Beta Subunit mRNA Levels Research Articles

Wastewater treatment plant effluent alters pituitary gland gonadotropin mRNA levels in juvenile coho salmon (Oncorhynchus kisutch)

It is well known that endocrine disrupting compounds (EDCs) present in wastewater treatment plant (WWTP) effluents interfere with reproduction in fish, including altered gonad development and induction of vitellogenin (Vtg), a female-specific egg yolk protein precursor produced in the liver. As a result, studies have focused on the effects of EDC exposure on the gonad and liver. However, impacts of environmental EDC exposure at higher levels of the hypothalamic-pituitary-gonad axis are less well understood. The pituitary gonadotropins, follicle-stimulating hormone (Fsh) and luteinizing hormone (Lh) are involved in all aspects of gonad development and are subject to feedback from gonadal steroids making them a likely target of endocrine disruption. In this study, the effects of WWTP effluent exposure on pituitary gonadotropin mRNA expression were investigated to assess the utility of Lh beta-subunit (lhb) as a biomarker of estrogen exposure in juvenile coho salmon (Oncorhynchus kisutch). First, a controlled 72-h exposure to 17α-ethynylestradiol (EE2) and 17β-trenbolone (TREN) was performed to evaluate the response of juvenile coho salmon to EDC exposure. Second, juvenile coho salmon were exposed to 0, 20 or 100% effluent from eight WWTPs from the Puget Sound, WA region for 72h. Juvenile coho salmon exposed to 2 and 10ng EE2L⿿1 had 17-fold and 215-fold higher lhb mRNA levels relative to control fish. Hepatic vtg mRNA levels were dramatically increased 6670-fold, but only in response to 10ng EE2L⿿1 and Fsh beta-subunit (fshb) mRNA levels were not altered by any of the treatments. In the WWTP effluent exposures, lhb mRNA levels were significantly elevated in fish exposed to five of the WWTP effluents. In contrast, transcript levels of vtg were not affected by any of the WWTP effluent exposures. Mean levels of natural and synthetic estrogens in fish bile were consistent with pituitary lhb expression, suggesting that the observed lhb induction may be due to estrogenic activity of the WWTP effluents. These results suggest that lhb gene expression may be a sensitive index of acute exposure to estrogenic chemicals in juvenile coho salmon. Further work is needed to determine the kinetics and specificity of lhb induction to evaluate its utility as a potential indicator of estrogen exposure in immature fish.

The influence of equol on the hypothalamic-pituitary-thyroid axis and hepatic lipid metabolic parameters in adult male rats.

Equol, the principal active metabolite of soy-derived phytoestrogen daidzein, has well-known estrogenic actions. Results of several studies indicate that equol may also have anti-androgenic activities. However, mechanisms of action of equol on hypothalamic-pituitary-thyroid axis (HPTA) and hepatic lipid metabolism in adult male rats have not been determined yet. Equol at two doses of 100 and 250mg/kgbodyweight(BW)/day was orally gavaged for 5days to groups of 4-month-old male rats. As a positive anti-androgenic control group, animals received 100mg of pure anti-androgenic drug flutamide/kgBW/day. Circulating concentrations of thyroid hormones and lipids, and expression levels of genes underlying HPTA function were determined by radioimmunoassay and TaqMan® real-time reverse transcription polymerase chain reaction, respectively. Flutamide significantly decreased relative prostate weight, whereas equol did not. Both equol and flutamide caused a significant increase in relative liver weights, and decreases in plasma levels of total tetraiodothyronine (T4) and triiodothyronine (T3), whereas free T4 and T3 concentrations were not reduced. Equol caused the marked down-regulation of hypothalamic thyrotropin-releasing hormone mRNA expression, whereas flutamide did not. Equol as well as flutamide significantly down-regulated the expression levels of pituitary thyrotropin beta-subunit mRNA, without altering thyrotropin secretion. Equol caused reductions in plasma levels of total cholesterol, high- and low-density lipoproteins and triglycerides, whereas flutamide exerted opposite effects. This is the first study to reveal that in male rats equol did not affect HPTA function and liver lipid metabolism through the anti-androgenic pathway, however, the intrinsic estrogenic actions of equol were observed.

Late, but Not Early, Inhibition of Soluble Guanylate Cyclase Decreases Mortality in a Rat Sepsis Model

Overproduction of nitric oxide and activation of soluble guanylate cyclase (sGC) are important in sepsis-induced hypotension and hyporesponsiveness to vasoconstrictors. A time course of the expression and activity of sGC in a sepsis model [cecal ligation and puncture (CLP)] was evaluated in rats. Soluble GC alpha-1 and beta-1 subunit mRNA levels increased in the lungs, but not in the aorta. However, in both tissues, the protein levels increased 24 h after sepsis and remained high for up to 48 h. Sodium nitroprusside-stimulated cGMP accumulation was higher 48 h after CLP in the lung and aorta. NOS-2 protein expression peaked 24 h after CLP, decreasing thereafter. The impact of inhibiting the expression of sGC early (8 h) or late (20 h) on vascular reactivity and the indexes of organ damage and mortality were also studied. Late administration of methylene blue (MB) or ODQ (1H-[1,2,4]-oxadiazole[4,3-a]quinoxalin-1-one) restored the blood pressure and vascular responsiveness to vasoconstrictors to normal levels but was ineffective in early sepsis. Late MB injection reduced the plasma levels of urea, creatinine, and lactate. MB improved the survival if administered late, but it increased the mortality when administrated early after sepsis onset. The increased sGC expression/activity may be relevant for the late hypotension and hyporesponsiveness to vasoconstrictors in sepsis. In accordance, MB increased survival if administered in late sepsis, but not in early sepsis. Therefore, differential responsiveness to sGC during the course of sepsis may determine the success or failure of treatment with sGC inhibitors.

Maxi-K+ Channel 1 Expression in Sleep Apnea Patients and Its Modulation by CPAP Treatment

Maxi-K(+) channels play a major vasodilator role in the regulation of arterial tone. Hypoxia downregulates the expression of the maxi-K(+) channel beta1-subunit in rat and human arterial myocytes, thus facilitating vasoconstriction. We have investigated the relationships among hypoxemia, arterial pressure, and the expression of the beta1-subunit in patients with severe obstructive sleep apnea-hypopnea syndrome (SAHS), a highly prevalent condition that predisposes to hypertension. We studied 20 male patients with strong clinical suspicion of SAHS. Overnight polysomnography and 24-h ambulatory blood pressure monitoring were performed in each patient. Evaluation of beta1-subunit mRNA expression was made by a simple blood test using peripheral blood leukocytes (PBLs). The last two determinations were repeated 3 months after initiation of continuous positive airway pressure (CPAP) treatment. In untreated obstructive SAHS patients, beta1-subunit mRNA levels were correlated with the minimum level of overnight blood O(2) saturation (r(2) = 0.56, P < 0.05) and systolic arterial pressure (r(2) = 0.64, P < 0.01). Notably, the correction of nocturnal hypoxemia with CPAP resulted in upregulation of beta1-subunit mRNA and to a parallel significant decrease of systolic and diastolic arterial pressures of 6.5 and 5.3 mm Hg, respectively. These observations, although preliminary, suggest that the maxi-K(+) beta1-subunit could contribute to vascular dysregulation in SAHS patients. Leukocyte beta1 expression may be an independent readout of hypoxemia that could be useful as molecular marker for impending hypertension.

Cell-specific signal transduction pathways regulating Na+-K+-ATPase. Focus on “Short-term effects of thyroid hormones on the Na+-K+-ATPase activity of chick embryo hepatocytes during development: focus on signal transduction”

the na+-k+-atpase is a complex integral membrane protein that carries out active transport of sodium and potassium across the cell plasma membrane, maintaining the ionic gradients. Each subunit has multiple isozymes that are expressed in tissue and developmental specific fashion. In addition to

Estrogen and progesterone regulate α, β, and γENaC subunit mRNA levels in female rat kidney

Estrogen and progesterone regulate alpha, beta, and gamma amiloride-sensitive epithelial sodium channel (ENaC) subunit mRNA levels in female rat kidney. Renal Na(+) handling differs between males and females. Further, within females Na(+) metabolism changes during the menstrual cycle and pregnancy. Electrolyte homeostasis and extracellular fluid volume are maintained primarily by regulated transport of Na(+) via the amiloride-sensitive Na(+) channel. This study examines the role of the female gender steroids in the regulation of expression of ENaC. We measured ENaC subunit mRNA levels in rat kidney using Northern blotting. Kidneys were taken from male and females at different ages and from adult ovariectomized rats treated with 17-beta-estradiol benzoate (estrogen) and/or progesterone for 8 or 24 hours. The abundance of alpha, beta, and gammaENaC mRNA was significantly higher in female compared to male rat kidneys from 10 weeks of age (P= 0.001, P= 0.004, and P= 0.02, N= 10, respectively). These differences were abolished in ovariectomized rats. Treatment of ovariectomized rats with estrogen increased alphaENaC mRNA abundance in the kidney at both 8 and 24 hours (P < 0.05, N= 6; and P < 0.05, N= 7, respectively). Progesterone inhibited the effect of estrogen on alphaENaC mRNA at 8 hours but when given alone increased gammaENaC mRNA (P < 0.05, N= 3). Neither hormone, alone or in combination, had any significant effect on betaENaC mRNA levels at 8 or 24 hours. Female gonadal steroids differentially modulate expression of ENaC subunit mRNA in the rat kidney.

Hypoxia decreases expression of soluble guanylate cyclase in cultured rat pulmonary artery smooth muscle cells.

Nitric oxide (NO) has an important role in modulating the pulmonary vascular tone. NO acts, in part, by stimulating soluble guanylate cyclase (sGC) to synthesize the intracellular second messenger cyclic GMP. In vascular smooth muscle cells, sGC is a heterodimer composed of alpha1 and beta1 subunits. The objective of this study was to test whether oxygen concentration regulates sGC expression in cultured rat pulmonary artery smooth muscle cells (rPaSMC). rPaSMC were exposed to 0, 3, and 20% oxygen for 1-48 h, and sGC subunit mRNA levels were measured. Compared with rPaSMC exposed to 20% oxygen, sGC alpha1 and beta1 subunit mRNA levels were markedly decreased in rPaSMC exposed to 0% and 3% oxygen. The decrease in sGC subunit mRNA levels in hypoxic rPaSMC was detected as early as 6 h of exposure. Compared with rPaSMC exposed to 20% oxygen, exposure of rPaSMC to 3% oxygen progressively decreased sGC subunit protein levels at 24 and 48 h. There was also a 30% and 50% decrease in sGC enzyme activity in cells exposed to hypoxia for 24 and 48 h (P < 0.05 and P < 0.001, respectively, as compared with cells maintained in normoxia). These results demonstrate that hypoxia decreases sGC expression in cultured pulmonary artery smooth muscle cells and suggest that, in hypoxic vascular smooth muscle, decreased cyclic GMP synthesis may limit the vasodilator response to NO.

Regulation of cell surface expression of voltage-dependent Nav1.7 sodium channels: mRNA stability and posttranscriptional control in adrenal chromaffin cells.

Regulated expression of Na+ channels is indispensable to physiological events, whereas dysregulated expression of otherwise silent or even normal Na+ channel isoforms causes Na+ channelopathies; however, the regulatory mechanisms remain unknown. In quiescent cultured bovine adrenal chromaffin cells, constitutive phosphorylation/activation of extracellular signal-regulated kinase-1 (ERK1) and ERK2 destabilized Nav l.7 Na+ channel alpha-subunit mRNA and decreased its level without altering alpha-subunit gene transcription, thus negatively regulating steady-state level of Na+ channels. Activation of protein kinase C (PKC) down-regulated Na+ channels via PKC isoform-specific mechanisms; conventional PKC-alpha promoted endocytic internalization of Na+ channels, whereas novel PKC-epsilon destabilized alpha-subunit mRNA without altering its gene transcription. Long-lasting (but not short-term) increase of cytoplasmic Ca2+ down-regulated Na+ channels; a slowly-developing moderate increase of Ca2+ activated PKC-alpha and calpain, promoting internalization of Na+ channels, whereas an immediate monophasic and salient plateau increase of Ca2+ lowered alpha- and beta1-subunit mRNA levels. Calcineurin, or FK506 binding protein- and rapamycin-associated protein (FRAP), a serine/threonine protein kinase, down-regulated, whereas insulin receptor tyrosine kinase or protein kinase A (PKA) up-regulated, Na+ channels via modulating Na+ channel internalization, and/or Na+ channel externalization from the trans-Golgi network. Neuroprotective, antiepiletic, antipsychotic, and local anesthetic drugs up-regulated Na+ channels via transcriptional/translational events.

Changes of renal cortical Na-K ATPase activity, protein, and mRNA expression in ureteral obstruction.

Decreased renal Na-K ATPase activity in ureteral obstruction contributes to tubular sodium reabsorption defect in obstructive nephropathy. The integrated changes on the enzyme activity, protein number, and mRNA level of renal cortical Na-K ATPase, in response to bilateral or unilateral ureteral obstruction (BUO & UUO), were studied in rabbits. Ouabain-sensitive Na-K ATPase activities of renal cortex were significantly decreased at 24 h after BUO and further decreased at 48 h. The unobstructed contra-lateral kidney had significantly higher Na-K ATPase activity compared to the obstructed side. Immunoblots of Na-K ATPase alpha and beta subunits protein were both decreased at 24 h of BUO and further decreased at 48 h. The levels of Na-K ATPase beta subunit mRNA showed to be significantly decreased at 12 h after obstruction and further decreased at 24 and 48 h. However, the levels of alpha subunit mRNA were not changed as that of beta subunit throughout the study period. This study also used a newly developed method for release of obstruction. All the parameters studied above recovered to variable extents after release of the ureteral obstruction. In summary, decreases in Na-K ATPase activity, protein, and mRNA in obstructed kidney are specific cellular responses to ureteral obstruction. The degree of down-regulation is related to the duration of obstruction. The reduced activity of Na-K ATPase can be explained by decreased enzyme protein. However, the discordant findings in Na-K ATPase subunits protein amounts and mRNA changes suggest that renal Na-K ATPase subunits have different cellular regulatory processes to obstruction, in which transcriptional, translational and even intra-cytoplasmic processing may be involved.

Segmental variability of ENaC subunit expression in rat colon during dietary sodium depletion.

In rat distal colon, aldosterone has little effect on Na(+) channel (ENaC) alpha-subunit levels but increases the expression of the beta- and gamma-subunits and stimulates electrogenic Na(+) transport. By contrast, the molecular basis of aldosterone's inability to stimulate electrogenic Na(+) transport in the proximal colon is unclear. We therefore compared the effects of hyperaldosteronism secondary to 10 days dietary Na(+) depletion on ENaC subunit expression in rat proximal and distal colon. Northern analyses revealed appreciable and similar levels of alpha-subunit mRNA throughout the colon in control and Na(+)-depleted animals. By contrast, Na(+) depletion substantially enhanced beta-subunit mRNA expression in the distal colon, but had no effect on the low expression levels of beta-subunit mRNA in the proximal colon. Expression of the gamma-subunit, evaluated by PCR, was also restricted to the distal colon of Na(+)-depleted animals. Western analyses demonstrated similar levels of alpha-subunit protein in the proximal and distal colon of both groups of animals, whereas beta-subunit and gamma-subunit proteins were detected solely or predominantly in the distal colon of the Na(+)-depleted animals. Immunocytochemistry confirmed that significant levels of all three subunit proteins only occurred in the apical membrane of surface cells in the distal colon of Na(+)-depleted animals. Our findings are consistent with previous studies demonstrating that aldosterone stimulates electrogenic Na(+) transport in rat distal colon by increasing the expression of beta- and gamma-subunit mRNA and protein, and thus the amount of functional heteromeric ENaC protein in the apical domain. They also show that aldosterone is incapable of stimulating electrogenic Na(+) transport in rat proximal colon (despite the presence of alpha-subunit mRNA and protein) because of its inability to enhance beta- and gamma-subunit expression in this segment.

Dynamics of messenger RNAs encoding inhibin/activin subunits and follistatin in the ovary during the rat estrous cycle.

Quantitative changes in ovarian inhibin/activin subunit and follistatin mRNAs during the rat estrous cycle were examined by ribonuclease protection assay using digoxygenin-labeled RNA probes. Levels of ovarian inhibin alpha subunit mRNA remained low throughout estrus, metestrus, and diestrus; abruptly increased on the morning of proestrus; then rapidly decreased when the primary gonadotropin surge occurred. A similar changing pattern was observed in inhibin/activin beta(A) subunit mRNA. On the other hand, inhibin/activin beta(B) subunit mRNA showed a different changing pattern. Levels of beta(B) subunit mRNA remained constant during metestrus and diestrus, abruptly decreased on the afternoon of proestrus, then quickly recovered from the nadir by 1100 h on estrus. Throughout the rat estrous cycle, especially during the periovulatory period, alpha subunit mRNA levels were considerably higher than beta(A) and beta(B) subunit mRNA levels. In addition, changes in plasma concentrations of inhibin A and inhibin B were very similar to that in ovarian beta(A) and beta(B) subunit mRNA levels, respectively, with several-hour delays. These results suggest that levels of beta subunit mRNAs restrict secretion of dimeric inhibins. Levels of follistatin mRNA remained low from the midnight of metestrus to the midnight of diestrus, then increased until initiation of the primary gonadotropin surge. Thereafter, follistatin mRNA decreased, reached the nadir at 0200 h on estrus, then increased abruptly at 1100 h on estrus. Afterward, follistatin mRNA levels remained high until the morning of metestrus. The changing pattern of ovarian follistatin mRNA was similar to, and preceded, the changes in plasma concentrations of progesterone, suggesting that ovarian follistatin may modulate progesterone secretion during the rat estrous cycle.

Vacuolar-type H(+)-ATPase and Na+, K(+)-ATPase expression in gills of Atlantic salmon (Salmo salar) during isolated and combined exposure to hyperoxia and hypercapnia in fresh water.

Changes in branchial vacuolar-type H(+)-ATPase B-subunit mRNA and Na+, K(+)-ATPase alpha- and beta-subunit mRNA and ATP hydrolytic activity were examined in smolting Atlantic salmon exposed to hyperoxic and/or hypercapnic fresh water. Pre-smolts, smolts, and post-smolts were exposed for 1 to 4 days to hyperoxia (100% O2) and/or hypercapnia (2% CO2). Exposure to hypercapnic water for 4 days consistently decreased gill vacuolar-type H(+)-ATPase B-subunit mRNA levels. Salmon exposed to hyperoxia had either decreased or unchanged levels of gill B-subunit mRNA. Combined hyperoxia + hypercapnia decreased B-subunit mRNA levels, although not to the same degree as hypercapnic treatment alone. Hyperoxia generally increased Na+, K(+)-ATPase alpha- and beta-subunit mRNA levels, whereas hypercapnia reduced mRNA levels in presmolts (beta) and smolts (alpha and beta). Despite these changes in mRNA levels, whole tissue Na+, K(+)-ATPase activity was generally unaffected by the experimental treatments. We suggest that the reduced expression of branchial vacuolar-type H(+)-ATPase B-subunit mRNA observed during internal hypercapnic acidosis may lead to reduction of functional V-type H(+)-ATPase abundance as a compensatory response in order to minimise intracellular HCO3- formation in epithelial cells.

Gonadotrophins inhibit and activin induces expression of inhibin/activin beta(B) subunit mRNA in cultured human granulosa-luteal cells.

During the human menstrual cycle, serum inhibin concentrations fluctuate in a cyclic fashion. To examine the regulation of inhibin/activin beta(B) subunit gene expression in ovarian granulosa-luteal cells, the levels of beta(B) subunit mRNA were determined in primary cultures of human granulosa-luteal cells treated with gonadotrophins and protein kinase modulators. Granulosa cells were obtained from women undergoing an IVF programme. The cells were enzymatically dispersed, separated from red blood cells, and maintained in culture for 5--10 days before addition of different agents. Northern blot analysis with specific oligonucleotide probes was performed to study inhibin/activin beta(B) subunit mRNA levels. Both LH and FSH reduced the accumulation of beta(B) subunit mRNA in a dose-dependent manner. The protein kinase A activator, (Bu)(2)cAMP, and the protein kinase inhibitor staurosporine also inhibited beta(B) subunit mRNA expression dose-dependently. Activin A increased dose-dependently beta(B) subunit mRNA expression. Our study suggests that activin-induced and gonadotrophin-inhibited beta(B) subunit expression in granulosa cells might be key factors in the transition from inhibin B to inhibin A dominance during the menstrual cycle.

Protein kinase C-alpha and -epsilon down-regulate cell surface sodium channels via differential mechanisms in adrenal chromaffin cells.

In cultured bovine adrenal chromaffin cells, our [3H]saxitoxin ([3H]STX) binding, immunoblot, and northern blot analyses specified protein kinase C (PKC) isoform-specific posttranscriptional and posttranslational mechanisms that direct down-regulation of cell surface Na channels. Immunoblot analysis showed that among 11 PKC isoforms, adrenal chromaffin cells contained only conventional (c)PKC-alpha, novel (n)PKC-epsilon, and atypical (a)PKC-zeta. Treatment of adrenal chromaffin cells with 100 nM 12-O-tetradecanoylphorbol 13-acetate (TPA) or 100 nM phorbol 12,13-dibutyrate (PDBu) caused a rapid (<15 min) and sustained (>15 h) translocation of PKC-alpha and -epsilon (but not -zeta) from cytosol to membranes, whereas a biologically inactive 4alpha-TPA had no effect. Thymeleatoxin (TMX), an activator of cPKC, produced similar membrane association of only PKC-alpha at 100 nM, with the potency of TMX being comparable with those of TPA and PDBu. Treatment with either 100 nM TPA or 100 nM TMX reduced cell surface [3H]STX binding to a comparable extent at 3, 6, and 12 h, whereas TPA lowered the binding to a greater extent than TMX at 15, 18, and 24 h; at 15 h, Gö6976, a specific inhibitor of cPKC, completely blocked TMX-induced decrease of [3H]STX binding while preventing by merely 57% TPA-induced decrease of [3H]STX binding. Treatment with 100 nM TPA lowered the Na channel alpha-subunit mRNA level between 3 and 12 h, with its maximum 52% fall at 6 h, and it was accompanied by a subsequent 61 % rise of the beta1-subunit mRNA level at 24 h. Gö6976 failed to prevent TPA-induced reduction of the alpha-subunit mRNA level; TMX did not change the alpha- and beta1-subunit mRNA levels throughout the 24-h treatment. Brefeldin A, an inhibitor of vesicular exit from the trans-Golgi network, augmented TPA- and TMX-induced decrease of [3H]STX binding at 1 and 3 h. Our previous and present studies suggest that PKC down-regulates cell surface Na channels without altering the allosteric gating of Na channels via PKC isoform-specific mechanisms; cPKC-alpha promotes Na channel internalization, whereas nPKC-epsilon decreases the alpha-subunit mRNA level by shortening the half-life of alpha-subunit mRNA without changing its gene transcription.

Protein kinase C and the opposite regulation of sodium channel alpha- and beta1-subunit mRNA levels in adrenal chromaffin cells.

Our previous [3H]saxitoxin binding and 22Na influx assays showed that treatment of cultured bovine adrenal chromaffin cells with 12-O-tetradecanoylphorbol 13-acetate (TPA) or phorbol 12,13-dibutyrate (PDBu), an activator of protein kinase C (PKC), decreased the number of cell surface Na channels (IC50 = 19 nM) but did not alter their pharmacological properties; Na channel down-regulation developed within 3 h, reached the peak decrease of 53% at 15 h, and was mediated by transcriptional/translational events. In the present study, treatment with 100 nM TPA lowered the Na channel alpha-subunit mRNA level by 34 and 52% at 3 and 6 h, followed by restoration to the pretreatment level at 24 h, whereas 100 nM TPA elevated the Na channel beta1-subunit mRNA level by 13-61% between 12 and 48 h. Reduction of alpha-subunit mRNA level by TPA was concentration-dependent (IC50 = 18 nM) and was mimicked by PDBu but not by the biologically inactive 4alpha-TPA; it was prevented by H-7, an inhibitor of PKC, but not by HA-1004, a less active analogue of H7, or by H-89, an inhibitor of cyclic AMP-dependent protein kinase. Treatment with cycloheximide, an inhibitor of protein synthesis, per se sustainingly increased the alpha-subunit mRNA level and decreased the beta1-subunit mRNA level for 24 h; also, the TPA-induced decrease of alpha-subunit mRNA and increase of beta1-subunit mRNA were both totally prevented for 24 h by concurrent treatment with cycloheximide. Nuclear run-on assay showed that TPA treatment did not alter the transcriptional rate of the alpha-subunit gene. A stability study using actinomycin D, an inhibitor of RNA synthesis, revealed that TPA treatment shortened the t(1/2) of alpha-subunit mRNA from 18.8 to 3.7 h. These results suggest that Na channel alpha- and beta-subunit mRNA levels are differentially down- and up-regulated via PKC; the process may be mediated via an induction of as yet unidentified short-lived protein(s), which may culminate in the destabilization of alpha-subunit mRNA without altering alpha-subunit gene transcription.

Preovulatory Changes in the Levels of Three Gonadotropin-Releasing Hormone- Encoding Messenger Ribonucleic Acids (mRNAs), Gonadotropin I-Subunit mRNAs, Plasma Gonadotropin, and Steroids in the Female Gilthead Seabream, Sparus aurata 1

Gilthead seabream females undergo daily cycles of final oocyte maturation (FOM), ovulation, and spawning throughout their spawning season. FOM consists of lipid droplet and yolk granule coalescence, germinal vesicle (GV) migration, and GV breakdown. Plasma maturational gonadotropin (GtH-II) levels fluctuate throughout the day, reaching a peak at 8 h before spawning, when the GV is at the periphery of the oocyte. The preovulatory GtH-II surge is accompanied by an increase in the plasma levels of 17alpha,20beta-dihydroxy-4-pregnen-3-one and estradiol, while testosterone and 17alpha,20beta,21-trihydroxy-4-pregnen-3-one levels remain unchanged. Concurrent with the preovulatory GtH-II surge, there is an increase in pituitary GtH-II beta subunit mRNA levels followed by an increase in GtH-Ibeta mRNA levels. Gilthead seabream brain contains three different forms of GnRH: salmon (s)GnRH, seabream (sb)GnRH, and chicken (c)GnRH-II. All three GnRH-encoding mRNAs fluctuate throughout the day, reaching highest levels 8 h before spawning, concurrent with the preovulatory GtH-II surge. On the basis of these correlations and of the anatomical organization of the three GnRH systems, it is hypothesized that in the daily-spawning gilthead seabream females, preovulatory GtH-II secretion, and probably synthesis, are induced by a surge of sbGnRH secretion. The involvement of the other two GnRH forms, sGnRH and cGnRH-II, in the control of ovulation and spawning is presumed, on the basis of the elevation of their mRNA levels at the time of the preovulatory GtH-II secretion and spawning.

Up‐Regulation of Sodium Channel Subunit mRNAs and Their Cell Surface Expression by Antiepileptic Valproic Acid: Activation of Calcium Channel and Catecholamine Secretion in Adrenal Chromaffin Cells

Treatment of cultured bovine adrenal chromaffin cells with a therapeutic concentration (0.6 mM) of valproic acid (VPA) for > 24 h caused a time-dependent (t1/2 = 74 h) increase in [3H]saxitoxin binding up to 1.4-fold without altering the KD value; it was prevented by the simultaneous treatment with cycloheximide (an inhibitor of protein synthesis). VPA also raised Na+ channel alpha- and beta 1-subunit mRNA levels 1.4- and 1.7-fold at 24 h, and 1.6- and 1.8-fold at 72 h, respectively. Chronic (but not acute) exposure to VPA enhanced 22Na+ influx caused by various concentrations of veratridine 1.4-2.1-fold, even when assayed in the presence of Na+,K(+)-ATPase inhibitor, but did not change the EC50 value of veratridine. Ptychodiscus brevis toxin-3 allosterically potentiated veratridine-induced 22Na+ influx by approximately 2-fold in VPA-treated cells as in nontreated cells. Long-term treatment with VPA augmented veratridine-induced 45Ca2+ influx via voltage-dependent Ca2+ channels and catecholamine secretion, but had no effect on 45Ca2+ influx and catecholamine secretion caused by high K+ (a direct activation of voltage-dependent Ca2+ channels). Chronic treatment with VPA also enhanced nicotine-induced 22Na+ influx via the nicotinic receptor-ion channel complex 1.2-1.4-fold with little change in the EC50 value of nicotine, thereby increasing the nicotine-induced 45Ca2+ influx via voltage-dependent Ca2+ channels and catecholamine secretion. These results suggest that chronic treatment with VPA up-regulates cell surface expression of Na+ channels via the transcription/translation-dependent mechanisms, and probably of nicotinic receptors, thereby resulting in the enhancement of Ca2+ channel gating and catecholamine secretion.

Beta-Hexosaminidase immunolocalization and alpha- and beta-subunit gene expression in the rat testis and epididymis.

beta-hexosaminidase is an essential lysosomal enzyme whose absence in man results in a group of disorders, the G(M2) gangliosidoses. beta-hexosaminidase activity is many times higher in the epididymis than in other tissues, is present in sperm, and is postulated to be required for mammalian fertilization. To better understand which cells are responsible for beta-hexosaminidase expression and how it is regulated in the male reproductive system, we quantitated the mRNA expression of the alpha- and beta-subunits of beta-hexosaminidase and carried out immunocytochemical localization studies of the enzyme in the rat testis and epididymis. beta-hexosaminidase alpha-subunit mRNA was abundant and differentially expressed in the adult rat testis and epididymis, at 13- and 2-fold brain levels, respectively. In contrast, beta-subunit mRNA levels in the testis and epididymis were 0.3- and 5-fold brain levels. During testis development from 7-91 postnatal days of age, testis levels of alpha-subunit mRNA increased 10-fold and coincided with the appearance of spermatocytes and spermatids in the epithelium; in contrast, beta-subunit mRNA was expressed at low levels throughout tests development. In isolated male germ cells, beta-hexosaminidase alpha-subunit expression was most abundant in haploid round spermatids, whereas the beta-subunit mRNA was not detected in germ cells. Within the epididymis both alpha- and beta-subunit mRNA concentrations were highest in the corpus, with 1.5-fold and 9-fold initial segment values, respectively. Light microscopic immunocytochemistry revealed that beta-hexosaminidase was localized to Sertoli cells and interstitial macrophages in the testis. In the epididymis, beta-hexosaminidase staining was most intense in narrow cells in the initial segment, principal cells in the caput and proximal corpus, and clear cells throughout the duct. Electron microscopic immunocytochemistry revealed that beta-hexosaminidase was predominantly present in lysosomes in Sertoli and epididymal cells. The cellular and regional specificity of beta-hexosaminidase immunolocalization suggest an important role for the enzyme in testicular and epididymal functions.

Increased Hepatic Na,K-ATPase Activity during Hepatic Regeneration Is Associated with Induction of the β1-Subunit and Expression on the Bile Canalicular Domain

Cellular and molecular mechanisms regulating the activity of the sodium pump or Na,K-ATPase during proliferation of hepatocytes following 70% liver resection have not been defined. Na,K-ATPase may be regulated by synthesis of its alpha- and beta-subunits, by sorting to either the sinusoidal or apical plasma membrane domains, or by increasing membrane lipid fluidity. This study investigated the time course of changes during hepatic regeneration for Na, K-ATPase activity, lipid composition and fluidity, and protein content of liver plasma membrane subfractions. As early as 4 h after hepatic resection, Na,K-ATPase activity was increased selectively in the bile canalicular fraction. It reached a new steady state at 12 h and remained elevated for 2 days. Although hepatic regeneration was associated with a reduced cholesterol/phospholipid molar ratio and increased fluidity, measured with two different probes, these changes in lipid metabolism were in the sinusoidal membrane domain. The Na,K-ATPase beta1-subunit, but not the alpha1-subunit, was increased selectively at the bile canalicular surface as shown by immunoblotting of liver plasma membrane subfractions and the morphological demonstration at both the light and electron microscopic levels. Furthermore, cycloheximide blocked the rise in beta1-subunit mRNA levels. Since the time course for beta1-subunit accumulation was similar to that for activation of Na,K-ATPase activity, this change implicated the beta1-subunit in activating sodium pump activity.

Cardiac sodium channels expressed in a peripheral neurotumor-derived cell line, RT4-B8.

RT4-B is one of several cell lines derived from a multipotent stem cell line, RT4-AC, which originated from a rat peripheral neurotumor. Based on Northern blot and ribonuclease protection experiments, RT4-B8 cells have been proposed to express rat cardiac Na channel mRNA as the major isoform. We report here direct electrophysiological evidence that the expressed voltage-gated Na channels in the RT4-B8 cell line are of the cardiac phenotype with no evidence for subpopulations expressing other Na channel isoforms. Current activation half point (conductance) was -41 +/- 5 mV (n = 7) and the steady-state voltage-dependent availability half point was -89 +/- 1 mV. As expected for cardiac Na channels, the half concentration of block for tetrodotoxin block was 0.74 microM, for saxitoxin (STX) was 0.15 microM, and for the class 2B divalent cation Cd2+ was 67 microM. Block was well described by single-site dose-response relationships with no indication of a subpopulation with "neuronal" affinity. Single-channel conductance (140 mM Na+) was 10 pS and predicted the average number of channels open at peak Na current to be 3 channels/microns2. [3H]STX binding data were also consistent with a single population of low-affinity STX binding sites and predicted channel density to be 11 sites/microns2. No inwardly or outwardly rectifying K or Ca currents were detected electrophysiologically, although in some cells a small time-independent Cl current was detected. Reverse transcription-polymerase chain reaction of mRNA isolated from RT4-B8 cells demonstrated the presence of rat cardiac (rH1) and brain IIa alpha-subunit mRNA, as well as mRNA for the Na channel beta 1-subunit. Northern blot analysis confirmed the predominance of the rat cardiac Na mRNA compared with brain IIa. The beta 1-subunit mRNA levels were significantly lower than those detected in rat brain and heart mRNA but were comparable to the low level of beta 1-subunit mRNA detected in isolated rat ventricular myocytes.