qPCR evidence for sex-dimorphic glutamate decarboxylase, estrogen receptor, and 5′-AMP-activated protein kinase alpha subunit mRNA expression by ventromedial hypothalamic nucleus GABAergic neurons

The inhibitory amino acid transmitter γ-aminobutryic acid (GABA) acts within the ventromedial hypothalamus to regulate systemic glucose homeostasis, but the issue of whether this neurochemical signal originates locally or is supplied by afferent innervation remains controversial. Here, combinatory in situ immunocytochemistry/laser-catapult microdissection/single-cell multiplex qPCR techniques were used to investigate the premise that ventromedial hypothalamic nucleus ventrolateral (VMNvl) and/or dorsomedial (VMNdm) division neurons contain mRNAs that encode glutamate decarboxylase (GAD)65 or GAD67 and metabolic-sensory biomarkers, and that expression of these genes is sex-dimorphic. In male and female rats, GAD65 mRNA was elevated in VMNvl versus VMNdm GAD65/67-immunopositive (-ir) neurons, yet the female exhibited higher GAD67 transcript content in VMNdm versus VMNvl GABAergic nerve cells. Estrogen receptor (ER)-alpha transcripts were lower in female versus male GABA neurons from either VMN division; ER-beta and G-protein-coupled ER-1 mRNA expression profiles were also comparatively reduced in cells from female versus male VMNvl. VMNvl and VMNdm GAD65/67-ir-positive neurons showed equivalent levels of glucokinase and sulfonylurea receptor-1 mRNA between sexes. 5′-AMP-activated protein kinase-alpha 1 (AMPKα1) and -alpha 2 (AMPKα2) transcripts were lower in female versus male VMNdm GABAergic neurons, yet AMPKα2 mRNA levels were higher in cells acquired from female versus male VMNvl. Current studies document GAD65 and -67 gene expression in VMNvl and VMNdm GAD65/67-ir-positive neurons in each sex. Results infer that GABAergic neurons in each division may exhibit sex differences in receptiveness to estradiol. Outcomes also support the prospect that energy sensory function by this neurotransmitter cell type may predominate in the VMNvl in female versus VMNdm in the male.

Iodoacetic Acid, a Water Disinfection Byproduct, Disrupts Hypothalamic, and Pituitary Reproductive Regulatory Factors and Induces Toxicity in the Female Pituitary.

Iodoacetic acid (IAA) is a water disinfection byproduct (DBP) formed by reactions between oxidizing disinfectants and iodide. In vitro studies have indicated that IAA is one of the most cyto- and genotoxic DBPs. In humans, DBPs have been epidemiologically associated with reproductive dysfunction. In mouse ovarian culture, IAA exposure significantly inhibits antral follicle growth and reduces estradiol production. Despite this evidence, little is known about the effects of IAA on the other components of the reproductive axis: the hypothalamus and pituitary. We tested the hypothesis that IAA disrupts expression of key neuroendocrine factors and directly induces cell damage in the mouse pituitary. We exposed adult female mice to IAA in drinking water in vivo and found 0.5 and 10 mg/l IAA concentrations lead to significantly increased mRNA levels of kisspeptin (Kiss1) in the arcuate nucleus although not affecting Kiss1 in the anteroventral periventricular nucleus. Both 10 mg/l IAA exposure in vivo and 20 μM IAA in vitro reduced follicle stimulating hormone (FSHβ)-positive cell number and Fshb mRNA expression. IAA did not alter luteinizing hormone (LHβ) expression in vivo although exposure to 20 μM IAA decreased expression of Lhb and glycoprotein hormones, alpha subunit (Cga) mRNA in vitro. IAA also had toxic effects in the pituitary, inducing DNA damage and P21/Cdkn1a expression in vitro (20 μM IAA) and DNA damage and Cdkn1a expression in vivo (500 mg/l). These data implicate IAA as a hypothalamic-pituitary-gonadal axis toxicant and suggest the pituitary is directly affected by IAA exposure.

Nociceptor Overexpression of NaV1.7 Contributes to Chronic Muscle Pain Induced by Early-Life Stress

Adult rats previously submitted to neonatal limited bedding (NLB), a model of early-life stress, display muscle mechanical hyperalgesia and nociceptor hyperexcitability, the underlying mechanism for which is unknown. Since voltage-gated sodium channel subtype 7 (NaV1.7) contributes to mechanical hyperalgesia in several preclinical pain models and is critical for nociceptor excitability, we explored its role in the muscle hyperalgesia exhibited by adult NLB rats. Western blot analyses demonstrated increased NaV1.7 protein expression in L4-L5 dorsal root ganglia (DRG) from adult NLB rats, and antisense oligodeoxynucleotide (AS ODN) targeting NaV1.7 alpha subunit mRNA attenuated the expression of NaV1.7 in DRG extracts. While this AS ODN did not affect nociceptive threshold in normal rats it significantly attenuated hyperalgesia in NLB rats. The selective NaV1.7 activator OD1 produced dose-dependent mechanical hyperalgesia that was enhanced in NLB rats, whereas the NaV1.7 blocker ProTx-II prevented OD1-induced hyperalgesia in control rats and ongoing hyperalgesia in NLB rats. AS ODN knockdown of extracellular signal-regulated kinase 1/2, which enhances NaV1.7 function, also inhibited mechanical hyperalgesia in NLB rats. Our results support the hypothesis that overexpression of NaV1.7 in muscle nociceptors play a role in chronic muscle pain induced by early-life stress, suggesting that NaV1.7 is a target for the treatment of chronic muscle pain. PerspectiveWe demonstrate that early-life adversity, induced by exposure to inconsistent maternal care, produces chronic muscle hyperalgesia, which depends, at least in part, on increased expression of NaV1.7 in nociceptors.

Deletion of Spata2 by CRISPR/Cas9n causes increased inhibin alpha expression and attenuated fertility in male mice.

As somatic cells in the testis seminiferous tubule, Sertoli cells provide the medium for spermatogenesis. One of the important functions of Sertoli cells is synthesizing and secreting cell factors to affect the production of sperm; however, much of those molecular regulation mechanisms remain unknown. Here, we confirm the localization of protein SPATA2 (spermatogenesis-associated protein 2), which had previously been shown to be highly expressed in Sertoli cells of the adult mouse testis. To further conduct a functional study, we generated SPATA2 global knockout mice via use of the CRISPR/Cas9n gene editing technology. The 120-day-old knockout mice testes showed almost a 40% decrease in size and weight and variations in the histomorphology of the seminiferous epithelium, with a 40% decrease in sperm count. Further examination revealed that the proliferation of germ cells in the seminiferous tubules was attenuated by 28%. In addition, we found that SPATA2 deletion led to an approximately 70% increase in the inhibin alpha-subunit mRNA and protein level in the testes compared to that of wild-type mice. Our data revealed the impact of SPATA2 on male fertility and suggested that SPATA2 ensures the normal secretory function of Sertoli cells.

Evaluation of in vivo estrogenic potency of natural estrogen-active chemical, puerarin, on pituitary function in gonadectomized female rats

AimsPrevious research has revealed that puerarin, the major phytoestrogen in tuberous roots of Pueraria lobata and Pueraria mirifica, acts as a selective estrogen receptor modulator that displays predominantly estrogenic potential for health benefit. However, little is known about the estrogenic potency of puerarin in pituitary, especially in the rat model of postmenopausal females. Main methodsPlasma prolactin and growth hormone levels as well as mRNA expression levels of pituitary estrogen-regulated genes, such as estrogen receptor (ER) subtypes alpha (ERα) and beta (ERβ), truncated ER product-1 (TERP-1) and -2 (TERP-2) and gonadotropin alpha subunit, were examined using radioimmunoassay and TaqMan® real-time PCR, respectively. The effects were compared with the potent ER agonist, 17β-estradiol-3-benzoate (E2B), and both substances were supplemented at low and high doses, i.e., 0.6 or 3g puerarin and 0.0043 or 0.0173g E2B per kilogram of phytoestrogens-free rat chow, and applied to ovariectomized rats (five groups; 11–12 rats per group) for 12weeks. Key findingsPuerarin possessed weak E2B-like activities on pituitary function by acting as ERβ and TERP-1/-2 agonists, which resulted in the downregulation and upregulation of ERβ and TERP-1/-2 mRNA expressions, respectively, and elevation of growth hormone levels. There were trends of decreased levels of alpha subunit mRNA transcripts and increased levels of prolactin in puerarin-treated rats as observed in E2B-treated animals. SignificanceThis is the first report in ovariectomized rats the effects of puerarin on somatotropes and pituitary estrogen-responsive mRNA expressions, which are very weakly estrogenic by acting through ERβ- and TERP-1/-2 mediated pathways.

Correction of vascular hypercontractility in spontaneously hypertensive rats using shRNAs-induced delta protein kinase C gene silencing

Potassium conductance in vascular smooth muscle (VSM) is known to be altered in arterial hypertension. High level of protein kinase C (PKC) activity is a common feature for hypertension of different genesis. The main goal of this study was to investigate the efficacy of the RNA interference (RNAi) technique targeting PKC delta-isoform gene as a possible pharmacological tool to restore vasodilator potential in spontaneously hypertensive rats (SHR). Experimental design of the study comprised RNAi and patch-clamp techniques, RT-PCR analysis and standard acetylcholine test. Total outward currents and acetylcholine-induced endothelium-dependent relaxant responses were blunted in SHR. BKCa alpha subunit mRNA expression in SHR was unchanged whereas KV and KATP mRNA expression appeared significantly increased. PKC inhibitor, chelerythrine (100nM), restored potassium channels activity in SHR. PKC-delta-isoform protein expression and PKC-delta-isoform mRNA expression are 2.5–4 fold increased in VSM from SHR. PKC gene silencing with the short hairpin RNAs (shRNAs)-plasmid delivery system administered intravenously led to an increment in maximal amplitude of acetylcholine-relaxation, restored outward K+ currents and PKC-delta-isoform mRNA and protein expression. Arterial blood pressure in SHR was normalized following shRNAs administration. We conclude that BKCa channels are likely to be the most PKC-dependent member of K+ channels family responsible for vascular hypercontractility in SHR while Kv and KATP channels may constitute a reserve mechanism for the maintenance of vasodilator potential under BKCa channelopathy. It is likely that RNAi technique is a good therapeutic approach to inactivate PKC gene and to normalize vascular functions and high arterial blood pressure in SHR.

Gonadotropin-induced changes in oviducal mRNA expression levels of sex steroid hormone receptors and activin-related signaling factors in the alligator

Oviducts respond to hormonal cues from ovaries with tissue proliferation and differentiation in preparation of transporting and fostering gametes. These responses produce oviducal microenvironments conducive to reproductive success. Here, we investigated changes in circulating plasma sex steroid hormones concentrations and ovarian and oviducal mRNA expression to an in vivo gonadotropin (FSH) challenge in sexually immature, five-month-old alligators. Further, we investigated differences in these observed responses between alligators hatched from eggs collected at a heavily-polluted (Lake Apopka, FL) and minimally-polluted (Lake Woodruff, FL) site. In oviducts, we measured mRNA expression of estrogen, progesterone, and androgen receptors and also beta A and B subunits which homo- or heterodimerize to produce the transforming growth factor activin. In comparison, minimal inhibin alpha subunit mRNA expression suggests that these oviducts produce a primarily activin-dominated signaling milieu. Ovaries responded to a five-day FSH challenge with increased expression of steroidogenic enzyme mRNA which was concomitant with increased circulating sex steroid hormone concentrations. Oviducts in the FSH-challenged Lake Woodruff alligators increased mRNA expression of progesterone and androgen receptors, proliferating cell nuclear antigen, and the activin signaling antagonist follistatin. In contrast, Lake Apopka alligators displayed a diminished increase in ovarian CYP19A1 aromatase expression and no increase in oviducal AR expression, as compared to those observed in Lake Woodruff alligators. These results demonstrate that five-month-old female alligators display an endocrine-responsive ovarian-oviducal axis and environmental pollution exposure may alter these physiological responses.

Anti-Müllerian Hormone (AMH) and Inhibin Alpha Subunit mRNA and Protein Are Differentially Expressed During Photoperiod-Induced Regression and Recrudescence in Siberian Hamster Ovaries.

Anti-Müllerian Hormone (AMH) and Inhibin Alpha Subunit mRNA and Protein Are Differentially Expressed During Photoperiod-Induced Regression and Recrudescence in Siberian Hamster Ovaries.

Mechanisms underlying off-target effects of the cholesteryl ester transfer protein inhibitor torcetrapib involve L-type calcium channels

The increased mortality observed with the cholesteryl ester transfer protein inhibitor torcetrapib is partly due to increased aldosterone production and blood pressure. The mechanisms underlying these effects were investigated. Cytochrome P450 subunit 11B2 (aldosterone synthase), extracellular signal-regulated kinase (p44/42) and voltage-gated Cachannel alpha subunit mRNA profiling, aldosterone production, cytosolic calcium and RNA interference were assessed in adrenocarcinoma human cells (H295R). Telemetry was conducted in spontaneously hypertensive rats. Torcetrapib and angiotensin II (Ang II) but not dalcetrapib (a structurally different cholesteryl ester transfer protein inhibitor) elevated both cytochrome P450 subunit 11B2 mRNA and aldosterone production in H295R cells at 6 h. At days 1-5, torcetrapib produced a sustained increase of cytochrome P450 subunit 11B2 mRNA, unlike Ang II. Although torcetrapib and Ang II potentiated the effect of 25-OH cholesterol and raised pregnenolone levels, torcetrapib increased neither cytosolic Ca at 5 min nor extracellular signal-regulated kinase1/2 phosphorylation, suggesting initially divergent pathways. Unlike Ang II, torcetrapib steroidogenesis was not affected by Ang II type 1 receptor antagonism or voltage-gated T-type Ca channel antagonism, but was blocked by several L-type Cachannel antagonists. In unbiased genome-wide screening, Ang II and torcetrapib modulated an overlapping but distinct set of genes in H295R cells. Torcetrapib, but not Ang II, upregulated mRNA levels of the L-type Ca channel alpha 1C subunit. In spontaneously hypertensive rat, torcetrapib had a potent hypertensive effect mediated by the L-type Ca channel. The unique steroidogenic and hypertensive side effects of torcetrapib may be linked and involve voltage-gated L-type Ca channels. Structurally unrelated cholesteryl ester transfer protein inhibitors such as dalcetrapib do not share this effect.

The effects of zolpidem treatment and withdrawal on the in vitro expression of recombinant α1β2γ2s GABAA receptors expressed in HEK 293 cells

Zolpidem, a widely used hypnotic drug which acts through benzodiazepine binding sites, is a positive allosteric modulator of gamma-aminobutyric acid (GABA) action with preferential affinity for GABA(A) receptors containing alpha1 subunit. The pharmacological profile of zolpidem is different from that of classical benzodiazepines. The aim of this study was to find out whether zolpidem treatment triggers adaptive changes in the recombinant alpha1 subunit-containing GABA(A) receptors other than those observed following treatment with classical benzodiazepine-diazepam. Radioligand binding studies showed that 2-day exposure of human embryonic kidney (HEK) 293 cells stably expressing recombinant alpha1beta2gamma2s GABA(A) receptors to zolpidem (10 muM) up-regulated the maximum number (B (max)) of [(3)H]flunitrazepam, [(3)H]muscimol, and [(3)H]t-butylbicycloorthobenzoate ([(3)H]TBOB) binding sites without changing their affinity (K (d)), suggesting an increase in total GABA(A) receptor number. Semi-quantitative RT-PCR analysis demonstrated increased levels of alpha1 subunit mRNA, while Western blot demonstrated up-regulated gamma2 subunit proteins, suggesting that zolpidem induced de novo synthesis of receptors proteins, at both the transcriptional and translational levels. GABA-induced potentiation of [(3)H]flunitrazepam binding to membranes obtained from zolpidem-treated cells was markedly reduced, indicating allosteric uncoupling between GABA and benzodiazepine binding sites. The number of benzodiazepine and convulsant binding sites as well as the functional coupling between GABA and benzodiazepine binding sites normalized in 24 h following discontinuation of zolpidem treatment. The results of our in vitro studies suggest that a 2-day exposure of recombinant alpha1 subunit-containing GABA(A) receptors stably transfected in HEK 293 cells to zolpidem induces adaptive changes in this selective GABA(A) receptor subtype, which are not substantially different from those obtained after prolonged exposure of cells to high concentrations of diazepam.

Long-term regulation of Na,K-ATPase pump during T-cell proliferation

The aim of the study was to elucidate the mechanism responsible for the proliferation-related regulation of Na,K-ATPase pump. Our data demonstrate that in mitogen-stimulated human blood lymphocytes, enhanced ouabain-sensitive Rb(K) fluxes in the middle/late stage of G(0)/G(1)/S transit are associated with the increased number of Na,K-ATPase pumps expressed at the cell surface (as determined by the [(3)H]ouabain binding). Analysis of total RNA (reverse transcription-polymerase chain reaction) and protein (Western blotting) showed a threefold increase in the level of Na,K-ATPase alpha1-subunit and beta1-subunit mRNAs and significant increase in the Na,K-ATPase alpha1-subunit protein during the first day of mitogen-induced proliferation. The elevated K transport as well as the increased expression of Na,K-ATPase is closely associated with the IL-2-dependent stage of T-cell response. The pharmacological inhibition of IL-2-induced MEK/ERK or JAK/STAT cascades suppressed the IL-2-induced proliferation and reduced the functional and protein expressions of Na,K-ATPase. It is concluded that during the lymphocyte transition from resting stage to proliferation, (1) long-term activation of Na,K-ATPase pump is due to the enhanced expression of Na,K-ATPase protein and mRNA, and (2) the cytokine signaling via the IL-2 receptor is necessary for the cell cycle-associated upregulation of Na,K-ATPase.

Carbon dioxide pneumoperitoneum, intraperitoneal pressure, and peritoneal tissue hypoxia: a mouse study with controlled respiratory support

Animal experiments have suggested that the laparoscopic peritoneal environment is hypoxic. This study aimed to investigate whether peritoneal tissue is hypoxic on a cellular level during a carbon dioxide (CO(2)) pneumoperitoneum at different intraperitoneal pressures (IPPs) and to determine the short-term effects of surgical injury on the hypoxia status of peritoneal tissue in the injured peritoneum and the distant noninjured peritoneum at cellular and molecular levels. Experiment 1: Mice were divided into five groups according to the following treatments: anesthesia alone, laparotomy, and CO(2) pneumoperitoneum at IPPs of 2, 8, or 15 mmHg. Over the course of each experiment, the peritoneal tissue-oxygen tension (PitO(2)) was continuously monitored. Experiment 2: On the first day, the mice were divided into three groups according to the following treatments: CO(2) pneumoperitoneum at an IPP of either 2 or 8 mmHg or laparotomy. The bilateral caudal epigastric arteries and uterine horns then were coagulated using a bipolar cautery device. On day 7, peritoneal tissue samples were collected for real-time reverse transcriptase-polymerase chain reaction (RT-PCR) and immunohistochemistry. In both experiments, pimonidazole hydrochloride was used to detect tissue hypoxia at a cellular level. Experiment 1: Peritoneal hypoxia at both tissue and cellular levels was detected only in the groups treated with an IPP of 15 mmHg (PitO(2): 5.2 ± 1.0 mmHg, mean ± SEM). Experiment 2: The percentage of pimonidazole immunostained mesothelial and stromal cells from the distant noninjured peritoneum was significantly higher in the group treated with an IPP of 8 mmHg than in the other groups. Hypoxia-inducible factor 1 alpha subunit mRNA expression in the distant noninjured peritoneum of the group treated with an IPP of 8 mmHg was significantly higher than in the control group (anesthesia alone). The CO(2) pneumoperitoneum itself did not cause peritoneal hypoxia at either a tissue or a cellular level in a mouse model when a low IPP was used.

Distribution of G alpha subunit mRNA in rat adrenal cortex and adrenal medulla

Mineralocorticoid release from adrenal cortex and catecholamine release from adrenal medulla contribute to the regulation of blood pressure. Release is controlled by actions of several G protein coupled receptors (GPCRs) including angiotensin and melanocortin receptors in the cortex and PACAP and opioid receptors in the medulla. Activation of a GPCR releases the Gá subunit (11 types) that activates downstream signaling components. Stimulatory Gás and inhibitory Gái/o/z subunits act via the cAMP signaling pathway. The aim of this study was to assess distribution of stimulatory and inhibitory Gá subunits in adrenal cortex and medulla to determine the primary signaling pathways utilized. The relative amount of Gá subunit mRNA was calculated in adrenal cortex and adrenal medulla of Wistar Kyoto rats (n=5) using quantitative RT‐PCR. All Gá subunits tested were detected in the cortex and medulla, with a greater abundance of each in the medulla. Abundance was normalized relative to Gz (least abundant subunit) as follows: Gs>Gi2>Gi3>Gi1>Go>Gz in cortex and Gs>>Gi2>Gi3>Go>Gi1>Gz in medulla. Gs and Gi2 were most abundant in both cortex and medulla, suggesting they are the primary stimulatory and inhibitory subunits involved in GPCR‐mediated signaling in adrenal glands. In situ hybrization is currently underway to examine distribution of these subunits in adrenal glands from normotensive and hypertensive animals.

Elevated Na+/K+-ATPase responses and its potential role in triggering ion reabsorption in kidneys for homeostasis of marine euryhaline milkfish (Chanos chanos) when acclimated to hypotonic fresh water

The milkfish (Chanos chanos) is an economic species in Southeast Asia. In Taiwan, the milkfish are commercially cultured in environments of various salinities. Na(+)/K(+)-ATPase (NKA) is a key enzyme for fish iono- and osmoregulation. When compared with gills, NKA and its potential role were less examined by different approaches in the other osmoregulatory organs (e.g., kidney) of euryhaline teleosts. The objective of this study was to investigate the correlation between osmoregulatory plasticity and renal NKA in this euryhaline species. Muscle water contents (MWC), plasma, and urine osmolality, kidney histology, as well as distribution, expression (mRNA and protein), and specific activity of renal NKA were examined in juvenile milkfish acclimated to fresh water (FW), seawater (SW 35 per thousand), and hypersaline water (HSW 60 per thousand) for at least two weeks before experiments. MWC showed no significant difference among all groups. Plasma osmolality was maintained within the range of physiological homeostasis in milkfish acclimated to different salinities, while, urine osmolality of FW-acclimated fish was evidently lower than SW- and HSW-acclimated individuals. The renal tubules were identified by staining with periodic acid Schiff's reagent and hematoxylin. Moreover, immunohistochemical staining showed that NKA was distributed in the epithelial cells of proximal tubules, distal tubules, and collecting tubules, but not in glomeruli, of milkfish exposed to different ambient salinities. The highest abundance of relative NKA alpha subunit mRNA was found in FW-acclimated milkfish rather than SW- and HSW-acclimated individuals. Furthermore, relative protein amounts of renal NKA alpha and beta subunits as well as NKA-specific activity were also found to be higher in the FW group than SW and the HSW groups. This study integrated diverse levels (i.e., histological distribution, gene, protein, and specific activity) of renal NKA expression and illustrated the potential role of NKA in triggering ion reabsorption in kidneys of the marine euryhaline milkfish when acclimated to a hypotonic FW environment.

Effects on the expression of GABAA receptor subunits by jujuboside A treatment in rat hippocampal neurons

To determine the effect of jujuboside A (JuA) in modulating the gamma-aminobutyric acid (GABA(A)) receptor subunits gene expression of hippocampal neurons at different terms in vitro. Hippocampal neurons of rat were cultured in vitro, treated with JuA or diazepam (DZP). Then GABA(A) receptor mRNAs were evaluated by semi-quantitative RT-PCR. JuA at the low dose of 41 microM (about 0.05 g/l) induced significant increase of GABA(A) receptor alpha1, alpha5, beta2 subunit mRNAs in both 24 and 72h treatments. JuA at the high dose of 82 microM (about 0.1g/l) significantly increased GABA(A) receptor alpha1, alpha5 subunit mRNA levels and decreased beta2 subunit mRNA level at 24h treatment, and decreased GABA(A) receptor subunit alpha1, beta2 mRNAs expression at 72h treatment. DZP of 10 microM significantly increased expression of GABA(A) receptor subunit alpha1, alpha5 and decreased expression of beta2 at 24h treatment, and decreased alpha1, alpha5, beta2 subunits gene expression at 72h treatment. Differences in alterations in GABA(A) receptor subunit mRNAs expression following JuA and DZP treatments could help to explain the differences in the pharmacological action of the two drugs.

Hypoxia is Important for Establishing Vascularization During Corpus Luteum Formation in Cattle

Hypoxia-inducible factor 1 (HIF1) has been demonstrated to have critical roles in angiogenesis via transcriptional regulation of angiogenic factors, such as vascular endothelial growth factor (VEGF). In the ovary, angiogenesis is known to occur after ovulation in the developing corpus luteum (CL) in mammals. To determine whether HIF1 participates in angiogenesis in bovine CL, the present study investigated the mRNA and protein expressions of the HIF1 alpha subunit (HIF1A) and VEGF in bovine CL during the estrous cycle. The effects of hypoxia on the expressions of HIF1A protein, VEGF mRNA and VEGF protein in bovine luteal cells were also examined by using a cell culture system. HIF1A mRNA expression was less at the regressed stage than at the other stages, whereas protein expression of HIF1A was highest at the early luteal stage and decreased thereafter. VEGF mRNA expression was highest at the developing luteal stage and decreased thereafter. VEGF protein expression was highest at the early luteal stage and decreased significantly at the regressed luteal stage. Hypoxia increased the amounts of HIF1A protein, VEGF mRNA and VEGF protein in cultured bovine luteal cells. Furthermore, we found that hypoxia inhibited progesterone production in the mid luteal cells, but not in the early luteal cells. The overall findings indicate that HIF1 is one of the factors promoting VEGF-induced angiogenesis during luteal development, and suggest that the hypoxic conditions formed after follicle rupture contribute to establishing luteal vascularization in cattle.

Trinitrobenzenesulphonic acid colitis alters Nav 1.8 channel expression in mouse dorsal root ganglia neurons

Visceral inflammation evokes hyperexcitability in nociceptive dorsal root ganglia (DRG) neurons and these changes are associated with increased voltage-gated sodium channel (Na(v)) 1.8 current density, but the molecular determinants of these changes are unclear. This study used Western blotting to measure changes in Na(v) 1.7, 1.8 and 1.9 protein expression during trinitrobenzenesulphonic acid (TNBS) colitis and quantitative polymerase chain reaction (PCR) to examine corresponding changes in mRNA. Colonic neurons were labelled with the retrograde tracer Fast Blue injected into the wall of the distal colon and quantitative PCR performed on laser-captured labelled colonic neurons from ganglia at T9-13 or unlabelled DRG neurons from the upper spinal cord. Immunohistochemistry and western blots were performed on whole DRG from the same sites. Fast Blue-labelled neurons demonstrated Na(v) 1.7, 1.8 and 1.9 immunoreactivity. On day 7 of colitis, which correlated with electrophysiological studies, there was a threefold increase in Na(v) 1.8 protein in ganglia from T9 to 13, but Na(v) 1.7 and 1.9 levels were unchanged. There was no corresponding change in the Na(v) 1.8 alpha-subunit mRNA levels. However, on days 2 and 4, Na(v) 1.8 mRNA was decreased 10-fold. Na(v) 1.8 protein and mRNA levels were unchanged in neurons isolated from ganglia in the upper spinal cord, where colonic neurons are not found. These findings suggest that the TNBS evoked increase in Na(v) 1.8 currents is associated with increased numbers of channels. The absence of corresponding changes in transcript suggests a translational or post-translational mechanism, but the 10-fold recovery of transcript preceding this time point also demonstrates a complex transcriptional regulation.

Cyclic AMP‐dependent activation of rhodopsin gene transcription in cultured retinal precursor cells of chicken embryo

The present study describes a robust 50-fold increase in rhodopsin gene transcription by cAMP in cultured retinal precursor cells of chicken embryo. Retinal cells isolated at embryonic day 8 (E8) and cultured for 3 days in serum-supplemented medium differentiated mostly into red-sensitive cones and to a lesser degree into green-sensitive cones, as indicated by real-time RT-PCR quantification of each specific opsin mRNA. In contrast, both rhodopsin mRNA concentration and rhodopsin gene promoter activity required the presence of cAMP-increasing agents [forskolin and 3-isobutyl-1-methylxanthine (IBMX)] to reach significant levels. This response was rod-specific and was sufficient to activate rhodopsin gene transcription in serum-free medium. The increase in rhodopsin mRNA levels evoked by a series of cAMP analogs suggested the response was mediated by protein kinase A, not by EPAC. Membrane depolarization by high KCl concentration also increased rhodopsin mRNA levels and this response was strongly potentiated by IBMX. The rhodopsin gene response to cAMP-increasing agents was developmentally gated between E6 and E7. Rod-specific transducin alpha subunit mRNA levels also increased up to 50-fold in response to forskolin and IBMX, while rod-specific phosphodiesterase-VI and rod arrestin transcripts increased 3- to 10-fold. These results suggest a cAMP-mediated signaling pathway may play a role in rod differentiation.

Altering the Relative Abundance of GABAA Receptor Subunits Changes GABA‐ and Ethanol‐Responses in Xenopus Oocytes

Variations in GABRA2 and GABRG3, genes encoding the alpha2 and gamma3 subunits of the pentameric GABA(A) receptor, are associated with the risk of developing alcoholism in adults, conduct disorder at younger ages, and with differences in electroencephalographic power in the beta frequency range. The SNPs associated with alcoholism did not alter the coding of these genes, and extensive DNA sequencing of GABRA2 did not find coding changes in the high-risk haplotypes. Therefore, we hypothesize that the associations arise from differences in gene expression. Here we report studies in Xenopus oocytes to examine the functional effects of altering the relative abundance of these 2 receptor subunits on GABA current and response to ethanol, as a model of potential effects of regulatory differences. When human alpha2beta2gamma3 subunits are co-expressed, increasing the amount of the alpha2 subunit mRNA increased GABA current; in contrast, increasing the amount of the gamma3 subunit decreased GABA currents. Acute ethanol treatment of oocytes injected with a 1:1:1 or 2:2:1 ratio of alpha2:beta2:gamma3 subunit mRNAs resulted in significant potentiation of GABA currents, whereas ethanol inhibited GABA currents in cells injected with a 6:2:1 ratio. Overnight treatment with ethanol significantly reduced GABA currents in a manner dependent on the ratio of subunits. These studies demonstrate that changes in relative expression of GABA(A) receptor subunits alter the response of the resulting channels to GABA and to ethanol.